Transposon end compositions and methods for modifying nucleic acids

The present application claims priority to provisional patent applications filed in the United States, serial numbers 61/108, 321, filed on October 24, 2008; 61 / 108,326, filed on October 24, 2008; 61 / 108,329, filed on October 24, 2008; 61 / 155,431, filed on February 25, 2009; and 61 / 184,530, filed on 5 June, 2009.

FIELD OF THE INVENTION [0002] The present invention relates to methods, sequences and kits for using transposase and transposon termination sequences to create a library of tagged DNA fragments from a target DNA. The resulting ssDNA fragments are used as standards, e.g. for various applications, including, e.g., massive parallel, high-throughput DNA sequencing, and / or multiplexed, high-throughput DNA sequencing.

BACKGROUND OF THE INVENTION [0003] There are a number of methods and uses for which it is desirable to create a library of fragmented and tagged DNA molecules from double-stranded target DNA (dsDNA) molecules. Often, the goal is to form smaller single-stranded DNA (ssDNA) molecules (e.g., DNA fragments) from larger dsDNA molecules for use as standards in DNA or RNA polymerase reactions (e.g., for use as standards in DNA sequencing reactions or in DNA amplification reactions or

RNA, where the primer connects to the tag and is extended by the polymerase).

[0004] Until recently, most DNA sequencing processes have been carried out using the Sanger chain dideoxy sequencing method, wherein the primer is extended by the polymerase using a sequenced DNA chain as template. Four reactions are carried out, each of which uses canonical nucleotides (dATP, dCTP, dGTP and dTTP) and one of four dideoxynucleotides terminating chain elongation (ddATP, ddCTP, ddGTP or ddTTP), whereby each nested fragment of the DNA chain is produced in each reaction. after the termination beginning with the primer and ending with the dideoxynucleotide. If such DNA molecules with the terminated chain are separated by electrophoresis in size, the sequence of ddNPT incorporation reflects the sequence of the template DNA. By using the present methods, a sequence of several hundred or several thousand bases can be determined from the primer side. Determination of more sequences requires the assembly of larger sequences from overlapping information from different clones.

[0005] Because these traditional methods require large amounts of DNA standards, and because these methods give unreliable results when large amounts of non-core DNA are present, DNA sequencing of the Sanger's dideoxy method is often carried out using cloned or amplified DNA. For example, most of the sequencing carried out in the Human Genome Project, officially launched in 1990 and ending with the announcement of the completion of the "initial version" of the human genome sequence in 2000 and the publication of the sequence of the last human chromosome in 2006, was based o gene libraries consisting of a population of host bacteria, each of which has incorporated a DNA molecule cloned into a DNA vector, so that a set of all DNA clones, each of which carried a fragment of genomic DNA, represented the entire genome. It was a tedious and highly iterative process involving the construction and banking of large amounts of DNA clones (e.g., BAC clones), which in turn were often subcloned to create libraries of smaller DNA clones used as sequential patterns. The primers used in these methods were often designed to combine with the vector in a way that would allow the prolongation of unknown cloned DNA in sequencing reactions. This approach allowed the same set of primers to be used for the analysis of many different clones. which in turn were often subcloned to create libraries of smaller DNA clones used as sequential patterns. The primers used in these methods were often designed to combine with the vector in a way that would allow the prolongation of unknown cloned DNA in sequencing reactions. This approach allowed the same set of primers to be used for the analysis of many different clones. which in turn were often subcloned to create libraries of smaller DNA clones used as sequential patterns. The primers used in these methods were often designed to combine with the vector in a way that would allow the prolongation of unknown cloned DNA in sequencing reactions. This approach allowed the same set of primers to be used for the analysis of many different clones.

[0006] In order to reduce the subcloning necessary for a human genome sequencing project, a method called "in vitro transposition" has been used from time to time. The in vitro transposition method includes the use of mobile genetic elements called transposons to insert a small piece of DNA of a known sequence into an unknown DNA. The method comprises incubating a DNA clone from a transposon genome library under conditions where a single transposon insertion occurs into a DNA clone, followed by transformation of E. coli cells by an in vitro transposition reaction and selection of a marker-containing cell such as an antibiotic-resistant marker, encoded by a transposon. . Thus, the in vitro transposition reaction allows the creation of a library of "insertive transposon clones" from the parent DNA clone, each of which will contain a transposon incorporated into a different location in the DNA clone. Each inserted clone will then be sequenced externally from each end of the transposon, using a different primer for each strand of DNA. As described above, the complete sequence of the parent DNA clone is generated by overlapping the sequences obtained from the various insertion clones. Examples of the use of such a transposon insertion method for the purposes of the Human Genome Project are described by Butterfield, YSN et al., Nucleic Acids Res 30: 2460-2468, 2002; Shevchenko, Y et al., Nucleic Acids Res. 30: 2469-2477, 2002; and Haapa, S et al., Genome Res 9: 308-315, 1999. The use of the in-vitro transposition process for the Human Genome Project project has facilitated the complete sequencing of both genomic DNA clones and cDNA clones created from mRNA encoded by genomic DNA. The disadvantage of the in vitro transposition method, however, is that it is not a full in vitro method because it requires transformation of E. coli cells, selection of E. coli colonies containing transposon inserts, and then isolation of DNA from insert transposon clones for sequencing.

[0007] In order to eliminate the need to transform E. coli cells by an in vitro transposition reaction and to grow E. coli cells on a selective medium to obtain insertive transposon clones, Teknanen et al. (US Patent No. 6,593,113) have developed completely transposable methods obtained in vitro, containing an in vitro transposition reaction and a PCR amplification reaction to select sequencing patterns. According to Teknanen et al., The DNA or target DNA used in their methods can range from several base pairs up to 40,000 base pairs, the only limiting factor in using longer DNA segments as target DNA is the inability of amplification reactions, such as PCR, to amplify longer segments. Hence, in some embodiments of creating sequencing patterns using this method, the DNA or target DNA of about 40 Kb to be tested is first subjected to an in vitro transposition reaction followed by PCR amplification using, as the first PCR primer, a constant primer complementary to the known sequence in the target DNA, or, if the target DNA is cloned in a vector, a solid primer complementary with the sequence in the vector, and as a second PCR primer, a selective primer complementary to the end of the transposon to which the target DNA is attached, plus, optionally, one to ten additional nucleotides with a known identity at the end of 3 '. In another embodiment, two selective primers are used for the PCR amplification step, of which at least one has one to ten additional nucleotides with a known identity at the 3 'end. The methods of Teknanen et al. Are preferred for Sanger sequencing because they eliminate the need to use E. coli cells for the selection of DNA molecules with the transposon being injected. These methods, however, are limited to target DNA of approximately 40 Kb and, due to the use of fixed or selective primers, these methods allow the selection of DNA molecules containing only part of the sequences contained in the target DNA. Accordingly, although these methods are useful for sequencing using the Sanger method, they are not suitable for creating sequential patterns for newer "next generation" DNA sequencing methods,

[0008] New generation sequencing platforms include the 454 FLX ™ or 454 TITANIUM ™ instruments (Roche), the SOLEXA ™ Genome Analyzer (Illumina) analyzer, the HELISCOPE ™ Single Molecule Sequencer (Helicos Biosciences) sequencer and the SOLID ™ DNA Sequencer DNA sequencer (Life Technologies / Applied Biosystems), as well as other platforms developed by companies such as Intelligent Biosystems and Pacific Biosystems. Although the chemical reactions that give information about the sequences vary depending on the new generation sequencing platform, all of them have a common feature - the creation of sequential data from a very large number of sequential patterns, where sequencing reactions occur in parallel. In general, data from all sequencing reactions are collected using a scanner, and then combined and analyzed by means of advanced bioinformatics programs. Sequencing reactions are performed, read, collected and analyzed in a mass parallel (MPS) or multiplexed manner. The use of MPS in instruments required a change in thinking about the type of sequential patterns needed and how to create them in order to obtain the maximum possible amount of sequential data from these advanced instruments. Hence, instead of relying on genomic libraries of DNA clones in E. coli, one should perceive a problem in terms of in vitro systems necessary to create libraries of DNA fragments containing a set or a population of DNA fragments derived from the target DNA in the sample, wherein the combination of all DNA fragments in the set or population comprises sequences representative in number and / or quality for the target DNA sequence from which the DNA fragments have been generated. In fact, in some cases a problem should be seen in terms of creating libraries of DNA fragments consisting of multiple libraries of genomic DNA fragments, each of which is labeled with a different tag or barcode to identify the source of each sequenced fragment.

[0009] In general, new-generation sequencing methods require the fragmentation of genomic DNA or double-stranded cDNA (obtained from RNA) into smaller ssDNA fragments and the addition of tags to at least one or, preferably, both strands of ssDNA fragments. In some methods, tags designate DNA sequencing sites using DNA polymerase. In some methods, tags also mean capture sites for fragments on a surface, e.g. beads (e.g., before PCR amplification by an emulsion technique for some methods, e.g. using methods described in US Patent No. 7,323,305). In most cases, the DNA fragment libraries used as the next generation sequencing patterns contain DNA fragments tagged at the 5 'and 3' ends or 'double tagged DNA fragments'. In general,

[0010] Current methods for creating next-generation sequential patterns are associated with many problems and ineffectiveness, as illustrated by the action scheme used at the Wellcome Trust Sanger Institute, one of the world's largest genomic research centers (e.g., described in Quail, MA et al., Nature Methods 5: 1005-1010, 2008). For example, Quail et al. Discovered that nebulization of genomic DNA for sequencing results in the loss of about half the DNA mass and about 5% of the original DNA consisting of fragments about 200 base pairs required for sequencing using the Illumina Genome Analyzer genome analyzer. . They invented an alternative method, called "adapted targeted acoustics", allowing for a larger amount of fragmented DNA and approx. 17% of the original DNA composed of fragments of the desired size of 200 base pairs, but even this process is not useful in terms of sampled or target DNA. In addition, the resulting DNA fragments often require size selection using gel electrophoresis, as well as additional steps to tag selected DNA fragments selected for size, which is a difficult task, laborious and time-consuming, and also expensive.

Hence, many of the methods currently used to fragment and tag double-stranded DNA for use in next-generation sequencing result in DNA loss, require expensive instruments for fragmentation, with procedures for fragmenting, tagging and recovering tagged DNA fragments difficult, tedious, time-consuming, inefficient , expensive and require a relatively large amount of sampled nucleic acids. In addition, many of these methods create tagged DNA fragments that are not fully representative of the sequences contained in the sampled nucleic acids. Therefore, in this field methods for creating libraries of double-tagged DNA fragments sequenced using the MPS method are necessary, and not overcoming the limitations posed by currently used methods.

[0012] Some of the new generation sequencing methods use circular ssDNA substrates in the sequencing process. For example, U.S. Patent Application No. 20090011943; 20090005252; 20080318796; 20080234136; 20080213771; 20070099208; and 20070072208 filed by Drmanac et al., disclose the formation of circular ssDNA patterns for the purposes of DNA sequencing using the MPS method. US Patent Application Publication No. 20080242560 filed by Gunderson and Steemers discloses methods comprising: creating digital circular DNA forms (see, e.g., FIG 8 in US Patent Application Publication No. 20080242560); and / or locus-specific cleavage and amplification of DNA, including by amplification with multiple displacements or whole genomic amplification (e.g., FIG. 17 therein) or with the use of hyperbranched RCA (e.g.

[0013] Improved methods, sequences and kits for creating tagged fragments of circular ssDNA from DNA from biological samples (e.g. from genomic DNA or mitochondrial or episomal DNA cloned in plasmids, BAC, fosmid, or other episomal vector) are required for use in amplification or DNA sequencing methods (such as the methods described in US Patent applications 20090011943; 20090005252; 20080318796; 20080234136; 20080213771; 20070099208; and 20070072208 reported by Drmanac et al. or in US Patent Application No. 20080242560 by Gunderson and Steemers or by Turner et al. Pacific Biosciences and published on their website <a href="http://www.pacificbiosciences.com">www.pacificbiosciences.com</a>).

[0014] In addition, some amplification methods, such as whole genome amplification, also require fragmentation and tagging of genomic DNA. Some of these methods have been evaluated in: Whole Genome Amplification, edited by S. Hughs and R. Lasken, 2005, Scion

Publishing Ltd (international site: <a href="http://www.scionpublishing.com">www.scionpublishing.com</a>).

[0015] Improved methods for creating libraries of DNA fragments from target DNA molecules are needed for the amplification of all or part of the genome from one organism (e.g. from a clinical trial) or from many organisms (e.g. metagenomic target DNA from an environmental sample) for further analysis (e.g., by real-time PCR, emulsion PCR, comparative genomic hybridization (CGH), comparative genomic sequencing (CGS), or for the preparation of probes for detection of specific DNA (e.g. chromosomal probes, e.g. chromosomal farms or e.g. son gene, e.g. for fluorescent in situ hybridization (FISH), for various purposes (e.g., for research, diagnostic and industrial purposes).

[0016] Hence, improved and more efficient methods for creating tagged DNA fragment libraries from target DNA for use in nucleic acid analysis methods, such as next-generation sequencing and amplification methods, are necessary in the art. Methods are needed to create libraries of DNA fragments that do not require specialized instruments, simpler, faster, less time-consuming and performed on smaller DNA samples and quantities, effective in tagging one or both ends of DNA fragments and forming tagged DNA fragments that are representative qualitatively and quantitatively for target nucleic acids in the sample from which they were generated. 503830871

SUMMARY OF THE INVENTION [0017] The present invention relates to methods for handling a nucleic acid and, in particular, methods and sequences for fragmenting and tagging DNA using a transposon sequence. The methods of the present invention are useful, for example, for creating libraries of tagged DNA fragments for use, e.g. in new generation sequencing methods, fluorescent in situ hybridization, and the like. In certain preferred embodiments, the present invention relates to the preparation of linear ssDNA fragments or tagged fragments of circular ssDNA (and their amplification products) from a target DNA containing dsDNA in the range of interest (including double-stranded cDNA obtained from RNA) from any source for genomic analysis subgenomic

[0018] In some embodiments, the present invention provides methods for creating a library of tagged DNA fragments derived from a target DNA comprising incubating a target DNA with a transposase and a transposon end or a transposon end sequence containing a transferable tag with a tagged domain at the 5 'end, under conditions where the transposition reaction is transposable catalyzed, and where the target DNA is fragmented to form multiple fragments of the target DNA, and the transferred transposon end or transposon end tag connects to the 5 'ends of each of the target DNA fragments to produce multiple 5' tagged fragments of the target DNA. In the present disclosure, the methods further comprise the incubation of a plurality of 5 'tagged at the end.

[0019] In some embodiments, the present invention provides methods for tagging a target DNA fragment comprising incubating a target DNA with transposase and a transposon end or transposon residue sequence comprising a transferred tag with a tagged domain at the 5 'end, under conditions where the transposition reaction is transposase catalyzed, and where the target DNA is fragmented and the transferred end-point of the transposon or the transposon end of the sequence is joined to the 5'-end of the target DNA fragment to produce the 5'-tagged target DNA fragment. In the present disclosure, the methods further comprise incubating the 5 'end tag of the target DNA fragment with a nucleic acid modifying enzyme under conditions where the 3' end tag connects to the 3 'end of the 5' tagged end. a fragment of the target DNA to create a double-tagged fragment of the target DNA. The methods are limited to the use of a specific nucleic acid modifying enzyme. For example, nucleic acid modification enzymes include polymerases, nucleases, ligases and the like. In the present disclosure, the nucleic acid modification enzyme comprises DNA polymerase, and the 3 'end tag is made by extending the 3' end of the tagged target DNA fragment at the 5 'end. In some embodiments, the DNA polymerase comprises a reference DNA polymerase, and in some other embodiments, the DNA polymerase comprises a pattern-independent DNA polymerase. In certain preferred embodiments, the DNA polymerase is a template-dependent polymerase having a thread transfer activity and / or a 5 'nuclease activity.

[0020] In some embodiments, the nucleic acid modification enzyme used in the present methods is a ligase, and the 3 'end tag is formed by ligation of the oligonucleotide to the 3' end of the tagged target DNA fragment at the 5 'end. In some embodiments of the disclosure, the ligase contains a standard-dependent ligase, while in some other examples, the ligase contains a standard-independent ligase.

[0021] In some embodiments, the transfer ends contain capture tag domains. In the present disclosure, tag domains comprise one or more restriction site tag domain, sequencing tag domain, amplification tag domain, discovery tag domain, addressing address domain, and transcriptional promoter domain. In some embodiments, the tag domains are sequencing tag domains comprising or consisting of sequencing tags selected from the Roche 454A and 454B sequencing sequences, ILLUMINA ™ sequencing tags, SOLEXA ™, SOLID ™ sequencing ™ labels from Applied Biosystems', Pacific Biosciences' SMRT sequencing tags'. ™, Pollon Polony sequencing tags or Complete Genomics sequencing tags.

[0022] Some embodiments of the disclosure further comprise amplifying one or more 5 'tagged fragments of the target DNA and / or double tagged fragments of the target DNA. In the disclosure, the amplification comprises the use of one or more PCR amplification reactions, a moving thread amplification (SDA) reaction, an RCA amplification reaction, a ligase chain reaction, a transcriptional amplification reaction (TMA) or a loop amplification (LMA) reaction. In certain preferred embodiments of the disclosure, the amplification includes non-selectively amplified tagged 5 'end fragments of a target DNA containing a DNA fragment library or double tagged DNA fragments containing a DNA fragment library.

[0023] In some embodiments of the present disclosure, a transposon sequence used to tag or fragment a library comprises a plurality of transferred strands differing in nucleic acid sequence by at least one nucleotide, and the amplification comprises selective amplification of double tagged DNA fragments based on nucleic acid sequences of tags. end of 5 'or tag domains. In other embodiments, the amplification comprises a polymerase chain reaction using a single oligonucleotide primer complementary to the 3 'end tag of the double tagged fragments of the target DNA. [0024] In some embodiments of the disclosure, the amplification comprises a moving thread amplification (SDA) reaction using a single oligonucleotide primer,

In some embodiments of the disclosure, the amplification comprises a polymerase chain reaction using the first and second oligonucleotide primers, each of which has a 3 'terminal portion, wherein at least a portion of the 3' end of the first PCR primer is complementary to the double tag tag. fragments of the target DNA, and wherein at least a portion of the 3 'end of the second PCR primer has a sequence of at least a portion of the 5' end tag or tag domain of the double tagged fragments of the target DNA. In embodiments of the disclosure, the first or second oligonucleotide primer comprises a 5 'end portion, wherein the PCR primer comprises a sequence of at least a portion of the 5' end tag or tag domain of the double tagged DNA fragments. In embodiments of the disclosure, the first or second oligonucleotide primer includes a 5 'terminal portion where at least a portion of the first 5' end is not complementary to the 3 'tag of the double tagged fragments of the target DNA, or where the second end 5' of the second primer does not contain a sequence of the least part of the 5 'end tag or tag domain of the double tagged fragments of the target DNA. In embodiments of the disclosure, the first and second oligonucleotide primers comprise a 5 'terminal portion where at least a portion of the 5' end of the first PCR primer is not complementary to the 3 'tag of the double tagged fragments of the target DNA, and / or where the 5' terminal portion of the second primer The PCR does not contain sequences of at least a portion of the 5 'end tag domain of the double tagged fragments of the target DNA.

[0026] In certain embodiments, the present disclosure provides methods for forming a population of tagged circular DNA fragments from a target DNA. In certain embodiments, this includes incubating the target DNA with a transposase and transposon end or a transposon end containing a transferable thread having a tag domain on the 5 'end portion and a transposon end at the 3' end, under conditions where the transposition reaction is transposase catalysed to subject The fragment DNA of the fragmentation to create multiple fragments of the target DNA and the transferred end of the transposon or transposon end of the transposon joined the 5 'end of each of the multiple fragments of the target DNA to produce a 5' tagged fragment of the target DNA.

[0027] In the disclosed embodiments, it is desirable to cleave such circular single-stranded DNA. Hence, in some embodiments of the methods of the present disclosure, the tag domain comprises a cleavage site sequence or structure and the method comprises: incubating tagged portions of circular single-stranded DNA with at least one enzyme comprising a sequence of cleavable enzymes, wherein the sequence of cleaving enzymes cleaves tagged fragments of circular single-stranded DNA on double-tagged fragments of linear single-stranded DNA. In the disclosed embodiments, the sequence of splitting enzymes comprises a restriction enzyme. In some embodiments, the tag domain comprises a sequence of restriction sites,

[0028] In some embodiments of the disclosure, amplification of the fragments and libraries of the invention is useful. Hence, some examples of the disclosure include the amplification of one or more tagged fragments of circular single-stranded DNA and / or double-tagged fragments of linear single-stranded DNA. In the disclosed embodiments, the amplification comprises a polymerase chain reaction employing a first and a second oligonucleotide primer, each of which has 3 'terminal portions, where at least a portion of the 3' end of the first PCR primer is complementary to at least a complement of the transfer portions in tagged circular single-stranded fragments. DNA or double tagged fragments of linear single-stranded DNA.

[0029] In the disclosed embodiments in which tagged circular single-stranded DNA fragments or double tagged fragments of circular single-stranded DNA are amplified, the first and second oligonucleotide primers comprise a 5'-end portion, where the 5'-end portion of the first PCR primer is not complementary to the sequence of the transferred strand in tagged circular single-stranded DNA fragments or double-tagged fragments of linear single-stranded DNA and wherein a portion of the 5'-end of the second PCR primer is not complementary with the complement of the transfer strand in tagged circular single-stranded DNA fragments or double-tagged linear DNA strand fragments.

each of a plurality of target DNA fragments to create a population of tagged target DNA fragments at the 5 'end. In some embodiments of the disclosure, the method further comprises filling gaps and ligation of cuts in DNA fragment fragments. In some embodiments of the disclosure, the above comprises incubating 5 'tagged with target-bound ligase and DNA polymerase without 5'3' exonuclease exonuclease, 3'5 exonuclease activity, and thread transfer activity, or one or more oligonucleotides with a random sequence. and one or different sizes that, alone or in combination, have the same length as single-hole gaps in the 5 'tagged DNA fragments resulting from transposase transposition reactions and the hairpin end of a transposon sequence, in conditions where single-stranded gaps in the 5 'tagged target DNA fragments are filled and the 3' end of the DNA tag tagged at the 5 'end is fused with the 5 'end of another 5' tagged DNA fragment containing a complementary portion of the target DNA to create tagged fragments of circular DNA containing the tag domain in the loop structures and both strands of the target DNA part. In some embodiments of the disclosure, padding and combining include incubation of DNA-tagged DNA fragments at the 5 'end, under conditions where the 3' end of each 5 'tagged at the end. The DNA fragment is elongated (extended) to form a population of extension products tagged at the 5 'end of the DNA fragments, and incubation of extension products tagged at the 5' end of the DNA fragments with a standard-dependent ligase under conditions where the products extend the 5 'tagged DNA fragments they are ligated to form tagged fragments of circular DNA. In embodiments of the disclosure, the DNA polymerase and ligase are provided in the form of a mixture, and the filling and ligation are carried out in a single reaction of the mixture. In certain embodiments of the disclosure, padding and ligation include incubation of 5'-tagged DNA fragments with oligonucleotides with a random sequence and one or more sizes, and a standard-dependent ligase under conditions,

[0031] In embodiments of the disclosure, the method further comprises separating tagged DNA circular fragments from linear DNA, non-ligated, random sequence oligonucleotides, and / or a hairpin-like transposon sequence not attached to the target DNA. In said embodiments, a reaction mixture containing tagged portions of the circular DNA is treated with T5 exonuclease to remove linear DNA, such as non-ligated fragments and oligonucleotides with a random sequence.

[0032] In embodiments of the disclosure, it is desirable to cleave tagged circular DNA molecules. For example, the method includes the step of: cleaving tagged portions of circular DNA in a loop structure to form blunted double-stranded DNA fragments, each tag having a tag portion at the 5 'end and a tag portion at the 3' end. Thus, the tag domain of the loop structures includes a sequence or cleavage site and the method further comprises: incubating tagged portions of circular single-stranded DNA with at least one enzyme containing a sequence of cleaving enzymes, where the sequence of cleavage enzymes cleaves tagged fragments of circular single-stranded DNA into blunt-ended double-stranded fragments GOUT.

[0033] In some embodiments of the disclosure, the sequence of cleavage enzymes comprises N-glycosyllase and an AP endonuclease. In said embodiments, the cleavage enzyme is N-glykozylase selected from uracil-N-glycosylase and endonuclease of AP and FPG protein, and the endonuclease of AP is selected from endonuclease III or E. coli endonuclease IV.

[0034] In embodiments of the disclosure, the sequence of splitting enzymes comprises a restriction enzyme. In said embodiments, the tag domain comprises a restriction site sequence and the method further comprises coupling tagged circular DNA fragment fragments complementary to the tag domain, and incubating tagged fragments of circular single-stranded DNA with a restriction endonuclease recognizing the restriction site, where restriction endonuclease cleaves tagged circular fragments. single-stranded DNA for blunt-ended portions of double-stranded DNA, each of which has a portion of the tag at the 5 'end and a portion of the tag at the 3' end.

[0035] Some of the disclosures further comprise denaturation of blunt-ended double-stranded DNA fragments to double-tagged linear-single-stranded DNA fragments.

[0036] The disclosures regarding circular and blunt DNA find use in methods comprising the use of DNA fragments as standards in a DNA sequencing method or in an amplification reaction. Hence, the methods of the present disclosure further comprise amplification of tagged DNA circular fragments, blunt-ended double-stranded DNA fragments, and / or double tagged fragments of linear single-stranded DNA. Amplification uses one or more PCR amplification reactions, a moving thread amplification reaction, an RCA amplification reaction, a ligase chain reaction, a transcriptional amplification reaction (TMA) or a loop-based amplification reaction (LMA). In the disclosure, the amplification comprises a polymerase chain reaction using the first and second oligonucleotide primers, each of which has end portions 4 'where at least a portion of the 3' end of the first PCR primer is complementary to at least a portion of the tag domain, and wherein at least a portion of the 3 'end of the second PCR primer comprises a sequence of at least a portion of the tag domain. In the disclosure, the first and second oligonucleotide primers comprise portions of the 5 'end, wherein the 5' end portion of the first PCR primer is not complementary to the tag sequence, and wherein the 5 'end portion of the second PCR primer does not contain the tag domain sequence.

[0037] The disclosure of any of the PCR amplifications described above includes amplifications, wherein the 5 'end portions of the first and / or the second PCR primers contain tag domains. In said disclosure, the tag domains comprise one or more restriction site domains, capture tag domain, sequencing tag domain, amplification tag domain, detection tag domain, addressing address domain, and transcriptional promoter domain.

[0038] In particularly preferred embodiments of the methods described herein, tag domains are sequencing tag domains comprising or consisting of sequencing tags selected from Roche 454A and 454B sequencing tags, ILLUMINA ™ sequencing tags, SOLEXA ™, SOLID ™ sequencing ™ tags from Applied Biosystems' , Pacific Biosciences' SMRT ™ sequencing tags, Pollonator Polony sequencing tags or Complete Genomics sequencing tags. In some embodiments, the present invention comprises methods for tagging a target DNA fragment comprising incubating the target DNA with transposase and a transposon end sequence comprising a transferred thread comprising, at the 5 'end, the tag domain sequence not being the end of the transposon and, in the 3' end part. .

[0040] In some embodiments, the present invention provides methods for creating libraries tagged at the 5'-end of DNA fragments containing a transposase target DNA incubation and a transposon termination sequence comprising transferring threads comprising, at the 5 'end portions, a sequence of one or more tag domains with specific and, at the 3 'end, the transposon end of the transfer sequence, under conditions where the transposition reaction is transposase catalyzed, where the target DNA is fragmented, and the transferred trans-end sequence strands are attached to the 5' ends of the target DNA fragments to form tagged on the 5 'end fragments of the target DNA, so that a number of 5' tagged endpoints result from the transposition reactionfragments of the target DNA containing the library of DNA fragments from the target DNA.

[0041] The present invention also encompasses sequences. For example, the present disclosure includes a sequence comprising a synthetic nucleic acid molecule having a 5 'end portion containing the tag domain and a 3' end portion comprising the transferred transposon end thread. In some embodiments, the disclosure comprises a plurality of synthetic nucleic acid molecules, wherein the nucleic acid molecules comprise 5 &amp; apos; end portions containing tag domains differing by at least one nucleotide, and end portions 3 &amp; apos; containing the transferred end of the transposon.

[0042] In the sequence disclosure described above, at least a portion of the 3 'terminus of the nucleic acid molecule is double-stranded DNA.

[0043] In embodiments, the end of the transposon is the end of the Tn5 transposon, while in other embodiments, the end of the transposon is the end of the transposon.

Him.

[0044] The tag sequence domain of nucleic acid molecules comprises a capture tag domain, or in other embodiments, a sequencing tag domain, amplification tag domain, discovery tag domain, addressing address domain, and a transcriptional promoter domain or restriction site domain. In particularly preferred embodiments, the tag domains comprise sequencing tags comprising or consisting of sequencing tags selected from Roche sequencing labels 454A and 454B, ILLUMINA ™ sequencing tags, SOLEXA ™, SOLID ™ sequencing ™ labels from Applied Biosystems ', Pacific Biosciences' SMRT sequencing labels ™, Pollon Polony sequencing tags or Complete Genomics sequencing tags.

[0045] In the disclosure, the sequence comprising the nucleic acid molecule further comprises purified transposase. In the disclosure, the transposase is selected from the Tn5 transposase and Mu transposase. In disclosure, the nucleic acid molecule and transposase are provided in a mixture. In disclosure, the mixture further comprises a nonionic detergent. In more preferred embodiments, the ionic detergent contains Nonidet P-40 and / or Tween-20. [0046] The present disclosure provides a kit comprising any of the sequences described above or elsewhere in the present invention. In said disclosure, the kit further comprises one or more ligases, a polymerase, and / or reagents for the amplification reaction. In said disclosure, the reagents for the amplification reaction contain reagents for the polymerase chain reaction. In the said disclosure, the reagents or the amplification reaction and / or the polymerase chain reaction contain at least one primer, and in particularly preferred embodiments, the reagents comprise a primer comprising a 3 'terminal portion complementary to the complement domain of the 5' terminal part of the nucleic acid molecule. In disclosure, the tag domain includes one or more restriction site domain, capture tag domain, sequencing tag domain, amplification tag domain, discovery tag domain, addressing domain domain and transcriptional promoter domain, and in particularly preferred embodiments, the tag domain comprises a sequencing tag domain comprising or consisting of sequencing tags selected from the Roche 454A and 454B sequencing tags, ILLUMINA ™ sequencing tags, SOLEXA ™,

[0047] In the disclosure, the kit of the present invention further comprises reagents for DNA sequencing reactions.

[0048] The disclosure comprises a reaction mixture comprising target double-stranded DNA and any of the nucleic acid molecules comprising the tagged end of the transposon and / or the transposase sequence described above.

[0049] The present disclosure provides a sequence comprising purified transposase and a plurality of synthetic transposon or sequence ends of the transposon. In said disclosure, the transposon end sequences contain hairpin-like ends of the transposon, while in some embodiments, the synthetic transposon ends or the transposon end sequences contain separate transferred threads and non-transferred threads. In disclosure, the transfer threads comprise 5 'end domain domains, e.g. containing one or more restriction site domains, capture tag domain, sequencing tag domain, amplification tag domain, detection tag domain, addressing address domain, and transcriptional promoter domain. In disclosure,

[0050] In the disclosure, the sequence containing the purified transposase comprises a plurality of synthetic transposon ends comprising at least two transferred threads differing from each other by at least one nucleotide, and in preferred embodiments, the transferred strands comprise the 5 'end portions and the 3' end portions, where the at least two parts of the 5 'end of the transferred strands contain such differing at least one nucleotide and where the 3' terminal portions of the transferred strands contain the same transposon end sequence.

[0051] The end of the transposon contains the Mu transposon ends, and the transposase is Mu transposase, and in some preferred embodiments, the 3 'terminal portions of the transferred strings contain a sequence from the Mu transpomon end, wherein the 5' terminal portions of the transferred strands are not from the Mu transposon.

[0052] In some embodiments, the transposon ends comprise Tn5 transposase ends , and the transposase is Tn5 transposase, and in some preferred embodiments, the 3 'end portions of the transferred strands contain a sequence from the Tn5 transposon end, with the 5' end portions of the transferred threads not being transposable. they come from the Tn5 transposon.

[0053] The present disclosure provides sequences comprising a library of DNA fragments, wherein the DNA fragment library comprises fragments of the target DNA having 5 'ends containing sequences from the transferred strands from the transposon ends or the sequence of the transposon ends. Sequences from the transferred strands contain 5 'end tag domains, and in more preferred embodiments, the DNA fragment library contains target DNA fragments containing 3' end tags' complementary to the transfer thread from the end of the transposon or sequence of the transposon ends. In disclosure, the DNA fragment library contains double-stranded fragments of the target DNA.

[0054] The present disclosure provides sequences containing a tagged fragment of circular DNA comprising a sequence comprising a sequence of non-transferred strands at the 5 'end of the part, a sequence of transferred strands at the 3' end of the part, a spacer loop sequence containing the tag domain, sequences of both strands of the target DNA part.

[0055] The present disclosure provides sequences containing a tagged fragment of circular single-stranded DNA comprising a sequence of transferred strands from the transposon end or sequence of the transposon ends as well as a single-stranded portion of the target DNA. In the aforementioned embodiments, the sequence of transferred threads includes the tag domain.

[0056] The present disclosure provides sequences comprising a blunt-ended (double-stranded) DNA strand derived from a target DNA, comprising a double stranded target DNA, each strand having a 5 'end containing at least a portion of the sequence of the transferred strands and containing at least a portion of sequence of non-transferred threads.

[0057] Embodiments of the invention are described in this summary and also in the Detailed Description of the Invention below, incorporated herein by reference.

[0058] Embodiments of the invention have been described in this summary and also in the Detailed Description of the Invention below.

[0059] Although the invention has been described with reference to specific embodiments, it should be understood that the invention, as patented, should not unduly be limited to such particular embodiments.

BRIEF DESCRIPTION OF THE DRAWINGS [0060] The following figures are part of the present specification and are included to demonstrate specific aspects of the present invention. The invention may be better understood by referring to one or more of the figures in combination with a detailed description of the particular embodiments illustrated herein.

FIG. 1 is a schematic diagram illustrating the insertion of a transposon into a target DNA in a transposition reaction.

FIG. 2 is a schematic diagram illustrating the fragmentation and tagging of target DNA by insertion of the transposon ends in the transposition reaction.

FIG. FIG. 3 is a schematic diagram of the products of two transposon transposon transposon transposon insertion events. Hence, the transposon transposon end-to-end transposon insertion product shown on the left of the figure shows the orientation of the transposon end, where the transfer of the transposon end of the sequence (i.e. where the transposon end transferred contains the sequence 5 'AGATGTGTATAAGAGACAG 3' (SEQ ID NO: 1), end 3 'attached to the target DNA) is located in the upper strand, and the insertion product of the transposon transposon end sequences shown on the right side of the figure shows the orientation of the transposon end, where the transferred thread is the bottom strand. Unprocessed thread is labeled with SEQ ID NO: 2.

FIG. 4 illustrates examples of two different tagged transposon ends, each of which contains an oligonucleotide of the transfer strand with a different tag on the 5 'termini portion for use in the generation of tagged DNA fragment library. Extending the 3 'ends of each strand using, e.g., DNA polymerase with 5' nuclease activity or strand-threading activity, creates double-tagged ssDNA fragments. The sequence of the transferred cord is shown as SEQ ID NO: 1; a non-transferred thread as SEQ ID NO: 2.

FIG. 5 is an image of an agarose gel and an illustration of the order of magnitude of transposition products tagged at the 5 'end of the DNA fragments using different transposome concentrations.

FIG. 6 shows an image of an agarose gel and an illustration of the order of magnitude of transposition products tagged at the 5'-end of DNA fragments obtained over five-minute reactions at different temperatures using different reaction buffers in the presence or absence of dimethylformamide (DMF).

FIG. 7 illustrates an example of a method where a DNA polymerase having a thread transfer activity and / or a 5'-do-3 'exonuclease activity is used to attach the complement of the transfer strand to 5' tagged DNA fragments from an in vitro transposition reaction to create a fragment library DNA containing double tagged ssDNA fragments. As shown, the strand displacement activity and / or 5'-to-3 'exonuclease activity of the DNA polymerase displaces or cleaves the DNA of the DNA polymerase extension product fused below, and the DNA polymerase extended fragment binds to the second tag containing or consisting of a DNA sequence complementary to the first tag inserted into the opposite thread. In some embodiments, the products of double tagged DNA fragments are amplified by PCR using oligonucleotides complementary to the complement of the transferred strand. The sequence of the transferred cord is shown as SEQ ID NO: 1; a non-transferred thread as SEQ ID NO: 2.

FIG. 8 illustrates an example of a method where a DNA polymerase having a thread transfer activity and / or a 5'-to-3 'exonuclease activity is used to attach a second tag to the 5' tagged DNA fragments from an in vitro transposition reaction to create a library formation target DNA fragments containing double tagged DNA fragments. As shown, the strand displacement activity and / or 5'-to-3 'exonuclease activity of the DNA polymerase displaces or cleaves the DNA of the DNA polymerase extension product fused below, and the DNA polymerase extended fragment binds to the second tag containing or consisting of a DNA sequence complementary to the first tag inserted into the opposite thread. In some embodiments, the products of double tagged ssDNA fragments are amplified by PCR using oligonucleotides complementary to the various sequences in the first and second tag respectively, as PCR primers. The sequence of the transferred cord is shown as SEQ ID NO: 1; a non-transferred thread as SEQ ID NO: 2.

FIG. 9 shows a comparison of the sequencing reading, accuracy and coverage of a single contiguous sequence using a DNA fragment library made in accordance with the embodiments of the present invention as compared to a control library formed by nebulization.

FIG. 10 is a schematic diagram showing the fragmentation and tagging of target DNA to form a Roche / 454 compatible library by inserting tagged transposon ends in a transposition reaction followed by PCR specific + / bar codes.

FIG. 11 is an image of an agarose gel showing input DNA (lane 2), an order of magnitude of transposition products tagged at the 5 'end of the DNA fragments (lane 3), an order of magnitude of PCR products (lane 4) and a control reaction (lane 5) for the preparation of the sequencing library labeled with barcodes and compatible with Roche / 454 FLX as shown in FIG. 10.

12 is an image of an agarose gel showing the order of magnitude of transposition products tagged at the 5'-end of DNA fragments (lane 2), the order of magnitude of PCR products (lane 3), and control reaction (lane 5) for the preparation of barcode-sequenced sequencing library and Roche-compatible sequencing / 454 FLX Titanium from amplicon DNA, similar to the method shown in FIG. 10.

FIG. 13 is a schematic diagram showing fragmentation and tagging of target DNA to create an Illumina / Solexa compatible library by inserting tagged transposon ends in a transposition reaction, followed by PCR specific + / bar codes.

FIG 14 is an image of an agarose gel showing input DNA (lane 2), an order of magnitude of transposition products tagged at the 5 'end of DNA fragments (lane 3), an order of magnitude of PCR products (lane 4) and a control reaction (lane 5) for the preparation of a sequencing library labeled with bar codes and compatible with Illumina GAII as shown in FIG. 13.

FIG. 15 compares the process and complexity of previous methods for preparing libraries known to the art to prepare a library of DNA fragments according to embodiments of the present invention.

FIG. 16. shows an example of a product obtained by the method of the invention after transposase incubation (e.g., EZ-Tn5 ™ transposase) and a hairpin transposon end (e.g., the EZ-Tn5 ™ hairpin transposon sequence shown herein) "PMETS-N-MENTS") by in vitro transposase reaction in the presence of double-stranded target DNA (e.g., genomic DNA or double-stranded cDNA).

FIG. 17. Fragmentation and tagging of the target DNA with the sequence of hairpin-like transposon ends. FIG. 17A is a schematic plot of a product (in a population of many such products) resulting from two transposase-mediated transposase insertion events of the hairpin to the target DNA sequence. Briefly, target DNA (e.g., containing double-stranded genomic DNA or cDNA) is incubated by in vitro transposase transposase containing transposase and a hairpin-like transposon sequence (e.g., the EZ-Tn5 ™ transposon shown herein and the transposon end of the hairpin EZ-Tn5 ™ hair). The sequence of the transferred end of each of the transposon end sequences is linked in a loop structure to the sequence of the non-transferred terminus of the transposon. The loop may have any arbitrary tag domain, including the restriction domain domain, capture tag domain, sequencing tag domain, detection tag domain, addressing tag domain, transcriptional promoter domain or amplification tag domain. For example, the domain of the sequencing tag may comprise the sequencing sequence of Roche 454A or 454B. For example, in the figure, the sequencing tag domain comprises one or more sequences in the loop between the complementary transposon end (stem) sequences. FIG. 17B shows a 1% SYBR Gold stained agarose gel for electrophoresis of 5 'tagging products and fragmentation of 1 μg of D7 D1 genomic dsDNA using 0, 0.5, 1, 2,

FIG. 18. Generation of patterns of 5 'circular DNA tagged at the 5' end. FIG 18A is a schematic diagram of one embodiment of the method. Briefly, the 9 nucleotide voids formed by the two insertions of the transposon end sequences into the target DNA are filled with the 3 'termination of the 5' tagged DNA fragments formed using DNA polymerase not containing 5'-to-3 exonuclease activity and thread displacement activity (e.g., polymerase T4 DNA) and single-stranded DNA in the gaps regions as standards, followed by DNA polymerase extension products tagged at the 5 'end of the DNA fragments are ligated using a template-dependent ligase (e.g., E. coli DNA ligase) to create tagged DNA circular fragments. . Tagged fragments of circular DNA are resistant to T5 exonuclease activity, used in the embodiment shown in the figure to remove un-ligated linear single-stranded and double-stranded DNA. FIG. 18B shows a 1% SYBR Gold stained agarose gel for electrophoresis of reaction products formed in the presence or absence of T4 DNA polymerase and / or E. coli DNA ligase. As shown, tagged fragments of circular DNA, resistant to T5 exonuclease activity, were formed only in the presence of both T4 DNA polymerase and E. coli DNA ligase.

FIG. 19 illustrates the use of terminal transferase to link the second tag (3 ') to 5' tagged at the 5 'end to generate a library of DNA fragments containing 5' and 3 'tagged (double-tagged) ssDNA fragments. In the illustrated embodiment, a domain of the sequencing tag containing such sequencing (SEQ) is added using a standard-dependent DNA polymerase.

FIG. 20 illustrates the use of a terminally-tagged oligonucleotide as a template for adding a second tag (3 ') to the 5'-tagged ssDNA fragments to create a library of DNA fragments containing double tagged ssDNA fragments.

FIG. 21 illustrates an embodiment where the 5 'tagged DNA fragments are incubated in the presence of a DNA ligase and a ligation tagged oligonucleotide comprising a 5' portion having a phosphate group at the 5 'end and containing a random sequence connecting to a gap or consisting of 9 bases or a single-stranded DNA region resulting from the insertion of an EZ-Tn5 transposon-catalyzed EZ-Tn5 transposon end of the EZ-Tn5 ME transposon into the target DNA, and a 3 'part containing the sequence of the second tag (tag # 2). In this example, the ligation tagged oligonucleotide has a 5 'end portion containing a random sequence of 6 nucleotides. A random sequence combines with single-stranded target DNA in the 9-base gap regions created by insertion of the transposon ends (e.g. the end of the EZ-Tn5 mosaic consisting of 19 base pairs or the transposon end of ME) to the double-stranded target DNA. Such ligation tagged oligonucleotides linking to single-stranded target DNA in gaps regions such that the phosphorylated 5 'end adheres to the 3' end of the tagged 5 'DNA fragment, and then combines with the nucleic acid ligase in a ligase dependent reaction. pattern, creating tagged 5 'and 3' endings of DNA fragments with the first tag at the 5 'end and the second tag at the 3' end. Hence, if insertions of the ends of the transposon appear on both strands of the target DNA at a short distance (eg in places on the target DNA in the range of about 50 Kb, about 40 Kb, about 30 Kb, about 20 Kb, about 10 Kb , about 5 Kb, about 1 Kb, about 500 base pairs or, preferably in the range of about 150 base pairs up to about

FIG. 22 (A) is a schematic diagram of an embodiment of DNA fragmentation, tagging and circularization of genomic DNA using the method of the invention. The labeled field with a black line represents the sequence of the transposon ends of p454.1MEDS. Dashed lines represent fragments of the D7 D111 genomic dsDNA. FIG. 22 (B) shows the agarose gel of 5 'tagging products and the D7 DDA dsDNA genomic fragmentation using the p454.1MEDS transposon transposon end and the EZ-Tn5 ™ transposase (EPICENTER Biotechnologies). One μg of D7 D111 genomic dsDNA was incubated with or without 0.5 μΜ of the transposon end of p454.1MEDS in 33 mM triacetate pH 7.6, 66 mM KOAc and 10 mM Mg (OAc) 2 for 1 hour at 37 ° C, both in the presence or absence of the EZ-Tn5 transposase, as indicated. Reaction products tagged at the 5 'end

FIG. 23 (A) shows a PCR amplification scheme of tagged fragments of circular ssDNA using oligonucleotides p454.1 and pMETS as PCR primers.

FIG. 23 (B) shows the agarose gel of the PCR amplification products obtained when the tagged circular ssDNA fragments obtained using the method of the invention were amplified by PCR using oligonucleotides pMETS and pc454.1 as PCR primers. First, the fragmented dsDNA tagged at the 5 'end, obtained as shown in FIG. 22 (B), denatured by heating at 95 ° C for 3 minutes and rapid cooling on ice. A portion of the resulting denatured 5'-labeled linear ssDNA fragments were incubated in 33 mM triacetate pH 7.6, 66 mM KOAc, 2.5 mM MnCl 2 and 1M betaine for 2 hours at 60 ° C in the presence or absence of 400 units of ssDNA ligase. CIRCLIGASE ™, according to Example 17 for the circularization of the linearized ssDNA fragments tagged at the 5 'end. The reaction products were incubated in exonuclease II and exonuclease III for 1 hour at 37 ° C to etch the linear ssDNA fragments prior to PCR amplification. The tagged fragments of circular ssDNA were further amplified by PCR using oligonucleotides pMETS and pc454.1 as PCR primers. The PCR products were dissolved in 1% agarose electrophoresis and visualized with SYBR Gold staining.

DEFINITIONS [0061] Unless the definitions or description provides otherwise, the following terms and descriptions related to the invention will be understood as set out below.

[0062] If the terms "e.g." "e.g.", "such as", "including", "including" or related are used in this application, these terms will not be construed as limiting and shall be construed as meaning "including, in a non-limiting manner "or" in particular ".

[0063] The use of definite and indefinite articles and the like in the context of the description of the invention (especially in the context of the following claims) will be interpreted so that these articles include both single and plural forms, unless otherwise indicated herein, or when the context indicates otherwise.

[0064] The use of the term "isolated", "isolated", "isolation", "purified", "purify", "purification" and their respective grammatical forms in this application, unless otherwise indicated, will refer to the reduction of the amount at least one contaminant (such as a protein and / or nucleic acid sequence) from the sample or source (e.g., cells) from which the material is isolated. Hence, purification leads to "enrichment", i.e. an increase in the amount of desired protein and / or nucleic acid sequences in the sample.

[0065] The use of the term "tag" in the present application will mean a non-target nucleic acid element, usually DNA, allowing the addressing of the nucleic acid fragment to which it binds. For example, in preferred embodiments, the tag comprises a nucleotide sequence that identifies, recognizes, and / or manipulates the molecular or biochemical DNA to which the tag is attached (e.g., by providing a site for attachment of an oligonucleotide, such as a primer for extension by DNA polymerase, or an oligonucleotide for capture or ligation reaction). The process of attaching a tag to a DNA molecule is sometimes referred to in the present application as "tagging" and DNA subjected to tagging or containing a tag called "tagged" (e.g., "tagged DNA").

[0066] As used in the present application, the term "ligase" will refer to a nucleic acid modifying enzyme that catalyzes the formation of intermolecular phosphodiester bonds between the 5 'end with the free phosphate residue and the 3' end with the free hydroxyl of the nucleic acid strand. Ligases include, e.g., standard-independent ligases, such as CIRCLIGASE ™ ssDNA ligase, capable of linking the ends of single-stranded RNA and DNA, and pattern-dependent ligases or homologous, repairing cuts in double-stranded DNA (example described below).

[0067] As used herein, the term "homologous ligase" or "reference-dependent ligase" means a DNA ligase catalyzing the formation of intramolecular or intermolecular phosphodiester bonds between the 5 'end with the free phosphate residue and the 3' end with the free hydroxyl group of DNA strands adjacent to each other. after attachment to a complementary polynucleotide. Some examples of intramolecular ligation include the formation of a circular molecule and are called "circularization." A polynucleotide to which both ends bind by ligation is referred to herein as a "ligation pattern" and the ligation is called "homologous ligation" or "standard-dependent ligation". The ligation pattern may be a complementary DNA sequence in genomic or other DNA in a biological sample (in which case it is often called a "target sequence") or it may also be a "bridged oligodeoxyribonucleotide" or an "oligodeoxy-ribonucleotide ligated using splint" (or a "splint" ligation ") synthesized and / or delivered specifically for use in a given study or method. Examples of homologous or pattern-dependent DNA ligases include NAD type DNA ligases, including E. coli DNA ligase, Tth DNA ligase, Tfl DNA ligase, and AMPLIGASE® DNA ligase (EPICENTER Biotechnologies, Madison, WI, USA), catalyzing intramolecular ligation of DNA molecules only in the presence of a ligation standard and ATP type DNA ligases, including T4 DNA ligase or ligation standard for ligation of blunt ends,

[0068] In some preferred embodiments, the reference-dependent ligase is derived from psychrophilic bacteria or a psychrophilic bacteriophage, allowing ligation at lower temperatures (e.g., when the oligonucleotide or polynucleotide sequences that make up the ligation bond have lower Tm's). The DNA ligase selected for use in the method is a ligase that is active at a temperature at which the DNA molecules to be joined (e.g., extension products at the 5 'end of DNA fragments or 5' tagged DNA fragments and oligonucleotides with random sequence) combine for sufficient to ligate with the ligase.

[0069] An important step in embodiments of the method of the present invention is the use of an in vitro transposition reaction to fragment and tag the target DNA to form tagged DNA fragments. The in vitro transposition reaction requires the use of transposase, transposon end sequences and appropriate reaction conditions.

[0070] "Transposase" means an enzyme capable of forming a functional complex with a sequence containing transposon ends (e.g., transposons, transposon ends, transposon termination sequences) and catalyzing insertions or transposition of a sequence containing transposon ends into double stranded target DNA in which it is incubated by in vitro transposition reactions.

[0071] The term "end of transposon" means double-stranded DNA having only nucleotide sequences ("transposon end sequences") necessary to form complexes with a transposase or integrase enzyme, functional in an in vitro transposition reaction. The end of the transposon forms a "complex" or "synapse complex" or "transposome complex" or "transposome sequence" with a transposase or integrase that recognizes and binds to the end of the transposon, which complex is capable of inserting or transposing the end of the transposon into the target DNA, in which it is incubated in an in vitro transposition reaction. The end of the transposon contains two complementary sequences consisting of a "transposed transfer end sequence", or a "transfer thread" and a "non-transferred end of the transposon" or "non-transferred thread". For example,

5 'AGATGTGTATAAGAGACAG 3' (SEQ ID NO: 1), and a non-transferred thread comprising the following "non-transferred sequence of the transposon end":

5 'CTGTCT CTTATACACATCT 3' (SEQ ID NO: 2).

[0072] The 3 'end of the transfer thread is joined or is transferred to target DNA by in vitro transposition reaction. The un-transferred thread, containing the transposon end sequence complementary to the transposon end of the transferred sequence, is not combined or not transferred to the target DNA by in vitro transposition reaction.

[0073] In some embodiments, the transfer thread and the non-transferred thread combine covalently. For example, in some embodiments, the sequences of the transferred and un-transferred threads are included on a single oligonucleotide, e.g. in a hairpin configuration. As such, although the free end of the non-transferred strand does not connect to the target DNA directly via the transposition reaction, the non-transferred thread attaches to the DNA fragment indirectly because it is connected to the thread transferred through the hairpin loop.

[0074] "Sequence of the transposon ends" means the sequence containing the transposon end (i.e., at least a double-stranded DNA segment capable of interacting with the transposase to carry out the transposition reaction), optionally with the additional sequence lb sequences, the 5'end of the transposon end of the transferred sequence, and / or the end of the 3 'unprocessed sequence of the transposon end. For example, the end of the transposon attached to the tag is the "sequence of the transposon ends". In some embodiments, the transposon termination sequence comprises or consists of two transposon end oligonucleotides comprising a "transposon endotransposed oligonucleotide" or "transferlable thread" and "non-transfer end endotracheal oligonucleotide" or "non-transferred strand" that, in combination, comprise an end sequence. transposon

[0075] The terms "transposon endsored oligonucleotide" and "transferlable thread" are used interchangeably and refer to the transferred portion of both "the transposon ends" and the "transposon end sequences", i.e. regardless of whether the end of the transposon was attached to the transposon end. tag or other group. Similarly, the terms "non-transferring oligonucleotide of the transposon end" and "non-transferlable strand" are used interchangeably and refer to the non -rans ported portion of both the "end of the transposon" and the "sequence of the transposon ends". In some embodiments, the transposon end sequence is the &amp; quot; hairpin &amp; apos; transposon sequence &amp; quot ;. The term "sequence of the hairpin-like transposon end" is used in this application. is a transposon end sequence consisting of a single oligodeoxyribonucleotide comprising a non-transferred end of the 5 'end of the transposon, a transposon endpoint transcribed at the 3' end, and any intermediate sequence between the non-transferred transposon end sequence and the transposon end-crossing sequence long enough to allow intramolecular creation stem structure - a loop, so that part of the transposon end can function in the transposition reaction. In some embodiments, the 5 'end of the hairpin transposon end has a phosphate group at the 5' position of the 5 'nucleotide. In some embodiments,

[0076] In some embodiments, the methods of the present invention form tagged fragments of circular ssDNA. In some embodiments, the tagged circular ssDNA fragments only contain the sequence of the transfered sequence thread of the transposon end, and the tagged circular ssDNA fragments do not contain the sequence of the non-transferred terminal of the transposon end.

[0077] In some embodiments, the transposon termination sequence used in the method of the present invention comprises the transposon-end oligonucleotides comprising only a transposon-ending sequence forming a complex with transposase or integrase necessary for the transposition reaction; in these embodiments, the tag in the tagged circular ssDNA fragments created using the method contains only the transposon end of the transfer sequence.

[0078] However, in some embodiments, the transposon termination sequence comprises or consists of at least one transposon end oligonucleotide comprising one or more nucleotide sequences apart from the transposon end sequences. Hence, in some embodiments, a transposon end sequence comprises a transferred thread comprising one or more 5 'nucleotide sequences of a transposed transfer termination sequence, wherein one or more other nucleotide sequences are also tagged. Hence, in addition to the transposon end of the transposed sequence, the tag may have one or more parts of the tag or tag domains.

[0079] The use of the term "tag portion" or "tag domain" in the present application means a portion or domain of a tag comprising a sequence for a desired, intended purpose or use. One part of the tag or tag domain is the "domain of the transposon end" where the tag portion or domain contains the transposon end of the transposed transposition sequence. In some embodiments, where the transferred thread also contains one or more other nucleotide sequences 5 'of the transposon end of the transfer sequence, the tag also has one or more &amp; quot; tag domains &amp; quot; in said portion 5', wherein each tag domain serves a defined purpose. For example, some embodiments of the invention comprise or consist of a transposon end sequence comprising or consisting of: (i) a transferred thread comprising one or more 5 'sequences of the transposon end tag sequence comprising or consisting of a tag domain selected from one or more restriction tag domain domain, capture tag domain, sequencing tag domain, amplification tag domain, detection tag domain, domain the addressing tag and the transcriptional promoter domain; and a non-transferred thread containing a non-transferred transposon end sequence. The invention includes embodiments of the method employing one or more of the mentioned transposon end sequences. sequencing tag domain, amplification tag domain, detection tag domain, addressing domain domain, and transcriptional promoter domain; and a non-transferred thread containing a non-transferred transposon end sequence. The invention includes embodiments of the method employing one or more of the mentioned transposon end sequences. sequencing tag domain, amplification tag domain, detection tag domain, addressing domain domain, and transcriptional promoter domain; and a non-transferred thread containing a non-transferred transposon end sequence. The invention includes embodiments of the method employing one or more of the mentioned transposon end sequences.

[0080] The use of the term "cleavage domain" in the present application refers to a nucleic acid sequence that is susceptible.

[0081] The use of the term "restriction site domain" in the present application means the domain of a tag comprising a sequence facilitating cleavage using a restriction endonuclease. For example, in some embodiments, the domain of a restriction site is used to create double tagged fragments of linear ssDNA. In some embodiments, the restriction domain domain is used to create a compatible double-stranded 5 'end in the tag domain so that the end can be ligated with another DNA molecule using a template dependent DNA ligase. In certain preferred embodiments, the domain of the restriction site comprises a restriction site sequence that is very rarely present in the target DNA (e.g., a rare cutnose restriction endonuclease site, such as NotI or AscI).

[0082] In some embodiments, where the transposon end of the transfer signal carries one or more restriction site domain 5 'transfer sequences of the transposon end, the method further comprises: attaching an oligodeoxyribonucleotide complementary to the single-stranded restriction site of the tagged circular ssDNA fragments followed by cleavage of tagged circular fragments ssDNA at the restriction site using the restriction endonuclease recognizing the restriction site. Hence, in some embodiments, the method comprises linearizing tagged circular ssDNA fragments to create double tagged fragments of linear ssDNA.

[0083] In some other embodiments, where the transposon endotransfered transfer tag contains one or more 5th restriction site domain domains of the transposon end of the transfer sequence, the transferred transposon end tag sequence comprises a double stranded hairpin structure containing a restriction site and the method further comprises cleavage steps. tagged fragments of the linear ssDNA at the restriction site using the restriction endonuclease recognizing the restriction site; however, in some embodiments, the method is not advantageous because the double-stranded hairpin structure forms a dsDNA location into which the transposon terminal sequence can be transposed using transposase or integrase.

[0084] In some preferred embodiments comprising (i) forming a double-stranded restriction site, either by attaching an oligodeoxyribonucleotide complementary to a single-stranded restriction site, or by using a transfer strand comprising a double-stranded hairpin structure and (ii) cleaving a restriction site by means of a restriction endonuclease recognizing the restriction endonuclease. a double-stranded restriction site, the method further comprising the step of ligating tagged linear ssDNA fragments cleaved with a restriction endonuclease to another DNA molecule with a compatible 3 'end.

[0085] The use of the term "capture tag domain" or "capture tag" in the present application means a tag domain comprising a sequence that facilitates capturing the ssDNA fragment to which the tag domain links (e.g., to create a binding site or affinity tag to capture tagged circular fragments DNA or double tagged linear DNA fragments on a spherical or other surface, e.g. where the point of attachment of the tag domain sequence enables capture by attachment to a specific sequence located on the surface, e.g. a probe or a ball or microchip or microarrays or on a sequential ball ") . In some embodiments of the method, after capturing the tagged circular ssDNA fragments or double tagged linear ssDNA fragments by attachment to a complementary probe on the surface, the capture tag domain creates a DNA synthesis initiation site using said tagged circular ssDNA fragments or double tagged linear ssDNA fragments (or complemented tagged circular ssDNA fragments) or double tagged fragments of linear ssDNA) as standards. In some other embodiments, the capture tag domain comprises a portion of the 5'-end of the transfer chain attached to a chemical group or group comprising or consisting of binding affinity molecules (e.g., where part 5; the transferred thread binds to the first molecule with a binding affinity, e.g. biotin, streptavidin,

[0086] The use of the term "sequencing tag domain" or "sequencing tag" in the present application means a tag domain comprising a sequence facilitating the sequencing of the ssDNA fragment to which the tag has been attached using the method to synthesize tagged fragments of circular ssDNA (e.g., to create the origin site sequencing by synthesizing or creating linking sites for ligation sequencing or creating hybridization site sequencing sites by hybridization). For example, in some embodiments, the sequencing tag domain comprises a site for initiating DNA synthesis of said ssDNA fragment or complement of said ssDNA fragment.

[0087] The use of the term &amp; quot; amplification tag domain &amp; quot; in the present application means a tag domain comprising a sequence facilitating the amplification of the nucleic acid to which said tag is attached. For example, in some embodiments, the amplification tag domain includes an activation site for a nucleic acid amplification reaction using DNA polymerase (e.g., PCR amplification or streaking amplification or RCA amplification reactions), or a ligase probe ligase pattern. a template dependent amplification of the nucleic acid amplification (e.g., ligation chain reaction).

[0088] The use of the term &amp; quot; detection tag domain &amp; quot; or &amp; quot; detection tag &amp; quot; as used herein refers to a tag domain comprising a sequence or a detectable chemical or biochemical group facilitating the detection of tagged fragments of circular ssDNA or double tagged linear ssDNA fragments (e.g., where the sequence or chemical group contains or is attached to a detectable molecule; such a detectable molecule selected from: visible, fluorescent, chemiluminescent, or other detectable dyes; an enzyme detectable in the presence of a substrate, e.g. alkaline phosphatase from NBT plus BCIP or peroxidase with a suitable substrate); a detectable protein, e.g. a green fluorescent protein; and molecules with a binding affinity associated with a detectable group or capable of creating a binding pair or specific binding pair with another detectable binding affinity molecule; or any other detectable molecules or systems known to the art). [0089] The use of the term "addressing domain domain" or "addressing tag" in the present application means a tag domain comprising a sequence allowing the identification of a given sample (e.g., where the transferred thread has a different address tag domain containing a different sequence for each sample).

[0090] The use of the term "transcription promoter domain" or "promoter domain" in the present application means a tag domain comprising a sense promoter sequence or an antisense RNA promoter promoter sequence. The use of the term "sense promoter sequence" in the present application means the sequence of an RNA polymerase promoter attached to a DNA strand serving as a template for transcription using RNA polymerase that binds the RNA polymerase promoter and initiates transcription under reaction conditions suitable for transcription. The use of the term "antisense promoter sequence" in the present application means an RNA polymerase promoter sequence complementary to the sense promoter sequence. In some embodiments, the sense promoter sequence contained in the transcriptional promoter domain serves RNA polymerase that binds the single stranded RNA polymerase promoter and initiates transcription, in which embodiments the sense promoter sequence is sufficient to act as an RNA polymerase promoter (e.g., for the N4 bacteriophage RNA polymerase). In some embodiments, the sense promoter sequence refers to a DNA polymerase polymerase that binds a double stranded RNA polymerase promoter and initiates transcription, in which embodiments a method comprising the RNA polymerase promoter's double-stranded (e.g. by attaching a sense oligodeoxyribonucleotide promoter sequence comprising an antisense promoter sequence that is complementary to the sense promoter sequence or using tagged DNA fragment fragments or double tagged linear ssDNA fragments as dsDNA synthesis standards comprising or consisting of a sense promoter sequence) prior to transcription using a binding RNA polymerase from and initiating transcription from the double stranded RNA polymerase promoter. In some embodiments, the sense promoter sequence refers to a T7 type RNA polymerase (e.g., selected from T7 RNA polymerase, T3 RNA polymerase, and SP6 RNA polymerase). The transcription promoter domain containing the sense promoter sequence enables RNA synthesis complementary to single-stranded target DNA, with which the transferred thread of the transposon end sequences is ligated using the method. The tagged fragments of circular ssDNA created using a transposon end sequence containing the transferlable thread having a transcriptional promoter domain containing the antisense promoter sequence can not be transcribed by DNA polymerase. However, in some embodiments, the dsDNA synthesized by extending the primer connecting to the tagged circular ssDNA fragments is used to transcribe by RNA polymerase combining with and initiating transcription from the double stranded RNA polymerase promoter; in these embodiments, the synthesized RNA contains the same sequence as the tagged fragments of circular ssDNA.

[0091] The names and descriptions of the various tag domains are for information purposes only, so as to facilitate understanding and discussion of the intended purposes and uses of the various parts or domains of the tags in various embodiments. However, the above names and descriptions are not intended to limit the use or application of the tag or any of the tag domains in any way. Hence, any particular tag or tag domain can be used for any purpose beyond, or in lieu of the original purpose or use. In addition, one tag domain may include two or more tag domains (e.g., the sequencing tag domain may include either a transposon end domain or another domain of the 5 'end of the transposon domain tag) or one tag domain may perform functions or serve for purposes or applications two or more different tag domains (e.g.

the transposon end domain can serve the purpose of the transposon end of the transposed site and perform the function or target domain of the sequencing tag and / or capture tag domain for a particular application). In addition, the tag does not have to be described in terms of one or more different domains to be used for a given purpose or application or function.

[0092] As used herein, the terms "amplify" or "amplify" in reference to a nucleic acid or a nucleic acid reaction, refers to in vitro methods for copying a given nucleic acid, including a target nucleic acid or tagged nucleic acid, e.g. through an embodiment of the present invention. Various nucleic acid amplification methods are known in the art, and the amplification reactions include polymerase chain reactions, ligase chain reactions, strand-type amplification reactions, RCA-type amplification reactions, transcription-amplification (TMA) methods, e.g. NASBA (e.g., US Patent Application No. 5,409,818), loop amplification methods (e.g., "LAMP" amplification using a loop-forming sequence, e.g. as described in U.S. Patent No. 6,410,278). The amplified nucleic acid can be DNA comprising, consisting of or derived from DNA or RNA or a mixture of DNA and RNA, including modified DNA and / or RNA. The amplification products of the nucleic acid molecule (s) (i.e., "amplification products), whether the starting nucleic acid is DNA or RNA, or a mixture of nucleosides or nucleotides of DNA and RNA, or containing nucleosides or nucleotides of modified DNA or RNA. A "copy" does not necessarily mean perfect complementarity of the sequence or its identity with the target sequence. For example, copies may include nucleotide analogues, such as deoxyinosine or deoxyuridine,

[0093] "Substances with binding affinity" or "molecules with binding affinity" means in the present application molecules with the ability to "bind" to one another under certain conditions, referred to as "binding conditions" to form a "specific binding pair". For example, biotin and streptavidin, biotin and avidin, or digoxeronin and a specific antibody combining digoxigenin are examples of "specific binding pairs", wherein the elements of each specific binding pair contain "binding affinity molecules" or "affinity binding substances" or "affinity molecules" . Molecules with binding affinities (e.g., biotin and / or streptavidin) can bind or covalently conjugate or bind non-covalently to other molecules (e.g., RNA or DNA) or to a solid surface using methods known in the art (e.g. using the reagents and methods described in Avidin-Biotin Chemistry: A Handbook, edited by D. Savage et al., Pierce Chemical Company, 1992, and in the Handbook of Fluorescent Probes and Research Products, ed. IX, ed. RP Hoagland, Molecular Probes, Inc., and in BIOCONJUGATE Techniques, edited by Greg T. Hermanson, ed. Academic Press, Inc., San Diego, CA, 1996). Affinity molecules conjugated to DNA or RNA can be synthesized using an oligonucleotide synthesizer using reagents and methods known in the art. San Diego, CA, 1996). Affinity molecules conjugated to DNA or RNA can be synthesized using an oligonucleotide synthesizer using reagents and methods known in the art. San Diego, CA, 1996). Affinity molecules conjugated to DNA or RNA can be synthesized using an oligonucleotide synthesizer using reagents and methods known in the art.

[0094] The term "binding" of the present invention means the interaction between an affinity molecule and a substance with binding capabilities as a result of non-covalent bonds, including, but not limited to, hydrogen bonds, hydrophobic interactions, van der Waals linkages, and ionic bonds. Without being limited to theory, it is recognized that in art the above types of non-covalent bonds result in binding, in part due to complementary shapes or structures of molecules in a specific binding pair. Based on the definition of "binding" and the diversity of molecules with binding affinities or specific binding pairs, it is clearly seen that the binding conditions differ for individual specific binding pairs. Those skilled in the art will easily detect or determine the conditions under which, in the trial, binding occurs between molecules with binding affinity. In particular, those skilled in the art can readily determine the conditions under which binding may occur between molecules with binding affinities that could be recognized in the art as "specific bonds". According to the understanding of art, such specificity is usually due to the higher affinity between molecules with binding affinities than to other substances and elements (e.g., vessel walls, solid bases) in the sample. In specific cases, the specificity may also include, or may result from, faster association of molecules with binding affinity than other substances and elements in the assay. in which binding may occur between molecules with binding affinities that could be considered in the art as "specific bonds". According to the understanding of art, such specificity is usually due to the higher affinity between molecules with binding affinities than to other substances and elements (e.g., vessel walls, solid bases) in the sample. In specific cases, the specificity may also include, or may result from, faster association of molecules with binding affinity than other substances and elements in the assay. in which binding may occur between molecules with binding affinities that could be considered in the art as "specific bonds". According to the understanding of art, such specificity is usually due to the higher affinity between molecules with binding affinities than to other substances and elements (e.g., vessel walls, solid bases) in the sample. In specific cases, the specificity may also include, or may result from, faster association of molecules with binding affinity than other substances and elements in the assay.

[0095] The terms "attach" or "hybridize" and "attach" or "hybridization" refer to the formation of complexes between nucleotide sequences sufficiently complementary to form complexes by base pairing according to the WatsonCrick method. With respect to the present invention, nucleic acid sequences "complementary to", "complementary to" or "hybridizing" or "fusing" with or among themselves should be capable of forming or forming "hybrids" or "complexes" sufficiently stable to serve for the intended purpose. It is not required that each nucleic acid base in the sequence consisting of one nucleic acid molecule be capable of base pairing or it evaporates or forms a complex with each nucleic acid base in the sequence of the second nucleic acid molecule, such that the two nucleic acid molecules or the corresponding sequences are &amp; quot; complementary &amp; quot; or &amp; quot; bound &amp; quot; or undergo &amp; quot; hybridization &amp; quot; with or between each other. The use of the terms "complementary" or "complementarity" in the present application refers to the nucleotide sequence defined by the base pairing rules. For example, the 5'-AGT-3 'sequence is complementary to the 3'-TCA-5' sequence. Complementarity may be "partial", where only some of the nucleic acid bases are matched according to rules of base pairing. It can also be "full" or "total". The degree of complementarity between nucleic acid strands is important for the efficiency and strength of hybridization between nucleic acid strands. This is of particular importance in the amplification reactions and methods for detecting hybridization dependent nucleic acids. The term "homology" refers to the degree of complementarity of one nucleic acid sequence with another nucleic acid sequence. There may be partial or total homology (ie complementarity). The partially complementary sequence is a sequence that at least partially inhibits the fully complementary sequence prior to hybridization to the target nucleic acid and is referred to by the functional term as "substantially homologous." Inhibition of the hybridization of a fully complementary sequence to the target sequence can be examined by hybridization assay (southern or northern hybridization, solution hybridization, etc.) under conditions of low specificity of the reaction. Indeed, a homologous sequence or probe will compete and inhibit the binding (i.e., hybridization) of the fully homologous sequence to the target sequence under conditions of low specificity. It should not be added that conditions of low specificity are conditions where such non-specific binding is possible; conditions of low specificity require that the binding of two sequences to each other is a specific (i.e., selective) interaction. The absence of non-specific binding can be determined using a second target material, non-complementary or with low complementarity (e.g., less than 30%). In the case where the specific binding is low or absent, the probe will not hybridize to the target nucleic acid. When used with reference to double-stranded nucleic acid sequences, e.g. cDNA or genomic clone, the term "substantially homologous" refers to any oligonucleotide or probe capable of hybridizing one or both of the double-stranded nucleic acid sequences under conditions of low specificity as described in the present application. The use of the terms "attaching" or "hybridization" in this application refers to the pairing of complementary nucleic acid strands. Hybridization and hybridization strength (i.e., the strength of association between nucleic acid strands) depends on many factors well known in the art, including the degree of complementarity between nucleic acids, specificity of conditions, including conditions such as salt concentration, Tm (melting temperature) formed hybrids, the presence of other elements (e.g. the presence or absence of polyethylene glycol or betaine), the molarity of the hybridizing strands and the G content:

[0096] In general, "cDNA" or "cDNA molecule" refers to "complementary DNA" synthesized by DNA polymerase-extended or DNA-dependent reverse transcriptase-binding primers that bind to the RNA molecule in the interest range using at least a portion of the molecule RNA in the range of interest as a pattern (which is also called "reverse transcription"). The synthesized cDNA molecules are "homologous to" or "basically paired with" or "form complex with" at least part of the standard.

[0097] The use of the term "population of DNA fragments" in the present application refers to a plurality or a collection of DNA fragments, e.g. from a target DNA. In some embodiments, the DNA fragment population comprises a library of DNA fragments comprising sequences that are representative in quality and / or quantitative for the target DNA sequence, while in other embodiments, the DNA fragment population comprises a subset of DNA libraries, e.g., which can not be representative of the target DNA sequence .

[0098] As used herein, the term "DNA fragment library" means a set or population of tagged DNA fragments (e.g., double tagged DNA fragments or tagged circular ssDNA fragments) formed from a target DNA, where the tagged DNA fragment combination in a set or population contains sequences, which are quantitatively and / or qualitatively representative of the target DNA sequence from which the tagged DNA fragments were generated, and where the tagged DNA fragments contained in the set or population have not been positively or negatively selected by the intentional application of the method by which the tagged DNA fragments have been included or excluded based on the nucleotide or sequence of the target DNA sequence. For various reasons, it is possible that the DNA fragment library does not contain a tagged DNA fragment representing each sequence contained in the target DNA. For example, in some embodiments, a library of tagged DNA fragments can not contain tagged DNA fragments containing the target DNA sequence sequences containing linear dsDNA (e.g., due to the low frequency of insertion of the two transposon end sequences into the terminal portions of the target DNA). In general, the lower frequency or absence of tagged DNA fragments containing the sequences of specific parts or regions of the target DNA is acceptable for the intended purpose or use. However, the invention also includes additional embodiments for situations where it is considered important or desirable for a particular purpose or use to create a library of DNA fragments, that the DNA fragment library will contain a tagged DNA fragment containing each target DNA sequence is amplification of the target DNA and then using the amplified target DNA in place of the target DNA to form a library of DNA fragments. In other embodiments, where the target DNA contains dsDNA formed from RNA using a reverse transcription reaction, the target DNA is amplified by amplification

RNA before conversion to dsDNA at reverse transcription. Some methods for amplifying RNA and DNA molecules that can be used to create the amplified target DNA have been disclosed in the present application. However, the invention is not limited to the method used to amplify the target DNA. In some embodiments, the target DNA is amplified using one of the methods disclosed in the present application, while in other embodiments, another method known in the art is used.

[0099] The use of the term "nucleic acid modifying enzyme" in the present application refers to an enzyme that acts on DNA to modify, e.g., cleave, ligation, polymerize, phosphorylate, etc. Nucleic acid modifying enzymes include, e.g., polymerases, nucleases , transferases, ligases, phosphorylases, phosphatases, metylases, transposases, etc. "DNA modification enzymes" include any enzyme that interacts with DNA, including those that also interact with other substrates, such as RNA.

[0100] The use of the term "DNA polymerase" in the present application refers to an enzyme that catalyzes the polymerization of deoxyribonucleotides into a DNA strand. The DNA polymerases contain "standard-dependent DNA polymerases" requiring the reference nucleic acid to determine the order in which the deoxyribonucleotides are added to the polymer, or "pattern-independent", catalyzing the polymerization without reference to the reference sequence.

[0101] "DNA-dependent DNA polymerase" is an enzyme that synthesizes a copy of complementary DNA ("cDNA") by extending the primer attached to the DNA standard. Some DNA-dependent DNA polymerases can also synthesize a copy of complementary DNA from the RNA template, which process is also referred to as "reverse transcription". DNA polymers capable of reverse transcription are also referred to as "reverse transcriptases."

[0102] In addition to the synthesis of DNA polymers, DNA polymerases may also have other features or functions. For example, DNA polymerase can be characterized as having 5 'to 3' exonuclease activity (also known as 5 'exonuclease activity or 5' nuclease activity), 3 'to 5' exonuclease activity, strand-shaping activity, and can to the degree of processivity or displacement, which is discussed in more detail below.

[0103] Certain DNA polymerases are also capable of displacing the complementary strand to the reference strand during the synthesis of a new strand of DNA by the polymerase. This process is referred to as "thread displacement" and DNA polymerases having this activity are referred to in the present application as "DNA polymerase displacing threads". The template for DNA synthesis with displacing the strand may be linear or circular double-stranded DNA (ssDNA) or double-stranded DNA (dsDNA). If the DNA template is a single-stranded circular DNA, the synthesis of the prepared DNA takes place in a circular structure, with a constant displacement of the strands before the replicating thread, which process is called "rolling circle replication" (RCR). The effect of rolling wheel replication is the synthesis of tandem copies of a circular pattern. In general, it is beneficial that DNA polymerase specific for the DNA template used in the method of the invention efficiently synthesizes DNA of a suitable length for the intended purpose without "falling out" of the pattern (or termination of DNA synthesis), which is termed the enzyme processiveness. The ability of the DNA polymerase to translocate the strand can be determined using the polymerase in a rolling circle type replication test, as described by Fire and Xu (Proc. Natl. Acad. Sci. USA 92: 4641-4645, 1995). The displacement of the strand and the processiveness of the DNA polymerase may also be determined by the methods described by Congo et al. (J. Biol. Chem. 268: 1965-1975, 1993). Terminal transferase is also defined in the present application as a DNA polymerase, which DNA polymerase is used as a sequence in some embodiments of the kits and methods of the present invention.

[0104] Some embodiments comprise a method using a DNA polymerase sequence having 5'-to-3'exonuclease activity to release a nucleotide labeled with a detectable group (e.g., a visible, fluorescent, chemiluminescent or other detectable moiety) as a means to determine DNA polymerization. and, thus, detection and / or quantification of the presence serving as a template of nucleic acid molecules in the assay (e.g., in a manner similar to the TaqMan® Applied Biosystems, Inc. designations). In certain embodiments, the present invention comprises a DNA polymerase sequence lacking 5'to-3 'exonuclease activity. &lt;

&gt; [0105] Some embodiments comprise methods using a DNA polymerase sequence lacking 5'-to-3 'exonuclease activity. For example, in some embodiments, a DNA polymerase sequence lacking 5'-do-3 'exonuclease activity is used for DNA sequencing. For example, in some other embodiments, a DNA polymerase sequence lacking 5'-to-3 'exonuclease activity is used to amplify the entire genome.

[0106] In certain embodiments, the present invention comprises a DNA polymerase sequence having 5'-to-3 'exonuclease activity. In certain preferred embodiments (e.g., where DNA polymerase is used, in addition to a reference-dependent ligase, for attachment in one of the methods described herein), the method uses a DNA polymerase sequence lacking 5 'nuclease activity (including 5'-exonuclease activity). up to 3 'and the 5' structure-dependent nuclease activity). For example, in some embodiments, a DNA polymerase sequence lacking 5'-to-3'exonuclease activity is used to fill gaps. Hence, in some embodiments of methods or kits,

[0107] Examples of strand-releasing DNA polymerases that may be used include, without limitation, RepliPHI ™ phi29 DNA polymerase, DisplaceAce ™ DNA polymerase, rGka DNA polymerase, SequiTherm ™ DNA polymerase, Taq DNA polymerase, Tfl DNA polymerase, and MMLV reverse transcriptase ( all available at EPICENTRE Biotechnologies, Madison, WI, USA). In some embodiments, a mixture of a DNA polymerase that does not have 3'-to-5 'exonuclease corrective activity with a DNA polymerase having such activity, such as FAILSAFE ™ DNA polymerase, is used as a thread transfer DNA polymerase. The enzyme mixture is useful in some embodiments due to better fidelity during DNA synthesis (i.e., synthesizes DNA with fewer nucleotides not complementary to the reference).

[0108] In general, in the thread displacement amplification method of the present invention, it is desirable that the amount of DNA polymerase displacing the thread used in the method is as high as possible without inhibiting or adversely affecting the reaction. For example, the DNA polymerase REPLIPHI ™ phi29 (EPICENTRE) can be used in an amount of about 1 microgram of protein in a 20 μΐ reaction and the DISPLACE ™ DNA polymerase (EPICENTRE) can be used in an amount of 50 - 300 units in a 50 μΐ reaction. Because the definitions of units are different for different DNA polymerases and even for similar DNA polymerases from different suppliers or sources, and because the activity of each enzyme varies at different temperatures and under different reaction conditions,

[0109] The movement of the thread can be facilitated by using a thread transfer medium, e.g. helicase, but since many DNA polymerases can be used in the present invention, such a thread displacement factor is not usually required. It is recognized that any DNA polymerase that can perform rolling wheel type replication in the presence of a thread displacement agent is suitable for use in embodiments of the invention including a thread displacement, even if the DNA polymerase does not replicate the rolling wheel type without such a factor. The thread displacing factors enabling rolling wheel type replication include, without limitation, the additional subunit of the BMRF1 polymerase (Tsurumi et al., J. Virology, 67: 7648-7653, 1993), the adenovirus DNA binding protein (Zijderveld and van der Vliet, J. Virology

68: 1158-1164, 1994), herpes simplex virus ICP8 (Boehmer and Lehman, J. Virology, 67: 711-715, 1993); Skaliter and Lehman, Proc. Natl. Acad. Sci. USA, 91: 10.66510,669, 1994), single-strand DNA binding proteins (SSB, Rigler and Romano, J. Biol. Chem., 270: 8910-8919, 1995), and calf thymus helix (Siegel et al. , J. Biol. Chem. 267: 13, 629, 1335, 1992).

[0110] As used herein, the term "mononucleoside" or "nucleoside" refers to a compound consisting of a purine base (guanine (G) or adenine (A)) or pyrimidine (thymine (T), cytidine (U) cytidine (C) )) covalently linked to the sugar of pentoses, while the "nucleotide" refers to a phosphorylated nucleoside on one of the hydroxyl groups of the pentose sugar. The term "canonical" is used to refer to four commonly-found nucleic acid bases: adenine, cytosine, guanine and thymine, which are elements of DNA or to the corresponding 2'-deoxyribonucleoside-5'-deoxyribonucleotides or triphosphates containing a canonical base. The term "non-canonical" is used to refer to the principles of nucleic acids in DNA other than the four canonical principles, or to the corresponding deoxyribonucleosides, 2'-deoxyribonucleoside-5'-deoxyribonucleotides or triphosphates containing a non-canonical base. For example, although uracil is a nucleic acid base often found in RNA, it is a non-canonical base in DNA. "Non-canonical principles" are found in nucleic acids by the incorporation of non-canonical nucleotides (e.g., by synthesis using an oligonucleotide synthesizer or synthesis using DNA polymerase) or in effect modifications of existing principles (canonical or non-canonical).

[0111] "Nucleic acid" or "polynucleotide" means a polymer molecule comprising a series of "mononucleosides", also called "nucleosides" in which the 3 'position of a single nucleoside pentose is linked internucleoside linkage, including, but not limited to, a phosphodiester bond, to the position 5 'of the next nucleoside pentose. The nucleoside attached to the phosphate group is called the "nucleotide". The nucleotide linking to the 5 'position of the next nucleotide in the series is called "5'" or "5 'nucleotide, and the nucleotide joining the 3' position of the 5 'nucleotide is called" 3' "or" 3 'nucleotide ". The use of the term "5" or "3" in this application refers to the position or orientation of a given chemical group, nucleotide, nucleotide sequence, or gene element (e.g. RNA polymerase promoter sequences) with respect to another chemical group, nucleotide, nucleotide sequence or gene element in a single strand of nucleic acid. If the first nucleic acid sequence will be the 3 'of the second sequence on one strand, the complement of the first sequence will be the complement of the second sequence on the complementary strand. The description of the invention will be understood with reference to the respective 5 'or 3' position and the orientation of the sequence or element of a gene on a given nucleic acid strand.

[0112] Linear nucleic acid molecules have a "terminus 5 '" (terminus 5') and "terminus 3" (terminus 3 ') because nucleic acid phosphodiester linkages are on the 5' carbon and the 3 'carbon of sugar groups of the following mononucleotides. The end of the polynucleotide in which the new bond would be formed with the 5 'carbon is the 5' terminal nucleotide. The end of the polynucleotide in which the new bond would be formed with the 3 'carbon is the 3' terminal nucleotide. The term "terminal nucleotide" as used in the present application means a nucleotide at the terminal 3 'or 5' end position.

[0113] The nucleic acid pentose may be a ribose, in which case the nucleic acid or polynucleotide is referred to as "RNA" or also 2'-deoxyribose, in which case the nucleic acid or polynucleotide is referred to as "DNA". Alternatively, especially if the nucleic acid is chemically synthesized, the nucleic acid may consist of both DNA and RNA mononucleotides. In both RNA and DNA, each pentose is covalently bound to one of the four common or "canonical" nucleic acid bases (each of which is called "base"). Three of the dominant, naturally occurring bases that combine with sugars (adenine, cytidine and guanine) are common to DNA and RNA, while one principle is different: DNA has an additional base, thymine, while RNA has an additional principle - uridine. In some cases, Uridine can occur as a base in DNA. Those skilled in the art will usually recognize a small polynucleotide as an "oligonucleotide." The use of the term "oligonucleotide" in the present application refers to a molecule consisting of two or more deoxyribonucleotides or ribonucleotides, preferably about 6 to 100 nucleotides, without a defined oligonucleotide length limit. The exact size will depend on many factors, which in turn depend on the basic function or use of the nucleotide. [0114] Furthermore, for a variety of reasons, the nucleic acid or polynucleotide of the invention may comprise one or more modified nucleic acid bases, sugar groups or internucleoside linkages. By way of example, some of the reasons for using nucleic acids or polynucleotides containing modified bases, sugar groups or internucleoside linkages include, without limitation: (1) modification of T m; (2) changing the susceptibility of a polynucleotide to one or more nucleases; (3) the presence of a group to attach the label; (4) the presence of a marker or marker quencher; or (5) the presence of a group, such as biotin, for attachment to another molecule of the solution being solution or bound to the surface. For example, in some embodiments, an oligonucleotide, such as a primer, may be synthesized such that the random portion contains one or more conformationally restricted ribonucleic acid analogs, including, but not limited to, nucleic acid analogs in which the ribose ring is &amp; quot; closed &amp; quot; methylene containing a 2'-O atom with a 4'-C atom (e.g., available from Exiqon, Inc. under the trademark "LNA ™"); such modified nucleotides result in an increase in the Tm or melting point of about 2 degrees to about 8 degrees Celsius on the nucleotide monomer. In the case of Tm increase, it is possible to reduce the number of random nucleotides in the 3 'random portion of the tagged terminal oligoronucleotide. However, a modified nucleotide, such as LNA, requires validation to function in the method for the intended purpose, as well as meeting other method criteria. For example, in some embodiments, where an oligonucleotide primer comprising ribonucleotides is used, one of the criteria for using the modified nucleotide in the method may be that the oligonucleotide comprising it may be digested by the RNA of a single stranded RNA. 2 steps to about 8 degrees Celsius on the nucleotide monomer. In the case of Tm increase, it is possible to reduce the number of random nucleotides in the 3 'random portion of the tagged terminal oligoronucleotide. However, a modified nucleotide, such as LNA, requires validation to function in the method for the intended purpose, as well as meeting other method criteria. For example, in some embodiments, where an oligonucleotide primer comprising ribonucleotides is used, one of the criteria for using the modified nucleotide in the method may be that the oligonucleotide comprising it may be digested by the RNA of a single stranded RNA. 2 steps to about 8 degrees Celsius on the nucleotide monomer. In the case of Tm increase, it is possible to reduce the number of random nucleotides in the 3 'random portion of the tagged terminal oligoronucleotide. However, a modified nucleotide, such as LNA, requires validation to function in the method for the intended purpose, as well as meeting other method criteria. For example, in some embodiments, where an oligonucleotide primer comprising ribonucleotides is used, one of the criteria for using the modified nucleotide in the method may be that the oligonucleotide comprising it may be digested by the RNA of a single stranded RNA. tagged terminal oligoronucleotide. However, a modified nucleotide, such as LNA, requires validation to function in the method for the intended purpose, as well as meeting other method criteria. For example, in some embodiments, where an oligonucleotide primer comprising ribonucleotides is used, one of the criteria for using the modified nucleotide in the method may be that the oligonucleotide comprising it may be digested by the RNA of a single stranded RNA. tagged terminal oligoronucleotide. However, a modified nucleotide, such as LNA, requires validation to function in the method for the intended purpose, as well as meeting other method criteria. For example, in some embodiments, where an oligonucleotide primer comprising ribonucleotides is used, one of the criteria for using the modified nucleotide in the method may be that the oligonucleotide comprising it may be digested by the RNA of a single stranded RNA.

[0115] In order to achieve the objectives of the invention, by way of example, nucleic acid bases in mononucleotides of one or more positions of a polynucleotide or oligonucleotide may comprise guanine, adenine, uracil, thymine or cytidine, or alternatively one or more nucleic acid bases may comprise a modified nucleic acid. the base, including, but not limited to, xanthine, alliamino-uracil, alliamino-thymidine, hypoxanthine, 2-amidoadenine, 5-prolynyluracil, 4-thiouracil, 6-thioguanine, azauracil and deazauracil, thymidine, cytosine, adenine or guanine. In addition, they may contain a nucleic acid base that forms a derivative with the biotin group, a digoxigenin group, a fluorescent or chemiluminescent group, a quenching group, or the like. The invention is not limited to the abovementioned nucleic acid bases; they have been replaced,

[0116] With respect to nucleic acids or polynucleotides of the invention, one or more sugar groups may contain 2'-deoxyribose, or alternatively, one or more sugar groups may be another sugar group, including, but not limited to, ribose, or 2'fluoro-2'-deoxyribose or 2'-O-methyl ribose, providing resistance to certain nucleases, or 2'-amino-2'-deoxyribose or 2'-azido-2'-deoxyribose, which can be determined by carrying out their reaction with visible, fluorescent, infrared fluorescent or other detectable dye or chemical substance having an electrophilic, photoreactive, alkynyl or other reactive chemical group.

[0117] The internucleoside linkages of nucleic acids or polynucleotides of the invention may be phosphodiester linkages, or alternatively, one or more internucleoside linkages may contain modified linkages, including, without limitation, phosphorothioate, phosphorodithioate, phosphoroselenate, or phosphorodiselenoate or phosphorodiselenoate linkages, resistant to certain nucleases. [0118] References to an oligonucleotide or portion of an oligonucleotide comprising a "random sequence" means that the oligonucleotide or portion thereof is synthesized (e.g., using an oligonucleotide synthesizer) using equal amounts of all four canonical nucleotide bases (A, G, C and T or U) ) for all nucleotide positions in the part of the random sequence. The effect of this method is the synthesis of an oligonucleotide mixture composed of (4 to the power of n) +1 different nucleotides, where "n" is the number of nucleotide positions in the sequence part of the sequence. Hence, in the present embodiments, the oligonucleotide comprises a mixture of a plurality of different oligonucleotides representing all possible sequences of the random portion of the sequence. References to an oligonucleotide or portion thereof containing a "semi-random sequence" means that a semi-random oligonucleotide or portion thereof is synthesized (e.g., using an oligonucleotide synthesizer) using equal amounts of all four canonical nucleotide bases (A, G, C and T or U) ( ie, these positions are "random" as described above), but one or more other positions in the semi-random part are synthesized using only one, two or three, rather than all four canonical nucleotide bases (A, G, C and T or U). In some embodiments, an oligonucleotide comprises one or more nucleotides with a "degenerate base", which is a nucleic acid base capable of base pairing with one or more bases of nucleic acids than according to standard base pairing principles in which A couples with T or U and G versus C, while "degenerative nucleotide" means a nucleotide containing a degenerate base. The terms "part" or "region" of a polynucleotide or oligonucleotide (including the primer) used herein interchangeably refer to a sequence of 2 adjacent or more bases. In other embodiments, the region or portion comprises at least 1, 2, 3, 5, 10, 15, 20, 25, 50, 75 or more contiguous nucleotides. rather than all four canonical nucleotide bases (A, G, C and T or U). In some embodiments, an oligonucleotide comprises one or more nucleotides with a "degenerate base", which is a nucleic acid base capable of base pairing with one or more bases of nucleic acids than according to standard base pairing principles in which A couples with T or U and G versus C, while "degenerative nucleotide" means a nucleotide containing a degenerate base. The terms "part" or "region" of a polynucleotide or oligonucleotide (including the primer) used herein interchangeably refer to a sequence of 2 adjacent or more bases. In other embodiments, the region or portion comprises at least 1, 2, 3, 5, 10, 15, 20, 25, 50, 75 or more contiguous nucleotides. rather than all four canonical nucleotide bases (A, G, C and T or U). In some embodiments, an oligonucleotide comprises one or more nucleotides with a "degenerate base", which is a nucleic acid base capable of base pairing with one or more bases of nucleic acids than according to standard base pairing principles in which A couples with T or U and G versus C, while "degenerative nucleotide" means a nucleotide containing a degenerate base. The terms "part" or "region" of a polynucleotide or oligonucleotide (including the primer) used herein interchangeably refer to a sequence of 2 adjacent or more bases. In other embodiments, the region or portion comprises at least 1, 2, 3, 5, 10, 15, 20, 25, 50, 75 or more contiguous nucleotides.

[0119] A "primer" is an oligonucleotide ("oligo"), with free 3'-OH that can be extended with nucleic acid polymerase. In the case of a template-dependent polymerase, generally the 3 'end of the oligo primer is complementary to the part of the template nucleic acid with which the oligonucleotide "binds" (or "forms a complex", "fused" or "hybridized") by hydrogen bonding and other molecular forces so as to form a primer / standard complex to initiate synthesis by DNA polymerase, and which is extended (i.e., "extended primer") by the addition of covalently attached bases attached at the 3 'end, complementary to the standard in the DNA synthesis process. The result is a primer extension product. The template-dependent DNA polymerases (including reverse transcriptases) generally require the formation of an oligonucleotide primer complex with a single-stranded pattern to initiate DNA synthesis ("initiation"), but the DNA polymerases generally do not require a primer to synthesize RNA complementary to the DNA template (transcription). [0120] "DNA-specific single-strand DNAase" means DNAse specifically digesting single-stranded DNA but non-digesting single-stranded RNA or RNA or DNA bound or complexed with complementary RNA or DNA, if said complementary RNA or DNA is part of another nucleic acid molecule (e.g. by intermolecular base pairing) or part of the same nucleic acid molecule (e.g., by intramolecular base pairing). DNAase specific for single-stranded DNA may be endonuclease or exonuclease, if it actively and specifically digest single-stranded DNA into monomers or short oligodeoxyribonucleotides. In certain preferred embodiments, the oligode-coucleotides, including primers, are removed from the reaction mixture after the method step in which they were used to digest DNAse specific for single-stranded DNA. Examples of DNAse specific for single-stranded DNA are Exonuclease I, Exonuclease VII, and Rec. J. exonuclease.

[0121] "T7 RNA polymerase" (RNAP) means in the present application a polymerase

T7 RNA, (e.g., see Studier, FW et al., Pp. 60-89 in: Methods in Enzymology, vol. 185, ed. Goeddel, DV, Academic Press, 1990) or RNAP derived from a "T7 type" bacteriophage, which means a bacteriophage with a similar genetic organization as the T7 bacteriophage organization.

The genetic organization of all T7 phages tested turned out to be largely the same as the T7 organization. Examples of T7 type bacteriophages of the invention include, without limitation, the phages T3, phi I, phi II, W31, H, Y, A1, 122, cro, C21, C22, and C23 Escherichia coli; phage gh-1 Pseudomonas putida; SP6 Salmonella typhimurium fag; fag IV Serratia marcescens; phage ViIII Citrobacter phage; and phage 11 Klebsiella (Hausmann, Current Topics in Microbiology and Immunology 75: 77-109, 1976, Korsten et al., J. Gen. Virol. 43: 57-73, 1975; Dunn, et al., Nature New Biology 230 : 94-96, 1971;

Towle, et al., J. Biol. Chem. 250: 1723-1733, 1975; Butler and Chamberlin, J. Biol. Chem. 257: 5772-5778, 1982), as well as mutant forms of such RNAPs (e.g., Sousa et al., US Patent Application No. 5,849,546; Padilla, R and Sousa, R, Nucleic Acids Res.,

15: e138, 2002; Sousa, R and Mukherjee, S, Prog Nucleic Acid Res Mol Biol., 73: 1-41, 2003; Guillerez, J, et al., US Patent Application No. 20040091854). In preferred embodiments of the invention, the promoter used is a wild or mutated promoter sequence recognized by T7 type RNA polymerase. In some embodiments, the promoter may be single-stranded, such as a pseudopromotor (e.g.

Ohmichi et al., Proc. Natl. Acad. Sci. USA 99: 54-59, 2002), or the N4 promoter of vRNAP, in which case a short chain protein containing the active amino acid domain 1,106 (corresponding to amino acids 9982103) of the v4 NN promoter is designed ("mini-vRNAP"; EPICENTRE Biotechnologies, Madison, WI, USA) (Kazmierczak, KM, et al., EMBO J., 21: 5815-5823, 2002).

[0122] The use in the present application of the term "target DNA" refers to any dsDNA in the scope of interest, transposed, e.g. to form a library of tagged DNA fragments (e.g., tagged at the 5 'and 3' end or double tagged linear ssDNA fragments) or dsDNA or tagged fragments of a spherical ssDNA).

[0123] "Target DNA" may be from an in vivo or in vitro source, including one or more cells, tissues, organs or organisms, whether living or dead, or any biological or environmental source (e.g., water, air, etc.). soils). For example, in some embodiments, the target DNA comprises or consists of eukaryotic and / or prokaryotic dsDNA derived or recovered from humans, animals, plants, fungi (e.g., mold or yeast), bacteria, viruses, viroids, mycoplasmas or other organisms. In some embodiments, the target DNA comprises or consists of genomic DNA, subgenomic DNA, chromosomal DNA (e.g., an isolated chromosome or a portion of a chromosome, e.g. one or more chromosomal genes or loci), mitochondrial DNA, chloroplast DNA, plasmid or other episomal DNA (or the recombinant DNA contained therein) or double-stranded cDNA created by reverse transcription of RNA using RNA-dependent DNA polymerase or reverse transcriptase to generate a first-strand cDNA followed by extension of the primer attached to the primary-cDNA to create dsDNA. In some embodiments, the target DNA comprises a plurality of dsDNA molecules comprised of or derived from nucleic acid molecules (e.g., a plurality of dsDNA molecules contained in or derived from genomic DNA or cDNA obtained from RNA in or from a biological source (e.g., cells, tissues, In some embodiments, the target DNA is from an in vitro source. molecular cloning of all or part of one or more nucleic acid molecules in a plasmid, fosmid, BAC or other vector sequentially replicated in a suitable host cell; or capturing one or more nucleic acid molecules by hybridization, including hybridizing to DNA probes on a matrix or microarrays (e.g., by "sequencing capture", e.g. using kits and / or ROCHE NIMBLEGEN, AGILENT, or FEBIT arrays). by "sequencing capture"; e.g. using sets and / or ROCHE NIMBLEGEN, AGILENT, or FEBIT matrices). by "sequencing capture"; e.g. using sets and / or ROCHE NIMBLEGEN, AGILENT, or FEBIT matrices).

[0124] In some embodiments, "target DNA" is defined as dsDNAs obtained or modified (e.g., using various biochemical or molecular biology techniques) prior to using tagged DNA fragments (e.g., tagged at the 5 'and 3' ends) for library creation, or double tagged fragments of linear ssDNA or dsDNA or tagged fragments of circular ssDNA). For example, the inventors of the present invention observed that the representation of the next generation sequential data from the ends of the target DNA containing dsDNA particles below 10 Kb was low compared to the representation of sequence data from the center of such target DNA. Without delving into theoretical considerations, one of the possible explanations for this observation is that that the probability of finding DNA fragments with two sequences of transposon inserts inserted in the reverse directions at the ends of the dsDNA linear molecule is lower than the probability of finding DNA fragments with two sequences of transposon inserts inserted in opposite directions in the middle of the linear dsDNA molecule. Thus, in some embodiments, to create libraries of double tagged DNA fragments or tagged circular DNA fragments better representing terminal sequences, the method further provides the target DNA for use in a dsDNA-containing method (e.g., double-stranded genomic DNA or cDNA obtained from RNA, such like mRNA), already having a tag at the 5 'and / or 3' end. For example, in some embodiments, the target DNA comprises a double-stranded cDNA obtained from RNA by: synthesis of the first-strand cDNA by extending the primer of the synthesis of the cDNA with a 3 'part and 5' part, where 3 'is complementary to the 3' end of the RNA part and 5 'contains the first tag and then the second tag is attached to the 3' end of the cDNA using the tagging of the terminal oligonucleotide and DNA polymerase as described in the present application followed by the use of DNA polymerase for the synthesis of double-stranded cDNA by extending the primer for synthesis of the second-strand cDNA that connects to the second tag. Alternatively, in other preferred embodiments, to create libraries of double tagged DNA fragments better representing terminal sequences, the target DNA used in the method to create double tagged DNA fragments or tagged circular DNA fragments comprises circular dsDNA obtained by intramolecular linear dsDNA ligation (e.g., obtained by intracellular ligation of double-stranded genomic DNA or double-stranded cDNA obtained from RNA, such as mRNA). Hence, in some embodiments, the method further comprises: ligating a linear dsDNA with a ligase (e.g., T4 DNA ligase) to form linear dsDNA for use as target DNA in the method. In some embodiments of a method comprising forming a circular dsDNA for use as target DNA by linear ligation of dsDNA, linear dsDNA is treated with T4 DNA polymerase and T4 polynucleotide kinase (e.g.

[0125] The use of the term "DNA fragment" in the present application means a part or fragment or segment of the target DNA cleaved or freed or detached from a longer DNA molecule in a manner completely detaching it from the parent molecule. The DNA fragment may be double stranded ("dsDNA fragment") or single stranded ("ssDNA fragment"), and the process of forming DNA fragments from the target DNA is called "fragmentation" of the target DNA. In certain preferred embodiments, the method is used to create a "DNA fragment library" containing a set or population of tagged DNA fragments.

[0126] A "pattern" is a nucleic acid molecule that is copied by a nucleic acid polymerase, e.g. DNA polymerase. Whether the nucleic acid molecule consists of two strands (i.e. "double-stranded") or one strand (i.e. "is single-stranded"), the strand of said nucleic acid molecule is used to determine the nucleotide sequence contained in the nucleic acid to be synthesized. is a "pattern" or "reference thread". The nucleic acid synthesized by the nucleic acid polymerase is complementary to the standard. Both RNA and DNA are always synthesized in the 5'-to-3 'direction, starting from the 3' end of the reference strand, and the two strands of double-stranded nucleic acid are so directed that the 5 'ends of both strands are on opposite sides of such double-stranded nucleic acid. acid (which also applies, necessarily, to the ends of 3 '). Both RNA and DNA templates require a primer to initiate synthesis using DNA polymerase, but the primer is not required for the initiation of synthesis mediated by DNA-dependent RNA polymerase, commonly referred to as "RNA polymerase". [0127] A "terminal transferase", also called a "terminal deoxynukeidyl transferase" or "TdT", is a DNA polymerase catalyzing a reference addition (or "tailing") of a deoxyribonucleoside triphosphates (dNTP) or a single deoxyribonucleoside triphosphate to the 3 'end with a hydroxyl group. GOUT. The common terminal transferase used in art available on the market is the transferase produced by the E. coli strain expressing the recombinant calf thymus gene. In some embodiments, the invention further comprises the step of incubating 5 'DNA fragments tagged at the 5' end, after denaturation, by TdT and dNTP under appropriate conditions and for a sufficient period of time, where 5 'and 3' tagged DNA fragments are synthesized having a second tag containing a homopolymer DNA tail. In some embodiments, the homopolymer DNA tail is used further as the initiation site for the synthesis of double-stranded cDNA. In some embodiments, the primer used to synthesize the second DNA strand has a 3 'part complementary to the second tag containing the homologous tail and a 5' portion having the desired sequence not complementary with the first tag, target DNA or a second tag containing the homopolymer tail. For example, in some embodiments, the 5 'part of the primer contains the antisense promoter sequence for the RNA polymerase promoter,

[0128] In some embodiments, the transposon end oligonucleotides used in the method of the present invention contain only the transposon end sequences necessary for the transposition reaction. However, in some embodiments, at least one transposon end oligonucleotide further comprises one or more nucleotide sequences of the 5 'end of the transposon end sequence. Hence, in some embodiments, a method or kit uses a transfer thread having a 3 'part and a 5' portion, wherein the 3 'portion comprises a transposon end transcript and the 5' part contains one or more additional sequences that do not participate in the formation of a transposase functional complex. There are no restrictions on the use of additional sequences to one or more additional sequences of the 5'-part of the transfer thread, which sequences can be used to accomplish any purpose. For example, in some embodiments, the 5 'portion of the transferred thread includes one or more additional tag sequences (e.g., a tag sequence that allows capture by connecting to a given sequence on a surface, such as a ball or probe on a microchip or array; e.g. spheres in next generation sequencing, e.g. 454A or 454B tag sequencing sequence for sequencing using a new generation sequencer Roche 454) or one or more sequences to identify, detect (e.g., fluorescent) or sort the products of the method. In some other instillation examples, the 5 'portion of the transfer thread contains one or more additional nucleotides or sequences, or a chemical group or group containing or consisting of a binding affinity (e.g., a sequence that allows uptake by joining a given sequence on a surface, such as a ball). or a probe on a microchip or array). In certain preferred embodiments, the size of one or more additional sequences in the 5 'portion of the transferred thread is minimized to reduce the probability or frequency of insertion of the transferred thread to that thread during the in vitro transposase reaction. For example, in some embodiments, the size of the 5 'portion of the transfer thread is less than about 150 nucleotides, less than about 100 nucleotides,

[0129] In some embodiments, the 5 'end of the transfer thread has a 5' monophosphate group. In some embodiments, both the transfer and non-transferred threads have a 5 'monophosphate group. In some preferred embodiments, only the 5 'end of the non-transferable thread has a 5' monophosphate group. In some other embodiments, the transfer thread does not have a 5 'monophosphate group at the 5' end.

[0130] The term "transposase" in reference to the present invention means an enzyme capable of forming a functional complex with the end of the transposon or the transposon end sequences necessary for the transposition reaction. The transposition of the invention also contains integrase with retrotransposons and retroviruses.

[0131] A "transposition reaction" is the reaction in which one or more of the ends of the transposon is inserted into the target DNA at random or near random locations. The main elements of the transposition reactions are the transposase and DNA oligonucleotides containing the nucleotide sequences of the transposon end, including the transposon end and complement sequence transcribed, the not-transferred transposon end sequence, and other elements necessary to form a functional transposition complex. The method of the present invention can be described by employing the transposition complex formed by the hyperactive Tn5 transposon and the end of the Tn5 transposon (Goryshin, I. and Reznikoff, WS, J. Biol. Chem., 273: 7367, 1998) or through the MuA transposase and the end of the transposon. Mu containing the R1 and R2 end sequences (Mizuuchi, K., Cell, 35: 785,

Savilahti, H, et al., EMBO J., 14: 4893, 1995). However, in the present invention, any transposition system capable of inserting the end of the transposon in a random or nearly random manner with sufficient efficiency to the 5 'end tag and a fragment of the target DNA can be used to accomplish the intended purpose. Examples of the transposition system known in the art that could be evaluated for the purposes of the present methods include, without limitation, Staphylococcus aureus Tn552 (Colegio OR et al., J Bacteriol., 183: 2384-8, 2001; Kirby C et al., Mol Microbiol. , 43: 173-86, 2002), Ty1 (Devine SE, and Boeke JD., Nucleic Acids Res., 22: 3765-72, 1994 and international patent application No. WO 95/23875), the transposon Tn7 (Craig, NL, Science 271: 1512, 1996; Craig, NL, Review in: Curr Top Microbiol Immunol., 204: 27-48, 1996), Tn10 and IS10 (Kleckner N, et al.

[0132] The method of inserting the transposon end into a target sequence may be performed in vitro using any transposon system for which an appropriate in vitro transposition system is available or may be developed based on available knowledge. in general, an appropriate in vitro transposition system for use in the methods of the present invention requires at least a transposase enzyme of sufficient purity, sufficient concentration, and sufficient in vitro transposition activity and end of the transposon with which the transposase will form a functional complex with a suitable transposase capable of catalyzing transposition reactions. A suitable transposon end-sequence transposition that can be used in the invention includes, without limitation, wild, derivatives or mutated transposon end sequences that form a complex with transposase selected from wild, derivative or mutated forms of transposase. Exemplary transposases that have been successfully used by Applicants in the methods of the present invention include wild or mutant forms of Tn5 transposase and MuA transposases (although the EZ-Tn5 transposase was much more efficient than the equivalent amount of white Mua transposase in 5 'tagged DNA fragments). in the methods of the present invention), but any other transposase, with known or newly developed composition and conditions for efficient in vitro transposition of certain transposon ends, can be used in the present methods. The end of the transozone sequences recognized by wild or mutant forms of Tn5 transposase or MuA transposases are preferred, and these transposon end sequences that will show the highest transposition efficiency after the complex is formed with transposase, once with the respective optimally active transposase enzymes that will form a complex with them, will be most preferred for use in the embodiments of the present invention. Preferably, the transposon is selected so that the transposon end sequence required by the transposase transposon is not too large and the transposon end sequences have the smallest possible size to allow them to function efficiently to the intended purpose and have sufficient size, and so that the same sequence occurs only rarely or, preferably, not present at the target DNA or sampled DNA. For example,

[0133] Suitable in vitro transposition systems that can be used to insert the end of the transposon into the target nucleic acid include, without limitation, systems using the Tn5 EZ-Tn5 ™ hyperactive transposase available from EPICENTRE Technologies, Madison, WI, or the hyperactive MuA HyperMu ™ transposase with EPICENTRE or other MuA transposase, including those available from Finnzymes Oy, Espoo, Finland. The transposon end oligonucleotides containing the sequences of the respective transposon ends can be synthesized using an oligonucleotide synthesizer or can be purchased from a commercial source based on information available from appropriate suppliers or information known in the art. For example, the nucleotide nucleotide sequences of the transposon hyperactive mosaic for EZ-Tn5 ™ transposase are shown in Example 1,<a href="http://www.EpiBio.com">www.EpiBio.com</a>, EPICENTER Biotechnologies, Madison, WI, USA.

[0134] In some embodiments, the insertion of the transposon end into the target DNA of the present invention may also be performed in vivo. If the transposition is performed in vivo, the transposition into the target DNA is preferably obtained by electroporation of the synaptic transposase complex and the appropriate transposon termination sequence into the host cell, as described in US Patent Application No. 6,159,736. An example of this transposition method is the use of the transposition complex formed by the hyperactive Tn5 transposase and the corresponding Tn5 transposon end sequence using methods similar to those described by (Goryshin, I. and Reznikoff, WS (J. Biol. Chem., 273: 7367, 1998) ) or the transposition complex formed by the hyperactive MuA HyperMu ™ transposase (EPICENTRE, Madison, WI) and the corresponding MuA transposon end sequence containing the R1 and R2 end sequences recognized by transposase. Suitable synaptic complexes or Transposome ™ complexes (EPICENTRE) between a transposase and transposase sequence can be prepared according to U.S. Patent No. 6,159,736 and related Goryshina and Reznikoff patents, or according to product literature for Tn5 EZ-Tn5 ™ Transposome ™ or HyperMu ™ complexes. MuA Transposome ™ from EPICENTRE Technologies, Madison, WI, with the exception of oligonucleotides containing only one end of transposon, used instead of a polynucleotide, or an oligonucleotide containing two ends of a transposon, usually at or near each end of the respective polynucleotide or oligonucleotide.

[0135] The invention also includes kits and individual sequences for any method of the invention. The kit is a combination of individual sequences useful for carrying out the method of the invention, wherein the sequences are optimized for use in the method. The sequence includes a particular component or mixture of components for at least one step of the method of the invention. The invention includes any set that can be obtained from a combination of any two sequences, and any new sequence used in a kit or method of the invention. Alternatively, the kit may be formed from a single component or sequence in a convenient application format, e.g. pre-split into single-use parts, and may optionally include a set of instructions regarding the use of the component or sequence.

DESCRIPTION OF THE INVENTION

Introduction [0136] The present invention relates to methods and sequences for treating nucleic acids, and in particular, methods and sequences for DNA fragmentation and tagging using a transposon sequence. The methods, sequences and kits of the present invention are useful for creating libraries of double tagged linear ssDNA fragments or tagged fragments of circular ssDNA (and their amplification products) from target DNA containing any dsDNA in the range of interest (including double-stranded cDNA obtained from RNA) from any source, for genomic, subgenomic, transcriptomic or metagenomic analysis or analysis of RNA expression (e.g., for use in the creation of a target for microarray analysis, e.g. for variations in copy number (CNV), detection and analysis of single nucleotide polymorphisms and for the identification of genes in environmental samples, such as soil or water samples). The methods are useful in many processes, including, but not limited to, whole genome amplification processes of one or more organisms, including one or more microbes or environmental organisms whose culture or growth conditions are unknown (e.g., whole genome amplification (WGA)) ), Real-time PCR, emulsion PCR, comparative genomic hybridization (CGH), comparative genomic sequencing (CGS), and to create DNA-specific probes (e.g., chromosome-specific probes, e.g. chromosomal inks, or e.g. probes specific for gene or locus) for applications such as in situ fluorescence hybridization (FISH). In some embodiments, methods are also used to create patterns for massive parallel DNA sequencing (so-called "next-generation sequencing"). Each of the above processes or applications is applicable in both research and molecular diagnostics.

[0137] The present invention includes methods, sequences and kits for creating a library of tagged DNA fragments from a target DNA comprising double-stranded DNA (dsDNA) contained in any sample in the scope of interest. The methods are simpler, faster, less labor intensive, can be performed on smaller samples and smaller amounts of sampled nucleic acids and are more efficient in tagging both ends of fragments and form double-tagged DNA fragments representative in quantity and / or quality for sampled nucleic acids from which they were created. The methods can be easily performed manually without the use of instruments, but they can also be easily adapted to automation in a high bandwidth environment.

Embodiments of the Methods [0138] All embodiments of the methods of the present invention disclosed in the present application use an in vitro transposition reaction to simultaneously cleave the target DNA into fragments and attach the tag to the 5 'end of each fragment. Since all methods are related, unless otherwise defined in relation to a particular embodiment, the method set forth in the present application with respect to one embodiment may also be applied to another embodiment described in the present application. All embodiments of the methods disclosed in the present application using the in vitro transposition reaction can be accomplished by using the reaction using both the transposase itself, as well as the transposon end sequence or a single transposome sequence containing a stable complex formed between the transposase and the transposon end sequence. Hence, it is understood that any method describing the use of transposase and transposon sequence may also utilize a transposon sequence formed from a transposase and a transposon end sequence, and any method describing the use of a transposome sequence may also use the transposase itself and the transposon end sequences from which the transposome sequence was created . The foregoing is illustrated by the following two descriptions of one general method of the invention. that any method describing the use of transposase and transposon termination sequences may also use a transposome sequence formed from a transposase and a transposon end sequence, and any method describing the use of a transposome sequence may also use the transposase itself and the transposon end sequences from which the transposome sequence was created. The foregoing is illustrated by the following two descriptions of one general method of the invention. that any method describing the use of transposase and transposon termination sequences may also use a transposome sequence formed from a transposase and a transposon end sequence, and any method describing the use of a transposome sequence may also use the transposase itself and the transposon end sequences from which the transposome sequence was created. The foregoing is illustrated by the following two descriptions of one general method of the invention.

One embodiment of the invention is a method of creating a library of tagged DNA fragments from a target DNA containing any dsDNA in the range of interest (e.g., for use as next generation sequencing or amplification patterns), the method comprising: incubating the target DNA in a transposition reaction in vitro with at least one transposase and a transposon end sequence in which the transposase forms a transposon complex, a transposon end sequence comprising (i) a transferred thread comprising the transposon end transcribed sequence and, optionally, an additional 5 'sequence of the transposon end-transcribed sequence, and (ii) a non-transferred thread containing a sequence complementary to the transposon end of the transfer sequence,under appropriate conditions and for a sufficient period of time during which many insertions into the target DNA occur, each resulting in the attachment of the first tag containing or consisting of the transferred strand to the 5 'end of the nucleotide in the target DNA, fragmenting the target DNA and forming a pool of tagged the end of the 5 'DNA fragments, each of which has the first tag at the 5' end, and then joining the 3 'ends tagged at the 5' end of the DNA fragments to the first or second tag, creating a library of tagged DNA fragments (e.g. containing both tagged fragments of circular ssDNA or tagged DNA fragments (or "double tagged DNA fragments") at the 5 'and 3' ends.each of which results in the attachment of the first tag containing or consisting of the transferred strand to the 5 'end of the nucleotide in the target DNA, fragmenting the target DNA and forming a population of attached 5' tagged DNA fragments, each of which has the first tag at the 5 'end, and then attach the 3 'ends tagged at the 5' end of the DNA fragments to the first or second tag, creating a library of tagged DNA fragments (e.g. containing both tagged fragments of circular ssDNA or 5 'and 3' tagged DNA fragments (or 'double tagged DNA fragments) ").each of which results in the attachment of the first tag containing or consisting of the transferred strand to the 5 'end of the nucleotide in the target DNA, fragmenting the target DNA and forming a population of attached 5' tagged DNA fragments, each of which has the first tag at the 5 'end, and then attach the 3 'ends tagged at the 5' end of the DNA fragments to the first or second tag, creating a library of tagged DNA fragments (e.g. containing both tagged fragments of circular ssDNA or 5 'and 3' tagged DNA fragments (or 'double tagged DNA fragments) ").each of which has a first tag at the 5 'end and then attach the 3' ends tagged at the 5 'end of the DNA fragments to the first or second tag, creating a library of tagged DNA fragments (e.g. containing both tagged fragments of circular ssDNA or tagged at the 5' end) and 3 'DNA fragments (or "double tagged DNA fragments").each of which has a first tag at the 5 'end and then attach the 3' ends tagged at the 5 'end of the DNA fragments to the first or second tag, creating a library of tagged DNA fragments (e.g. containing both tagged fragments of circular ssDNA or tagged at the 5' end) and 3 'DNA fragments (or "double tagged DNA fragments").

[0140] In one preferred embodiment, as described immediately above, the method is performed using the transposase transposon alone and sequence, while in some other preferred embodiments, the method is carried out using a transposome sequence comprising a complex formed between the transposase and the transposon end sequence.

Hence, one preferred embodiment of the invention is a method of creating a library of tagged DNA fragments from a target DNA in an in vitro transposition reaction comprising any dsDNA in the range of interest (e.g., for use as next generation sequencing or amplification patterns), the method comprising : incubation of the target DNA by in vitro transposition reaction with one or more transposomes, each comprising a complex between transposase and the end of the transposon, where the transposase forms a transposition complex, a transposon end sequence containing (i) the transferred thread containing the transferred end sequence the transposon and, optionally, the additional 5 'sequence of the transposon end of the transfer sequence,and (ii) a non-transferred thread comprising a sequence complementary to the transposon end of the transposed sequence, under suitable conditions and for a sufficient period of time during which multiple insertions into the target DNA occur, each resulting in the attachment of the first tag containing or consisting of the transferred thread to the a nucleotide in the target DNA, fragmenting the target DNA and forming a population of attached 5 'tagged DNA fragments, each of which has a first tag at the 5' end, and then joining the 3 'ends tagged at the 5' end of the DNA fragments to the first or second tag by creating a library of tagged DNA fragments (e.g. containing both tagged fragments of circular ssDNA or tagged at the 5 'and 3' end)DNA fragments (or "double tagged DNA fragments").

[0142] In some embodiments of any method of the invention, the amount of transposon or transposon sequence or transposome sequence used in the in vitro transposition reaction is in the range of 1 pmol and about 25 pmol for 50 ng of target DNA per 50 μΐ reaction. In certain preferred embodiments of any method of the invention, where the transposase is a hypernaonal Tn5 transposase and the transposon end sequence comprises the MEDS transposon end sequence, or wherein the transposom sequence comprises said hyper-active Tn5 transposase and transposon end sequence comprising the MEDos transposon end, the amount of said transposase and end sequence The transposon or said transposome sequence used in the in vitro transposition reaction is in the range of about 5 pmol to about 25 pmol per 50 ng of target DNA for a 50 μl reaction. In certain preferred embodiments of any method of the invention, where the transposase is a hyper-active Tn5 transposase or MuA transposase, the final concentrations of the transposase and transposon end or transposome sequences used in the in vitro transposition reaction are at least 250 nM; In some other embodiments, the final concentrations of the Tn5 hyperactive transposase or MuA transposition and the corresponding transposon or transposome sequence sequence are at least 500 nM. are at least 250 nM; In some other embodiments, the final concentrations of the Tn5 hyperactive transposase or MuA transposition and the corresponding transposon or transposome sequence sequence are at least 500 nM. are at least 250 nM; In some other embodiments, the final concentrations of the Tn5 hyperactive transposase or MuA transposition and the corresponding transposon or transposome sequence sequence are at least 500 nM.

[0143] In some embodiments of any method of the invention, the in vitro transposition reaction reaction time is two hours or less, one hour or less, 30 minutes or less, or 15 minutes or less. In certain preferred embodiments of any method of the invention, the reaction time for an in vitro transposition reaction is 5 minutes or less. In certain preferred embodiments of any method of the invention, the transposome sequence comprises a Tn5 hyperactive transposase and a transposon end sequence comprising a MEDS transposon end, reaction time for the reaction. in vitro transposition is 5 minutes or less.

[0144] In some embodiments, the method further comprises a non-selective amplification step of tagged DNA fragments comprising double tagged DNA fragments or tagged circular ssDNA fragments using a thermostable DNA polymerase and at least one primer complementary with the first tag or the second tag. In some preferred embodiments of the method, where only one transposome is used in an in vitro transposition reaction, the step of amplifying tagged DNA fragments comprises amplifying double tagged DNA fragments or tagged DNA core fragments using a single primer containing the sequence of at least a portion of the transfer thread. In some embodiments, the step of amplifying tagged DNA fragments using a single primer contains a PCR reaction or roller type replication reaction. In some embodiments, the 5 'portion of the primer used for the amplification comprises or consists of a sequencing tag domain.

[0145] In certain preferred embodiments of any method of the invention, a DNA fragment library is used to create standards for DNA sequencing or ampli- fication of nucleic acids.

[0146] The invention includes several embodiments for creating a library of tagged DNA fragments comprising both double tagged DNA fragments or tagged fragments of circular ssDNA, as discussed below.

Use of DNA polymerase with strand transfer activity or 5 'nuclease activity for generation of tagged DNA fragments comprising double tagged DNA fragments [0147] One preferred embodiment of the method comprises: incubating the target DNA by in vitro transposition reaction with at least one transposome under appropriate conditions and for a sufficient period of time to create a population of linked DNA fragments tagged at the 5 'end; followed by incubation of a pool of 5 'tagged DNA fragments linked to a DNA polymerase having a strand displacement or 5' nuclease activity under non-thermocycling conditions, and wherein the linked 5 'tagged DNA fragments do not denature, where the DNA polymerase lengthens the 3' end any thread of connected tagged at the end of the 5 '

One preferred embodiment of the method comprises: incubating the target DNA in an in vitro transposition reaction with at least one transposome under appropriate conditions and for a sufficient period of time to create a population of linked 5 'DNA fragments; incubating a population of fused 5 'tagged DNA fragments with a DNA polymerase having a strand displacement or 5' nuclease activity to create double tagged dsDNA fragments; and denaturation of double tagged dsDNA fragments to create a library of tagged DNA fragments containing double tagged ssDNA fragments (e.g., by heating to 95^aboutC and rapid cooling). In one preferred version of this embodiment of the method, a library of tagged DNA fragments comprising double tagged ssDNA fragments is generated from the target DNA in a single tube without a purification step from the spacer sequences.

[0149] In some embodiments of a method comprising creating a library of tagged DNA fragments comprising double tagged DNA fragments using DNA polymerase having a strand displacement or 5 'nuclease activity, the method further comprises a step of amplifying tagged DNA fragments comprising double tagged DNA fragments using a thermostable polymerase. DNA and at least one prmer complementary to the second tag. In certain preferred embodiments of the present method, the amplification step of library of tagged DNA fragments containing double tagged DNA fragments comprises amplification of tagged DNA fractions with PCR using one oligodeoxyribonucleotide containing the sequence of at least part of the transferred strand as PCR primer and double tagged DNA fragments as standards. Hence, this embodiment is a PCR amplification method using a single tagged tag library of DNA tags containing double tagged DNA fragments formed from the target DNA. If the target DNA contains the total genomic DNA of the organism, the present embodiment is a method of non-selective amplification of the entire genome. the present embodiment is a method of amplifying PCR using a single tagged tag library of DNA tags containing double tagged DNA fragments formed of the target DNA. If the target DNA contains the total genomic DNA of the organism, the present embodiment is a method of non-selective amplification of the entire genome. the present embodiment is a method of amplifying PCR using a single tagged tag library of DNA tags containing double tagged DNA fragments formed of the target DNA. If the target DNA contains the total genomic DNA of the organism, the present embodiment is a method of non-selective amplification of the entire genome.

[0150] In some preferred embodiments, the single transposon termination sequence is used to transpose a method comprising creating a library of tagged DNA fragments comprising double tagged DNA fragments using DNA polymerase with either 5+ nuclease activity or 5 'nuclease activity and further amplification of double tagged fragments Two different PCR primers are used for PCR-generated DNA, each of which contains a sequence of at least a portion of the transposon end of the transposon end containing the transposon end sequence. In certain preferred embodiments, each PCR primer comprises a 3 'part and a 5' part, wherein the 3 'part contains a suitable transposon end-transition sequence and a 5' portion.

[0151] In certain preferred embodiments of any method comprising creating a library of tagged DNA fragments comprising double tagged DNA fragments using DNA polymerase with a strand displacement or 5'-nuclease activity, at least one transposome in an in vitro transposition reaction comprises or consists of two various transposomes. In some preferred embodiments, where two different transposomes are used, each of the two transposomes has the same transposase but a different sequence of the transposon ends. In some preferred embodiments, where two different transposomes are used, each of them has the same transposase, and the transposon end sequences contain distinctly transferred threads. In some preferred embodiments, where two different transposomes are used, each of them contains various transposase enzymes and various transposon end sequences, each of which forms a functional complex with appropriate transposase. In certain preferred embodiments of the method, where two different transoseon end sequences are used in an in-transposition reaction, and where a tagged DNA library library of double tagged ssDNA fragments is generated using DNA polymerase with either 5 or 5 nuclease activity, the first tag it contains the sequence of the transferred thread of one transposon end sequence, and the second tag contains the sequence of the non-transferred strand of the second sequence of the transposon ends. each of which forms a functional complex with appropriate transposase. In certain preferred embodiments of the method, where two different transoseon end sequences are used in an in-transposition reaction, and where a tagged DNA library library of double tagged ssDNA fragments is generated using DNA polymerase with either 5 or 5 nuclease activity, the first tag it contains the sequence of the transferred thread of one transposon end sequence, and the second tag contains the sequence of the non-transferred strand of the second sequence of the transposon ends. each of which forms a functional complex with appropriate transposase. In certain preferred embodiments of the method, where two different transoseon end sequences are used in an in-transposition reaction, and where a tagged DNA library library of double tagged ssDNA fragments is generated using DNA polymerase with either 5 or 5 nuclease activity, the first tag it contains the sequence of the transferred thread of one transposon end sequence, and the second tag contains the sequence of the non-transferred strand of the second sequence of the transposon ends.

[0152] In some preferred embodiments of the method comprising creating a library of tagged DNA fragments comprising double tagged DNA fragments using DNA polymerase with a strand or 5 'nuclease activity, where two different transposon end sequences are used in the in vitro transposition reaction, and the method further comprises a step of amplifying double-tagged DNA fragments generated by PCR, two different PCr primers are used, one of which contains sequences of at least a portion of the transfer thread containing one sequence of the transposon ends and the other PCR primer contains the sequence of at least a portion of the transfer thread containing the second sequence the ends of the transposon. In some preferred embodiments,

The use of Terminal Transferase to create Tagged DNA Fragments

Containing Double Tagged DNA Fragments [0153] A further embodiment of the method comprises: incubating the target DNA in an in vitro transposition reaction using at least one transposome to create dsDNA tagged at the 5 'end; denature the 5 'dsDNA fragments tagged at the 5' end to create ssDNA tagged at the 5 'end using a terminal transferase DNA polymerase and at least one dNTP substrate for terminal transferase under appropriate conditions and for a sufficient period of time where the terminal transferase attaches the second fold tag with poly (dNMP) to the 3 'end of the 5'-tagged DNA fragments, thereby creating a library of tagged DNA fragments containing double tagged DNA fragments (e.g., FIG. 3).

[0154] A further embodiment of the method comprises: incubating the target DNA in an in vitro transposition reaction using at least one transposome to create 5 'tagged DNA fragments; incubation of the 5 'tagged DNA fragments without a prior denaturation step using a terminal transferase DNA polymerase and at least one dNTP substrate for terminal transferase under suitable conditions and for a sufficient period of time where the terminal transferase attaches the second poly (dNMP) tag to the end of 3 'tagged at the 5' end of the DNA fragments, creating a library of tagged DNA fragments containing double tagged DNA fragments. In some embodiments of the present method, the 3 'endof a transposome having a dideoxy nucleotide or 3'-O-methyl-nucleotide as the terminal 3 'nucleotide).

Application of DNA polymerase and Terminal Tagged Oligonucleotide to

Creating Tagged DNA Fragments Containing Double Tagged Fragments

DNA [0155] A further embodiment of the method comprises: incubating the target DNA in an in vitro transposition reaction using at least one transposome to form tagged dsDNA fragments at the 5 'end; denaturation of the 5 'dsDNA fragments tagged at the 5' end to form ssDNA tagged at the 5 'end (e.g. by heating to 95 ° C and rapid cooling); and attaching the second tag to the 5'-tagged ssDNA fragments using DNA polymerase and a terminally tagged oligonucleotide (e.g., FIG. 4), thereby creating a library of tagged DNA fragments containing double tagged DNA fragments. In some preferred embodiments, the step of attaching the second tag to the 3 'end tagged at the 5' end

(1) Providing a terminally tagged oligonucleotide comprising or consisting of a 5 'portion and a 3' portion, wherein the 5 'portion comprises a sequence complementary to the sequence of the second tag desired to attach to the 4' end of the ssDNA tagged at the 5 'end, and part 3 'comprises a random sequence comprising or consisting of three to eight (e.g., 3, 4, 5, 6, 7 or 8 random nucleotides from which the 3' terminal nucleotide is blocked, such that it is not extended by DNA polymerase;

(2) attaching the 5'-tagged ssDNA fragments to a terminally tagged oligonucleotide under appropriate conditions and for a sufficient period of time where the terminally tagged oligonucleotide binds to the 5'-tagged ssDNA fragments; and (3) attachment of the 5'-tagged ssDNA fragments to which the terminally tagged oligonucleotide binds using DNA polymerase in the reaction mixture and under DNA polymerization conditions and for a sufficient period of time where the 3 'ends of the ssDNA tagged at the 5' end are extended with a terminally tagged oligonucleotide as a pattern, while the second tag is attached to their 4 'ends and ssDNA fragments tagged at the 3' end are created. In some embodiments,

[0156] In certain embodiments of the present embodiment, the terminally tagged oligonucleotide comprises or consists of deoxyribonucleotides. In certain embodiments of the present embodiment, the terminally tagged oligonucleotide comprises or consists of ribonucleotides, in which embodiments the DNA polymerase is an RNA dependent DNA polymerase. In certain preferred embodiments, the 3 'portion of the terminally tagged oligonucleotide comprises seven random nucleotides. The method where the terminally tagged oligonucleotide is used to attach the second tag to the ssDNA tagged at the 5 'end, the second tag is not complementary to the first tag.

The application of the dependent (or Homologous) Ligase and Tagged on the way

Ligation of the Oligonucleotide to Create Tagged DNA Fragments Containing

Double Tagged DNA Fragments [0157] One preferred embodiment of the method comprises: incubating the target DNA in an in vitro transposition reaction using at least one transposome under appropriate conditions and for a sufficient period of time to create a population of tagged 5 'DNA fragments; followed by incubation of the 5 'tagged fragments using a template dependent (or homologous) DNA ligase and a tagged ligation oligonucleotide comprising lu consisting of the 3' part and the 5 'part, wherein the 3' portion contains a second tag containing the desired sequence for attach to the 3 'end of the population of tagged DNA fragments attached at the 5' end (e.g., a random sequence), and the 5 'part has a 5' monophosphate group and a random sequence, where, under appropriate conditions and for a sufficient period of time, the second tag is attached to the 5 'tagged DNA fragments fused to the library, forming a library of DNA fragments containing double tagged DNA fragments. In certain preferred embodiments, the method further comprises denaturing a library of DNA fragments comprising linked double tagged DNA fragments (e.g., by heating to 95 ° C and rapid cooling), creating a library of DNA fragments containing double tagged ssDNA fragments.

[0158] In certain preferred embodiments, the ligation tagged oligonucleotide comprises a 5 'portion comprising a random sequence of three to eight nucleotides. In certain preferred embodiments, the ligation tagged oligonucleotide comprises a random sequence of four nucleotides. In certain preferred embodiments, the reference-dependent ligase is an E. coli DNA ligase. In one preferred version of this embodiment of the method, a library of tagged DNA fragments comprising double tagged ssDNA fragments is generated from the target DNA in a single tube without a purification step from the spacer sequences.

Application of Transposon Tip Sequences with the structure of Hairpins and Ligase dependent on the Pattern for Creating a Library of Tagged DNA Fragments Containing

Tagged Pieces of Circular DNA, Pieces of Blunted dsDNA or Double

not long enough to allow the creation of an intramolecular stem-loop structure; under appropriate conditions and for a sufficient period of time, where the insertion of the hairpin transposon end sequences to the target DNA forms a population of linked 5 'DNA fragments encoded at the 5' end; followed by incubation of the attached 5 'tagged DNA fragments by one or more oligonucleotide-5'-phosphates which, alone or in combination, have the same length as the single-stranded gaps in the 5' tagged DNA fragments generated in the 5 'end. effect of in vitro transposition reactions, under appropriate conditions and for a sufficient period of time, where single-stranded gaps in the attached population tagged at the 5 'end DNA fragments are filled by linking oligonucleotides with a random sequence to the target DNA in single-stranded voids; then, incubating the population of the tagged 5 'tagged DNA fragments with single-stranded standard-patterned ligases, under appropriate conditions and for a sufficient period of time, where the random sequence oligonucleotides attached are ligated to each other or to the 5' terminal adjacencies tagged at the 5th 'DNA fragments, creating a library of tagged fragments of circular DNA.

to allow the creation of the intramolecular structure of the stem - a loop; under appropriate conditions and for a sufficient period of time, where the insertion of the hairpin end of the transposon sequence into the target DNA forms a population of linked, tagged at the 5 'end of the DNA fragments; then, incubation of the 5'-tagged DNA-attached DNA fragment with non-exonuclease DNA polymerase with 5'-to-3 'activity and 5'-nuclease with structure-dependent activity and thread-spinning activity, under appropriate conditions and for a sufficient period of time, where the single-stranded gaps present in the population of fused 5 'tagged DNA fragments after in vitro transposition reactions are filled with the 3' end of each attached 5 'tag. a DNA fragment by DNA polymerase; and then, incubation of the population population attached to the 5'-labeled DNA fragments with single-stranded standard-patterned gaps, under appropriate conditions and for a sufficient period of time where the 3 'ends of the DNA polymerase extension products are ligated to the tagged adjacently bound terminals the 5 'end of DNA fragments, thereby creating a library of tagged circular DNA fragments.

[0161] In some preferred embodiments of the present method, both the DNA polymerase and the reference-dependent ligase are provided in one reaction mixture and both the extension of the DNA polymerase and the reference-based ligation are carried out in one reaction mixture.

[0162] In some embodiments of any method of creating a library of tagged DNA ring fragments, the method further comprises, after an incubation step, with a standard-dependent ligase to form a tag library of circular DNA fragments, one or more steps to remove the un-ligated linear ssDNA and dsDNA (e.g., including random sequence oligonucleotides, linear target DNA and / or hairpin terminus sequences not attached to the target DNA). In one preferred embodiment, the removal of the non-ligated linear ssDNA and dsDNA, the method further comprises: subjecting the reaction mixture containing tagged circular DNA fragments to T5 exonuclease activity.

[0163] In some preferred embodiments of any method of creating a library of tagged DNA ring fragments, the method further comprises: cleaving tagged portions of circular DNA in each of the loop structures obtained from the hairpin end of the transposon sequence to form blunt-ended dsDNA fragments, with each thread has a part of the tag at the 5 'end and a part of the tag at the end of 3'. In some embodiments, the cleavage step of tagged circular DNA fragments in each loop structure includes: subjecting the tagged circular DNA fragments to the sequence of cleavage enzymes under appropriate conditions and for a sufficient period of time where the tagged circular DNA fragments are cleaved at cleavage sites to form blunt ends fragments of dsDNA. In some embodiments, the cleavage step of tagged circular DNA fragments in each loop structure includes: attachment of tagged oligodeoxyribonucleotide ring DNA fragments to the tag restriction site, followed by restriction endonuclease incubation resulting in cleavage at a double-stranded restriction site, under appropriate conditions and for sufficient period of time to create a library of blunt dsDNA fragments. In some preferred embodiments, the cleavage step of tagged circular DNA fragments in each loop structure includes: subjecting the tagged DNA nucleus to DNA glycosylase and endonuclease AP, where the DNA glycyclase removes the nucleic acid base from a non-canonical nucleotide (e.g., dUMP or 8-oxo-dGMP ) present in the tag, and the AP endonuclease cleaves tagged fragments of circular ssDNA at the resulting non-basic site; in some embodiments, the DNA glycosylase is selected from uracil-N-glycosylase and FPG protein, and the AP endonuclease is selected from E. coli endonuclease III or IV.

[0164] In some preferred embodiments of any method for creating a library of blunt-ended dsDNA fragments, the method further comprises the step of: denaturing a library of blunt-ended dsDNA fragments to create a library of double tagged fragments of linear ssDNA.

The use of Pattern-independent Ligase to create Tagged DNA Fragments

Containing Tagged Pieces of Circular ssDNA or Double Tagged Fragments

Linear ssDNA [0165] One preferred embodiment of the method comprises: incubating the target DNA in an in vitro transposition reaction using at least one transposome where the 5 'end of the transfer-containing transposable-containing cord has a 5' phosphate group, under appropriate conditions and for a sufficient period of time , to form a population of attached 5 'DNA fragments tagged at the 5' end; subsequent denaturation of the 5 'tagged DNA fragments attached to the 5'-end of the ssDNA fragments (e.g., by heating to 95^aboutC and rapid cooling); followed by incubation of the 5'-termed ssDNA fragments by ligation reaction with a template-independent (or non-homogeneous) ligase under appropriate conditions and for a sufficient period of time where the 5'-tagged ssDNA fragments are subjected to an intramolecular ligation to create a tagged circular fragment library ssDNA, each of which contains the sequence of the target part of the DNA and the sequence of the tag.

[0166] In one preferred version of this embodiment of the method, a library of tagged DNA fragments comprising a tagged fragment of a circular ssDNA is created from the target DNA in one tube without a purification step from the spacer sequences. In one preferred embodiment, the standard-independent ligase is selected from the thermostable RNA ligase of the TS2126 bacteriophage and the archaeal RNA ligase (e.g., Methanobacterium thermoautotrophicum RNA ligase). In certain preferred embodiments, the reference-dependent ligase is provided in an adenylated form, and the incubation step of the 5'-labeled ssDNA-tagged, pattern-dependent ligase is performed without the addition of ATP or NAD to the ligation reaction.

[0167] In some preferred embodiments, the method further comprises: cleaving tagged fragments of circular ssDNA at a site in the region of the tag, thereby creating a library of tagged DNA fragments comprising double tagged fragments of linear ssDNA. In some embodiments, the cleavage step comprises the attachment of an oligodeoxyribonucleotide complementary to the single-stranded restriction site in the region of the tagged tag of circular ssDNA fragments followed by cleavage of tagged portions of circular ssDNA at the restriction endonuclease restriction restriction site recognizing the restriction site. In some other embodiments, the cleavage step includes subjecting the tagged DNA ring fragments to DNA glycosylase and endonuclease, wherein DNA glycosyllase removes a nucleic acid base from a non-canonical nucleotide (e.g., dUMP or 8-oxo-dGMP) present in the tag, and the endonuclease cleaves tagged fragments of circular ssDNA at the resulting non-basic site; in some embodiments, the DNA glycosylase is selected from uracil-N-glycosylase and FPG protein, and the AP endonuclease is selected from E. coli endonuclease III or IV.

[0168] In some embodiments, the method further comprises the step of amplifying a library of tagged DNA fragments comprising tagged circular ssDNA fragments or double tagged linear ssDNA fragments thereby forming an amplified library of tagged DNA fragments. In some preferred embodiments, the tagging library library amplification step comprises the step of amplifying the tag library of DNA fragments by polymerase chain reaction (PCR), thereby creating an amplified library of tagged DNA fragments containing the amplified double tagged DNA fragments. In certain preferred embodiments, the PCR reaction is performed using the first PCR primer and the second PCR primer, each of which has a 3 'part and a 5' part, where the 3 'part

Ways to create libraries of DNA fragments with Enhanced Sequence Representation on

The ends of the Target DNA [0169] The inventors observed specific sequential data showing that the representation of the next generation sequential data from the ends of the target DNA containing dsDNA particles below 10 Kb is low compared to the representation of sequence data from the center of such target DNA. Without delving into theoretical considerations, one possible explanation for this observation is that the probability of finding DNA fragments with two sequences of transposon inserts inserted in reverse directions at the ends of the linear dsDNA molecule is lower than the probability of finding DNA fragments with two sequences of transposon inserts inserted in opposite directions in the middle of the dsDNA linear molecule. In order to solve this problem,

each of which has a first so consisting of or containing a thread at the 5 'end; denaturation of the tagged dsDNA fragments at the 5 'end to release the ssDNA tagged at the 5' end; followed by circularization of the ssDNA tagged at the 5'-end with an intramolecular ligase that is independent of the standard, ligating ssDNA (e.g., bacteriophage TS2126 RNA ligase; e.g., thermostable CIRCLIGASE ™ ssDNA ligase, EPICENTRE, Madison, WI, USA), thereby creating a library tagged circular ssDNA fragments. In some embodiments, at least one transposase and at least one sequence of the transposon ends is added to the reaction as separate components and not as a single component comprising the transposom sequence. In some embodiments, tagged circular ssDNA fragments are used as next-generation sequencing patterns, or after determination, as ssDNA targets for linking to probes on matrices or microarrays or for other uses described in this application. In some other embodiments, the method further comprises the step of linearizing the tagged circular ssDNA fragments in the first tag, forming double-tagged fragments of the linear ssDNA. In some other embodiments involving the linearization of tagged circular ssDNA fragments, the first tag comprises a plurality of tag domains, wherein the first tag linearization stage generates one part of the first tag at the 5 'end and the second part of the first tag at the 3' end. For example, in some embodiments, the transferred transcript of the transposon ends contains the first so many multi-domain tags (e.g., both Roche 454A sequencing and Roche 454B sequencing domains) of which at least one tag domain is attached to the 3 'end of the double tagged ssDNA fragments formed in the linearization step tagged circular ssDNA fragments. For example, in some embodiments, 5'-tagged DNA fragments are created using a transposon sequence containing a transferred thread containing one or more nucleotides to allow cleavage at the sites of said nucleotides, and the linearization step of the tagged circular ssDNA fragments in the tag comprises the cleavage of tagged circular ssDNA fragments at said nucleotide or more nucleotides. For example, in some embodiments, the transferred thread contains one or more deoxyuridine nucleotides or one or more 849 oxoguanine nucleotides (e.g. synthesized using an oligonucleotide synthesizer), and the step of linearizing the tagged circular ssDNA fragments in the tag comprises cleavage of tagged fragments of the uRDy circular ssDNA DNA with glycosylation or formamidopyrimidine-DNA glycolase, as well as DNA-splitting endonuclease at a non-basic site (e.g., endonuclease IV). For example, in some other embodiments, tagged circular ssDNA fragments are linearized in the tag by attaching a complementary oligonucleotide to the tag and linearizing with restriction endonuclease recognizing the restriction site in the double-stranded tag. In some other embodiments comprising the linearization of tagged circular ssDNA fragments, the method further comprises purifying double tagged ssDNA fragments (e.g., using a Qiagen PCR purification column); in some embodiments, the double tagged ssDNA fragments are used as the next generation sequencing patterns or, after labeling, as target ssDNA for attachment to probes on the array or microarrays, or for other applications described in the present application.

Amplification of Tagged DNA Fragments and Other Embodiments [0171] In some embodiments of any method of forming a library of tagged DNA fragments, the method further comprises: amplifying a tag library of DNA fragments containing double tagged DNA fragments, tagged circular ssDNA fragments, or blunt DNA fragments .

[0172] In some embodiments of any method, the method further comprises the step of: amplifying a library of double tagged linear ssDNA fragments, tagged portions of circular DNA, or blunt-ended DNA fragments using a polymerase chain reaction (PCR). Hence, in some embodiments, the method further comprises (a) providing (i) a first and a second PCR primer, wherein at least the 3 'end of the first PCR primer is complementary to at least a portion of the tagged tag sequence of the circular DNA fragments or at least a portion of the tag. tag sequence attached to the 3 'end of blunt dsDNA fragments or with at least a portion of the tag sequence attached to the 3' end of the ssDNA linear fragments, and where at least the 3 'end

[0173] In some embodiments, where the method comprises amplifying tagged DNA core fragments using PCR, the first PCR primer is complementary to the tag sequence in at least a portion of the loop structure of the hairpin transposon endnotes inserted into the target DNA tagged circular fragments. The DNA and / or the second PCR primer contains a sequence identical to at least a portion of the structure of the hairpin transposon terminus inserted into the target DNA of tagged DNA ring fragments. In some embodiments, the first PCR primer is complementary to at least a portion of the transposed transfer sequence or non-transferred transposon end sequence,

In some embodiments, the 5 'portion of the first PCR primer or the 5' portion of the second PCR primer additionally or additionally consists of the addressing tag domain or other tag domain for a particular purpose. In other embodiments, the tagged tag of circular DNA fragments includes a sequencing tag for next-generation sequencing using a particular platform.

[0175] In embodiments of the method, where the library of tagged DNA fragments comprising double tagged DNA fragments is generated using a DNA polymerase having 5 'nuclease activity or thread-transfer activity, the library amplification step involves the use of a single oligodeoxyribonucleid complement primer complementary to the second tag to amplify the library tagged DNA fragments by PCR. In some embodiments, the single primer used for PCR contains at least a portion of the transposon end of the transfer sequence. In some other embodiments, the single primer used for PCR comprises at least a portion of the 5'sequence sequence of the transposon end oligonucleotide transferred. In some other preferred embodiments, the step of library amplification or tagged DNA fragments comprising double tagged DNA fragments using a single oligonucleotide primer comprises: providing a single oligonucleotide primer complementary to the second tag at the 3 'end of the tagged DNA fragments and a thermostable DNA polymerase suitable for PCR; incubating the library of tagged DNA fragments with the oligonucleotide primer and thermostable DNA polymerase under PCR amplification conditions for a sufficient period of time, where a library of tagged DNA fragments is subjected to PCR amplification, creating a library of amplified tagged DNA fragments. providing a single oligonucleotide primer complementary to the second tag at the 3'end of tagged DNA fragments and a thermostable DNA polymerase suitable for PCR; incubating the library of tagged DNA fragments with the oligonucleotide primer and thermostable DNA polymerase under PCR amplification conditions for a sufficient period of time, where a library of tagged DNA fragments is subjected to PCR amplification, creating a library of amplified tagged DNA fragments. providing a single oligonucleotide primer complementary to the second tag at the 3'end of tagged DNA fragments and a thermostable DNA polymerase suitable for PCR; incubating the library of tagged DNA fragments with the oligonucleotide primer and thermostable DNA polymerase under PCR amplification conditions for a sufficient period of time, where a library of tagged DNA fragments is subjected to PCR amplification, creating a library of amplified tagged DNA fragments.

a second PCR primer complementary to at least a complement of the second tag of double tagged DNA fragments or blunt DNA fragments or with at least a portion of the tag complement in the tagged circular ssDNA fragments; and (b) a thermostable DNA polymerase suitable for PCR; and (2) incubating the library of tagged DNA fragments with PCR primers and thermostable DNA polymerase under PCR amplification conditions and for a sufficient period of time where a library of tagged DNA fragments is amplified to create a library of amplified tagged DNA fragments. In some embodiments, the first and second PCR primers comprise a 3 'portion and a 5' portion, where the 5 'portion is not complementary to the sequence of the appropriate tag or complement thereof in the tagged DNA fragments and a 3' portion is complementary to the sequence of the appropriate tag or its complement. In some embodiments, the first and second PCR primer portion 5 'comprises or consists of appropriate first and second sequencing tags allowing them to be used to create patterns for next generation sequencing (e.g., Roche 454A and 454B sequencing tags or the corresponding first and second tag) sequencing for another sequencing platform, including, but not limited to, Illumina Solexa or Applied Biosystems Solid platforms).

[0177] A range of enzymes and kits are available for carrying out the amplification reaction by PCR. For example, in some embodiments, PCR amplification is performed using the FAILSAFE ™ PCR System or MASTER-AMP ™ Extra-Long PCR System from EPICENTER Biotechnologies, Madison, WI, as described by the manufacturer. These systems enable rapid optimization of the PCR reaction conditions thanks to a series of 2 Preixix Preixix premixes supplied with each system to identify the optimal pre-mix for a given standard and pair of primers. However, the invention is not limited to the use of these products or conditions in the amplification reaction and any suitable thermostable DNA polymerase and reaction mixture allowing the amplification of the sequence between the primer may be used therein,

[0178] The invention is also not limited to the use of PCR for amplifying a library of tagged DNA fragments. Any suitable amplification method may be used in the embodiments of the present invention (e.g., rolling circle amplification, amplification with a riboprimeter (e.g., US Patent No. 7,413,857), ICAN, UCAN, fish-SPIA, terminal tagging (US Patent Application No. 20050153333 ), EBERwine-type amplification of aRNA or thread-displacement amplification) that amplifies the same sequence and creates the appropriate sequence and amount of amplification product for the intended purpose. For example, some of the transfer methods described in PCT Patent Publication Nos. WO 02/16639 may be used; WO 00/56877; and AU 00/29742; Takara Shuzo Company, Kyoto, Japan; U.S. Patent Application No. 5,523,204; 5,536,649; 5,624,825; 5,631,147; 5,648,211; 5,733,752; 5,744,311; 5,756,702; and 5,916,779 Becton Dickinson and Company; U.S. Patent Application No. 6,238,868; 6,309,833; and 6,326,173 Nanogen / Becton Dickinson Partnership; U.S. Patent Application No. 5,849,547; 5,874,260; and 6,218,151 Bio Merieux; U.S. Patent Application No. 5,786,183; 6,087,133; and 6,214,587 Gen-Probe, Inc .; U.S. Patent Application No. 6,063,604 to Wick et al; U.S. Patent Application No. 6,251,639 to Kurn; U.S. Patent Application 6,410,278; and PCT Publication No. WO 00/28082 by Eiken Kagaku Kabushiki Kaishi, Tokyo, Japan; U.S. Patent Application No. 5,591,609; 5,614,389; 5,773,733; 5,834,202; and 6,448,017 Auerbach; and U.S. Patent Application No. 6,124,120; and 6,

[0179] In preferred embodiments of the invention, the selection of 5 'tagged DNA fragments formed by the in vitro transposition reaction or the final library of tagged DNA fragments in size is not necessary. In the case of size or purification necessary for specific applications, the 5 'tagged DNA fragments can be sized for size by agarose gel electrophoresis (e.g. using a low melting and non-denaturing agarose gel suitable for the desired order of agarose). the size of DNA fragments) and purify (e.g., remove unsuccessful transposon end oligonucleotides, other reaction products and agarose gel, e.g. by digesting a portion of an agarose gel containing the desired order of magnitude tagged at the 5 'end. DNA fragments by GELase ™ agarose gel digesting enzyme, EPICENTER Biotechnologies, Madison, WI, USA, followed by alcohol precipitation and other purification steps as indicated for GELase, or by using a different purification process (purification) known in the art). In some embodiments, a purification step comprising polyethylene glycol (PEG) precipitation is used to precipitate a library of tagged DNA fragments without precipitating contaminants (e.g., without limitation, tagged by ligation of oligonucleotides not ligated or other reaction components). In some embodiments, a centrifugal column or other purification method known in the art is used. and then by alcohol precipitation and other purification steps as indicated for the GELase product, or by using a different purification method (purification) known in the art). In some embodiments, a purification step comprising polyethylene glycol (PEG) precipitation is used to precipitate a library of tagged DNA fragments without precipitating contaminants (e.g., without limitation, tagged by ligation of oligonucleotides not ligated or other reaction components). In some embodiments, a centrifugal column or other purification method known in the art is used. and then by alcohol precipitation and other purification steps as indicated for the GELase product, or by using a different purification method (purification) known in the art). In some embodiments, a purification step comprising polyethylene glycol (PEG) precipitation is used to precipitate a library of tagged DNA fragments without precipitating contaminants (e.g., without limitation, tagged by ligation of oligonucleotides not ligated or other reaction components). In some embodiments, a centrifugal column or other purification method known in the art is used. In some embodiments, a purification step comprising polyethylene glycol (PEG) precipitation is used to precipitate a library of tagged DNA fragments without precipitating contaminants (e.g., without limitation, tagged by ligation of oligonucleotides not ligated or other reaction components). In some embodiments, a centrifugal column or other purification method known in the art is used. In some embodiments, a purification step comprising polyethylene glycol (PEG) precipitation is used to precipitate a library of tagged DNA fragments without precipitating contaminants (e.g., without limitation, tagged by ligation of oligonucleotides not ligated or other reaction components). In some embodiments, a centrifugal column or other purification method known in the art is used.

[0180] In some embodiments, tagged portions of circular DNA are used as DNA sequencing standards.

[0181] In some embodiments, tagged DNA fragments are used as DNA sequencing standards.

[0182] In some embodiments, a library of tagged DNA fragments is used as a template for amplification reactions (e.g., PCR amplification reactions using PCR primers complementary to first and second tagged DNA fragments containing double tagged DNA fragments or blunt DNA fragments or complementary fragments) with tagged tagged DNA fragments containing tagged circular ssDNA fragments). In some preferred embodiments, the library of amplified tagged DNA fragments contains the majority or consists virtually entirely of the target DNA sequence. In some embodiments, where the target DNA contains the genomic DNA of the organism, the amplification reaction is a whole genomic amplification reaction.

[0183] In some embodiments of a method comprising amplifying tagged DNA fragments, the amplified fragments are determined by incorporating a tagged nucleotide during one or more steps of the amplification method (e.g., a PCR amplification reaction method). In some embodiments, a library of amplified tagged DNA fragments is used to detect or capture or to detect and capture amplified tagged DNA fragments containing a tag for a particular application.

[0184] Some embodiments of any method of forming a library of tagged DNA fragments (e.g., double tagged DNA fragments) comprise forming a library of &amp; quot; tagged &amp; quot; tagged DNA fragments comprising one or more groups (e.g., one or more molecules with binding affinity) to allow labeled capture. tagged DNA fragments on the surface, or one or more detectable groups capable of detecting tagged tagged DNA fragments (e.g., combining with complementary DNA, such as complementary DNA on a chromosome). In addition, some embodiments of any method of the invention further comprise amplifying a library of tagged DNA fragments comprising one or more groups (e.g., one or more molecules with binding affinity),

DNA (e.g., combining with complementary DNA, such as complementary DNA on a chromosome). In some embodiments, a library of tagged tagged DNA fragments or labeled amplified DNA fragments is generated using at least one tagged oligonucleotide (e.g., a tagged transposon end oligonucleotide tagged, a tagged oligonucleotide tagged by ligation or at least one labeled amplification primer, such as least one (or more than one) PCR primer). In some other embodiments, a library of tagged amplified tagged DNA fragments is created by incorporating labeled dNTPs incorporated into the amplification products during the amplification reaction. Labeled dNTPs may have any tag known in the art, which can be used to create labeled amplified tagged DNA fragments, e.g. by direct or indirect labeling. By "direct labeling" we understand that the capture group or detectable label is attached directly to the amplified tagged DNA fragments without the presence of another group between the capture group or the detectable group, and the tagged DNA fragment or tagged DNA fragment amplified. By "indirect labeling" we understand that there is at least one other group between the capture group or the detectable group, and the tagged DNA fragment or the amplified tagged DNA fragment. One example of direct labeling is the inclusion of a tagged nucleotide into tagged DNA fragments, and an example of indirect labeling is incorporation of a biotin-labeled nucleotide into tagged DNA fragments followed by labeling of tagged DNA fragments by a group of dye-labeled group detectable by incubating a dye-labeled streptavidin under conditions where the streptavidin-labeled dye binds to biotin labeled nucleotides. The present invention includes the use of any suitable method for creating a library of tagged DNA fragments or tagged tagged DNA fragments, wherein the label is then used to capture or detect (detect). where streptavidin-labeled dye binds to biotin labeled nucleotides. The present invention includes the use of any suitable method for creating a library of tagged DNA fragments or tagged tagged DNA fragments, wherein the label is then used to capture or detect (detect). where streptavidin-labeled dye binds to biotin labeled nucleotides. The present invention includes the use of any suitable method for creating a library of tagged DNA fragments or tagged tagged DNA fragments, wherein the label is then used to capture or detect (detect).

[0185] In some other embodiments, the tagged DNA fragments in a library created using the method of the invention are sequentially labeled, directly or indirectly, by subjecting the library of tagged DNA fragments to a reactive dye molecule (e.g., reactive fluorescent dyes comprising an N-hydroxysuccinimidyl ester or " NHS "Molecular Probes, Eugene, OR) or a reactive molecule with binding affinities (e.g., a reactive biotinylation reagent such as a biotin-HNS compound, Pierce Chemical Company, Rockford, IL). For example, in some embodiments, a library of tagged tagged DNA fragments is created by including a dNTP containing an allylamine group in the amplification process, and then a library of amplified tagged DNA fragments containing an allylamyl group is treated with labeled fluorescent NHS ester or biotin-NHS ester to generate the tagged fluorescent dye labeled amplified DNA fragments or the amplified tagged DNA fragments labeled with biotin, respectively. Those skilled in the art will know or have knowledge about how to identify additional methods and reagents, including kits, e.g. from Molecular Probes, designed to label amplified tagged DNA fragments for a particular purpose (e.g., to allow surface capture or detection). For example, examples may include one or more modified nucleotides having an allylamino group, a propyl group, a biotin group, a fluorescent dye or other detectable dye, or other detectable molecule or combination of molecules known in the art, including quantum dots, an enzyme (e.g., phosphatase, peroxidase or pyrophosphatase) or a detectable protein (e.g., phycobiliprotein, phycoerythrin). In some other embodiments, a library of labeled amplified tagged DNA fragments is created by incorporating one or more dNTP modified tagged molecules with a binding affinity or a detectable group in an amplification reaction, e.g. in a PCR amplification reaction, e.g. by incorporating one or more modified ones. dNTPs having an allylamino group, a biotin group, a fluorescent dye or other detectable dye, or other detectable molecule or combination of molecules known in the art, including quantum dots, an enzyme (e.g.

[0186] In some embodiments, tagged DNA fragments (e.g., double tagged DNA fragments used to create tagged DNA fragments for hybridization to surface bound probes (e.g., as labeled target DNA for hybridizing to DNA probes on a matrix or microarrays)) . In some embodiments, tagged 9np DNA fragments. containing double tagged DNA fragments) are used for hybridization to chromosomes or parts of chromosomes in fixed cells or tissue sections (e.g., fluorescent in situ hybridization or FISH).

[0187] In some embodiments, a method comprising forming tagged tagged DNA fragments or tagged amplified tagged DNA fragments (e.g., tagged double tagged DNA fragments or labeled amplified double tagged DNA fragments) for use in chromosomal hybridization (e.g., labeled tagged fragments DNA is derived from a DNA target containing DNA from one or more specific chromosomes for use as "chromosomal paints" (e.g., for hybridization to one or more chromosomes in fixed cells or tissue sections, e.g. for fluorescent in situ hybridization or FISH for such applications such as chromosomal typing, or for research purposes, medical diagnosis, gender identification or other applications in molecular biology).In some embodiments, the method comprises forming labeled tagged DNA fragments or labeled amplified tagged DNA fragments from a target DNA containing parts of chromosomes (e.g., tagged DNA fragments are formed from DNA encoding one or more specific genes or loci of one or more chromosomes ( e.g. for hybridization to one or more chromosomes in fixed cells or tissue slices, e.g. using in situ fluorescence or FISH hybridization or for use as genospecific or loci specificist probes in in vitro assays for applications such as analytical or specific studies diagnostic for medical, industrial, environmental or molecular applications or molecular biology research).the method comprises forming labeled tagged DNA fragments or labeled amplified tagged DNA fragments from a target DNA containing parts of chromosomes (e.g. where tagged DNA fragments are formed from DNA encoding one or more specific genes or loci of one or more chromosomes (e.g., for hybridization) to one or more chromosomes in fixed cells or tissue sections, e.g. using in situ fluorescence or FISH fluorescence or for use as genospecific or loci-specific probes in in vitro tests for applications such as analytical or diagnostic tests for medical applications, industrial, environmental or molecular research or molecular biology research).the method comprises forming labeled tagged DNA fragments or labeled amplified tagged DNA fragments from a target DNA containing parts of chromosomes (e.g. where tagged DNA fragments are formed from DNA encoding one or more specific genes or loci of one or more chromosomes (e.g., for hybridization) to one or more chromosomes in fixed cells or tissue sections, e.g. using in situ fluorescence or FISH fluorescence or for use as genospecific or loci-specific probes in in vitro tests for applications such as analytical or diagnostic tests for medical applications, industrial, environmental or molecular research or molecular biology research).

[0188] In some embodiments, hybridization of labeled tagged DNA fragments to surface probes (e.g., matrix or microarrays, test strip, quantum dot, bead, microchannel in a microfluidic device) is used to detect, quantify, determine relative sizes or characteristics of one or more DNA molecules or parts of DNA contained in or derived from a natural source (e.g., genomic DNA from a cell, e.g. human DNA for the variation of copy number (CNV), or DNA from bacterial, fungal, mycoplasma, viral or nematode cells pathogen) or an in vitro source (e.g., double-stranded cDNA obtained by reverse transcription of RNA, such as mRNA or non-coding RNA or viral RNA,isolated from a natural source or amplified from a natural source using a method for amplifying nucleic acids, such as amplification of DNA or RNA).

[0189] In some other embodiments, wherein the method comprises amplifying tagged DNA fragments, the method comprises generating tagged amplified tagged DNA fragments by incorporating one or more modified dNTPs having an allylamino group, a biotin group, a fluorescent dye or other detectable dye or other group enabling its detection, directly or indirectly, after labeling with another detectable molecule or combination of molecules known in the art, including quantum dots or an enzyme or a detectable protein (e.g., phycobiliprotein, phycoerythrin) connected to a molecule with binding affinity (e.g., streptavidin, antibody).

phycoerythrin) combined with a molecule with binding affinity (e.g., streptavidin, antibody). In some embodiments, suitable products are used to create tagged nucleic acid fragments to hybridize to surface bound probes (e.g., as labeled target nucleic acid for hybridizing to DNA probes on a matrix or microarrays). In some embodiments, suitable labeled products are used to hybridize to chromosomes or parts thereof in fixed cells or tissue fragments (e.g., for in situ fluorescence or FISH hybridization). In some embodiments, the hybridization of labeled products to probes on a surface is used to detect, quantify,

[0191] In some embodiments of methods comprising forming a library of tagged DNA ring fragments, the transferred transposon end oligonucleotide, further comprising a transposon end sequence at the 3 'end, also contains a single strand RNA polymerase promoter sequence in the 5' part. In some embodiments of methods comprising forming a library of double tagged DNA fragments using a tagged oligonucleotide and a template-dependent ligase, the tagged ligation oligonucleotide comprises the single strand sequence of the double stranded RNA polymerase promoter in the 3 'part. In some embodiments of the methods, wherein the transferred transposon end oligonucleotide or oligonucleotide tagged by ligation does not contain an RNA polymerase promoter sequence, the method further comprises PCR amplification of double tagged DNA fragments using at least one PCR primer being a "promoter primer". The promoter promoter has a "5 'end" or "5' tail" that does not associate with double tagged DNA fragments and contains a single strand double strand RNA polymerase promoter sequence and a 3 'portion joining with the first or second tag tagged at the 5' end and 3 'DNA fragments or their genes.

[0192] In certain preferred embodiments, where the transposon end oligonucleotide to be tagged by ligation or PCR primer contains the RNA polymerase promoter sequence, the RNA polymerase promoter is a T7 type RNA polymerase promoter, and the method further comprises a 5 '5'-end transcription step and 3 'in vitro DNA fragments using T7 type RNA polymerase recognizing the promoter. Most preferably, the RNA polymerase and promoter are selected from RNAP T7, RNAP T3 and RNAP SP6 and their corresponding related promoters. However, the steps of transcription of the method of the invention may use RNAP for which a suitable promoter sequence is known which enables transcription with high specificity or for which such a sequence may be obtained. Kits and enzymes for in vitro transcription are commercially available from many suppliers and used for appropriate reaction mixtures and under appropriate conditions to carry out the steps of the present invention containing in vitro transcription as described by the manufacturer. For example, in vitro transcription using RNAP T7 can be performed using the AMPLISCRIBE ™ Transcription Kit T7-Flash ™ Transcription Kit or the AMPLISCRIBE ™ T7 High Yield Transcription Kit, EPICENTER Biotechnologies, Madison, WI, as described in the product description. Similarly, if RNAP T3 or RNAP SP6 is used for in-vitro transcription in the method of the invention, it may be used as described herein, respectively.

[0193] In some embodiments, the transposon end grafted oligonucleotide, ligation tagged oligonucleotide or PCR primer contains, besides the RNA polymerase promoter sequence, additional translation sequences, including, but not limited to, a ribosome binding site and a translation initiation codon (hereinafter referred to as "the"). also a &amp; quot; start signal of translation &amp; quot;) and the method further comprises translation of transcribed RNA. In some embodiments, the method also comprises an in vitro translation step of the resulting RNA transcripts. Systems and kits for translating in vitro RNA transcripts are also available commercially from many sources and can be used in the present invention. By way of example, but not limited to, the present invention can be rabbit reticulocyte lysate, wheat germ extract and E. extraction systems. coli S30 from PROMEGA Corporation, Madison, WI. In addition, commercially available in vitro conjugated transcription and in vitro translation kits such as TNT® Quick Coupled Transcription / Translation Systems conjugated transcription and translation systems from Promega can be used.

[0194] In some preferred embodiments of the method, a library of double tagged DNA fragments formed from a target DNA containing a DNA sample from the entire genome of a cell or organism is subjected to PCR amplification (i.e., the method comprises or consists of a whole genome amplification method). In some embodiments, the whole genome amplification method is used to amplify the entire genome from one cell. In some preferred embodiments of the whole genome amplification method in the present application, a library of tagged DNA fragments is created from a DNA sample from the entire genome of a cell or organism being PCR amplified using a single oligonucleotide primer (or PCR primer) complementary to the second tag.

[0195] In some embodiments, tagged DNA fragments formed according to the method of the invention are formed from a target DNA comprising or consisting of genomes and / or double stranded cDNA formed from RNA derived from all organisms (e.g., many organisms) present in an environmental trial (e.g. for metagenomic or metatranscryptomic applications, including for industrial, medical or research applications).

[0196] In some other embodiments of the method, a library of tagged DNA fragments is formed from a target DNA comprising DNAs consisting of or consisting of a single chromosome or part of a chromosome. In some embodiments, the method comprises PCR amplification of tagged DNA fragments formed from a target DNA assembled or containing a single chromosome DNA or a portion of a chromosome, including a portion comprising one or more genes or gene loci under conditions where the PCR amplified products are labeled with a detectable group ( e.g. a fluorescent dye, an infrared fluorescent, a chemiluminescent, visible or other detectable dye, e.g. using a dNTP dye-labeled PCR. In some embodiments, PCR amplified products labeled with a detectable group are used to stain fixed cells in situ (e.g., PCR amplification products are used as chromosomal inks). Hence, in some preferred embodiments, the method comprises or consists of a method for forming chromosomal or subchromosome paints or chromosomal markers.

[0197] In some embodiments, tagged DNA fragments or amplified tagged DNA fragments formed using the method are used as target DNA for a second round of fragmentation and tagging using the method of the invention. In some embodiments, the same transposomes are used in both the first and second method rounds. In some embodiments, a second, different transposase, and different ends of the transposon are used for the second round.

[0198] In some embodiments, the tagged DNA fragments or amplified tagged DNA fragments formed using the method are cloned in a vector (e.g., in a COPYCONTROL ™ fosmid vector, EPICENTER Biotechnologies, Madison, WI, USA). In some embodiments, the method further comprises cloning tagged DNA fragments or amplified tagged DNA fragments, and wherein tagged DNA fragments or amplified tagged DNA fragments (e.g., tagged DNA fragments PCR amplified) contain a DNA polymerase promoter, the method further includes transcription at least one strand of cloned tagged DNA fragments or amplified tagged DNA fragments. In some embodiments, cloned tagged DNA fragments are transcribed in vitro using an RNA polymerase that recognizes the RNA polymerase promoter. In some embodiments, cloned tagged DNA fragments or amplified tagged DNA fragments are transcribed in vivo in a host cell capable of inducing the expression of an RNA polymerase recognizing the RNA polymerase promoter followed by transcription of DNA templates containing a promoter to which the RNA polymerase binds (e.g. pET is widely used for the expression of in vivo proteins from the induced T7 RNA polymerase). In certain preferred embodiments, the RNA polymerase for expression and transcription in vitro or in vivo in a T7 type RNA polymerase is initiated from the corresponding related T7 type RNAP promoter. In some preferred embodiments,

[0199] In some embodiments of any method, either a transposon end oligonucleotide, a ligation tagged oligonucleotide or a PCR primer, contains or is coupled to an affinity molecule (e.g., biotin or digoxigenin), and the method further comprises the step of: providing a solid coated surface covalently or non-covalently a substance with a binding affinity capable of specifically binding and forming a specific pair of bonds with an affinity molecule (e.g., streptavidin or avidin to bind biotin or an antibody to bind digoxigenin); and before or after the stage in which it is involved, treatment with products formed using the transposon end of the transfer oligonucleotide,

[0200] The invention is not limited to a given solid surface that can be porous or non-porous, and a sequence, size or shape suitable for a given method and use. For example, but not limited to, a solid surface can be selected from the group consisting of: magnetic beads, coated beads, microscope slides, microtitre plate walls, test tubes and test strips containing glass, plastic (e.g., latex or polystyrene), silicon, Teflon and other suitable material. The purpose of the solid surface is to cover it with a substance with binding affinity to allow manipulation (e.g., scavenging and washing to remove other molecules in the reaction mixture), isolating and binding the transferred oligonucleotide to the end of the transposon, an oligonucleotide tagged by a ligation or PCR primer chemically bound to an affinity molecule or to allow manipulation, isolation and capture of 5 'tagged DNA fragments, tagged at the 5' and 3 'ends of DNA fragments or PCR products formed therefrom. In order to prevent non-specific binding, in some embodiments, the solid surface is subjected to a large amount of a substance selected from the group consisting of DNA-free tRNA; proteins (e.g., BSA), polysaccharide (e.g., glycogen, dextran sulfate or heparin). The invention is also not limited to a specific affinity molecule or a substance with binding affinity as long as they are capable of specifically binding and forming a specific pair of bonds. isolating and capturing 5 'tagged DNA fragments tagged at the 5' and 3 'ends of the DNA fragments or PCR products formed therefrom. In order to prevent non-specific binding, in some embodiments, the solid surface is subjected to a large amount of a substance selected from the group consisting of DNA-free tRNA; proteins (e.g., BSA), polysaccharide (e.g., glycogen, dextran sulfate or heparin). The invention is also not limited to a specific affinity molecule or a substance with binding affinity as long as they are capable of specifically binding and forming a specific pair of bonds. isolating and capturing 5 'tagged DNA fragments tagged at the 5' and 3 'ends of the DNA fragments or PCR products formed therefrom. In order to prevent non-specific binding, in some embodiments, the solid surface is subjected to a large amount of a substance selected from the group consisting of DNA-free tRNA; proteins (e.g., BSA), polysaccharide (e.g., glycogen, dextran sulfate or heparin). The invention is also not limited to a specific affinity molecule or a substance with binding affinity as long as they are capable of specifically binding and forming a specific pair of bonds. in some embodiments, the solid surface is subjected to a large amount of a substance selected from the group consisting of DNA-free tRNA; proteins (e.g., BSA), polysaccharide (e.g., glycogen, dextran sulfate or heparin). The invention is also not limited to a specific affinity molecule or a substance with binding affinity as long as they are capable of specifically binding and forming a specific pair of bonds. in some embodiments, the solid surface is subjected to a large amount of a substance selected from the group consisting of DNA-free tRNA; proteins (e.g., BSA), polysaccharide (e.g., glycogen, dextran sulfate or heparin). The invention is also not limited to a specific affinity molecule or a substance with binding affinity as long as they are capable of specifically binding and forming a specific pair of bonds.

[0201] Hence, in some embodiments, tagged DNA fragments or amplified tagged DNA fragments are captured, isolated, purified, or used in a different method by binding to a solid surface, the method comprising the steps of: contacting tagged DNA fragments or amplified tagged DNA fragments containing an affinity molecule with a solid surface in the presence of reagents and conditions to facilitate binding to a substance having a binding affinity attached to a solid surface, where the tagged DNA fragments or amplified tagged DNA fragments are bound to the surface.

[0202] In some preferred embodiments, the affinity molecule is biotin, and the substance having a binding affinity of avidin or streptavidin, or an affinity molecule is digoxigenin, and a substance having an affinity binding antibody that specifically binds digoxigenin.

[0203] As used herein, the terms "transposase" and "DNA polymerase" and "ligase" refer to protein molecules or complexes of protein molecules responsible for the catalysis of specific biological and chemical reactions. In general, the method, sequence, set of the invention is not limited to the use of a specific transposase enzyme or DNA polymerase from a particular source, but rather comprise any enzyme, transposase or polymerase.DNA from any source that has equivalent enzymatic activity to the enzyme activity disclosed in the present invention with respect to a particular method, sequence or kit. In addition, the methods of the present invention also include embodiments where each particular enzyme provided and used in the process step is replaced by a combination of two or more enzymes that, when used in combination, used alone or in combination or together, simultaneously in the reaction mixture, produce results. identical to the results obtained using one particular enzyme. The methods, buffer solutions and reaction conditions set forth in the present application are currently preferred for embodiments of the methods, sequences and kits of the present invention. However, other buffer solutions for storage are known in the art,

Embodiments of Sequences and Sets [0204] The invention also includes kits and sequences for the method of the invention. The kit is a combination of individual sequences useful for carrying out the method of the invention, wherein the sequences are optimized for the combined use in the method. The sequence consists of components for at least one step of the method of the invention. The invention includes a kit that can be formed from a combination of any two new sequences or sets of the invention, or from any new sequence used in the kit. In some embodiments, a kit or sequence comprises or consists of a subset of the set or sequences described in the present application, in a suitable combination and for any reason,

Embodiments of the Sequence [0205] One embodiment of the sequence of the invention is a transposome sequence comprising (i) a transferred thread having a 3 'portion comprising the transposon end transcribed sequence and a 5' portion comprising the tag domain sequence, and (ii) a non-transferlable 5 'phosphate thread containing also only the transposon end of the transfer sequence, where the transposase forms a complex with the transposon final sequence in an in vitro transposition reaction. In some embodiments, the tag domain is the tag domain used in next generation sequencing or amplification. In some embodiments, the tag domain is selected from a restriction site domain, a capture tag domain, a sequencing tag domain, a capture tag domain, an addressing tag domain,

[0206] Another sequence of the invention is the transferred transposon end sequence, where the transfer thread comprises a 3 'and 5' part, wherein the 3 'part contains the transposon end transcribed sequence and the 5' part contains the transcriptional promoter domain containing the RNA polymerase promoter sequence.

[0207] Another sequence of the invention is a hairpin-like transposon sequence or comprises an oligonucleotide with a 5 'phosphate group containing a non-transferred transposon endpoint at the 5' end, a transposon end sequence transcribed at the 3 'end, and a binaural random tag sequence between both non-transferable and transferred the transposon end sequence, long enough to allow the intramolecular loop structure to be formed. In certain preferred embodiments, the hairpin end of the transposon sequence contains the transposon end sequences of the hyperactive Tn5 transposon. In some other embodiments, the hairpin end of the transposon sequence is more often adenylated at the 5 'end than does the 5' phosphate group.

[0208] The invention also comprises methods of making the methods. For example, one sequence of the invention is an oligonucleotide comprising a 3 'part and a 5' part, wherein the 3 'part contains the transposon terminal sequence transferred and the 5' part comprises a restriction site domain (e.g., a rarely cut restriction endonuclease such as NotI or AscI, or for type II restriction endonuclease such as FokI). For example, another sequence of the invention is an oligonucleotide comprising a 3 'part and a 5' part, wherein the 3 'part contains the transposon terminal sequence transferred, and the 5 part contains the RNA polymerase promoter sequence (e.g., for T7, T3, SP6 or N4 phage RNA polymerase). In certain preferred embodiments, the RNA polymerase promoter sequence is a promoter sequence for any of the aforementioned RNA polymerases. In some other embodiments, the RNA polymerase promoter sequence is the antisense promoter sequence for any of the above-mentioned RNA polymerases. Another sequence of the invention is an oligonucleotide comprising a 3 'part and a 5' part, wherein the 3 'part contains the transposon endransferred sequence, and the 5' part comprises a tag domain selected from the sequencing tag domain, amplification tag domain, capture tag domain, addressing domain domain, Discovery tag domain and tag domain of the restriction site.

[0209] In some preferred embodiments, the transposon end-of-transposition sequence transferred is the MEDS or pMEDS transposon end of the EZ-Tn5 ™ transposase (EPICENTRE). In some preferred embodiments, the sequencing tag domain comprises a sequencing tag suitable for the ROCHE 454 sequencing platform, ILLUMINA ™ SOLEXA ™ sequencing platform, LIFE TECHNOLOGIES / APPLIED BIOSYSTEMS 'SOLID ™ sequencing platform, PACIFIC BIOSCIENCES' SMRT ™ sequencing platform, Polon POLONONT sequencing platform, COMPLETE GENOMICS sequencing platforms, INTELLIGENT BIOSYSTEMS 'sequencing platforms or HELICOS sequencing platforms.

whereas the reaction buffer contains dimethylformamide in the amount resulting from the in vitro transposition in the final concentration of 10%. [0211] In some embodiments, the panel further comprises at least one other enzyme component selected from: DNA polymerase with 5 'nuclease activity or thread displacement; DNA polymerase with no 5 'nuclease activity or thread displacement, pattern-dependent NAD ligase, and pattern-independent ligase. In some embodiments, the at least one enzyme component is selected from: a mixture of FAILSAFE ™ DNA polymerases; Taq, Tfl DNA polymerase, T4 DNA polymerase, E. coli DNA ligase, TS2126 thermostable RNA ligase, Mth Rn 1 thermostable RNA ligase, and CIRCLIGASE ™ thermostable Ligt ssDNA ligase.

[0212] In certain preferred embodiments, wherein the at least one enzyme in the kit is a reference-dependent ligase (e.g., E. coli DNA ligase), a large proportion of the ligase molecules are adenylated and the kit does not contain ATP. In some embodiments, wherein the at least one enzyme in the kit is a pattern-dependent ligase (e.g. E. coli DNA ligase), the kit further comprises a tagged ligation oligonucleotide comprising a 3 'part and a 5' part, wherein the 3 'part comprises a sequence tag domain, and part 5 'contains a random sequence of from three to about eight nucleotides. In certain preferred embodiments, the ligation tagged oligonucleotide comprises a 5 'portion comprising a random sequence of four nucleotides. In some other embodiments, wherein the at least one enzyme in the kit is a reference-dependent ligase, the kit further comprises a hairpin-like transposon sequence. In some embodiments, wherein the hairpin end of the transposon sequence has an adenylated 5 'end, less than 50% of the molecules comprising the nucleic acid ligase depending on the standard included in the kit are adenylated and the kit does not contain ATP or NAD.

[0213] In certain preferred embodiments, wherein the at least one enzyme in the kit is a pattern-independent ligase selected from the thermostable TS2126 RNA ligase of the bacteriophage, the thermostable RNA ligase Mth Rn 1 and the thermostable CIRCLIGASE ™ ssDNA ligand, the standard-independent ligase is provided in highly adenylated and the kit does not contain ATP.

[0214] In one preferred embodiment of the kit, the transposome comprises a wild or hyper-active TnP transposase or MuA transposase provided at a concentration where the transposome final concentration in the in-transposition reaction is at least 250 nM. In some other embodiments, the concentration of the terminal wild or hyperactive Tn5 transposase or MuA transposition is at least 500 nM.

[0215] One preferred embodiment is a kit for forming tagged circular ssDNA fragments using EZ-Tn5 ™ transposase and E. coli DNA ligase, the kit containing: (1) a wild or mutant Tn5 transposase form (e.g., EZ- transposase; Tn5 ™); (2) a transposon end sequence comprising a transferred thread comprising the trans-end termination sequence and a non-transferred thread comprising a non-transferred transposon endpoint for the EZ-Tn5 transposase; (3) reaction buffer for EZ-Tn5 transposase; and (4) a nucleic acid ligase independent of a standard that is able to catalyze intramolecular lysation of ssDNA in the absence of a ligation standard (e.g., selected from the TS2126 hot-tube RNA ligase (U.S. Patent Application No. 7,303,901); a thermostable CIRCLIGASE ™ ssDNA ligase (EPICENTER Biotechnologies, Madison, WI, USA); and ligase 1 RNA Mth). In one preferred embodiment, the transposase in the kit is a wild or mutated form of the Tn5 transposase (e.g., EZ-Tn5 ™ transposase) at a concentration greater than or equal to: about 5 units per μΐ, about 10-20 units per μΐ; about 20-40 units per μΐ; approx. 40-60 units per μΐ; approx. 60-80 units per μΐ; or about 80-100 units per μΐ. In one preferred embodiment of the kit comprising the EZ-Tn5TM5 transposase and HGase independent of the standard, the EZTn5 pMEDS end sequence contains both the transferred EZ-Tn5 pMETS strand having a 5 'monophosphate group and a non-transferable EZ-Tn5 pMENTS strand having a 5' monophosphate group. In one preferred embodiment, the transposase in the kit is a wild or mutated form of the Tn5 transposase (e.g., EZ-Tn5 ™ transposase) at a concentration greater than or equal to: about 5 units per μΐ, about 10-20 units per μΐ; about 20-40 units per μΐ; approx. 40-60 units per μΐ; approx. 60-80 units per μΐ; or about 80-100 units per μΐ. In one preferred embodiment of the kit comprising the EZ-Tn5TM5 transposase and HGase independent of the standard, the EZTn5 pMEDS end sequence contains both the transferred EZ-Tn5 pMETS strand having a 5 'monophosphate group and a non-transferable EZ-Tn5 pMENTS strand having a 5' monophosphate group. In one preferred embodiment, the transposase in the kit is a wild or mutated form of the Tn5 transposase (e.g., EZ-Tn5 ™ transposase) at a concentration greater than or equal to: about 5 units per μΐ, about 10-20 units per μΐ; about 20-40 units per μΐ; approx. 40-60 units per μΐ; approx. 60-80 units per μΐ; or about 80-100 units per μΐ. In one preferred embodiment of the kit comprising the EZ-Tn5TM5 transposase and HGase independent of the standard, the EZTn5 pMEDS end sequence contains both the transferred EZ-Tn5 pMETS strand having a 5 'monophosphate group and a non-transferable EZ-Tn5 pMENTS strand having a 5' monophosphate group. 20-40 units per μΐ; approx. 40-60 units per μΐ; approx. 60-80 units per μΐ; or about 80-100 units per μΐ. In one preferred embodiment of the kit comprising the EZ-Tn5TM5 transposase and HGase independent of the standard, the EZTn5 pMEDS end sequence contains both the transferred EZ-Tn5 pMETS strand having a 5 'monophosphate group and a non-transferable EZ-Tn5 pMENTS strand having a 5' monophosphate group. 20-40 units per μΐ; approx. 40-60 units per μΐ; approx. 60-80 units per μΐ; or about 80-100 units per μΐ. In one preferred embodiment of the kit comprising the EZ-Tn5TM5 transposase and HGase independent of the standard, the EZTn5 pMEDS end sequence contains both the transferred EZ-Tn5 pMETS strand having a 5 'monophosphate group and a non-transferable EZ-Tn5 pMENTS strand having a 5' monophosphate group.

[0216] In one preferred embodiment, the transposase in the kit is a wild-type mutant form of Tn5 transposase (e.g., EZ-Tn5 ™ transposase) at a concentration greater kb of: about 5 units per μΐ, about 10-20 units per μΐ; about 20-40 units per μΐ; approx. 40-60 units per μΐ; approx. 60-80 units per μΐ; kb about 80-100 units per μΐ. In one preferred embodiment, the kit contains the transposase EZ-Tn5 ™ and the nucine acid hygrase non-master, with the transpomon end of the EZ-Tn5 pMEDS containing both the transferred EZ-Tn5 pMETS thread having a 5 'monophosphate group and the non-transferable EZ-Tn5 thread pMENTS having a 5 'monophosphate group.

[0217] The methods, sequences and kits of the follow-up are useful for creating tagged fragments of kbd DNA k blunt-ended dsDNA bib fragments of double tagged hDNA ssDNA fragments (and their amphfection products) from target DNA from a source source for genomic, subgenomic or metagenomic anaHz ( e.g. for use in the creation of tagged target DNA for analysis on microarrays, e.g. for the analysis of copy number polymorphism (CNV), anemia detection of single-nucleotide ohmorphisms, and for the identification of genes from environmental samples, such as samples derived from gkby kb water). The methods are useful in the age of processes, including the process of amphbridization of the entire genome of one kb of the greater Hobby of organisms, including one or more microbial or environmental organisms with unknown growing or growing conditions (e.g., whole genome or WGA amplification), real time PCR, emulsion PCR, comparative genomic hybridization (CGH), comparative genomic sequencing (CGS), and the creation of DNA-specific probes (e.g., chromosomal specific probes, e.g. chromosomal paints) for applications such as fluorescence in situ hybridization (FISH). In some embodiments, the methods are also used to create patterns of massive parallel DNA sequencing (so-called "next-generation sequencing"). Each of these processes or applications is used in molecular research and diagnostics. comparative genomic hybridization (CGH), comparative genomic sequencing (CGS), and to create DNA-specific probes (e.g., chromosomal specific probes, e.g. chromosomal dyes) for applications such as fluorescent in situ hybridization (FISH). In some embodiments, the methods are also used to create patterns of massive parallel DNA sequencing (so-called "next-generation sequencing"). Each of these processes or applications is used in molecular research and diagnostics. comparative genomic hybridization (CGH), comparative genomic sequencing (CGS), and to create DNA-specific probes (e.g., chromosomal specific probes, e.g. chromosomal dyes) for applications such as fluorescent in situ hybridization (FISH). In some embodiments, the methods are also used to create patterns of massive parallel DNA sequencing (so-called "next-generation sequencing"). Each of these processes or applications is used in molecular research and diagnostics. methods are also used to create patterns of massive parallel DNA sequencing (the so-called "next-generation sequencing"). Each of these processes or applications is used in molecular research and diagnostics. methods are also used to create patterns of massive parallel DNA sequencing (the so-called "next-generation sequencing"). Each of these processes or applications is used in molecular research and diagnostics.

DETAILED DESCRIPTION OF EMBODIMENTS OF THE INVENTION [0218] A detailed description of exemplary embodiments of the invention is provided in the following sections:

I. Fragmentation and Double DNA Tagging with the use of DNA transposase and Polymerase

III. Fragmentation, Tagging and Single-processing Amplification of Target DNA

II. DNA fragmentation and tagging with the use of Transposase and Ligase

IV. Generation of Tagged Sections of Circular ssDNA from Target dsDNA using Transposase and Ligase

V. Fragmentation and Tagging of dsDNA in the In vitro Transposition Path of the Transposon Terminus Sequences with Structure Hairpins

I. DNA fragmentation and double tagging with the use of DNA Transposase and Polymerase [0219] The present invention includes methods, sequences and transposase kits to create 5 'tagged fragments from a target DNA comprising or consisting of one or more double-stranded molecules DNA (dsDNA) followed by the attachment of a second tag containing a different DNA sequence than in the first tag to the 3 'ends of said 5'-tagged DNA fragments. The first tag contains the sequence of the transposon recognition transposon terminus and is optionally contains one or more other 5 'terminal sequences of the sequence of said transposable end of the transposon. The second tag connects to the 3 'ends tagged at the end of the 5'

One method of the invention comprises: incubating the transposon and the end of the transposon with which it forms a transposable transposition complex in vitro with the target DNA under suitable conditions and for a sufficient period of time where the transferred end of the transposon is attached to the target DNA to form tagged at the 5 'end, DNA fragments having the first tag at the 5' ends; incubation of DNA-tagged DNA fragments at DNA-end conditions under DNA polymerization conditions, which conditions do not include thermocycling, for a sufficient period of time, where a second tag containing a sequence different from the first tag attaches to the 3 'ends of the 5' tagged DNA fragments, creating tagged 5 'and 3' endings

GOUT. In some embodiments, the method further comprises amplifying tagged 5 'and 3' ends of DNA fragments using DNA polymerase and at least one primer complementary to the second tag. The target DNA may comprise or consist of double-stranded DNA from any source in vivo or in vitro, such as genomic DNA, subgenomic DNA, plasmid or other episomal DNA or recombinant DNA, or double-stranded cDNA created by reverse transcription of RNA. Genomic DNA can contain or consist of one or more genomes from a biological or environmental source. Therefore, the methods, sequences and sets of the invention are useful for the generation of 5 'and 3' tagged DNA fragments and, optionally, amplifications tagged at the 5 'and 3' ends.

The use of DNA polymerase with Nicot and / or DNA polymerase transferase activity

Nuclease activity 5 'to create DNA fragments that have 5' and 3 'tags

Containing Sequences of Different Transposone Terminals [0221] In certain preferred embodiments, the present invention provides a method of forming a library of DNA fragments comprising 5 'and 3' tagged DNA fragments from a target DNA comprising or consisting of one or more double-stranded DNA molecules (dsDNA) ), the method comprising:

Delivery:

[0222]

1. a target DNA comprising or consisting of one or more double-stranded DNA (dsDNA) molecules (e.g., eukaryotic and / or prokaryotic genomic DNA or double-stranded cDNA generated by reverse transcription of RNA),

2. transposases (e.g., wild or mutant transposase, e.g., wild or mutant Tn5 transposase, e.g. EZ-Tn5 ™ transposase or e.g. MuA HYPERMU ™ transposase, EPICENTER Biotechnologies, Madison, WI, USA), and

3. the end of a transposon capable of forming a functional complex with transposase in a transposition reaction (e.g., consisting of nineteen base pairs of the transposon end "OE"), end of the transposon ("IE") or "mosaic end" ("ME") ) transposon end recognized by a wild or mutant Tn5 transposase, e.g. by EZ-Tn5 ™ transposase or the end of R1 and R2 transposon, e.g. by MuA HYPERMU ™ transposase), said transposon end containing double-stranded DNA consisting of a transfer thread and a non-transferable strands which, in combination, comprise sequences of the double-stranded end of the transposon, where the end transferred comprises the sequence of the first tag;

and

4. DNA polymerase transferring or cleaving DNA attached to a thread pattern below the 3 'terminus of the DNA molecule extended with said DNA polymerase (i.e., DNA polymerase with thread transfer activity and / or DNA polymerase with 5' nuclease activity; e.g. Taq DNA polymerase, polymerase Tf1 DNA, mixture of FAILSAFE ™ DNA polymerases, phi29 DNA polymerase, E. coli DNA polymerase I and DISPLACEACE ™ DNA polymerase, all available from EPICENTER Biotechnologies, Madison, WI, USA);

[0223] Incubation of the target DNA with transposase and the end of the transposon under appropriate conditions and for a sufficient period of time where transposon insertion of the transposase-catalyzed end into both strands of the target DNA forms 5 'tagged DNA fragments, each of which has the first tag at the 5' end ( e.g., FIG. 2); and [0224] Incubation of 5 'tagged DNA fragments by in vitro transposition reaction with DNA polymerase under appropriate conditions and for a sufficient period of time where the DNA polymerase extends the 3' ends of the 5 'tagged DNA fragments by attaching a second tag containing at least one part of the non-transcribed end of the transposon end to the 5'-tagged DNA fragments and forming fragments tagged at the 5 'and 3' ends.

[0225] In some preferred embodiments, the transfer thread comprises only the transposon end of the transfer sequence and, therefore, the first tag present in the 5 'tagged DNA fragments only contains the transposon end of the transposed sequence. In some other embodiments, the transferred thread comprises or consists of a 3 'part and a 5' part, where the 3 'portion contains the transposon end of the transfer sequence and the 5' part contains another desired sequence, in which embodiments the first tag comprises or consists of from both the 3 'part and the 5' part, and the non-transferred thread may or may not include a complementary sequence to the 5 'part of the transfer thread.

[0226] In some embodiments, where the transfer thread comprises or consists of the 3 'part and the 5' part, the 5 'part comprises a sequencing tag (e.g., Roche 454A sequencing label, as shown in the graph, e.g. in FIG 10) and part 3 'contains the sequence of the transposon end of the transfer thread. As a result, tagged DNA fragments are created at the 5 'end with the first tag that contains or consists of a sequencing tag (e.g., Roche 454A sequencing tag). Thereafter, the DNA polymerase is used to attach a second tag that contains or consists of another sequencing tag (e.g., Roche 454B sequencing tag) to the 5 'tagged DNA fragments to form a library of DNA fragments containing 5' and 3 tagged ends. ' DNA fragments with both sequencing tags (e.g., 454A and 454B, as shown in the scheme in FIG. 10). The 5 'and 3' tagged DNA fragments in the desired size range are used as the next generation sequencing patterns using the Roche 454 FLX genome sequencer. In other embodiments, the 5 'and 3' tagged DNA fragments in the library are created using the first and second tag, suitable for use as next generation sequencing sequencing tags using other sequencing platforms (eg using the ROCHE 454 sequencing platform, ILLUMINA ™ SOLEXA ™ sequencing platforms, LIFE TECHNOLOGIES / APPLIED BIOSYSTEMS 'SOLID ™ sequencing platforms) , PACIFIC BIOSCIENCES 'SMRT ™ sequencing platforms,

[0227] In some embodiments, a plurality of double-stranded transposon ends are used for one particular transposase or a plurality of different transposon ends recognized by various transposase enzymes. In some preferred embodiments, where a DNA polymerase with a strand transfer activity or a DNA polymerase with 5 'nuclease activity is used, two different transposon ends are inserted side by side on opposing strands of the target DNA, followed by DNA polymerase to extend the 3' ends tagged at the 5 DNA fragments using the opposite strand as a template and forming a library of 5 'and 3' tagged DNA fragments with tags containing other end of the transposon at the 3 'end than at the 5' end (e.g.

[0228] Hence, in some embodiments, the method further comprises:

Delivery in addition:

[0229]

5. a second transposon recognizing another end of the transposon from the transposon end recognized by the first transposase (hereinafter referred to in the present embodiment as the "first end of the transposon" recognized by the "first transposase"); and

6. a second end of a transposon capable of forming a functional complex with a second transposase in a transposition reaction, wherein said end of a transposon comprising a transfer thread and a non-transferred strand which, in combination, comprises a double-stranded transposon end sequence, wherein the transferred strand comprises a complementary sequence to a second sequence. tag; and [0230] Incubation of target DNA with the first transposase and first transposon end and a second transposase and second transposon end under appropriate conditions and for a sufficient period of time where insertions of first and second transposon-catalyzed first and second transposase into the target DNA form DNA fragments, each of which contains a sequence of the transfer of a first-end transposon or second end of the transposon at the 5 'end; and [0231] Incubation of DNA-tagged DNA fragments at DNA polymerization conditions, which conditions do not involve denaturation of dsDNA or thermocycling, and for a sufficient period of time where the DNA polymerase extends the 3 'ends of the 5' tagged DNA fragments, by attaching the second tag to the 5 'tagged DNA fragments and creating a library of tagged DNA fragments (e.g., tagged at the 5' and 3 'end of DNA fragments) without performing amplification reactions.

[0232] In some embodiments, the method comprises simultaneously incubating the target DNA with both a first transposase and oligonucleotides of the first end of the transposon and a second transposase and oligonucleotides of the second end of the transposon in the same reaction mixture. In some other embodiments, the method is performed sequentially by first incubating the target DNA with the first transposase and oligonucleotides of the first end of the transposon, followed by incubating the products of this reaction with a second transposase and oligonucleotides of the other end of the transposon. In some embodiments, where the method is performed sequentially,

[0233] In some embodiments of the method, where the transfer thread comprises or consists of the 3 'part and the 5' part, the 5 'part comprises the sequencing sequence sequence (e.g., the first Roche 454 sequencing tag, e.g. Roche 454A), and part 3 'contains the sequence of the transposon end of the transfer thread. The use of the term "sequencing tag" in the present application means a tag fused to the 5 'end or the 3' end of a single-stranded DNA fragment formed from a target DNA molecule, which tag serves to facilitate the sequencing of said DNA fragment. For example, in some embodiments, a sequencing tag provides a site for capturing said DNA strand fragment on a surface and / or initiating DNA synthesis of said DNA fragment and / or complementing said DNA fragment (e.g. as Roche454A and 454B sequencing tags for the Roche 454 FLX genome sequencer). Hence, when the 5 'part of the transfer thread contains the sequence of the sequencing tag, the DNA fragments tagged at the 5' end have a first tag that contains or consists of a sequencing tag (e.g., Roche 454 sequencing tag). Thereafter, the DNA polymerase binds a second tag that comprises or consists of a second sequencing tag (e.g., Roche 454B sequencing tag) to the 5 'tagged DNA fragments with sequencing tags at each end (e.g., sequencing tags 454A and Roche 454B). The DNA fragments tagged at the 5 'and 3' ends in a library of the desired size order are used as the next generation sequencing patterns using the Roche 454 FLX genome sequencer.

[0234] In some embodiments, each of the distinct double-stranded ends of the transposon comprises a distinct transfer thread comprising a 5 'part and a 3' part, wherein the 5 'portion of each different transferred thread contains a different desired tag sequence, and the 3' part comprises a suitable transfer end sequence. transposon. In some embodiments, e.g. as shown in the example shown in FIG. 8, the 5 'and 3' tagged DNA fragments in the library have both the first tag at the 5 'end and the second tag at the 3' end. The various ends of the transposon are presented in FIG. 8 were inserted at different locations during separate in vitro transposition events catalyzed by the same transposase. However, in some other embodiments, various transposases are used that form functional transposition complexes with various transposon ends. The different tags in portions 5 'of the transfer thread of each transposon end may contain the desired sequences for the desired purpose. For example, in some embodiments, the first and second tagged 5 'and 3' tagged DNA fragments contain a sequence of Roche sequencing tags 454A and 454B which, after isolation of fragments in the desired size order, are used as the next generation sequencing patterns using the Roche 454 FLX genome sequencer. Similarly, in other embodiments, 5 'and 3' tagged DNA fragments, after isolating fragments of the desired size order,

Polon POLONATOR, COMPLETE GENOMICS sequencing platforms, INTELLIGENT BIOSYSTEMS 'sequencing platforms, or HELICOS sequencing platforms). In certain preferred embodiments, the 5 'and 3' tagged DNA fragments are generated using the present method from a target DNA comprising the entire genome of a cell or organism.

[0235] In some embodiments, the transfer thread of the first end of the transposon or second end of the transposon is labeled with a molecule with a binding affinity (e.g., biotin) or a detectable molecule (e.g., a fluorescent dye) that allows capture (e.g., using the surface to which it is bound) is streptavidin for the capture of biotinylated molecules) or detection of tagged 5 'and 3' ends of tag-containing DNA fragments with a molecule with binding affinity or a detectable molecule at the 5 'end.

[0236] Adding the tag domain to the 5 'and 3' tagged DNA fragments: in some embodiments, the oligonucleotide DNA polymerase containing the pattern for the tag domain is used to add the tag domain to the 5 'ends tagged at the 5' and 3 'ends of the fragments DNA in the library of tagged fragments (e.g., FIG. 19). In some embodiments, the DNA polymerase used to add the tag domain is a thermostable DNA polymerase, and the oligonucleotide is a PCR primer, and the tag domain is attached to the second tag by performing PCR.

III. Fragmentation, Tagging and Single Target Amplification of Target DNA [0237] One of the preferred methods of the invention involves: incubating a transposase-containing transposon complex and end of the transposon with which it forms a transposition complex with the target DNA in an in vitro transposition reaction under appropriate conditions and for a sufficient period of time, where the transferred end of the transposon is inserted into a plurality of sites in both strands of the target DNA; incubating the in vitro transposition reaction products with a DNA polymerase having a thread transfer activity and / or 5'-nuclease activity under suitable conditions and for a sufficient period of time where the 3 'end of each strand of the target DNA having the transposon end grafted attached to its 5' end is extended from using the opposite strand of the target DNA as a template, wherein each said DNA polymerase extended spacer displaces or cleaves the non-transferible end of the transposon joined to the subsequent adjacent transposed end of the transposon attached to the opposite strand of the target DNA, thereby creating a library of double tagged DNA fragments, each consisting of a different portion of the target DNA with the thread transferred at the 5 'end and a non-transferlable thread at the 3' end, where the population of all double tagged DNA fragments is significantly representative of the target DNA sequence from which they were created; and incubating a library of double tagged DNA fragments with a thermostable DNA polymerase and a single primer containing at least a portion of the transposon end of the transfer sequence under PCR cycliccycling conditions, thus creating an amplified library of double DNA fragments. In certain preferred embodiments, the method comprises creating an amplified library of double tagged DNA fragments in the presence of one or more dNTP labeled substrates, used as substrates by thermostable DNA polymerase, thereby forming an amplified library of double tagged DNA fragments. [0238] Hence, one embodiment of the invention is an in vitro method for the single-PCR amplification of DNA fragments formed from a target DNA, the method comprising: performing a transposition reaction in the presence of a target DNA and in the presence of an end of a transposon containing the transferred thread, containing the not-transferred transposon end sequence . wherein the effect of said transposition reaction is the insertion age including attachment of the transposon transposed end to each strand of DNA, thereby creating tagged 5 'DNA fragments associated with each other, each having a first tag at the 5' end containing the transposon end of the transfer sequence. ; extension of the 3 'ends of the 5' tagged DNA fragments using DNA poHasmase with thread-shifting activity and / or 5 'nuclease activity using opposite strands to which the 5' tagged DNA fragments are attached as standards; and performing a PCR amplification reaction using a single primer containing at least a portion of the transposon end of the transfer sequence,

[0239] In some preferred embodiments, tagged DNA is selected from eukaryotic and / or prokaryotic genomic DNA or double-stranded cDNA created by reverse transcription of RNA.

[0240] In some preferred embodiments, the transposom is a compilation of wild or hyperactive mutant transposase selected from Tn5 transposase, MuA transposase, Sleeping Beauty transposase, Mariner transposase, Tn7 transposase, Tn10 transposase, Ty1 transposase and Tn552 transposase and transposon end with which transposase forms an active complex in the transposition reaction.

[0241] In some preferred embodiments, the single enzyme enzyme kb enzyme is used both as a DNA pohmerase with a thread displacement activity and / or a 5'-nuclease activity and a DNA poHmerase thermostabil, which DNA kb mix is ​​selected from wild recombinant kb forms DNA polymerase TAQ DNA polymerase, Tfl DNA polymerase, Tth DNA polymerase and FAILSAFE ™ DNA poHmerase mixture.

[0242] In some preferred embodiments, at least one labeled dNTP containing a label, e.g. a cyanine, (e.g. Cy5.5, Cy5, Cy3, Cy2), FITC, Alexa Fluors (e.g., 647, 594), Texas Red. , JOE, 5-FAM, 6-FAM, VIC, HEX, 6-ROX, Rhodamine, Lissamine, Cyan 500, etc. (See, e.g., Handbook of Molecular Probes, R. Haughland, Molecular Probe, Eugene, OR., ).

[0243] In some preferred embodiments, the biB kraft of an amphified BIA protein tagged at the 5 'and 3' ends of the DNA fragments formed using the present method is derived from a kb-containing DNA consisting of the entire genome of the kb cell. In some embodiments, the kb of the amphified BIO protein of doubly tagged DNA fragments formed using the present method is derived from DNA-specific DNA containing kb consisting of genomes and / or double-stranded cDNA from all organisms (e.g., organisms) present in an environmental trial (e.g. for studies of kb metagenomic or metatranscryptomic applications).

[0244] In some preferred embodiments of the method, the transferred thread contains only the transposon end of the transfer sequence and, consequently, the first tag present in the 5 'tagged DNA fragments only contains the transposon end of the transposed sequence. In some other embodiments, the transferred thread comprises kb consisting of a portion 3 and a portion 5, wherein the 4 'portion comprises the transposon end transcribed sequence and the 5' portion comprises another desirable kb nucleotide sequence of nucleotides, in which embodiments the first tag contains kb it consists of both parts 3, and 5 '. In embodiments, where the transfer thread contains kb consists of parts 3 'and 5', the non-transferable strand may, ak does not have to include a compre-ment sequence with a 5 'part transferred thread. In certain preferred embodiments, where the transfer thread comprises or consists of a 3 'portion and a 5' portion, the 5 'portion contains at least one nucleotide containing the capture domain (e.g., a biotin group containing nucleotide that can be captured by a streptavidin group bound to the cell). a surface, or e.g. another molecule with binding affinity).

II. DNA fragmentation and tagging using Transposase and Ligase [0245] The present invention includes methods, sequences, and transposase kits to create 5 'tagged fragments from a target DNA comprising or consisting of one or more double-stranded DNA molecules (dsDNA). and then attaching a second tag containing a different DNA sequence than in the first tag to the 3 'ends of the tagged DNA fragments mentioned at the 5' end. The first tag contains the sequence of the transposon recognition transposon terminus and is optionally contains one or more other 5 'terminal sequences of the sequence of said transposable end of the transposon. The second tag connects to the 3 'ends tagged at the end of the 5'

One method of the invention comprises: incubating the transposon and the end of the transposon with which it forms a transposable transposition complex in vitro with the target DNA under suitable conditions and for a sufficient period of time where the transferred end of the transposon is attached to the target DNA to form tagged at the 5 'end, DNA fragments having the first tag at the 5' ends; incubating 5 'tagged DNA fragments with a nucleic acid ligase and a tagged oligonucleotide that contains or consists of a second tag under appropriate conditions and for a sufficient period of time where the tagged oligonucleotide is attached to the 3' ends tagged on the 5 'end of DNA fragments, forming tagged 5' and 3 'DNA fragments. In some embodiments, the method further comprises amplifying tagged 5 'and 3' ends of DNA fragments using DNA polymerase and at least one primer complementary to the second tag. In some embodiments, the library amplification step tagged at the 5 'end and 3' of DNA fragments using DNA polymerase comprises PCR amplification using a thermostable DNA polymerase, a first PCR primer complementary to the second tag, and a second PCR primer containing a sequence identical to at least a portion of the sequence contained in the first tag.

One of the preferred embodiments of the present invention is a method of creating a library of tagged DNA fragments comprising tagged 5 'and 3' ends of DNA fragments derived from a target DNA comprising or consisting of one or more double-stranded DNA molecules (dsDNA), wherein the method includes:

Delivery:

[0248]

1. a target DNA comprising or consisting of one or more double-stranded DNA (dsDNA) molecules (e.g., eukaryotic and / or prokaryotic genomic DNA or double-stranded cDNA generated by reverse transcription of RNA)

2. transposase (e.g., wild or mutant transposase, e.g., wild or mutant Tn5 transposase, e.g. EZ-Tn5 ™ transposase or, e.g., MuA HYPERMU ™ transposase,

EPICENTER Biotechnologies, Madison, WI, USA), and

3. the end of a transposon capable of forming a functional complex with transposase in a transposition reaction (e.g., consisting of nineteen base pairs of the transposon end "OE"), end of the transposon ("IE") or "mosaic end" ("ME") ) transposon end recognized by a wild or mutant Tn5 transposase, e.g. by EZ-Tn5 ™ transposase or the end of R1 and R2 transposon, e.g. by MuA HYPERMU ™ transposase), said transposon end containing double-stranded DNA consisting of a transfer thread and a non-transferable strands which, in combination, comprise sequences of the double-stranded end of the transposon, where the end transferred comprises the sequence of the first tag, and

4. a ligation tagged oligonucleotide having a 5 'terminus capable of ligation with the hydroxyl group at the 3' end of the DNA molecule and containing the sequence of the second tag, and

5. nucleic acid ligase;

[0249] Incubation of the target DNA with transposase and the end of the transposon under appropriate conditions and for a sufficient period of time where transposon transposon insertion into the target DNA creates the 5 'tagged DNA fragments, each of which has the first tag at the 5' end [0250] Incubation of 5 'tagged DNA fragments with a nucleic acid ligase and oligonucleotide tagged by ligation under appropriate conditions and for a sufficient period of time, where the second tag is attached to their 3' ends and a library of 5 'and 3' tagged DNA fragments is created in which each tagged DNA fragment has the first tag at the 5 'end and the second tag at the 3' end.

[0251] In some preferred embodiments, the transfer thread comprises only the transposon end transcribed sequence and, therefore, the first tag present in the 5 'tagged DNA fragments only contains the transposon end of the transposed sequence. In some other embodiments, the transferred thread comprises or consists of a 3 'part and a 5' part, where the 3 'portion contains the transposon end of the transfer sequence and the 5' part contains another desired sequence, in which embodiments the first tag comprises or consists of from both the 3 'part and the 5' part. In embodiments where the transfer thread includes or consists of part 3 and part 5 ', the non-transferring thread may or may not include a complementary sequence to the 5' part.

[0252] In some embodiments, where the transfer thread comprises or consists of the 3 'part and the 5' part, the 5 'part comprises the sequencing sequence sequence (e.g., the first Roche 454 sequencing tag, e.g. Roche 454A), and the 3' portion contains the sequence of the transposon end of the transfer thread. The use of the term "sequencing tag" in the present application means a tag fused to the 5 'end or the 3' end of a single-stranded DNA fragment formed from a target DNA molecule, which tag serves to facilitate the sequencing of said DNA fragment. For example, in some embodiments, a sequencing tag provides a site for capturing said DNA strand fragment on a surface and / or initiating DNA synthesis of said DNA fragment and / or complementing said DNA fragment (e.g. as Roche454A and 454B sequencing tags for the Roche 454 FLX genome sequencer). Hence, when the 5 'part of the transfer thread contains the sequence of the sequencing tag, the DNA fragments tagged at the 5' end have a first tag that contains or consists of a sequencing tag (e.g., Roche 454 sequencing tag). The nucleic acid ligase then ligates the tagged ligation oligonucleotide that has a second tag that contains or consists of a second sequencing tag (e.g., Roche 454B sequencing tag) to 5 'tagged DNA fragments with sequencing tags at each end (e.g. with sequencing tags 454A and Roche 454B). Tagged at the 5 'and 3' ends

Second Tag Pattern Ligation [0253] In certain preferred embodiments, the method comprises providing a tagged ligation oligonucleotide, comprising or consisting of a 3 'part and a 5' part, wherein the 3 'part comprises a second tag that contains or consists of from any sequence that should bind to the 3 'end of the tagged DNA fragments (i.e., a random sequence), and the 5' portion has a 5 'monophosphate group and contains a random sequence (e.g., a random sequence containing about three to four). about eight nucleotides) at the 5 'end. In certain preferred embodiments, the ligation tagged oligonucleotide has a 5 'portion containing a random sequence of four nucleotides (e.g., in certain embodiments described in the present application,

[0254] The invention is not limited to a ligation tagged oligonucleotide having a 5 'portion containing a random sequence of about three to about eight nucleotides. For example, the invention also comprises methods wherein the tagged oligonucleotide has a 5 'portion which: comprises a random sequence consisting of only two nucleotides; contains a random sequence of more than eight nucleotides; it contains a semi-random sequence, not a completely random sequence; or contains a sequence consisting of one or more degenerate nucleotides (e.g., inosine nucleotides) rather than a completely random sequence. However, a ligation tagged oligonucleotide having a 5 'portion containing a random sequence of about three to about eight nucleotides is preferred.

[0255] In some preferred embodiments, the ligation of the oligonucleotide tagged by ligation to the 3 'end of the tagged 5' DNA fragments only occurs in the presence of a DNA template containing the sequence fully complementary to the ligation bond; in such embodiments, the template to which two ligated nucleic acid molecules bind to will be referred to as the "ligation pattern" and the ligation will be referred to as the "standard-dependent ligation". In some embodiments, where the ligation occurs only in the presence of a ligation standard, the nucleic acid ligase is a DNA ligase requiring a ligation pattern and will be referred to as a "pattern-dependent ligase" (e.g., a DNA ligase dependent on the NAD standard including, but not limited to, a ligase) E. coli DNA, Tth DNA ligase, Tf DNA ligase or AMPLIGASE® DNA ligase, available at EPICENTER Biotechnologies, Madison, WI, USA). In some other embodiments, where the ligation is only in the presence of a ligation standard, the nucleic acid ligase is a DNA ligase that, if it does not require a ligation ligation, catalyzes the ligation more efficiently in the presence of a ligation standard than in its absence (e.g., a ligase). DNA dependent on the ATP standard, including, but not limited to, T4 DNA ligase or FASTLINK ™ DNA ligase, available from EPICENTER Biotechnologies, Madison, WI, USA). If the ligation is on the ligation pattern, the ligation is called "pattern-dependent ligation" in the present application, even if the ligase can also catalyze a ligation independent of the standard. In preferred embodiments, the random oligonucleotide sequence tagged by ligation is short, in which embodiments, a nucleic acid that catalyzes a reference-specific ligase at a lower temperature (e.g., equal to or lower than about 40 ° C, equal to or lower than about 37 ° C, equal to or lower than about 30 ° C) is preferred, equal to or less than approx. 25 ° C, or equal to or less than approx. 20 ° C). In certain preferred embodiments, the reference-dependent ligase is an E. coli DNA ligase.

[0256] The invention is not limited to the ligation method used except that, with reference to embodiments comprising a pattern-dependent ligation, the ligation should occur efficiently in the presence of the target sequence to which the tagged ligation oligonucleotide is attached and tagged at the end. 5 'DNA fragments, and the ligation should occur rarely or in inches if there is no target sequence. The use of the term "reference-dependent ligation" in the present application refers to any suitable method of attaching the 5 'and 3' adjacent oligonucleotides tagged by ligation and tagged at the 5 'end of the DNA fragments, adjacent or tangentially adjacent to or adjacent to each other during the process. connecting to the target sequence.

[0257] Inserting the transposon transposon end into both strands of the target DNA results in fragmentation of the target DNA, attaching the transposon end of the transfer to each strand of the target DNA, and creating a 9-base single-stranded target DNA region at the 3 'attachment site of the transposon end, resulting in a gap on the opposite strand of the target DNA (e.g., FIG. 3). The single-stranded region of the target DNA below the transposon end of the transfer may serve as a ligation pattern for attachment of a random portion of the tagged oligonucleotide. Of all sequences represented by a ligation tagged oligonucleotide comprising a random sequence in the 5 'part (including all possible sequences), at least one of them contains a 5' end sequence. capable of linking to the single-stranded region of the target DNA, such that the phosphorylated 5'-end of such tagged oligonucleotide adheres to and adjoins the 3 'end of the target DNA complementary to the 5'-tagged DNA fragment, in which case the nucleic acid ligase can catalyze an oligonucleotide-labeled ligation ligation to said 3 'end. In some embodiments, the transferred transposon end thread is inserted into opposite strands of target DNA at two locations located relatively close to each other to form two 5 'tagged DNA fragments as shown in FIG. 2, and two DNA fragments tagged at the 5 'and 3' ends as shown in FIG. 7. After denaturation of two tagged at the end of 5 'and 3'

[0258] In some embodiments of a method comprising using a nucleic acid ligase and a template-dependent ligation to attach a second tag to the 5 'tagged DNA fragments, 5' and 3 'tagged at the 5' end have the first 5 'tag containing the transposon end sequence and a second 3'-tag that does not contain the end of the transposon sequence (although the 3'-part of the oligonucleotide tagged by ligation may contain or consist of a second tag containing the desired sequence, including the transposon end sequence). For example, in some embodiments, the 3 'portion of the tagged oligonucleotide comprises a sequence of the Roche 454 sequencing tag or a sequencing tag for another sequencing platform (e.g., the ROCHE 454 sequencing platform,

[0259] As a further example, in some embodiments, the second tag in the 3'-part of the tagged oligonucleotide comprises an RNA polymerase promoter sequence (e.g., a T7 type RNA polymerase promoter; e.g., the T7, T3, SP6 or phage RNA polymerase promoter) N4 MINI-V ™; EPICENTRE Biotechnologies, Madison, WI, USA); in general, if the RNA polymerase requires a double stranded RNA polymerase promoter, the second tag in such embodiments contains the "promoter RNA polymerase sequence", which is the RNA polymerase promoter sequence attached to the 3 'end of the template DNA strand transcribed with RNA polymerase, in which examples performance, the complementary "antisense RNA polymerase promoter sequence" must also be provided or synthesized at the process step to create a double stranded RNA polymerase promoter recognized by an RNA polymerase. In some embodiments, the second tag in the 3'-part of the ligation tagged oligonucleotide also contains the sequence of "addressing tag", which is a sequence allowing identification of a given sample (e.g., by using an addressing tag in an oligonucleotide tagged by a ligation containing a different sequence for each target DNA sample) ). In some embodiments, the 3 'portion of the ligation tagged oligonucleotide also comprises a sequence of one or more other tags for a particular purpose in the method. the second tag in the ligation tagged oligonucleotide portion 3 &amp; apos; also includes the &amp; quot; addressing tag &amp; quot; sequence, which is a sequence allowing identification of a given assay (e.g., by using an addressing tag in an oligonucleotide tagged by a ligation containing a different sequence for each target DNA sample). In some embodiments, the 3 'portion of the ligation tagged oligonucleotide also comprises a sequence of one or more other tags for a particular purpose in the method. the second tag in the ligation tagged oligonucleotide portion 3 &amp; apos; also includes the &amp; quot; addressing tag &amp; quot; sequence, which is a sequence allowing identification of a given assay (e.g., by using an addressing tag in an oligonucleotide tagged by a ligation containing a different sequence for each target DNA sample). In some embodiments, the 3 'portion of the ligation tagged oligonucleotide also comprises a sequence of one or more other tags for a particular purpose in the method.

[0260] In certain preferred embodiments, the 5 'portion of the ligation tagged oligonucleotide is a random sequence of length capable of linking to single-stranded portions of the 5' tagged DNA fragments formed by insertion of the transposase transposable ends into both strands of the target DNA. The random sequence in the 5'-part of the oligonucleotide tagged by ligation is combined with a single-stranded gap adjacent to the 3 'end of the 5'-tagged DNA fragments serving as a ligation pattern for ligation of the oligonucleotide-labeled ligation to the 3' ends of the complementary target strand. GOUT. In the examples using the EZ-Tn5 ™ transposase, the insertion of the EZ-Tn5 ™ transpozon-consisting of 19 base pairs of oligonucleotides into both strands of the target DNA creates tagged 5 'ends DNA fragments containing 9-base gaps composed of single-stranded regions opposite to the insertion sites of the transposon ends transferred. However, the size of the gap to which the tagged oligonucleotide can attach may differ for different transposase enzymes. For example, the MuA transposase forms a single-stranded region of the target DNA downstream of the insertion site of the transposon end containing only five nucleotides. In some embodiments, a random ligation tagged oligonucleotide sequence comprises or consists of about three to about eight random nucleotides. However, the length of the random portion of the tagged oligonucleotide sequence may differ for different transposase enzymes based on the size of the single-stranded region formed and other factors, such as the length of the random sequence that most effectively ligation with the appropriate nucleic acid ligase and the ligation conditions used. For example, Applicants have observed that, using the 5 'tagged DNA fragments using the EZ-Tn5 ™ transposase, an oligonucleotide tagged by ligation with a 5' portion containing a random sequence of four nucleotides creates a large number of 5 'and 3' tagged DNA fragments. using E. coli DNA ligase as nucleic acid ligase. However, such an oligonucleotide tagged by ligation to a 5 'portion containing a random sequence of four nucleotides is not effectively ligated with thermostable DNA-dependent ligases, such as a thermostable AMPLIGASE ligase under similar ligation conditions. In preferred embodiments, tagged at the 5 'and 3' ends

[0261] In some embodiments, the template-dependent nucleic acid ligase is a ligase using NAD as a cofactor. In some embodiments, the ligand-dependent ligand ligase ligase is selected from the following NAD DNA ligases: E. coli DNA ligase, Tth ligase, Tf1 DNA ligase, and Ampligase® DNA ligase DNA (all available in EPICENTER Biotechnologies, Madison, WI). , USA), as well as Tsc DNA ligases (Roche Applied Systems, Indianapolis, IN, USA). In some embodiments, the nucleic acid ligase for a pattern-dependent ligation is ATP DNA ligase. In some embodiments, the ATP-type DNA ligase is selected from: T4 DNA ligase and FASTLINK ™ DNA ligase (EPICENTER Biotechnologies, Madison, WI, USA). In some preferred embodiments, The nucleic acid ligase is selected from an E. coli DNA ligase or other mesophilic bacterial DNA ligase using NAD as a cofactor. In some preferred embodiments, size dependent selection and purification of 5 'tagged DNA fragments selected on a size basis are accomplished to improve the ligation efficiency of the oligonucleotide tagged by ligation to the 5' tagged DNA fragments using a standard DNA ligase. DNA (e.g., E. coli DNA ligase).

Ligating the Second Tag Independent of Pattern [0262] In some embodiments of the method of attaching the second tag to the 3 'end of the tagged 5' DNA fragments using a nucleic acid ligase, the tagged ligation oligonucleotide containing the second tag is ligated directly to the 3 'end tagged at the 5 'end of the DNA fragments without the linkage of the oligonucleotide tagged by ligation to the ligation pattern adjacent to the 3' ends tagged at the 5 'end of the DNA fragments. In these embodiments, the ligation tagged oligonucleotide does not contain a random sequence, but rather only contains the sequence of the second tag, desired to connect to the 5'-tagged DNA fragments. In such embodiments, the ligation tagged oligonucleotide is ligated directly to the 3 'ends of the tagged 5' DNA fragments without the use of a ligation standard. In these embodiments of the method, the nucleic acid ligase is a nucleic acid ligase capable of ligation of a single-stranded DNA molecule with a 3 'hydroxyl group to a single-stranded DNA molecule with a 5' monophosphate group in the absence of binding to a complementary sequence in a ligation bond (e.g. selected from RNA ligase 1) T4, T4 RNA ligase 2, thermostable TS2126 RNA ligase, as well as CIRCLIGASE ™ DNA ligase, EPICENTER Biotechnologies, Madison, WI, USA); 5 'tagged DNA fragments with nucleic acid ligase and tagged ligation oligonucleotide. In these embodiments of the method, the nucleic acid ligase is a nucleic acid ligase capable of ligation of a single-stranded DNA molecule with a 3 'hydroxyl group to a single-stranded DNA molecule with a 5' monophosphate group in the absence of binding to a complementary sequence in a ligation bond (e.g. selected from RNA ligase 1) T4, T4 RNA ligase 2, thermostable TS2126 RNA ligase, as well as CIRCLIGASE ™ DNA ligase, EPICENTER Biotechnologies, Madison, WI, USA); 5 'tagged DNA fragments with nucleic acid ligase and tagged ligation oligonucleotide. In these embodiments of the method, the nucleic acid ligase is a nucleic acid ligase capable of ligation of a single-stranded DNA molecule with a 3 'hydroxyl group to a single-stranded DNA molecule with a 5' monophosphate group in the absence of binding to a complementary sequence in a ligation bond (e.g. selected from RNA ligase 1) T4, T4 RNA ligase 2, thermostable TS2126 RNA ligase, as well as CIRCLIGASE ™ DNA ligase, EPICENTER Biotechnologies, Madison, WI, USA); 5 'tagged DNA fragments with nucleic acid ligase and tagged ligation oligonucleotide. in the absence of binding to a complementary sequence in a ligation bond (e.g., selected from T4 RNA ligase 1, T4 RNA ligase 2, thermostable TS2126 RNA ligase, as well as CIRCLIGASE ™ DNA ligase, EPICENTER Biotechnologies, Madison, WI, USA); 5 'tagged DNA fragments with nucleic acid ligase and tagged ligation oligonucleotide. in the absence of binding to a complementary sequence in a ligation bond (e.g., selected from T4 RNA ligase 1, T4 RNA ligase 2, thermostable TS2126 RNA ligase, as well as CIRCLIGASE ™ DNA ligase, EPICENTER Biotechnologies, Madison, WI, USA); 5 'tagged DNA fragments with nucleic acid ligase and tagged ligation oligonucleotide.

[0263] The invention is not limited to a particular nucleic acid ligase, and methods comprising 5 'tagged DNA fragments with nucleic acid ligase under suitable conditions and for a sufficient period of time, where the second tag is attached to their 3' ends and formedis a library of tagged DNA fragments at the 5 'and 3' ends, will be understood to also include the use of other sequences in place of a nucleic acid ligase for linking dependent or pattern independent. For example, other ligation methods include, but are not limited to, using a tagged ligation oligonucleotide containing a topoisomerase group, wherein the ligation includes ligation with the tomoposomerase (e.g., U.S. Patent No. 5,766,891) even though ligation with topoisomerase is not preferred in most embodiments .

IV. Generation of Tagged Fragments of Circular ssDNA from Target dsDNA using Transposase and Ligase (30842) [0264] The present invention includes methods, sequences and kits for creating a library containing a population of tagged circular ssDNA fragments from the target DNA in the assay for use as standards in DNA sequencing or reactions amplification of nucleic acids. In general, each tagged fragment of the circular ssDNA in the library contains the adjacent sequence of the target DNA and tag portion. [0265] Briefly, in some embodiments, the method comprises: incubating the target DNA, which is mostly dsDNA, transposase, and the transposon termination sequence in an in vitro transposition reaction to simultaneously fragment and tag the target DNA, thereby creating a tagged DNA fragment population; followed by denaturation of the tagged DNA fragments to create 5'-tagged ssDNA fragments at the 5'-ends, followed by incubation of the 5D-labeled ssDNA fragments with a template-independent or non-homogenous nucleic acid ligase capable of catalyzing an intramolecular lysylation independent of the standard ssDNA in the to create a library of tagged circular ssDNA fragments. In some embodiments, the tagged circular ssDNA fragments are linearized by attaching an oligodeoxy-gen nucleotide linking to the restriction site in the tag and then subjecting to restriction endonuclease to form linear ssDNA fragments having a portion of the tag at the 5 'end and the remainder of the tag at the 3' ends.

[0266] In some embodiments, tagged fragments of circular ssDNA are used as DNA standards in nucleic acid amplification and / or DNA sequencing reactions. In some embodiments, the method further comprises the step of amplifying and / or sequencing a library of tagged DNA ring fragments (e.g., for amplifying or determining the target DNA sequence). In some embodiments, the method further comprises a DNA sequencing step complementary to the target DNA obtained by amplifying tagged portions of circular DNA or double tagged fragments of linear ssDNA. In some embodiments, at least a portion of the target DNA in each of the tagged circular ssDNA fragments is DNA polymerase sequencing and at least one complementary primer with the tag (e.g. sequencing by synthesis). In some embodiments, at least a portion of the target DNA in each of the tagged fragments of circular ssDNA or double tagged linear ssDNA fragments is sequenced using a standard-dependent ligase to ligate at least one oligodeoxyribonucleotide complementary to the tag and at least one other oligodeoxyribonucleotide combining with part of the target sequence (e.g., ligation sequencing). In some embodiments, at least a portion of the target DNA in each of the tagged fragments of circular ssDNA or double tagged linear ssDNA fragments is sequenced by attaching oligodeoxyribonucleotides that link to or hybridize to the tag and to a portion of the target sequence (e.g. sequencing by hybridization). In some embodiments, the DNA complementary to tagged fragments of circular ssDNA is sequenced using sequencing by synthesis, ligation sequencing or sequencing by hybridization.

[0267] Hence, one of the preferred embodiments of the present invention is a method of creating a library comprising a population of tagged circular ssDNA fragments from the target DNA in the assay for use as standards in DNA sequencing or nucleic acid amplification reactions, of which each of the tagged circular ssDNA fragments comprises a sequence the target DNA part and the tag sequence attached to the target portion of the sequence, the method comprising:

Delivery:

[0268]

1. a target DNA comprising or consisting of one or more double-stranded DNA (dsDNA) molecules (e.g., eukaryotic and / or prokaryotic genomic DNA or double-stranded cDNA generated by reverse transcription of RNA using DNA dependent DNA polymerase or reverse transcriptase to creation of the first-strand cDNA and extension of the primer attached to the primary-strand cDNA to form dsDNA)

2. transposases (e.g., wild or mutant transposase, e.g., wild or mutant Tn5 transposase, e.g. EZ-Tn5 ™ transposase or e.g. MuA HYPERMU ™ transposase, EPICENTER Biotechnologies, Madison, WI, USA), and

3. a transposon end sequence capable of forming a functional complex with transposase in a transposition reaction (e.g., consisting of nineteen base pairs of the transposon end "OE"), end of the transposon ("IE") or "mosaic end" ("ME") ") the transposon end recognized by the wild or mutant Tn5 transposase, e.g. by EZ-Tn5 transposase, or the R1 and R2 transposon end, e.g. by MuA HYPERM ™ ™ transposase, wherein said sequence of transposon ends consists of a transfer thread and a non-transferlable thread , which, in combination, contain sequences of the double-stranded end of the transposon, where the end transferred contains the sequence of the tag,

4. Nucleic acid ligase independent of the standard or non-homologous lysine capable of intramolecular ligation independent of the ssDNA pattern or circularization containing a 5 'monophosphate group and a 3' hydroxyl group (e.g., thermostable RNA ligase, e.g. where a large proportion of the RNA ligase molecules are adenylated)

Incubation of the target DNA with transposase and the transposon termination sequence under appropriate conditions and for a sufficient period of time, where the insertion of the transferred transposase-catalyzed strand into the target DNA forms 5'-tagged DNA fragments (e.g., FIG. 2); and

Denaturation of the target DNA containing the 5 'DNA tagged at the 5' end to yield ssDNA fragments tagged at the 5 'end; and [0269] Incubation of the 5'-tagged ssDNA fragments of the nucleic acid ligase under suitable conditions and for a sufficient period of time, where the 5'-tagged ssDNA fragments are intramolecularly ligated to form a library of tagged circular ssDNA fragments, each of which contains a sequence of parts target DNA and tag.

[0270] In some embodiments, prior to the ligation step, the method further comprises one or more steps to remove the target DNA untagged during the transposition and / or removal of the components of the transposon end sequences not attached to the DNA of interest.

[0271] In some embodiments, the method further comprises submitting bibhatin containing tagged fragments of the ssDNA with exonergase I to remove non-Hd Nanovirus ssDNA. In some embodiments, the method further comprises the step of subjecting the reaction mixture to exonuccinate I and exonuclease III (EPICENTER Biotechnokgies, Madison, WI) to remove unbound linear ssDNA. Egzokukkaza III helps remove the part of Hni sdNANA by digesting double-stranded regions of Histoxa ssDNA molecules resulting from intermolecular molecule binding. In certain preferred embodiments, the method comprises the subjecting of tagged ssDNA fragments to T5 exonuccinate (EPICENTER Biotechnokgies, Madison, WI) for the removal of unhamped cultivated ssDNA and dsDNA (e.g.

[0272] In some embodiments, the method comprises the d ampyation of tagged fragments of the ssDNA kb of double tagged Hatin sHNA fragments by transcription, the method comprising: (a) combining with a sense sequence of a .homoisoxyribonucleotide sense promoter containing a compliant antisense promoter sequence, kb also attaching to tagged fragments of the coherent ssDNA kb of double tagged fragments of the Histological ssDNA complementary primer with them and extension of the primer with DNA poHmerase under conditions where the double-stranded DNA poinoma promoter is synthesized; and (b) incubating the dsDNA products with RNA RNA binding to the RNA polymerase promoter under RNA synthesis conditions. [0273] In certain preferred embodiments, where the transferable kb strand PCR primer contains the RNA polymerase promoter sequence, the RNA-RNA promoter is a T7-type RNA-based promoter, and the method further comprises a step of transcription of tagged ssDNA fragments in vitro using a T7-type RNA polymerase that recognizes the promoter. Most preferably, the RNA messenger and promoter are selected from T7 RNAP, T3 RNAP and SP6 RNAP and related related promoters. However, the steps of transcription of the process of the invention may use a RNAP jug, the appropriate promoter sequence of which allows transcription with high specificity to be known kb that can be obtained. Kits and enzymes for in vitro transcription are commercially available from the age of suppliers, and the appropriate reaction mixtures and conditions for carrying out the steps of the present invention containing in vitro transcription may use these products as described by the manufacturers. For example, In vitro transcription using T7 RNAP can be performed using the AMPLISCRIBE ™ Transcription Kit T7-FLASH ™ Transcription Kit kb AMPLISCRIBE ™ T7 High Yak Transcription Kit from EPICENTER Biotechnologies, Madison, WI as described in the product literature. Similarly, if T3 RNAP or SP6 RNAP is used for the in vitro transcription method, the AMPLISCRIBE ™ T3-FLASH ™ High Yink Transcription Kit kb kit of AMPLISCRIBE ™ SP6 High Yink Transcription Kit (EPICENTRE Biotechnologies, Madison, WI) can be used, respectively. as described.

[0274] In some embodiments, the transferable strand, oHgonuccotide tagged by Hgination kb PCR primer contains, in addition to the RNA polymerase promoter sequence, additional translation sequences, including, but not limited to, a ribosome binding site and a transcription initiation codon (also referred to as "start signal"). In some embodiments, the method comprises a subsequent in vitro transcription of the resulting RNA transcripts. Systems and kits for in vitro translation of RNA transcripts are also commercially available from the age of sources and can be used for the present invention. In addition, reticulocyte Hetteat, Wheat Germ Extract and E. coli S30 Extraction Systems from PROMEGA Corporation, Madison, WI can be broken down in the present invention.commercial in vitro conjugated transcription kits and in vitro translation systems such as TNT® Quick Coupled Transcription / Translation Systems conjugated transcription and translation systems from Promega can be used.

[0275] In some other embodiments, the method further comprises a step of amplifying and / or sequencing the DNA in tagged fragments of circular ssDNA using DNA polymerase and at least one primer complementary with the tag. In some embodiments, the step of amplifying tagged fragments of circular ssDNA using DNA polymerase comprises rolling circle replication. In some embodiments, the step of amplifying tagged fragments of circular ssDNA using DNA polymerase comprises PCR amplification using a thermostable DNA polymerase and a first PCR primer complementary to at least a portion of the complement of the tag. In some embodiments, the method further comprises the step of amplifying tagged fragments of circular ssDNA using RNA polymerase.

[0276] Hence, in some other embodiments, the method further comprises amplifying tagged fragments of circular rollingDNADNA replication by a rolling circle method (RCR), the method comprising: (a) attaching a primer complementary to tagged fragments of circular ssDNA; and (b) extension of the primer attached to the tagged circular ssDNA fragments using DNA polymerase having a thread transfer activity (e.g., a large fragment of phi29 DNA polymerase or rBst DNA polymerase (EPICENTRE) or DISPLACEACE ™ DNA polymerase (EPICENTRE).) In these embodiments, the products RCR amplifications are concatameric ssDNA molecules complementary to tagged fragments of circular ssDNA In some embodiments, where the tagged circular ssDNA fragments contain the antisense promoter sequence,

[0277] In some preferred embodiments, the sequence of the transposon ends comprises a transferred thread containing only the transposon end of the transfer sequence and, consequently, a tag comprising only the transposon end of the transposed sequence. In some other embodiments, the sequence of the transposon ends includes a transfer thread that contains or consists of a 3 'part and a 5' portion, where the 3 'portion contains the transposon end of the transfer sequence and the 5' part contains another desired sequence, in which examples the implementation of the tag contains or consists of both the 3 'part and the 5' part. In some embodiments, where the sequence of the transposon ends comprises a transfer thread, which comprises or consists of the 3 'part and the 5' part, the non-transferred thread contains a complementary sequence with the 5 'part of the transfer thread. However, in some preferred embodiments of the transposon termination sequence, the not-transferred thread does not contain a complementary sequence with the 5 'portion of the transfer thread. In some preferred embodiments, the non-transferred thread contains only the non-transferred transposon end sequence. In certain preferred embodiments, the non-transfered strand comprises a non-complementary sequence with the 3 'transfer of the non-transferred end of the transposon sequence. the non-transferred thread does not contain a sequence complementary to the 5 'part of the transferred thread. In some preferred embodiments, the non-transferred thread contains only the non-transferred transposon end sequence. In certain preferred embodiments, the non-transfered strand comprises a non-complementary sequence with the 3 'transfer of the non-transferred end of the transposon sequence. the non-transferred thread does not contain a sequence complementary to the 5 'part of the transferred thread. In some preferred embodiments, the non-transferred thread contains only the non-transferred transposon end sequence. In certain preferred embodiments, the non-transfered strand comprises a non-complementary sequence with the 3 'transfer of the non-transferred end of the transposon sequence.

[0278] In some embodiments, where the transposon termination sequence comprises a transferred thread that comprises or consists of a 5 'portion and a 3' portion, the 5 'portion comprises the sequence of the sequencing tag domain or capture tag domain (e.g., sequencing tag domain or domain) the capture tag of the Roche 454 FL000 genomic sequencer, e.g. when Roche 454A and 454B are used for sequencing using the Roche 454 FLX genome sequencer), and the 3 'part contains the transposon end of the transposed sequence. Hence, if the sequence of the transposon ends contains a transfer thread having a sequencing tag domain or capture tag domain, tagged circular ssDNA fragments or double tagged linear ssDNA fragments have a tag containing a sequencing tag domain or capture tag domain (e.g., Roche 454A or 454B tag used for tag sequencing using a Roche 454 FL000 genomic sequencer). The tagged circular ssDNA fragments or double tagged linear ssDNA fragments are created in the desired size order and used as the next generation sequencing patterns using the Roche 454 FLX genome sequencer. In other embodiments, tagged circular ssDNA fragments or double tagged linear ssDNA fragments are created and contain one or more restriction domain domains, sequencing tag domain, capture tag domain, amplification tag domain,

[0279] There are no restrictions on the use of additional sequences for one or more additional sequences in the 5 'portion of the transfer thread or in the 3' portion of the non-transfer thread, which sequences can be used to accomplish the intended purpose. In some embodiments, the 5 'portion of the transfer thread or the 3' portion of the non-transfer thread comprises one or more tag domain sequences.

[0280] In some embodiments, the method further comprises the steps of elongating the transferred strands containing 5'-tagged DNA fragments formed by the DNA polymerase without thread-transfer activity and 5'-to-3'-endonuclease activity (e.g., DNA polymerases). T4, EPICENTRE), followed by using a template-dependent DNA ligase (e.g., E. coli DNA ligase) to ligate the 3 'end of each DNA extension product to the 5' end of the non-transferlased tag containing tagged DNA fragments using the reverse strand as a ligation standard; in such embodiments, the 5 'ends of the non-transferred strands of the transposon end of the sequence have a 5' monophosphate group. This embodiment of the method allows the creation of double tagged ssDNA fragments.

[0281] The work carried out during the development of the embodiments of the present invention led to the observation that transposition occurs in dsDNA. Therefore, in some preferred embodiments, the sequence of the transposon ends comprises or consists of a non-transfer thread that only contains a non-transfer transposon end sequence so that the 5 'portion of the transferred thread is single-stranded (e.g., to minimize the likelihood or frequency of insertion of the transferred thread to its double stranded parts by in vitro transposition reaction). In some preferred embodiments, where the not-transferred thread includes a 3 'part complementary to the 5' portion of the transfer thread, the size of the 5 'part the transferred thread is minimized in order to minimize the probability or frequency of insertion of the transferred thread to itself during in vitro transposition reactions. For example, in some embodiments, the size of the 5 'portion of the transfer thread (and the complementary 3' portion of the non-transfer thread) is less than 150 nucleotides, less than 100 nucleotides, less than 75 nucleotides, less than 50 nucleotides, less than 25 nucleotides, or less than 15 nucleotides.

[0282] In some preferred embodiments, the 5 'end of the transfer strand of the transposon end of the sequence has a 5' monophosphate group. In some preferred embodiments, the 5 'end of the non-transfer group has a 5' monophosphate group. In some preferred embodiments, both the transferred and the non-transferred thread have a 5 'monophosphate group. In the examples where the transferred thread lacks a 5 'monophosphate group, the method further comprises a phosphorylation step of the 5' end of the transposon end of the transferred oligonucleotide (e.g., using a polynucleotide kinase, e.g., T4 polynucleotide kinase) prior to the method ligation step.

[0283] Inserting the transposon end of the transposase catalyzed DNA into the target DNA results in the transposon end of the one-stranded DNA being attached to the 5'-end of the strand and splitting or fragmentation of the strand at the site where the transposon end transferred sequence is attached to the target DNA, simultaneously forming a 9-base the single-stranded region of the target DNA located at the 3 'end of the attachment site of the transferred end of the tranposone to the target DNA due to the 9-base region of the loops on the opposite strand of the target DNA. For example, FIG. 1 shows the results of two independent insertion events of the transposon end transferred to the opposite strands of the target DNA. As shown in FIG. 2 independent insertions of the transposon transposed end into opposite strands of the target DNA are sometimes found in locations in the target DNA located in relative proximity, forming two 5 'tagged DNA fragments at the 5' end. After denaturation, two ssDNA fragments tagged at the 5 'end are released.

Pattern-independent Ligation of 5'-Tagged ssDNA Fragments [0284] In some embodiments, template-independent nucleic acid ligases or non-homologous nucleic acid ligases (e.g., which perform intramolecular ligation, i.e. circularization of ssDNA, which has a 3'-hydroxyl and 5 'group) monophosphate) is used in the method of carrying out the invention for the circularization of 5'-linearly labeled ssDNA fragments. In some preferred embodiments, the nucleic acid ligase is a thermostable RNA ligase (e.g., selected from the TS2126 bacteriophage DNA thermostable ligase (U.S. Patent No. 7,303,901 and Blondal et al., Nucleic Acids Res 33: 135-142, 2005), CIRCLIGASE ™ ssDNA ligases ( EPICENTER Biotechnologies, Madison, WI, USA) and Archaea RNA ligases (e.g., Methanobacterium RNA thermoautotrophicum RNA ligands or "MthRnl"; Torchia, C et al., Nucleic Acids Res 36: 6218-6227, 2008). "Non-standard ligase" or "nonhomogeneous ligase" means a ligase that leads to ssDNA ligation in the absence of attachment of a complementary sequence to the ssDNA ends to be linked or ligated (i.e., the two ends are not attached to the complementary sequence to keep them past) at the ligation stage). In these embodiments, the method comprises the step of: denaturation of the tagged 5 'DNA fragments generated by the in vitro transposition reaction by incubating the tagged 5' linear ssDNA fragments with a nucleic acid ligase. By "intramolecular ligation" we mean that the two ends of one ssDNA molecule are ligated together to form circular ssDNA fragments rather than ligation to the ends of other DNA molecules. Nucleic Acids Res 36: 6218-6227, 2008). "Non-standard ligase" or "nonhomogeneous ligase" means a ligase that leads to ssDNA ligation in the absence of attachment of a complementary sequence to the ssDNA ends to be linked or ligated (i.e., the two ends are not attached to the complementary sequence to keep them past) at the ligation stage). In these embodiments, the method comprises the step of: denaturation of the tagged 5 'DNA fragments generated by the in vitro transposition reaction by incubating the tagged 5' linear ssDNA fragments with a nucleic acid ligase. By "intramolecular ligation" we mean that the two ends of one ssDNA molecule are ligated together to form circular ssDNA fragments rather than ligation to the ends of other DNA molecules. Nucleic Acids Res 36: 6218-6227, 2008). "Non-standard ligase" or "nonhomogeneous ligase" means a ligase that leads to ssDNA ligation in the absence of attachment of a complementary sequence to the ssDNA ends to be linked or ligated (i.e., the two ends are not attached to the complementary sequence to keep them past) at the ligation stage). In these embodiments, the method comprises the step of: denaturation of the tagged 5 'DNA fragments generated by the in vitro transposition reaction by incubating the tagged 5' linear ssDNA fragments with a nucleic acid ligase. By "intramolecular ligation" we mean that the two ends of one ssDNA molecule are ligated together to form circular ssDNA fragments rather than ligation to the ends of other DNA molecules. "Non-standard ligase" or "nonhomogeneous ligase" means a ligase that leads to ssDNA ligation in the absence of attachment of a complementary sequence to the ssDNA ends to be linked or ligated (i.e., the two ends are not attached to the complementary sequence to keep them past) at the ligation stage). In these embodiments, the method comprises the step of: denaturation of the tagged 5 'DNA fragments generated by the in vitro transposition reaction by incubating the tagged 5' linear ssDNA fragments with a nucleic acid ligase. By "intramolecular ligation" we mean that the two ends of one ssDNA molecule are ligated together to form circular ssDNA fragments rather than ligation to the ends of other DNA molecules. "Non-standard ligase" or "nonhomogeneous ligase" means a ligase that leads to ssDNA ligation in the absence of attachment of a complementary sequence to the ssDNA ends to be linked or ligated (i.e., the two ends are not attached to the complementary sequence to keep them past) at the ligation stage). In these embodiments, the method comprises the step of: denaturation of the tagged 5 'DNA fragments generated by the in vitro transposition reaction by incubating the tagged 5' linear ssDNA fragments with a nucleic acid ligase. By "intramolecular ligation" we mean that the two ends of one ssDNA molecule are ligated together to form circular ssDNA fragments rather than ligation to the ends of other DNA molecules. which leads to the ligation of ssDNA in the absence of attachment of the complementary sequence to the ssDNA ends to be linked or ligated (i.e., the two ends are not attached to the complementary sequence to keep them side by side in the ligation step). In these embodiments, the method comprises the step of: denaturation of the tagged 5 'DNA fragments generated by the in vitro transposition reaction by incubating the tagged 5' linear ssDNA fragments with a nucleic acid ligase. By "intramolecular ligation" we mean that the two ends of one ssDNA molecule are ligated together to form circular ssDNA fragments rather than ligation to the ends of other DNA molecules. which leads to the ligation of ssDNA in the absence of attachment of the complementary sequence to the ssDNA ends to be linked or ligated (i.e., the two ends are not attached to the complementary sequence to keep them side by side in the ligation step). In these embodiments, the method comprises the step of: denaturation of the tagged 5 'DNA fragments generated by the in vitro transposition reaction by incubating the tagged 5' linear ssDNA fragments with a nucleic acid ligase. By "intramolecular ligation" we mean that the two ends of one ssDNA molecule are ligated together to form circular ssDNA fragments rather than ligation to the ends of other DNA molecules. the two ends are not attached to the complementary sequence to keep them side by side at the ligation stage). In these embodiments, the method comprises the step of: denaturation of the tagged 5 'DNA fragments generated by the in vitro transposition reaction by incubating the tagged 5' linear ssDNA fragments with a nucleic acid ligase. By "intramolecular ligation" we mean that the two ends of one ssDNA molecule are ligated together to form circular ssDNA fragments rather than ligation to the ends of other DNA molecules. the two ends are not attached to the complementary sequence to keep them side by side at the ligation stage). In these embodiments, the method comprises the step of: denaturation of the tagged 5 'DNA fragments generated by the in vitro transposition reaction by incubating the tagged 5' linear ssDNA fragments with a nucleic acid ligase. By "intramolecular ligation" we mean that the two ends of one ssDNA molecule are ligated together to form circular ssDNA fragments rather than ligation to the ends of other DNA molecules. linear ssDNA fragments with nucleic acid ligase. By "intramolecular ligation" we mean that the two ends of one ssDNA molecule are ligated together to form circular ssDNA fragments rather than ligation to the ends of other DNA molecules. linear ssDNA fragments with nucleic acid ligase. By "intramolecular ligation" we mean that the two ends of one ssDNA molecule are ligated together to form circular ssDNA fragments rather than ligation to the ends of other DNA molecules.

[0285] In certain preferred embodiments, the non-homologous ligation reaction is performed in an "improved ligation reaction mixture", which herein is a ligation reaction mix that includes: a) tagged linear ssDNA fragments; B) buffer maintaining the pH; B) Mn cations²⁺; and (c) a sequence of thermostable RNA ligase molecules in which a large portion of thermostable RNA-ligase molecules are adenylated; wherein the concentration of adenylated thermostable RNA ligase molecules is at least equal to the molarity of linear ssDNA fragments

2+ and no ATP or Mg cations are added²⁺ to the ligation reaction mixture.

[0286] The finding that "a significant proportion of thermostable RNA ligase molecules are adenylated" means that at least about 50% of all thermostable RNA ligase molecules in the improved ligation reaction mixture are adenylated. In some embodiments of the improved reaction mixture, over 60% of all thermostable RNA ligase molecules are adenylated to ligate. In some embodiments of the improved reaction mixture for ligation adenylated more than about 70% of all thermostable RNA ligase molecules. In some embodiments of the improved ligation reaction mixture, more than about 80% of all thermostable RNA ligase molecules are adenylated to the ligation. In some preferred embodiments, the improved ligation reaction mixture is adenylated over approx. 90% of all thermostable RNA ligase molecules. In some preferred embodiments of the improved ligation reaction mixture, over 95% of all thermostable RNA ligase molecules are adenylated. In certain preferred embodiments, the thermostable RNA ligase is adenylated to produce a sequence in which a large proportion of thermostable RNA ligase molecules are adenylated by incubating the enzyme with ATP during or after the purification process. One of the protocols that can be used to adenylate a thermostable RNA ligase is to incubate the enzyme in a solution containing 50 mM TrisHCl, pH 8.0, 2 mM MgCl 2, 100 mM NaCl and 0.5 mM ATP for 15 minutes at room temperature. In some preferred embodiments of the improved ligation reaction mixture, over 95% of all thermostable RNA ligase molecules are adenylated. In certain preferred embodiments, the thermostable RNA ligase is adenylated to produce a sequence in which a large proportion of thermostable RNA ligase molecules are adenylated by incubating the enzyme with ATP during or after the purification process. One of the protocols that can be used to adenylate a thermostable RNA ligase is to incubate the enzyme in a solution containing 50 mM TrisHCl, pH 8.0, 2 mM MgCl 2, 100 mM NaCl and 0.5 mM ATP for 15 minutes at room temperature. In some preferred embodiments of the improved ligation reaction mixture, over 95% of all thermostable RNA ligase molecules are adenylated. In certain preferred embodiments, the thermostable RNA ligase is adenylated to produce a sequence in which a large proportion of thermostable RNA ligase molecules are adenylated by incubating the enzyme with ATP during or after the purification process. One of the protocols that can be used to adenylate a thermostable RNA ligase is to incubate the enzyme in a solution containing 50 mM TrisHCl, pH 8.0, 2 mM MgCl 2, 100 mM NaCl and 0.5 mM ATP for 15 minutes at room temperature. In certain preferred embodiments, the thermostable RNA ligase is adenylated to produce a sequence in which a large proportion of thermostable RNA ligase molecules are adenylated by incubating the enzyme with ATP during or after the purification process. One of the protocols that can be used to adenylate a thermostable RNA ligase is to incubate the enzyme in a solution containing 50 mM TrisHCl, pH 8.0, 2 mM MgCl 2, 100 mM NaCl and 0.5 mM ATP for 15 minutes at room temperature. In certain preferred embodiments, the thermostable RNA ligase is adenylated to produce a sequence in which a large proportion of thermostable RNA ligase molecules are adenylated by incubating the enzyme with ATP during or after the purification process. One of the protocols that can be used to adenylate a thermostable RNA ligase is to incubate the enzyme in a solution containing 50 mM TrisHCl, pH 8.0, 2 mM MgCl 2, 100 mM NaCl and 0.5 mM ATP for 15 minutes at room temperature.

50 ° C; then stop the reaction by adding EDTA to a final concentration of 5 mM; then removing the reaction components by dialysis or gel filtration. The percentage of adenylated thermostable RNA ligase can be estimated by SDS-PAGE analysis. In certain preferred embodiments, the thermostable RNA ligase in which a large portion of thermostable RNA ligase molecules are adenylated is a thermostable RNA ligase of the TS2126 bacteriophage. In some embodiments of the improved ligation reaction mixture, the buffer maintains a pH in the range of 6.5 and 8.0. In some preferred embodiments of the improved ligation reaction mixture, the buffer maintains a pH in the range of 7.0 to 8.0. In some preferred embodiments of the improved ligation reaction mixture, the buffer maintaining the pH between pH 7.0 and 8.0 is the Tris buffer.²⁺ is from 0.5 to 10 mM. In some embodiments, improved

2+ ligation reaction mixture, the concentration of Mn cations²⁺ is from 1 to 10 mM. In some embodiments, the improved ligation reaction is concentration

2 _) _ cations Mn²⁺ is from 1 to 5 mM. In some preferred embodiments of the improved ligation reaction mixture, the concentration of Mn cations²⁺ is 2.5 mM. In certain preferred embodiments of the improved ligation reaction mixture, Mn cations²⁺ are provided as MnCl². In some embodiments, the concentration of adenylated thermostable RNA ligase molecules in the improved ligation reaction mix has at least twice the molarity of the linear ssDNA fragments. In some embodiments, the concentration of adenylated thermostable RNA ligase molecules in the improved ligation reaction mixture is at least a five-fold greater in molarity than the linear ssDNA fragments. In some embodiments, the concentration of adenylated thermostable RNA ligase molecules in the improved ligation reaction mix has at least a ten-fold greater molarity than the linear ssDNA fragments. In some preferred embodiments, the improved ligation reaction mixture further comprises a salt such as potassium chloride or potassium acetate (e.g., at a concentration of about 50 to about 100 mM). In some preferred embodiments, the improved ligation reaction mixture additionally contains a reducing agent such as dithiothreitol (DTT) (e.g., at a concentration of about 0.5 or 1 mM). In some embodiments, the improved ligation reaction mixture additionally contains zwitterion trimethylglycine (betaine) at a concentration of 0.25 to 5.2 M. In some embodiments, the improved ligation reaction mixture further comprises zwitter ion trimethylglycine (betaine) at a concentration of 0.5 up to 2 M. In certain embodiments, the improved ligation reaction mixture further comprises zwitter ion trimethylglycine (betaine) at a concentration of about 1 M. 5 or 1 mM). In some embodiments, the improved ligation reaction mixture additionally contains zwitterion trimethylglycine (betaine) at a concentration of 0.25 to 5.2 M. In some embodiments, the improved ligation reaction mixture further comprises zwitter ion trimethylglycine (betaine) at a concentration of 0.5 up to 2 M. In certain embodiments, the improved ligation reaction mixture further comprises zwitter ion trimethylglycine (betaine) at a concentration of about 1 M. 5 or 1 mM). In some embodiments, the improved ligation reaction mixture additionally contains zwitterion trimethylglycine (betaine) at a concentration of 0.25 to 5.2 M. In some embodiments, the improved ligation reaction mixture further comprises zwitter ion trimethylglycine (betaine) at a concentration of 0.5 up to 2 M. In certain embodiments, the improved ligation reaction mixture further comprises zwitter ion trimethylglycine (betaine) at a concentration of about 1 M.

[0287] In some preferred embodiments, the improved ligation reaction mixture comprises: a) linear ssDNA fragments that have 5'-phosphoryl and 3'-hydroxy groups (e.g., 0.5 micromolar); B) 33 mM TRIS acetate, pH 7.8; B) 2.5 mM Mn cations²⁺; and (c) a sequence of thermostable RNA-ligase molecules, wherein&gt; 70% of thermostable RNA-ligase molecules are adenylated; wherein the concentration of adenylated thermostable RNA ligase molecules is at least equal to the molarity of linear ssDNA fragments (e.g., about 1 micromole of adenylated thermostable RNA ligase for 0.5 micromolar tagged linear ssDNA fragments) and no ligation is added to the ligation reaction ATP or Mg cations²⁺. In some preferred embodiments, the concentration of adenylated thermostable RNA ligase molecules is at least 5 times greater, at least 10 times greater and at least 20 times greater than the molarity of linear ssDNA fragments (e.g., about 2.5 micromolar, about 5 micromolar or about 10 micromoles of adenylated thermostable RNA ligase for 0.5 micromolar tagged linear ssDNA fragments). In certain preferred embodiments, the improved ligation reaction mixture further comprises 66 mM potassium acetate and 0.5 mM DTT. In certain preferred embodiments, the improved ligation reaction mixture additionally contains 1 M betaine.

[0288] In certain preferred embodiments, the intramolecular ligation of 5'-labeled linear ssDNA fragments for the synthesis of tagged circular ssDNA fragments is carried out in the ligation reaction mixture at a temperature of about 40 ° C to about 70 ° C for a sufficient period (e.g. about one hour to about 72 hours), with round ssDNA fragments synthesized. In some preferred embodiments, the intramolecular ligation is carried out at a reaction temperature of about 60 ° C for a sufficiently long time, with round ssDNA fragments synthesized. [0289] The invention is not limited to only the nucleic acid ligases described herein. It will be understood by those skilled in the art that the intramolecular ligase can be performed using any nucleic acid ligase,

V. Ds-DNA Fragmentation and Labeling by In Vitro Transposition of Hairpin-shaped Endospecies (30963) [0290] In short, in some embodiments, the method includes: incubating the target DNA, which is dsDNA, with transposition and transposon sequence in the shape of a hair clip in an in-transposition reaction in order to simultaneously fragment and tag the target DNA, thus creating a library containing a population of tagged DNA fragments; then combining the 3 'end of each tagged 5' DNA fragment containing one strand of target DNA to the 5 'end of another tagged 5' DNA fragment containing the complementary portion (i.e., the opposite strand of the target DNA), thus generating a library of covalently closed tagged circular DNA fragments (e.g. exhibiting single-stranded round or dumbbell-shaped structures). In certain preferred embodiments, the linking step comprises: extending the 3 'ends of tagged 5' DNA fragments with a DNA polymerase that has no 5'to 3 'exonuclease (including structure-dependent 5' nuclease) and mixing of the strands to produce tagged extension products. 'DNA fragments and ligation of the 3' end of each of the 5'-tagged extension products on the end of the complementary 5 'tagged extension product with a template dependent DNA ligase (e.g., E. coli DNA ligase or template-dependent DNA ligase) psychophilic bacteria or psychrophilic bacteriophages).

[0291] Thus, one preferred embodiment of the invention is a method of generating a library comprising a population of tagged circular DNA fragments from double-stranded target DNA for use as a template in DNA sequencing or nucleic acid amplification reactions, each tagged round DNA fragment having both strand sequences of the target part. The DNA and the sequence of the tag, the method comprising:

Providing:

[0292]

1. a target DNA comprising or consisting of one or more double-stranded molecules (dsDNA) (e.g., genomic, mitochondrial, chloroplast or other dsDNA from a eukaryotic cell and / or genomic or episomal DNA from a prokaryotic cell or double-stranded cDNA prepared by reverse transcription of RNA from a eukaryotic and / or prokaryotic cell to produce a first-strand cDNA followed by extension of the primer attached to the first-strand cDNA);

2. transposases (e.g., wild-type or mutant, e.g., wild-type or mutant Tn5 transposases, e.g. EZ-Tn5 ™ transposases, e.g. HYPERMU ™ MuA transposases, EPICENTER Biotechnologies, Madison, Wisconsin, USA); and

3. a transposon end sequence in hairpin formation that is capable of forming a functional transposase-mediated complex in a transposition reaction, and which has a tag sequence, wherein said hairpin-like sequence of the hairpin transposon includes or consists of an oligonucleotide containing 5 ' a phosphate that exhibits an non-transferred sequence of the transposon ends at its 5 'end (e.g., &amp; quot; MENTS &amp; quot; relative to the transposon sequence of the non-transferable EZ-Tn5 ™), the transposon terminal sequence moved at its 3' end (e.g. "METS" for the transposon end sequence not transferred by EZ-Tn5 ™) and the intervening sequence (for any purpose, e.g.for providing a tag) between the non-transferred transposon final sequence and the transposon terminator that is long enough to allow intramolecular core loop formation, the core having a double-stranded transposon end sequence with which the transposon forms a transposable complex (e.g. wherein the core has a 19-bp transposon end ("OE") sequence, an internal 19-bp ("IE") or "19-bp" patch ("ME") recognized by wild-type or mutant Tn5 transposase, e.g. EZ-Tn5 transposase) (or, for example, the ends of the R1 and R2 transposon MuA for MuA transposase), and the loop has an intervention sequence that can be any sequence;wherein the core has a sequence of double-stranded transposon ends with which the transposase forms a transposable complex (e.g., wherein the core has a 19-bp transposon end ("OE") sequence, an internal 19-bp ("IE") end or "end" mosaic 19-bp ("ME") recognized by wild-type or mutant Tn5 transposase, e.g. by transposase EZ-Tn5 ™) (or, e.g., the transposable ends of R1 and R2 MuA for MuA transposase), and the loop has an intervention sequence that may be any sequence;wherein the core has a sequence of double-stranded transposon ends with which the transposase forms a transposable complex (e.g., wherein the core has a 19-bp transposon end ("OE") sequence, an internal 19-bp ("IE") end or "end" mosaic 19-bp ("ME") recognized by wild-type or mutant Tn5 transposase, e.g. by transposase EZ-Tn5 ™) (or, e.g., the transposable ends of R1 and R2 MuA for MuA transposase), and the loop has an intervention sequence that may be any sequence;the ends of the transposon R1 and R2 MuA for the MuA transposase), and the loop has an intervention sequence that can be any sequence;the ends of the transposon R1 and R2 MuA for the MuA transposase), and the loop has an intervention sequence that can be any sequence;

4. (a) a DNA polymerase that does not contain 5 'nucleases (including exonuclease 5'-3' and 5'-dependent exonuclease activity) and strand displacement activity (e.g., T4 DNA polymerase); or (b) random number oligonucleotides of one or more sizes, which alone or in combination have the same length as single-strand gaps in the tagged 5 'DNA fragments that result from the transposition reaction with the transposase and the hairpin-like sequence of the hairpin transposon ends; and

5. a reference-dependent ligase (e.g., E. coli DNA ligase or a standard-dependent ligase from psychrophilic bacteria or psychrophilic bacteriophages);

Incubation of target DNA in an in transposition reaction with transposase and a hairpin-like transposon sequence under conditions and for a sufficiently long period, wherein insertion of hairpin-like ends of the hairpin transposon to a target DNA generates a population of tagged 5 'DNA fragments (see e.g., FIG 2 and FIG 3);

Incubation of tagged 5 'DNA fragments under conditions and for a sufficiently long period in which single-strand gaps in DNA fragments are filled and the 3' end of each tagged 5 'DNA fragment is extended and fused to the 5' end of another tagged 5 'DNA fragment that it contains a complementary portion of the target DNA, thus generating a library of tagged circular DNA fragments, each of which exhibits sequences of both strands of the target DNA part and the sequence of the tag.

[0293] In some preferred embodiments of the method (as depicted in the scheme, e.g., in Figures 7 and 8), the linking step includes: (1) incubating the tagged 5 'DNA fragment with a DNA polymerase that does not show 5' nuclease activity in conditions in which the 3 'end of each tagged 5' DNA fragment is extended to produce a population of 5 'tagged extension products; and (2) incubating the 5 'tagged extension products with the pattern-dependent ligase under conditions and for a sufficient period of time to lengthen the tagged 5' DNA fragmentation products, thereby creating a library of tagged circular DNA fragments. In some embodiments, a DNA polymerase that lacks 5 'nuclease activity

[0294] In some other preferred embodiments of the method, the linking step comprises: incubating the tagged 5 'DNA fragments with one or more oligonucleotide sizes with a random sequence and pattern-dependent ligase under conditions and for a sufficiently long period, wherein the random sequence oligonucleotides attach and fill in the regions of the single-strand breaks in tagged 5 'DNA fragments, and wherein said random-sequence oligonucleotides are ligated together or with the adjacent 5'-tagged DNA fragment, thereby forming tagged circular DNA fragments.

[0295] In some embodiments, the method further comprises, after the ligation step, one or more steps performed to remove oligonucleotides having a random sequence, a linear target DNA, and / or a hairpin-like transposon sequence that is not linked to the target DNA .

[0296] In some preferred embodiments, the method further comprises: treating a reaction mixture comprising tagged circular DNA fragments of T5 exonuclease (EPICENTER Biotechnologies, Madison, Wisconsin) to remove un-ligated linear ssDNA and dsDNA (e.g., DNA fragments that are incised and / or contain single-stranded regions).

[0297] In some embodiments, the method further comprises: cleaving tagged circular DNA fragments in each of the loop structures to generate linear double-stranded DNA fragments, and each strand of DNA fragment has a portion of the tag at its 5 'end and a portion of the tag at its 3 end. '(which are linear DNA fragments are referred to herein as' blunt ended 'double-tagged fragments of linear dsDNA' or 'blunt-ended dsDNA fragments').

[0298] In some embodiments, the cleavage method comprises: attaching to tagged circular DNA fragments of an oligodeoxyribonucleotide that attaches to the restriction site within the tag, and then incubates with the restriction endonuclease that cleaves at the double-stranded restriction site to generate the dsDNA fragments.

incubation with tagged circular DNA fragments by means of the splitting enzyme sequence under conditions and for a sufficiently long period of time in which tagged circular DNA fragments are cleaved at fissile sites to generate blunt-ended dsDNA fragments. In some embodiments of the method, a non-canonical non-nucleotide is used to provide a cleavable site, and another N-glycosylase is used in the cleavage enzyme sequence. The hairpin-like sequence of the hairpin transposon is synthesized (e.g., using an oligonucleotide synthesizer) to provide a cleavage site consisting of a non-canonical nucleotide instead of a canonical nucleotide (e.g., dUMP as a non-canonical nucleotide instead of TMP,

[0300] Therefore, in some preferred embodiments, wherein the transposon termination sequence has one or more cleavable site the cleavage enzyme sequence uses N-glycosyllase (or "DNA glycosylation") to create a abrasion or apirimidine / apurbinid (AP) site. As defined herein, "N-glycosyllase" is an enzyme that catalyzes the hydrolysis of the bond between a non-canonic nucleic acid base and sugar in DNA to form a site of abrasion (AP). Such enzymes are found in many species. An example from Escherichia coli is uracil-N-glycosyllase (UNG), also called uracil-DNA glycosylase (UDG). UNG catalyzes the cleavage of base uracil from deoxyribose in sugar in DNA (Lindahl, Prog. Nucl. Acid Res. Mol. Biol. 22: 135-192, 1979), but it does not catalyze the cleavage of uracil from free dUTP, free deoxyuridine or RNA (Duncan, in The Enzymes, Boyer ed., pp. 565-586, 1981). Other examples of N-glycosyls that can be used as cleavage enzymes are described in Demple and Harison (Annu. Rev. Biochem. 63: 915-48, 1994) and in Duncan ("DNA Glycosylases," in The Enzymes, Boyer ed., pp. 565-586, 1981). By "N-Glycosyllase" or "DNA Glycosylase" is meant an enzyme having N-glycosylase activity regardless of whether the enzyme is formally referred to as glycosylase or glycosyllase activity in combination with another enzymatic activity. Glycosylases are sometimes referred to as "glycosidases" and therefore the definition of N-glycosyllase includes N-glycosidases. For example, the FPG protein as defined herein is also N-glycosyllase. 1994) and catalyzes the cleavage of the N-glycosyl linkage between the modified base and the backbone of the deoxyribo- phosphodiester in DNA, generating an AP site. In addition, the FPG protein also has AP lyase activity. The enzymatic activity of lyase AP catalyzes the deletion of beta, delta, leaving a gap of one nucleotide in the DNA (Bailly, et al., Biochem. J. 261: 707-713, 1989). The FPG protein and the 8-hydroxyguanine DNA glycosylase proved to be identical (Chung, MH et al., Mutation Research 254: 112, 1991). DNA treatment with methylene blue and visible light (Floyd, et al., Arch Biohem Biophys 273: 106-111, 1989) or Bengal red and ultraviolet light (Friedmann and Brown, Nucleic Acids Research 5: 615-622, 1978) induces a specific guanine modification, which is cleavable by the FPG protein. Other specific N-glycosylates will be available and known to those skilled in the art. To determine if the N-glycosyllase is suitable for the present invention, a non-canonical nucleotide is first introduced into the DNA and determines whether the non-canonical base can be specifically removed by a given N-glycosyllase in a similar manner to the removal of uracil or 8-oxo-guanine, respectively UNG and FPG protein. In the field, various methods for separating the abnormal site are known after the creation of the abnormal site or the AP site. You can use heat and / or basic conditions to split a DNA molecule at abrasive sites. For example, the following protocol may be used: Nucleic acids containing ablation sites (AP) after removing non-canonical bases are heated in an amine-containing buffer solution, for example, 25 mM Tris-HCl and from 1 to 5 mM magnesium ions for a period of 10 to 30 minutes at 70 ° C to 95 ° C. Alternatively, the following treatment can be used to break the DNA at abrasive sites: 1.0 M piperidine, base, is added to the DNA precipitated with ethanol and dried in vacuo. The solution is then heated for 30 minutes at 90 ° C and lyophilized to remove the piperidine. In some preferred embodiments, the enzyme treatment using apurin / apirimidine endonuclease (AP endonuclease) known in the industry (Lindahl, Prog. Nucl. Acid Res. Mol. Biol. 22: 135-192, 1979; Demple and Harison Annu. Rev. . to DNA precipitated with ethanol and dried in vacuo. The solution is then heated for 30 minutes at 90 ° C and lyophilized to remove the piperidine. In some preferred embodiments, the enzyme treatment using apurin / apirimidine endonuclease (AP endonuclease) known in the industry (Lindahl, Prog. Nucl. Acid Res. Mol. Biol. 22: 135-192, 1979; Demple and Harison Annu. Rev. . to DNA precipitated with ethanol and dried in vacuo. The solution is then heated for 30 minutes at 90 ° C and lyophilized to remove the piperidine. In some preferred embodiments, the enzyme treatment using apurin / apirimidine endonuclease (AP endonuclease) known in the industry (Lindahl, Prog. Nucl. Acid Res. Mol. Biol. 22: 135-192, 1979; Demple and Harison Annu. Rev. .

Biochem. 63: 915-48, 1994) is used to break the poHmer DNA at the abused site. As defined herein, endonuclease AP is any enzyme that catalyzes DNA cleavage at abrasion sites (AP). Such enzymes occur in age species. Examples of E. coli AP endonuclease include, but are not limited to, endonuclease III and endonuclease IV. Also exonuclease III of E. coli in the presence of calcium ions is the endonucleotide of AP. The enzymes useful in the present invention include messenger enzymes with an AP-like endonuclease activity, regardless of whether it is specific to one or another name.

[0301] In some preferred embodiments, the method further comprises: denaturing the blunt-ended dsDNA fragments to generate a library of double tagged Hni ssDNA fragments (e.g., for use as a template for DNA sequencing or DNA amplification).

[0302] In some embodiments, tagged kb double-tagged circular DNA fragments of ssDNA fragments in bibHotece generated using methods are used as DNA template in nucleic acid amplification and / or DNA sequencing reactions. In some embodiments, the method further comprises the step of amplifying and / or sequencing the target DNA in tagged circular kb DNA fragments of double tagged Hat IHN fragment fragments. In some embodiments, the method further comprises the step of amp- typing i / kb DNA sequencing, which is compliant with respect to the exact DNA obtained by amp- typing tagged circular kb DNA fragments of double tagged Hat IHN fragment. In some embodiments, at least a portion of the DNA attenuated in each of the tagged kb double-tagged DNA kb fragment fragments is sequenced using DNA DNA and at least one primer that is compliant to the tag (e.g., for synthesis sequencing). In some embodiments, at least a portion of the DNA attenuated in each of the tagged circular kb DNA fragments of double tagged Hni DNA fragments is sequenced using Hgase to clump from the pattern of at least one ohgodexyribonucleotide that is complementary to the tag and at least one other. oHgodeoxyribonucleotide that attaches to the sequence part of the sequence (e.g., for sequencing by Hgation). In some embodiments, at least a portion of the DNA that is attenuated in each of the tagged circular kb DNA fragments of double tagged Hni DNA fragments of ssDNA is sequenced by annealing of oligodeoxyribonucleotides that attach to the kb hybridizes to the tag and part of the downstream sequence (e.g., for hybridization). In some embodiments, the controversial DNA relative to the tagged circular kb DNA fragments of double tagged Hni DNA fragments of ssDNA is sequenced by synthesis, sequencing by Hygrination of kb by hybridization. for sequencing by hybridization). In some embodiments, the controversial DNA relative to the tagged circular kb DNA fragments of double tagged Hni DNA fragments of ssDNA is sequenced by synthesis, sequencing by Hygrination of kb by hybridization. for sequencing by hybridization). In some embodiments, the controversial DNA relative to the tagged circular kb DNA fragments of double tagged Hni DNA fragments of ssDNA is sequenced by synthesis, sequencing by Hygrination of kb by hybridization.

[0303] For example, in some preferred embodiments, the transposon transposed transposon sequence conveyed by a sequence of hairpin-shaped ends, provided in a kb kit used in the method of the present invention, is a transferred transposon sequence recognized by Tn5 transposon. In some preferred embodiments, the transposon terminal transfered sequence is a sequence recognized by the EZ-Tn5 ™ transposase (EPICENTER Biotechnologies, Madison, Wisconsin, USA).

[0304] Substantially tagged circular DNA fragments, blunt-ended dsDNA fragments, and double tagged HNI DNA fragments produced using the hairpin-shaped terminal sequences of the hairpin processes in the methods of the present invention exhibit both the transposon-transposed nucleotide sequence and the non-transferred transposon sequence and additional sequences including or derived from a non-complementary portion of the loop hairpin-like sequence of the hairpin transposon. Thus, in some embodiments, the hairpin-like sequence of the hairpin transposon ends shows one or more other 5 'nucleotide sequences of the transposon transposed transposition sequence and 3' non-ported transposon termination sequence one or more of the other nucleotide sequences are also expressed by the tag.

[0305] In some embodiments, wherein the hairpin-like clipper sequence comprises one or more restriction site domains, the method further comprises: annealing an oligodeoxyribonucleotide that is complementary to the restriction site of tagged circular DNA fragments followed by cleavage of tagged circular fragments DNA at the restriction site using restriction endonuclease that recognizes the restriction site. Thus, in some embodiments, the method comprises lining tagged circular DNA fragments to generate blunt-ended dsDNA fragments or, after denaturation, double-tagged linear ssDNA fragments.

[0306] In some embodiments, the method further comprises the step of ligating linear restriction endonuclease cleaved ssDNA fragments to one or more other DNA molecules (e.g., for combining the tag).

[0307] Thus, in some embodiments, the method further comprises: amplifying tagged circular DNA fragments or blunt-ended dsDNA fragments or double-tagged linear ssDNA fragments by transcription, and the method includes: (a) attaching to the sense oligodeoxyribonucleotide sense promoter that has a complementary sequence an antisense promoter or attachment to tagged circular DNA fragments or blunt-ended dsDNA fragments or double tagged linear ssDNA primer fragments that is complementary thereto and primer extension using DNA polymerase under conditions wherein dsDNA is synthesized, including a double stranded RNA polymerase promoter; and (b) incubating dsDNA products with an RNA polymerase that binds the RNA polymerase promoter under conditions,

[0308] In some preferred embodiments, wherein the hairpin-like transposon sequence or PCR primer sequence has an RNA polymerase promoter sequence, the RNA polymerase promoter is a T7 RNA polymerase promoter, and the method further comprises the step of transcribing tagged circular DNA fragments in vitro using T7 type RNA polymerase that recognizes the promoter. Preferably, the RNA polymerase and promoter are selected from T7 RNAP, T3 RNAP and SP6 RNAP and related related promoters. However, the transcription steps of the method of the invention may use any RNAP for which the appropriate promoter sequence allowing high specificity transcription is known or can be obtained. Sets and enzymes for in vitro transcription are available on the market from many manufacturers, and suitable reaction mixtures and conditions for performing the steps of the present invention comprising in vitro transcription may be carried out using these products as described by the manufacturers. For example, in vitro transcription using T7 RNAP can be performed using the AMPLISCRIBE ™ T7-FLASH ™ transcription kit or the AMIDISCRIBE ™ T7 High Yield transcription kit from EPICENTER Biotechnologies, Madison, Wisconsin, as described in the product literature. Similarly, if in vitro transcription uses T3 RNAP or SP6 RNAP in the method of the invention, the AMPLISCRIBE ™ T3-FLASH ™ High Yield transcription kit or AMPLISCRIBE ™ kit may be used as described, respectively. In vitro transcription using T7 RNAP can be carried out using the AMPLISCRIBE ™ T7-FLASH ™ transcription kit or the AMIDISCRIBE ™ T7 High Yield Transcription Kit from EPICENTER Biotechnologies, Madison, Wisconsin as described in the product literature. Similarly, if in vitro transcription uses T3 RNAP or SP6 RNAP in the method of the invention, the AMPLISCRIBE ™ T3-FLASH ™ High Yield transcription kit or AMPLISCRIBE ™ kit may be used as described, respectively. In vitro transcription using T7 RNAP can be carried out using the AMPLISCRIBE ™ T7-FLASH ™ transcription kit or the AMIDISCRIBE ™ T7 High Yield Transcription Kit from EPICENTER Biotechnologies, Madison, Wisconsin as described in the product literature. Similarly, if in vitro transcription uses T3 RNAP or SP6 RNAP in the method of the invention, the AMPLISCRIBE ™ T3-FLASH ™ High Yield transcription kit or AMPLISCRIBE ™ kit may be used as described, respectively.

SP6 High Yield (EPICENTER Biotechnologies, Madison, Wisconsin).

[0309] In some other embodiments, the method further comprises the step of amplifying and / or sequencing the target DNA in tagged circular DNA fragments using DNA polymerase and at least one primer that is complementary to the tag. In certain embodiments, the method further comprises: amplifying tagged circular DNA fragments by rolling circle type replication using DNA polymerase that translocates the strand. In some other embodiments, the method further comprises: amplifying tagged circular DNA fragments by PCR using a thermostable DNA polymerase, a first PCR primer that is complementary to at least a portion of the tag, and a second PCR tag that is complementary to at least a portion of the complement tag.

[0310] In some embodiments, the method further comprises: amplifying tagged circular DNA fragments by rolling circle type (RCR) replication, the method comprising: (a) attaching a primer that is complementary to tagged circular DNA fragments; and (b) prolonging the primer attached to tagged circular DNA fragments using DNA strand DNA polymerase (e.g., phi29 DNA polymerase, a large proportion of rBst DNA polymerase or DISPLACEACE ™ DNA Polymerase (EPICENTRE). In these embodiments, RCR amplification products are concatameric ssDNA molecules , which are complementary to the tagged circular DNA fragments, in some embodiments in which the tagged circular DNA fragments have the antisense promoter sequence,

[0311] In some preferred embodiments, the hairpin portion of the hairpins transposon end core only shows the transferred and non-transferred transposon termination sequences, and the loop is single-stranded (e.g., to minimize the likelihood or frequency of insertion of the hairpin end of the transposon end. double-stranded parts during in vitro transposition reaction). In some other embodiments, the hairpin transposon terminal sequence shows, in addition to the transposon ends transferred and not transferred, additional sequences that are directly 5'-transposed to the transposon end and directly to the 3 'non-ported sequence of the transposon ends. However, in these embodiments, the size of the additional sequences at the portion of the stem of the hairpin-like transposon end is minimized to minimize the likelihood or frequency of introducing the hairpin-like nailpod sequence of the transpomon during in vitro transposition reactions. For example, in some embodiments, the trunk length at the hairpin transposon end sequence is less than about 75 nucleotides; less than about 50 nucleotides; or less than about 30 nucleotides. For example, in some embodiments, the trunk length at the hairpin transposon end sequence is less than about 75 nucleotides; less than about 50 nucleotides; or less than about 30 nucleotides. For example, in some embodiments, the trunk length at the hairpin transposon end sequence is less than about 75 nucleotides; less than about 50 nucleotides; or less than about 30 nucleotides.

[0312] In some embodiments, the loop portion of the hairpin transposon end sequence has a sequence tag domain domain or capture tag domain (e.g., a sequencing tag domain and / or capture tag domain for a Roche 454 Genome Sequencer FLX genome sequencer system, e.g. they show the sequence sequences of the Roche 454A and 454B sequence tag domains used for sequencing using the Roche 454 Genome Sequencer FLX system). In some embodiments, in which a portion of the hairpin transposon end sequence has a sequencing tag domain or capture tag domain, tagged circular DNA fragments, or bluntly completed dsDNA fragments, or bi-tagged linear ssDNA fragments possess a tag that includes the sequencing tag domain and / or the capture tag domain (e.g. Roche 454A or 454B tag used for sequencing using the Roche 454 Genome Sequencer FLX sequencing system). After isolating tagged circular DNA fragments or blunt-ended dsDNA fragments or bi-tagged linear ssDNA fragments in the desired size range, they are used as the next generation sequencing standard using the Roche 454 Genome Sequencer FLX system. In other embodiments, generated tagged circular DNA fragments or blunt-ended dsDNA fragments or bi-tagged linear ssDNA fragments have one or more restriction site domain, sequencing tag domain, amplification tag domain, capture tag domain, detection and / or address tag domain (e.g. using the ROCHE 454 sequential platform, ILLUMINA ™ SOLEXA ™ sequential platform,

[0313] In some embodiments, a hairpin-like nail transposon end sequence has one or more tag domain sequences, which sequences may be used to achieve the desired purpose. There is no limit to which additional sequences are used for one or more additional sequences in the loop portion of the hairpin-like sequence of the hairpin transposon.

[0314] In some preferred embodiments, the 5'-end of the hairpin-like transposon end has a 5'-monophosphate group. In embodiments in which the hairpin-like sequence of the hairpin transposon ends lacks a 5'-monophosphate group, the method further comprises the step of phosphorylating the hairpin-like nail sequence of the hairpins (e.g., using a polynucleotide kinase, e.g., T4 polynucleotide kinase) before the ligation stage as part of the method.

[0315] The target transposase-catalyzed insertion of the transposon ends results in the 3 'end of the transposon end of the transposed transposition to the 5' position of the nucleotide in one strand of the target DNA, leading to rupture or fragmentation of the strand at the site where the transposon end of the transposed sequence is linked to target DNA and simultaneously producing a single-stranded region of a 9 base pair target DNA located at the 3'-position of the transposon end-to-target attachment site of the target DNA due to a 9 base pair negative region in the target strand of the target DNA. For example, FIG. 16 shows one possible outcome of two independent events for introducing a hairpin-like nailpipe sequence sequence into the target DNA. As shown in FIG. 16 the independent insertion of the hairpin-like ends of the hairpin into the opposite strands of the target DNA sometimes occurs at the target DNA sites, generating two 5 'tagged DNA fragments, as shown in FIG. 16. After attachment, a tagged round DNA fragment is generated.

[0316] The invention is not limited only to the nucleic acid ligases described herein. It will be understood by those of skill in the art that any nucleic acid ligase based on a standard that exhibits similar activity to the enzymes described herein, and methods and conditions for using these enzymes for reference-based ligation are known and are readily available.

EXPERIMENTAL EXAMPLES [0317] The present invention is further defined in the following Examples. It should be remembered that the Examples, while indicating preferred embodiments of the invention, are given for illustrative purposes only. Based on the foregoing discussion and Examples, those skilled in the art can determine the essential features of the present invention and, without departing from its scope and scope, may make various changes and modifications of the invention to suit different applications and conditions.

[0318] Standard molecular biology techniques are well known in the art and are described in Sambrook, J., Fritsch, EF and Maniatis, T., Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989).

Definitions, Naming and Abbreviations used in the Examples:

[0319] "pMETS" is a 19 base pairs transposon oligonucleotide containing a 5'-phosphate that has the EZ-Tn5 ™ transposon end sequence:

5 'pAGA TGT GTA TAA GAG ACAG 3' (SEQ ID NO: 1) 'METS' means oligonucleotide of a 19 base pairs transposon end that has the EZ-Tn5 ™ transposon terminal sequence:

5 'AGA TGT GTA TAA GAG ACAG 3' (SEQ ID NO: 1) 'pMENTS' is a 19 base pairs transposon oligonucleotide containing 5'-phosphate that has the sequence of the EZ-Tn5 ™ transposon ends:

5 'pCTG TCT CTT ATA CAC ATCT 3' (SEQ ID NO: 2) 'pMEDS' means the end of a double-stranded transposon with 19 base pairs of EZ-Tn5 ™, with both 5 'ends containing phosphates:

5 'pGA TGT GTA TAA GAG ACAG 3' (SEQ ID NO: 1)

3 'TCT ACA CAT ATT CTC TGTCp 5' (SEQ ID NO: 2) [0320] The end of the pMEDS EZ-Tn5 ™ transposon is produced by attaching the pMETS transposon oligonucleotide to the oligonucleotide of the pAYS transposon oligonucleotide.

[0321] "MEDS" means the end of a double stranded transposon with 19 base pairs of EZTn5 ™, with only the non-transferable strand (pMENTS) containing 5'-phosphate:

5 'AGA TGT GTA TAA GAG ACAG 3' (SEQ ID NO: 1)

3 'TCT ACA CAT ATT CTC TGTCp 5' (SEQ ID NO: 2) [0322] The end of the MEDS EZ-Tn5 TM transposon is produced by attaching the METS transposon oligonucleotide to the oligonucleotide of the pAYS transposon oligonucleotide.

[0323] "p454.1METS" is a 36 base pairs of 5'-phosphate containing a single-stranded transferring thread that includes a 5 'portion consisting of a Roche 454 sequencer tag showing the following sequence attached to the 5' end of the underlined transposon end of the sequence by 19 EZ-Tn5 ™ base pairs (pMETS):

5'pGCC TTG CCA GCC CGC TCA GAT GTG TAT AAG AGA CAG 3 '(SEQ ID NO: 4) [0324] The sequence of the "p454.1MEDS" EZ-Tn5 ™ transposon is created by attaching the transfer thread through p454.1METS (SEQ ID No. SEQUENCE: 4) to the untreated pMENTS strand (SEQ ID NO: 2):

5'pGCC TTG CCA GCC CGC TCA GAT GTG TAT AAG AGA CAG

3'T CTA CAC ATA TTC TCT GTCp '

[0325] "pc454.1" means a single-stranded oligonucleotide having a 18 base paired 5'-phosphate that is complementary to the 5 'part of p454.1METS and has the sequence:

5 'pTGA GCG GGC TGG CAA GGC 3' (SEQ ID NO: 5) [0326] &amp; quot; A-METS &amp; quot; is a 38-base single-stranded transfer thread that contains a 5 'portion consisting of a Roche 454 sequence tag showing the sequence below attached to the 5 'end of the underlined transposon end segment with 19 base pairs of EZ-Tn5 ™ (METS):

5 'GCC TCC CTC GCG CCA TCA GAG ATG TGT ATA AGA GAC AG 3' (SEQ ID NO: 7) [0327] The sequence of the "E-Tn5 ™" A-MEDS transposon ends is obtained by attaching the A-METS-transferred strand (NR SEQUENCE ID: 7) to the untranslated pMENTS strand (SEQ ID NO: 2):

5 'GCC TCC CTC GCG CCA TCA GAG ATG TGT ATA AGA GAC AG 3' 3'TC TAC ACA TAT TCT CTG TCp 5 '[0328] &amp; quot; B-METS &amp; quot; means a 38-basic single-strand transferring thread that includes a 5' part consisting of from the sequence tag Roche 454, showing the following sequence attached to the 5 'end of the 19-EZ-Tn5 ™ 19-base transposon underlined transposon sequence (METS):

5 'GCC TTG CCA GCC CGC TCA GAG ATG TGT ATA AGA GAC AG 3' (SEQ ID NO: 8) [0329] The sequence of the "B-MEDS" EZ-Tn5 ™ transposon ends is obtained by attaching the B-METS transfer thread (NR SEQUENCE ID: 8) to the untranslated pMENTS strand (SEQ ID NO: 2):

5 'GCC TTG CCA GCC CGC TCA GAG ATG TGT ATA AGA GAC AG 3' 3 'TC TAC ACA TAT TCT CTG TCp 5' [0330] 'FLX-A' is a 19 base pairs oligonucleotide consisting of the Roche 454 sequence tag which contains the following sequence:

5 'GCC TCC CTC GCG CCA TCA G 3' (SEQ ID NO: 9) [0331] &amp; quot; FLX-B &amp; quot; is a 19 base pairs oligonucleotide consisting of a Roche 454 sequence tag that includes the sequence below:

5 'GCC TTG CCA GCC CGC TCA G 3' (SEQ ID NO: 10) [0332] &amp; quot; A-MID2-METS &amp; quot; is a single stranded 48 base pairs which includes a 5 'portion consisting of the Roche 454 sequence tag and a bar code sequence (MID2, cursive), showing the following sequence attached to the 5 'end of the underlined transposon end of the 19 base pairs EZ-Tn5 ™ (METS):

5 'GCC TCC CTC GCG CCA TCA G ACGCTCGACA AG ATG TGT ATA AGA GAC AG 3' (SEQ ID NO: 11) [0333] &amp; quot; Ti A-METS &amp; quot; is a single stranded 49-pairs base-containing thread that includes a 5 'part consisting of from the sequence tag Roche 454, showing the following sequence attached to the 5 'end of the underlined transposon end of the 19 base pairs EZ-Tn5 ™ (METS):

5 'CCA TCT CAT CCC TGC GTG TCT CCG ACT CAG AGA TGT GTA TAA GAG ACA G 3' (SEQ ID NO: 12) [0334] &amp; quot; Ti B-METS &amp; quot; is a single strand transferable 49 base pairs which includes a 5 part 'consisting of the Roche 454 sequencer tag, showing the following sequence attached to the 5' end of the underlined transposon end segment with 19 EZ-Tn5 ™ base pairs (METS):

5 'CCT ATC CCC TGT GTG CCT TGG CAG TCT CAG AGA TGT GTA TAA GAG ACA G 3' (SEQ ID NO: 13) [0335] &amp; quot; Ti A &amp; quot; is a 26 base pairs 26-base oligonucleotide consisting of a Roche 454 sequencing tag, which contains the following sequence:

5 'CCA TCT CAT CCC TGC GTG TCT CCG AC 3' (SEQ ID NO: 14) [0336] &amp; quot; Ti B &amp; quot; is a 26 base pairs 26-base oligonucleotide consisting of a Roche 454 sequence tag that includes the sequence below:

5 'CCT ATC CCC TGT GTG CCT TGG CAG TC 3' (SEQ ID NO: 15) [0337] &amp; quot; BP1-A &amp; quot; is a 48 base pair oligonucleotide that comprises a 5 'portion consisting of an Illumina bridge sequence tag exhibiting the following sequence attached to the FLX-A sequence (underlined below):

5 'AAT GAT ACG GCG ACC ACC GAG ATC TAC ACG CCT CCC TCG CGC CAT CAG 3' (SEQ ID NO: 16) [0338] "BP2-A" is a 49 base pairs oligonucleotide that contains a 5 'part consisting of from the Illumina PCR bridge tag, showing the following sequence attached to the FLX-B sequence (underlined below):

5 'CAA GCA GAA GAC GGC ATA CGA GAT CGG TCT GCC TTG CCA GCC CGCTCAG 3' (SEQ ID NO: 17) [0339] "BP2-ID1-B" is a 49 base pairs oligonucleotide that contains a 5 'part consisting of from the Illumina PCR bridge tag and the barcode sequence (ID2, italics), showing the following sequence attached to the FLX-B sequence (underlined below):

5 'CAA GCA GAA GAC GGC ATA CGA GAT GCATGT CGG TCT GCC TTG CCA GCC CGC TCA G 3' (SEQ ID NO: 18) [0340] "BP1" means a 20 base pairs oligonucleotide consisting of an bPCR Illumina adapter tag, which contains the following sequence:

5 'AAT GAT ACG GCG ACC ACC GA 3' (SEQ ID NO: 19) [0341] "BP2" is a 21 base pairs oligonucleotide consisting of the bPCR Illumina adapter tag, which contains the sequence below:

5 'CAA GCA GAA GAC GGC ATA CGA 3' (SEQ ID NO: 20) [0342] &amp; quot; pMETS-N-MENTS &amp; quot; means the hairpin-like sequence of a hairpin transposon, comprising or consisting of: an oligonucleotide containing 5'-phosphate which shows the non-transferred sequence of the transposon ends of the non-seeded EZ-Tn5 ™ at the 5 'end and the transferred sequence of the EZ-Tn5 ™ transposon ends at the 3' end, joined by any intervening sequence shown as "(N) x". The intervening sequence between the METS and MENTS sequences consists of a sufficient number of nucleotides to allow formation of the parent loop:

5 'PCTGTCTCTTATACACATCT- (N) x-AGATGTGTATAAGAGACAG 3' (ID No

SEQ ID NO: 3) [0343] The intramolecular linkage of the transposed pMETS transposon endpoint to the non-transferring pTERS transposon peptide sequence in the pMETS-N-MENTS oligonucleotide, creating the EZ-Tn5 ™ hairpin terminal sequence of the EZ-Tn5 hairpin. For example, if x = 6;

NN

N AGATGTGTATAAGAGACAG 3 '

N TCTACACATATTCTCTGTCp 5 '

NN (SEQ ID NO: 3) [0344] "TSase" means the overactive transposase EZ-Tn5 ™ Tn5 (EPICENTER Biotechnologies, Madison, Wisconsin, USA) in 50 mM Tris pH 7.5, 50% glycerol, 0.1 mM EDTA, 1 mM DTT, 500 mM sodium chloride, 0.5% v / v NP-40, 0.5% v / v Tween-20. [0345] "Transposom" means the hyperactive transpozase of EZ-Tn5 ™ Tn5 (EPICENTER Biotechnologies, Madison, Wisconsin, USA) pre-incubated with double-stranded DNA transposon under conditions that support the formation of non-covalent complexes. The double-stranded DNA transposon may include, without limitation, Tn5 DNA, a portion of Tn5 DNA, a transposon sequence, a sequence of transposon ends or other double-stranded DNAs capable of interacting with the over-active EZTn5 ™ transposase.

10X reaction buffer TA 330 mM Tris acetate, pH 7.8

100 mM magnesium acetate 660 mM potassium acetate

5X TA-DMF reaction buffer: 165 mM Tris acetate, pH 7.8 mM magnesium acetate 330 mM potassium acetate 50% by volume dimethylformamide

10X TMgCl reaction buffer: 100 mM Tris chloride, pH 8.0 mM magnesium chloride

5X TMgCl-DMF reaction buffer: 50 mM Tris chloride, pH 8.0 mM magnesium chloride 50% by volume dimethylformamide

10X reaction buffer TMgAc: 100 mM Tris acetate, pH 7.6 mM magnesium chloride

5x TMgAc-DMF reaction buffer: 50 mM Tris acetate, pH 7.6 25 mM magnesium chloride 50% by volume dimethylformamide [0346] "Target DNA" means the DNA to be transposed. In the following example, bacteriophage T7D111 was used as the target DNA.

[0347] "TSase" means hyperactive transposase EZ-Tn5 ™ Tn5 (EPICENTRE

Biotechnologies, Madison, Wisconsin, USA).

[0348] 10X Transposase reaction buffer:

330 mM Tris acetate, pH 7.8 100 mM magnesium acetate 660 mM potassium acetate

EXAMPLE 1

DNA fragmentation mediated by Transposition in vitro, and 5'-Tagase using Transposase EZ-Tn5 ™ and Transpozon EZ-Tn5 ™ Terminals [0349] The following reaction mixture was formed:

x microliters micrograms microliters 2 microliters microliters of water to the final volume 50 microliters 10X EZ-Tn5 ™ Transpose buffer target DNA from 1 to 40 microliters pMEDS (25 micromolar) *

EZ-Tn5 ™ transposase (at 10 units per microliter) [0350] * In some embodiments, two different ends of the pMEDS transposon, each of which additionally exhibits another arbitrary sequence in its respective 5 'part of the transposon transposed end, 5' transposon transposition sequence (FIGURE 4).

[0351] After mixing, the reaction was incubated for 1 hour at 37 ° C. The reaction was stopped at 10 microHtrs retention solution (15% sucrose, 66 mM EDTA, 20 mM TRIS, pH 8.0, 0.1% SDS, 0.9% Orange G [Sigma O-7252] and proteinase K in 100 micrograms). on m ^, stirred and heated at 50 ° C for 10 minutes.

[0352] DNA was anaerated by agarose 1% agarose electrophoresis in TAE buffer. LMP agarose was used to induce DNA in the kksach zarodach. The bud was stained with SYBR Gok dye and the DNA was coupled with light of a frequency other than UV. The flocks that H LMP were incubated at 70 ° C for 5 minutes in the liquefaction agent. After 5 minutes at 37 ° C, one hundredth volume of Gekse ™ agarose digestion solution (EPICENTRE Biotechnokgies) was added. The reaction mixture was mixed and incubated for 1 hour at 37 ° C.

[0353] The DNA assay was fragmented in a similar range and at similar times as described in Examples 3 and 4, using comparisons and EZ-Tn5 ™ Tn5 transposase concentrations and transposon ends. The DNA from the sizing procedure was used in EXAMPLE 2 for tagging the 3 'ends and tagged 5 DNA fragments.

EXAMPLE 2

Size Range of Transposition Products Tagged with 5 'DNA Fragments Using Different Concentrations Ez-Tn5 ™ Tn5 Transposase.

[0354] Hyperactive Tn5 Ez-Tn5 ™ transposase (EPICENTRE) at a concentration of 90 units per microHtr was diluted to final concentrations of 45, 22.5, 11.3 and 9 units per microHtr. Two microcresses of the enzyme at each concentration were incubated with 1 microgram of DNA T7 D111 phage (approximately 39 Kbp) and 1 micromax of pMEDS transposon ends in TA buffer in a final volume of 50 microHtra reaction mixture for 1 hour at 37 ° C.

The reaction mixtures were stopped with 10 microHtra of a retaining solution containing 15% sucrose, 66 mM EDTA, 20 mM TRIS, pH 8.0, 0.1% SDS, 0.9% Orange G [Sigma O-7252] and proteinase K in 100 micrograms per ml · After mixing and incubating at 50 ° C for 10 min. aliquots of 10 microH samples were subjected to ecoprophoresis per 1% agarose in TAE buffer for 1 hour at 100 ° C. The gel was colored with SYBR Gok and photographed with the A340 transcriptizer.

[0356] A final concentration of approximately 0.9 units per Tn5 transposase microHtr in the reaction mixture resulted in maximal fragmentation of the DNA of the T7 D111 phage. Higher concentrations of Tn5 transposase inhibited and lower concentrations increased the fragment's size. At the final concentration of approximately 0.9 Tn5 transposase units per microHter, the majority of the T7 D111 phage DNA was subdivided into DNA that migrated to a gel between about 150 bp and about 1.5 Kbp on the basis of marker bands. At the final concentration of approximately 0.45 Tn5 transposase units per microHter, the majority of the T7 D111 phage DNA was subdivided into DNA that migrated to a gel between about 400 bp and about 3.5 Kbp, based on the marker bands.

EXAMPLE 3

Size Range of Transposition Products Tagged with 5 'DNA Fragments Using Different Concentration of PMEDS Transposons.

[0357] 25 micromoles of the pMEDS transposon ends were serially diluted 2, 4 and 8-fold with T10E1 buffer. Then, 2 microliters of each final transposon dilution and control of the buffer without transposon ends were incubated in 50 microliter reaction mixtures containing 1X TA buffer, 1 microgram of target 7 D111 phage DNA and 0.4 units on Tn5 hyperactive transposase microlitre for 1 hour at room temperature. 37 ° C.

[0358] The reactions were stopped and the samples were analyzed by electrophoresis in a 1% agarose gel as described in EXAMPLE 2.

[0359] A four-fold dilution of 25 μΜ stock that resulted in a final concentration of 0.25 micromolar pMEDS transposon ends in the reaction mixture resulted in good fragmentation of the target DNA and was probably the most efficient in the use of pMEDS transposon ends. At this concentration, most of the Target DNA of the T7 D111 phage was divided into DNA that migrated to a gel in the size between about 400 bp and about 3.5 Kbp based on the marker bands. At a concentration of 0.5 and 1 micromole of the pMEDS transposon end size divided into DNA fragments decreased slightly to about 200-300 bp and about 3 Kbp.

EXAMPLE 4

Size Range of Transposition Products Tagged with 5 'DNA Fragments Using Different Transposome Concentrations.

[0360] "A-MEDS transposomes" and "B-MEDS transposomes" were formed by pre-incubating 12.5 μΜ TSase with either 12.5 μΜ A-MEDS or 12.5 μΜ B-MEDS transposon sequence for 60 minutes in 37 ° C. The A-MEDS and B-MEDS transposomes were combined in equal proportions to form "Transposomes A / B".

[0361] The transposomes were then used at 12.5 μΜ or diluted to 10 μΜ,

7.5 μΜ, 5 μΜ, 2.5 μΜ or 1 μΜ with a storage buffer (50 mM Tris pH 7.5, 50% glycerol, 0.1 mM EDTA, 1 mM DTT, 500 mM sodium chloride, 0 , 5% v / v NP-40, 0.5% v / v Tween-20).

[0362] E. coli genomic DNA was tagged with 5 'and fragmented using A / B transposomes in the following reactions:

<td>Reagent</td><td>Volume</td>

<td>5X TMgCl-DMF reaction buffer</td><td>4 gl</td>

<td>50 ng / en E. coli genomic DNA</td><td>1 ml</td>

<td>Transposome A / B (12.5, 10, 7.5, 5, 2.5, or 1μΜ)</td><td>1 μl</td>

<td>water</td><td>14 μl</td>

<td>Final volume:</td><td>20 μΐ</td>

[0363] Reactions were incubated for 5 minutes at 55 ° C. The reaction mixtures were then stopped with 5 microliters of stop solution (15% sucrose, 66 mM EDTA, 20 mM TRIS, pH 8.0, 0.1% SDS, 0.9% Orange G [Sigma O-7252] and proteinase K in 100 micrograms per ml), mixed and heated at 70 ° C for 10 minutes.

[0364] DNA was analyzed by electrophoresis in a 1% agarose gel in TAE buffer. The gels were stained with SYBR Gold dye, and the DNA was visualized using light with a frequency other than UV.

[0365] The degree of fragmentation of the target DNA is proportional to the amount of transposome added to a 12.5-fold dilution of 12.5 μΜ transposome. At high transposome concentrations, most of the DNA fragments migrate in the gel in sizes below 1000 bp (Figure 5, lanes 3 and 9). At low concentrations of transposomes, the DNA fragments migrated in the gel mainly in sizes from 500 bp to 6000 bp (Figure 5, lanes 8 and 14). The block arrow indicates the migration of the free sequence of the transposon ends in the gel.

EXAMPLE 5

Target DNA fragmentation and 5 'Tagging at 55 ° C and 37 ° C in the Presence of Dimethylformamide.

[0366] To study the effect of dimethylformamide on target DNA fragmentation and 5 'tagging, HeLa genomic DNA was fragmented and tagged with ME transpomomes or A / B transposomes in the following manner.

[0367] "Transposomes ME" were formed by pre-incubation with 12.5 μM TSase z

12.5 μM MEDS transposon end sequence for 60 minutes at 37 ° C.

[0368] Duplicate reactions were set up as follows:

<td>Reagent</td><td>TA</td><td>TA-DMF</td><td>TMgCl</td><td>TMgCl-DMF</td>

<td>10X reaction buffer TA</td><td>2 μl</td><td>-</td><td>-</td><td>-</td>

(continued)

<td>Reagent</td><td>TA</td><td>TA-DMF</td><td>TMgCl</td><td>TMgCl-DMF</td>

<td>5X TA-DMF reaction buffer</td><td>-</td><td>4 μΐ</td><td>-</td><td>-</td>

<td>10X TMgCl reaction buffer</td><td>-</td><td>-</td><td>2 μΐ</td><td>-</td>

<td>5X TMgCl-DMF reaction buffer</td><td>-</td><td>-</td><td>-</td><td>4 μΐ</td>

<td>50 ng / μΐ HeLa genomic DNA</td><td>1 μΐ</td><td>1 μΐ</td><td>1 μΐ</td><td>1 μΐ</td>

<td>ME Transpozom (12,5 μΜ)</td><td>1 μΐ</td><td>1 μΐ</td><td>1 μΐ</td><td>1 μΐ</td>

<td>water</td><td>16 μΐ</td><td>14 μΐ</td><td>16 μΐ</td><td>14 μΐ</td>

<td>Final volume:</td><td>20 μΐ</td><td>20 μΐ</td><td>20 μΐ</td><td>20 μΐ</td>

[0369] "A-MEDS transposomes" and "B-MEDS transposomes" were formed by pre-incubating 12.5 μM TSase with 12.5 μM A-MEDS or 12.5 μM B-MEDS transposon end-end for 60 minutes, respectively. 37 ° C. The A-MEDS and B-MEDS transposomes were combined in equal proportions to form "Transposomes A / B".

[0370] Duplicate reactions were set up as follows:

<td>Reagent</td><td>TA</td><td>TA-DMF</td><td>TMgCl</td><td>TMgCl-DMF</td>

<td>10X reaction buffer TA</td><td>2 μΐ</td><td>-</td><td>-</td><td>-</td>

<td>5X TA-DMF reaction buffer</td><td>-</td><td>4 μΐ</td><td>-</td><td>-</td>

<td>10X TMgCl reaction buffer</td><td>-</td><td>-</td><td>2 μΐ</td><td>-</td>

<td>5X TMgCl-DMF reaction buffer</td><td>-</td><td>-</td><td>-</td><td>4 μΐ</td>

<td>50 ng / μΐ HeLa genomic DNA</td><td>1 μΐ</td><td>1 μΐ</td><td>1 μΐ</td><td>1 μl</td>

<td>MA / B Transpozom (12.5 μM)</td><td>1 μΐ</td><td>1 μΐ</td><td>1 μΐ</td><td>1 μΐ</td>

<td>water</td><td>16 μΐ</td><td>14 μΐ</td><td>16 μΐ</td><td>14 μΐ</td>

<td>Final volume:</td><td>20 μΐ</td><td>20 μΐ</td><td>20 μΐ</td><td>20 μΐ</td>

[0371] Reactions were incubated for 5 minutes at 37 ° C and for 5 minutes at 55 ° C. The reaction mixtures were stopped at 5 microliters of stop solution (15% sucrose, 66 mM EDTA, 20 mM TRIS, pH 8.0, 0.1% SDS, 0.9% Orange G [Sigma O-7252] and proteinase K in 100 micrograms per ml), mixed and heated at 70 ° C for 10 minutes.

[0372] DNA was analyzed by electrophoresis in a 1% agarose gel in TAE buffer. The gels were stained with SYBR Gold dye, and the DNA was visualized using light with a frequency other than UV.

[0373] Dimethylformamide improved the efficiency of 5 'fragmentation and tagging reactions, as judged by the decrease in the distribution of the MW reaction products (Figure 6, compare tracks 4, 6, 8, 10, 13, 15, 17, and 19 with paths 3, respectively), 5, 7, 9, 12, 14, 16, and 18). Similarly, reactions in the presence of a TMgCl reaction buffer were more efficient than reactions in the presence of a TA reaction buffer. Finally, reactions at 55 ° C appeared to improve overall reaction efficiency compared to reactions at 37 ° C (Figure 6, compare lanes 4-10 with lanes 12-19). The block arrow indicates the migration of the free end of the transposon to the gel.

EXAMPLE 6

Determination of Target DNA Fragmentation Using the MuA Transposase.

[0374] HyperMu ™ MuA transposase (EPICENTRE) at a final concentration of 1 unit per microliter followed by a concentration range of MuA transposase proteins between about 0.01 micrograms and about 0.5 micrograms protein per microliter of the reaction mixture was incubated in 50 microliters of reaction mixture containing buffer for the MuA transposase reaction (EPICENTRE), 1 microgram of Target T7 D111 Target DNA and 1 micromole of MuR transpylone pR1R2 for 1 hour at 37 ° C.

[0375] The reaction was stopped and the products were analyzed by agarose gel electrophoresis as described in EXAMPLE 3.

[0376] Fragmentation of the target DNA of T7 D111 phage was much less than observed using the EZ-Tn5 ™ Tn5 transposase at all levels of the MuA transposase under test. A very small fragmentation scale was observed only at the highest concentration of the tested MuA transposase. Thus, the use of MuA transposase and MuA pR1R2 transposon ends was significantly less effective in the 5 'fragmentation and tagging of target DNA than EZ-Tn5 ™. Hyperactive Tn5 transposase and end of the EZTn5 ™ Tn5 ME transposon.

EXAMPLE 7

Tagged Ends 3 'and Tagged 5' DNA Fragments

A. Two-way PCR [0377] In order to tag the 3 'ends of the DNA fragments produced by transposition and tagged 5' DNA fragments with the transferred sequence of the transposon ends, the following reaction is carried out:

microliters selected in terms of 0.5-1 Kbp tagged

100

5 'transpose products Failsafe ™ PCR PreMix C DNA polymerase FailSafe ™ (EPICENTRE) 5 micromic microliters of each oligonucleotide PCR primer, one of which is complementary to the 5' part of each of the 5 'portions of the two different transposon end portions.

microliters of the total volume of the reaction mixture [0378] Since the FailSafe DNA polymerase exhibits the property of displacing the 5 'nuclease and nuclease, the polymerization of the method is done by incubating the reaction mixture for 10 minutes at 70 ° C (3' DNA polymerase extension step), thereby generating tagged 5'- and 3'- DNA fragments (FIG 8).

[0379] The reaction is then incubated at 94 ° C for 5 minutes to denature the DNA.

[0380] Amplification of 5'- and 3 'tagged DNA fragments is accomplished by PCR amplifying 5'- and 3'-tagged DNA fragments using two PCR primers, each of which is complementary to the 5' part of one of the two different transferred transposon ends.

[0381] The above PCR reaction mixture is subjected to PCR for 20 cycles with the following cyclic change conditions:

94 ° C 10 sec.

55 ° C 10 sec.

72 ° C 2 min.

[0382] Gel analysis showed that PCR products are being produced with the expected size range (0.5 - 1 Kbp).

[0383] Control reactions are also performed: If the size-transposed transposition products are subjected to thermal denaturation before PCR and without the 3 'DNA polymerase extension step, PCR products with 0.5-1 Kbp size are not produced.

B. Single PCR primer [0384] In order to tag the 3 'ends of the fragments generated by transposition and tagged 5' ME tagged fragments in the ME sequence, the following reaction is performed:

Microliters microliters 1 microliters 1 microliters microliters selected in size 0.5-1 Kbp tagged 5 'transposition products

Failsafe ™ PCR PreMix C

FailSafe ™ DNA Polymerase (EPICENTRE)

5-micromolar pMETS as oligonucleotide primer

PCR [0385] Since FailSafe DNA polymerase has the property of displacing the 5 'nuclease and nuclease, the method has been carried out by incubating the reaction mixture through

101 minutes at 70 ° C (the 3 'DNA polymerase extension step), thereby generating tagged 5' and 3 'DNA fragments (FIGURE 7).

[0386] Subsequently, the reaction mixture was incubated at 94 ° C for 5 minutes to denature the DNA.

[0387] Amplification of 5'- and 3 'tagged DNA fragments was performed by PCR amplifying 5'- and 3'-tagged DNA fragments using pMET as the only oligonucleotide PCR primer. The above PCR reaction mixture was subjected to PCR for 20 cycles with the following conditions of cyclic changes:

94 ° C 10 sec.

55 ° C 10 sec.

72 ° C 2 min.

[0388] Gel analysis showed that PCR products were produced with the expected size range (0.5 - 1 Kbp).

[0389] Control reactions are also performed: If the size-transposed transposition products were subjected to thermal denaturation before PCR and without the 3 'DNA polymerase extension step, PCR products 0.5-1 Kbp were not produced.

EXAMPLE 8

Amplification and Deep Sequencing of the DNA Fragments Library [0390] To generate a nonselective library of DNA fragments that can be amplified prior to library preparation, DNA fragments were made and tagged at the 3 'end and the 5' end, using "ME transpozomes".

[0391] "ME transposomes" were formed by pre-incubating 10 μΜ of TSase with 10 μΜ of the MEDS transposon end sequence for 10 minutes on ice.

43 kb cosmid DNA was fragmented and tagged with 5 'in the following reaction:

<td>Reagent</td><td>Volume</td>

<td>10X reaction buffer TA</td><td>5 μl</td>

<td>142 ng / g 43 kb Cosmid DNA</td><td>7 gl</td>

<td>ME Transpozom (10 μM)</td><td>5 gl</td>

<td>water</td><td>33 gl</td>

<td>Final volume:</td><td>50 μΐ</td>

[0392] The reaction was incubated for 2 hours at 37 ° C. An additional 5 μl of 10 μM ME Transposome was added to the reaction and incubated for an additional 2 hours at 37 ° C.

[0393] To tag the 3 'ends of the DNA fragments generated by transposition and tagged 5' DNA fragments with the transposon end of the transferred sequence, the reaction products were incubated with a mixture of thread transfer polymerase (FailSafe ™) and dNTP. [0394] A portion of the DNA fragments generated in transposition and tagged 5 'were diluted 1:10 before tagging the 3' ends and amplification to characterize the amplification of 4 ng of the DNA library template. For non-selective amplification of the entire population of tagged DNA fragments using a single PCR primer, the following reaction was performed with the METS PCR primer that hybridized only to the transposon end sequences and did not contain additional information about the 3 'sequence.

102

<td>Reagent</td><td>Volume</td>

<td>2X FailSafe ™ PCR Buffer E</td><td>12.5 gl</td>

<td>tagged 5 'DNA fragments (diluted 1:10)</td><td>2 gl</td>

<td>METS PCR starter (25 gM)</td><td>1 gl</td>

<td>FailSafe ™ PCR enzyme, 2.5 U / g</td><td>1 gl</td>

<td>water</td><td>8.5 gl</td>

<td>Final volume:</td><td>25 μΐ</td>

[0395] The reaction was incubated as follows:

• 72 ° C / 2: 00 * • 98 ° C / 1: 00 • 25 cycles (98 ° C / 0: 10, 55 ° C / 0: 10, 72 ° C / 1: 00) • 4 ° C holding [0396] * - To tag the 3 'ends of the DNA fragments generated by transposition and tagged 5' with the transposon end of the transferred sequence, the reaction products were incubated with a mixture of thread transfer polymerase (FailSafe ™) and dNTPs prior to the denaturation step (see FIG 7).

[0397] Amplified and unamplified reaction products were purified using the QIAGEN PCR-Clean-up cleaning column according to the manufacturer's instructions and used as material for step 3.4 of the standard Roche / 454 FLX preparation protocol according to the manufacturer's instructions (USM00048.A, October 2008 r.).

[0398] Deep sequencing of libraries of elements fragmented by transposons produced a single contig of the expected size with the reading length, accuracy and range comparable to the control library generated by nebulization (Figure 9). These data are consistent with non-selective and massively parallel amplification of the DNA fragment library.

EXAMPLE 9

Preparation of Sequential Libraries with Barcodes Compatible with Roche / 454 FLX by Addition of Additional 5 'and 3' Sequence Information with PCR with Adapter Oligonucleotides [0399] To generate a barcode DNA fragment library that can be used directly in emPCR for sequencing the 454 GS FLX, genomic lambda DNA was fragmented and tagged with 5 'ME transposomes. The non-selective adapter oligonucleotides were used to attach the DNA library during PCR to 454 FLX emPCR and the sequencing adapter and barcode sequence (FIGURE 10).

[0400] "Transposomes ME" were formed by pre-incubation 12.5 μM TSase z

12.5 μM MEDS transposon end sequence for 60 minutes at 37 ° C.

[0401] The lambda genomic DNA was fragmented and tagged with 5 'in the following reaction:

<td>Reagent</td><td>Volume</td>

<td>10X reaction buffer TA</td><td>5 gl</td>

103

<td>500 ng / g Lambda DNA</td><td>2 gl</td>

<td>ME Transpozom (12.5 gM)</td><td>2 gl</td>

<td>water</td><td>39 gl</td>

<td>Final volume:</td><td>48 μΐ</td>

[0402] The reaction was incubated for 2 hours at 37 ° C. An additional 2 gl was added to the reaction

12.5 gM ME Transposome and incubated for an additional 2 hours at 37 ° C. [0403] The reaction products were purified using the QIAGEN PCR-Clean-Up column according to the manufacturer's instructions.

[0404] For the purpose of non-selective amplification and attachment of a library of DNA fragments to adapters compatible with emPCR Roche / 454 FLX and sequencing, PCR was performed using adapter oligonucleotides that hybridize to the sequence of the transposon ends and do not contain additional information about the 3 'sequence (FIGURE 10) .

<td>Reagent</td><td>Volume</td>

<td>2X FailSafe ™ PCR buffer</td><td>25 gl</td>

<td>tagged 5 'DNA fragments (20 ng / ml)</td><td>0.5 gl</td>

<td>A-MID2-METS PCR primer (2.5 gM)</td><td>1 gl</td>

<td>B-METS PCR primer (2.5 gM)</td><td>1 gl</td>

<td>PCR FLX-A primer (50 gM)</td><td>1 gl</td>

<td>PCR FLX-B primer (50 gM)</td><td>1 gl</td>

<td>FailSafe ™ PCR enzyme, 2.5 U / g</td><td>1 gl</td>

<td>water</td><td>24.5 degrees</td>

(continued)

<td>Reagent</td><td>Volume</td>

<td>Final volume:</td><td>50 μΐ</td>

[0405] The reaction was incubated as follows:

• 72 ° C / 5: 00 * • 98 ° C / 2: 00 • 4 cycles (98 ° C / 0: 10, 37 ° C / 0: 30, 72 ° C / 3:00) • 6 cycles (98 ° C / 0: 10, 64 ° C / 3: 00) • 4 ° C holding [0406] * - For tagging the 3 'ends of the DNA fragments made by transposition and tagged with the transposon tagged sequence, the reaction products were incubated with with a mixture of thread transfer polymerase (FailSafe ™) and dNTPs prior to the denaturation step (see FIG. 7).

[0407] Control reactions omitted the A-MID2-METS and B-METS PCR primers and contained 20 ng tagged 5 'DNA fragments.

[0408] The PCR reaction produced an emPCR compatible library with the expected MW distribution and was similar to the library of 5 'DNA fragments produced by transposition and tagged (Figure 11, lanes 3 and 4). The lack of detectable amplification products in reaction mixtures without adapter primers (A-MID2-METS and B-METS) is

104 consistent with the specific amplification of the FLX-A and FLX-B tagged DNA library (Figure 11, lane 5).

EXAMPLE 10

Deep Sequencing of Roche / 454 FLX Titanium Corresponding Library with 50 ng cDNA of Viral Amplon [0409] To generate a library of non-selective DNA fragments that can be used directly in the emPCR for the 45X GS FLX Titanium sequencing, the DNA amplicon was fragmented and tagged 'ME transposomes. The non-selective adapter oligonucleotides were used to attach the DNA library to Roche / 454 FLX Titanium emPCR and sequencing the adapter sequence (FIGURE 10).

[0410] "ME transposomes" were prepared by pre-incubating 12.5 μΜ TSase with 12.5 μΜ of the MEDS transposon end sequence for 60 minutes at 37 ° C. Stocks were diluted to 7.5 μΜ in TSase storage buffer (50 mM Tris pH 7.5, 50% glycerol, 0.1 mM EDTA, 1 mM DTT, 500 mM sodium chloride, 0.5% v / v NP- 40, 0.5% v / v Tween-20).

[0411] The virus cDNA amplicon was fragmented and tagged with 5 'in the following reaction:

<td>Reagent</td><td>Volume</td>

<td>10X reaction buffer TA</td><td>5 μΐ</td>

<td>Viral cDNA amplicon 9.4 ng ^ L</td><td>5.5 μΐ</td>

<td>ME Transpozom (7.5 μΜ)</td><td>1 μΐ</td>

<td>water</td><td>38.5 μΐ</td>

<td>Final volume:</td><td>50 μΐ</td>

[0412] The reaction was incubated for 15 minutes at 55 ° C. An additional 1 μl 7.5 μΜ of ME Transposome was added to the reaction and incubated for an additional 15 minutes at 55 ° C.

[0413] The reaction products were purified using a QIAGEN PCR-Clean-Up column according to the manufacturer's instructions using two combined elutions of 11 μΐ.

[0414] For the purpose of non-selective amplification and attachment of a library of DNA fragments to adapters compatible with emPCR Roche / 454 FLX Titanium and sequencing, PCR was performed using adapter oligonucleotides that hybridize to the sequence of the transposon ends and do not contain additional information about the 3 'sequence (FIGURE 10) ).

<td>Reagent</td><td>Volume</td>

<td>2X PCR FailSafe ™ Buffer</td><td>25 μΐ</td>

(continued)

<td>Reagent</td><td>Volume</td>

<td>Tagged 5 'DNA fragments (~ 20 μΐ recovered)</td><td>5 μΐ</td>

<td>PCR Starter Ti A-METS (0.5 pM)</td><td>1 μΐ</td>

<td>PCR primer Ti B-METS (0.5 pM)</td><td>1 μΐ</td>

105

<td>PCR Starter Ti A (10 pM)</td><td>1 μΐ</td>

<td>Starter Ti B (10 μΜ)</td><td>1 μΐ</td>

<td>FailSafe ™ PCR enzyme, 2.5 U / μΐ</td><td>1 μΐ</td>

<td>water</td><td>15 μΐ</td>

<td>Final volume:</td><td>50 μΐ</td>

[0415] The reaction was incubated as follows:

• 72 ° C / 5: 00 * • 98 ° C / 2: 00 • 4 cycles (98 ° C / 0: 10, 55 ° C / 0: 30, 72 ° C / 3:00) • 6 cycles (98 ° C / 0: 10, 64 ° C / 3: 00) • 4 ° C holding [0416] * - To tag the 3 'ends of the DNA fragments made by transposition and tagged with 5' with the transposon end of the transfer sequence, the reaction products were incubated with with a mixture of thread transfer polymerase (FailSafe ™) and dNTPs prior to the denaturation step (see FIG. 7).

[0417] The PCR reaction produced an emPCR compatible library with the expected MW distribution and was similar to the library of 5 'DNA fragments produced by transposition and tagged (Figure 12, lanes 2 and 3).

[0418] Deep library sequencing on the 1/8 Roche / 454 FLX Titanium plate provided about 80,000. readings with approximation of approx. 95% of readings to the reference image of the virus genome with the expected range (data not shown). These data are consistent with non-selective and massively parallel amplification of the DNA fragment library.

EXAMPLE 11

Preparation of Sequential Libraries with Barcodes Compatible with

Illumina GAII by Addition of Additional 5 'and 3' Sequence Information by PCR with Adapter Oligonucleotides [0419] To generate a barcode DNA fragment library that can be used directly in the bPCR for Illumina GAII sequencing, genomic lambda DNA was fragmented and tagged with 5 'A / B transposomes. The non-selective adapter oligonucleotides were used to attach the DNA library to the bPCR Illumina GAII adapter and barcode sequence (FIG 13).

[0420] "A-MEDS transposomes" and "B-MEDS transposomes" were formed by pre-incubation 12.5 μΜ TSase with either 12.5 μΜ A-MEDS or 12.5 μΜ B-MEDS transposon sequence for 60 minutes in 37 ° C.

[0421] The lambda genomic DNA was fragmented and tagged with 5 'in the following reaction:

<td>Reagent</td><td>Volume</td>

<td>10X reaction buffer TA</td><td>5 μΐ</td>

<td>500 ng / μΐ Lambda DNA</td><td>2 μΐ</td>

<td>A-MEDS transposom (12.5 μΜ)</td><td>2 μΐ</td>

<td>B-MEDS transposom (12.5 μΜ)</td><td>2 μΐ</td>

106

<td>water</td><td>39 gl</td>

<td>Final volume:</td><td>50 μΐ</td>

[0422] The reaction was incubated for 2 hours at 37 ° C. An additional 2 μl was added to the reaction

12.5 gM ME Transposome and 2 μl 12.5 μM B-MEDS Transposome and incubated for an additional 2 hours at 37 ° C.

[0423] The reaction products were purified using the QIAGEN PCR-Clean-Up column according to the manufacturer's instructions.

[0424] For the purpose of non-selective amplification and attachment of a library of DNA fragments to adapters compatible with Illumina GAII PCR bridge and sequencing, PCR was performed using adapter oligonucleotides that hybridize to the sequence of the transposon ends and do not contain additional information about the 3 'sequence (FIG 13).

<td>Reagent</td><td>Volume</td>

<td>2X FailSafe ™ PCR buffer</td><td>25 gl</td>

<td>tagged 5 'DNA fragments (20 ng / ml)</td><td>0.5 gl</td>

<td>PCR primer BP1-A (0.5 gM)</td><td>1 gl</td>

<td>PCR primer BP2-ID1-B (0.5 gM)</td><td>1 gl</td>

<td>PCR BP1 primer (10 gM)</td><td>1 gl</td>

<td>BP2 PCR primer (10 gM)</td><td>1 gl</td>

<td>FailSafe ™ PCR enzyme, 2.5 U / g</td><td>1 gl</td>

<td>water</td><td>24.5 degrees</td>

<td>Final volume:</td><td>50 μΐ</td>

[0425] The reaction was incubated as follows:

• 72 ° C / 5: 00 * • 98 ° C / 2: 00 • 10 cycles (98 ° C / 0: 10, 58 ° C / 0: 30, 72 ° C / 3:00) • 4 ° C holding [0426] * - For tagging the 3 'ends of the DNA fragments generated by transposition and tagged 5' with the transferred transposon sequence, the reaction products were incubated with a thread transfer polymerase mixture (FailSafe ™) and dNTPs before the denaturation step (see FIG 8).

[0427] Control reactions omitted BP1-A and BP2-ID1-B PCR primers and contained 20 ng tagged 5 'DNA fragments.

[0428] The PCR reaction produced a library compatible with bPCR with the expected MW distribution and was similar to the library of 5 'DNA fragments produced by transposition and tagged (Figure 14, lanes 3 and 4). The lack of detectable amplification products in reaction mixtures lacking adapter primers (BP1-A and BP2-ID1-B) is consistent with the specific amplification of tagged library BP1 and BP2 (Figure 14, lane 5).

EXAMPLE 12

Deep Sequencing of the ILUMINA GAII Consecutive Library

[0429] As to the generation of bibHunctions of non-specific DNA fragments that can be used directly in the bPCR for IHumin GAII sequencing, genomic E. coH CC118 DNA was fragmented and tagged with 5 'A / B transposomes. The non-selective oHgonucocotides of the adapter were used to attach the bibHoteki DNA to the bPCR Illumina GAII adapter (FIG 13).

E. coH CC11 genomic DNA was fragmented, tagged and purified as described in EXAMPLE D2.

[0430] In order to inactively amplify and add bibHoteka DNA fragments to the IHumina GAII-companion PCR adapter and sequencing, PCR was performed using oHgonucleotides of the adapter that hybridize to the sequence of the transposon ends and do not contain additional information about the 3 'sequence.

<td>Reagent</td><td>Volume</td>

<td>2X PCR E FaHSafe ™ Buffer</td><td>25 μΐ</td>

<td>tagged 5 'DNA fragments (20 ng / μΐ)</td><td>0.5 μΐ</td>

(dakzy string)

<td>Reagent</td><td>Volume</td>

<td>PCR primer BP1-A (0.5 pM)</td><td>1 μΐ</td>

<td>BP2-B PCR primer (0.5 pM)</td><td>1 μΐ</td>

<td>BP1 PCR primer (10 pM)</td><td>1 μΐ</td>

<td>BP2 PCR primer (10 pM)</td><td>1 μΐ</td>

<td>The FaHSafe ™ PCR enzyme, 2.5 U / μΐ</td><td>1 μΐ</td>

<td>water</td><td>24.5 μΐ</td>

<td>Final volume:</td><td>50 μΐ</td>

[0431] The reaction was incubated as follows:

• 72 ° C / 5: 00 * • 98 ° C / 2: 00 • 10 cyclesH (98 ° C / 0: 10, 58 ° C / 0: 30, 72 ° C / 3:00) • 4 ° C holding [0432] * - At the tagging of the 3 'ends of the DNA fragments generated by transposition and tagged 5' with the transferred transposon sequence, the reaction products were incubated with the thread transfer poWder mixture (FaHSafe ™) and dNTPs prior to the denaturation step (see FIG 8).

[0433] Deep sequencing of the generated bibHoteki reached a genome reach of approximately 4.6 Mb with an average depth of ~ 115X (data not shown). These data are consistent with the non-inert and massively uniform amphibition of the bibHoteka DNA fragments, which is a compatib with emPCR Roche / 454 FLX Titanium and sequencing.

EXAMPLE 13

Comparison of Methods of State of Technology with Embodiments of Invention and

108

Set Protocols.

[0434] The workflow and the term of preparing the library of tagged DNA fragments by the methods provided for in the present invention are compared with the workflow and the timing of preparation of such libraries using the usual methods. A table comparing the process steps and the time needed in each step is shown in Figure 15. The methods of the present invention require fewer steps, less time spent directly in the procedures, and generally less time.

EXAMPLE 14

DNA fragmentation mediated by In vitro transposition and Tagging using Transposase EZ-Tn5 ™ and the EZ-Tn5 ™ Transposon Tip Sequence in the Shape of Hair Clips [0435] The following ingredients were mixed to form transposomes ™ hair clips transposomes ( i.e., transposase hairpin transposon end frame with transposase) at a final concentration of 25 micromolar (see FIG 16):

microliters microliters 63 microliters 100 microliters

EZTn5 ™ Hairpin End-to-end Transpozon sequence (250 micromolar)

EZ-Tn5 ™ transposase (91.4 micromolar)

Transposase Storage Buffer [0436] The following reaction mixture was formed:

x microliters of water to the final volume of 50 microliters 10X EZ-Tn5 ™ Transposition buffer micrograms of target DNA from 1 to 40 microliters

0, 1, 2, 4, or 6 microliters 25 micromolar shaped transposomes

Hairpin Transposomes ™ hairpin clips microliters [0437] After mixing, the reaction was incubated for 2 hours at 37 ° C. The reaction was stopped at an equal volume of stop solution (15% sucrose, 66 mM EDTA, 20 mM TRIS, pH 8.0, 0.1% SDS, 0.9% Orange G [Sigma O-7252] and proteinase K in 100 micrograms). per ml), stirred and heated at 70 ° C for 10 minutes.

[0438] DNA was analyzed by electrophoresis in a 1% agarose gel in TAE buffer. The gels were stained with SYBR Gold dye, and the DNA was visualized using light with a frequency other than UV.

[0439] Fragmentation of the target DNA is proportional to the amount of Transposome added (FIG 17).

EXAMPLE 15

The tagged circular DNA fragments are resistant to T5 exonuclease. [0440] The 5 'tagged DNA fragments of EXAMPLE 14 were isolated using PCR Clean-up NucleoTraP®CR (Macherey-Nagel, GmbH) according to the manufacturer's instructions. IN

For the production of tagged circular DNA fragments, the recovered DNA was incubated with a non-multimeric polymerase with no 5'-to-3 exonuclease activity (e.g., T4 DNA polymerase) and a standard-dependent ligase (e.g., E. coli ligase) in the presence of dNTPS and β- ABOVE.

[0441] The following reaction mixture was formed:

microliters 2 microliters 1 microliters 1 microliters 1 microliters 1 microliters 20 microliters

Tagged 5 'DNA Fragments from Example 1 10X EZ-Tn5 ™ Transposition Buffer 2 mM β-NAD mM dNTPs

E. coli DNA ligation (10 U / g)

T4 DNA polymerase (0.5 U / g) [0442] The reaction was incubated for 15 minutes at ambient temperature. The reactions were stopped by incubating 20 minutes at 75 ° C. Part of the reaction (10 gl) was incubated with 10 T5 exonuclease units for 5 minutes at 37 ° C to degrade the linear (non-circulated) DNA fragments.

[0443] All reaction mixtures were treated with 2 g retention solution (15% sucrose, 66 mM EDTA, 20 mM TRIS, pH 8.0, 0.1% SDS, 0.9% Orange G [Sigma O7252]), mixed and heated at 70 ° C for 5 minutes.

[0444] DNA was analyzed by electrophoresis in a 1% agarose gel in TAE buffer. The gels were stained with SYBR Gold dye, and the DNA was visualized using light with a frequency other than UV.

[0445] Treatment with T4 DNA polymerase and E. coli ligase transformed part of the DNA fragments into particles of T5 exonuclease-tolerant DNA fragments that are easily detected (FIG 18, lane 9). In addition, the molecular weight distribution of T5 exonuclease-resistant DNA is comparable to the introduced DNA, indicating no molecular weight deviation in this reaction.

[0446] Control reactions are also performed: When DNA fragments were not treated, treated with T4 DNA polymerase alone or treated with E. coli alone, no tagged fragments of T5 exonuclease-resistant DNA fragments were detected (Figure 18, lanes 3, 5 and 7 ).

EXAMPLE 16

Tagged 3 'Ends generated by Transposition of 5' Tagged DNA Fragments using Nucleic Acid Ligase and Oligonucleotide Labeling Ligation.

[0447] The 5 'tagged divided into DNA fragments from the above dimensioning procedure (Example 1) was used to tag the 3' ends and tagged 5 DNA fragments using a nucleic acid ligase and a ligation marking oligonucleotide (FIG 21).

[0448] To tag the 3 'ends of the fragments produced by transposition and tagged 5' fragments with a second tag containing the Roche 454 sequence tag (4N454B), the following reaction was performed.

microliters of 0.5-1 Kb selected 5'-labeled DNA fragments EXAMPLE 1 5 microliters 10X Ligase Reaction Buffer (0.2 M Tris-HCl pH 8.3, 100 mM MgCk, 250 mM KCl, 5 mM β- NAD) micromole of oligonucleotide 4N454B

110 (5 'pNNNNCTGAGCGGGCTGGCAAGGC 3' (SEQ ID NO: 6)) 1 microliter E. coli DNA ligase (10 units per microl) [0449] After mixing, the reaction was incubated for 1 hour at room temperature. Then the reaction was stopped, Hgase was inactivated and the DNA denatured by incubation at 95 ° C for 5 minutes. After that, the reaction mixture was immediately cooled in a water bath with fod.

[0450] An anaerobe PCR was performed in cebi showing that the 5 'ends of the DNA fragments displayed the transposed EZ-Tn5 transposon sequence and the 3' ends exhibited the Roche 454 sequence tag (4N454B).

[0451] The following PCR reaction was performed as follows:

microHtra 1 microliter microliter 1 microliter double tagged water 5 'and tagged 3' DNA fragments

5-micromolar Starter 1 PCR (pMETS) 5-micromolar Starter 2 PCR (5 'ATA GGC GCG CCG CCT TGC CAG CCC GCT CAG 3' 0 (SEQ ID NO: 21) microliter 25 microHiters 50 microHtra

PoHmerase DNA FaHSafe ™ FaHSafe ™ 2X PCR PreMix C [0452] PCR was performed in 20 CM in the following conditions:

94 ° C 10 sec.

55 ° C 10 sec.

72 ° C 2 min.

[0453] AnaHza-rib has shown that PCR products have been made with an expected range of 0.5-1 Kbp (data not shown).

[0454] Control reactions were also carried out. When the binding reaction was performed without Hgase bib without the Roche 454 sequencing tag (4N454B), no PCR products were generated. When PCR primer 1 bib primer 2 PCR pMETS was omitted in the PCR reaction, no 0.5-1-KB products were obtained. When the bending reaction was performed without hgase bib without the Roche 454 sequencing tag (4N454B), no PCR products were generated. When PCR primer 1 bib PCR primer 2 PCR pMETS was omitted from the PCR reaction, no 0.5-1KB products were obtained (data not shown).

EXAMPLE 17

Circulation of Tagged ssDNA Fragments from DNA Inhibition mediated by Transposition in vitro, and 5 'Tagging using the Transposon End of p454.1MEDS and Transposase EZ-Tn5 ™. [0455] T7 D111 genomic DNA was tagged with 5' and fragmented using sequences the transposon ends of p454MEDS EZ-Tn5 ™ in the following reaction:

x water to the final volume of 50 microHtra microHtra 10X EZ-Tn5 ™ Transpose buffer 1 microgram docef DNA in the HEP from 1 to 40 microHtra

111 microliters. Transposon end of p454.1MEDS (25 pM) 2 microliters Transposase EZ-Tn5 ™ (at 10 units per myrroleine) _ microHeests Final volume of the reaction mixture [0456] After mixing, the reaction was incubated for 1 hour at 37 ° C. The reaction mixture was then stopped at 10 microliters of stopping solution (15% sucrose, 66 mM EDTA, 20 mM TRIS, pH 8.0, 0.1% SDS, 0.9% Orange G [Sigma O-7252] and proteinase K in HCI). 100 micrograms per m 2, mixed and heated at 50 ° C for 10 minutes.

[0457] The DNA was ana? Elled by the ecotrophoresis method in 1% agarose in TAE buffer. The bud was stained with SYBR Gok dye and the DNA was coupled with light of a frequency other than UV.

[0458] The DNA attachment was subjected to fragmentation in a similar range and in similar amounts as described in Example 3, using comparisons of the EZ-Tn5Tn5 transposase and transposon Tn5 transposon and the pMEDS transposon ends (FIG 22B, lane 5). The data indicate that extension of oHgo pMETS with the additional Roche 454 sequence tag does not significantly affect the efficiency of DNA fragmentation and tagging by EZ-Tn5 ™ transposase. Omission of the transposon end of the EosTn5 ™ transposase transposon end of the p384.1MEDS graft did not trigger detectable DNA fragmentation (FIG.22, lanes 3 and 4).

[0459] The 5 'tagged fragmented DNA of FIG. 22, lane 5 was subjected to thermal denaturation and circulation with a non-pattern Hgase (see FIG 22A) in the following reaction:

x microliter 1 microliter 4 microliters 10 microHit microliters 20 microHertz water to final volume 20 microHtra

330 mM Tris acetate pH 7.8, 660 mM KOAc mM MnCl₂

M betaine ί ^ / ηΗ denatured 5'-tagged fragment into DNA fragments

100 U / μΐ ssIR HLase CIRCLIGASE ™ (EPICENTRE) Final volume of the reaction mixture [0460] The reaction mixture was incubated for 2 hours at 60 ° C. Thereafter, the reaction products were treated with 18 Exo I units and 20 Exo III units for 1 hour at 37 ° C in a non-rounded, Hni's DNA uptake.

EXAMPLE 18

PCR analysis of Round Labeled ssDNA [0461] Anaerobic PCR was performed using pMETS and pc454.1 as primers for demonstrating the circulatoryization; Only a rounded ssDNA can be amp-rated to generate a dsDNA linear product corresponding to the round shape of ssDNA. The PCR reaction was carried out as follows:

mikroHtrów 1 microliter microliter 1 microliter water

Reaction mix of CircLigase treated with exonuccinase (1: 1000) pM oHgonuckotide pMETS as 5 pM primer oHgonucleotide pc454.1 as primer

112 microliters FailSafe ™ DNA polymerase FailSafe ™ 2X PCR PreMix C_ microliters Final volume of the reaction mixture [0462] PCR was performed in 29 cycles under the following conditions:

94 ° C 10 sec.

50 ° C 10 sec.

72 ° C 1 min.

[0463] Gel analysis showed that the size range of PCR products produced was comparable to the 5'-tagged DNA fragment (FIG 23).

[0464] Control reactions were also carried out. When the CIRCLIGASE ™ ssDNA ligase was bypassed in the ligation reaction, PCR products were generated that indicated the circularization of the p454.1METS transferred strand but did not point to the 5 'tagged linear ssDNA fragments (FIG 23, lane 1). When the pMETS oligonucleotide (as PCR primer) or PC454.1 PCR primer was omitted in the PCR reaction, no products were obtained (data not shown).

[0465] The fact that the PCR products had the same size distribution as the 5 'tagged fragments of the linear ssDNA (see Figure 22, lane 5 and Figure 23, lane 2) indicates that: 1) the transposon sequence of the p454.1MEDS can efficiently tag 5 'and divided into DNA target fragments; 2) attached complementary tagged 5 'linear ssDNA fragments can be thermally denominated to yield denatured tagged linear ssDNA fragments that are substrates for libraries that are independent of the standard; and 3) the tagged 5 'linear ssDNA fragments can be efficiently transformed into tagged circular ssDNA fragments without detectable loading (confirmed by PCR amplification after exonuclease I and exonuclease III treatment).

SEQUENCE LIST [0466] &lt;110&gt; Epicentre Technologies Corporation Jendrisak, Jerome Dahl, Gary

Grunenwald, Haiying Li Caruccio, Nicholas &lt;120&gt; Transposon Tip Sequences and Modes of Nucleic Acid Modification &lt;130&gt; EPICE-30705 / US-2 / ORD &lt;140&gt; US 12 / 605,337 &lt;141&gt; 2009-10-24 &lt;150&gt; US 61 / 108,321 &lt;151&gt; 2008-10-24 &lt;150&gt; US 61 / 108,326 &lt;151&gt; 2008-10-24

113 &lt;150&gt; US 61 / 108,329 &lt;151&gt; 2008-10-24 &lt;150&gt; US 61 / 155,431 &lt;151&gt; 2009-02-25 &lt;150&gt; US 61 / 184,530 &lt;151&gt; 2009-06-05 &lt;160&gt; 21 &lt;170&gt; PatentIn version 3.5 &lt;210&gt; 1 &lt;211&gt; 19 &lt;212&gt; DNA &lt;213&gt; Artificial sequence &lt;220&gt;

&lt;223&gt; Synthetic &lt;400&gt; 1 agatgtgtat aagagacag 19 &lt;210&gt; 2 &lt;211&gt; 19 &lt;212&gt; DNA &lt;213&gt; Artificial sequence &lt;220&gt;

&lt;223&gt; Synthetic &lt;400&gt; 2 ctgtctctta tacacatct 19 &lt;210&gt; 3 &lt;211&gt; 44 &lt;212&gt; DNA &lt;213&gt; Artificial sequence &lt;220&gt;

&lt;223&gt; Synthetic &lt;220&gt;

&lt;221&gt; misc_feature &lt;222&gt; (20) .. (25) &lt;223&gt; n is a, c, g, or t &lt;400&gt; 3 ctgtctctta tacacatctn nnnnnagatg tgtataagag acag

114 &lt;210&gt; 4 &lt;211&gt; 36 &lt;212&gt; DNA &lt;213&gt; Artificial sequence &lt;220&gt;

&lt;223&gt; Synthetic &lt;400&gt; 4 gccttgccag cccgctcaga tgtgtataag agacag &lt;210&gt; 5 &lt;211&gt; 18 &lt;212&gt; DNA &lt;213&gt; Artificial sequence &lt;220&gt;

&lt;223&gt; Synthetic &lt;400&gt; 5 tgagcgggct ggcaaggc 18 &lt;210&gt; 6 &lt;211&gt; 23 &lt;212&gt; DNA &lt;213&gt; Artificial sequence &lt;220&gt;

&lt;223&gt; Synthetic &lt;220&gt;

&lt;221&gt; misc_feature &lt;222&gt; (1) .. (4) &lt;223&gt; n is a, c, g, or t &lt;400&gt; 6 nnnnctgagc gggctggcaa ggc 23 &lt;210&gt; 7 &lt;211&gt; 38 &lt;212&gt; DNA &lt;213 &gt; Artificial sequence &lt;220&gt;

&lt;223&gt; Synthetic &lt;400&gt; 7 gcctccctcg cgccatcaga gatgtgtata agagacag &lt;210&gt; 8

115 &lt;211&gt; 38 &lt;212&gt; DNA &lt;213&gt; Artificial sequence &lt;220&gt;

&lt;223&gt; Synthetic &lt;400&gt; 8 gccttgccag cccgctcaga gatgtgtata agagacag &lt;210&gt; 9 &lt;211&gt; 19 &lt;212&gt; DNA &lt;213&gt; Artificial sequence &lt;220&gt;

&lt;223&gt; Synthetic &lt;400&gt; 9 gcctccctcg cgccatcag 19 &lt;210&gt; 10 &lt;211&gt; 19 &lt;212&gt; DNA &lt;213&gt; Artificial sequence &lt;220&gt;

&lt;223&gt; Synthetic &lt;400&gt; 10 gccttgccag cccgctcag 19 &lt;210&gt; 11 &lt;211&gt; 48 &lt;212&gt; DNA &lt;213&gt; Artificial sequence &lt;220&gt;

&lt;223&gt; Synthetic &lt;400&gt; 11 gcctccctcg cgccatcaga cgctcgacaa gatgtgtata agagacag &lt;210&gt; 12 &lt;211&gt; 49 &lt;212&gt; DNA &lt;213&gt; Artificial sequence &lt;220&gt;

&lt;223&gt; Synthetic

116 &lt;400&gt; 12 ccatctcatc cctgcgtgtc tccgactcag agatgtgtat aagagacag &lt;210&gt; 13 &lt;211&gt; 49 &lt;212&gt; DNA &lt;213&gt; Artificial sequence &lt;220&gt;

&lt;223&gt; Synthetic &lt;400&gt; 13 cctatcccct gtgtgccttg gcagtctcag agatgtgtat aagagacag &lt;210&gt; 14 &lt;211&gt; 26 &lt;212&gt; DNA &lt;213&gt; Artificial sequence &lt;220&gt;

&lt;223&gt; Synthetic &lt;400&gt; 14 ccatctcatc cctgcgtgtc tccgac 26 &lt;210&gt; 15 &lt;211&gt; 26 &lt;212&gt; DNA &lt;213&gt; Artificial sequence &lt;220&gt;

&lt;223&gt; Synthetic &lt;400&gt; 15 cctatcccct gtgtgccttg gcagtc 26 &lt;210&gt; 16 &lt;211&gt; 48 &lt;212&gt; DNA &lt;213&gt; Artificial sequence &lt;220&gt;

&lt;223&gt; Synthetic &lt;400&gt; 16 aatgatacgg cgaccaccga gatctacacg cctccctcgc gccatcag &lt;210&gt; 17 &lt;211&gt; 49 &lt;212&gt; DNA &lt;213&gt; Artificial sequence

117 &lt;220&gt;

&lt;223&gt; Synthetic &lt;400&gt; 17 caagcagaag acggcatacg agatcggtct gccttgccag cccgctcag 49 &lt;210&gt; 18 &lt;211&gt; 55 &lt;212&gt; DNA &lt;213&gt; Artificial sequence &lt;220&gt;

&lt;223&gt; Synthetic &lt;400&gt; 18 caagcagaag acggcatacg agatgcatgt cggtctgcct tccagcccg ctcag &lt;210&gt; 19 &lt;211&gt; 20 &lt;212&gt; DNA &lt;213&gt; Artificial sequence &lt;220&gt;

&lt;223&gt; Synthetic &lt;400&gt; 19 aatgatacgg cgaccaccga 20 &lt;210&gt; 20 &lt;211&gt; 21 &lt;212&gt; DNA &lt;213&gt; Artificial sequence &lt;220&gt;

&lt;223&gt; Synthetic &lt;400&gt; 20 caagcagaag acggcatacg a 21 &lt;210&gt; 21 &lt;211&gt; 30 &lt;212&gt; DNA &lt;213&gt; Artificial sequence &lt;220&gt;

&lt;223&gt; Synthetic &lt;400&gt; 21 ataggcgcgc cgccttgcca gcccgctcag

118