Single molecule arrays for genetic and chemical analysis

188142/2

SINGLE MOLECULE ARRAYS FOR GENETIC

AND CHEMICAL ANALYSIS 5 Field of the Invention

The present invention relates to methods and compositions for high-throughput analysis of populations of individual molecules, and more particularly, to methods and compositions related to fabrication of single molecule arrays and applications thereof, especially in high-throughput nucleic acid sequencing and genetic analysis, 10

BACKGROUND

Large-scale molecular analysis is central to understanding a wide range of biologicalphenomena related to states of health and disease both in humans and in a host of economicallyimportant plants and animals, e.g. Collins et al (2003), Nature, 422: 835-847; Hirschhom et al (2005), 15 Nature Reviews Genetics, 6: 95-108; National Cancer Institute, Report of Working Group on

Biomedical Technology, “Recommendation for a Human Cancer Genome Project,” (February, 2005).Miniaturization has proved to be extremely important for increasing the scale and reducing the costsof such analyses, and an important route to miniaturization has been the use of microatrays of probesor analytes. Such arrays play a key role in most currently available, or emerging, large-scale genetic 20 analysis and proteomic techniques, including those for single nucleotide polymorphism detection,copy number assessment, nucleic acid sequencing, and the like, e.g. Kennedy et al (2003), NatureBiotechnology, 21: 1233-1237; Gunderson et al (2005), Nature Genetics, 37: 549-554; Pinkel andAlbertson (2005), Nature Genetics Supplement, 37: SI 1 -S17; Leamon et al (2003), Electrophoresis,24: 3769-3777; Shendure et al (2005), Science, 309:1728-1732; Cowie et al (2004), Human 25 Mutation, 24: 261-271; and the like. However, the scale of microarrays currently used in suchtechniques still falls short of that required to meet the goals of truly low cost analyses that wouldmake practical such operations as personal genome sequencing, environmental sequencing to usechanges in complex microbial communities as an indicator of states of health, either personal orenvironmental, studies that associate genomic features with complex traits, such as susceptibilities to 30 cancer, diabetes, cardiovascular disease, and the like, e.g. Collins et al (cited above); Hirschhom et al(cited above); Tringe et al (2005), Nature Reviews Genetics, 6: 805-814; Service (2006), Science,311: 1544-1546.

Increasing the scale of analysis in array-based schemes for DNA sequencing is particularlychallenging as the feature size of the array is decreased to molecular levels, since most schemes 35 require not only a procedure for forming high density arrays, but also repeated cycles of complex biochemical steps that complicate the problems of array integrity, signal generation, signal detection, and the like,, e.g. Metzker (2005), Genome Research, 15: 1767-1776; Shendure et al (2004), Nature

Reviews Genetics, 5: 335-344; Weiss (1999), Science, 283; 1676-1683. Some approaches have-1 - 188142/2 employed high density arrays of unamplified target sequences, which present serious signal-to-noisechallenges, when “sequencing by synthesis” chemistries have been used, e.g. Balasubramanian et al,U.S. patent 6,787.308. Other approaches have employed in situ amplification of randomly disposedtarget sequences, followed by application of “sequencing by synthesis” chemistries. Such approaches 5 also have given rise to various difficulties, including (i) significant variability in the size of target sequence clusters, (ii) gradual loss of phase in extension steps carried out by polymerases, (iii) lack ofsequencing cycle efficiency that inhibits read lengths, and the like, e.g. Kartalov et al, Nucleic AcidsResearch, 32: 2873-2879 (2004); Mitra et al, Anal. Biochem., 320: 55-65 (2003); Metzker (citedabove). US 2002/0012930 discloses a method of sequencing a nucleic acid. 10

In view of the above, it would be advantageous for the medical, life science, and agriculturalfields if there were available molecular arrays and arraying techniques that permitted efficient andconvenient analysis of large numbers of individual molecules, such as DNA fragments coveringsubstantially an entire mammalian-sized genome, in parallel in a single analytical operation. 15

SUMMARY OF THE INVENTION

In one aspect, the invention provides high density single molecule arrays, methods of makingand using such compositions, and kits for implementing such methods. Compositions of the inventionin one form include random arrays of a plurality of different single molecules disposed on a surface, 20 where the single molecules each comprise a macromolecular structure and at least one analyte, suchthat each macromolecular structure comprises a plurality of attachment functionalities that are capableof forming bonds with one or more functionalities on the surface. In one aspect, the analyte is acomponent of the macromolecular structure, and in another aspect, the analyte is attached to themacromolecular structure by a linkage between a unique functionality on such structure and a reactive 25 group or attachment moiety on the analyte. In another aspect, compositions of the invention includerandom arrays of single molecules disposed on a surface, where the single molecules each comprise aconcatemer of at least one target polynucleotide and each is attached to the surface by linkages formedbetween one or more functionalities on the surface and complementary functionalities on theconcatemer. In another form, compositions of the invention include random arrays of single 30 molecules disposed on a surface, where the single molecules each comprise a concatemer of at leastone target polynucleotide and at least one adaptor oligonucleotide and each is attached to such surfaceby the formation of duplexes between capture oligonucleotides on the surface and the attachment -2- WO 2006/138257 PCT/US2006/022895 oligonucleotides in the concatemer. In still another form, compositions of the invention includerandom arrays of single molecules disposed on a surface, where each single molecule comprises abifunctional macromolecular structure having a unique functionality and a plurality ofcomplementary functionalities, and where each single molecule is attached to the surface by linkages 5 between one or more functionalities on the surface and complementary functionalities on thebifunctional macromolecular structure, the unique functionality having an orthogonal chemicalreactivity with respect to the complementary functionalities and being capable of forming a covalentlinkage with an analyte. In regard to the above compositions, in another aspect, such single moleculesare disposed in a planar array randomly distributed onto discrete spaced apart regions having defined 10 positions. Preferably, in this aspect, the discrete spaced apart regions each have an area that permitsthe capture of no more than a single molecule and each is surrounded by an inter-regional space that issubstantially free of other single molecules.

In one aspect, the invention includes an array of polymer molecules comprising: (a) a supporthaving a surface; and (b) a plurality of polymer molecules attached to the surface, wherein each 15 polymer molecule has a random coil state and comprises a branched or linear structure of multiplecopies of one or more linear polymeric units, such that the polymer molecule is attached to the surfacewithin a region substantially equivalent to a projection of the random coil on the surface andrandomly disposed at a density such that at least thirty percent of the polymer molecules areseparately detectable. As discussed more fully below, whenever the polymer molecules are linear, in 20 one embodiment, “substantially equivalent” in reference to the above proj ection means a substantiallycircular region with a diameter equal to the root mean square of the end-to-end distance of such linearpolymer, hi another embodiment, for linear or branched polymers, “substantially equivalent” means asubstantially circular region having a diameter that is one half or less than the total length of thepolymer; or in another embodiment one tenth or less; or in another embodiment, one hundredth or 25 less.

In another aspect, the invention includes an array of polynucleotide molecules comprising: (a) a support having a surface; and (b) a plurality of polynucleotide molecules attached to the surface,wherein each polynucleotide molecule has a random coil state and comprises a concatemer ofmultiple copies of a target sequence such that the polynucleotide molecule is attached to the surface 30 within a region substantially equivalent to a projection of the random coil on the surface and randomly disposed at a density such that at least thirty percent of the polynucleotide molecules have anearest neighbor distance of at least fifty nm. A method of making arrays of provided polymer molecules wherein each polymer moleculehas a random coil or similar or other three-dimensional state and comprises a branched or linear 35 structure of multiple copies of one or more linear polymeric units, such that the existing polymer molecule is attached to the surface within a region substantially equivalent to a projection of therandom coil on the surface or a region having size that is one half or less, one tenth or less or one -3- WO 2006/138257 PCT/US2006/022895 hundredth or less of the total length of the polymer, and randomly disposed at a density such that atleast twenty or at least thirty percent of the polymer molecules are separately detectable.

In still another aspect, the invention provides an array of single' molecules comprising: (a) asupport having a planar surface having a regular array of discrete spaced apart regions, wherein each 5 discrete spaced apart region has an area of less than 1 pm2 and contains reactive functionalitiesattached thereto; and (b) a plurality of single molecules attached to the surface, wherein each singlemolecule comprises a macromolecular structure and at least one analyte having an attachment moiety, such that each macromolecular structure comprises a unique functionality and a plurality ofattachment functionalities that are capable of forming linkages with the reactive functionalities of the 10 discrete spaced apart regions, and such that the analyte is attached to the macromolecular structure bya linkage between the unique functionality and the attachment moiety of the analyte, wherein theplurality of single molecules are randomly disposed on the discrete spaced apart regions such that atleast a majority of the discrete spaced apart regions contain only one single molecule.

In another aspect, the invention provides an array of polynucleotide molecules comprising: (a) 15 a support having a surface with capture oligonucleotides attached thereto; and (b) a plurality of polynucleotide molecules attached to the surface, wherein each polynucleotide molecule comprises aconcatemer of multiple copies of a target sequence and an adaptor oligonucleotide such that thepolynucleotide molecule is attached to the surface by one or more complexes formed between captureoligonucleotides and adaptor oligonucleotides, the polynucleotide molecules being randomly 20 disposed on the surface at a density such that at least a maj ority of the polynucleotide molecules havea nearest neighbor distance of at least fifty nm. In one embodiment of this aspect, the surface is aplanar surface having an array of discrete spaced apart regions, wherein each discrete spaced apartregion has a size equivalent to that of the polynucleotide molecule and contains the captureoligonucleotides attached thereto and wherein substantially all such regions have at most one of the 25 polynucleotide molecules attached.

The invention further includes, a method of making an array of polynucleotide moleculescomprising the following steps: (a) generating a plurality of polynucleotide molecules eachcomprising a concatemer of a DNA fragment from a source DNA and an adaptor oligonucleotide; and(b) disposing the plurality of polynucleotide molecules onto a support having a surface with capture 30 oligonucleotides attached thereto so that the polynucleotide molecules are fixed to the surface by oneor more complexes formed between capture oligonucleotides and adaptor oligonucleotides and so thatthe polynucleotide molecules are randomly distributed on the surface at a density such that a majorityof the polynucleotide molecules have a nearest neighbor distance of at least fifty nm, thereby formingthe array of polynucleotide molecules. 35 In another aspect, the invention provides a method of determining a nucleotide sequence of a target polynucleotide, the method comprising the steps of: (a) generating a plurality of targetconcatemers from the target polynucleotide, each target concatemer comprising multiple copies of a -4- WO 2006/138257 PCT/US2006/022895 fragment of the target polynucleotide and the plurality of target concatemers including a number of fragments that substantially covers the target polynucleotide; (b) forming a random array of targetconcatemers fixed to a surface at a density such that at least a majority of the target concatemers areoptically resolvable; (c) identifying a sequence of at least a portion of each fragment in each targetconcatemer; and (d) reconstructing the nucleotide sequence of the target polynucleotide fromthe identities of the sequences of the portions of fragments of the concatemers. In a preferredembodiment of this aspect, the step of identifying includes the steps of (a) hybridizing one or moreprobes from a first set of probes to the random array under conditions that permit the formation ofperfectly matched duplexes between the one or more probes and complementary sequences on targetconcatemers; (b) hybridizing one or more probes from a second set of probes to the random arrayunder conditions that permit the formation of perfectly matched duplexes between the one or moreprobes and complementary sequences on target concatemers; (c) ligating probes from the first andsecond sets hybridized to a target concatemer at contiguous sites; (d) identifying the sequences of theligated first and second probes; and (e) repeating steps (a through (d) until the sequence of the targetpolynucleotide can be determined from the identities of the sequences of the ligated probes.

In another aspect, the invention includes kits for making random arrays of the invention andfor implementing applications of the random arrays of the invention, particularly high-throughputanalysis of one or more target polynucleotides.

The present invention provides a significant advance in the microanray field by providingarrays of single molecules comprising linear and/or branched polymer structures that may incorporateor have attached target analyte molecules. In one form, such single molecules are concatemers oftarget polynucleotides arrayed at densities that permit efficient high resolution analysis ofmammalian-sized genomes, including sequence determination of all or substantial parts of suchgenomes, sequence determination of tagged fragments from selected regions of multiple genomes,digital readouts of gene expression, and genome-wide assessments of copy number patterns,methylation patterns, chromosomal stability, individual genetic variation, and the like.

Brief Description of the Drawings

Figs. 1A-1I illustrate various embodiments of the methods and compositions of the invention.

Figs. 2A-2B illustrate methods of circularizing genomic DNA fragments for generatingconcatemers of polynucleotide analytes.

Fig. 3 is an image of a glass surface containing a disposition of concatemers of E. colifragments.

Fig. 4 is an image of concatemers derived from two different organisms that are selectivelylabeled using oligonucleotide probes.

Fig. 5 is an image of concatemers of DNA fragments that contain a degenerated base, each ofwhich is identified by a specific ligation probe. -5- WO 2006/138257 PCT/US2006/022895

Fig. 6 is an image of concatemers of DNA fragments that contain a segment of degeneratebases, pairs of which are identified by specific probes.

Fig. 7 is a scheme for identifying sequence differences between reference sequences and testsequences using enzymatic mismatch detection and for constructing DNA circles therefrom. 5 Fig. 8 is another for identifying sequence differences between a reference sequence and a test sequence using enzymatic mismatch detection and for constructing DNA circles therefrom. DETAILED DESCRIPTION OF THE INVENTIONThe practice of the present invention may employ, unless otherwise indicated, conventional 10 techniques and descriptions of organic chemistry, polymer technology, molecular biology (includingrecombinant techniques), cell biology, biochemistry, and immunology, which are within the skill ofthe art. Such conventional techniques include polymer array synthesis, hybridization, ligation, anddetection of hybridization using a label. Specific illustrations of suitable techniques can be had byreference to the example herein below. However, other equivalent conventional procedures can, of

15 course, also be used. Such conventional techniques and descriptions can be found in standardlaboratory manuals such as Genome Analysis: A Laboratory Manual Series (Vols. I-IV), UsingAntibodies: A Laboratory Manual, Cells: A Laboratory Manual, PCR Primer: A Laboratory Manual,and Molecular Cloning: A Laboratory Manual (all from Cold Spring Harbor Laboratory Press),Stryer, L. (1995) Biochemistry (4th Ed.) Freeman, New York, Gait, “Oligonucleotide Synthesis: A 20 Practical Approach ” 1984, IRL Press, London, Nelson and Cox (2000), Lehninger, Principles of

Biochemistry 3ri Ed., W. H. Freeman Pub., New York, N. Y. and Berg et al. (2002) Biochemistry, 5thEd., W. H. Freeman Pub., New York, N.Y., all of which are herein incorporated in their entirety byreference for all purposes. 25 The invention provides random single molecule arrays for large-scale parallel analysis of populations of molecules, particularly DNA fragments, such as genomic DNA fragments. Generally,single molecules of the invention comprise an attachment portion and an analyte portion. Theattachment portion comprises a macromolecular structure that provides for multivalent attachment toa surface, particularly a compact or restricted area on a surface so that signals generated from it or an 30 attached analyte are concentrated. That is, the macromolecular structure occupies a compact andlimited region of the surface. Macromolecular structures of the invention may be bound to a surfacein a variety of ways. Multi-valent bonds may be covalent or non-covalent. Non-covalent bondsinclude formation of duplexes between capture oligonucleotides on the surface and complementarysequences in the macromolecular structure, and adsorption to a surface by attractive noncovalent 35 interactions, such as Van der Waal forces, hydrogen bonding, ionic and hydrophobic interactions, andthe tike. Multi-valent covalent bonding may be accomplished, as described more fully below, byproviding reactive functionalities on the surface that can reactive with a plurality of complementary -6- WO 2006/138257 PCT/US2006/022895 functionalities in the macromolecular structures. An analyte portion may be attached to amacromolecular structure by way of a unique linkage or it may form a part of, and be integral with,the macromolecular structure. Single molecules of the invention are disposed randomly on a surfaceof a support material, usually from a solution; thus, in one aspect, single molecules are uniformly 5 distributed on a surface in close approximation to a Poisson distribution. In another aspect, singlemolecules are disposed on a surface that contains discrete spaced apart regions in which singlemolecules are attached. Preferably, macromolecular structures, preparation methods, and areas ofsuch discrete spaced apart regions are selected so that substantially all such regions contain at mostonly one single molecule. Preferably, single molecules of the invention, particularly concatemers, are 10 roughly in a random coil configuration on a surface and are confined to the area of a discrete spacedapart region. In one aspect, the discrete space apart regions have defined locations in a regular array,which may correspond to a rectilinear pattern, hexagonal pattern, or the like. A regular array of suchregions is advantageous for detection and data analysis of signals collected from the arrays during ananalysis. Also, single molecules confined to the restricted area of a discrete spaced apart region 15 provide a more concentrated or intense signal, particularly when fluorescent probes are used inanalytical operations, thereby providing higher signal-to-noise values. Single molecules of theinvention are randomly distributed on the discrete spaced apart regions so thai a given region usuallyis equally likely to receive any of the different single molecules. In other words, the resulting arraysare not spatially addressable immediately upon fabrication, but may be made so by carrying out an 20 identification or decoding operation. That is, the identities of the single molecules are discernable,but not known. As described more fully below, in some embodiments, there are subsets of discretespaced apart regions that receive single molecules only from corresponding subsets, for example, asdefined by complementary sequences of capture oligonucleotides and adaptor oligonucleotides.

Macromolecular structures of the invention comprise polymers, either branched or linear, and

25 may be synthetic, e.g. branched DNA, or may be derived from natural sources, e.g linear DNA fragments from a patient’s genomic DNA. Usually, macromolecular structures comprise concatemersof linear single stranded DNA fragments that can be synthetic, derived from natural sources, or can bea combination of both. As used herein, the term “target sequence” refers to either a synthetic nucleicacid or a nucleic acid derived from a natural source, such as a patient specimen, or the like. Usually, 30 target sequences are part of a concatemer generated by methods of the invention, e.g. by RCR, butmay also be part of other structures, such as dendrimers, and other branched structures. When targetsequences are synthetic or derived from natural sources, they are usually replicated by variousmethods in the process of forming macromolecular structures or single molecules of the invention. Itis understood that such methods can introduce errors into copies, which nonetheless are encompassed 35 by the term “target sequence.”

Particular features or components of macromolecular structures may be selected to satisfy avariety of design objectives in particular embodiments. For example, in some embodiments, it may -7- 07 WO 2006/138257 PCT/US2006/022895 be advantageous to maintain an analyte molecule as far from the surface as possible, e.g. by providingan inflexible molecular spacer as part of a unique linkage. As another example, reactivefunctionalities may be selected as having a size that effectively prevents attachment of multiplemacromolecular structures to one discrete spaced apart region. As still another example, 5 macromolecular structures may be provided with other functionalities for a variety of other purposes, e.g. enhancing solubility, promoting formation of secondary structures via hydrogen bonding, and thelike.

In one aspect, macromolecular structures are sufficiently large that their size, e.g. a lineardimension (such as a diameter) of a volume occupied in a conventional physiological saline solution, 10 is approximately equivalent to that a discrete spaced apart region. For macromolecular structures thatare linear polynucleotides, in one aspect, sizes may range from a few thousand nucleotides, e.g. 10,000, to several hundred thousand nucleotides, e.g. 100-200 thousand. As explained more fullybelow, in several embodiments, such macromolecular structures are made by generating circularDNAs and then replicating them in a rolling circle replication reaction to form concatemers of 15 complements of the circular DNAs.

The above concepts are illustrated more fully in the embodiments shown schematically inFigs. 1A-1G. After describing these figures, elements of the invention are disclosed in additionaldetail and examples are given. As mentioned above, in one aspect, macromolecular structures of theinvention are single stranded polynucleotides comprising concatemers of a target sequence or 20 fragment. In particular, such polynucleotides may be concatemers of a target sequence and an adaptoroligonucleotide. For example, source nucleic acid (1000) is treated (1001) to form single strandedfragments (1006), preferably in the range of from 50 to 600 nucleotides, and more preferably in therange of from 300 to 600 nucleotides, which are then ligated to adaptor oligonucleotides (1004) toform a population of adaptor-fragment conjugates (1002). Source nucleic acid (1000) may be 25 genomic DNA extracted from a sample using conventional techniques, or a cDNA or genomic libraryproduced by conventional techniques, or synthetic DNA, or the like. Treatment (1001) usually entailsfragmentation by a conventional technique, such as chemical fragmentation, enzymatic fragmentation,or mechanical fragmentation, followed by denaturation to produce single stranded DNA fragments.Adaptor oligonucleotides (1004), in this example, are used to form (1008) a population (1010) of 30 DNA circles by the method illustrated in Fig. 2A. In one aspect, each member of population (1010)has an adaptor with an identical primer binding site and a DNA fragment from source nucleic acid(1000). The adapter also may have other functional elements including, but not limited to, taggingsequences, attachment sequences, palindromic sequences, restriction sites, functionalizationsequences, and the like. In other embodiments, classes of DNA circles may be created by providing 35 adaptors having different primer binding sites. After DNA circles (1010) are formed, a primer androlling circle replication (RCR) reagents may be added to generate (1011) in a conventional RCRreaction a population (1012) of concatemers (1015) of the complements of the adaptor oligonucleotide -8- WO 2006/138257 PCT/US2006/022895 and DNA fragments, which population can then be isolated using conventional separation techniques.Alternatively, RCR may be implemented by successive ligation of short oligonucleotides, e.g. 6-mers,from a mixture containing all possible sequences, or if circles are synthetic, a limited mixture ofoligonucleotides having selected sequences for circle replication. Concatemers may also be generated 5 by ligation of target DNA in the presence of a bridging template DNA complementary to both beginning and end of the target molecule. A population of different target DNA may be converted inconcatemers by a mixture of corresponding bridging templates. Isolated concatemers (1014) are thendisposed (1016) onto support surface (1018) to form a random array of single molecules. Attachmentmay also include wash steps of varying stringencies to remove incompletely attached single molecules 10 or other reagents present from earlier preparation steps whose presence is undesirable or that arenonspecifrcally bound to surface (1018). Concatemers (1020) can be fixed to surface (1018) by avariety of techniques, including covalent attachment and non-covalent attachment. In oneembodiment, surface (1018) may have attached capture oligonucleotides that form complexes, e.g.double stranded duplexes, with a segment of the adaptor oligonucleotide, such as the primer binding 15 site or other elements. In other embodiments, capture oligonucleotides may comprise oligonucleotideclamps, or like structures, that form triplexes with adaptor oligonucleotides, e.g. Gryaznov et al, U.S.patent 5,473,060. In another embodiment, surface (1018) may have reactive functionalities that reactwith complementary functionalities on the concatemers to form a covalent linkage, e.g. by way of thesame techniques used to attach cDNAs to microarrays, e.g. Smirnov et al (2004), Genes, 20 Chromosomes & Cancer, 40: 72-77; Beaucage (2001), Current Medicinal Chemistry, 8:1213-1244,which are incorporated herein by reference. Long DNA molecules, e.g. several hundred nucleotidesor larger, may also be efficiently attached to hydrophobic surfaces, such as a clean glass surface thathas a low concentration of various reactive functionalities, such as —OH groups. Concatemers ofDNA fragments may be further amplified in situ after disposition of a surface. For example after 25 disposition, concatemer may be cleaved by reconstituting a restriction site in adaptor sequences by hybridization of an oligonucleotide, after which the fragments are circularized as described below andamplified in situ by a RCR reaction.

Fig. IB illustrates a section (1102) of a surface of a random array of single molecules, such assingle stranded polynucleotides. Such molecules under conventional conditions (a conventional DNA 30 buffer, e.g. TE, SSC, SSPE, or the like, at room temperature) form random coils that roughly fill aspherical volume in solution having a diameter of from about 100 to 300 nm, which depends on thesize of the DNA and buffer conditions, in a manner well known in the art, e.g. Edvinsson, “On thesize and shape of polymers and polymer complexes," Dissertation 696 (University of Uppsala, 2002).One measure of the size of a random coil polymer, such as single stranded DNA, is a root mean 35 square of the end-to-end distance, which is roughly a measure of the diameter of the randomly coiledstructure. Such diameter, referred to herein as a “random coil diameter," can be measured by lightscatter, using instruments, such as a Zetasizer Nano System (Malvern Instruments, UK), or like -9- ID? ? " . - - : · xj.. fe. : r ,, =' · · .....-..-=-...^-:-~ ·. - •-•--Χ-, WO 2006/138257 PCT/US2006/022895 instrument. Additional size measures of macromolecular structures of the invention includemolecular weight, e.g. in Daltons, and total polymer length, which in the case of a branched polymeris the sum of the lengths of all its branches. Upon attachment to a surface, depending on theattachment chemistry, density of linkages, the nature of the surface, and the like, single stranded . 5 polynucleotides fill a flattened spheroidal volume that on average is bounded by a region (1107)defined by dashed circles (1108) having a diameter (1110), which is approximately equivalent to thediameter of a concatemer in random coil configuration. Stated another way, in one aspect,macromolecular structures, e.g. concatemers, and the like, are attached to surface (1102) within aregion that is substantially equivalent to a projection of its random coil state onto surface (1102), for 10 example, as illustrated by dashed circles (1108). An area occupied by a macromolecular structure can vary, so that in some embodiments, an expected area may be within the range of from 2-3 times thearea of projection (1108) to some fraction of such area, e.g. 25-50 percent. As mentioned else where,preserving the compact form of the macromolecular structure on the surface allows a more intensesignal to be produced by probes, e.g. fluorescently labeled oligonucleotides, specifically directed to 15 components of a macromolecular structure or concatemer. The size of diameter (1110) of regions (1107) and distance (1106) to the nearest neighbor region containing a single molecule are twoquantities of interest in the fabrication of arrays. A variety of distance metrics may be employed formeasuring the closeness of single molecules on a surface, including center-to-center distance ofregions (1107), edge-to-edge distance of regions (1007), and the like. Usually, center-to-center 20 distances are employed herein. The selection of these parameters in fabricating arrays of the inventiondepends in part on the signal generation and detection systems used in the analytical processes.Generally, densities of single molecules are selected that permit at least twenty percent, or at leastthirty percent, or at least forty percent, or at least a majority of the molecules to be resolvedindividually by the signal generation and detection systems used. In one aspect, a density is selected 25 that permits at least seventy percent of the single molecules to be individually resolved. In one aspect,whenever scanning electron microscopy is employed, for example, with molecule-specific probeshaving gold nanoparticle labels, e.g. Nie et al (2006), Anal. Chem., 78:1528-1534, which isincorporated by reference, a density is selected such that at least a majority of single molecules have anearest neighbor distance of 50 nm or greater; and in another aspect, such density is selected to ensure 30 that at least seventy percent of single molecules have a nearest neighbor distance of 100 nm or greater.In another aspect, whenever optical microscopy is employed, for example with molecule-specificprobes having fluorescent labels, a density is selected such that at least a majority of single moleculeshave a nearest neighbor distance of 200 nm or greater; and in another aspect, such density is selectedto ensure that at least seventy percent of single molecules have a nearest neighbor distance of 200 nm 35 or greater. In still another aspect, whenever optical microscopy is employed, for example with molecule-specific probes having fluorescent labels, a density is selected such that at least a majorityof single molecules have a nearest neighbor distance of300 nm or greater; and in another aspect, such -10- WO 2006/138257 PCT/US2006/022895 density is selected to ensure that at least seventy percent of single molecules have a nearest neighbordistance of 300 nm or greater, or 400 nm or greater, or 500 nm or greater, or 600 nm or greater, or 700nm or greater, or 800 nm or greater. In still another embodiment, whenever optical microscopy isused, a density is selected such that at least a majority of single molecules have a nearest neighbor 5 distance of at least twice the minimal feature resolution power of the microscope. In another aspect, polymer molecules of the invention are disposed on a surface so that the density of separatelydetectable polymer molecules is at least 1000 per pm2, or at least 10,000 per pm2, or at least 100,Ό00per pm2.

In another aspect of the invention, illustrated for a particular embodiment in Fig. 1C, the 10 requirement of selecting densities of randomly disposed single molecules to ensure desired nearest neighbor distances is obviated by providing on a surface discrete spaced apart regions that aresubstantially the sole sites for attaching single molecules. That is, in such embodiments the regionson the surface between the discrete spaced apart regions, referred to herein as “inter-regional areas,”are inert in the sense that concatemers, or other macromolecular structures, do not bind to such 15 regions. In some embodiments, such inter-regional areas may be treated with blocking agents, e.g.DNAs unrelated to concatemer DNA, other polymers, and the like As in Fig. 1 A, source nucleic acids(1000) are fragmented and adaptored (1002) for circularization (1010), after which concatemers areformed by RCR (1012). Isolated concatemers (1014) are then applied to surface (1120) that has aregular array of discrete spaced apart regions (1122) that each have a nearest neighbor distance (1124) 20 that is determined by the design and fabrication of surface (1120). As described more fully below,arrays of discrete spaced apart regions (1122) having micron and submicron dimensions forderivatizing with capture oligonucleotides or reactive functionalities can be fabricated usingconventional semiconductor fabrication techniques, including electron beam lithography, nanoimprint technology, photolithography, and the like. Generally, the area of discrete spaced apart 25 regions (1122) is selected, along with attachment chemistries, macromolecular structures employed,and the like, to correspond to tlie size of single molecules of the invention so that when singlemolecules are applied to surface (1120) substantially every region (1122) is occupied by no more thanone single molecule. The likelihood of having only one single molecule per discrete spaced apartregion may be increased by selecting a density of reactive functionalities or capture oligonucleotides 30 that results in fewer such moieties than their respective complements on single molecules. Thus, asingle molecule will “occupy” all linkages to tlie surface at a particular discrete spaced apart region,thereby reducing the chance that a second single molecule will also bind to tlie same region. Inparticular, in one embodiment, substantially all the capture oligonucleotides in a discrete spaced apartregion hybridize to adaptor oligonucleotides a single macromolecular structure. In one aspect, a 35 discrete spaced apart region contains a number of reactive functionalities or capture oligonucleotidesthat is from about ten percent to about fifty percent of the number of complementary functionalities oradaptor oligonucleotides of a single molecule. Tlie length and sequence(s) of capture -11- . - - --- WO 2006/138257 PCT/US2006/022895 oligonucleotides may vary widely, and may be selected in accordance with well known principles,e.g. Wetmur, Critical Reviews in Biochemistry and Molecular Biology, 26:227-259 (1991); Brittenand Davidson, chapter 1 inHames et al, editors, Nucleic Acid Hybridization: A Practical Approach(IRL Press, Oxford, 1985). In one aspect, the lengths of capture oligonucleotides are in a range of 5 from 6 to 30 nucleotides, and in another aspect, within a range of from 8 to 30 nucleotides, or from 10to 24 nucleotides. Lengths and sequences of capture oligonucleotides are selected (i) to provideeffective binding of-macTomolecular structures to a surface, so that losses of macromolecularstructures are minimized during steps of analytical operations, such as washing, etc., and (ii) to avoidinterference with analytical operations on analyte molecules, particularly when analyte molecules are 10 DNA fragments in a concatemer. In regard to (i), in one aspect, sequences and lengths are selected toprovide duplexes between capture oligonucleotides and their complements that are sufficiently stableso that they do not dissociate in a stringent wash. In regard to (ii), if DNA fragments are from aparticular species of organism, then databases, when available, may be used to screen potentialcapture sequences that may form spurious or undesired hybrids with DNA fragments. Other factors in 15 selecting sequences for capture oligonucleotides are similar to those considered in selecting primers,hybridization probes, oligonucleotide tags, and the like, for which there is ample guidance, asevidenced by the references cited below in the Definitions section. In some embodiments, a discretespaced apart region may contain more than one kind of capture oligonucleotide, and each differentcapture oligonucleotide may have a different length and sequence. In one aspect of embodiments 20 employing regular arrays of discrete spaced apart regions, sequences of capture oligonucleotides areselected so that sequences of capture oligonucleotide at nearest neighbor regions have differentsequences. In a rectilinear array, such configurations are achieved by rows of alternating sequencetypes. In other embodiments, a surface may have a plurality of subarrays of discrete spaced apartregions wherein each different subarray has capture oligonucleotides with distinct nucleotide 25 sequences different from those of the other subarrays. A plurality of subarrays may include 2 subarrays, or 4 or fewer subarrays, or 8 or fewer subarrays, or 16 or fewer subarrays, or 32 or fewersubarrays, or 64 of fewer subarrays. In still other embodiments, a surface may include 5000 or fewersubarrays. In one aspect, capture oligonucleotides are attached to the surface of an array by a spacermolecule, e.g. polyethylene glycol, or like inert chain, as is done with microarrays, in order to 30 minimize undesired affects of surface groups or interactions with the capture oligonucleotides or otherreagents.

In one aspect, the area of discrete spaced apart regions (1122) is less than 1 pm2; and inanother aspect, the area of discrete spaced apart regions (1122) is in the range of from 0.04 pm2 to 1pm2; and in still another aspect, the area of discrete spaced apart regions (1122) is in the range of from 35 0.2 pm2 to 1 pm2. In another aspect, when discrete spaced apart regions are approximately circular or square in shape so that their sizes can be indicated by a single linear dimension, the size of suchregions are in the range of from 125 nra to 250 ran, or in the range of from 200 nm to 500 nm. In one -12-

Sil Η·j : ' · . --^χν-χςς_.<2x-Eb.\vvg> -/-': ::-:-- - ΖΜ WO 2006/138257 PCT/US2006/022895 aspect, center-to-center distances of nearest neighbors of regions (1122) are in the range of from 0.25pm to 20 pm; and in another aspect, such distances are in the range of from 1 pm to 10 pm, or in. therange from 50 to 1000 nm. in one aspect, regions (1120) may be arranged on surface (1018) invirtually any pattern in which regions (1122) have defined locations, i.e. in any regular array, which - 5 makes signal collection and data analysis functions more efficient. Such patterns include, but are notlimited to, concentric circles of regions (1122), spiral patterns, rectilinear patterns, hexagonal patterns,and the like. Preferably, regions (1122) are arranged in a rectilinear or hexagonal pattern.

As illustrated in Fig. ID, in certain embodiments, DNA circles prepared from source nucleicacid (1200) need not include an adaptor oligonucleotide. As before, source nucleic acid (1200) is 10 fragmented and denatured (1202) to form a population of single strand fragments (1204), preferably inthe size range of from about 50 to 600 nucleotides, and more preferably in the size range of fromabout 300 to 600 nucleotides, after which they are circularized in a non-template driven reaction withcircularizing ligase, such as CircLigase (Epicentre Biotechnologies, Madison, WI), or the like. Afterformation of DNA circles (1206), concatemers are generated by providing a mixture of primers that 15 bind to selected sequences. The mixture of primers may be selected so that only a subset of the totalnumber of DNA circles (1206) generate concatemers. After concatemers are generated (1208), theyare isolated and applied to surface (1210) to form a random array of the invention.

As mentioned above, single molecules of the invention comprise an attachment portion and ananalyte portion such that the attachment portion comprises a macromolecular structure that provides 20 multivalent attachment of the single molecule to a surface. As illustrated in Fig. IE, macromolecularstructures may be concatemers made by an RCR reaction in which the DNA circles in the reaction aresynthetic. An analyte portion of a single molecule is then attached by way of a unique functionalityon the concatemer. Synthetic DNA circles of virtually any sequence can be produced using well-known techniques, conveniently, in sizes up to several hundred nucleotides, e.g. 200, and with more 25 difficulty, in sizes of many hundreds of nucleotides, e.g. up to 500, e.g. Kool, U.S. patent 5,426,180;

Dolinnaya et al (1993), Nucleic Acids Research, 21: 5403-5407; Rubin et al (1995), Nucleic AcidsResearch, 23:3547-3553; and the like, which are incorporated herein by reference. Synthetic DNAcircles (1300) that comprise primer binding sites (1301) are combined with primer (1302) in an RCRreaction (1306) to produce concatemers (1308). Usually, in this embodiment, all circles have the 30 same sequence, although different sequences can be employed, for example, for directing subsets ofconcatemers to preselected regions of an array via complementary attachment moieties, such asadaptor sequences and capture oligonucleotides. Primer (1302) is synthesized with a functionality(1304, designated as “R”) at its 5’ end that is capable of reacting with a complementary functionalityon an analyte to form a covalent linkage. Exemplary functionalities include amino groups, sulfhydryl 35 groups, and the like, that can be attached with commercially available chemistries (e.g. Glen

Research). Concatemers (1308) are applied to surface (1310) to form an array (1314), after whichanalytes (1312) having an attachment moiety are applied to array (1310) where a linkage is formed -13- WO 2006/138257 PCT/US2006/022895 with a concatemer by reaction of unique functionalities, R (1311) and attachment moiety (1312).Alternatively, prior to application to array (1310), concatemers (1308) may be combined with analytes(1312) so that attachment moieties and unique functionalities can react to form a linkage, after whichthe resulting conjugate is applied to array (1310). There is abundant guidance in the literature inselecting appropriate attachment moieties and unique functionalities for linking concatemers (1308)and many classes of analyte. In one aspect, for linking protein or peptide analytes to concatemers,many homo- and heterobifunctional reagents are available commercially (e.g. Pierce) and aredisclosed in references such as Hermanson, Bioconjugate Techniques (Academic Press, New York,1996), which is incorporated by reference. For example, whenever the unique functionality is anamino group, then concatemers (1308) can be linked to a sufhydiyl group on an analyte using N-succinimidyl 3-(2-pyridyldithio)propionate (SPDP), succinimidyloxycarbonyI-a-methyl-a-(2-pyridyldithio)toluene (SMPT), succinimidy I-4-(N-maleimidomethyl)cyclohexane-1 -carboxylate(SMCC), m-maleimidobenzoyl-N-hydroxy succinimide ester (MBS), N-succinimidyl(4- iodoacetyl)aminobenzoate (SIAB), succinimidyl 6-((iodoacetyl)amino)hexanoate (SIAX), and likereagents. Suitable complementary functionalities on analytes include amino groups, sulfhydrylgroups, carbonyl groups, which may occur naturally on analytes or may be added by reaction with asuitable homo- or heterobifiinctional reagent. Analyte molecules may also be attached tomacromolecular structures by way of non-covalent linkages, such as biotin-streptavidin linkages, theformation of complexes, e.g. a duplexes, between a first oligonucleotide attached to a concatemer anda complementary oligonucleotide attached to, or forming part of, an analyte, or like linkages.

Analytes include biomolecules, such as nucleic acids, for example, DNA or RNA fragments,polysaccharides, proteins, and the like.

As mentioned above, macromolecular structures of the invention may comprise branchedpolymers as well as linear polymers, such as concatemers of DNA fragments. Exemplary branchedpolymer structures are illustrated in Figs. IF and IG. In Fig. IF, a branched DNA structure isillustrated that comprises a backbone polynucleotide (1400) and multiple branch polynucleotides(1402) each connected to backbone polynucleotide (1400) by their 5’ ends to form a comb-likestructure that has all 3’ ends, except for a single 5’ end (1404) on backbone polynucleotide (1400),which is derivatized to have a unique functionality. As mentioned below, such unique functionalitymay be a reactive chemical group, e.g. a protected or unprotected amine, sulfhydiyl, or the like, or itmay be an oligonucleotide having a unique sequence for capturing an analyte having anoligonucleotide with a complementary sequence thereto. Likewise, such unique functionality may bea capture moiety, such as biotin, or the like. Such branched DNA structures are synthesized usingknown techniques, e.g. Giyaznov, U.S. patent 5,571,677; Urdea et al, U.S. patent 5,124,246; Seemanet al, U.S. patent 6,255,469; and the like, which are incorporated herein by reference. Whenever suchmacromolecular structures are polynucleotides, the sequences of components thereof may be selectedfor facile self-assembly, or they may be linked by way of specialized linking chemistries, e.g. as -14- WO 2006/138257 PCT/US2006/022895 disclosed below, in which case sequences are selected based on other factors, including, in someembodiments, avoidance of self-annealing, facile binding to capture oligonucleotides on a surface,and the like. In Fig. 1G, a dentrimeric structure is illustrated that comprises oligonucleotide (1406),which is derivatized with multiple tri-valent linking groups (1408) that each have two functionalities. 5 (1410, designated by “R”) by which additional polymers (1407), e.g. polynucleotides, can be attached to form a linkage to oligonucleotide (1406) thereby forming macromolecular structure (1409), which,in turn, if likewise derivatized with multivalent linkers, can form a nucleic acid dendrimer. Trivalentlinkers (1408) for use with oligonucleotides are disclosed in Iyer et al, U.S. patent 5,916,750, which isincorporated herein by reference. As illustrated in Fig. IH, once such dendrimeric or branched 10 structures (1411) are constructed, they can be attached to array (1420) as described above for linearpolynucleotides, after which analytes (1430) can be attached via unique functionalities (1410).Optionally, unreacted unique functionalities (1422) may be capped using conventional techniques.Alternatively, dendrimeric or branched structures (1411) may be combined with analytes (1430) first,e.g. in solution, so that conjugates are formed, and then the conjugates are disposed on array (1420). 15 When the analyte is a polynucleotide (1440) with a free 3’ end, as shown in Fig. II, such end may be extended in an in situ RCR reaction to form either concatemers of target sequences or other sequencesfor further additions. Likewise, polynucleotide analytes may be extended by ligation usingconventional techniques. 20 Source Nucleic Acids and Circularization of Target Sequences

In one aspect of the invention, macromolecular structures comprise concatemers ofpolynucleotide analytes, i.e. target sequences, which are extracted or derived from a sample, such asgenomic DNA or cDNAs from a patient, an organism of economic interest, or the like. Random 25 arrays of the invention comprising such single molecules are useful in providing genome-wideanalyses, including sequence determination, SNP measurement, allele quantitation, copy numbermeasurements, and the like. For mammalian-sized genomes, preferably fragmentation is carried outin at least two stages, a first stage to generate a population of fragments in a size range of from about100 kilobases (Kb) to about 250 kilobases, and a second stage, applied separately to each 100-250Kb 30 fragment, to generate fragments in the size range of from about 50 to 600 nucleotides, and more preferably in the range of from about 300 to 600 nucleotides, for generating concatemers for a randomarray. In some aspects of the invention, the first stage of fragmentation may also be employed toselect a predetermined subset of such fragments, e.g. fragments containing genes that encode proteinsof a signal transduction pathway, or the like. The amount of genomic DNA required for constructing 35 arrays of the invention can vary widely. In one aspect, for mammalian-sized genomes, fragments aregenerated from at least 10 genome-equivalents of DNA; and in another aspect, fragments are -15- WO 2006/138257 PCT/US2006/022895 generated from at least 30 genome-equivalents of DNA; and in another aspect, fragments aregenerated from at least 60 genome-equivalents of DNA.

Genomic DNA is obtained using conventional techniques, for example, as disclosed inSambrook et al., supra, 1999; Current Protocols in Molecular Biology, Ausubel et al,, eds.(John Wiley 5 and Sons, Inc., NY, 1999), or the like, Important factors for isolating genomic DNA include thefollowing: 1) the DNA is free of DNA processing enzymes and contaminating salts; 2) the entiregenome is equally represented; and 3) the DNA fragments are between about 5,000 and 100,000 bp inlength. In many cases, no digestion of the extracted DNA is required because shear forces createdduring lysis and extraction will generate fragments in the desired range. In another embodiment, 10 shorter fragments (1-5 kb) can be generated by enzymatic fragmentation using restriction endonucleases. In one embodiment, 10-100 genome-equivalents of DNA ensure that the populationof fragments covers the entire genome. In some cases, it is advantageous to provide carrier DNA, e.g.unrelated circular synthetic double- stranded DNA, to be mixed and used with the sample DNAwhenever only small amounts of sample DNA are available and there is danger of losses through 15 nonspecific binding, e.g. to container walls and the like.

In generating fragments in either stage, fragments may be derived from either an entire genome or it may be derived from a selected subset of a genome. Many techniques are available forisolating or enriching fragments from a subset of a genome, as exemplified by the followingreferences that are incorporated by reference: Kandpal et al (1990), Nucleic Acids Research, 18: 20 1789-1795; Callow et al, U.S. patent publication 2005/0019776; Zabeau et al, U.S. patent 6,045,994;

Deugau et al, U.S. patent 5,508,169; Sibson, U.S. patent 5,728,524; Guilfoyle et al, U.S. patent5,994,068; Jones et al, U.S. patent publication 2005/0142577; Gullberg et al, U.S. patent publication2005/0037356; Matsuzaki et al, U.S. patent publication 2004/0067493; and the like.

For mammalian-sized genomes, an initial fragmentation of genomic DNA can be achieved by 25 digestion with one or more “rare” cutting restriction endonucleases, such as Not I, Asc I, Bae I, CspCI, Pac I, Fse I, Sap I, Sfi I, Psr I, or the like. The resulting fragments can be used directly, or forgenomes that have been sequenced, specific fragments may be isolated from such digested DNA forsubsequent processing as illustrated in Fig. 2B. Genomic DNA (230) is digested (232) with a rarecutting restriction endonuclease to generate fragments (234), after which the fragments (234) are 30 further digested for a short period (i.e. the reaction is not allowed to run to completion) with a 5’ single stranded exonuclease, such as λ exonuclease, to expose sequences (237) adjacent to restrictionsite sequences at the end of the fragments. Such exposed sequences will be unique for each fragment.Accordingly, biotinylated primers (241) specific for the ends of desired fragments can be annealed toa capture oligonucleotide for isolation; or alternatively, such fragments can be annealed to a primer 35 having a capture moiety, such as biotin, and extended with a DNA polymerase that does not havestrand displacement activity, such as Taq polymerase Stoffel fragment. After such extension, the 3’end of primers (241) abut the top strand of fragments (242) such that they can be ligated to form a -16- WO 2006/138257 PCT/US2006/022895 continuous strand. The latter approach may also be implemented with a DNA polymerase that doeshave strand displacement activity and replaces the top strand (242) by synthesis. In either approach,the biotinylated fragments may then be isolated (240) using a solid support (239) derivatized withstreptavidin. 5 In another aspect, primer extension from a genomic DNA template is used to generate a linear amplification of selected sequences greater than 10 kilobases surrounding genomic regions of interest.For example, to create a population of defined-sized targets, 20 cycles of linear amplification isperformed with a forward primer followed by 20 cycles with a reverse primer. Before applying thesecond primer, the first primer is removed with a standard column for long DNA purification or 10 degraded if a few uracil bases are incorporated. A greater number of reverse strands are generatedrelative to forward strands resulting in a population of double stranded molecules and single strandedreverse strands. The reverse primer may be biotinylated for capture to streptavidin beads which can beheated to melt any double stranded homoduplexes from being captured. All attached molecules willbe single stranded and representing one strand of the original genomic DNA. 15 The products produced can be fragmented to 0.2-2 kb in size, or more preferably, 0.3-0.6 kb in size (effectively releasing them from the solid support) and circularized for an RCR reaction. In onemethod of circularization, illustrated in Fig. 2A, after genomic DNA (200) is fragmented anddenatured (202), single stranded DNA fragments (204) are first treated with a terminal transferase(206) to attach a poly dA tails (208) to 3-prime ends. This is then followed by ligation (212) of the 20 free ends intra-molecularly with the aid of bridging oligonucleotide (210). that is complementary tothe poly dA tail at one end and complementary to any sequence at the other end by virtue of asegment of degenerate nucleotides. Duplex region (214) of bridging oligonucleotide (210) contains atleast a primer binding site for RCR and, in some embodiments, sequences that provide complementsto a capture oligonucleotide, which may be the same or different from the primer binding site 25 sequence, or which may overlap the primer binding site sequence. The length of capture oligonucleotides may vary widely, In one aspect, capture oligonucleotides and their complements in abridging oligonucleotide have lengths in the range of from 10 to 100 nucleotides; and morepreferably, in the range of from 10 to 40 nucleotides. In some embodiments, duplex region (214) maycontain additional elements, such as an oligonucleotide tag, for example, for identifying the source 30 nucleic acid from which its associated DNA fragment came. That is, in some embodiments, circles oradaptor ligation or concatemers from different source nucleic acids may be prepared separately duringwhich a bridging adaptor containing a unique tag is used, after which they are mixed for concatemerpreparation or application to a surface to produce a random array. The associated fragments may beidentified on such a random array by hybridizing a labeled tag complement to its corresponding tag 35 sequences in the concatemers, or by sequencing the entire adaptor or the tag region of the adaptor.

Circular products (218) may be conveniently isolated by a conventional purification column, digestionof non-circular DNA by one or more appropriate exonucleases, or both. -17- WO 2006/138257 PCT/US2006/022895

As mentioned above, DNA fragments of the desired sized range, e.g. 50-600 nucleotides, canalso be circularized using circularizing enzymes, such as CircLigase, as single stranded DNA ligasethat circularizes single stranded DNA without the need of a template. CircLigase is used inaccordance with the manufacturer’s instructions (Epicentre, Madison, WI). A preferred protocol for 5 forming single stranded DNA circles comprising a DNA fragment and one or more adapters is to usestandard ligase such as T4 ligase for ligation an adapter to one end of DNA fragment and than to useCircLigase to close the circle, as described more fully below.

An exemplary protocol for generating a DNA circle comprising an adaptor oligonucleotideand a target sequence using T4 ligase. The target sequence is a synthetic oligo TIN (sequence : 5- 10 ‘ NNNNNNNNGCATANCACGANGTCAINATCGTNCAAACGTCAGTCCANGAATCNAGATCCACTTAGANTGNCGNNNNNNNN-3r)(SEQ ID NO: 1). The adaptor is made up of 2 separate oligos.The adaptor oligo that joins to the 5’ end of TIN is BR2-ad (sequence : 5’- TATCATCTGGATGTTAGGAAGACAAAAGGAAGCTGAGGACATTAACGGAC-3’) (SEQ EDNO: 2) and the adaptor oligo that joins to the 3' end of TIN is UR3-ext (sequence: 5'- 15 ACCTTCAGACCAGAT-3 *) (SEQ ID NO: 3) UR3-ext contains a type Ils restriction enzyme site (Acu I: CTTCAG) to provide a way to linearize the DNA circular for insertion of a second adaptor.BR2-ad is annealed to BR2-temp (sequence 5-NNNNNNNGTCCGTTAATGTCCTCAG-3') (SEQ IDNO: 4) to form a double-stranded adaptor BR2 adaptor. UR3-ext is annealed to biotinylated UR3-temp (sequence 5'-[BIOTIN]ATCTGGTCTGAAGGTNNNNNNN-3f) (SEQ ID NO: 5) to form a 20 double-stranded adaptor UR3 adaptor. 1 pmol of target TIN is ligated to 25 pmol of BR2 adaptor and10 pmol of LJR3 adaptor in a single ligation reaction containing 50mM Tris-Cl, pH7.8, 10% PEG,lmM ATP, 50 mg/L BSA, lOmM MgCl2, 0.3 unit/μΐ T4 DNA ligase (Epicentre Biotechnologies, WI)and 10 mM DTT) in a final volume of 10 ul. The ligation reaction is incubated in a temperaturecycling program of 15°C for 11 min, 37°C for 1 min repeated 18 times. The reaction is terminated by

25 heating at 70°C for 10 min. Excess BR2 adaptors are removed by capturing the ligated products withstreptavidin magnetic beads (New England Biolabs, MA). 3.3 ul of 4x binding buffer (2M NaCl, 80mM Tris HCI pH7.5) is added to the ligation reaction which is then combined with 15pg ofstreptavidin magnetic beads in lx binding buffer (0.5M NaCl, 20 mM Tris HCI pH7.5). After 15 minincubation in room temperature, the beads are washed twice with 4 volumes of low salt buffer (0.15M 30 NaCl, 20 mM Tris HCI pH7.5). Elution buffer (10 mM Tris HCI pH7.5) is pre-warmed to 70 deg, 10μϊ of which is added to the beads at 70’C for 5 min. After magnetic separation, the supernatant isretained as primary purified sample. This sample is further purified by removing the excess UR3adaptors with magnetic beads pre-bound with a biotinylated oligo BR-rc-bio (sequence: 5'-[BIOTINJCTTTTGTCTTCCTAACATCC-3') (SEQ ID NO: 6) that is reverse complementary to 35 BR2-ad similarly as described above. The concentration of the adaptor-target ligated product in thefinal purified sample is estimated by urea polyacrylamide gel electrophoresis analysis. Thecircularization is carried out by phosphoiylating the ligation products using 0.2unit/pl T4 -18- WO 2006/138257 PCT/US2006/022895 polynucleotide kinase (Epicentre Biotechnologies) in 1 mM ATP and standard buffer provided by thesupplier, and circularized with ten-fold molar excess of a splint oligo UR3-closing-88 (sequence 5'-AGATGATAATCTGGTC-31) (SEQ ID NO: 7) using 0.3 unit/pl of T4 DNA ligase (EpicentreBiotechnologies) and ImM ATP. The circularized product is validated by performing RCR reactions 5 as described below.

Generating Polynucleotide Concatemers by Rolling Circle Replication

In one aspect of the invention, single molecules comprise concatemers of 10 polynucleotides, usually polynucleotide analytes, i.e. target sequences, that have been produce in aI conventional rolling circle replication (RCR) reaction. Guidance for selecting conditions and reagents ! for RCR reactions is available in many references available to those of ordinary skill, as evidence by the following that are incorporated by reference: Kooi, U.S. patent 5,426,180; Lizardi, U.S. patents5,854,033 and 6,143,495; Landegren, U.S. patent 5,871,921; and the like. Generally, RCR reaction I 15 components comprise single stranded DNA circles, one or more primers that anneal to DNA circles, a ί DNA polymerase having strand displacement activity to extend the 3 * ends of primers annealed to DNA circles, nucleoside triphosphates, and a conventional polymerase reaction buffer. Suchcomponents are combined under conditions that permit primers to anneal to DNA circles and beextended by the DNA polymerase to form concatemers of DNA circle complements. An exemplary 20 RCR reaction protocol is as follows: In a 50 pL reaction mixture, the following ingredients areassembled: 2-50 pmol circular DNA, 0.5 units/pL phage φ29 DNA polymerase, 0.2 pg/pL BSA, 3mM dNTP, IX φ29 DNA polymerase reaction buffer (Amersham). The RCR reaction is carried outat 30°C for 12 hours. In some embodiments, the concentration of circular DNA in the polymerasereaction may be selected to be low (approximately 10-100 billion circles per ml, or 10-100 circles per 25 picoliter) to avoid entanglement and other intermolecular interactions.

Preferably, concatemers produced by RCR are approximately uniform in size; accordingly, in some embodiments, methods of making arrays of the invention may include a step of size-selectingconcatemers. For example, in one aspect, concatemers are selected that as a population have acoefficient of variation in molecular weight of less than about 30%; and in another embodiment, less 30 than about 20%. In one aspect, size uniformity is further improved by adding low concentrations ofchain terminators, such ddNTPs, to the RCR reaction mixture to reduce the presence of veiy largeconcatemers, e.g. produced by DNA circles that are synthesized at a higher rate by polymerases. Inone embodiment, concentrations of ddNTPs are used that result in an expected concatemer size in therange of from 50-250 Kb, or in the range of from 50-100 Kb. In another aspect, concatemers may be / 35 enriched for a particular size range using a conventional separation techniques, e.g. size-exclusion j chromatography, membrane filtration, or the like.

I r. -19- 188142/2

Generation of Macromolecular Structures

Comprising Branched Polymers and DNA Assemblies

In one aspect of the invention, macromolecular structures comprise polymers having at least5 one unique functionality, which for polynucleotides is usually a functionality at a 5’ or 3’ end, and a plurality of complementary functionalities that are capable of specifically reacting with reactivefunctionalites of the surface of a solid support. Macromolecular structures comprising branchedpolymers, especially branched polynucleotides, may be synthesized in a variety of ways, as disclosedby Gryaznov (cited above), Urdea (cited above), and like references. In one aspect, branched 10 polymers of the invention include comb-type branched polymers, which comprise a linear polymericunit with one or more branch points located at interior monomers and/or linkage moieties. Branchedpolymers of the invention also include fork-type branched polymers, which comprise a linearpolymeric unit with one or two branch points located at terminal monomers and/or linkage moieties.Macromolecular structures of the invention also include assemblies of linear and/or branched 15 polynucleotides bound together by one or more duplexes or triplexes. Such assemblies may be self-assembled from component linear polynucleotide, e.g. as disclosed by Goodman et al, Science, 310:1661-1665 (2005); Birac etal, J. Mol. Graph Model, (April 18, 2006); Seemanet al, U.S. patent6,255,469; and the like, which are incorporated herein by reference. In one aspect, linear polymericunits of the invention have the form: (M-L)n - ‘ wherein L is a linker moiety and M is a monomer 20 that may be selected from a wide range of chemical structures to provide a range of functions fromserving as an inert non-sterically hindering spacer moiety to providing a reactive functionality whichcan serve as a branching point to attach other components, a site for attaching labels; a site forattaching oligonucleotides or other binding polymers for hybridizing or binding to amplifier strands orstructures, e.g. as described by Urdea et al, U.S. Pat. No. 5,124,246 or Wang et al, U.S. Pat. No. 25 4,925,785; a site for attaching "hooks", e.g. as described in Whiteley et al, U.S. Pat, No. 4,883,750; or as a site for attaching other groups for affecting solubility, promotion of duplex and/or triplexformation, such as intercalators, alkylating agents, and the like. The following references discloseseveral phosphoramidite and/or hydrogen phosphonate monomers suitable for use in the presentinvention and provide guidance for their synthesis and inclusion into oligonucleotides: Newton et al, 30 Nucleic Acids Research, 21:1155-1162 (1993); Griffin et al, J. Am. Chem. Soc., 114:7976-7982(1992); Jaschke et al, Tetrahedron Letters, 34:301-304 (1992); Ma et al, International publicationWO/1993/006122; Zon et al, International publication WO/1991/007092 ; Durand et al, NucleicAcids Research, 18:6353-6359 (1990); Salunkhe et al, J, Am. Chem. Soc., 114:8768-8772 (1992);Urdea et al, U.S. Pat. No. 5,093,232; Ruth, U.S. Pat. No. 4,948,882; Cruickshank, U.S. Pat. No. 35 5,091,519; Haralambidis et al, Nucleic Acids Research, 15:4857-4876 (1987); and the like. More particularly, M is a straight chain, cyclic, or branched organic molecular structure containing from 1to 20 carbon atoms and from 0 to 10 heteroatoms selected from the group consisting of oxygen,nitrogen, and -20- WO 2006/138257 PCT/US2006/022895

sulfur. Preferably, M is alkyl, alkoxy, alkenyl, or aryl containing from 1 to 16 carbon atoms;heterocyclic having from 3 to 8 carbon atoms and from 1 to 3 heteroatoms selected from the groupconsisting of oxygen, nitrogen, and sulfur; glycosyl; or nucleosidyl. More preferably, M is alkyl,alkoxy, alkenyl, or aryl containing from 1 to 8 carbon atoms; glycosyl; or nucleosidyl. Preferably, L 5 is aphosphorus(V) linking group which may be phosphodiester, phosphotrxester, methyl or ethyl phosphonate, phosphorothioate, phosphorodithioate, phosphoramidate, or the like. Generally, linkagesderived from pho sphoramidite or hydrogen phosphonate precursors are preferred so that the linearpolymeric units of tire invention can be conveniently synthesized with commercial automated DNAsynthesizers, e.g. Applied Biosystems, Inc. (Foster City, Calif.) model 394, or the like, n may vary 10 significantly depending on the nature ofM andL. Usually, n varies from about 3 to about 100. WhenM is a nucleoside or analog thereof or a nucleoside-sized monomer and L is a phosphorus(V) linkage,then n varies from about 12 to about 100. Preferably, when M is a nucleoside or analog thereof or anucleoside-sized monomer and L is a phosphorus(V) linkage, then n varies from about 12 to about 40.Polymeric units are assembled by forming one or more covalent bridges among them. In one aspect, 15 bridges are formed by reacting thiol, phosphorothioate, or phosphorodithioate groups on one or morecomponents with haloacyl- or haloalkylamimo groups on one or more other components to form oneor more thio- or dithiophosphorylacyl or thio- or dithiophosphoiylalkyi bridges. Generally, suchbridges have one of the following forms: —NHRSP(M)(O')—' OR —NHRS—, wherein R is alkyl oracyl and Z is sulfur or oxygen. The assembly reaction may involve from 2 to 20 components 20 depending on the particular embodiment; but preferably, it involves from 2 to 8 components; andmore preferably, it involves from 2 to 4 components. Preferably, the haloacyl. or haloalkylaminogroups are haloacetylamino groups; and more preferably, the haloacetylamino groups arebromoacetyl amino groups. The acyl or alkyl moieties of the haloacyl- or haloalkylamino groupscontain from 1 to 12 carbon atoms; and more preferably, such moieties contain from 1 to 8 carbon 25 atoms. The reaction may take place in a wide range of solvent systems; but generally, the assemblyreaction takes place under liquid aqueous conditions or in a frozen state in ice, e.g. obtained bylowering the temperature of a liquid aqueous reaction mixture. Alternatively, formation ofthiophosphorylacetylamino bridges in DMSO/H2O has been reported by Thuong et al, TetrahedronLetters, 28:4157-4160 (1987); and Francois etal, Proc. Natl. Acad. Sci., 86:9702-9706 (1989). 30 Typical aqueous conditons include 4 μΜ of reactants in 25 mM NaCl and 15 mM phosphate buffer(pH 7.0). The thio- or dithiophosphorylacyl- or thio- or dithiophosphorylalky] amino bridges arepreferred because they can be readily and selectively cleaved by oxidizing agents, such as silvernitrate, potassium iodide, and the like. Preferably, the bridges are cleaved with potassium iodide, Kl3,at a concentration equivalent to about a hundred molar excess of tire bridges. Usually, a I<I3 Is 35 employed at a concentration of about 0.1M. The facile cleavage of these bridges is a great advantagein synthesis of complex macromolecular structures, as it provides a convenient method for analyzingfinal products and for confirming that the structure of the final product is correct. A 3’-haloacyl- or -21 - Γ.—-.w. r '-: v··;_._··: ,7* ·:τ·:?ν=ζ<- --. -. W Wv <·· W^·· U--W- W···'^.: 188142/2 haloalkylamino (in this example, haloacetylamino) derivatized oligonucleotide 1 is reacted with a 5’-phosphorothioate derivatized oligonucleotide 2 according to the following scheme: 5’-BBB ... B-NHC(=O)CH2X + (1) 5 S"P(=O)(O-)-BBB ... B-3’ _» (2) 5’-BBB ... B-NHC(=O)CH2SP(=O)(O-)O-BBB ... B-3’ wherein X is halo and B is a nucleotide. It is understood that the nucleotides are merely exemplary ofthe more general polymeric units, (M-L)n described above. Compound 1 can be prepared by reacting 10 N-succimmidyl haloacetate in Ν,Ν-dimethylformamide (DMF) with a 3'-aminodeoxyribonucleotideprecursor in a sodium borate buffer at room temperature. After about 35 minutes the mixture is diluted(e.g. with H2 O), desalted and, purified, e.g. by reverse phase HPLC. The Y- aminodeoxyribonucleotide precursor can be prepared as described in Gryaznov and Letsinger, NucleicAcids Research, 20:3403-3409 (1992). Briefly, after deprotection, the 5’ hydroxyl of a 15 deoxythymidine linked to a support via a standard succinyl linkage is phosphitylated by reaction withchloro-(diisopropylethy1amino)-methoxyphosphine in an appropriate solvent, such asdichloromethane/diisopropylethylamme. After activation with tetrazole, the 5-phosphitylatedthymidine is reacted with a 5’-trityl-O-3'-amino-3'-deoxynucleoside to form a nucleoside-thymidinedimer wherein the nucleoside moieties are covalently joined by a phosphoramidate linkage. The 20 remainder of the oligonucleotide is synthesized by standard phosphoramidite chemistry. Aftercleaving the succinyl linkage, the oligonucleotide with a 3' terminal amino group is generated bycleaving the phosphoramidate link by acid treatment, e.g. 80% aqueous acetic acid for 18-20 hours atroom temperature. 5’-monophosphorothioate oligonucleotide 2 is formed as follows: A 5'monophosphate is attached to the 5’ end of an oligonucleotide either chemically or enzymatically with 25 a kinase, e.g. Sambrook et al, Molecular Cloning: A Laboratory Manual, 2nd Edition (Cold SpringHarbor Laboratory, New York, 1989). Preferably, as a final step in oligonucleotide synthesis, amonophosphate is added by chemical phosphorylation as described by Thuong and Asscline, Chapter12 in, Eckstein, editor, Oligonucleotides and Analogues (IRL Press, Oxford, 1991) or by Horn andUrdea, Tetrahedron Lett., 27:4705 (1986) (e.g. using commercially available reagents such as 5' 30 Phosphate-ON.TM. from Clontech Laboratories (Palo Alto, Calif.)). The S'-monophosphate is thensulfurized using conventional sulfurizing agents, e.g. treatment with a 5% solution of S8 inpyfidine/CS2 (1:1, v/v, 45 minutes at room temperature); or treatment with sulfurizing agent describedin U.S. Pat. Nos. 5,003,097; 5,151,510; or 5,166,387. Monophosphorodithioates are prepared byanalogous procedures, e.g. Froehler et al, European patent publication 0 360 609 A2; Caruthers et al, 35 International publication WO/1989/01I486; and the like. Likewise to the above, a 5’-haloacetylaminoderivatized oligonucleotide 3 is reacted with a 3’-monophosphorothioate oligonucleotide 4 accordingto the following scheme: -22- WO 2006/138257 PCT/US2006/022895 3’-ΒΒΒ ... B-NHCOOJCHaX + (3) S-P^OXCQO-BBB ... Β-5’ -> (4) 3’-ΒΒΒ ... B-NHC(=O)CH2SP(=OXO')-BBB ... Β-5’ 5 wherein the symbols are defined the same as above, except that the nucleotides monomers of the j-and k-mers are in opposite orientations. In this case, Compound 3 can be prepared by reacting N-succinimidyl haloacetate in Ν,Ν-dimethylformamide (DMF) with a 5-aminodeoxyribonucleotideprecursor in a sodium borate buffer at room temperature, as described above for the 3'-amino 10 oligonucleotide. 5'-aminodeoxynucleosides are prepared in accordance with Glinski et al, J. Chem.Soc. Chem. Comm., 915-916 (1970); Miller etal, J. Org. Chem. 29:1772 (1964); Ozols et al,Synthesis, 7:557-559 (1980); and Azhayev et al, Nucleic Acids Research, 6:625-643 (1979); whichare incorporated by reference. The 3'-monophosphorothioate oligonucleotide 4 can be prepared asdescribed by Thuong and Asscline (cited above). Oligonucleotides 1 and 4 and 2 and 3 may be 15 reacted to form polymeric units having either two 5’ termini or two 3’ termini, respectively.

Reactive functionalities for the attachment of branches may be introduced at a variety of sites.

Preferably, amino functionalities are introduce on a polymeric unit or loop at selected monomers orlinking moieties which are then converted to haloacetylamino groups as described above. Amino-derivatized bases of nucleoside monomers may be introduced as taught by Urdea et al, U.S. Pat. No. 20 5,093,232; Ruth U.S. Pat. No. 4,948,882; Haralatnbidis et al, Nucleic Acids Research, 15:4857-4876 (1987); or the like. Amino functionalities may also be introduced by a protected hydroxyaminephosphoramidite commercially available from Clontech Laboratories (Palo Alto, Calif.) asAminomodifier TI.TM.. Preferably, amino functionalities are introduced by generating a derivatizedphosphoramidate linkage by oxidation of a phosphite linkage with L and an alkyldiamine, e.g. as 25 taught by Agrawal et al, Nucleic Acids Research, 18:5419-5423 (1990); and Jager et al, Biochemistry,27:7237-7246 (1988). Generally, for the above procedures, it is preferable that the haloacyl- orhaloalkyl amino derivatized polymeric units be prepared separately from the phosphorothioatederivatized polymeric units, otherwise the phosphorothioate moieties require protective groups. 30 Solid Phase Surfaces for Constructing

Random Arrays A wide variety of supports may be used with the invention. In one aspect, supports are rigidsolids that have a surface, preferably a substantially planar surface so that single molecules to beinterrogated are in the same plane. The latter feature permits efficient signal collection by detection 35 optics, for example. In another aspect, solid supports of the invention are nonporous, particularlywhen random arrays of single molecules are analyzed by hybridization reactions requiring smallvolumes. Suitable solid support materials include materials such as glass, polyaciylamide-coated -23- 7W- WO 2006/138257 PCT/US2G06/022895 glass, ceramics, silica, silicon, quartz, various plastics, and the like. In one aspect, the area of a planarsurface may be in the range of from 0.5 to 4 cm2. In one aspect, the solid support is glass or quartz,such as a microscope slide, having a surface that is uniformly silanized. This may be accomplishedusing conventional protocols, e.g. acid treatment followed by immersion in a solution of 3- 5 glycidoxypropyl trimethoxysilane, Ν,Ν-diisopropylethylamine, and anhydrous xylene (8:1:24 v/v) at80oC, which forms an epoxysilanized surface, e.g. Beattie et a (1995), Molecular Biotechnology, 4:213. Such a surface is readily treated to permit end-attachment of capture oligonucleotides, e.g. byproviding capture oligonucleotides with a 3’ or 5’ triethylene glycol phosphoryl spacer (see Beattie etal, cited above) prior to application to the surface. Many other protocols may be used for adding 10 reactive fiinctionalites to glass and other surfaces, as evidenced by the disclosure in Beaucage (citedabove).

Whenever enzymatic processing is not required, capture oligonucleotides may comprise non-natural nucleosidic units and/or linkages that confer favorable properties, such as increased duplexstability; such compounds include, but not limited to, peptide nucleic acids (PNAs), locked nucleic 15 acids (LNA), oligonucleotide N3'-»P5' phosphoramidates, oligo-2'-0-alkylribonucleotides, and thelike.

In embodiments of the invention in which patterns of discrete spaced apart regions arerequired, photolithography, electron beam lithography, nano imprint lithography, and nano printingmay be used to generate such patterns on a wide variety of surfaces, e.g. Pirrung et al, U.S. patent 20 5,143,854; Fodor et al, U.S. patent 5,774,305; Guo, (2004) Journal of Physics D: Applied Physics, 37: R123-141; which are incorporated herein by reference.

In one aspect, surfaces containing a plurality of discrete spaced apart regions are fabricated byphotolithography. A commercially available, optically flat, quartz substrate is spin coated with a 100-500nm thick layer of photo-resist. The photo-resist is then baked on to the quartz substrate. An 25 image of a reticle with a pattern of regions to be activated is projected onto the surface of the photo-resist, using a stepper. After exposure, the photo-resist is developed, removing the areas of theprojected pattern which were exposed to the UV source. This is accomplished by plasma etching, adry developing technique capable of producing very fine detail. The substrate is then baked tostrengthen the remaining photo-resist. After baking, the quartz wafer is ready for functionalization. 30 The wafer is then subjected to vapor-deposition of 3-aminopropyldimethyl ethoxysilane. The densityof the amino functionalized monomer can be tightly controlled by varying the concentration of themonomer and the time of exposure of the substrate. Only areas of quartz exposed by the plasmaetching process may react with and capture the monomer. The substrate is then baked again to curethe monolayer of amino-functionalized monomer to die exposed quartz. After baking, the remaining 35 photo-resist may be removed using acetone. Because of the difference in attachment chemistrybetween the resist and silane, aminosilane-functionalized areas on the substrate may remain intactthrough the acetone rinse. These areas can be further functionalized by reacting them with p- -24- WO 2006/138257 PCT/US2006/022895 phenylenediisothiocyanate in a solution of pyridine and N-N-dimethlyformamide. The substrate isthen capable of reacting with amine-modified oligonucleotides. Alternatively, oligonucleotides canbe prepared with a 5’-carboxy-modifier-cl0 linker (Glen Research). This technique allows theoligonucleotide to be attached directly to the amine modified support, thereby avoiding additional 5 functionalization steps.

In another aspect, surfaces containing a plurality of discrete spaced apart regions arefabricated by nano-imprint lithography (NIL). For DNA array production, a quartz substrate is spincoated with a layer of resist, commonly called the transfer layer. A second type of resist is thenapplied over the transfer layer, commonly called the imprint layer. The master imprint tool then 10 makes an impression on the imprint layer. The overall thickness of the imprint layer is then reduced by plasma etching until the low areas of the imprint reach tlie transfer layer. Because the transferlayer is harder to remove than the imprint layer, it remains largely untouched. The imprint andtransfer layers are then hardened by heating. The substrate is then put into a plasma etcher until thelow areas of the imprint reach the quartz. The substrate is then derivatized by vapor deposition as 15 described above.

In another aspect, surfaces containing a plurality of discrete spaced apart regions arefabricated by nano printing. This process uses photo, imprint, or e-beam lithography to create amaster mold, which is a negative image of the features required on the print head. Print heads are .usually made of a soft, flexible polymer such as polydimethylsiloxane (PDMS). This material, or 20 layers of materials having different properties, are spin coated onto a quartz substrate. The mold isthen used to emboss the features onto the top layer of resist material under controlled temperature andpressure conditions. The print head is then subjected to a plasma based etching process to improvethe aspect ratio of the print head, and eliminate distortion of the print head due to relaxation over timeof the embossed material. Random array substrates are manufactured using nano-printing by 25 depositing a pattern of amine modified oligonucleotides onto a homogenously derivatized surface.

These oligo-nucleotides would serve as capture probes for the RCR products. One potentialadvantage to nano-printing is the ability to print interleaved patterns of different capture probes ontothe random array support. This would be accomplished by successive printing with multiple printheads, each head having a differing pattern, and all patterns fitting together to form tlie final structured 30 support pattern. Such methods allow for some positional encoding of DNA elements within therandom array. For example, control concatemers containing a specific sequence can be bound atregular intervals throughout a random array.

In still another aspect, a high density array of capture oligonucleotide spots of sub micron sizeis prepared using a printing head or imprint-master prepared from a bundle, or bundle of bundles, of 35 about 10,000 to 100 million optical fibers with a core and cladding material. By pulling and fusingfibers a unique material is produced that has about 50-1000 nm cores separated by a similar or 2-5fold smaller or larger size cladding material. By differential etching (dissolving) of cladding material -25- WO 2006/138257 PCT/US2006/022895 a nano-printing head is obtained having a veiy large number of nano-sized posts. This printing headmay be used for depositing oligonucleotides or other biological (proteins, oligopeptides, DNA,aptamers) or chemical compounds such as silane with various active groups. In one embodiment theglass fiber tool is used as a patterned support to deposit oligonucleotides or other biological or 5 chemical compounds, In this case only posts created by etching may be contacted with material to bedeposited. Also, a flat cut of the fused fiber bundle may be used to guide light through cores and allowlight-induced chemistry to occur only at the tip surface of the cores, thus eliminating the need foretching. In both cases, the same support may then be used as a light guiding/collection device forimaging fluorescence labels used to tag oligonucleotides or other reactants. This device provides a 10 large field of view with a large numerical aperture (potenti al ly > 1). Stamping or printing tools that perform active material or oligonucleotide deposition may be used to print 2 to 100 differentoligonucleotides in an interleaved pattern. This process requires precise positioning of the print headto about 50-500 nm. This type of oligonucleotide array may be used for attaching 2 to 100 differentDNA populations such as different source DNA. They also may be used for parallel reading from sub-

15 light resolution spots by using DNA specific anchors or tags. Information can be accessed by DNA specific tags, e.g. 16 specific anchors for 16 DNAs and read 2 bases by a combination of 5-6 colorsand using 16 ligation cycles or one ligation cycle and 16 decoding cycles. This way of making arraysis efficient if limited information (e.g. a small number of cycles) is required per fragment, thusproviding more information per cycle or more cycles per surface. 20 In one embodiment “inert” concatemers are used to prepare a surface for attachment of test concatemers. The surface is first covered by capture oligonucleotides complementary to the bindingsite present on two types of synthetic concatemers; one is a capture concatemer, the other is a spacerconcatemer. The spacer concatemers do not have DNA segments complementary to the adapter usedin preparation of test concatemers and they are used in about 5-50, preferably 10 x excess to capture 25 concatemers. The surface with capture oligonucleotide is “saturated” with a mix of synthetic concatemers (prepared by chain ligation or by RCR) in which the spacer concatemers are used inabout 10 -fold (or 5 to 50-fold) excess to capture concatemers. Because of the -10:1 ratio betweenspacer and capture concatemers, the capture concatemers are mostly individual islands in a sea ofspacer concatemers. The 10:1 ratio provides that two capture concatemers are on average separated by 30 two spacer concatemers. If concatemers are about 200 nm in diameter, then two capture concatemersare at about 600 nm center-to-center spacing. This surface is then used to attach test concatemers orother molecular structures that have a binding site complementary to a region of the captureconcatemers but not present on the spacer concatemers. Capture concatemers may be prepared to haveless copies than the number of binding sites in test concatemers to assure single test concatemer 35 attachment per capture concatemer spot. Because the test DNA can bind only to capture concatemers,an array of test concatemers may be prepared that have high site occupancy without congregation.

Due to random attachment, some areas on the surface may not have any concatemers attached, but -26- w —-W. ATT·'— WO 2006/138257 PCT/US2006/022895 these areas with free capture oligonucleotide may not be able to bind test concatemers since they aredesigned not to have binding sites for the capture oligonculeotide. An array of individual testconcatemers as described would not be arranged in a grid pattern. An ordered grid pattern shouldsimplify data collection because less pixels are needed and less sophisticated image analysis systems 5 are needed also.

In one aspect, multiple arrays of the invention may be place on a single surface. For example,patterned array substrates may be produced to match die standard 96 or 384 well plate format. Aproduction format can be an 8 x 12 pattern of 6mm x 6mm arrays at 9mm pitch or 16x24 of 3.33mm x3.33mm array at 4.5mm pitch, on a single piece of glass or plastic and other optically compatible 10 material. In one example each 6mm x 6mm array consists of 36 million 250-500nm square regions at1 micrometer pitch. Hydrophobic or other surface or physical barriers may be used to prevent mixingdifferent reactions between unit arrays.

By way of example, binding sites (i.e. discrete spaced apart regions) for DNA samples areprepared by silanization of lithographically defined sites on silicon dioxide on silicon, quartz, or glass 15 surfaces with 3-aminopropyldimethylethoxysilane or similar silanization agent followed by derivatization with p-phenylenediisothiocyanate or similar derivatization agent. For example, thebinding sites may be square, circular or regular/irregular polygons produced by photolithography,direct-write electron beam, or nano-imprint lithography. Minimization of non-specific binding inregions between binding site The wetability (hydrophobic v.hydrophilic) and reactivity of the field

20 surrounding the binding sites can be controlled to prevent DNA samples from binding in the field;that is, in places other than the binding sites. For example, the field may be prepared withhexamethyldisilazane (HMDS), or a similar agent covalently bonded to the surface, to be hydrophobicand hence unsuitable to hydrophilic bonding of the DNA samples. Similarly, the field may be coatedwith a chemical agent such as a fluorine-based carbon compound that renders it unreactive to DNA 25 samples.

For the three surface fabrication processes listed in the prior paragraph, the follow exemplarysteps are followed. For photolithography; 1) Clean glass wafer

2) Prime surface with HMDS 30 3) Pattern binding sites in photoresist

4) Reactive ion etch binding site surface with oxygen to remove HMDS 5) Silanize with .3% 3-aminopropyldimethylethoxysilane 6) Coat with photoresist to protect wafer during sawing 7) Saw wafer into chips 35 8) Strip photoresist 9) Derivatize binding sites with solution of 10% pyridine and 90% Ν,Ν-Dimethylformaide (DMF)using 2.25mg p-phenylenediisothiocyanate (PDC) per ml of solution for 2h followed by methanol, -27- WO 2006/138257 PCT/US2006/022895 acetone, and water rinses

For direct write electron beam surface fabrication:

J) Clean glass wafer. 2) Prime surface with HMDS 5 3) Pattern binding sites in PMMA with electron beam

4) Reactive ion etch binding site surface with oxygen to remove HMDS 5) Silanize with .3% 3-aminopropyldimethylethoxysilane 6) Coat with photoresist to protect wafer during sawing ' 7) Saw wafer into chips 10 8) Strip photoresist 9) Derivatize binding sites with solution of 10% pyridine and 90% N,N Dimethylformaide (DMF)using 2.25mg p-phenylenediisothiocyanate (PDC) per ml of solution for 2h followed by methanol,acetone, and water rinses.

For nano imprint lithography surface fabrication: 15 1) Clean glass wafer

2) Prime surface with HMDS 3) Coat wafer with transfer layer 4) Contact print pattern with nano imprint template and photopolymer on top of transfer layer 5) Dry etch pattern into transfer layer

20 6) Reactive ion etch binding site surface with oxygen to remove HMDS 7) Silanize with .3% 3-aminopropyIdimethylethoxysilane 8) Coat with photoresist to protect wafer during sawing 9) Saw wafer into chips 10) Strip photoresist 25 11) Derivatize binding sites with solution of 10% pyridine and 90% N,N Dimethylformaide (DMF) using 2.25mg p-phenylenediisothiocyanate (PDC) per ml of solution for 2h followed by methanol,acetone, and water rinses.

As mentioned above, a glass surface may also be used for constructing random arrays of theinvention. For example, a suitable glass surface may be constructed from microscope cover slips. 30 Microscope cover slips (22mm sq ~170um thick) are placed in Teflon racks. They are soaked in 3molar KOH in 95% ethanol/water for 2 minutes. They are then rinsed in water, followed by anacetone rinse. This removes surface contamination and prepares the glass for silanization. Plasmacleaning is an alternative to KOH cleaning. Fused silica or quartz may also be substituted for glass.The clean, dry cover slips are immersed in .3% 3-aminopropyldimethylethoxysilane, .3% water, in 35 acetone. They are left to react for 45 minutes. They are then rinsed in acetone and cured at 100°C for1 hour. 3-aminopropyldimethylethoxysilane may be used as a replacement for 3-aminopropyltriethoxy silane because it forms a mono-layer on the glass surface. The monolayer -28- WO 2006/138257 PCT/US2006/022895 surface provides a lower background. The silanization agent may also be applied using vapordeposition. 3-aminopropyltriethoxysilane tends to form more of a polymeric surface when deposited in solutionphase. The amino modified silane is then terminated with a thiocyanate group. This is done in a 5 solution of 10% pyridine and 90% Ν,Ν-Dimethylformaide (DMF) using 2.25mg p- phenylenediisothiocyanate (PDC) per ml of solution. The reaction is run for 2 hours, then the slide iswashed in methanol, followed by acetone, and water rinses. The cover slips are then dried and readyto bind probe. There are additional chemistries that can be used to modify the amino group at the endof the silanization agent. For example, glutaraldehyde can be used to modify the amino group at the 10 end of the silanization agent to a aldehyde group which can be coupled to an amino modified oligonucleotide. ^Capture oligonucleotides are bound to the surface of the cover slide by applying asolution of 10-50 micromolar capture oligonucleotide in 100 millimolar sodium bicarbonate in waterto the surface. The solution is allowed to dry, and is then washed in water.

It may be beneficial to avoid terminating the 3-amino group with PDC and perform a direct 15 conj ugation (of the 3-amino end) to the capture oligonucleotide which has been modified with either a carboxyl group or an aldehyde group at the 5’ end. In the case of the carboxyl group, theoligonucleotide is applied in a solution that contains EDC ( l-Ethyl-3-(3-dimethyl aminopropyl)-carbodiimide ). In the case of the aldehyde group, the oligo is kept wet for 5-10 minutes then thesurface is treated with a 1% solution of sodium borohydride. 20 In another aspect of the invention, random arrays are prepared using nanometer-sized beads.

Sub-micron glass or other types of beads (e.g. in the 20-50nm range) are used which are derivatizedwith a short oligonucleotide, e.g. 6-30 nucleotides, complementary to an adaptor oligonucleotide inthe circles used to generate concatemers. The number of oligonucleotides on the bead and the lengthof the sequence can be controlled to weakly bind the concatemers in solution. Reaction rate of the 25 beads should be much faster than that of the solid support alone.

After binding concatemers, the beads are then allowed to settle on the surface of an array substrate.The array substrate has longer, more stable, more numerous oligonucleotides, such that conditionsmay be selected to permit preferential binding to the surface, thereby forming a spaced array ofconcatemers. If the beads are magnetic, a magnetic field can be used to pull them to the surface, it 30 may also be used to move them around the surface. Alternatively, a centrifuge may be used to concentrate the beads on the surface. An exemplary protocol is as follows: 1. A preparation of 20 ulof concatemer solution with one million concatemers per lul is mixed with 20 million nano-beadswith about 500 capture oligonucleotides about 8 bases in length (6-16 bases may be use underdifferent conditions). A 100 nm nano-bead there is approximately 40,000 nm2 and can hold up to 35 4000 short oligonucleotides. One way to control the density of capture probes is to mix in this case about 8 times more of a 2-4 bases long oligonucleotieds with the same attachment chemistry with thecapture probe. Also, much smaller nano-beads (20-50 nm) may be used. 2. Reaction conditions -29- WO 2006/138257 PCT/US2006/022895 (temperature, pH, salt concentration) are adjusted so that concatemers with over 300 copies will attachto nanobeads in significant numbers. 3. The reaction is applied under the same stringent conditions toa support with 4x4 mm of patterned surface with 16 million active sites about 200 nm in size, andnanobeads are allowed or forced to settle on the substrate surface bringing large concatemers with 5 them. The largest distance that a nano-bead-concatemer has to travel is about 1mm. The vertical movement of beads minimizes number of potential concatemer-concatemer encounters. The reactionsolution may be applied in aliquots, e.g. 4 applications 5 ul each. In this case the thickness of theapplied solution (e.g. the nano-bead maximal travel distance) is only about 250 microns. 4. Furtherincrease stringency of the reaction to release concatemers from nano-beads and attach them to active 10 sites on the support with ~300 capture oligonucleotides 20-50 bases in length. 5. Concatemers attached to nano-beads will predominately settle initially between active sites on the support becausethere are 25 times more inactive than active surface. Slight horizontal movement force (e.g. substratetilting, and other forces), may be applied to move nano-bead-concatemers about one to a few micronsaround. 15

Detection Instrumentation

As mentioned above, signals from single molecules on random arrays made in accordancewith the invention are generated and detected by a number of detection systems, including, but notlimited to, scanning electron microscopy, near field scanning optical microscopy (NSOM), total 20 internal reflection fluorescence microscopy (TIRFM), and the like. Abundant guidance is found inthe literature for applying such techniques for analyzing and detecting nanoscale structures onsurfaces, as evidenced by the following references that are incorporated by reference: Reimer et al,editors, Scanning Electron Microscopy: Physics of Image Formation and Microanalysis, 2nd Edition(Springer, 1998); Nie et al, Anal. Chem., 78:1528-1534 (2006); Hecht et al, Journal Chemical 25 Physics, 112: 7761-7774 (2000); Zhu et al, editors, Near-Field Optics: Principles and Applications(World Scientific Publishing, Singapore, 1999); Drmanac, International patent publication WO2004/076683; Lehr et al, Anal. Chem., 75:2414-2420 (2003); Neuschafer et al, Biosensors &Bioelectronics, 18: 489-497 (2003); Neuschafer et al, U.S. patent 6,289,144; and the like. Ofparticular interest is TIRFM, for example, as disclosed by Neuschafer et al, U.S. patent 6,289,144; 30 Lehr et al (cited above); and Drmanac, International patent publication WO 2004/076683. In oneaspect, instruments for use with arrays of the invention comprise three basic components: (i) afluidics system for storing and transferring detection and processing reagents, e.g. probes, washsolutions, and the like, to an array; (ii) a reaction chamber, or flow cell, holding or comprising anarray and having flow-through and temperature control capability; and (iii) an illumination and 35 detection system. In one embodiment, a flow cell has a temperature control subsystem with ability tomaintain temperature in the range from about 5-95°C, or more specifically 10-85°C, and can changetemperature with a rate of about 0.5-2°C per second. -30- WO 2006/138257 PCT/US2006/022895

In one aspect, a flow cell for 1’’square 170 micrometer thick cover slips can be used that hasbeen derivatized to bind macromolecular structures of the invention. The cell encloses the “array” bysandwiching the glass and a gasket between two planes. One plane has an opening of sufficient sizeto permit imaging, and an indexing pocket for the cover slip. The other plane has an indexing pocket 5 . for the gasket, fluid ports, and a temperature control system. One fluid port is connected to a syringepump which “pulls” or ’’pushes” fluid from the flow cell the other port is connected to a funnel likemixing chamber. The chamber, in turn is equipped with a liquid level sensor. The solutions aredispensed into the funnel, mixed if needed, then drawn into the flow cell. When the level sensor readsair in the funnels connection to the flow cell the pump is reversed a known amount to back the fluid 10 up to the funnel. This prevents air from entering the flow cell. The cover slip surface may besectioned off and divided into strips to accommodate fluid flow/capillaiy effects caused bysandwiching. Such substrate may be housed in an “open air” I “open face” chamber to promote evenflow of the buffers over the substrate by eliminating capillary flow effects. Imaging may beaccomplished with a lOOx objective using TIRF or epi illumination and a 1.3 mega pixel Hamamatsu 15 orca-er-ag on a Zeiss axiovert 200, or like system. This configuration images RCR concatemersbound randomly to a substrate (non-ordered array). Imaging speed may be improved by decreasingthe objective magnification power, using grid patterned arrays and increasing the number of pixels ofdata collected in each image.

For example, up to four or more cameras may be used, preferably in the 10-16 megapixel range. 20 Multiple band pass filters and dichroic mirrors may also be used to collect pixel data across up to fouror more emission spectra. To compensate for the lower light collecting power of the decreasedmagnification objective, the power of the excitation light source can be increased. Throughput can beincreased by using one or more flow chambers with each camera, so that the imaging system is notidle while the samples are being hybridized/reacted. Because the probing of arrays can be non- 25 sequential, more than one imaging system can be used to collect data from a set of arrays, furtherdecreasing assay time.

During the imaging process, the substrate must remain in focus. Some key factors inmaintaining focus are the flatness of the substrate, orthogonality of the substrate to the focus plane,and mechanical forces on the substrate that may deform it. Substrate flatness can be well controlled, 30 glass plates which have better than Ά wave flatness are readily obtained. Uneven mechanical forceson the substrate can be minimized through proper design of tire hybridization chamber. Orthogonalityto the focus plane can be achieved by a well adjusted, high precision stage. Auto focus routinesgenerally take additional time to run, so it is desirable to run them only if necessary. After each imageis acquired, it will be analyzed using a fast algorithm to determine if the image is in focus. If the 35 image is out of focus, the auto focus routine will run. It will then store the objectives Z positioninformation to be used upon return to that section of that array during the next imaging cycle. By -31- WO 2006/138257 PCT/US2006/022895 mapping the objectives Z position at various locations on the substrate, we will reduce the timerequired for substrate image acquisition. A suitable illumination and detection system for fluorescence-based signal is a Zeiss Axiovert200 equipped with a TURF slider coupled to a 80 milliwatt 532 nm solid state laser. The slider 5 illuminates the substrate through the objective at the correct TIRF illumination angle. TIRF can alsobe accomplished without the use of the objective by illuminating the substrate though a prismoptically coupled to the substrate. Planar wave guides can also be used to implement TIRF on thesubstrate Epi illumination can also be employed. The light source can be rastered, spread beam,coherent, incoherent, and originate from a single or multi-spectrum source. 10 One embodiment for the imaging system contains a 20x lens with a 1.25mm field of view, with detection being accomplished with a 10 megapixel camera. Such a system images approx 1.5million concatemers attached to the patterned array at 1 micron pitch. Under this configuration thereare approximately 6.4 pixels per concatemer. The number of pixels per concatemer can be adjustedby increasing or decreasing the field of view of the objective. For example a 1mm field of view 15 would yield a value of 10 pixels per concatemer and a 2mm field of view would yield a value of 2.5pixels per concatemer. The field of view may be adjusted relative to the magnification and NA of theobjective to yield the lowest pixel count per concatemer that is still capable of being resolved by theoptics, and image analysis software.

Both TIRF and EPI illumination allow for almost any light source to be used. One 20 illumination schema is to share a common set of monochromatic illumination sources (about 4 lasersfor 6-S colors) amongst imagers. Each imager collects data at a different wavelength at any given timeand the light sources would be switched to the imagers via an optical switching system. In such anembodiment, the illumination source preferably produces at least 6, but more preferably 8 differentwavelengths. Such sources include gas lasers, multiple diode pumped solid state lasers combined 25 through a fiber coupler, filtered Xenon Arc lamps, tunable lasers, or the more novel Spectralum LightEngine, soon to be offered by Tidal Photonics. The Spectralum Light Engine uses prism to spectrallyseparate light. The spectrum is projected onto a Texas Instruments Digital Light Processor, which canselectively reflect any portion of the spectrum into a fiber or optical connector. This system is capableof monitoring and calibrating the power output across individual wavelengths to keep them constant 30 so as to automatically compensate for intensity differences as bulbs age or between bulb changes.

The following table represent examples of possible lasers, dyes and filters. laser excitation filter emission filter Dye 407nm 405/12 436/12 AIexa-405 401/421 407nm 405/12 546/10 cascade yellow 409/558 488nm 488/10 514/11 Alexa-488 492/517 543nm 546/10 540/565 Tamra 540/565 -32- WO 2006/138257 PCT/US2O06/022895 543nm 546/10 620/12 Bodipy 577/618 577/618 546/10 620/12 Alexa-594 594/613 635nm 635/11 650/11 Alexa-635 632/647 635nm 635/11 . Alexa700 702/723

Successfully scoring 6 billion concatemers through ~350 (~60 per color) images per regionover 24 hours may require a combination of parallel image acquisition, increased image acquisitionspeed, and increased field of view for each imager. Additionally, the imager may support between six 5 to eight colors. Commercially available microscopes commonly image a ~lmm field of view at 20xmagnification with an NA of 0.8. At the proposed concatemer pitch of 0.5 micron, this translates intoroughly 4 million concatemers per image. This yields approximately 1,500 images for 6 billion spotsper hybridization cycle, or 0.5 million images for 350 imaging cycles. In a large scale sequencingoperation, each imager preferably acquires —200,000 images per day, based on a 300 millisecond 10 exposure time to a 16 mega pixel CCD, Thus, a preferred instrument design is 4 imager modules eachserving 4 flow cells (16 flow cells total). The above described imaging schema assumes that eachimager has a CCD detector with 10 million pixels and be used with an exposure time of roughly 300milliseconds. This should be an acceptable method for collecting data for 6 fluorophor labels. Onepossible drawback to this imaging technique is that certain fluorophors may be unintentionally photo 15 bleached by the light source while other fluorophores are being imaged. Keeping the illumination power low and exposure times to a minimum would greatly reduce photo bleaching. By usingintensified CCDs (ICCDs) data could be collected of roughly the same quality with illuminationintensities and exposure times that are orders of magnitude lower than standard CCDs. ICCDs aregenerally available in the 1 -1.4 megapixel range. Because they require much shorter exposure times, 20 a one megapixel ICCD can acquire ten or more images in the time a standard CCD acquires a singleimage. Used in conjunction with fast filter wheels, and a high speed flow cell stage, a one mega pixelICCD should be able to collect the same amount of data as a 10 megapixel standard CCD.

Optics capable of imaging larger fields of view with high numerical apertures can bemanufactured as custom lens assemblies. Indications are that 20x optics capable of imaging a 3mm 25 field of view with a NA >0.9 can be fabricated. Two such imaging systems, in combination with highpixel count CCD’s or CCD mosaic arrays should be able to image the complete eight flow cell assayin roughly 14 hours. As described, further gains can be realized by using 16 flow cells. Doubling thenumber of flow cells would reduce imaging time to 9 hours by reducing the number of images pereach field of view. 30 The reaction efficiency on the concatemer and other random DNA arrays may depend on the efficient use of probes, anchors or primers and enzymes. This may be achieved by mixing liquids(such as pooling liquid back and forth in the flow through chamber), applying agitations or using -33- ·'· - · WO 2006/138257 PCT/US2006/022895 horizontal or vertical electric fields to bring DNA from different parts of the reaction volume in theproximity of the surface. One approach for efficient low cost assay reaction is to apply reaction mixesin a thin layer such as droplets or layers of about one to a few microns, but preferably less than 10microns, in size/thickness. In a lxlxl micron volume designated for a lxlmicron spot area, in 5 lpmol/lul (luM concentration) there would be about 1000 molecules of probe in close proximity to1-1000 copies of DNA. Using up to 100-300 molecules of probes would not significantly reduce theprobe concentration and it would provide enough reacted probes to get significant signal. Thisapproach may be used in an open reaction chamber that may stay open or closed for removal andwashing of the probes and enzyme. 10 As mentioned above, higher throughput can be achieved by using multiple cameras and multiple flow cells. A single robotic liquid handling gantry may service, for example, 16 flow cells.In addition, all components of the system may share a common temperature control system, and set ofreagents. For combinatorial SBH sequencing operations, the robot may prepare probe pools andligation buffers to be dispensed into the flow cell funnels. Dedicated syringe pumps may dispense 15 wash and hybridization buffers directly into the funnel ports for each flow cell. Each imager may service a group of 2-4 flow cells. Each group of flow cells may be positioned on an XY motionplatform, similar to the automated plate stages commonly found on research microscopes. Systemcontrol and coordination between all system components may be performed via software running on amaster computer. The control software may run assay cycles asynchronously, allowing each imager to 20 run continuously throughout the assay. Flow cells are connected to a temperature control system withone heater and one chiller allowing for heating or cooling on demand of each flow cell or 2-4 blocksof cells independently. Each flow cell temperature may be monitored, and if a flow cell temperaturedrops below a set threshold, a valve may open to a hot water recirculation. Likewise, if a flow celltemperature is above the set threshold a valve may open to a cold water recirculation. If a flow cell is 25 within a set temperature range neither valve may open. The hot and cold recirculation water runsthrough the aluminum flow cell body, but remains separate and isolated from the assay buffers andreagents.

Sequence Analysis of Random Arrays 30 of Target Sequence Concatemers

As mentioned above, random arrays of biomolecules, such as genomic DNA fragments or cDNA fragments, provides a platform for large scale sequence determination and for genome-widemeasurements based on counting sequence tags, in a manner similar to measurements made by serialanalysis of gene expression (SAGE) or massively parallel signature sequencing, e.g. Velculescu, et al, 35 (1995), Science 270,484-487; and Brenner et al (2000), Nature Biotechnology, 18: 630-634. Such genome-wide measurements include, but are not limited to, determination of polymorphisms,including nucleotide substitutions, deletions, and insertions, inversions, and the like, determination of -34- WO 2006/138257 PCT/US2006/022895 methylation patterns, copy number patterns, and the like, such as could be carried out by a wide rangeof assays known to those with ordinary skill in the art, e.g. Syvanen (2005), Nature GeneticsSupplement, 37: S5-S10; Gunderson et al (2005), Nature Genetics, 37: 549-554; Fan et al (2003),

Cold Spring Harbor Symposia on Quantitative Biology, LXVIII: 69-78; and U.S. patents 4,883,750; 5 6,858,412; 5,871,921; 6,355,431; and the like, which are incorporated herein by reference. A variety of sequencing methodologies can be used with random arrays of the invention, including, but not limited to, hybridization-based methods, such as disclosed in Drmanac, U.S.patents 6,864,052; 6,309,824; and 6,401,267; and Drmanac et al, U.S. patent publication2005/0191656, which are incorporated by reference, sequencing by synthesis methods, e.g. Nyren et 10 al, U.S. patent 6,210,891; Ronaghi, U.S. patent 6,828,100; Ronaghi et al (1998), Science, 281:363- ·365; Balasubramanian, U.S. patent 6,833,246; Quake, U.S. patent 6,911,345; Li et al, Proc. Natl.

Acad. Sci., 100:414-419 (2003), which are incorporated by reference, and ligation-based methods,e.g. Shendure et al (2005), Science, 309:1728-1739, which is incorporated by reference. In oneaspect, a method of determining a nucleotide sequence of a target polynucleotide in accordance with 15 the invention comprises the following steps: (a) generating a plurality of target concatemers from thetarget polynucleotide, each target concatemer comprising multiple copies of a fragment of the targetpolynucleotide and the plurality of target concatemers including a number of fragments thatsubstantially covers the target polynucleotide; (b) forming a random array of target concatemers fixedto a surface at a density such that at least a majority of the target concatemers are optically resolvable; 20 (c) identifying a sequence of at least a portion of each fragment in each target concatemer; and (d) reconstructing the nucleotide sequence of the target polynucleotide from the identities of thesequences of the portions of fragments of the concatemers. Usually, “substantially covers” means thatthe amount of DNA analyzed contains an equivalent of at least two copies of the targetpolynucleotide, or in another aspect, at least ten copies, or in another aspect, at least twenty copies, or 25 in another aspect, at least 100 copies. Target polynucleotides may include DNA fragments, includinggenomic DNA fragments and cDNA fragments, and RNA fragments.

In one aspect, a sequencing method for use with the invention for determining sequences in aplurality of DNA or RNA fragments comprises the following steps: (a) generating a plurahty ofpolynucleotide molecules each comprising a concatemer of a DNA or RNA fragment; (b) forming a 30 random array of polynucleotide molecules fixed to a surface at a density such that at least a majorityof the target concatemers are optically resolvable; and (c) identifying a sequence of at least a portionof each DNA or RNA fragment in resolvable polynucleotides using at least one chemical reaction ofan optically detectable reactant. In one embodiment, such optically detectable reactant is anoligonucleotide. In another embodiment, such optically detectable reactant is a nucleoside 35 triphosphate, e.g. a fluorescently labeled nucleoside triphosphate that may be used to extend an oligonucleotide hybridized to a concatemer. In another embodiment, such optically detectable reagentis an oligonucleotide formed by ligating a first and second oligonucleotides that form adjacent -35- WO 2006/138257 PCT/US2006/022895 duplexes on a concatemer. In another embodiment, such chemical reaction is synthesis of DNA orRNA, e.g. by extending a primer hybridized to a concatemer. In yet another embodiment, the aboveoptically detectable reactant is a nucleic acid binding oligopeptide or polypeptide or protein.

In one aspect, parallel sequencing of polynucleotide analytes of concatemers on a random 5 array is accomplished by combinatorial SBH (cSBH), as disclosed by Drmanac in the above-citedpatents. In one aspect, a first and second sets of oligonucleotide probes are provide, wherein each setshas member probes that comprise oligonucleotides having every possible sequence for the definedlength of probes in the set. For example, if a set contains probes of length six, then it contains 4096(=46) probes. In another aspect, first and second sets of oligonucleotide probes comprise probes 10 having selected nucleotide sequences designed to detect selected sets of target polynucleotides.

Sequences are determined by hybridizing one probe or pool of probe, hybridizing a second probe or asecond pool of probes, ligating probes that form perfectly matched duplexes on their target sequences,identifying those probes that are ligated to obtain sequence information about the target sequence,repeating the steps until all the probes or pools of probes have been hybridized, and determining the 15 nucleotide sequence of the target from the sequence infonnation accumulated during the hybridization and identification steps.

For sequencing operation, in some embodiments, the sets may be divided into subsets that areused together in pools, as disclosed in U.S. patent 6,864,052. Probes from the first and second setsmay be hybridized to target sequences either together or in sequence, either as entire sets or as 20 subsets, or pools. In one aspect, lengths of the probes in the first or second sets are in the range offrom 5 to 10 nucleotides, and in another aspect, in the range of from 5 to 7 nucleotides, so that whenligated they form ligation products with a length in the range of from 10 to 20, and from 10 to 14,respectively.

In another aspect, using such techniques, the sequence identity of each attached DNA 25 concatemer may be determined by a “signature” approach. About 50 to 100 or possibly 200 probesare used such that about 25-50% or in some applications 10-30% of attached concatemers will have afull match sequence for each probe. This type of data allows each amplified DNA fragment within aconcatemer to be mapped to the reference sequence. For example, by such a process one can score 64 4-mers (i.e. 25% of all possible 256 4-mers) using 16 hybridization/stripoff cycles in a 4 colors 30 labeling schema. On a 60-70 base fragment amplified in a concatemer about 16 of 64 probes will bepositive since there are 64 possible 4mers present in a 64 base long sequence (i.e. one quarter of allpossible 4mers). Unrelated 60-70 base fragments will have a veiy different set of about 16 positivedecoding probes. A combination of 16 probes out of 64 probes has a random chance of occurrence in1 of every one billion fragments which practically provides a unique signature for that concatemer. 35 Scoring 80 probes in 20 cycles and generating 20 positive probes create a signature even more likelyto be unique: occurrence by chance is 1 in billion billions. Previously, a “signature” approach wasused to select novel genes from cDNA libraries. An implementation of a signature approach is to sort -36- WO 2006/138257 PCT/US2006/022895 obtained intensities of all tested probes and select up to a predefined (expected) number of probes thatsatisfy the positive probe threshold. These probes will be mapped to sequences of all DNA fragments(sliding window of a longer reference sequence may be used) expected to be present in the array. Thesequence that has all or a statistically sufficient number of the selected positive probes is assigned as 5 the sequence of the DNA fragment in the given concatemer. In another approach an expected signalcan be defined for all used probes using their pre measured full match and mismatchhybridization/Iigation efficiency. In this case a measure similar to the correlation factor can becalculated. A preferred way to score 4-mers is to ligate pairs of probes, for example: N^BBB with 10 BN(7-9), where B is the defined base and N is a degenerate base. For generating signatures on longer DNA concatemer probes, more unique bases will be used. For example, a 25% positive rate in afragment 1000 bases in length would be achieved by Ν(4.6)ΒΒΒΒ and BBN(C_g). Note that longerfragments need the same number of about 60-80 probes (15-20 ligation cycles using 4 colors).

In one embodiment all probes of a given length (e.g. 4096 N2-4BBBBBBN24) or all ligation 15 pairs may be used to determine complete sequence of the DNA in a concatemer. For example, 1024combinations of N(5_7)B3 and BBN^gj may be scored (256 cycles if 4 colors are used) to determinesequence of DNA fragments of up to about 250 bases, preferably up to about 100 bases.

The decoding of sequencing probes with large numbers of Ns may be prepared from multiplesyntheses of subsets of sequences at degenerated bases to minimize difference in the efficiency. Each 20 subset is added to the mix at a proper concentration. Also, some subsets may have more degeneratedpositions than others. For example, each of 64 probes from the set Ν^,ηΒΒΒ may be prepared in 4different synthesis. One is regular all 5-7 bases to be fully degenerated; second is N0-3(A,T)5BBB;third is N0-2(A,T)(G,C)(A,T)(G,C)(A,T)BBB, and the fourth is N0- 2(G,C)(A,T)(G,C)(A,T)(G,C)BBB. 25 Oligonucleotide preparation from the three specific syntheses is added in to regular synthesis in experimentally determined amounts to increase hybrid generation with target sequences that havein front of the BBB sequence an AT rich (e.g. AATAT) or (A or T) and (G or C) alternating sequence(e.g. ACAGT or GAGAC). These sequences are expected to be less efficient in forming a hybrid. All1024 target sequences can be tested for the efficiency to form hybrid with Νο^ΝΝΝΝΝΒΒΒ probes 30 and those types that give the weakest binding may be prepared in about 1-10 additional synthesis andadded to the basic probe preparation.

Decoding by Signatures: a smaller number of probes for small number of distinct samples: 5-7 positive out of 20 probes (5 cycles using 4 colors) has capacity to distinct about 10-100 thousanddistinct fragments 35 Decoding of 8-20mer RCR products. In this application arrays are formed as random distributions of unique 8 to 20 base recognition sequences in the form of DNA concatemers. Theprobes need to be decoded to determine the sequence of the 8-20 base probe region. At least two -37- WO 2006/138257 PCT/US2006/022895 options are available to do this and the following example describes the process for a 12 mer. In thefirst, one half of the sequence is determined by utilizing the hybridization specificity of short probesand the ligation specificity of fully matched hybrids. Six to ten bases adjacent to the 12 mer arepredefined and act as a support for a 6mer to 10-mer oligonucleotide. This short 6mer will ligate at its 5 3-prime end to one of 4 labeled 6-mers to 10-mers. These decoding probes consist of a pool of 4oligonucleotides in which each oligonucleotide consists of 4-9 degenerate bases and 1 defined base.This oligonucleotide will also be labeled with one of four fluorescent labels. Each of the 4 possiblebases A, C, G, or T will therefore be represented by a fluorescent dye. For example these 5 groups of4 oligonucleotides and one universal oligonucleotide (Us) can be used in the ligation assays to 10 sequence first 5 bases of 12-mers: B=each of 4 bases associated with a specific dye or tag at the end: UUUUUUUU.BNNNNNNN*

UUUUUUUU.NBNNNNNN

UUUUUUUU.NNBNNNNN

15 UUUUUUUU.NNNBNNNNUUUUUUUU.NNNNBNNN

Six or more bases can be sequences with additional probe pools. To improve discrimination atpositions near the center of the 12mer the 6mer oligonucleotide can be positioned further into the 20 12mer sequence. This will necessitate the incorporation of degenerate bases into the 3-prime end ofthe non-labeled oligonucleotide to accommodate the shift. This is an example of decoding probes forposition 6 and 7 in the 12-mer.

UULOJUUNN.NNNBNNNN

25 UUUUUUNN.NNNNBNNN

In a similar way the 6 bases from the right side of the 12mer can be decoded by using a fixedoligonucleotide and 5-prime labeled probes. In the above described system 6 cycles are required todefine 6 bases of one side of the 12mer. With redundant cycle analysis of bases distant to the ligation 30 site this may increase to 7 or 8 cycles. In total then, complete sequencing of the 12mer could be accomplished with 12-16 cycles of ligation. Partial or complete sequencing of arrayed DNA bycombining two distinct types of libraries of detector probes. In this approach one set has probes of thegeneral type N3^B« (anchors) that are ligated with the first 2 or 3 or 4 probes/probe pools from theset BN6.a, NBN5.7, N2BN4_6, and N3BN3.5.The main requirement is to test in a few cycles a probe from 35 the first set with 2-4 or even more probes from the second set to read longer continuous sequence suchas 5-6+3-4=8-10 in just 3-4 cycles. In one example, the process is: -38- WO 2006/138257 PCT/US2006/022895 1) Hybridize 1-4 4-mers or more 5-mer anchors to obtain 70-80% 1 or 2 anchors per DNA.One way to discriminate which anchor is positive from the pool is to mix specific probes with distincthybrid stability (maybe different number of Ns in addition). Anchors may be also tagged to determinewhich anchor from the pool is hybridized to a spot. Tags, as additional DNA segment, may be used 5 for adjustable displacement as a detection method. For example, EEEEEEEENNNAAAAA and FFFFFFFFNNNCCCCC probes can be after hybridization or hybridization and ligation differentiallyremoved with two corresponding displacers: EEEEEEEENNNNN and FFFFFFFFNNNNNNNN where the second is more efficient.

Separate cycles may be used just to determine which anchor is positive. For this purpose anchors 10 labeled or tagged with multiple colors may be ligated to unlabeled N7-N10 supporter oligonucleotides. 2) Hybridize BNNNNNNNN probe with 4 colors corresponding to 4 bases; washdiscriminatively (or displace by complement to the tag) to read which of two scored bases isassociated to which anchor if two anchors are positive in one DNA. Thus, two 7-10 base sequences 15 can be scores at the same time.

In 2-4 cycles extend to 4-6 base anchor for additional 2-4 bases run 16 different anchors pereach array (32-64 physical cycles if 4 colors are used) to determine about 16 possible 8-mers (-100bases total) per each fragment (more then enough to map it to the reference (probability that a 100-mer will have a set of 10 8-mers is less than 1 in trillion trillions; (10exp-28). By combining data from 20 different anchors scored in parallel on the same fragment in another array complete sequence of thatfragment and by extension to entire genomes may be generated from overlapping 7-10-mers.

Tagging probes with DNA tags for larger multiplex of decoding or sequence determinationprobes Instead of directly labeling probes they can be tagged with different oligonucleotidesequences made of natural bases or new synthetic bases (such as isoG and isoC), Tags can be 25 designed to have very precise binding efficiency with their anti-tags using different oligonucleotidelengths (about 6-24 bases) and/or sequence including GC content. For example 4 different tags maybe designed that can be recognized with specific anti-tags in 4 consecutive cycles or in onehybridization cycle followed by a discriminative wash. In the discriminative wash initial signal isreduced to 95-99%, 30-40%, 10-20% and 0-5% for each tag, respectively. In this case by obtaining 30 two images 4 measurements are obtained assuming that probes with different tags will rarelyhybridize to the same dot. Another benefit of having many different tags even if they areconsecutively decoded (or 2-16 at a time labeled with 2-16 distinct colors) is the ability to use a largenumber of individually recognizable probes in one assay reaction. This way a 4-64 times longer assaytime (that may provide more specific or stronger signal) may be affordable if the probes are decoded 35 in short incubation and removal reactions.

The decoding process requires the use of48-96 or more decoding probes. These pools will be further combined into 12-24 or more pools by encoding them with four fluorophores, each having -39- 1 - ίΗ·- WO 2006/138257 PCT/US2006/022895 different emission spectra. Using a 20x objective, each 6mm x 6mm array may require roughly 30images for full coverage by using a 10 mega pixel camera with. Each of 1 micrometer array areas isread by about 8 pixels. Each image is acquired in 250 milliseconds, 150ms for exposure and 100ms tomove tiie stage. Using this fast acquisition it will take -7,5 seconds to image each array, or 12 5 minutes to image the complete set of 96 arrays on each substrate. In one embodiment of an imagingsystem, this high image acquisition rate is achieved by using four ten-megapixel cameras, eachimaging the emission spectra of a different fluorophore. The cameras are coupled to the microscopethrough a series of dichroic beam splitters. The autofocus routine, which takes extra time,,runs only ifan acquired image is out of focus. It will then store the Z axis position information to be used upon 10 return to that section of that array during the next imaging cycle. By mapping the autofocus positionfor each location on the substrate we will drastically reduce the time required for image acquisition.

Each array requires about 12-24 cycles to decode. Each cycle consists of a hybridization,wash, array imaging, and strip-off step. These steps, in their respective orders, may take for the aboveexample 5,2,12,and 5 minutes each, for a total of 24 minutes each cycle, or roughly 5-10 hours for 15 each array, if the operations were performed linearly. The time to decode each array can be reducedby a factor of two by allowing the system to image constantly. To accomplish this, the imaging oftwo separate substrates on each microscope is staggered. While one substrate is being reacted, theother substrate is imaged.

An exemplary decoding cycle using cSBH includes the following steps: (i) set temperature of 20 array to hybridization temperature (usually in the range 5-25°C); (ii) use robot pipetter to pre mix asmall amount of decoding probe with the appropriate amount of hybridization buffer; (iii) pipettemixed reagents into hybridization chamber; (iv) hybridize for predetermined time; (v) drain reagentsfrom chamber using pump (syringe or other); (vi) add a buffer to wash mismatches of non-hybrids;(vii) adjust chamber temperature to appropriate wash temp (about 10-40 °C); (viri) drain chamber; (ix) 25 add more wash buffer if needed to improve imaging; (x) image each array, preferably with a mid power (20x) microscope objective optically coupled to a high pixel count high sensitivity ccd camera,or cameras; plate stage moves chambers (or perhaps flow-cells with input funnels) over object, orobjective-optics assembly moves under chamber; certain optical arrangements, using di-chroicmirrors/beam-splitters can be employed to collect multi-spectral images simultaneously, thus 30 decreasing image acquisition time; arrays can be imaged in sections or whole, depending onarray/image size/pixel density; sections can be assembled by aligning images using statisticallysignificant empty regions pre-coded onto substrate (during active site creation) or can be made using amulti step nano-printing technique, for example sites (grid of activated sites) can be printed usingspecific capture probe, leaving empty regions in the grid; then print a different pattern or capture

35 probe in that region using separate print head; (xi) drain chamber and replace with probe strip buffer(or use the buffer already loaded) then heat chamber to probe stripoff temperature (60-90 °C); high pH -40- WO 2006/138257 PCT/US2006/022895 buffer may be used in the strip-off step to reduce stripoff temperature; wait for the specified time; (xii)remove buffer; (xifi) start next cycle with next decoding probe pool in set.

Labels and Signal Generation by Probes Directed 5 to Polynucleotides on Arrays of the Invention

The oligonucleotide probes of the invention can be labeled in a variety of ways, including the direct or indirect attachment of radioactive moieties, fluorescent moieties, colorimetric moieties,chemiluminescent moieties, and the like. Many comprehensive reviews of methodologies forlabeling DNA and constructing DNA adaptors provide guidance applicable to constructing 10 oligonucleotide probes of the present invention. Such reviews include Kricka, Ann. Clin. Biochem.,39: 114-129 (2002); Schaferling et al, Anal. Bioanal. Chem., (April 12,2006); Matthews et al, Anal.Biochem., Vol 169, pgs. 1-25 (1988); Haugland, Handbook of Fluorescent Probes and ResearchChemicals, Tenth Edition (Invitrogen/Molecular Probes, Inc., Eugene, 2006); Keller and Manak, DNA Probes, 2nd Edition (Stockton Press, New York, 1993); and Eckstein, editor, Oligonucleotides 15 and Analogues: A Practical Approach (IRL Press, Oxford, 1991); Wetmur, Critical Reviews inBiochemistry and Molecular Biology, 26: 227-259 (1991); Hermanson, Bioconjugate Techniques(Academic Press, New York, 1996); and the like. Many more particular methodologies applicable tothe invention are disclosed in the following sample of references: Fung et al, U.S. patent 4,757,141;Hobbs, Jr., et al U.S. patent 5,151,507; Cruickshank, U.S. patent 5,091,519; (synthesis of 20 functionalized oligonucleotides for attachment of reporter groups); Jablonski et al, Nucleic Acids

Research, 14: 6115-6128 (1986)(enzyme-oligonucleotide conjugates); Ju et al, Nature Medicine, 2:246-249 (1996); Bawendi et al, U.S. patent 6,326,144 (derivatized fluorescent nanocrytals); Bruchezet al, U.S. patent 6,274,323 (derivatized fluorescent nanocrystals); and the like.

In one aspect, one or more fluorescent dyes are used as labels for the oligonucleotide probes, 25 e.g. as disclosed by Menchen et al, U.S. patent 5,188,934 (4,7-dichlorofluorscein dyes); Begot et al,U.S. patent 5,366,860 (spectrally resolvable rhodamine dyes); Lee et al, U.S. patent 5, 847,162 (4,7-dichlororhodamine dyes); Khanna et al, U.S. patent 4,318,846 (ether-substituted fluorescein dyes);Lee et al, U.S. patent 5,800,996 (energy transfer dyes); Lee et al, U.S. patent 5,066,580 (xanthenedyes): Mathies et al, U.S. patent 5,688,648 (energy transfer dyes); and the like. Labeling can also be 30 carried out with quantum dots, as disclosed in the following patents and patent publications, incorporated herein by reference: 6,322,901; 6,576,291; 6,423,551; 6,251,303; 6,319,426; 6,426,513;6,444,143; 5,990,479; 6,207,392; 2002/0045045; 2003/0017264; and the like. As used herein, theterm "fluorescent signal generating moiety" means a signaling means which conveys informationthrough the fluorescent absorption and/or emission properties of one or more molecules. Such 35 fluorescent properties include fluorescence intensity, fluorescence life time, emission spectrumcharacteristics, energy transfer, and tire like.

Commercially available fluorescent nucleotide analogues readily incorporated into thelabeling oligonucleotides include, for example, Cy3-dCTP, Cy3-dUTP, Cy5-dCTP, Cy5-dUTP -41- Κ : WO 2006/138257 PCT/US2006/022895 (Amersham Biosciences, Piscataway, New Jersey, USA), fluorescein-12-dUTP,tetramethylrhodamine-6-dUTP, Texas Red®-5-dUTP, Cascade Blue®-7-dUTP, BODIPY® FL- 14-dUTP, BODIPY®R-14-dUTP, BODIPY® TR-14-dUTP, Rhodamine Green™-5-dUTP,Oregon Green® 488-5-dUTP, Texas Red®-12-dUTP, BODIPY® 630/650-14-dUTP, BODIPY® 5 650/665-14-dUTP, Alexa Fluor® 488-5-dUTP, Alexa Fluor® 532-5-dUTP, Alexa Fluor® 568-5-dUTP, Alexa Fluor® 594-5-dUTP, Alexa Fluor® 546-14-dUTP, fluorescein-12-UTP,tetramethylrhodamine-6-UTP, Texas Red®-5-UTP, Cascade Blue©-7-UTP, BODIPY® FL-14-UTP,BODIPY® TMR-14-UTP, BODIPY® TR-14-UTP, Rhodamine Green™-5-UTP, Alexa Fluor®488-5-UTP, Alexa Fluor® 546-14-UTP (Molecular Probes, Inc. Eugene, OR, USA). - Other 10 fluorophores available for post-synthetic attachment include, inter alia, Alexa Fluor® 350, AlexaFluor® 532, Alexa Fluor® 546, Alexa Fluor® 568, Alexa Fluor® 594, Alexa Fluor® 647, BODIPY493/503, BODIPY FL, BODIPY R6G, BODIPY 530/550, BODIPY TMR, BODIPY 558/568,BODIPY 558/568, BODIPY 564/570, BODIPY 576/589, BODIPY 581/591, BODIPY 630/650,BODIPY 650/665, Cascade Blue, Cascade Yellow, Dansyl, lissamine rhodamine B, Marina Blue, 15 Oregon Green 488, Oregon Green 514, Pacific Blue, rhodamine 6G, rhodamine green, rhodamine red,tetramethylrhodamine, Texas Red (available from Molecular Probes, Inc., Eugene, OR, USA), andCy2, Cy3.5, Cy5.5, and Cy7 (Amersham Biosciences, Piscataway, NJ USA, and others). FRETtandem fluorophores may also be used, such as PerCP-Cy5.5, PE-Cy5, PE-Cy5.5, PE-Cy7, PE-TexasRed, and APC-Cy7; also, PE-Alexa dyes (610, 647, 680) and APC-AIexa dyes. Biotin, or a derivative 20 thereof, may also be used as a label on a detection oligonucleotide, and subsequently bound by adetectably labeled avidin/streptavidin derivative (e.g. phycoerythrin-conjugated streptavidin), or adetectably labeled anti-biotin antibody. Digoxigenin may be incorporated as a label and subsequentlybound by a detectably labeled anti-digoxigenin antibody (e.g. fluoresceinated anti-digoxigenin). Anaminoallyl-dUTP residue may be incorporated into a detection oligonucleotide and subsequently 25 coupled to an N-hydroxy succinimide (NHS) derivitized fluorescent dye, such as those listed supra.

In general, any member of a conjugate pair may be incorporated into a detection oligonucleotideprovided that a detectably labeled conjugate partner can be bound to permit detection. As used herein,the term antibody refers to an antibody molecule of any class, or any subfragment thereof, such as anFab. Other suitable labels for detection oligonucleotides may include fluorescein (FAM), 30 digoxigenin, dinitrophenol (DNP), dansyl, biotin, bromodeoxyuridine (BrdU), hexahistidine (6xHis),phosphor-amino acids (e.g. P-tyr, P-ser, P-thr), or any other suitable label. In one embodiment thefollowing hapten/antibody pairs are used for detection, in which each of the antibodies is derivatizedwith a detectable label: biotin/a-biotin, digoxigenin/a-digoxigenin, dinitrophenol (DNP)Za-DNP, 5-Carboxyfluorescein (FAM)/a-FAM. As described in schemes below, probes may also be indirectly 35 labeled, especially with a hapten that is then bound by a capture agent, e.g. as disclosed in Holtke etal, U.S. patent 5,344,757; 5,702,888; and 5,354,657; Huber et al, U.S. patent 5,198,537; Miyoshi, U.S.patent 4,849,336; Misiura and Gait, PCT publication WO 91/17160; and the like. Many different -42- WO 2006/138257 PCT/US2006/022895 hapten-capture agent pairs are available for use with the invention. Exemplary, haptens include,biotin, des-biotin and other derivatives, dinitrophenol, dansyl, fluorescein, CY5, and other dyes,digoxigenin, and the like. For biotin, a capture agent may be avidin, streptavidin, or antibodies.Antibodies may be used as capture agents for the other haptens (many dye-antibody pairs being 5 commercially available, e.g. Molecular Probes).

Kits of the Invention

In the commercialization of the methods described herein, certain kits for construction ofrandom arrays of die invention and for using the same for various applications are particularly useful. 10 Kits for applications of random arrays of the invention include, but are not limited to, kits for determining the nucleotide sequence of a target polynucleotide, kits for large-scale identification ofdifferences between reference DNA sequences and test DNA sequences, kits for profiling exons, andthe like. A kit typically comprises at least one support having a surface and one or more reagentsnecessary or useful for constructing a random array of the invention or for canying out an application 15 therewith. Such reagents include, without limitation, nucleic acid primers, probes, adaptors, enzymes,and the like, and are each packaged in a container, such as, without limitation, a vial, tube or bottle, ina package suitable for commercial distribution, such as, without limitation, a box, a sealed pouch, ablister pack and a carton. The package typically contains a label or packaging insert indicating theuses of the packaged materials. As used herein, "packaging materials" includes any article used in the 20 packaging for distribution of reagents in a kit, including without limitation containers, vials, tubes,bottles, pouches, blister packaging, labels, tags, instruction sheets and package inserts.

In one aspect, the invention provides a kit for making a random array of concatemers of DNAfragments from a source nucleic acid comprising the following components: (i) a support having asurface; and (ii) at least one adaptor oligonucleotide for ligating to each DNA fragment and forming a 25 DNA circle therewith, each DNA circle capable of being replicated by a rolling circle replication reaction to form a concatemer that is capable of being randomly disposed on the surface. In such kits,the surface may be a planar surface having an array of discrete spaced apart regions, wherein eachdiscrete spaced apart region has a size equivalent to that of said concatemers. The discrete spacedapart regions may form a regular array with a nearest neighbor distance in the range of from 0.1 to 20 30 pm. The concatemers on the discrete spaced apart regions may have a nearest neighbor distance suchthat they are optically resolvable. The discrete spaced apart regions may have captureoligonucleotides attached and the adaptor oligonucleotides may each have a region complementary tothe capture oligonucleotides such that the concatemers are capable of being attached to the discretespaced apart regions by formation of complexes between the capture oligonucleotides and the 35 complementary regions of the adaptor oligonucleotides. In some embodiments, the concatemers arerandomly distributed on said discrete spaced apart regions and the nearest neighbor distance is in therange of from 0.3 to 3 pm. Such kits may further comprise (a) a terminal transferase for attaching a homopolymer tail to said DNA fragments to provide a binding site for a first end of said adaptor-43- WO 2006/138257 PCT/US2006/022895 oligonucleotide, (b) a ligase for ligating a strand of said adaptor oligonucleotide to ends of said DNAfragment to form said DNA circle, (c) a primer for annealing to a region of the strand of said adaptoroligonucleotide, and (d) a DNA polymerase for extending the primer annealed to the strand in arolling circle replication reaction. The above adaptor oligonucleotide may have a second end having a 5 number of degenerate bases in the range of from 4 to 12.

In another aspect the invention provides kits for sequencing a target polynucleotide comprising the following components: (i) a support having a planar surface having an array ofoptically resolvable discrete spaced apart regions, wherein each discrete spaced apart region has anarea of less than 1 pm2; (ii) a first set of probes for hybridizing to a plurality of concatemers randomly 10 disposed on the discrete spaced apart regions, the concatemers each containing multiple copies of aDNA fragment of the target polynucleotide; and (iii) a second set of probes for hybridizing to theplurality of concatemers such that whenever a probe from the first set hybridizes contiguously to aprobe from the second set, the probes are ligated. Such kits may further include a ligase, a ligasebuffer, and a hybridization buffer. In some embodiments, the discrete spaced apart regions may have 15 capture oligonucleotides attached and the concatemers may each have a region complementary to thecapture oligonucleotides such that said concatemers are capable of being attached to the discretespaced apart regions by formation of complexes between the capture oligonucleotides and thecomplementary regions of said concatemers.

In still another aspect, the invention provides kits for constructing a single molecule array 20 comprising the following components: (i) a support having a surface having reactive functionalities;and (ii) a plurality of macromolecular structures each having a unique functionality and multiplecomplementary functionalities, the macromolecular structures being capable of being attachedrandomly on the surface wherein the attachment is formed by one or more linkages formed byreaction of one or more reactive functionalities with one or more complementary functionalities; and 25 wherein the unique functionality is capable of selectively reacting with a functionality on an analytemolecule to form the single molecule array. In some embodiments of such kits, the surface is a planarsurface having an array of discrete spaced apart regions containing said reactive functionalities andwherein each discrete spaced apart region has an area less than 1 pm2. In further embodiments, tirediscrete spaced apart regions form a regular array with a nearest neighbor distance in the range of 30 from 0.1 to 20 pm. In further embodiments, the concatemers on the discrete spaced apart regionshave a nearest neighbor distance such that they are optically resolvable. In still further embodiments,the macromolecular structures may be concatemers of one or more DNA fragments and wherein theunique functionalities are at a 3’ end or a 5’ end of the concatemers.

In another aspect, the invention includes kits for circularizing DNA fragments comprising the 35 components: (a) at least one adaptor oligonucleotide for ligating to one or more DNA fragments andforming DNA circles therewith (b) a terminal transferase for attaching a homopolymer tail to saidDNA fragments to provide a binding site for a first end of said adaptor oligonucleotide, (c) a ligase for -44- WO 2006/138257 PCT/l)S2006/022895 ligating a strand, of said adaptor oligonucleotide to ends of said DNA fragment to form said DNAcircle, (d) a primer for annealing to a region of the strand of said adaptor oligonucleotide, and (e) aDNA polymerase for extending the primer annealed to the strand in a Tolling circle replicationreaction. In an embodiment of such kit, the above adaptor oligonucleotide may have a second end 5 having a number of degenerate bases in the range of from 4 to 12. The above kit may further include reaction buffers for the terminal transferase, ligase, and DNA polymerase. Iii still another aspect, theinvention includes a kit for circularizing DNA fragments using a Circligase enzyme (EpicentreBiotechnologies, Madison, WI), which kit comprises a volume exclusion polymer. In another aspect,such kit further includes the following components: (a) reaction buffer for controlling pH and 10 providing an optimized salt composition for Circligase, and (b) Circligase cofactors. In another aspect, a reaction buffer for such kit comprises 0.5 M MOPS (pH 7.5), 0.1 M KC1, 50 mM MgCI2, and10 mM DTT. In another aspect, such kit includes Circligase, e.g. 10-100 pL Circligase solution (at100 unit/pL). Exemplary volume exclusion polymers are disclosed in U.S. patent 4,886,741, andinclude polyethylene glycol, polyvinylpyrrolidone, dextran sulfate, and like polymers. In one aspect, 15 polyethylene glycol (PEG) is 50% PEG4000. In one aspect, a kit for circle formation includes thefollowing:

Amount_Component Final Cone. 2 pL Circligase 1 OX reaction buffer IX 0.5 pL 1 mM ATP 25 pM 0.5 pL 50 mM MnCl2 1.25 mM 4 pL 50% PEG4000 10% 2 pL Circligase ssDNA ligase (100 units/pL) 10 units/pL single stranded DNA template 0.5-1 Opmol/pL sterile water

Final reaction volume: 20 pL. The above components are used in the following protocol: 20 1. Heat DNA at 60- 96°C depending on the length of the DNA (ssDNA templates that have a 5’- phosphate and a 3-hydroxyl group). 2. Preheat 2.2X reaction mix at 60°C for about 5-10 min. 3. If DNA was preheated to 96°C cool it down at 60°C. 4. Mix DNA and buffer at 60°C without cooling it down and incubate for 2-3h. 25 5. Heat inactivate enzyme to stop the ligation reaction.

Large-Scale Mutation Discovery by Mismatch Enzyme CleavageArrays and sequencing methods of the invention used may be used for large-scale 30 identification of polymorphisms using mismatch cleavage techniques. Several approaches tomutation detection employ a heteroduplex in which the mismatch itself is utilized for cleavagerecognition. Chemical cleavage with piperidine at mismatches modified with hydroxyl amine or -45- -- WO 2006/138257 PCT/US2006/022895 osmium tetroxide provides one approach to release a cleaved fragment. In a similar way theenzymesT7 endonuclease I or T4 endonuclease VII have been used in the enzyme mismatch cleavage(EMC) techniques, e.g. Youil et al, Proc, Natl. Acad. Sci., 92: 87-91 (1995); Mashal et al, NatureGenetics, 9: 177-183 (1995); Babon et at, Molecular Biotechnology, 23:73-81 (2003); Ellis et al, 5 Nucleic Acids Research, 22:2710-2711 (1994); and the like, which are incorporated herein by reference. Cleavase is used in the cleavage fragments length polymorphism (CFLP) technique whichhas been commercialized by Third Wave Technologies. When single stranded DNA is allowed to foldand adopt a secondary structure the DNA will form internal hairpin loops at locations dependent uponthe base sequence of the strand. Cleavase will cut single stranded DNA five-prime of the loop and the 10 fragments can then be separated by PAGE or similar size resolving techniques. Mismatch bindingproteins such as Mut S and Mut Y also rely upon the formation of heteroduplexes for their ability toidentify mutation sites. Mismatches are usually repaired but the binding action of the enzymes can beused for the selection of fragments through a mobility shift in gel electrophoresis or by protectionfrom exonucleases, e.g. Ellis et al (cited above). 15 Templates for heteroduplex formation are prepared by primer extension from genomic DNA.

For the same genomic region of the reference DNA, an excess of the opposite strand is prepared in thesame way as the test DNA but in a separate reaction. The test DNA strand produced is biotinylatedand is attached to a streptavidin support. Homoduplex formation is prevented by heating and removalof the complementary strand. The reference preparation is now combined with the single stranded test 20 preparation and annealed to produce heteroduplexes. This heteroduplex is likely to contain a numberof mismatches. Residual DNA is washed away before the addition of the mismatch endonuclease,which, if there is a mismatch every 1 kb would be expected to produce about 10 fragments for a lOkbprimer extension. After cleavage, each fragment can bind an adapter at each end and enter themismatch-fragment circle selection process. Capture of mismatch cleaved DNA from Large genomic 25 fragments. The 5-10 kb genomic fragments prepared from large genomic fragments as describedabove are biotinylated by the addition of a biotinylated di deoxy nucleotide at the 3-prime end withterminal transferase and excess biotinylated nucleotide are removed by filtration. A reference BACclone that covers the same region of sequence is digested with the same six-base cutter to match thefragments generated from the test DNA. The biotinylated genomic fragments are heat denatured in 30 the presence of the BAC reference DNA and slowly annealed to generate biotinylated heteroduplexes.The reference BAC DNA is in large excess to the genomic DNA so the majority of biotinylatedproducts will be heteroduplexes. The biotinylated DNA can then be attached to the surface forremoval of the reference DNA. Residual DNA is washed away before the addition of the mismatch .endonuclease. After cleavage, each fragment can bind an adapter at each end and enter the mismatch 35 circle selection process as follows, (a) DNA is cleaved on both sides of the mismatch, (b) 5-primeoverhangs are generated that can be ligated. (3’ overhangs are also created by digesting with anappropriate restriction endonuclease having a four base recognition site.) (c) An adapter is introduced -46- WO 2006/138257 PCT/US2006/022895 that contains an active overhang at one side, (d) An adapter is ligated to each of the two generatedfragments (only ligation to the right from the 5’ phosphate after addition of sequences to ihe 3’ end ofthe top strand), (e) The molecule is phosphorylated and a bridging oligonucleotide is used to ligate thetwo ends of the single stranded molecule, (f) After circularization, a concatemer is generated by 5 extending a primer in a RCR reaction.

Circle Formation from Mismatch Cleavage ProductsMethod I. The heteroduplexes generated above can be used for selection of small DNA circles, asillustrated in Figs. 7 and 8. As shown in Fig. 7, in this process, heteroduplex (700) of a sample is 10 treated with the mismatch enzyme to create products cleaved on both strands (704 and 706) surrounding the mutation site (702) to produce fragments (707) and (705). T7 endonuclease I orsimilar enzyme cleaves 5-prime of the mutation site to reveal a 5-prime overhang of vaiying length onboth strands surrounding the mutation. The next phase is to capture the cleaved products in a formsuitable for amplification and sequencing. Adapter (710) is ligated to the overhang produced by the 15 mismatch cutting (only fragment (705) shown), but because the nature of the overhang is unknown, atleast three adapters are needed and each adapter is synthesized with degenerate bases to accommodateall possible ends. The adapter can be prepared with an internal biotin (708) on the non-circularizingstrand to allow capture for buffer exchange and sample cleanup, and also for direct amplification onthe surface if desired. 20 Because the intervening sequence between mutations does not need to be sequenced and reduces the sequencing capacity of the system it is removed when studying genomic-derived samples.Reduction of sequence complexity is accomplished by a type Ils enzyme that cuts the DNA at a pointaway from the enzyme recognition sequence. In doing so, the cut site and resultant overhangs will bea combination of all base variants. Enzymes that can be used include Mmel (20 bases with 2 base 3’ 25 overhang) and Eco P15I (with 25 bases and 2 base 5 s overhang). Tire adapter is about 50 bp in lengthto provide sequences for initiation of rolling circle amplification and also provide stuffer sequence forcircle formation, as well as recognition site (715) for a type Ils restriction endonuclease. Once theadapter has been ligated to the fragment the DNA is digested (720) with the type Ils restrictionenzyme to release all but 20-25 bases of sequence containing the mutation site that remains attached 30 to the adapter.

The adaptered DNA fragment is now attached to a streptavidin support for removal of excessfragment DNA. Excess adapter that did not ligate to mismatch cleaved ends will also bind to thestreptavidin solid support. The new degenerate end created by the type Ils enzyme can now be ligatedto a second adapter through the phosphorylation of one strand of the second adapter. The other strand 35 is non-phosphorylated and blocked at the 3-prime end with a dideoxy nucleotide. The structureformed is essentially the genomic fragment of interest captured between two different adapters. Tocreate a circle from this structure would simply require both ends of the molecule coming together -47-

UiU ' h? . : Uh 'Hv?·

iSirwH WO 2006/138257 PCT/US2006/022895 and ligating, e.g. via formation of staggered ends by digesting at restriction sites (722) and (724),followed by intra-molecular ligation. Although this event should happen efficiently, there is also thepossibility that the end of an alternative molecule could ligate at the other end of the moleculecreating a dimer molecule, or greater multiples of each unit molecule. One way to minimize this is to ' 5 perform the ligation under dilute conditions so only intra-molecular ligation is favored, then re-concentrating die sample for future steps. An alternative strategy to maximize the efficiency of circleformation without inter-molecular ligation is to block excess adapters on the surface. This can beachieved by using lambda exonuclease to digest the lower strand. If second adapter has been attachedthen it will be protected from digestion because there is no 5-prime phosphate available. If only the 10 first adapter is attached to the surface then the 5-prime phosphate is exposed for degradation of thelower strand of the adapter. This will lead to loss of excess first adapter from the surface.

After lambda exonuclease treatment the 5 prime end of the top strand of the first adapter isprepared for ligation to the 3-prime end of the second adapter. This can be achieved by introducing arestriction enzyme site into the adapters so that re-circularization of the molecule can occur with 15 ligation. Amplification of DNA captured into the circular molecules proceeds by a rolling circle amplification to form long linear concatemer copies of the circle. If extension initiates 5-prime of thebiotin, the circle and newly synthesized strand is released into solution. Complementaryoligonucleotides on the surface are responsible for condensation and provide sufficient attachment fordownstream applications. One strand is a closed circle and acts as the template. The other strand, with 20 an exposed 3-prime end, acts as an initiating primer and is extended.

Method II. This method, illustrated in Fig. 8, is similar to the procedure above with the following modifications. 1) The adapter can be prepared with a 3-prime biotin (808) on the non-circularized strand to allow capture for buffer exchange and sample cleanup. 2) Reduction ofsequence complexity of the 10 kb heteroduplex fragments described above occurs through the use of 25 4-base cutting restriction enzymes, e.g. with restriction sites (810), (812), and (814), Use of 2 or 3enzymes in the one reaction could reduce the genomic fragment size down to about 100 bases. Theadapter-DNA fragment can be attached to a streptavidin support for removal of excess fragmentDNA. Excess adapter that did not ligate to mismatch cleaved ends will also bind to the streptavidinsolid support. The biotinylated and phosphorylated strand can now be removed by lambda 30 exonuclease which will degrade from the 5-prime end but leave the non-phosphorylated strand intactTo create a circle from this structure now requires both ends of the molecule coming together andligating to form the circle. Several approaches are available to form the circle using a bridgingoligonucleotide, as described above. A polynucleotide can be added to the 3-prime end with terminaltransferase to create a sequence for one half of a bridge oligonucleotide (818) to hybridize to, shown 35 as polyA tail (816). The other half will bind to sequences in the adapter. Alternatively, before

addition of the exonuclease, an adapter can be added to the end generated by the 4-base cutter whichwill provide sequence for the bridge to hybridize to after removal of one strand by exonuclease. A -48- Νίίί:Λ^Ν>72Γ WO 2006/138257 PCT/US2006/022895 key aspect of this selection procedure is the ability to select the strand for circularization andamplification. This ensures that only the strand with the original mutation (from the 5-primeoverhang) and not the strand from the adapter is amplified. If the 3-prime recessed strand wasamplified then a mismatch from the adapter could create a false base call at the site of or near to the 5 mutation. Amplification of DNA captured into the circular molecules proceeds by a rolling circleamplification to form linear concatemer copies of the circle.

Alternative applications of mis-match derived circles. The mis-match derived small circularDNA molecules may be amplified by other means such as PCR. Common primer binding sites can beincorporated into the adapter sequences The amplified material can be used for mutation detection by 10 methods such as Sanger sequencing or array based sequencing.

Cell-free clonal selection of cDNAs. Traditional methods of cloning have several drawbacksincluding the propensity of bacteria to exclude sequences from plasmid replication and the timeconsuming and reagent-intensive protocols required to generate clones of individual cDNAmolecules. Linear single-stranded can be made from amplifications of DNA molecules that have been 15 closed into a circular form. These large concatemeric, linear forms arise from a single molecule and can act as efficient, isolated targets for PCR when separated into a single reaction chamber, in muchthe same way a bacterial colony is picked to retrieve the cDNA containing plasmid. We plan todevelop this approach as a means to select cDNA clones without having to pass through a cell-basedclonal selection step. The first step of this procedure will involve ligating a gene specific 20 oligonucleotide directed to the 5-prime end with a poly dA sequence for binding to the poly dTsequence of the 3-prime end of the cDNA. This oligonucleotide acts as a bridge to allow T4 DNAligase to ligate the two ends and form a circle.

The second step of the reaction is to use a primer, or the bridging oligonucleotide, for a stranddisplacing polymerase such as Phi 29 polymerase to create a concatemer of the circle. The long linear 25 molecules will then be diluted and arrayed in 1536 well plates such that wells with single moleculescan be selected. To ensure about 10 % of the wells contain 1 molecule approximately 90% wouldhave to be sacrificed as having no molecules. To detect the wells that are positive a dendrimer thatrecognizes a universal sequence in the target is hybridized to generate 10K-100K dye molecules permolecule of target, Excess dendrimer is removed through hybridization to biotinylated capture oligos. 30 The wells are analyzed with a fluorescent plate reader and the presence of DNA scored. Positivewells are then re-arrayed to consolidate the clones into plates with complete wells for furtheramplification

Splice Variant Detection and Exon Profiling 35 The process described is based on random DNA arrays and "smart" probe pools for the identification and quantification of expression levels of thousands of genes and their splice variants.

In eukaryotes, as the primary transcript emerges from the transcription complex, spliceosomes -49- WO 2006/138257 PCT/US2006/022895 interact with splice sites on the primary transcript to excise out the introns, e.g. Maniatis et al, Nature,418:236-243 (2002). However, because of either mutations that alter the splice site sequences, orexternal factors that affect spliceosome interaction with splice sites, alternative splice sites, or crypticsplice sites, could be selected resulting in expression of protein variants encoded by mRNA with 5 different sets of exons. Surveys of cDNA sequences from large scale EST sequencing projectsindicated that over 50 % of the genes have known splice variants. In a recent study using amicroarray-based approach, it was estimated that as high as 75% of genes are alternatively spliced,e.g. Johnson et al, Science, 302:2141-2144 (2003).

The diversity of proteins generated through alternative splicing could partially contribute to 10 the complexity of biological processes in higher eukaryotes. This also leads to the implication that theaberrant expression of variant protein forms could be responsible for pathogenesis of diseases.

Indeed, alternative splicing has been found to associate with various diseases like growth hormonedeficiency, Parkinson's disease, cystic fibrosis and myotonic dystrophy, e.g. Garcia-Blanco et al,Nature Biotechnology, 22: 535-546 (2004). Because of the difficulty in isolating and characterizing 15 novel splice variants, the evidence implicating roles of splice variants in cancer could represent the tipof the iceberg. With the availability of tools that could rapidly and reliably characterize splicingpatterns of mRNA, it would help to elucidate the role of alternative splicing in cancer and in diseasedevelopment in general.

In one aspect, methods of the invention permit large-scale measurement of splice variants 20 with the following steps: (a) Prepare full length first strand cDNA for targeted or all mRNAs. (b)Circularize the generated full length (or all) first strand cDNA molecules by incorporating an adaptersequence, (c) By using primer complementary to the adapter sequence perform rolling circlereplication (RCR) of cDNA circles to form concatemers with over 100 copies of initial cDNA. (d)Prepare random arrays by attaching RCR produced “cDNA balls” to glass surface coated with capture 25 oligonucleotide complementary to a portion of the adapter sequence; with an advanced submicronpatterned surface one mm2 can have between 1-10 million cDNA spots; note that the attachment is amolecular process and does not require robotic spotting of individual “cDNA balls” or concatemers.(e) Starting from pre-made universal libraries of 4096 6-mers and 1024 labeled 5-mers, use asophisticated computer program and a simple robotic pipettor to create 40-80 pools of about 200 6- 30 mers and 20 5-mers for testing all 10,000 or more exons in targeted 1000 or more up to all knowngenes in the sample organism/tissue. (f) In a 4-8 hour process, hybridize/ligate all probe pools in 40-80 cycles on the same random array using an automated microscope-like instrument with a sensitive10-mega pixel CCD detector for generating an array image for each cycle, (g) Use a computerprogram to perform spot signal intensity analysis to identify which cDNA is on which spot, and if any 35 of the expected exons is missing in any of the analyzed genes. Obtain exact expression levels for eachsplice variant by counting occurrences in the array. -50- --- WO 2006/138257 PCT/US2006/022895

This system provides a complete analysis of the exon pattern on a single transcript, instead ofmerely providing information on the ratios of exon usage or quantification of splicing events over theentire population of transcribed genes using the current expression arrays hybridized with labeledmRNA/cDNA. At the maximum limit of its sensitivity, it allows a detailed analysis down to a single 5 molecule of a mRNA type present in only one in hundreds of other cells; this would provide unique potentials for early diagnosis of cancer cells. The combination of selective cDNA preparation with an"array of random arrays" in a standard 384-well format and with "smart" pools of universal shortprobes provides great flexibility in designing assays; for examples, deep analysis of a small number ofgenes in selected samples, or more general analysis in a larger number of samples, or analysis of a 10 large number of genes in smaller number of samples. The analysis provides simultaneously 1) detection of each specific splice variant, 2) quantification of expression of wild type and alternativelyspliced mRNAs. It can also be used to monitor gross chromosomal alterations based on the detectionof gene deletions and gene translocations by loss of heterozygosity and presence of two sub-sets ofexons from two genes in the same transcript on a single spot on the random array. The exceptional 15 capacity and informativeness of this assay is coupled with simple sample preparation from very small quantities of mRNA, fully-automated assay based on all pre-made, validated reagents includinglibraries of universal labeled and unlabeled probes and primers/adapters that will be ultimatelydeveloped for all human and model organism genes. The proposed splice variant profiling process isequivalent to high throughput sequencing of individual full length cDNA clones; rSBH throughput 20 can reach one billion cDNA molecules profiled in a 4-8 hour assay. This system will provide apowerful tool to monitor changes in expression levels of various splice variants during diseaseemergence and progression. It can enable discovery of novel splice variants or validate known splicevariants to serve as biomarkers to monitor cancer progression. It can also provide means to furtherunderstanding the roles of alternative splice variants and their possible uses as therapeutic targets. 25 Universal nature and flexibility of this low cost and high throughput assay provides great commercialopportunities for cancer research and diagnostics and in all other biomedical areas. This high capacitysystem is ideal for service providing labs or companies.

Preparation of templates for in vitro transcription. Exon sequences are cloned into themultiple cloning sites (MCS) of plasmid pBluescript, or like vector. For the purposes of 30 demonstrating the usefulness of the probe pools, it is not necessary to clone the contiguous full-lengthsequence, nor to maintain the proper protein coding frame. For genes that are shorter than 1 kb, PCRproducts are generated from cDNA using gene specific oiigos for the full length sequence. For longergenes, PCR products are generated comprising about 500 bp that corresponding to contiguous blockof exons and ordered the fragments by cloning into appropriate cloning sites in the MCS of 35 pBluescript. This is also the approach for cloning the alternative spliced versions, since the desiredvariant might not be present in the cDNA source used for PCR. -51 - WO 2006/138257 FCT/US2006/022895

The last site of the MCS is used to insert a string of 40 A's to simulate the polyA tails ofcellular mRNA. This is to control for the possibility that the polyA tail might interfere with thesample preparation step described below, although it is not expected to be a problem since a poly-dAtail is incorporated in sample preparation of genomic fragments as described. T7 RNA polymerase 5 will be used to generate the run-off transcripts and the RNA generated will be purified with thestandard methods.

Preparation of samples for arraying. Because the probe pools are designed for specific genes,cDNA is prepared for those specific genes only. For priming tlie reverse transcription reactions, gene-specific primers are used, therefore for 1000 genes, 1000 primers are used. The location of the 10 priming site for the reverse transcription is selected with care, since it is not reasonable to expect thesynthesis of cDNA >2kb to be of high efficiency. It is quite common that the last exon would consistof the end of the coding sequence and a long 3' untranslated region. In the case of CD44 for example,although the frill-length mRNA is about 5.7 kb, the 3' UTR comprises of 3kb, while the coding regionis only 2.2 kb. Therefore the logical location of the reverse transcription primer site is usually 15 immediately downstream of the end of the coding sequence. For some splice variants, the alternativeexons are often clustered together as a block to create a region of variability. In the case of TenascinC variants (8.5kb), the most common isoform has a block of 8 extra exons, and there is evidence tosuggest that there is variability in exon usage in that region. So for Tenascin C, the primer will belocated just downstream of that region. Because of the concern of synthesizing cDNA with length 20 >2kb, for long genes, it might be necessary to divide the exons into blocks of 2 kb with multiple primers.

Reverse transcription reactions may be carried out with commercial systems, e.g. SuperscriptIII system from Invitrogen (Carlsbad, CA) and the StrataScript system from Stratagene (La Jolla, CA). Once single stranded cDNA molecules are produced, the rest of the procedures involved putting 25 on the adaptor sequence, circularization of the molecule and RCR as described above. The 5' ends ofthe cDNAs are basically the incorporated gene-specific primers used for initiating the reverse .transcription. By incorporating a 7 base universal tag on the 5' end of the reverse-transcriptionpriming oligos, all the cDNA generated will carry the same 7 base sequence at tlie 5' end. Thus asingle template oligonucleotide that is complementary to both the adaptor sequence and the universal 30 tag can be used to ligate the adaptor to all the target molecules, without using the template oligonucleotide with degenerate bases. As for the 3' end of the cDNA (5' end of the mRNA) which isusually ill-defined, it may be treated like a random sequence end of a genomic fragment. Similarmethods of adding a polyA tail will be applied, thus the same circle closing reaction may also be used.

Reverse transcriptases are prone to terminate prematurely to create truncated cDNAs. 35 Severely truncated cDNAs probably will not have enough probe binding sites to be identified with agene assignment, thus would not be analyzed. cDNA molecules that are close, but not quite full-length, may show up as splice variant with missing 5' exons. If there are no corroborating evidence -52- WO 2006/138257 PCT/US2006/022895 from a sequence database to support such variants, they may be discounted. A way to avoid suchproblem is to select for only the full-length cDNA (or those with the desired 3' end) to be compatiblewith circle closing reaction, then any truncated molecules will not be circularized nor replicated. Firsta dideoxy-cytosine residue can be added to the 3' end of all the cDNA to block ligation, then by using 5 a mismatch oligo targeting the desired sequence, a new 3' end can be generated by enzyme mismatchcleavage using T4 endonuclease VII. With the new 3’ end, the cDNA can proceed with the adding apoly-dA tail and with the standard protocols of circularization and replication.

Replicated and arrayed concatemers of the exon fragments may be carried out usingcombinatorial SBH, as described above. The algorithm of the following steps may be used to select 10 5-mer and 6-mer probes for use in the technique:

Step 1: Select 1000-2000 shortest exons (total about 20 - 50 kb), and find out matching sequences for each of 1024 available labeled 5-mers. On average each 5-mer will occur 20 times over20 kb, but some may occur over 50 or over 100 times. By selecting the most frequent 5-mer, thelargest number of short exons will be detected with the single labeled probe. A goal would be to 15 detect about 50-100 short exons (10%-20% of 500 exons) per cycle. Thus less than 10 labeled probesand 50-100 unlabeled 6-mers would be sufficient. Small number of labeled probes is favorablebecause it minimizes overall fluorescent background.

Step 2. Find out all 6-mers that are contiguous with all sites in all 1000 genes that arecomplementary to 10 selected 5-mers. On average 20 such sites will exist in each 2kb gene. Total 20 number of sites would be about 20,000, e.g., each 6-mer on average will occur 5 times. Sort 6-mersby the hit frequency. The most frequent may have over 20 hits, e.g. such 6-mer will detect 20 genesthrough combinations with 10 labeled probes. Thus, to get a single probe pair for each of the 500genes a minimum of 25 6-mer probes would be required. Realistically, 100 to 200 6-mers may berequired. 25 Due to benefits of combinatorial SBH that uses pre-made libraries of 6-mer and 5-mer probes 40 probe pools are readily prepared with about 200 probes per pool using established pipettingrobotics. The information generated is equivalent to having over 3 probes per exon, therefore the useof 8000 5-mers and 6-mers effectively replaces the 30,000 longer exons specific probes required for asingle set of 1000 genes. 30 Exon profiling. The profiling of exons can be performed in two phases : the gene identification phase and the exon identification phase. In die gene identification phase, eachconcatemer on the array can be uniquely identified with a particular gene. In theory, 10 probe poolsor hybridization cycles will be enough to identify 1000 genes using the following scheme. Each geneis assigned a unique binary code. The number of binary digits thus depends on the total number of 35 genes: 3 digits for 8 genes, 10 digits for 1024 genes. Each probe pool is designed to correspond to adigit of the binary code and would contain probes that would hit a unique combination of half of thegenes and one hit per gene only. Thus for each hybridization cycle, an unique half of the genes will -53- WO 2006/138257 PCT/US2006/022895 score a 1 for that digit and the other half will score zero. Ten hybridization cycles with 10 probepools will generate 1024 unique binary codes, enough to assign 1000 unique genes to all theconcatemers on the array. To provide redundancy in the identification data, 15-20 cycles would beused. If 20 cycles are used, it would provide 1 million unique binary codes and there should be 5 enough information to account for loss of signals due to missing exons or gene deletions. It will alsobe equivalent to having 10 data points per gene (20 cycles of 500 data point each give 10,000 datapoints total), or one positive probe-pair per exon, on average. At this point after 20 cycles, thissystem is capable of making assignment of 1 million unique gene identities to the ampliots. Thereforeby counting gene identities of the ampliots, one can determine quantitatively the expression level of 10 all the genes (but not sub-typing of splice variants) in any given samples.

After identifying each ampliot with a gene assignment, its exon pattern will be profiled in the exon identification phase. For the exon identification phase, one exon per gene in all or most of thegenes is tested per hybridization cycle. In most cases 10-20 exon identification cycles should besufficient. Thus, in the case of using 20 exon identification cycles we will obtain information of 2 15 probes per each of 10 exons in each gene. For genes with more than 20 exons, methods can be developed so that 2 exons per gene can be probed at the same cycle. One possibility is using multiplefluorophores of different colors, and another possibility is to exploit differential hybrid stabilities ofdifferent ligation probe pairs.

In conclusion, a total of about 40 assay cycles will provide sufficient information to obtain 20 gene identity at each spot and to provide three matching probe-pairs for each of 10,000 exons withenough informational redundancy to provide accurate identification of missing exons due toalternative splicing or chromosomal deletions.

Example 1 25 Glass Cover Slip as Random Array Support:

Derivatization Protocol

In this example, a glass cover slip is prepared for use as a support for disposing DNAconcatemers. The following materials are used:

Millipore DI water 30 2.5 ml of 3 - Aminopropyldimethyl ethoxysilane (Gelest) 1.6 grams p-phenylenediisothiocyanate (Acros Organics / fisher) 210 grams KOH (VWR)

Ethanol (VWR)

Methanol (VWR) 35 Pyridine (VWR) Ν,Ν-dimethylformamide (VWR)

Acetone (VWR) -54- WO 2006/138257 PCT/US2006/022895

Equipment100c ovenmagnetic stir plate1 2s’x.5” magnetic stir bar 5 2 4 liter Nunc beaker 7 4”x8”x4” glass containers1 liter graduated cylinder1 100 ml graduated cylinder1 lab scale 10 1 Metzler scale 1 large weigh boat1 small weigh boat1 pair thick nitrile gloves1 large funnel 15 1 ml pipettman with filter tips 1 nalgene stir bar1 airtight container (tupperware)

Using die large graduated cylinder measure 950ml of ethanol, add to the 4 liter Nunc beaker.

Measure 50ml of DI water in the small graduated cylinder and add to the same nunc beaker. Measure 20 out 210 grams of KOH pellets in a weigh boat on the lab scale. Add stir bar and KOH pellets to thebeaker. Place beaker on stir plate and stir at low speed until KOH is completely dissolved. WhileKOH is dissolving, lay out 6 pre-washed glass containers, fill containers 2-5 with DI water until %inch from top (~800ml). Fill container 6 with acetone to top. Carefully pour dissolved KOHsolution into container 1 until 16” to top. Add racked cover slips to container 1 wait 3 minutes, 25 remove racks from container 1 and wash in containers 2-5 leaving racks in each container a minimumof 15 seconds. Submerse racks briefly in container 6. Set aside racks, dispose the solutions fromcontainers 1 and 2 in the basic waste container using the large funnel and thick nitrile gloves, cleanarid dry labware. Lay out 7 clean and dry glass containers. Add 775 ml of acetone to container 1 add2.5 ml of DI water to container 1. stir container 1 with pipette tip for 20 seconds. With a new pipette 30 tip add 2.5 ml of 3-aminopropyldimethylethoxysilane to container 1. Stir with pipette tip for 10seconds. Immerse all 5 racks of cover slips into container 1. Cover container 1 with polypropylenebox top. Wait 45 minutes. 15 minutes prior to the completion of the reaction, fill containers 2-4 until!4” to top with acetone, fill container 5 with water 14” to top. Fill container 6 until to top withacetone. Upon reaction completion (45 minutes) transfer cover slip racks 1-5 from container 1 to 35 container 2, wait 15 seconds. Repeat this though container 6. Place racks into empty container 7 andput in 100c oven. Wait one hour. -55-

-HH WO 2006/138257 PCT/US2006/022895

Lay out 7 glass containers. After racks come out of oven, use the Meltzer scale to weigh out 1.6grams of p-phenylenediisothiocy anate (PDC) in the small weigh boat. Pour 720 mldimethylformaniide into the cleaned 1 liter graduated cylinder, fill to 800ml with pyridine. Pour 50%this solution into a clean class container then pour it hack into the cylinder to mix (repeat once). Fill 5 container 1 until 14” to top with this solution. Add the PDC from the weigh boat to container L Usestir bar to mix solution. Crush PDC clumps that refuse to dissolve, then stir again. Cover slip racksshould be cool by now. Place all 5 racks into container one. Cover with polypropylene box top. Wait2 hours. 10 minutes prior to reaction completion fill containers 2 and 3 with methanol until Ά” fromtop. Fill containers 4 and 5 with acetone until from top. Fill container 6 with 65% acetone 35% 10 water until VP from top. Fill container 7 with acetone. Successively transfer racks through all containers, waiting 15 seconds between each transfer. Remove racks from container 7 dump contentsof containers 1-7 into organic waste drum. Replace racks to container 7 and dry in oven for 15minutes. Place dry racks into airtight container, they are now ready for attachment. 15 Example 2

Preparation of RCR Products form E. coli Genomic DNA and Disposition onto a Glass Cover Slip E.coli genomic DNA (32 ug) (Sigma Chemical Co) was fragmented with 0.16 U of Dnasei(Epicentre) at 37°C for 10 min and then heat inactivated at 95°C for 10 min. Reaction products were 20 distributed with an average size of 200 bp as determined by agarose gel electrophoresis. If reaction products did not meet the required size distribution they were further digested with the addition offresh enzyme. The final concentration was 200 ng/ul of genomic DNA.

The Dnase digested DNA (26 ng/ul) was reacted with Terminal deoxynucleotide transferase(0.66 U/ul) from New England Biolabs (NEB) in reaction buffer supplied by NEB. The reaction 25 contained dATP (2 mM) and was performed at 37C for 30 min and then heat inactivated at 70 C for10 min. The DNA sample was then heated to 95 C for 5 min before rapid cooling on ice. A synthetic DNA adapter was then ligated to the 5’ end of the genomic DNA by first forminga hybrid of a 65-base oligonucleotide

(TATCATCTACTGCACTGACCGGATGTTAGGAAGACAAAAGGAAGCTGAGGGTCACATTA 30 ACGGAC)(SEQ ID NO: 8) with a second oligonucleotide (NNNNNNNGTCCGTTAATGTGAC 3’2’3’ddC) (SEQ ID NO: 9) at the 3’ end of the 65mer in which the 7 “Ns” form an overhang. Theshorter oligo will act as a splint for ligation of the 65mer to the 5’ end of the genomic fragments. Thesplint molecule consists of 7 degenerate bases at its 5* end to hybridize to variable bases at the 5’ endof the genomic DNA. The adapter hybrid was formed by slowly hybridizing 1200 pmol of adapter 35 with 1200 pmol of splint in 52 ul from 95C to room temperature over Ihr. T4 DNA Ligase (0.3 U/ul) was combined with genomic DNA (17 ng/ul) and adapter-splint (0.5 uM) in IX ligase reaction buffer supplied by NEB. The ligation proceeded at 15C for 30 min, -56- WGG" -???*·· <Μ7:--~—Μ§Βί W0 2006/138257 PCT/US2006/022895 20C for 30 min and then inactivated at 70C for 10 min. A second splint molecule(AGATGATATTTTTTTT 3' 2'3'ddC) (SEQ ID NO: 10) (0.6 uM) was then added to the reaction andthe mix was supplemented with more ligase buffer and T4 DNA ligase (0.3 U/ul). The reactionproceeded at 15C for 30 min and then at 20C for 30 min before inactivation for 10 min at 70C. 5 The ligation mix was then treated with exonuclease I (NEB) (1 U/ui) at 37 C for 60 min, followed by inactivation at 80C for 20 min

Rolling circle replication was performed in reaction buffer supplied by NEB with BSA (0.1Lig/ul), 0.2 mM each dNTP, an initiating primer (TCAGCTTCCTTTTGTCTTCCTAAC) (SEQ IDNO: 11) at 2 fmol/ul, exonuclease treated ligation of genomic DNA at 24 pg/ul, and Phi 29 10 polymerase (0.2 U/ul). The reaction was performed for 1 hr at 30C and then heat inactivated at 70Cfor 10 min. RCR reaction products were attached to the surface of cover slips by first attaching aminemodified oligonucleotides to the surface of the cover slips. A capture probe ([AMINOC6][SP-C18][SP“C18]GGATGTTAGGAAGACAAAAGGAAGCTGAGG) (SEQ ID NO: 12) (50 uM) was 15 added to the DITC derivatized cover slips in 0.1 uM NaHCO3 and allowed to diy at 40 C for about 30min. The cover slips were rinsed in DDI water for 15 min and dried. RCR reaction products (4.5 ul)were then combined with 0.5 ul of 20 X SSPE and added to the center of the slide. The sample wasallowed to air dry and non-attached material was washed off for 10 min in 3x SSPE and then brieflyin DDI water. The slide was then dried before assembly on the microscope. Attached RCR products 20 were visualized by hybridizing an 1 lmer TAMRA labeled probe that is complementary to a region ofthe adapter RCR reaction products were fonned from a single stranded SOmer synthetic DNA target(NNNNNNNNGCATANCACGANGTCATNATCGTNCAAACGTCAGTCCANGAATCNAGATCCACTTAGANTAAAAAAAAAAAA) (SEQ ID NO: 13) as above but without poly A addition with 25 TDT. The RCR reaction contained target molecules at an estimated 12.6 fmol/ul. Reaction products(5 ul) were combined with SSPE (2X) and SDS (0.3%) in a total reaction volume of 20 ul. Thesample was applied to a cover-slip in which lines of capture probe ([AMINOC6][SP-C18][SP-C18]GGATGTTAGGAAGACAAAAGGAAGCTGAGG),deposited in a solution of 50 uM with 0.1uM NaHCO3, were dried onto the surface and left in a humid chamber for 30 min. The solution was 30 then washed off in 3x SSPE for 10 min and then briefly in water.

Various reaction components were tested for their effect upon RCR product formation, The addition of Phi 29 to the RCR reaction at a final concentration of 0.1 U/ul rather than 0.2 U/ul wasfound to create a greater proportion of RCR products that were of larger intensity after detection probehybridization. The addition of initiating primer at 10 to 100 fold molar ratio relative to estimated 35 target concentration was also found to be optimal. Increased extension times produced more intensefluorescent signals but tended to produce more diffuse concatemers. With the current attachment -57- η - —β to----- :«ΗΰββΗΤτ WO 2006/138257 PCT/US2006/022895 protocols a 2hr extension time produced enhanced signals relative to a lhr incubation with minimaldetrimental impact upon RCR product morphology.

Further optimization of RCR products have been achieved by reducing the estimatedconcentration of synthetic and genomic targets to 0.1 to 0.25 finol/ul in the RCR reaction. This 5 typically results in distinct and unique RCR products on the surface of the microscope slide usingmethod 1 for attachment. For synthetic targets in which a higher concentration of targets in the RCRreaction may be present (e.g. >5 fmol/ul), RCR products may be attached by method 2.

Attachment method 1. RCR reaction products (4.5 ul) were combined with 0.5 ul of 20 X SSPE andadded to the center of the slide. The sample was allowed to air dry and non-attached material was 10 washed off for 10 min in 3x SSPE and then briefly in DDI water. The slide was then dried beforeassembly on the microscope. Attached RCR products were visualized by hybridizing an 1 lmerTAMRA labeled probe that is complementary to a region of the adapter. Attachment method 2. RCRreaction products (1 ul) were combined with 50 ul of 3XSSPE and added to the center of the coverslip with capture probe attached. Addition of SDS (0.3%) was found to promote specific attachment 15 to the capture probes and not to the derivatized surface. The sample was incubated at room temperature for 30 min and non-attached material was washed off for 10 min in 3x SSPE and thenbriefly in DDI water. The slide was then dried before assembly on the microscope. Attached RCRproducts were visualized by hybridizing an 1 lmer TAMRA labeled probe that is complementaiy to aregion of the adapter. The above protocols provide RCR product densities of about 1 RCR product 20 per 2-4 micron square. Exemplaiy image of a resulting cover slip is shown in Fig. 3.

Example 3

Distinguish RCR Products on Random Arrays

Using Fluorescently Labeled Probes 25 PCR products from diagnostic regions of Bacillus anthracis and Yersinia pestis were converted into single stranded DNA and attached to a universal adaptor. These two samples were thenmixed and replicated together using RCR and deposited onto a glass surface as a random array.Successive hybridization with amplicon specific probes showed that each spot on the arraycorresponded uniquely to either one of the two sequences and that they can be identified specifically 30 with the probes, as illustrated in Fig. 4. This result demonstrates sensitivity and specificity of

identifying DNA present in submicron sized DNA concatemers having about 100-1000 copies of aDNA fragment generated by the RCR reaction. A 155 bp amplicon sequence from B. anthracis and a275 bp amplicon sequence from Y. pestis were amplified using standard PCR techniques with PCRprimers in which one primer of the pair was phosphorylated. A single stranded form of the PCR 35 products was generated by degradation of the phosphorylated strand using lambda exonuclease. The5' end of the remaining strand was then phosphoiylated using T4 DNA polynucleotide kinase toallow ligation of the single stranded product to the universal adaptor. The universal adaptor was -58- WO 2006/138257 PCT/US2006/022895 ligated using T4 DNA ligase to the 5' end of the target molecule, assisted by a templateoligonucleotide complementary to the 5’ end of the targets and 3’ end of the universal adaptor. Theadaptor ligated targets were then circularized using bridging oligonucleotides with basescomplementary to the adaptor and to the 3' end of the targets. Linear DNA molecules were removed 5 by treating with exonuclease I. RCR products (DNA concatemers) were generated by mixing thesingle-stranded samples and using Phi29 polymerase to replicate around the circularized adaptor-target molecules with the bridging oligonucleotides as the initiating primers.

To prepare the cover slips for attaching amine-modified oligonucleotides, the cover slips werefirst cleaned in a potassium/ethanol solution followed by rinsing and drying. They were then treated 10 with a solution of 3-aminopropyldimethylethoxysilane, acetone, and water for 45 minutes and curedin an oven at 100’C for 1 hour. As a final step, the cover slips were treated with a solution ofp-phenylenediisothiocyanate (PDC), pyridine, and dimethylformamide for 2 hours. The captureoligonucleotide (sequence 5'-GGATGTTAGGAAGACAAAAGGAAGCTGAGG-3') (SEQ ID NO:14) is complementary to the universal adaptor sequence, and is modified at the 5' end with an amine 15 group and 2 C-18 linkers. For attachment, 10 μϊ of the capture oligo at 10 μΜ in 0.1 M NaHCO3 wasspotted onto the center of the derivatized cover slip, dried for 10 minutes in a70°C oven and rinsedwith water. To create an array of DNA concatemers, the RCR reaction containing the DNAconcatemers was diluted 10-folds with 3X SSPE, 20 μϊ of which was then deposited over theimmobilized capture oligonucleotides on the cover slip surface for 30 minutes in a moisture saturated 20 chamber. Tire cover slip with the DNA concatemers was then assembled into a reaction chamber andwas rinsed by 2 ml of 3X SSPE. Arrayed target concatemer molecules derived from B. anthracis andY. pestis PCR amplicons were probed sequentially with TAMRA-labeled oligomer: probe BrPrb3(sequence : 5'-CATTAACGGAC-3' (SEQ ID NO: 15), specifically complementary to the universaladaptor sequence), probe Ba3 (sequence : 5'-TGAGCGATTCG-3' (SEQ ID NO: 16), specifically 25 complementary to the Ba3 amplicon sequence), probe Yp3 (sequence : 5'-GGTGTCATGGA-3’, specifically complementary to the Yp3 amplicon sequence). The probes were hybridized to the arrayat a concentration of 0.1 μΜ for 20 min in 3X SSPE at room temperature. Excess probes werewashed off with 2 ml of 3X SSPE. Images were taken with the TERF microscope. The probes werethen stripped off with 1 ml of 3X SSPE at 80°C for 5 minutes to prepare the arrayed target molecules 30 for the next round of hybridization.

By overlaying the images obtained from successive hybridization of 3 probes, as shown inFig. 4, it can be seen that most of the arrayed molecules that hybridized with the adaptor probe wouldonly hybridize to either the amplicon 1 probe (e.g. “A” in Fig. 4) or the amplicon 2 probe (e.g. “B” inFig. 4), with very few that would hybridize to both. This specific hybridization pattern demonstrates 35 that each spot on the array contains only one type of sequence, either the B anthracis amplicon or theY. pestis amplicon. -59- '-v WO 2006/138257 PCT/US2006/022895

Example 4

Decoding a Base Position in Arrayed Concatemers Created

From a Synthetic 80-Mer Oligonucleotide Containing 10 a Degenerated Base

Individual molecules of a synthetic oligonucleotide containing a degenerate base can be divided into 4 sub-populations, each may have either an A, C, G or T base at that particular position.An array of concatemers created from this synthetic DNA may have about 25% of spots with each ofthe bases. Successful identification of these sub-populations of concatemers was demonstrated by four 15 successive hybridization and ligation of pairs of probes, specific to each of the 4 bases, as shown in

Fig. 5. A 5' phosphoiylated, 3' TAMRA-labeled pentamer oligonucleotide was paired with one of thefour hexamer oligonucleotides. Each of these 4 ligation probe pairs should hybridize to either an A, C,G or T containing version of the target. Discrimination scores of greater than 3 were obtained for mosttargets, demonstrating the ability to identify single base differences between the nanoball targets. The 20 discrimination score is the highest spot score divided by the average of the other 3 base-specific signals of the same spot. By adjusting the assay conditions (buffer composition, concentrations of allcomponents, time and temperature of each step in the cycle) higher signal to background and fullmatch to mismatch ratios are expected. This was demonstrated with a similar ligation assay performedon the spotted arrays of 6-mer probes. In this case full-match/background ratio was about 50 and the 25 average full match/mismatch ratio was 30. The results further demonstrate the ability to determinepartial or complete sequences of DNA present in concatemers by increasing the number ofconsecutive probe cycles or by using 4 or more probes labeled with different dyes per each cycle.Synthetic oligonucleotide (TIA : 5'-

NNNNNNNNGCATANCACGANGTCATNATCGTNCAAACGTCAGTCCANGAATCNAGATCC 30 ACTTAGANTAAAAAAAAAAAA-3') (SEQ ID NO: 13) contains at position 32 a degenerate base.Universal adaptor was ligated to this oligonucleotide and the adaptor-Tl A DNA was circularized asdescribed before. DNA concatemers made using the rolling circle replication (RCR) reaction on thistarget were arrayed onto the random array. Because each spot on this random array corresponded totandemly replicated copies originated from a single molecule of TIA, therefore DNA in a particular 35 arrayed spot would contain either an A, or a C, or a G, or a T at positions corresponding to position 32of TIA. To identify these sub-populations, a set of 4 ligation probes specific to each of the 4 baseswas used. A 5' phosphorylated, 3' TAMRA-labeled pentamer oligonucleotide corresponding to -60- ΜΗ WO 2006/138257 PCT/US2006/022895 position 33-37 of Τ1Α with sequence CAAAC (probe TlA9b) was paired with one of the followinghexamer oligonucleotides corresponding to position 27-32 : ACTGTA (probe Tl A9a), ACTGTC(probe TlAlOa), ACTGTG (probe T1A1 la), ACTGTT (probe Tl A 12a). Each of these 4 ligationprobe pairs should hybridize to either an A, C, G or T containing version of Tl A. For each 5 hybridization cycle, the probes were incubated with die array in a ligation/hybridization buffer containing T4 DNA ligase at 20" C for 5 minutes. Excess probes were washed off at 20"C and imageswere taken with a TIRF microscope. Bound probes were stripped to prepare for the next round ofhybridization.

An adaptor specific probe (BrPrb3) was hybridized to the array to establish the positions of all 10 the spots. The 4 ligation probe pairs, at 0.4 μΜ, were then hybridized successively to the array withthe base identifications as illustrated for four spots in Fig. 5. It is clear that most of the spots areassociated with only one of the 4 ligation probe pairs, and thus the nature of the base at position 32 ofTl A can be determined specifically. 15 Example 5

Decoding Two Degenerate Bases at the End of a Synthetic 80-Mer Oligonucleotide

The same synthetic oligonucleotide described above contains 8 degenerate bases at the 5' endto simulate random genomic DNA ends. The concatemers created from this oligonucleotide may have 20 these 8 degenerate bases placed directly next to the adaptor sequence. To demonstrate the feasibilityof sequencing the two unknown bases adjacent to the known adaptor sequence, a 12-meroligonucleotide (UKO-12 sequence 5-ACATTAACGGAC-3') (SEQ ID NO: 17) with a specificsequence to hybridize to the 3' end of the adaptor sequence was used as the anchor, and a set of 16TAMRA-labeled oligonucleotides in the form of BBNNNNNN were used as the sequence-reading 25 probes. For each hybridization cycle, 0.2uM of UK0-12 anchor probe and 0.4uM of the BBNNNNNN probe were incubated with the array in a ligation/hybridization buffer containing T4DNA ligase at 20" C for 10 minutes. Excess probes were washed off at 20° C and images were takenwith a TIRF microscope. Bound probes were stripped to prepare for the next round of hybridization.Using a subset of the BBNNNNNN probe set (namely GA, GC, GG and GT in the place of BB), spots 30 were able to be identified spots on the concatemer array created from targets that specifically bind toone of these 4 probes, with an average full match/mismatch ratio of over 20, as shown in Fig. 6.

DEFINITIONS

Terms and symbols of nucleic acid chemistry, biochemistry, genetics, and molecular biologyused herein follow those of standard treatises and texts in the field, e.g. Kornberg and Baker, DNA 35 Replication, Second Edition (W.H. Freeman, New York, 1992); Lehninger, Biochemistry, SecondEdition (Worth Publishers, New York, 1975); Strachan and Read, Human Molecular Genetics,

Second Edition (Wiley-Liss, New York, 1999); Eckstein, editor, Oligonucleotides and Analogs: A -61- WO 2006/138257 PCT/US2006/022895

Practical Approach (Oxford University Press, New York, 1991); Gait, editor, OligonucleotideSynthesis: A Practical Approach (IRL Press, Oxford, 1984); and the like. “Amplicon” means the product of a polynucleotide amplification reaction. That is, it is apopulation of polynucleotides, usually double stranded, that are replicated from one or more starting 5 sequences. The one or more starting sequences may be one or more copies of the same sequence, orit may be a mixture of different sequences. Amplicons may be produced by a variety ofamplification reactions whose products are multiple replicates of one or more target nucleic acids.Generally, amplification reactions producing amplicons are “template-driven” in that base pairing ofreactants, either nucleotides or oligonucleotides, have complements in a template polynucleotide 10 that are required for the creation of reaction products. In one aspect, template-driven reactions areprimer extensions with a nucleic acid polymerase or oligonucleotide ligations with a nucleic acidligase. Such reactions include, but are not limited to, polymerase chain reactions (PCRs), linearpolymerase reactions, nucleic acid sequence-based amplification (NASBAs), rolling circleamplifications, and the like, disclosed in the following references that are incorporated herein by 15 reference: Mullis et al, U.S. patents 4,683,195; 4,965,188; 4,683,202; 4,800,159 (PCR); Gelfand etal, U.S. patent 5,210,015 (real-time PCR with “taqman” probes); Wittwer et al, U.S. patent6,174,670; Kacian et al, U.S. patent 5,399,491 (“NASBA”); Lizardi, U.S. patent 5,854,033; Aono etal, Japanese patent publ. JP 4-262799 (rolling circle amplification); and the like. In one aspect,amplicons of the invention are produced by PCRs. An amplification reaction may be a “real-time” 20 amplification if a detection chemistry is available that permits a reaction product to be measured asthe amplification reaction progresses, e.g. “real-time PCR” described below, or “real-time NASBA”as described in Leone et al, Nucleic Acids Research, 26:2150-2155 (1998), and like references. Asused herein, the term “amplifying” means performing an amplification reaction. A “reactionmixture” means a solution containing all the necessary reactants for performing a reaction, which 25 may include, but not be limited to, buffering agents to maintain pH at a selected level during areaction, salts, co-factors, scavengers, and the like. “Complementary or substantially complementary” refers to the hybridization or base pairingor the formation of a duplex between nucleotides or nucleic acids, such as, for instance, between thetwo strands of a double stranded DNA molecule or between an oligonucleotide primer and a primer 30 binding site on a single stranded nucleic acid. Complementary nucleotides are, generally, A and T (orA and U), or C and G. Two single stranded RNA or DNA molecules are said to be substantiallycomplementary when the nucleotides of one strand, optimally aligned and compared and withappropriate nucleotide insertions or deletions, pair with at least about 80% of the nucleotides of theother strand, usually at least about 90% to 95%, and more preferably from about 98 to 100%. 35 Alternatively, substantial complementarity exists when an RNA or DNA strand will hybridize underselective hybridization conditions to its complement. Typically, selective hybridization will occurwhen there is at least about 65% complementary over a stretch of at least 14 to 25 nucleotides, -62- WO 2006/138257 PCT/US2006/022895 preferably at least about 75%, more preferably at least about 90% complementary. See, M. KanehisaNucleic Acids Res. 12:203 (1984), incorporated herein by reference. “Duplex” means at least two oligonucleotides and/or polynucleotides that are fully orpartially complementary undergo Watson-Crick type base pairing among all or most of their 5 nucleotides so that a stable complex is formed. The terms “annealing” and ‘hybridization” are usedinterchangeably to mean the formation of a stable duplex. "Perfectly matched" in reference to aduplex means that the poly- or oligonucleotide strands making up the duplex form a double strandedstructure with one another such that every nucleotide in each strand undergoes Watson-Crickbasepairing with a nucleotide in the other strand. The term “duplex” comprehends the pairing of 10 nucleoside analogs, such as deoxy inosine, nucleosides with 2-aminopurine bases, PNAs, and thelike, that may be employed. A "mismatch" in a duplex between two oligonucleotides orpolynucleotides means that a pair of nucleotides in the duplex fails to undergo Watson-Crickbonding. “Genetic locus,” or “locus” in reference to a genome or target polynucleotide, means a 15 contiguous subregion or segment of the genome or target polynucleotide. As used herein, geneticlocus, or locus, may refer to the position of a nucleotide, a gene, or a portion of a gene in a genome,including mitochondrial DNA, or it may refer to any contiguous portion of genomic sequencewhether or not it is within, or associated with, a gene. In one aspect, a genetic locus refers to anyportion of genomic sequence, including mitochondrial DNA, from a single nucleotide to a 20 segment of few hundred nucleotides, e.g. 100-300, in length. “Genetic variant” means a substitution, inversion, insertion, or deletion of one or more nucleotides at genetic locus, or a translocation of DNA from one genetic locus to another geneticlocus. In one aspect, genetic variant means an alternative nucleotide sequence at a genetic locus thatmay be present in a population of individuals and that includes nucleotide substitutions, insertions, 25 and deletions with respect to other members of the population. In another aspect, insertions ordeletions at a genetic locus comprises the addition or the absence of from 1 to 10 nucleotides atsuch locus, in comparison with the same locus in another individual of a population. “Hybridization” refers to the process in which two single-stranded polynucleotides bind non-covalently to form a stable double-stranded polynucleotide. The term “hybridization” may also refer 30 to triple-stranded hybridization. The resulting (usually) double-stranded polynucleotide is a “hybrid”or “duplex.” “Hybridization conditions” will typically include salt concentrations of less than about1M, more usually less than about 500 mM and less than about 200 mM. A “hybridization buffer” is abuffered salt solution such as 5X SSPE, or the like. Hybridization temperatures can be as low as 5°C., but are typically greater than 22° C., more typically greater than about 30° C., and preferably in 35 excess of about 37° C. Hybridizations are usually performed under stringent conditions, i.e. conditions under which a probe will hybridize to its target subsequence. Stringent conditions aresequence-dependent and are different in different circumstances. Longer fragments may require -63- WO 2006/138257 PCT/XJS2006/022895 higher hybridization temperatures for specific hybridization. As other factors may affect thestringency of hybridization, including base composition and length of the complementaiy strands,presence of organic solvents and extent of base mismatching, the combination of parameters is moreimportant than the absolute measure of any one alone. Generally, stringent conditions are selected to 5 be about 5° C. lower than the Tm for the specific sequence at s defined ionic strength and pH.

Exemplary stringent conditions include salt concentration of at least 0.01 M to no more than 1 MNaion concentration (or other salts) at a pH 7.0 to 8.3 and a temperature of at least 25° C. For example,conditions of 5*SSPE (750 mM NaCl, 50 mM NaPhosphate, 5 mM EDTA, pH 7.4) and atemperature of 25-30° C. are suitable for allele-specific probe hybridizations. For stringent 10 conditions, see for example, Sambrook, Fritsche and Maniatis. “Molecular Cloning A laboratory

Manual” 2nd Ed. Cold Spring Harbor Press (1989) and Anderson “Nucleic Acid Hybridization” 1st Ed.,BIOS Scientific Publishers Limited (1999), which are hereby incorporated by reference in its entirelyfor all purposes above. “Hybridizing specifically to” or “specifically hybridizing to” or likeexpressions refer to the binding, duplexing, or hybridizing of a molecule substantially to or only to a 15 particular nucleotide sequence or sequences under stringent conditions when that sequence is presentin a complex mixture (e.g., total cellular) DNA or RNA. “Ligation" means to form a covalent bond or linkage between the termini of two or morenucleic acids, e.g. oligonucleotides and/or polynucleotides, in a template-driven reaction. The natureof the bond or linkage may vary widely and the ligation may be carried out enzymatically or 20 chemically. As used herein, ligations are usually carried out enzymatically to form a phosphodiesterlinkage between a 5’ carbon of a terminal nucleotide of one oligonucleotide with 3’ carbon of anotheroligonucleotide. A variety of template-driven ligation reactions are described in the followingreferences, which are incorporated by reference: Whitely et al, U.S. patent 4,883,750; Letsinger et al,U.S. patent 5,476,930; Fung et al, U.S. patent 5,593,826; Kool, U.S. patent 5,426,180; Landegren et 25 al, U.S. patent 5,871,921; Xu and Kool, Nucleic Acids Research, 27:875-881 (1999); Higgins et al,Methods in Enzymology, 68: 50-71 (1979); Engler et al, The Enzymes, 15: 3-29 (1982); andNamsaraev, U.S. patent publication 2004/0110213. Enzymatic ligation usually takes place in a ligasebuffer, which is a buffered salt solution containing any required divalent cations, cofactors, and thelike, for the particular ligase employed. 30 “Microarray" or “array” refers to a solid phase support having a surface, usually planar or substantially planar, which carries an array of sites containing nucleic acids, such that each membersite of the array comprises identical copies of immobilized oligonucleotides or polynucleotides andis spatially defined and not overlapping with other member sites of the array; that is, the sites arespatially discrete. In some cases, sites of a microarray may also be spaced apart as well as discrete; 35 that is, different sites do not share boundaries, but are separated by inter-site regions, usually free ofbound nucleic acids. Spatially defined hybridization sites may additionally be “addressable" in thatits location and the identity of its immobilized oligonucleotide are known or predetermined, for -64- WO 2006/138257 PCT/US2006/022895 example, prior to its use. In some aspects, the oligonucleotides or polynucleotides are singlestranded and are covalently attached to the solid phase support, usually by a 5’-end or a 3 ’-end. Inother aspects, oligonucleotides or polynucleotides are attached to the solid phase support non-covalently, e.g. by a biotin-streptavidin linkage, hybridization to a capture oligonucleotide that is 5 covalently bound, and the like. Conventional microarray technology is reviewed in the followingreferences: Schena, Editor, Microarrays: A Practical Approach (IRL Press, Oxford, 2000);

Southern, Current Opin. Chem. Biol., 2: 404-410 (1998); Nature Genetics Supplement, 21: 1-60(1999). As used herein, “random array” or “random microarray” refers to a microarray whosespatially discrete regions of oligonucleotides or polynucleotides are not spatially addressed. That is, 10 the identity of the attached oligonucleoties or. polynucleotides is not discernable, at least initially, from its location, but may be determined by a particular operation on the array, e.g, sequencing,hybridizing decoding probes, or the like. Random microarrays are frequently formed from a planararray of microbeads, e.g. Brenner et al, Nature Biotechnology, 18: 630-634 (2000); Tulley et al, U.S. patent 6,133,043; Stuelpnagel et al, U.S. patent 6,396,995; Chee et al, U.S. patent 6,544,732; 15 and the like. "Mismatch" means a base pair between any two of the bases A, T (or U for RNA), G, and Cother than the Watson-Crick base pairs G-C and A-T. The eight possible mismatches are A-A, T-T,G-G, C-C, T-G, C-A, T-C, and A-G. “Mutation" and "polymorphism" are usually used somewhat interchangeably to mean a20 DNA molecule, such as a gene, that differs in nucleotide sequence from a reference DNA sequence, or wild type sequence, or normal tissue sequence, by one or more bases, insertions, and/or deletions.In some contexts, the usage of Cotton (Mutation Detection, Oxford University Press, Oxford, 1997)is followed in that a mutation is understood to be any base change whether pathological to anorganism or not, whereas a polymorphism is usually understood to be a base change with no direct 25 pathological consequences. “Nucleoside" as used herein includes the natural nucleosides, including 2’-deoxy and 2'-hydroxyl forms, e.g. as described in Kornberg and Baker, DNA Replication, 2nd Ed. (Freeman, SanFrancisco, 1992). "Analogs" in reference to nucleosides includes synthetic nucleosides havingmodified base moieties and/or modified sugar moieties, e.g. described by Scheit, Nucleotide Analogs 30 (John Wiley, New York, 1980); Uhlman and Peyman, Chemical Reviews, 90: 543-584 (1990), or thelike, with Hie proviso that they are capable of specific hybridization. Such analogs include syntheticnucleosides designed to enhance binding properties, reduce complexity, increase specificity, and thelike. Polynucleotides comprising analogs with enhanced hybridization or nuclease resistanceproperties are described in Uhlman and Peyman (cited above); Crooke et al, Exp. Opin. Ther. Patents, 35 6: 855-870 (1996); Mesmaeker et al, Current Opinion in Structual Biology, 5:343-355 (1995); and the like. Exemplaty types of polynucleotides that are capable of enhancing duplex stability includeoligonucleotide N3'-»P5' phosphoramidates (referred to herein as "amidates"), peptide nucleic acids -65- WO 2006/138257 PCT/US2006/022895 (referred to herein as "PNAs"), oligo-2'-O-alkylribonucleotides, polynucleotides containing C-5propynylpyrimidines, locked nucleic acids (LNAs), and like compounds. Such oligonucleotides areeither available commercially or maybe synthesized using methods described in the literature. “Polymerase chain reaction,” or “PCR,” means a reaction for the in vitro amplification of 5 specific DNA sequences by the simultaneous primer extension of complementary strands of DNA. Inother words, PCR is a reaction for making multiple copies or replicates of a target nucleic acidflanked by primer binding sites, such reaction comprising one or more repetitions of the followingsteps: (i) denaturing the target nucleic acid, (ii) annealing primers to the primer binding sites, and (iii)extending the primers by a nucleic acid polymerase in the presence of nucleoside triphosphates. 10 Usually, the reaction is cycled through different temperatures optimized for each step in a thermalcycler instrument. Particular temperatures, durations at each step, and rates of change between stepsdepend on many factors well-known to those of ordinary skill in the art, e.g. exemplified by thereferences: McPherson et al, editors, PCR: A Practical Approach and PCR2: A Practical Approach(IRL Press, Oxford, 1991 and 1995, respectively). For example, in a conventional PCR using Taq 15 DNA polymerase, a double stranded target nucleic acid may be denatured at a temperature >90°C,primers annealed at a temperature in the range 50-75°C, and primers extended at a temperature in therange 72-78°C. The term “PCR” encompasses derivative forms of the reaction, including but notlimited to, RT-PCR, real-time PCR, nested PCR, quantitative PCR, multiplexed PCR, and the like.Reaction volumes range from a few hundred nanoliters, e.g. 200 nL, to a few hundred pL, e,g. 200 20 pL. “Reverse transcription PCR,” or “RT-PCR,” means a PCR that is preceded by a reverse transcription reaction that converts a target RNA to a complementary single stranded DNA, which isthen amplified, e.g. Tecott et al, U.S. patent 5,168,038, which patent is incorporated herein byreference. “Real-time PCR” means a PCR for which the amount of reaction product, i.e. amplicon, ismonitored as the reaction proceeds. There are many forms of real-time PCR that differ mainly in the 25 detection chemistries used for monitoring the reaction product, e.g. Gelfand et al, U.S. patent 5,210,015 (“taqman”); Wittwer et al, U.S. patents 6,174,670 and 6,569,627 (intercalating dyes); Tyagiet al, U.S. patent 5,925,517 (molecular beacons); which patents are incorporated herein by reference.Detection chemistries for real-time PCR are reviewed in Mackay et al, Nucleic Acids Research, 30:1292-1305 (2002), which is also incorporated herein by reference. “Nested PCR” means a two-stage 30 PCR wherein the amplicon of a first PCR becomes the sample for a second PCR using a new set of primers, at least one of which binds to an interior location of the first amplicon. As used herein,“initial primers” in reference to a nested amplification reaction mean the primers used to generate afirst amplicon, and “secondary primers” mean the one or more primers used to generate a second, ornested, amplicon. “Multiplexed PCR” means a PCR wherein multiple target sequences (or a single 35 target sequence and one or more reference sequences) are simultaneously carried out in the same reaction mixture, e.g. Bernard et al, Anal. Biochem., 273: 221-228 (1999)(two-color real-time PCR).Usually, distinct sets of primers are employed for each sequence being amplified. -66- WO 2006/138257 PCT/US2006/022895 “Quantitative PCR” means a PCR designed to measure the abundance of one or more specific targetsequences in a sample or specimen. Quantitative PCR includes both absolute quantitation and relativequantitation of such target sequences. Quantitative measurements are made using one or morereference sequences that may be assayed separately or together with a target sequence. The reference 5 sequence may be endogenous or exogenous to a sample or specimen, and in the latter case, maycomprise one or more competitor templates. Typical endogenous reference sequences includesegments of transcripts of the following genes: β-actin, GAPDH, pk-microglobulin, ribosomal RNA,and the like. Techniques for quantitative PCR are well-known to those of ordinary skill in the art, asexemplified in the following references that are incorporated by reference: Freeman et al, 10 Biotechniques, 26:112-126 (1999); Becker-Andre et al, Nucleic Acids Research, 17: 9437-9447(1989); Zimmerman et al, Biotechniques, 21: 268-279 (1996); Diviacco et al, Gene, 122:3013-3020(1992); Becker-Andre et al, Nucleic Acids Research, 17: 9437-9446 (1989); and the like. “Polynucleotide” or “oligonucleotide” are used interchangeably and each mean a linearpolymer of nucleotide monomers. As used herein, tire terms may also refer to double stranded 15 forms. Monomers making up polynucleotides and oligonucleotides are capable of specificallybinding to a natural polynucleotide by way of a regular pattern of monomer-to-monomerinteractions, such as Watson-Crick type of base pairing, base stacking, Hoogsteen or reverseHoogsteen types of base pairing, or the like, to form duplex or triplex forms. Such monomers andtheir intemucleosidic linkages may be naturally occurring or may be analogs thereof, e.g. naturally 20 occurring or non-naturally occurring analogs. Non-naturally occurring analogs may include PNAs,phosphorothioate intemucleosidic linkages, bases containing linking groups permitting theattachment of labels, such as fluorophores, or haptens, and the like. Whenever the use of anoligonucleotide or polynucleotide requires enzymatic processing, such as extension by apolymerase, ligation by a ligase, or the like, one of ordinary skill would understand that 25 oligonucleotides or polynucleotides in those instances would not contain certain analogs of intemucleosidic linkages, sugar moities, or bases at any or some positions, when such analogs areincompatable with enzymatic reactions. Polynucleotides typically range in size from a fewmonomeric units, e.g. 5-40, when they are usually referred to as “oligonucleotides,” to severalthousand monomeric units. Whenever a polynucleotide or oligonucleotide is represented by a 30 sequence of letters (upper or lower case), such as "ATGCCTG," it will be understood that the nucleotides are in 5’—>3’ order from left to right and that "A" denotes deoxyadenosine, "C" denotesdeoxycytidine, "G" denotes deoxyguanosine, and "T" denotes thymidine, “I” denotes deoxyinosine,“U” denotes uridine, unless otherwise indicated or obvious from context. Unless otherwise notedthe terminology and atom numbering conventions will follow those disclosed in Strachan and Read, 35 Human Molecular Genetics 2 (Wiley-Liss, New York, 1999). Usually polynucleotides comprise thefour natural nucleosides (e.g. deoxyadenosine, deoxycytidine, deoxyguanosine, deoxy thymidine forDNA or their ribose counterparts for RNA) linked by phosphodiester linkages; however, they may -67- ggffSj WO 2006/138257 PCT/US2006/022895 also comprise non-natural nucleotide analogs, e.g. including modified bases, sugars, orintemucleosidic linkages. It is clear to those skilled in the art that where an enzyme has specificoligonucleotide or polynucleotide substrate requirements for activity, e.g. single stranded DNA,RNA/DNA duplex, or the like, then selection of appropriate composition for the oligonucleotide or 5 polynucleotide substrates is well within the knowledge of one of ordinary skill, especially with guidance from treatises, such as Sambrook et al, Molecular Cloning, Second Edition (Cold SpringHarbor Laboratory, New York, 1989), and like references. "Primer” means an oligonucleotide, either natural or synthetic, that is capable, uponforming a duplex with a polynucleotide template, of acting as a point of initiation of nucleic acid 10 synthesis and being extended from its 3 ’ end along the template so that an extended duplex is formed. The sequence of nucleotides added during the extension process are determined by thesequence of the template polynucleotide. Usually primers are extended by a DNA polymerase.Primers usually have a length in the range of from 9 to 40 nucleotides, or in some embodiments,from 14 to 36 nucleotides. 15 “Readout” means a parameter, or parameters, which are measured and/or detected that can be converted to a number or value. In some contexts, readout may refer to an actual numericalrepresentation of such collected or recorded data. For example, a readout of fluorescent intensitysignals from a mlcroarray is the position and fluorescence intensity of a signal being generated at eachhybridization site of the microarray; thus, such a readout may be registered or stored in various ways, 20 for example, as an image of the microarray, as a table of numbers, or the like. "Solid support”, “support”, and "solid phase support” are used interchangeably and refer to a material or group of materials having a rigid or semi-rigid surface or surfaces. In many embodiments,at least one surface of the solid support will be substantially flat, although in some embodiments itmay be desirable to physically separate synthesis regions for different compounds with, for example, 25 wells, raised regions, pins, etched trenches, or the like. According to other embodiments, the solidsupports) will take the form of beads, resins, gels, microspheres, or other geometric configurations.Microarrays usually comprise at least one planar solid phase support, such as a glass microscope slide. “Reference sequence” or "reference population" of DNA refers to individual DNA sequencesor a collection of DNAs (or RNAs derived from it) which is compared to a test population of DNA or 30 RNA, (or "test DNA sequence,” or "test DNA population”) by the formation of heteroduplexesbetween the complementary strands of the reference DNA population and test DNA population. Ifperfectly matched heteroduplexes form, then the respective members of the reference and testpopulations are identical; otherwise, they are variants of one another. Typically, the nucleotidesequences of members of the reference population are known and the sequences typically are listed in 35 sequence databases, such as Genbank Embl, or the like. In one aspect, a reference population of DNAmay comprise a cDNA library or genomic library from a known cell type or tissue source. Forexample, a reference population of DNA may comprise a cDNA libraiy or a genomic library derived -68- W0 2006/138257 PCT/US2006/022895 from the tissue of a healthy individual and a test population of DNA may comprise a cDNA library orgenomic library derived from the same tissue of a diseased individual. Reference populations ofDNA may also comprise an assembled collection of individual polynucleotides, cDNAs, genes, orexons thereof, e.g. genes or exons encoding all or a subset of known p53 variants, genes of a signal 5 transduction pathway, or the like. "Specific” or “specificity” in reference to the binding of one molecule to another molecule,such as a labeled target sequence for a probe, means the recognition, contact, and formation of a stablecomplex between the two molecules, together with substantially less recognition, contact, or complexformation of that molecule with other molecules. In one aspect, “specific” in reference to the binding 10 of a first molecule to a second molecule means that to the extent the first molecule recognizes andforms a complex with another molecules in a reaction or sample, it forms the largest number of thecomplexes with the second molecule. Preferably, this largest number is at least fifty percent.Generally, molecules involved in a specific binding event have areas on their surfaces or in cavitiesgiving rise to specific recognition between the molecules binding to each other. Examples of specific 15 binding include antibody-antigen interactions, enzyme-substrate interactions, formation of duplexes ortriplexes among polynucleotides and/or oligonucleotides, receptor-ligand interactions, and the like.

As used herein, “contact” in reference to specificity or specific binding means two molecules are closeenough that weak noncovalent chemical interactions, such as Van der Waal forces, hydrogen bonding,base-stacking interactions, ionic and hydrophobic interactions, and the like, dominate the interaction 20 of the molecules.

As used herein, the term “Tin” is used in reference to the “melting temperature.” The meltingtemperature is the temperature at which a population of double-stranded nucleic acid moleculesbecomes half dissociated into single strands. Several equations for calculating the Tm of nucleic acidsare well known in the art. As indicated by standard references, a simple estimate of the Tm value may 25 be calculated by the equation. Tm = 81.5 + 0.41 (% G + C), when a nucleic acid is in aqueous solutionat 1 M NaCl (see e.g., Anderson and Young, Quantitative Filter Hybridization, in Nucleic AcidHybridization (1985). Other references (e.g., Allawi, H.T. & SantaLucia, J., Jr., Biochemistry 36,10581-94 (1997)) include alternative methods of computation which take structural andenvironmental, as well as sequence characteristics into account for the calculation of Tm. 30 “Sample” usually means a quantity of material from a biological, environmental, medical, or patient source in which detection, measurement, or labeling of target nucleic acids is sought. On theone hand it is meant to include a specimen or culture (e.g., microbiological cultures). On the otherhand, it is meant to include both biological and environmental samples. A sample may include aspecimen of synthetic origin. Biological samples may be animal, including human, fluid, solid (e.g., 35 stool) or tissue, as well as liquid and solid food and feed products and ingredients such as dairy items,vegetables, meat and meat by-products, and waste. Biological samples may include materials takenfrom a patient including, but not limited to cultures, blood, saliva, cerebral spinal fluid, pleura] fluid, -69-

:- ^¾. y < W WO 2006/138257 PCT/US2006/022895 milk, lymph, sputum, semen, needle aspirates, and the like. Biological samples may be obtained fromall of the various families of domestic animals, as well as feral or wild animals, including, but notlimited to, such animals as ungulates, bear, fish, rodents, etc. Environmental samples includeenvironmental material such as surface matter, soil, water and industrial samples, as well as samples 5 obtained from food and dairy processing instruments, apparatus, equipment, utensils, disposable andnon-disposable items. These examples are not to he construed as limiting the sample types applicableto the present invention.

The above teachings are intended to illustrate the invention and do not by their details limitthe scope of the claims of the invention. While preferred illustrative embodiments of the present 10 invention are described, it will be apparent to one skilled in the art that various changes and modifications may be made therein without departing from the invention, and it is intended in theappended claims to cover all such changes and modifications that fall within the true spirit and scopeof the invention. 15 -70-