De novo enzymatic production of nucleic acid molecules

0001The present invention is related to methods for the manufacture of a nucleic acid molecule.

0002<i>De novo</i> generation of nucleic acid molecules is increasingly used in biopharmaceutical research to replace the often quite complex cloning procedures necessary to produce desired DNA constructs with optimised properties, e.g. high level protein expression in suitable in <i>vivo</i> or <i>in vitro</i> systems. There are a variety of methods known to synthesise such DNA molecules. Practically all of these procedures rely on the synthesis, annealing and subsequent ligation of synthetic single-stranded oligonucleotides to assemble larger double-stranded DNA molecules that typically consist of more than one hundred up to several thousand base pairs. However, the efficiency of these methods is limited by several factors: (i) the quality of the oligonucleotides used, (ii) the size of the desired construct and (iii) the proportion of "difficult" sequences, e.g. those with self-complementary regions, high GC content, G tetrads, DNA kinks or repetitive sequence blocks. The oligonucleotide building blocks themselves are contaminated with various termination products and internal deletions. Especially problematic are n-1 products (oligonucleotides containing internal one nucleotide deletions occurring as a result of incomplete capping reactions), which can hardly be separated from the desired full-length oligonucleotide. As many oligonucleotides have to be assembled in order to generate a complete gene, the probability of obtaining an error-free clone, i.e. not incorporating even one defective oligonucleotide with a base change or an internal deletion approaches 0%. For example, if a gene were assembled from fifty oligonucleotides each having a purity of 90%, the probability of creating an error-free product would be roughly 0.9⁵⁰ = 0.005. Generally, tedious error correction procedures must be employed in order to obtain a 100% error-free construct. In many cases, defective synthesis products cannot be tolerated because mistakes in the coding sequence may cause the generation of shortened transcription or translation products due to e.g. a frame shift of the open reading frame. Whereas the first two problems can be alleviated by the use of oligonucleotides of very high purity, the formation of unwanted secondary structures that may cause deletions in the synthesis product can in many cases only be suppressed if alterations are allowed in the DNA sequence.

0003In the prior art a variety of methods are known to produce synthetic DNA. More than 20 years ago, the pioneering work of Khorana and colleagues (Sekiya et al., 1979) demonstrated the complete <i>de novo</i> synthesis of a suppressor tRNA gene via ligation of pairs of annealed oligonucleotides. In this and related methods, complementary single-stranded oligonucleotides comprising the complete desired DNA sequence are annealed in pairs to yield double-stranded fragments, which are aligned in the correct order by virtue of complementary single-stranded overhangs (Stabinsky, <patcit id="pcit0001" dnum="US4652639A"><text>US patent 4,652,639</text></patcit>). The resulting fragments are then ligated either sequentially or in a one-tube-reaction (Jayaraman, <patcit id="pcit0002" dnum="US5132215A"><text>US patent 5,132,215</text></patcit>) either enzymatically or chemically. After purification and/or cloning these gene fragments may be joined together to form larger DNA constructs. In the so-called "cassette synthesis", each pair of annealed oligonucleotides is separately cloned in a plasmid vector before joining the fragments using restriction endonucleases (Richards et al., <patcit id="pcit0003" dnum="US5093251A"><text>US patent 5,093,251</text></patcit>).

0004Alternatively, DNA constructs can be assembled from partially annealed oligonucleotides, which after hybridisation contain single-stranded gaps that must be filled by DNA polymerases; this method is commonly referred to as "gap filling" method. According to this method a variety of partially overlapping oligonucleotides are synthesised, purified and subsequently hybridised usually in pairs or in subgroups. After the synthesis of the respective opposite strands using a DNA polymerase the individual fragments are ligated to each other. The double stranded ligation products generated in this way may be either cloned as partial fragments or amplified in a polymerase chain reaction (PCR) with terminal oligonucleotide primers. However, this method is plagued by frequent mispriming events and internal deletions due to the formation of secondary structures.

0005Both methods are of limited use as with increasing length of the nucleic acid molecule to be synthesised the probability increases that one or several oligonucleotides with an incorrect sequence will be incorporated into the final product. Such errors are then copied in the DNA polymerase reaction. In addition, sequence errors may also be introduced during the PCR reaction.

0006A combination of the above methods is described in <patcit id="pcit0004" dnum="US6472184B"><text>US patent 6,472,184</text></patcit> in which a series of linkable oligonucleotides representing adjoining regions in one strand of the target sequence are hybridised with non-linkable oligonucleotides that are complementary to the 3' or 5' ends of the linkable oligonucleotides that are to be connected. This method is relatively simple and straightforward but is also plagued by the common problems shared by all procedures that use single-stranded oligonucleotides as building blocks: the formation of unwanted secondary structures and the incorporation of n-x oligonucleotides, which both lead to internal deletions.

0007Besides these standard procedures, there are further methods known in the art for the production of synthetic DNA molecules. International patent application <patcit id="pcit0005" dnum="WO9815567A"><text>WO 98/15567</text></patcit> and <patcit id="pcit0006" dnum="US6110668A"><text>US patent 6,110,668</text></patcit> teach a template-directed method of coupling oligonucleotides to yield synthetic DNA constructs by ligating a plurality of oligonucleotides that are at least partially complementary to the single-stranded template DNA and the ends of said oligonucleotides are ligated in the correct order in successive annealing and denaturation steps. However, a precondition for the application of this method is the prior existence of a suitable template DNA excluding its use in <i>de novo</i> synthesis.

0008International patent application <patcit id="pcit0007" dnum="WO9947536A"><text>WO 99/47536</text></patcit> discloses a solid phase gene synthesis method in which single-stranded oligonucleotides are sequentially ligated to an immobilised starter molecule in a defined orientation. A disadvantage of this method is that many steps are required to synthesise larger genes resulting in reduced yield and enrichment of defective sequences. Also, this method is difficult to automate which is a prerequisite for a rapid, standardised synthesis.

0009<patcit id="pcit0008" dnum="WO0075368A"><text>International patent application WO 00/75368</text></patcit> discloses a combinatorial solid phase synthesis of nucleic acids using a library of double-stranded oligonucleotides as standardised building blocks. The use of standardised building blocks makes it unnecessary to synthesise a new set of oligonucleotides for each new synthesis. These double-stranded library oligonucleotides generally share an identical overall structure and thus avoid common synthesis problems caused by the formation of alternative secondary structures of the oligonucleotide building blocks such as the introduction of deletions. In one preferred version, they contain a terminal loop, a double-stranded stem and a short single-stranded overhang. There are two different classes of library oligonucleotides, which are characterised by the presence of different recognition sites for type IIS restriction enzymes within their sequence and the presence or absence or the type of an internal modification. The nucleotides in the overhang and the directly adjacent region form the variable portion that actually contributes to the nucleic acid to be synthesize d; the remaining sequence is generally identical in all oligonucleotides belonging to the same class.

0010To build up a double-stranded nucleic acid, its sequence is first broken down into smaller fragments (usually between 6 and 30 base pairs each). These so-called elongation blocks are then synthesised in parallel reactions. In one such reaction, two double-stranded library oligonucleotides, one of each class, are ligated via matching single-stranded overhangs. The ligation products thereof are subsequently cleaved with the type IIS restriction enzyme, which is specific for the oligonucleotide that donates nucleotides. The net effect of such a ligation/restriction cycle is the addition of a small number of base pairs (typically between one to five) to the starting oligonucleotide. This process is then repeated until the synthesis of the desired elongation block is completed.

0011In a second reaction phase, the so-called transposition, those elongation blocks that are adjacent in the nucleic acid to be synthesised are ligated in a pair wise fashion after each block has been cleaved with a different type IIS restriction enzyme. By repeating this procedure several times the length of the transposition intermediates doubles in each step whereas the number of reactions is cut in half. Thus a defined nucleic acid molecule can be generated in very few cycles. The advantage of this method resides in the combinatorial pair wise assembly of the fragments of the nucleic acid molecule to be synthesised, in a sequence independent manner. Any desired elongation block may thus be generated from a standardised nucleic acid library with a defined number of elements.

0012The number of the elements of such a library depends on the length of the overhangs generated by the individual type IIS restriction enzyme as well as the number of nucleotides that are added to the growing oligonucleotides in each elongation cycle.

0013This method offers a number of advantages: it can be completely automated since there is no need to synthesise and purify new oligonucleotides to build large genes or DNA fragments, the building blocks are prepared in a large scale and can be used to assemble many different constructs until the supply is used up thus reducing the cost for oligonucleotides by one to two orders of magnitude. However, an inherent disadvantage of this method is the fact that the individual intermediates to be ligated in a pair wise fashion may be produced in different yield. In some cases, the resulting uneven stoichiometry of the ligation partners may lead to the formation of unwanted side products that can further decrease the yield of subsequent ligations.

0014International patent application <patcit id="pcit0009" dnum="WO030044193A"><text>WO 03/0044193</text></patcit> (<patcit id="pcit0010" dnum="EP0213154W"><text>PCT/EP02/13154</text></patcit>) is related to methods for the production of a nucleic acid linking various partial sequences in a sequence-independent manner.

0015The problem underlying the present invention is to provide a method for the manufacture of a nucleic acid molecule which allows for an increased yield and/or a nucleic acid molecule having a more accurate sequence compared to the methods according to the prior art.

0016This any other problems are solved by the subject matter of the independent claims. Preferred embodiments may be taken from the dependent claims.

0017In an embodiment of the method according to the first aspect the single-stranded overhang of the capping oligonucleotide, or part thereof; is essentially complementary to the partial sequence of the further oligonucleotide, or part thereof.

0018In an embodiment of the method according to the first aspect the ligation reaction between the further oligonucleotide and the capping oligonucleotide is preferred to the ligation of the further oligonucleotide and the first and second oligonucleotide, respectively.

0019In an embodiment of the method according to the first aspect the capping oligonucleotide is not ligating to the first and second oligonucleotide in the ligation reaction.

0020In an embodiment of the method according to the first aspect the capping oligonucleotide is contained in the ligation reaction in excess, preferably 2-10fold.

0021In an embodiment of the method according to the first aspect the capping oligonucleotide comprises a loop structure, preferably a loop structure at the end opposite to the single-stranded overhang.

0022More specifically, according to the present invention the problem is solved in a first aspect by a method for the manufacture of a nucleic acid molecule, comprising the steps of <ol id="ol0001" compact="compact"><li>a) providing a first at least partially double-stranded oligonucleotide which comprises a recognition site for a first type IIS restriction enzyme which cuts outside its recognition site, and which oligonucleotide comprises a single-stranded overhang;</li><li>b) providing a second at least partially double-stranded oligonucleotide which comprises a modification allowing the oligonucleotide to be coupled to a surface, whereby the oligonucleotide further comprises a recognition site for a second type IIS restriction enzyme which cuts outside its recognition site, and which second oligonucleotide comprises a single-stranded overhang;</li><li>c) ligating the first and the second oligonucleotide, via their overhangs generating a first ligation product;</li><li>d) immobilising the first ligation product on a surface via the modification contributed by the second oligonucleotide;</li><li>e) cutting the immobilised first ligation product with the second type IIS restriction enzyme thus releasing an elongated first oligonucleotide having an overhang and a shortened second oligonucleotide, which remains bound to the surface;</li><li>f) providing a further at least partially double-stranded oligonucleotide which has a modification allowing the further oligonucleotide to be specifically coupled to a surface, whereby the oligonucleotide contains a recognition site for a second or a further type IIS restriction enzyme and a single-stranded overhang which is complementary to the overhang of the elongated first oligonucleotide;</li><li>g) ligating the further at least partially double-stranded oligonucleotide with the elongated first oligonucleotide via their overhangs generating a second level ligation product;</li><li>h) cutting the second level ligation product with the second or further type IIS restriction enzyme thus generating a second level elongated oligonucleotide having an overhang and a shortened further oligonucleotide;</li><li>i) immobilising the shortened further oligonucleotide;</li><li>j) repeating steps f) to i) at least once, generating in step g) a higher level ligation product, whereby in the last repetition the incoming further oligonucleotide comprises a recognition site for a <u>type IIS</u> restriction enzyme which upon cleavage produces a single-stranded overhang identical in length to the overhang generated by the <u>first type IIS restriction</u> enzyme specific for the first oligonucleotide, and steps h) and i) are replaced by the steps k) and l);</li><li>k) immobilising the higher level ligation product via the modification provided by the further oligonucleotide; and</li><li>l) cutting the higher level ligation product with the further type IIS restriction enzyme, leaving the part of the nucleic acid to be manufactured attached to the first oligonucleotide, which is preferably released into the supernatant, and more preferably allowing its transfer to a new reaction vessel.</li></ol>

0023More specifically, according to the present invention the problem is solved in a second aspect by a method for the manufacture of a nucleic acid molecule, comprising the steps of <ol id="ol0002" compact="compact"><li>a) providing a first at least partially double-stranded oligonucleotide which comprises a recognition site for a first type IIS restriction enzyme which cuts outside its recognition site, and which oligonucleotide comprises a single-stranded overhang;</li><li>b) providing a second at least partially double-stranded oligonucleotide which comprises a modification allowing the oligonucleotide to be coupled to a surface, whereby the oligonucleotide further comprises a recognition site for a second type IIS restriction enzyme which cuts outside its recognition site, and which second oligonucleotide comprises a single-stranded overhang;</li><li>c) ligating the first and the second oligonucleotide via their overhangs generating a first ligation product;</li><li>d) immobilising the first ligation product on a surface via the modification contributed by the second oligonucleotide;</li><li>e) cutting the immobilised first ligation product with the second type IIS restriction enzyme thus releasing an elongated first oligonucleotide having an overhang and a shortened second oligonucleotide, which remains bound to the surface;</li><li>f) providing a further at least partially double-stranded oligonucleotide which has a modification allowing the further oligonucleotide to be specifically coupled to a surface, whereby the oligonucleotide contains a recognition site for a second or further type IIS restriction enzyme and a single-stranded overhang which is complementary to the overhang of the elongated first oligonucleotide;</li><li>g) ligating the further at least partially double-stranded oligonucleotide with the elongated first oligonucleotide via their overhangs generating a second level ligation product;</li><li>h) cutting the second level ligation product with the second or further type IIS restriction enzyme thus generating a second level elongated oligonucleotide having an overhang and a shortened further oligonucleotide;</li><li>i) immobilising the shortened further oligonucleotide;</li><li>j) repeating steps f) to i) at least once, generating in step g) a higher level ligation product, whereby in the last repetition the incoming further oligonucleotide comprises a recognition site for a type IIS restriction enzyme which upon cleavage produces a single-stranded overhang identical in length to the overhang generated by the first type IIS restriction enzyme specific for the first oligonucleotide, and steps h) and i) are replaced by the steps k) and l)</li><li>k) immobilising the higher level ligation product via the modification provided by the further oligonucleotide; and</li><li>l) cutting the immobilised higher level ligation product with the type IIS restriction enzyme specific for the first oligonucleotide, leaving the part of the nucleic acid to be manufactured attached to the further oligonucleotide, which is immobilised on a surface.</li></ol>

0024In an embodiment of the method according to the first and/or second aspect as step m) the cut immobilised higher level ligation product of step l) of the method according to the second aspect of the present invention is ligated with the cut higher level ligation product of step l) of the method according to the first aspect of the present invention.

0025In an embodiment of the method according to the first and/or second aspect the cut higher level ligation product of step l) is cleaved with the second type IIS restriction enzyme prior to the ligation step m).

0026In an embodiment of the method according to the first and/or second aspect the number of repetitions in step j) is two, three, four, five or six.

0027In an embodiment of the method according to the first and/or second aspect the overhang is a 5' or a 3' overhang.

0028In an embodiment of the method according to the first and/or second aspect the overhang is selected from the group comprising a one nucleotide overhang, a two nucleotides overhang, a three nucleotides overhang, a four nucleotides overhang, and a five nucleotides overhang.

0029In an embodiment of the method according to the first and/or second aspect the at least partially double-stranded oligonucleotide comprises a constant region and a variable region, whereby the constant region contains the recognition site for a type IIS restriction enzyme, and the variable region contains a nucleic acid sequence which corresponds to a part of the nucleic acid sequence of the nucleic acid molecule to be manufactured.

0030In an embodiment of the method according to the first and/or second aspect the further type IIS restriction enzyme is the second type IIS restriction enzyme.

0031In an embodiment of the method according to the first and/or second aspect the elongated oligonucleotide is transferred to a different reaction vessel.

0032In an embodiment of the method according to the first and/or second aspect the second level elongated oligonucleotide is transferred to a different reaction vessel.

0033In an embodiment of the method according to the first and/or second aspect the second level elongated oligonucleotides is used as the elongated oligonucleotide in step g).

0034In an embodiment of the method according to the first and/or second aspect the modification of the elongated oligonucleotide and/or of the higher level ligation product is provided by the further at least partially double-stranded oligonucleotide.

0035The present inventors have surprisingly found that the design of building blocks which are used in combinatorial synthesis of a nucleic acid using a library of double-stranded oligonucleotides as standardised building blocks such as described in international patent application <patcit id="pcit0011" dnum="WO0075368A"><text>WO 00 /75368</text></patcit> or international patent application <patcit id="pcit0012" dnum="EP0311551W"><text>PCT/EP03/11551</text></patcit>, allows for a further optimisation of the synthesis of nucleic acid molecules using this kind of synthesis strategy. Although the various embodiments of said combinatorial solid phase synthesis has proven a valuable tool for providing nucleic acid molecules, the need for an even more efficient synthesis using less chemicals, more particularly less amounts of enzymes, still exists. This need is met by the methods and tools for carrying out such methods.

0036Type IIS restriction enzymes as used in connection with any aspect of the present invention are preferably outside cutters, i.e. restriction enzymes which are characterised by the fact that they interact with two discrete sites of a double-stranded DNA. One of said two sites is the non-palindromic recognition site for said restriction enzyme which typically has a length of four to seven base pairs. The other site is the cleavage site which is typically zero to twenty base pairs apart from the recognition site. The recognition sites of the restriction enzymes are either completely or partially asymmetric. It is a preferred feature of outside cutters that their cleavage site is beyond, i.e. outside their recognition site. As used herein in preferred embodiments, the at least partially double-stranded oligonucleotides comprise a recognition site for one of at least two different outside cutters which may be either completely or partially part of the oligonucleotide. To allow for the proper functioning of the method according to the present invention, the type IIS restriction enzyme the recognition site of which is contained in the first and further at least partially double-stranded oligonucleotide, respectively, and which is also referred to herein as the first type IIS restriction enzyme, respectively, and the type IIS restriction enzyme the recognition site of which is contained in the second at least partially double-stranded oligonucleotide and which is also referred to herein as the second or further type IIS restriction enzyme, must be different.

0037The following table provides some possible combinations of recognition sequences of type IIS restriction enzymes. <tables id="tabl0001" num="0001"><table frame="all"><tgroup cols="2"><colspec colnum="1" colname="col1" colwidth="91mm" /><colspec colnum="2" colname="col2" colwidth="71mm" /><thead><row><entry valign="top"><b>Recognition site for first oligonucleotide</b></entry><entry valign="top"><b>Recognition site for second oligonucleotide</b></entry></row></thead><tbody><row><entry>CGTCTCN^NNNN_(Esp3I, BsmBI) (SEQ.ID.No1)</entry><entry>GGTCTCN^NNNN_(BsaI, Eco31I...)</entry></row><row><entry>GGTCTCN^NNNN_(BsaI, Eco31I,..) (SEQ.ID.No. 2)</entry><entry>CGTCTCN^TTNNN_(Esp3I, BsmB1)</entry></row><row><entry>GAAGACNN^NNNN_(BbsI, BpiI...) (SEQ.ID.No.3)</entry><entry>ACCTGCNNNN^NNNN_(BspMI, Acc36I)</entry></row><row><entry>ACCTGCNNNN^NNNN_(BspMI, Acc36I) (SEQ.ID.No.4)</entry><entry>GAAGACNN^NNNN_(BbsI, BpiI...)</entry></row><row><entry>GCAGTG_NN^ (BtsI) (SEQ.ID.No.5)</entry><entry>GCAATG_NN^ (BsrDI, Bse3DI, ..)</entry></row><row><entry>GCAATG_NN^ (BsrDI, Bse3DI, ..) (SEQ.ID.No.6)</entry><entry>GCAGTG_NN^ (BtsI)</entry></row><row><entry>GTATCCNNNNN_N^ (BciVI, BfuI) (SEQ.ID.No.7)</entry><entry>ACTGGGNNNN_N^ (BfiI, BmrI)</entry></row><row><entry>ACTGGGNNNN_N^ (BfiI, BmrI) (SEQ.ID.No.8)</entry><entry>GTATCCNNNNN_N^ (BciVI, BfuI)</entry></row><row><entry>GGCGGANNNNNNNNN_NN^ (EciI) (SEQ.ID.No.9)</entry><entry>GAGGAGNNNNNNNN_NN^ (BseRI)</entry></row><row><entry>GAGGAGNNNNNNNN_NN^ (BseRI) (SEQ.ID.No.10)</entry><entry>GGCGGANNNNNNNNN_NN^ (EciI)</entry></row><row><entry>CACCTGCNNNN^NNNN_ (AarI) (SEQ.ID.No.11)</entry><entry>CAGCTCNNNNNNN^NNN_ (AceIII)</entry></row><row><entry>CAGCTCNNNNNNN^NNN_ (AceIII) (SEQ.ID.No.12)</entry><entry>CACCTGCNNNN^NNNN_ (AarI)</entry></row><row><entry>GCTCTTCN^NNN_ (SapI) (SEQ.ID.No.13)</entry><entry>- (adapter linker necessary)</entry></row><row><entry>CTCTTCN^NNN_ (Eam1104I, Ksp6321, EarI) (SEQ.ID.No.14)</entry><entry>- (adapter linker necessary)</entry></row></tbody></tgroup></table></tables> whereby N = any of the nucleotides A, G, C or T; ^ is the cleavage site in the upper strand, i. e. 5'->3' from left to right, and - the cleavage site in the lower strand, i. e. 5'->3' from right to left.

0038Preferred combinations of the first and second (and further) type IIS restriction enzyme to be used in connection with the present invention are Eco31I/Esp3I (37°C), BsaI/BsmBI (50°C), BsmBI/BsaI (55°C), BbsI/BspMI (3-7°C), BspMI/BbsI (37°C) BsrDI/BtsI (65°C), BtsI/BsrDI (37°C), BciVI/BmrI (37°C), AarI/AceIII (37°C), EciI/BseRI (37°C) und BmrI/BciVI (37°C). Temperatures in brackets indicate the incubation temperatures used for each of the pairs. The isoschizomers of these enzymes (BsaI: Bso31, Eco31I; BsmBI: Esp3I; BbsI: BpiI, BpuAI; BspMI: Acc36I; BsrDI:Bse3DI, BseMI; BmrI: BfiI) are potential alternatives.

0039It is within a preferred embodiment of any aspect of the present invention that any of the second and further oligonucleotides having an at least partially double-stranded structure, a single-stranded overhang and a recognition site for a type IIS restriction enzyme generally comprises at least one nucleotide which can be cleaved off from the oligonucleotide upon digestion with said restriction enzyme. More preferably, such nucleotide(s) are part of a nucleic acid to be manufactured.

0040As used herein, the expression 'that the modification allows for a specific binding' preferably means that the binding occurs only under certain circumstances or reaction conditions but avoids an unintended binding of the molecule comprising such modification. Because of this, depending on the reaction conditions or circumstances existing or realized, the modification comprising molecule can bind to an interaction partner or not. Preferably such binding partner is attached to a surface, more preferably a solid phase. Any modification which provides for these characteristics can be used as a modification which allows for a specific binding. This kind of modification can be a modification as described herein or in any of international patent applications <patcit id="pcit0013" dnum="DE0001863W"><text>PCT/ DE 00/01863</text></patcit>, <patcit id="pcit0014" dnum="EP0213154W"><text>PCT/EP 02/13154</text></patcit> or international patent application <patcit id="pcit0015" dnum="EP0311551W"><text>PCT/EP 03/11551</text></patcit>.

0041According to the first and/or second aspect the present invention is related to a method for the manufacture of a nucleic acid molecule. It will be acknowledged that this method is a variant of the RSPS method such as described in international patent application <patcit id="pcit0016" dnum="EP0311551W"><text>PCT/EP 03/11551</text></patcit> and the RLPS method which is also described in said international patent application. By combining both methods the method according to the first aspect of the present invention, the overall efficacy of the method is considerably increased. This method is also referred to herein as S4LS method. In short, S4LS comprises one solid phase reaction cycle, followed by four liquid phase reaction cycles, which are again followed by a final solid phase reaction cycle. The term "solid phase reaction cycle" implies that a ligation product in an elongation step is first bound to a surface where after the elongated first oligonucleotide is cleaved off this surface, yielding a practically pure product. The term "liquid phase reaction cycle" means that in an elongation step the ligation product is first cleaved by the second type IIS restriction enzyme in the liquid phase where after the reaction products containing a modification are bound to a suitable surface and thereby removed before entering a new cycle.

0042Without wishing to be bound by any theory, the increased yield observed in connection with the S4LS method, mainly arises from the fact that the kinetics of enzymatic reactions, particularly the cleavage reactions are much faster in solution than on a solid phase where diffusion pathways are much longer. The incubation times can therefore be shortened, thus increasing the throughput of the <i>Sloning</i> process. In contrast to the solid phase reaction cycle, however, there is no selection possible that excludes non-ligated first oligonucleotides from being transferred into a new reaction cycle.

0043The present invention is now further illustrated by the following figures and example which are given for purpose of example but not for purpose of limitation. From said figures and examples further features, embodiments and advantages of the various aspects of the present invention may be taken either alone or in any combination irrespective of whether such single feature or combination of feature is literally disclosed. <dl id="dl0001"><dt>Figs 1 and 2</dt><dd>show a schematic illustration of the S4LS procedure;</dd><dt>Fig. 3</dt><dd>shows a gel depicting the generation of three different elongation blocks using either the S4LS procedure or the reverse solid phase synthesis procedure; and</dd><dt>Fig. 4</dt><dd>shows a diagram indicating the relative yield of the different elongation blocks using either the S4LS procedure of the reverse solid phase synthesis procedure (<figref idref="f0004">Fig. 4A</figref>), and the yield of various elongation blocks in the S4LS procedure (<figref idref="f0004">Fig. 4B</figref>), both based on a densitometric analysis of the gel depicted in <figref idref="f0003">Fig. 3</figref>.</dd></dl>

0044<figref idref="f0001">Figs. 1</figref> and <figref idref="f0002">2</figref> outline the elongation cycles of the S4LS procedure, in which the first reaction cycle is carried out as a solid phase cycle, i.e. the ligation product of the first and the second oligonucleotide is first bound to the solid phase as in step d) and then digested with the type IIS restriction enzyme the recognition site of which is contained in the second oligonucleotide as in step e), thereby releasing an elongated first oligonucleotide. In the subsequent steps f) to i) the elongation cycles are repeated but the order of the binding and cleavage step is reversed. Such a procedure takes advantage of the higher cleavage efficiency of restriction enzymes in solution at the expense of accumulating non-reacted first oligonucleotide or elongated first oligonucleotides. However, since in the final repetition, in steps k) and 1) the order of the binding and cleavage step is again reversed, a purified elongation product is obtained because any of the unreacted first or elongated first oligonucleotides will not bind to the solid phase since these molecules lack the modification contributed by the second or further oligonucleotide.

0045<figref idref="f0003">Figs. 3</figref> and <figref idref="f0004">4</figref> show a comparison between three different elongation blocks generated with the S4LS method as described herein and the same elongation blocks generated with the RSPS (reverse solid phase synthesis) method. The major difference between the two methods is the reversion of the order of the binding and cleavage steps as described above. As theoretically predicted, the overall yield is significantly increased at the expense of the accumulation of side products. In the fifth step, however, the last ligation product is bound to the solid phase before restriction takes place resulting in both cases in a pure elongation product. The bands of the stained gels in <figref idref="f0003">Fig. 3</figref> were quantitated using a densitometric screen and their relative intensities depicted in <figref idref="f0004">Fig. 4</figref>.

Example 1: Semi half inverted transposition with Eco31I and Esp3I (not covered by the claims)

0046Elongation blocks (E1-E4) were generated using standard methods, i.e. ligating the respective components such as anchors and splinkers, in 150 µl at 25° C for 15 minutes (1 x buffer Y⁺, 10 mM DTT, 0,5 mM ATP), cleaving the ligation products with the respective outside cutting endonucleases in the same buffer supplemented with 100 µg/ml BSA for 60 to 90 minutes at 37°C and using 15-100 U enzyme, and binding to suitable surfaces in the same buffer for 20 minutes at 25°C. Elongation blocks E1 and E4 were cut with Esp3I (by adding 1 µl equalling 100 U). All blocks were then bound to the appropriate plates. After binding blocks E2 and E3 were cut with Eco 31I (1150 µl containing 1 µl 100 U/ml Eco31I, 15 µl buffer Y⁺ and 134 µl H₂O). The supernatants from blocks E2 and E3 were pipetted into the wells containing blocks E1 and E4, respectively, and 1 µl T4 DNA ligase (30 U/µl) 15 µl 5 mM ATP, 15 µl 10 mM DTT was added. The ligation was allowed to proceed for 1 hour at 25° C. This gives the T1 products T1.1 and T1.2.

0047Then the ligated blocks T1.1 and T1.2 were cut with Esp3I (in 150 µl reaction volume containing 1 µl 100 U/µl Esp3I, 15 µl buffer Y⁺, 15 µl 100 mM DTT and 129 µl H₂O). After washing block T1.2, it was additionally cut with Eco31I (150 µl containing 1 µl 100 U/µl Eco31I, 15 µl buffer Y⁺ and 134 µl H₂O). The supernatant from block T1.2 was pipetted into the well containing block T1.1, and 1 µl T4 DNA ligase (30 U/µl) 15 µl 5 mM ATP, 15 µl 100 mM DTT was added. The ligation was allowed to proceed for 1 hour at 25° C to produce the T2 product; T2.1. Correspondingly, another second order transposition product is generated by the same course of reactions for a fifth to eighth elongation block. This molecule is referred to as T2.2 and is cleaved with Eco31I. The cut T2.2 is released from the solid phase and transferred to the T2.1 reaction, preferably the reaction vessel which the T2.1 intermediate is contained in. Upon ligation, a third order transposition product is formed, which is called T3.1.

Example 2: Alternative semi inverted transposition with Eco31I and Esp3I (not covered by the claims)

0048Using the <i>Sloning</i> procedure as described in patent application <patcit id="pcit0017" dnum="EP0311551W"><text>PCT/EP 03/11551</text></patcit>, transposition anchors and splinkers were first assembled in several ligation/restriction/binding cycles (ligation: 150 µl at 25° C for 15 to 30 minutes (1 x buffer Y⁺, 10 mM DTT, 0,5 mM ATP), restriction in the same buffer supplemented with 100 µg/ml BSA with 15 to 100 units Earn11041 for 60 to 90 minutes at 37°C, binding in the same buffer for 15 minutes at 25°C) to yield the elongation blocks (E1-4). Elongation blocks 1 and 4 were cut with Esp3I in 1 x Y⁺ (by adding 200 U enzyme). All blocks were then bound to the appropriate plates. After binding, blocks E2 and E3 were cut with Eco31I (in 150 µl reaction volume containing 1 µl 100 U/µl Eco 31I, 15 µl 10 x buffer Y⁺ and 134 µl H₂O). The supernatants from blocks E2 and E3 were pipetted into the wells containing blocks E1 and E4, respectively, and 1 µl T4 DNA ligase (30 U/µl) 15 µl 5 mM ATP, 15 µl 10 mM DTT and 2 µl Esp3I was added. The ligation and the Esp3I digestion were allowed to proceed simultaneously for 1 hour at 37° C. This is to ensure that the short double-stranded pieces (15 nucleotides on each strand, but only 11 base pairs overlapping) are not denatured by an inactivation step of 10 min at 65° C. The rest of the procedure is as that for the semi inverted transposition.

Literature citations

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0050The features of the present invention disclosed in the specification, the claims and/or the drawings may both separately and in any combination thereof be material for realising the invention in various forms thereof.