US 2012O142004A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2012/0142004 A1 Battersby et al. (43) Pub. Date: Jun. 7, 2012

(54) UNIVERSAL TAGS WITH NON-NATURAL Related U.S. Application Data NUCLEOBASES (60) Provisional application No. 61/180.272, filed on May 21, 2009. (75) Inventors: Thomas Battersby, El Cerrito, CA O O (US); Toumy Guettouche, Stuttgart Publication Classification (DE); James Hnatyszyn, Coral (51) Int. Cl. Gables, FL (US) CI2O I/68 (2006.01) CI2P 19/34 (2006.01) (73) Assignee: SEMENS HEALTHCARE C7H 2L/00 (2006.01) DIAGNOSTICS INC., Tarrytown, (52) U.S. Cl...... 435/6.12:536/24.33: 536/23.1; NY (US) 435/91.2 (57) ABSTRACT (21) Appl. No.: 13/318,634 The present invention relates to amplification primers with a universal tag and sequencing primers comprising at least one (22) PCT Filed: May 19, 2010 non-natural nucleobase capable of hybridizing to a comple mentary non-natural nucleobase. The present invention fur (86). PCT No.: PCT/US 10/35339 ther relates to amplification methods of nucleic acid amplifi cation and sequencing using an amplification primer with a S371 (c)(1), universal tag and sequencing primers, as well as kits and solid (2), (4) Date: Nov. 3, 2011 Supports comprising Such primers and tags. US 2012/0142004 A1 Jun. 7, 2012

UNIVERSAL TAGS WITH NON-NATURAL can be combined with gene-specific PCR primers containing NUCLEOBASES non-natural nucleobases to facilitate sequencing of geneti cally diverse targets. TECHNICAL FIELD OF THE INVENTION DETAILED DESCRIPTION OF THE INVENTION 0001. The present invention relates generally to the field of nucleic acids, and more particularly to primer-based amplifi 0008 Unless defined otherwise, technical and scientific cation and sequence determination of polynucleotides. terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Units, prefixes, and symbols may be BACKGROUND OF THE INVENTION denoted in their SI accepted form. Unless otherwise indi 0002 DNA and RNA constitute the key molecular com cated, nucleic acid sequences are written left to right in 5' to ponents of all genetic processes, and have similar structural 3' orientation. Numeric ranges recited herein are inclusive of components. DNA typically exists as a complex of two anti the numbers defining the range and include and are Supportive parallel linear Strands or sequences of deoxyribonucleotide of each integer within the defined range. Amino acids may be structural units, each unit of which consists of a nitrogenous referred to herein by either their commonly known three letter base (adenine (A), thymidine (T), cytosine (C) or guanine symbols or by the one-letter symbols recommended by the (G)), a pentose Sugar (a 5-carbon Sugar), and a phosphate IUPAC-IUBMB Nomenclature Commission. Nucleotides, group. RNA is typically single stranded, and uses uracil (U) in likewise, may be referred to by their commonly accepted place of thymidine (T). Moreover, the pentose sugar in DNA single-letter codes. Unless otherwise noted, the terms “a” or “an are to be construed as meaning “at least one of.” The is 2-deoxyribose, while the pentose sugar in RNA is ribose. section headings used herein are for organizational purposes The nitrogenous bases of DNA and RNA are of two classes: only and are not to be construed as limiting the Subject matter the larger nine-member double-ring purines, A and G, and described. All documents, or portions of documents, cited in Smaller six-member single-ring pyrimidines, C.T and U. this application, including but not limited to patents, patent 0003. The polymerase chain reaction (PCR) presents a applications, articles, books, and treatises, are hereby very effective method for selectively amplifying specific expressly incorporated by reference in their entirety for any DNA fragments. In the PCR procedure, oligonucleotides purpose. In the case of any amino acid or nucleic acid complementary to known segments of the target DNA frag sequence discrepancy within the application, the figures con ment are added as “primers. The primers serve as starting trol. One skilled in the art will recognize many methods and point for DNA replication enabling PCR amplification. materials similar or equivalent to those described herein, Often, sequencing tags can be included in the primeras away which could be used in the practice of the present invention. to identify and or track a gene transcript. The present invention is in no way limited to the methods and 0004 Sequencing of DNA and RNA is an important ana materials described herein, and it is understood that other lytical technique for generating genetic information from embodiments of the invention may exist that are not expressly biological sources. Sequencing has made possible the deter described herein. mination of DNA and RNA sequences of entire genomes. It is an important diagnostic tool in the clinic, where the rapid detection of a single nucleobase change or a few nucleobase DEFINITIONS changes can be used to detect, for example, a genetic disease. 0009 For purposes of the present invention, the following terms are defined below. SUMMARY OF THE INVENTION 0010. The term “purine-pyrimidine Watson-Crick interac tion” as used herein means the interaction of a purine nucleo 0005. The present invention is generally directed to ampli base and a pyrimidine nucleobase joined through hydrogen fication and sequencing primers having universal tags com bonding in which the N-1 nitrogen atom of the purine is prising at least one non-natural nucleobase. directly opposite the N-3 nitrogenatom of the pyrimidine, the 0006. The primers of the invention can be used to amplify functional group at C-2 (if present) on the purine is directly polynucleotide templates and incorporate the universal tag in opposite the functional group at C-2 (if present) on the pyri the amplified product. The universal tag can then be used as midine, and the functional group at C-6 (if present) on the the template for a universal sequencing primer. purine is directly opposite the functional group at C-4 (if 0007. In one particular embodiment of the invention, non present) on the pyrimidine. These interactions can exist in natural isoforms, such as isocytosine (iC) and isoguanine both natural and non-natural nucleobases such as purine and (iG), are added to the 5' tail of sequence-specific amplification pyrimidine analogs in which atoms not directly involved in primers. Base pairs comprising non-natural isoforms can the base-pairing interaction have been Substituted. impart improved stability to duplex nucleic acids and permit 0011. The term “nucleobase' as used herein means any the generation of highly specific 5' tails that can be reduced in nitrogen-containing heterocyclic moiety capable of forming length (for example, to 10-bases, versus 15-20 bases). A Watson-Crick-type hydrogen bonds and stacking interactions plurality of contiguous non-natural isoforms (such as iC and in pairing with a complementary nucleobase or nucleobase iG) also prevents DNA polymerase from completing primer analog (i.e., derivatives of nucleobases) when that nucleobase extension, thereby preventing sequencing of proprietary 5' is incorporated into a polymeric structure. “Heterocyclic' tails used in commercial kits by unauthorized parties. Uni refers to a molecule with a ring system in which one or more Versal sequencing tags can be used to sequence any PCR ring atom is a heteroatom, e.g., nitrogen, oxygen, or Sulfur amplicon. The universal sequence tags incorporating non (i.e., not carbon), such as a purine, pyrimidine, or an analog natural isoforms are highly specific. Non-natural isoforms thereof. US 2012/0142004 A1 Jun. 7, 2012

0012. A large number of nucleobases, nucleobase analogs osphate ester group may include Sulfur Substitutions for the and nucleobase derivatives are known. Non-limiting various oxygen moieties, e.g., C.-thio-nucleotide 5'-triphos examples of nucleobases include purines and pyrimidines, phates. Nucleotides can exist in the mono-, di-, or tri-phos and modified forms, e.g., 7-deazapurine. Typical nucleobases phorylated forms. The carbon atoms of the ribose present in are the naturally occurring nucleobases adenine, guanine, nucleotides are designated with a prime character () to dis cytosine, uracil, thymine, and analogs (Seela, U.S. Pat. No. tinguish them from the backbone numbering in the bases. For 5,446,139) of the naturally occurring nucleobases, e.g., a review of polynucleotide and nucleic acid chemistry, see 7-deaZaadenine, 7-deazaguanine, 7-deaza-8-azaguanine, Shabarova, Z. and Bogdanov, A. Advanced Organic Chemis 7-deaza-8-azaadenine, inosine, nebularine, nitropyrrole try of Nucleic Acids, VCH, New York, 1994. The term (Bergstrom, J. Amer. Chem. Soc., 117:1201-1209 1995), “nucleic acid as used herein means a nucleobase polymer nitroindole, 2-aminopurine, 2-amino-6-chloropurine, 2,6-di having a backbone of alternating Sugar and phosphate units in aminopurine, hypoxanthine, pseudouridine, pseudocytosine, DNA and RNA. “Nucleic acid” and “polynucleotide' are pseudoisocytosine, 5-propynylcytosine, isocytosine, isogua considered to be equivalent and interchangeable. Nucleic nine (Seela, U.S. Pat. No. 6,147.199), 7-deazaguanine (Seela, acids are commonly in the form of DNA or RNA. U.S. Pat. No. 5,990.303), 2-azapurine (Seela, WO 01/16149), 0016. The term “nucleic acid includes polynucleotides of 2-thiopyrimidine, 6-thioguanine, 4-thiothymine, 4-thiou genomic DNA or RNA, cDNA, semisynthetic, or synthetic racil. 0-6-methylguanine, N-6-methyladenine, O-4-meth origin. Nucleic acids may also substitute standard nucleotide ylthymine, 5,6-dihydrothymine, 5,6-dihydrouracil, 4-meth bases with nucleotide isoform analogs, including, but not ylindole, pyrazolo 3,4-Dipyrimidines, “PPG’ (Meyer, U.S. limited to iso-C and iso-G bases, which may hybridize more Pat. Nos. 6,143,877 and 6,127,121; Gall, WO 01/38584), and or less permissibly than standard bases, and which will pref ethenoadenine (Fasman (1989) in Practical Handbook of Bio erentially hybridize with complementary isoform analog chemistry and Molecular Biology, pp. 385-394, CRC Press, bases. Many such isoform bases are described, for example, Boca Raton, Fla.). by Benner et al., (1987) Cold Spring Harb. Symp. Quant. 0013 The term “nucleoside' as used herein means a Biol. 52, 53-63. In representations of degenerate primers or nucleobase linked to a carbohydrate. A nucleobase is coupled mixture of different strands having mutations in one or sev to a carbohydrate, for example D-ribose (in RNA) or eral positions, the symbol R refers to either G or A, the symbol 2'-deoxy-D-ribose (in DNA), through an N-glycosidic bond Y refers to either T/U or C, the symbol Mrefers to either A or between the anomeric carbon of the carbohydrate (1'-carbon C, the symbol Krefers to either G or T/U, the symbol Srefers atom of the carbohydrate) and the nucleobase. When the to G or C, the symbol W refers to either A or T/U, the symbol nucleobase is purine, e.g., A or G, the ribose Sugar is generally B refers to “notA, the symbol D refers to “not C” the symbol attached to the N9-position of the nucleobase. When the H refers to “not G.,” the symbol V refers to “not T/U” and the nucleobase is pyrimidine, e.g., C., T or U, the Sugar is gener symbol N refers to any nucleotide. ally attached to the N1-position of the nucleobase. The car (0017 “Polynucleotide' and “oligonucleotides” are used bohydrate may be substituted or unsubstituted. Substituted interchangeably and mean single-stranded and double ribose Sugars include, but are not limited to, those in which Stranded polymers of nucleotide monomers, including one or more of the carbon atoms, for example the 2-carbon 2'-deoxyribonucleotides (DNA) and ribonucleotides (RNA) atom, is substituted with one or more of the same or different linked by internucleotide phosphodiester bond linkages, Cl, F. —R, —OR, —NR or halogen groups, where each Ris (e.g., 3'-5", and 2'-5'), inverted internucleotide phosphodiester independently H, C-C alkyl or Cs-C aryl. Ribose bond linkages (e.g., 3'-3' and 5'-5'), branched structures, or examples include ribose, 2'-deoxyribose, 2',3'-dideoxyri internucleotide analogs. A "polynucleotide sequence” refers bose, 2-haloribose, 2'-fluororibose, 2'-chlororibose, and to the sequence of nucleotide monomers along the polymer. 2'-alkylribose, e.g. 2'-O-methyl, 4-alpha-anomeric nucle “Polynucleotides are not limited to any particular length of otides, 1-alpha-anomeric nucleotides (Asseline et al., Nucl. nucleotide sequence, as the term “polynucleotides’ encom Acids Res., 19:4067-74 (1991), 2-4'- and 3'-4'-linked and passes polymeric forms of nucleotides of any length. Poly other “locked' or “LNA. bicyclic sugar modifications (WO nucleotides that range in size from about 5 to about 40 mono 98/22489; WO 98/39352; WO 99/14226). meric units are typically referred to in the art as 0014 Carbohydrates (also called sugars) can include oligonucleotides. Polynucleotides that are several thousands modifications at the 2- or 3'-position Such as methoxy, or more monomeric nucleotide units in length are typically ethoxy, allyloxy, isopropoxy, butoxy, isobutoxy, methoxy referred to as nucleic acids. Polynucleotides can be linear, ethyl, alkoxy, phenoxy, azido, amino, alkylamino, fluoro, branched linear, or circular molecules. Polynucleotides also chloro and bromo. Nucleosides and nucleotides include the have associated counter ions, such as H, NH, trialkylam natural D configurational isomer (D-form), as well as the L monium, Mg", Na" and the like. configurational isomer (L-form) (Beigelman, U.S. Pat. No. 0018 Polynucleotides that are formed by 3'-5' phosphodi 6,251,666; Chu, U.S. Pat. No. 5,753,789: Shudo, ester linkages are said to have 5'-ends and 3'-ends because the EP0540742; Garbesi et al., Nucl. Acids Res., 21:4159-4165 mononucleotides that are reacted to make the polynucleotide (1993); Fujimori, J. Amer. Chem. Soc., 112:7435 (1990); are joined in Such a manner that the 5' phosphate of one Urata, (1993) Nucleic Acids Symposium Ser. No. 29:69-70). mononucleotide pentose ring is attached to the 3' oxygen (i.e., 0.015 The term “nucleotide' as used herein means a hydroxyl) of its neighbor in one direction via the phosphodi nucleoside in a phosphorylated form—a phosphate ester of a ester linkage. Thus, the 5'-end of a polynucleotide molecule nucleoside, as a monomer unit or within a polynucleotide has a free phosphate group or a hydroxyl at the 5' position of polymer. “Nucleotide 5'-triphosphate” refers to a nucleotide the pentose ring of the nucleotide, while the 3' end of the with a triphosphate ester group at the 5' position, sometimes polynucleotide molecule has a free phosphate or hydroxyl denoted as “NTP, or “dNTP and “ddNTP to particularly group at the 3' position of the pentose ring. Within a poly point out the structural features of the ribose sugar. The triph nucleotide molecule, a position or sequence that is oriented 5' US 2012/0142004 A1 Jun. 7, 2012 relative to another position or sequence is said to be located dyads, such as pentose Sugar backbones, phosphate groups, “upstream,” while a position that is 3' to another position is detectable labels, and cross-linking agents, etc. said to be “downstream.” This terminology reflects the fact 0027. The term “corresponding when used to refer to two that polymerases proceed and extend a polynucleotide chain nucleotide sequences or two nucleobases within a sequence in a 5' to 3' fashion along the template strand. means having the same or nearly the same relationship with 0019. A polynucleotide may be composed entirely of respect to position and complementarity, or having the same deoxyribonucleotides, entirely of ribonucleotides, or chi meric mixtures thereof. Polynucleotides may be composed of or nearly the same relationship with respect to structure, internucleotide nucleobase and Sugar analogs. Unless function, or genetic coding (for example, as between a gene denoted otherwise, whenever a polynucleotide sequence is and the “corresponding protein encoded by the gene). For represented, it will be understood that the nucleotides are in 5' example, a nucleotide sequence “corresponds' to a region of to 3' orientation from left to right. a polynucleotide template if the two sequences are comple 0020. The term "heteropolynucleotide' means a poly mentary or have portions that are complementary. Similarly, nucleotide comprising more than one nucleobase type. a nucleobase of an oligomer'corresponds to a nucleobase of 0021. The term “duplex’ means a bimolecular nucleic a polynucleotide template when the two nucleobases occupy acid complex, usually formed through association of a series positions such that when the oligomer and the polynucleotide of interacting nucleobase dyads, one from each molecule of hybridize the two nucleobases pair opposite each other. The the complex. A single nucleic acid molecule may also have term "corresponding is generally used herein in reference to regions of duplex association by folding back onto itself and the positional relationship between two polynucleotide intramolecularly hybridizing to a complementary sequence. sequences or two nucleobases. The term "corresponding 0022. The term “complementary' means that two nucleo does not imply complementarity; thus, corresponding bases are capable of associating in a Watson-Crick interaction nucleobases may be complementary, or may be non-comple of potential hydrogen bonding functionality without repul mentary. sive interaction(s). “Complementary also means that a 0028. The term “dyad” means two nucleobases or analogs nucleobase of a polynucleotide is capable of hybridizing to a paired within a duplex, one from each opposing strand of the corresponding nucleobase in a different polynucleotide. As duplex. used herein, the term “complementary' is not limited to 0029. The term “backbone' means a repeating linear poly canonical Watson-Crick base pairs with A/T, G/C and U/A. merto which nucleobases or analogs are attached. In DNA the Thus, nucleobase pairs may be considered to be "complemen backbone is formed by alternating 2'-deoxy-D-ribose and tary’ if one or both of the nucleobases is a nucleobase other phosphate units. In RNA the backbone is formed by alternat than A, G, C, or T. The term “complementary also refers to ing D-ribose and phosphate units. antiparallel strands of polynucleotides (as opposed to a single 0030 The phrase “hydrogen bonding pattern' means the nucleobase pair) that are capable of hybridizing. Thus, hydrogen bonding pattern of acceptor (A) and donor (D) complementary strands have a sufficient number of comple groups of a pyrimidine or pyrimidine analog (py) and a purine mentary nucleobases to enable hybridization of the two or purine analog (pu) molecule, designated using the nomen Strands. It should be understood that complementary Strands clature of Benner (Lutz, et al. Differential discrimination of of polynucleotides can include Some corresponding base DNA polymerases for variants of the non-standard nucleo pairs that are non-complementary. Accordingly, it should also base pair between Xanthosine and 2,4-diaminopyrimidine, be understood that corresponding nucleobase pairs need not two components of an expanded genetic alphabet, Nucleic be complementary. The term “complementary' as used in Acids Res. 24: 1308-1313 (1996)). The term “pu' as used reference to two nucleotide sequences or two nucleobases, herein denotes a carbon/nitrogen heterocycle isosteric to the implies that the nucleotide sequences or nucleobases are “cor ring system of purines (i.e., adenine and guanine) with a responding.” nitrogenatomat position 1. Functionality capable of acting as 0023 The terms “capable of hybridizing” mean that at hydrogen bond donors or acceptors in Watson-Crick interac least two nucleobases can form a dyad or that a polynucle tion may be present at the carbon atoms of positions 2 and 6. otide has a Sufficient number of nucleobases complementary The nature of this functionality, if present, is indicated by a to another polynucleotide that they can anneal and form a series of three symbols representing positions moving around duplex. the puring from position 6 to position 1 to position 2 (“D’=H 0024. The term “target-specific nucleotide sequence' bond donor. 'A'-H bond acceptor. “ —no functionality). means a nucleic acid sequence that is the native sequence of Similarly, the term "py' as used herein denotes a carbon/ interest. It may be a gene, a regulatory sequence, genomic nitrogen heterocycle isosteric to the ring system of pyrim DNA, mRNA, or others or a portion of any of the foregoing. idines (i.e., thyminefuracil and cytosine) with a nitrogenatom It may be any length, with the understanding that longer at position 3. Functionality capable of acting as hydrogen sequences are more specific. bond donors or acceptors in Watson-Crick interaction may be 0025. The term “comprising” means the listed elements, present at the carbonatoms of positions 2 and 4. The nature of plus any additional unspecified elements. this functionality, if present, is indicated by a series of three 0026. The term “consisting essentially of means the symbols representing positions moving around the py ring listed elements, plus any additional unspecified elements that from position 4 to position 3 to position 2 (“D’-H bond do not function as a nucleobase dyad. Thus, with respect to donor, 'A'-H bond acceptor. “ —no functionality). claims reciting polynucleotide duplexes "consisting essen 0031. The hydrogen bonding patterns of the natural tially of a plurality of complementary purine-purine nucleo purines are denoted as puPA (adenine) and puADD (gua base dyads, the term "consisting essentially of is used to nine) Similarly, the hydrogen bonding patterns of the natural characterize only the nucleobase dyads, and thus the claim is pyrimidines are pyDAA (cytosine) and pyADA (thymine? open to the inclusion of other elements that are not nucleobase uracil). Thus, the notation representing cytosine-guanine US 2012/0142004 A1 Jun. 7, 2012 bonding pattern is pyDAA-puADD, and the thymine/uracil 0040. The practice of the present invention will employ, adenine bonding pattern is pyADA-puPA . unless otherwise indicated, conventional techniques of 0032. The term “ribose' as used herein means a pentose molecular biology, microbiology, recombinant DNA tech Sugar of the general formula C5HoOs occurring as a compo niques, and oligonucleotide synthesis which are within the nent of riboflavin, nucleotides, and nucleic acids. skill of the artisan. Such techniques are explained fully in the 0033. The term “deoxyribose as used herein means any literature. Enzymatic reactions and purification techniques of certain pentose carbohydrates derived from ribose by the are performed according to manufacturer's specifications or replacement of a hydroxyl group with a hydrogenatom of the as commonly accomplished in the art or as described herein. general formula C5HoO. The foregoing techniques and procedures are generally per formed according to conventional methods well known in the 0034. The term “non-natural nucleobase' means a nucleo art and as described in various general and more specific base other than G, C, TWU or A. In some embodiments, the references that are cited and discussed throughout the present non-natural nucleobases for Watson-Crick complementary specification. See, e.g., Sambrooketal. Molecular Cloning: A nucleobase pair analogs having hydrogen bonding interac Laboratory Manual (2d ed., Cold Spring Harbor Laboratory tions that can be discriminated from natural nucleobase pairs. Press, Cold Spring Harbor, N.Y. (1989)): Oligonucleotide It should be understood that non-natural nucleobases can Synthesis (M. J. Gait, ed., 1984); Nucleic Acid Hybridization complement one another without engaging in Watson-Crick (B. D. Hames & S. J. Higgins, eds., 1984); A Practical Guide complementarity. to Molecular Cloning (B. Perbal, 1984); and a series, Meth 0035. The term “phosphate' as used herein means a salt or ods in Enzymology (Academic Press, Inc.), the contents of all ester of phosphoric acid. of which are incorporated herein by reference. 0036. The abbreviation “Tm' as used herein means the 0041 Universal Tags Incorporating Non-Natural Nucleo "melting temperature. The melting temperature is the tem bases perature at which half of a population of double-stranded 0042. As is known in the art, DNA (e.g., a DNA template) polynucleotide molecules or nucleobase oligomers, in homo is amplified by the polymerase chain reaction process, using duplexes or heteroduplexes, become dissociated into single two oligonucleotide extension primers—one that is comple strands. The Tm of a double-stranded nucleobase oligomeric mentary to a first locus on one strand of the DNA, and a molecule is influenced by the types of bases, the base second that is complementary to a different or second locus sequence, structure of the oligomeric linkages, and the pres on the other strand of the DNA. The region between the two ence of non-natural features in the sequence, which would loci is the region of the original DNA template that is ampli included, for example, artificial linkages. Methods for calcu fied. The amplified product includes a DNA sequence lating or experimentally determining Tm are known in the art. between and including the two primers that hybridize to the See, for example, Breslauer et al. Proc. Natl. Acad. Sci. USA original template (i.e. amplicons). Primers may also include 83: 3746-3750 (1986); Baldino et al. Methods in Enzymol. tags, which can be incorporated into the amplified product. 168: 761-777 (1989); and Breslauer Methods in Enzymol. 0043. The oligonucleotide amplification primers of the 259: 221-242 (1995). present invention include improved universal tags that com 0037. The term “antiparallel' is used to refer to interaction prise non-natural nucleobases. In some embodiments, the in which an oligonucleotide strand oriented in the 5'-3' direc primers include (i) a target-specific nucleotide sequence and tion is hybridized to a complementary strand oriented in the (ii) a 5' non-target-specific universal tag comprising one or 3'-5' direction. more non-natural nucleobases. Amplification of a template 0038. The term “stable' as used in reference to a het polynucleotide results in amplification products that incorpo eropolynucleotide duplex, means that the duplex remains rate the universal tag having the non-natural nucleobases. The hybridized essentially exclusively in the form of a duplex non-natural nucleobases are capable of complementary bind under typical salt and temperature conditions used in nucleic ing to a complementary second non-natural nucleobase. acid diagnostic applications. 0044) For example, in the first cycle of amplification, the 0039. The term “universal tag as used herein means an double-stranded DNA is separated to provide two DNA tem oligonucleotide sequence having at least one (i.e. one or plates (the sense strand and the complementary anti-sense more) non-natural nucleobase. The universal tag can be rep Strand). The target-specific sequence of one amplification licated during amplification prior to a Subsequent sequencing primer initially binds to its complementary first locus on one reaction. A portion of the universal tag may also be replicated of these strands, while the target-specific sequence of the during sequencing until the replication reaches a first non other amplification primer binds to its complementary second natural nucleobase in the tag. A universal tag can refer to both locus on the other strand. The 5' non-target-specific universal the sequence and its complement created during amplifica tag regions of the primers do not participate in hybridization tion. Universal tags can be used to simplify sequencing or act to the DNA template, since the tags, by definition, are “non as a marker for sequence identification or purification. Uni target specific' (i.e., they are not complementary) and cannot Versal tags can be used to identify amplicons. Universal tags therefore hybridize to any region of the template. Primer can also be used to purify and/or isolate amplicons with the extension of both amplification primers is initiated (using a universal tag. For example, a complementary oligonucleotide DNA polymerase and appropriate ddNTP nucleotide build to the universal tag may be immobilized on a surface for ing blocks) to add additional nucleotides to the primer down hybridization to the tag, thereby separating the amplicon from stream of the target-specific sequence, thereby producing a mixture of oligonucleotide sequences. A universal tag newly synthesized second strands that are complementary to should not be complementary to any sequence of a polynucle the target DNA and which also include the 5' non-target otide template or any other template present in a sample. The specific universal tag. universal tag is non-target specific in relation to the target 0045. In the second cycle of amplification, this second template. strand then becomes a template for the other amplification US 2012/0142004 A1 Jun. 7, 2012

primer which hybridizes to the other locus downstream of the bases following the target-specific nucleotide sequence, pre universal tag on the newly synthesized DNA strand. Primer venting the polymerase sequencing enzyme from entering a extension of both amplification primers is again initiated. region of the universal tag downstream of the non-natural Because the 5' non-target-specific universal tag is now part of nucleobases and preventing the user from obtaining the the DNA template (derived from the newly synthesized DNA nucleotide sequence of the universal tag. This approach Strand from the first cycle), primer extension of the target enables manufacturers of kits to protect the identity of pro specific nucleotide sequence (using a primer specific for the prietary universal tag sequences, and prevent users from other DNA locus and also having a 5' non-target-specific using the universal tag sequence without a manufacturer's universal sequencing tag) adds nucleotides corresponding to permission. the target DNA region, as well as a 5' non-target-specific 0049. In some embodiments, amplification is followed by universal tag. The newly synthesized DNA sequence from sequencing. The oligonucleotide amplification primers used this second cycle now includes the sequence between and in these processes include a universal sequencing tag, which including the two primers in the original template, including incorporates the universal sequencing tag into the amplifica the universal tag sequence at each end of the newly synthe tion products. The amplified products derived from primers sized DNA sequence, corresponding to the universal tag with the universal sequencing tag include the DNA sequence sequences of each of the two primers. In Subsequent ampli between and including the two primers that hybridize to the fication cycles, the DNA templates will include the sequence original template, as well as the universal tags at the end of between and including the two primers in the original tem each amplification product. Thus, the primers include (i) a plate and the universal tag sequences at each end, providing target-specific nucleotide sequence and (ii) a 5' non-target complementary sequence to which the 5' non-target-specific specific universal sequencing tag comprising one or more universal tags of the primers can hybridize. non-natural nucleobases. The non-natural nucleobases are 0046 Methods for using universal tags and applications capable of complementary binding to a complementary sec are disclosed, for example, in U.S. Patent Publication 2003/ ond non-natural nucleobase. The universal sequencing tag 02919751 A1, Nov. 27, 2003. can then be used to identify and/or isolate the amplification 0047 A 5' non-target-specific sequencing tag comprises products, or to sequence the amplification products using one or more non-natural nucleobases capable of complemen sequencing primers that are complementary to the sequenc tary binding to a complementary second non-natural nucleo ing tags and include non-natural nucleobases that are comple base. Incorporation of non-natural nucleobases in the 5' non mentary to the non-natural nucleobases of the universal tags target-specific universal tag has numerous advantages. One incorporated into the amplification products. Sequencing can advantage is that certain non-natural nucleobase pairs hybrid be performed via monodirectional orbidirectional sequenc ize with higher affinity than natural nucleobase pairs. The ing. Bidirectional sequencing involves simultaneous higher affinity of non-natural nucleobase pairs (compared to sequencing on each of the component strands of a duplex. natural nucleobase pairs GC and AT) increases the melting Thus, from the perspective of a given Strand, information is temperature of duplexes that include the non-natural nucleo obtained for the 5' to 3" direction and for the 3' to 5' direction base pairs. Certain non-natural nucleobase pairs, such as iC of the duplex. iG pairs, for example, have greater thermodynamic duplex 0050. In accordance with the present invention, primers stability than natural nucleobase pairs G-C or A-T. Since the with a 3" target-specific nucleotide sequence and a 5' non stringency of binding is controlled by temperature, the tem target-specific universal tag comprising one or more non perature can be adjusted so that the specificity of the primer natural nucleobase(s) capable of hybridizing to a comple including non-natural bases will be higher than a primer mentary non-natural nucleobase are described. In one without non-natural bases, thereby allowing discrimination embodiment, the non-natural nucleobases include nucleo of duplexes having the non-natural nucleobases from bases that can associate through Watson-Crick pairing of duplexes having only natural nucleobases. In addition, at a purine-pyrimidine nucleic acid duplexes. In another embodi given temperature, a shorter primer length may be used when ment, the non-natural nucleobases include nucleobases that non-natural nucleobase pairs (such as iC-iG) are incorporated can associate through non-Watson-Crick pairing such as than when they are not. Another advantage of using universal those described in Leconte, A.M., et al., “Discovery, charac sequencing tags arises from obviating the need to performany terization, and optimization of an unnatural base pair for screening of sequencing reaction conditions. expansion of the genetic alphabet.” J. Am. Chem. Soc. 130 0048. Another advantage of incorporating a plurality of (7):2336-43; and Hirao, K., et al., Nat. Methods 2006, 3, non-natural isoforms into the universal tag is that it prevents 729-735. These non-natural nucleobases form a base pair that DNA polymerase from completing primer extension, which is replicatable, that is they are a matched pair to the poly property can be used, for example, to prevent unauthorized merase in use. Non-Waston-Crick pairing non-natural parties from sequencing proprietary 5' tails used in commer nucleobases have good affinity in a duplex structure and are cial kits. Thus, in Some embodiments, a universal tag may replicatable by a polymerase. include a plurality of contiguous non-natural nucleobase iso 0051. The purine-pyrimidine nucleic acid duplexes can forms. The plurality of contiguous non-natural nucleobase include a N3-H tautomer of isoguanine. isoforms are incorporated at or near the junction of (i.e., 0052. In one aspect, a universal tag comprises at least one between) the (i) a target-specific nucleotide sequence and (ii) (i.e. one or more) non-natural nucleobase capable of forming a 5' non-target-specific universal sequencing tag. Extension purine-pyrimidine nucleobase dyads. The nucleic acids may of the target-specific primer from the 3' to the 5' direction further comprise a carbohydrate backbone of ribose or deox (new ddNTP dNTP and NTP nucleotides are added to the yribose, and phosphate. primer in the 5' to 3’ direction of the primer but in the 3' to the 0053. In another aspect, a universal tag is disclosed con 5' direction of the template) terminates when the polymerase sisting essentially of a plurality of non-natural nucleobases enzyme reaches the block of contiguous non-natural nucleo capable of forming complementary purine-pyrimidine US 2012/0142004 A1 Jun. 7, 2012 nucleobase dyads. The nucleic acids may further comprise a comprise a plurality of not natural purine nucleobases carbohydrate backbone of ribose or deoxyribose, and phos capable of forming purine-purine nucleobase dyads and a phate. Suitable carbohydrate backbones include, for plurality of non-natural purine nucleobases capable of form example, D-ribose and 2'-deoxy-D-ribose. ing purine-pyrimidine nucleobase dyads. 0054 The universal tag can form one or more nucleobase 0059. In some embodiments, one or more of the non dyads with other nucleobases through purine-pyrimidine natural nucleobases are independently selected from the pairing through complementary Watson-Crick interactions. group consisting of isocytosine, isoguanine and 5-methyliso In some embodiments, the universal tag can form comple cytosine. In one embodiment, as least one non-natural mentary purine-pyrimidine nucleobase dyads that are con nucleobase is isocytosine. In one embodiment, as least one tiguous. In some embodiments, the universal tag can form non-natural nucleobase is isoguanine. In one embodiment, as complementary purine-pyrimidine nucleobase dyads that are least one non-natural nucleobase is 5-methylisocytosine. not contiguous. 0060. In some embodiments, one or more of the non 0055. The universal tag can include a nucleobase adopting natural nucleobases are contiguous to the target-specific a tautomer that is a minor species of the nucleobase present in nucleotide sequence. aqueous solution (i.e., an unhybridized nucleobase) in the 0061. In some embodiments, one or more of the non absence a Watson-Crick interaction. natural nucleobases are positioned 3' of a least a portion of a 0056. In some embodiments, one or more of the non target-specific nucleotide sequence. natural nucleobases are selected from the group of purine and 0062. In some embodiments, the non-natural nucleobases pyrimidine analogs capable of Watson-Crick pairing through are selected from the group consisting of Watson-Crick formation of 2 or 3 hydrogen bonds. For example, non-natural complementary nucleobase analogs having hydrogen bond nucleobases may form nucleic acid duplexes with one or ing interactions that can be discriminated from natural more purine-pyrimidine nucleobase dyads may have the fol nucleobase pairs. lowing hydrogen bond donor and acceptor pairings: 0063 Amplified Polynucleotide Templates 0064. In another aspect, an amplified polynucleotide tem plate comprises (a) a 3' target-specific nucleotide sequence and (b) a 5' universal tag comprising at least one (i.e one or puADD-pyDAA puAD -py DAA puAD -py DA puADD-py AA pu DD-py AA more) non-natural nucleobase, each non-natural nucleobases puDAD-py ADA puldA -py ADA puldA -py AD of which is complementary to a non-natural nucleobase of a pu AD-py ADA pu AD-py DA complementary polynucleotide. In some embodiments, the 5' puDDA-py AAD pu DA-py AAD pu DA-py AD universal tag comprises a plurality of non-natural nucleo puldDA-py AA puCD -py AA puDAA-py ADD pu AA-py ADD pu AA-py DD bases, each non-natural nucleobases of which is complemen puCAA-py AD puldA -py AD tary to a non-natural nucleobase of a complementary poly puADA-pyDAD puADA-py AD pu DA-py AD nucleotide. In some embodiments, the polynucleotide puADA-pyl)A puAD -py DA template is a sequencing template with a 5' universal sequenc puAAD-pyDDA puAA -py DDA puAA -py DD puAAD-py DA pu AD-py DA ing tag. 0065. Nucleic Acid Duplex 0066. In another aspect, a nucleic acid duplex comprises The universal tag can form a nucleic acid duplex can com an oligonucleotide primer comprising (a) a 3" target-specific prising nucleobase dyads having the above hydrogen bond nucleotide sequence and (b) a 5' non-target-specific universal donor and acceptor pairings with one or both of the nucleo tag comprising at least one (i.e. one or more) non-natural bases of the dyad adopting a tautomer that is a minor species nucleobase capable of complementary binding to a corre present in aqueous solution in the absence a Watson-Crick sponding second non-natural nucleobase wherein the oligo interaction. nucleotide is hybridized to a polynucleotide target. 0057. In some embodiments, the universal tags incorpo 0067. In one embodiment, a nucleic acid duplex comprises rating one or more non-natural nucleic acids can comprise a (a) a universal tag further comprising (i) a target-specific plurality of contiguous purine-pyrimidine dyads with non nucleotide sequence and (ii) a 5' universal tag comprising a natural nucleobases. In one embodiment, the universal tags non-natural nucleobase complementary to a corresponding incorporating one or more non-natural nucleic acids comprise non-natural nucleobase hybridized to (b) an amplified poly at least 3 contiguous purine-pyrimidine nucleobase dyads nucleotide template comprising (i) a target polynucleotide with non-natural nucleobases. In another embodiment, the and (ii) a 5' universal tag comprising at least one (i.e. one or universal tags incorporating one or more non-natural nucleic more) non-natural nucleobase. In some embodiments, the acids comprise at least 4 contiguous purine-pyrimidine polynucleotide template is a sequencing template with a 5' nucleobase dyads with non-natural nucleobases. In still universal sequencing tag. another embodiment, the universal tags incorporating one or 0068 Amplification more non-natural nucleic acids comprise at least 5 contiguous 0069. In another aspect, a method of amplifying a poly purine-pyrimidine nucleobase dyads with non-natural nucleotide molecule includes providing a polynucleotide nucleobases. molecule, contacting the polynucleotide molecule with an 0058. In some embodiments, the universal tags incorpo oligonucleotide primer, wherein the oligonucleotide primer rating a plurality of non-natural nucleic acids can comprise at comprises a 3' nucleotide sequence complementary to the least one non-natural purine capable of forming a dyad with polynucleotide molecule and a 5' non-target-specific univer another purine nucleobase and a plurality of non-natural sal tag comprising at least one non-natural nucleobase purine nucleobases capable of forming purine-pyrimidine capable of hybridizing to a complementary non-natural nucleobase dyads. In another embodiment, the universal tags nucleobase. Amplicons prepared in this manner will have the incorporating a plurality of non-natural nucleic acids can universal tag. In some embodiments, the universal tag will be US 2012/0142004 A1 Jun. 7, 2012 a universal sequencing tag so that the amplicons derived a stable anti-parallel nucleic acid duplex having a plurality of therefrom can be sequenced with universal sequencing tag. purine-pyrimidine dyads. In some embodiments, the plurality 0070 Hybridization of non-natural purine nucleotides comprising at least one 0071. In another aspect, a polynucleotide molecule with a non-natural nucleobase contains a plurality of non-natural universal tag can be hybridized to a complementary poly nucleobases. In some embodiments, the non-natural nucleo nucleotide molecule. The first polynucleotide molecule pos bases are contiguous. In some embodiments, the non-natural sesses a first universal tag. The first polynucleotide molecule nucleobases are not contiguous. hybridizes to a second polynucleotide molecule by contacting 0076. In some embodiments, a method of hybridizing two a second polynucleotide molecule that is complementary to nucleic acid molecules includes providing a first polynucle the first polynucleotide molecule, and the second polynucle otide molecule comprising a first universal tag having one or otide molecule has a second universal tag complementary to more regions consisting of a plurality of purine and pyrimi the first universal tag present in the first polynucleotide mol dine nucleotides coupled to a backbone of ribose or deoxyri ecule. bose, and phosphate, and hybridizing a second polynucle 0072. In one embodiment, the first polynucleotide mol otide molecule comprising a second universal tag having one ecule comprises a region consisting of a plurality of non or more regions consisting of a plurality of pyrimidine and natural purine and non-natural pyrimidine nucleotides purine nucleotides coupled to a backbone consisting of ribose coupled to a backbone consisting of ribose or deoxyribose, or deoxyribose, and phosphate, wherein the one or more and phosphate. The first polynucleotide molecule after con regions of a plurality of purine and pyrimidine nucleotides of tacting the second polynucleotide molecule hybridizes to the the first universal tag complement the pyrimidine and purine second polynucleotide molecule comprising a plurality of nucleotides of the second universal tag, thereby forming a complementary non-natural pyrimidine and non-natural stable anti-parallel nucleic acid duplex having a plurality of purine nucleotides coupled to a backbone consisting of ribose purine-pyrimidine dyads. In some embodiments, the plurality or deoxyribose, and phosphate, wherein the nucleotides of the of purine and pyrimidine nucleotides comprising at least one first polynucleotide molecule are complementary to the non-natural nucleobase contains a plurality of non-natural nucleotides of the second polynucleotide molecule, thereby nucleobases. In some embodiments, the non-natural nucleo forming a stable anti-parallel nucleic acid duplex. bases are contiguous. In some embodiments, the non-natural 0073. In one embodiment the first polynucleotide mol nucleobases are not contiguous. ecule comprises a region consisting of a plurality of non (0077 Labeling of Oligonucleotides natural purine nucleotides coupled to a backbone consisting 0078 Universal tags with a target-specific nucleotide of ribose or deoxyribose, and phosphate. The first polynucle sequence and a 5' universal tag comprising at least one non otide molecule after contacting the second polynucleotide natural nucleobase complementary to a corresponding non molecule hybridizes to the second polynucleotide molecule natural nucleobase can also be labeled with a suitable label/ comprising a plurality of pyrimidine nucleotides coupled to a reporter moiety. For example, the universal tags may be backbone consisting of ribose or deoxyribose, and phosphate, labeled with a label or with multiple labels selected from the wherein the purine nucleotides of the first polynucleotide group of labels consisting of dyes, fluorescent labels, lumi molecule are complementary to the plurality of non-natural nescent labels, radioactive labels, antigens, haptens, pyrimidine nucleotides of the second polynucleotide mol enzymes, enzyme Substrates, protecting groups, and chemi ecule, thereby forming a stable anti-parallel nucleic acid cally reactive groups. Other labels may also be used, in addi duplex. tion to, or in conjunction with, these labels. 0074. In another embodiment the first polynucleotide 0079. As used herein, the term “label in reference to molecule comprises a region consisting of a plurality of non universal tags refers to any moiety that can be attached to the natural pyrimidine nucleotides coupled to a backbone con tag and: (i) provides a detectable signal, where the signal can sisting of ribose or deoxyribose, and phosphate. The first be in the visible wavelength spectrum or any other wave polynucleotide molecule after contacting the second poly length or particle type, e.g., a radioisotope decay particle; (ii) nucleotide molecule hybridizes to the second polynucleotide interacts with a second label to modify the detectable signal molecule comprising a plurality of purine nucleotides provided by the second label, i.e., energy transfer label pairs, coupled to a backbone consisting of ribose or deoxyribose, e.g., FRET pairs; (iii) stabilizes hybridization, i.e., duplex and phosphate, wherein the pyrimidine nucleotides of the first formation; (iv) confers a capture function, e.g., hydrophobic polynucleotide molecule are complementary to the plurality affinity, antibody/antigen, ionic complexation, or (V) changes of non-natural purine nucleotides of the second polynucle a physical property, such as electrophoretic mobility, hydro otide molecule, thereby forming a stable anti-parallel nucleic phobicity, hydrophilicity, solubility, or chromatographic acid duplex. behavior. Labeling can be accomplished using any one of a 0075. In some embodiments, a method of hybridizing two large number of known techniques employing known labels, nucleic acid molecules includes providing a first polynucle linkages, linking groups, reagents, reaction conditions, and otide molecule comprising a first universal tag having one or analysis and purification methods. Labels include light-emit more regions consisting of a plurality of purine nucleotides ting or light-absorbing compounds which generate or quench coupled to a backbone of ribose or deoxyribose, and phos a detectable fluorescent, chemiluminescent, or biolumines phate, and hybridizing a second polynucleotide molecule cent signal (Kricka, L. in Nonisotopic DNA Probe Tech comprising a second universal tag having one or more regions niques (1992), Academic Press, San Diego, pp. 3-28). As used consisting of a plurality of pyrimidine nucleotides coupled to herein, the terms “label' and “reporter may in some cases be a backbone consisting of ribose or deoxyribose, and phos used interchangeably. phate, wherein the one or more regions of a plurality of purine 0080. It is contemplated that the universal tags can be nucleotides of the first universal tag complement the pyrimi labeled with any labeling moiety or technique currently dine nucleotides of the second universal tag, thereby forming known in the art for labeling nucleic acids, modified nucleic US 2012/0142004 A1 Jun. 7, 2012

acids or nucleic acid analogs. It is not intended that the inven rescein, biotin, 2,4-dinitrophenyl, digoxigenin, tion be limited in any way to any particular labeling method. lipopolysaccharide; apotransferrin; ferrotransferrin; insulin; Techniques for labeling of nucleic acids, modified nucleic a cytokine; gp120; B-actin; leukocyte function-associated acids and nucleic acid analogs are widely known in the art, antigen 1 (LFA-1; CD11a/CD18); Mac-1 (CD11b/CD18); and thorough discussion and detailed protocols for labeling glycophorin; laminin; collagen; fibronectin; vitronectin; an are available from many sources. For example, see, "Non integrin, ankyrin, fibrinogen, Factor X; inter-cellular adhe Radioactive Labeling, A Practical Introduction.” Garman, sion molecule 1 (ICAM-1); inter-cellular adhesion molecule Academic Press, San Diego, Calif. (1997). 2 (ICAM-2); spectrin, fodrin: CD4; a cytokine receptor; an 0081. Non-limiting examples of reporter/label moieties insulin receptor, a transferrin receptor, Fe+++; polymyxin B; suitable for the direct labeling of oligonucleotides include, endotoxin-neutralizing protein (ENP); an antibody-specific but are not limited to, a quantum dot, a dextran conjugate, a antigen; avidin; Streptavidin; and biotin. Non-radioactive branched nucleic acid detection system, a chromophore, a labeling methods, techniques, and reagents are reviewed in: fluorophore, a quencher, a spin label, a radioisotope, an Non-Radioactive Labeling, A Practical Introduction, Garman enzyme, a hapten, an acridinium ester and a chemilumines (1997) Academic Press, San Diego. In some embodiments, cent compound. Quenching moieties are also considered the terms “label' and “reporter are used interchangeably. labels. Other suitable labeling reagents and preferred meth I0086 Sequencing ods of label attachment would be recognized by those of I0087 Universal tags with a 5' universal tag comprising at ordinary skill in the art. Any examples cited herein are least one non-natural nucleobase complementary to a corre intended to be merely illustrative and are non-limiting sponding non-natural nucleobase can also be used in 0082. A label or reporter moiety can be linked to any sequencing processes. Sequencing processes determine the position within a nucleobase oligomers within the universal order of the nucleotide bases in a DNA or RNA sequence. For tag. A label can reside at a terminus of the universal tag or at example, chain-termination processes start with a single a position internal to the universal tags (e.g., within or Stranded DNA (or RNA) template, a sequencing primer, a attached to the nucleobases). The nucleobase can be labeled polymerase, ddNTPs, and modified nucleotides that termi either following synthesis of the complete universal tag or nate elongation (for example ddNTPs). The modified nucle incorporated during amplification of a target nucleotide otides may be added at a lower concentration than the dNTPs Sequence. to allow strand elongation, and the concentration of dNTPs 0083) Non-limiting examples of fluorescent reporter dyes and ddNTPs is determined based on their relative preference useful for labeling biomolecules (fluorophores) include, but as a substrate for the polymerase enzyme. are not limited to, 5(6)-carboxyfluorescein (Flu), 2',4', 1,4- I0088. After elongation, the newly synthesized fragments tetrachlorofluorescein; and 2',4',5'7", 1,4-hexachlorofluo are heat denatured and separated by size. The separate frag resc-ein, other fluorescein dyes (see, e.g., U.S. Pat. Nos. ments are then imaged for identification of the terminal nucle 5,188,934; 6,008.379; 6,020,481, incorporated herein by ref. otide base that terminated elongation. In this manner, the erence), 6-((7-amino-4-methylcoumarin-3-acetyl)amino) specific nucleotide base is identified for an entire strand. hexanoic acid (Cou), 5(and 6)-carboxy-X-rhodamine (Rox), I0089. In on such technique, nucleic acid fragments can be other rhodamine dyes (see, e.g., U.S. Pat. Nos. 5.366,860; tagged with radioactive elements for radiolabelling. In 5,847,162; 5,936,087; 6,051,719; 6,191,278; 6,248,884, another technique, the sequencing primer may be labeled at incorporated herein by reference), benzophenoxazines (see, the 5' end with a fluorescent dye for tagging (dye-primer e.g., U.S. Pat. No. 6,140,500, incorporated herein by refer sequencing). In yet another technique, the chain terminators ence) Cyanine 2 (Cy2) Dye, Cyanine 3 (Cy3) Dye, Cyanine can be labeled with a dye (dye-terminator sequencing). These 3.5 (Cy3.5) Dye, Cyanine 5 (Cy5) Dye, Cyanine 5.5 (Cy5.5) and other sequencing processes can be adapted for use with Dye Cyanine 7 (Cy7) Dye, Cyanine 9 (Cy9) Dye (Cyanine the universal tags. dyes 2, 3, 3.5, 5 and 5.5 are available as NHS esters from 0090 Articles of Manufacture Amersham, Arlington Heights, Ill.), other cyanine dyes 0091. The present invention provides articles of manufac (Kubista, WO 97/45539), 6-carboxy-4,5'-dichloro-2',7- ture (e.g., kits) comprising at least one universal tag. In cer dimethoxyfluorescein (JOE), 5(6)-carboxy-tetramethyl tain embodiments, kits serve to facilitate the performance of rhodamine (Tamara), Dye 1 Dye 2 or the Alexa dye series a process, method, assay, analysis or manipulation of interest (Molecular Probes, Eugene, Oreg.). by assembling two or more components used to carry out the 0084. Non-limiting examples of enzymes that can be used methods. Kits can contain any chemical reagent, enzyme, or as labels include, but are not limited to, alkaline phosphatase equipment required for use of the method. In certain embodi (AP), horseradish peroxidase (HRP), soy bean peroxidase ments, kits contain components in pre-measured amounts to (SBP), ribonuclease and protease. minimize the need for measurements by end-users. In certain 0085. Another class of labels includes hybridization-sta embodiments, kits include instructions for performing one or bilizing moieties which serve to enhance, stabilize, or influ more methods. In certain embodiments, the kit components ence hybridization of duplexes and cross-linking functional are optimized to operate in conjunction with one another. groups (Blackburn and Gait, Eds., “DNA and RNA Struc 0092. Other components of kits include an amplification ture' in Nucleic Acids in Chemistry and Biology, 2nd Edi primer with a sequencing tag, a corresponding sequencing tion, (1996) Oxford University Press, pp. 15-81). Yet another primer, an amplification polymerase, sequencing poly class of labels affects the separation or immobilization of a merase, dNTP mix (for amplification), ddNTP/DNTP mix molecule by specific or non-specific capture, for example (for sequencing), and Suitable buffers. In some embodiments, biotin, digoxigenin, and other haptens (Andrus, "Chemical a second amplification primer and corresponding sequencing methods for 5' non-isotopic labeling of PCR probes and prim primer may also be present. ers” (1995) in PCR 2: A Practical Approach, Oxford Univer 0093. When used in kits, the amplification primer contain sity Press, Oxford, pp. 39-54). Suitable haptens include fluo ing the universal tag can be made sequence-specific for a US 2012/0142004 A1 Jun. 7, 2012 given target sequence. The sequencing primer can be labeled 0100. In still another aspect, there are kits comprising a or unlabeled. If the sequencing primer is labeled, the label universal tagged primer comprising a plurality of purine chosen will be suitable for use in the intended application. nucleotides, where at least one purine nucleotide comprises a The primers can be packaged in Suitable containers, such as non-natural nucleobase, coupled to a backbone consisting of tubes or ampules, and can be packaged in a dried (e.g., lyo ribose or deoxyribose, and phosphate, wherein the purine philized) form, or in an aqueous form. If necessary, the nucleotides of the universal tagged primer are complemen articles of manufacture in the kits can be chilled or frozen tary to a plurality of pyrimidine nucleotides of a polynucle during shipping and/or storage. Any article of manufacture otide template, where at least one pyrimidine nucleotide com comprising universal tag can further include a description of prises a non-natural nucleobase complementary to the non the product, specifications of the product, or instructions for natural purine nucleobase. Kits comprising universal tagged use of the product. primers may further include other reagents necessary for 0094. In addition, kits can also include, for example but primer-initiated synthesis, including dNTPs and suitable are not limited to, apparatus and reagents for sample collec buffer components. tion and/or purification, apparatus and reagents for product 0101. In another aspect, there are kits comprising a uni collection and/or purification, Sample tubes, holders, trays, Versal tagged primer comprising one or more regions having racks, dishes, plates, instructions to the kit user, Solutions, a plurality of purine and/or pyrimidine nucleotides, where at buffers or other chemical reagents, suitable samples to be least one purine and/or pyrimidine nucleotide is a non-natural used for standardization, normalization, and/or control nucleobase, coupled to a backbone of ribose or deoxyribose, samples. Kits can also be packaged for convenient storage and phosphate, wherein the purine nucleotides of the univer and shipping, for example, in a box having a lid. sal tagged primer are complementary to a polynucleotide 0095. The sequencing primer provided in the kits may or template comprising one or more regions having a plurality of may not be labeled. In other embodiments, the invention purine and/or pyrimindine nucleotides, where at least one provides kits comprising sequencing primers as well as purine and/or pyrimidine nucleotide is a non-natural pyrimi means for labeling the oligomers. In other embodiments, the dine nucleobase complementary to at least one other non invention provides kits comprising a labeled or unlabeled natural nucleobase present in the primer, coupled to a back universal tag as well as some means (for example an appara bone of ribose or deoxyribose, and phosphate. tus or reagent) for the visualization or detection the tags. Such kits may be useful in diagnostic and research settings. 0096. The invention also provides kits to facilitate use of 0102) Applications and Methods of Use the universal tags in various methods, e.g., any method that (0103) The described compositions and methods find use in involves sequence-specific hybridization. Materials and a variety of applications. It is not intended that the invention reagents to carry out these methods can be provided in kits to find use in only the few applications discussed herein, as one facilitate execution of the methods. A kit comprises at least familiar with the art will immediately recognize a variety of one universal tagged primer and at least one sequencing uses for the universal tags with at least one non-natural primer, and optionally can additionally comprise a number of nucleobase. The uses cited herein are intended to be exem additional components, including but not limited to (i) one or plary and not limiting, and Such examples are not exhaustive. more buffers; (ii) one or more nucleotide triphosphates; (iii) a It is understood that use is not limited to any particular appli nucleic acid amplification master mix; (iv) one or more poly cation cited herein, as the invention finds use with any proto merase enzymes, or (V) reagents or equipment Suitable for the col that incorporates universal tags as probes or primers. isolation/purification of a nucleic acid product. In one embodiment, the kit comprises at least two universal tagged 0104 Use in Hybridization Reactions primers suitable for use as primers in a PCR reaction. 0105. The nucleobase oligomers find use in any method 0097. In some embodiments, the present invention pro involving hybridization, i.e., the forming of a complex vides kits for conducting real-time PCR analysis. These kits between two complementary nucleobase sequences. The can include, for example but are not limited to, reagents for complementarity need not be 100%, as effective hybridiza the collection of a sample, a reverse transcriptase, primer tions can occur when there is less than 100% complementa suitable for reverse transcriptase initiation and first strand rity. cDNA synthesis, at least one universal tagged primer, a DNA 0106 The potential uses of the universal tags are not in any dependent DNA polymerase, free deoxyribonucleotide triph way limited. Thus, one familiar with the art recognizes that osphates, and reagents Suitable for the isolation/purification the specific conditions to be used in hybridization reactions as of the cDNA molecules produced by the reaction. practiced using compositions are similarly unlimited, and are 0098. In one embodiment providing kits, a single univer dependent on the particular application and the primary sal tagged primer is provided that is specific for a single target sequence of the universal tags used. A wide variety of sources sequence. In other embodiments, multiple universal tagged are available that describe hybridization conditions for par primers specific for a plurality of targets are provided in the ticular application; see, e.g., Ausubel et al. (eds.), Current kit. In some embodiments, kits are provided having the uni Protocols in Molecular Biology, Chapter 22, “Nucleic Acid Versal tagged primers affixed to a solid phase or Surface. In Arrays.” John Wiley & Sons, Inc., New York 1994; and M. certain embodiments, the kits may be used to sequence at Schena, (ed.), Microarray Biochip Technology, BioTech least one target nucleic acid template. nique Books, Eaton Publishing, Natick, Mass. 2000. 0099. In still other embodiments, there are kits for the 0107 Immobilization on a Solid Support (Arrays) analysis of gene expression using the universal sequencing 0108. In one aspect, there are compositions and methods primers. These kits can include multiple universal tagged for making and using nucleobase oligomers with a universal primers affixed to a Suitable array or chip configuration, as tag that are affixed to a solid support. A wide variety of solid well as reagents required for the detection/visualization of Supports find use with the invention, and it is not intended that hybridized complexes. the invention be limited to the use of any particular type of US 2012/0142004 A1 Jun. 7, 2012

Solid Support. Similarly, it is not intended that the manner in anates and malimide groups are commercially available. which the nucleobase oligomers are affixed to the solid sup Non-limiting examples of Suitable Solid Supports include port be limited in any way. chips of any type (e.g., arrays), membranes, glass, controlled 0109. In one embodiment, the support-bound nucleobase pore glass, polystyrene particles (beads), silica and gold oligomers with a universal tag form an array (e.g., a chip) of nanoparticles. All of the above recited methods of immobili oligomers. Detailed methods for making and using arrays Zation are not intended to be limiting in any way but are comprising polymeric nucleobase structures (e.g., nucleic merely provided by way of illustration. acid, modified nucleic acids, nucleic acid analogs, or chi 0115 Detection/Identification of Biological Organisms meric structures) are well-known in the art and are described 0116. The nucleobase oligomers with universal tags find in many sources. See, e.g., Ausubel et al. (eds.), Current use in the detection, identification and/or enumeration of Protocols in Molecular Biology, Chapter 22, “Nucleic Acid biological organisms, and especially, pathogens. Such organ Arrays.” John Wiley & Sons, Inc., New York 1994; and M. isms can include viruses, bacteria and eucarya in food, bev Schena, (ed.), Microarray Biochip Technology, BioTech erages, water, pharmaceutical products, personal care prod nique Books, Eaton Publishing, Natick, Mass. 2000. Any ucts, dairy products or in Samples of plant, animal, human or methods for the synthesis and use of nucleic acids, modified environmental origin. The nucleobase oligomers with univer nucleic acids and nucleic acid analogs with Solid Supports, sal tags find use in the analysis of raw materials, equipment, and more specifically arrays, can be used. products or processes used to manufacture or store food, 0110 Because the location and sequence of each support beverages, water, pharmaceutical products, personal care bound oligomer with a universal tag is known, arrays can be products dairy products or environmental samples. Addition used to simultaneously detect, identify and/or quantitate the ally, the nucleobase oligomers find use in the detection of presence or amount of one or more target sequences in a pathogens (e.g., various bacteria, viruses and eucarya) in sample. For example, a target sequence can be captured by the clinical specimens, equipment, fixtures or products used to complementary nucleobase oligomer on the array Surface and treat humans or animals as well as in clinical samples and then the complex containing the target sequence can be clinical environments. For example, the analysis for micro detected. Since the sequence of the nucleobase oligomer is organisms of interest can be performed using FISH or multi known at each location on the Surface of the array, the plex FISH using probes generated by the invention described sequence of target sequence(s) can be directly detected, iden herein (See: BP U.S. application Ser. Nos. 09/335,629 and tified and/or quantitated by determining the location of a 09/368,089). detectable signal generated on the array. Thus, arrays are 0117 The compositions, methods, kits, libraries and useful in diagnostic applications or in Screening compounds, arrays with a universal tag are particularly useful in areas Such e.g., during development of therapeutic compounds. as expression analysis, single nucleotide polymorphism 0111. In one embodiment, the oligomers with a universal (SNP) analysis, genetic analysis of humans, animals, fungi, tag can be synthesized on the Surface in a manner Suitable for yeast, viruses, and plants (including genetically modified deprotection but not cleavage from the synthesis Support (see, organisms), therapy monitoring, pharmacogenomics, phar e.g., Weiler et al., Hybridization based DNA screening on macogenetics, epigenomics, and high throughput Screening peptide nucleic acid (PNA) oligomer arrays. Nucl. Acids operations. Res., 25(14):2792-2799 (1997)). In still another embodi ment, one or more nucleobase oligomers with a universal tag EXAMPLES can be covalently linked to a surface by the reaction of a Example 1 Suitable functional group on the oligomer or the universal tag with a functional group of the Surface (see, e.g., Geiger et al., 0118. A universal sequencing protocol was tested by PNAArray technology in molecular diagnostics, Nucleosides amplifying and sequencing the gene for the carboxyl termi & Nucleotides 17(9-11):1717-1724 (1998)). This method is nus of Hsc70-interacting protein (CHIP). Two sets of ampli advantageous since the oligomers immobilized on the Surface fication primers were examined. The length of the 5'-over can be highly purified and attached using a defined chemistry, hang (or tag) used to create a primer binding region for the thereby possibly minimizing or eliminating non-specific sequencing reaction was changed between the two sets (Table interactions. 1). 2'-Deoxyisocytidine (F) and 2'-deoxyisoguanosine (J) 0112 Methods for the chemical attachment of nucleobase were included in the tag sequences. The CHIP-specific por oligomers with universal tags to Solid Support Surfaces can tions of the amplification primers (underlined in Table 1) involve the reaction of a nucleophilic group, (e.g., an amine or were identical in both sets. thiol) of the oligomer or the universal tag to be immobilized, with an electrophilic group on the Solid Support Surface. TABLE 1. Alternatively, the nucleophile can be present on the Support and the electrophile (e.g., activated carboxylic acid) can be Amplification and Sequencing Primers present on the oligomer. Primer Name Primer Type Sequence 0113 Conditions suitable for the immobilization of a nucleobase oligomer with a universal tag to a surface are LF Amp Forward CGFTGACTFTTACAAGGAGCAGG widely known in the art. The immobilization reaction to a Amplification GCAATCGTC Solid Support is analogous to a labeling reaction, where the SF Amp Forward CFGFCTFTTACAAGGAGCAGGGC label is substituted with the surface to which the polymer is to Amplification AATCGTC be linked. It is not intended that the invention be limited to any particular immobilization chemistry or method. LF Seq Sequencing CGFTGACTFTTAC 0114. Numerous types of solid supports derivatized with SF Seq Sequencing CFGFCTFJJAC amino groups, carboxylic acid groups, isocyantes, isothiocy US 2012/0142004 A1 Jun. 7, 2012

I0121 Sequencing TABLE 1 - continued 0.122 Cycle sequencing was performed with the DYEnamicTM ET Dye Terminator Kit on a MegaBACE 1000 Amplification and Sequencing Primers Sequencer (GE Healthcare). Bidirectional sequencing was performed in two reactions with DYEnamic ET reagent pre Primer Name Primer Type Sequence mix (8 uI), 5 uM individual sequencing primer (1 uL), and purified amplicon (1 uL of 1 to 40 dilution with water) in a LR. Amp Rewerse AGTTGAGFGCFFJAGCAGGTAGT total volume of 20 uL. The cycling conditions were 95°C. (20 Amplification CGGGGATGTCTC s), 50° C. (15 s), 60° C. (60 s), repeated 20-30 times. The extension step worked with a 50° C. step replacing the 60° C. SR Amp Rewerse GFGFGCFFJAGCAGGTAGTCGGG step, as well. Either the LF/LR or the SF/SR sequencing Amplification GATGTCTC primers were used. Sequencing reactions were purified with LR. Seq Sequencing AGTTGAGFGCFFJAG Illustra autoSeq G50 Spin columns and analyzed as recom mended on the MegaBACE 1000. SR Seq Sequencing GFGFGCFFJAG (0123 Results 0.124 Amplification product lengths were verified with a 2100 Bioanalyzer (Agilent) or a Trugene System (Siemens) 0119) Amplification using fragment length analysis Software for reactions using 0120. A 606 nucleotide fragment of CHIP in the green either the LF/LR or the SF/SR amplification primer sets. fluorescent protein fusion vector pBGFP-C3 (Ballinger, Sequencing analysis gave the expected CHIP sequence with 1999) was amplified with either the LF/LR amplification either LF/LR sequencing primers or the SF/SR sequencing primer pair or the SF/SR amplification primer pair and Tita primers. The sequencing reaction always aborted when encountering For J positions introduced to the amplicon by nium Taq polymerase (BD BioSciences), using a Mastercy the sequencing primers. cler ep gradient S thermal cycler (Eppendorf). Thermal 0.125. It is to be understood that the foregoing descriptions cycling conditions were 95°C. (60s) followed by 40 cycles of of embodiments of the present invention are exemplary and 95°C. (30 s), 60° C. (60s). Reaction solutions contained 10 explanatory only, are not restrictive of the invention, as pM each amplification primer (LF/LR or SF/SR), 50 ng plas claimed, and merely illustrate various embodiments of the mid, 2.5 mM each dNTP (including dFTP and dJTP), 0.4 uL invention. It will be appreciated that other particular embodi of Titanium Taq in Titanium Taq buffer (20 u, total volume). ments consistent with the principles described in the specifi Reactions were purified with MinElute PCR Purification spin cation but not expressly disclosed may fall within the scope of columns. the claims.

SEQUENCE LISTING

<16 Os NUMBER OF SEO ID NOS: 8

<21 Os SEQ ID NO 1 &211s LENGTH: 32 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: primer 22 Os. FEATURE: <221s NAMEAKEY: misc feature <222s. LOCATION: (3) . . (3) <223> OTHER INFORMATION: n is isocytosine or isoguanine 22 Os. FEATURE: <221s NAMEAKEY: misc feature <222s. LOCATION: (9) . . (11) <223> OTHER INFORMATION: n is isocytosine or isoguanine

<4 OOs SEQUENCE: 1

cgntgactinn nacaaggagc agggcaatcg tc 32

<21 Os SEQ ID NO 2 &211s LENGTH: 30 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: primer 22 Os. FEATURE: <221s NAMEAKEY: misc feature <222s. LOCATION: (2) ... (2) <223> OTHER INFORMATION: n is isocytosine or isoguanine 22 Os. FEATURE: <221s NAMEAKEY: misc feature US 2012/0142004 A1 Jun. 7, 2012 12

- Continued LOCATION: (4) ... (4) OTHER INFORMATION: n is isocytosine or isoguanine FEATURE: NAMEAKEY: misc feature LOCATION: (7) ... (9) OTHER INFORMATION: n is isocytosine or isoguanine

SEQUENCE: 2

Cngnctnnna Caaggagcag ggcaatcgt.c 3 O

SEQ ID NO 3 LENGTH: 13 TYPE: DNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: primer FEATURE: NAMEAKEY: misc feature LOCATION: (3) ... (3) OTHER INFORMATION: n is isocytosine or isoguanine FEATURE: NAMEAKEY: misc feature LOCATION: (9) . . (11) OTHER INFORMATION: n is isocytosine or isoguanine

SEQUENCE: 3 cgntgactinn nac 13

SEQ ID NO 4 LENGTH: 11 TYPE: DNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: primer FEATURE: NAMEAKEY: misc feature LOCATION: (2) ... (2) OTHER INFORMATION: n is isocytosine or isoguanine FEATURE: NAMEAKEY: misc feature LOCATION: (4) ... (4) OTHER INFORMATION: n is isocytosine or isoguanine FEATURE: NAMEAKEY: misc feature LOCATION: (7) ... (9) OTHER INFORMATION: n is isocytosine or isoguanine

SEQUENCE: 4 cingnctnnna c 11

SEO ID NO 5 LENGTH: 35 TYPE: DNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: primer FEATURE: NAMEAKEY: misc feature LOCATION: (3) ... (3) OTHER INFORMATION: n is isocytosine or isoguanine FEATURE: NAMEAKEY: misc feature LOCATION: (8) ... (8) OTHER INFORMATION: n is isocytosine or isoguanine FEATURE: NAMEAKEY: misc feature LOCATION: (11) . . (13) OTHER INFORMATION: n is isocytosine or isoguanine

SEQUENCE: 5 US 2012/0142004 A1 Jun. 7, 2012 13

- Continued agntgagngc nnnagCaggit agt cqgggat gtctic 35

SEQ ID NO 6 LENGTH: 31 TYPE: DNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: primer FEATURE: NAMEAKEY: misc feature LOCATION: (2) ... (2) OTHER INFORMATION: n is isocytosine or isoguanine FEATURE: NAMEAKEY: misc feature LOCATION: (4) ... (4) OTHER INFORMATION: n is isocytosine or isoguanine FEATURE: NAMEAKEY: misc feature LOCATION: (7) ... (9) OTHER INFORMATION: n is isocytosine or isoguanine

SEQUENCE: 6 gngngcnnna gcaggtag to ggggatgtct C 31

SEO ID NO 7 LENGTH: 15 TYPE: DNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: primer FEATURE: NAMEAKEY: misc feature LOCATION: (3) ... (3) OTHER INFORMATION: n is isocytosine or isoguanine FEATURE: NAMEAKEY: misc feature LOCATION: (8) ... (8) OTHER INFORMATION: n is isocytosine or isoguanine FEATURE: NAMEAKEY: misc feature LOCATION: (11) . . (13) OTHER INFORMATION: n is isocytosine or isoguanine

SEQUENCE: 7 agntgagngc nnnag 15

SEQ ID NO 8 LENGTH: 11 TYPE: DNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: primer FEATURE: NAMEAKEY: misc feature LOCATION: (2) ... (2) OTHER INFORMATION: n is isocytosine or isoguanine FEATURE: NAMEAKEY: misc feature LOCATION: (4) ... (4) OTHER INFORMATION: n is isocytosine or isoguanine FEATURE: NAMEAKEY: misc feature LOCATION: (7) ... (9) OTHER INFORMATION: n is isocytosine or isoguanine

SEQUENCE: 8 gngngcnnna g 11 US 2012/0142004 A1 Jun. 7, 2012 14

1. An oligonucleotide primer comprising: 16. The oligonucleotide primer according to claim 1, (a) a 3' segment comprising a target-specific nucleotide wherein at least one non-natural nucleobase is at the 3' end of Sequence; the tag. (b) a 5' segment comprising a non-target-specific universal 17. The oligonucleotide primer according to claim 1, sequencing tag comprising at least one non-natural wherein at least one non-natural nucleobase is not at the 5' end nucleobase capable of hybridizing to a complementary of the tag. non-natural nucleobase and capable of being replicated 18. An oligonucleotide sequencing primer, comprising a 5' during amplification. non-target-specific universal sequencing tag comprising at 2. The oligonucleotide primer according to claim 1, least one non-natural nucleobase capable of hybridizing to a wherein the at least one non-natural nucleobase of the uni complementary non-natural nucleobase and capable of being Versal sequencing tag is capable of hybridizing to a comple replicated during amplification. 19.-32. (canceled) mentary non-natural nucleobase with a higheraffinity relative 33. An amplified polynucleotide sequencing template, to the natural nucleobase pairs G-C and A-T. comprising: 3. The oligonucleotide primer according to claim 1, (a) a polynucleotide sequence complementary to a region wherein the 5' universal sequencing tag comprises a plurality of a target polynucleotide; of non-natural nucleobases capable of hybridizing to comple (b) a 5' universal sequencing tag comprising a plurality of mentary non-natural nucleobases. non-natural nucleobases capable of hybridizing to a 4. The oligonucleotide primer according to claim 1, complementary non-natural nucleobase and capable of wherein the 5' universal sequencing tag comprises a plurality being replicated during amplification. of contiguous non-natural nucleobases capable of hybridiz 34.-48. (canceled) ing to complementary non-natural nucleobases. 49. A nucleic acid duplex, comprising an oligonucleotide 5. The oligonucleotide primer according to claim 1, primer comprising: wherein the 5' universal sequencing tag comprises at least (a) a 3" target-specific nucleotide sequence; three contiguous non-natural nucleobases capable of hybrid (b) a 5' non-target-specific universal tag comprising at least izing to complementary non-natural nucleobases. one non-natural nucleobase capable of complementary 6. The oligonucleotide primer according to claim 1, binding to a corresponding second non-natural nucleo wherein the 5' universal sequencing tag comprises a plurality base and capable of being replicated during amplifica of contiguous non-natural nucleobases capable of hybridiz tion, wherein the oligonucleotide is hybridized to a poly ing to complementary non-natural nucleobases, and wherein nucleotide target. the contiguous non-natural nucleobases are contiguous to the 50. A nucleic acid duplex, comprising: target-specific nucleotide sequence. (a) a universal tagged primer comprising 7. The oligonucleotide primer according to claim 1, (i) a target-specific nucleotide sequence and wherein the 5' universal sequencing tag comprises a plurality (ii) a 5' universal tag comprising at least one non-natural of contiguous non-natural nucleobases capable of hybridiz nucleobase capable of being replicated during ampli ing to complementary non-natural nucleobases, and wherein fication and complementary to at least one corre the contiguous non-natural nucleobases are positioned 5' of at sponding non-natural nucleobase, least a portion of the target-specific nucleotide sequence. wherein the universal tagged primer is hybridized to 8. The oligonucleotide primer according to claim 1, (b) an amplified polynucleotide template comprising wherein the non-natural nucleobases are selected from the (i) a polynucleotide sequence complementary to a group consisting of non-Watson-Crick complementary region of a target polynucleotide and nucleobase analogs. (ii) a 3' universal tag comprising at least one non-natural 9. The oligonucleotide primer according to claim 1, nucleobase capable of being replicated during ampli wherein the non-natural nucleobases are selected from the fication. group consisting of Watson-Crick complementary nucleo 51.-66. (canceled) base analogs having hydrogen bonding interactions that can 67. A method for replicating a polynucleotide target, com be discriminated from natural nucleobase pairs. prising the step of hybridizing to a polynucleotide target an 10. The oligonucleotide primer according to claim 1, amplification primer comprising a 3" target-specific nucle wherein the plurality of non-natural nucleobases of the ampli otide sequence and a 5' universal sequencing tag comprising fication primer are independently selected from the group at least one non-natural nucleobase, wherein the non-natural consisting of isocytosine, isoguanine, and 5-methylisocy nucleobase is capable of being replicated during amplifica tosine. tion, and extending the amplification primer under PCR con 11. The oligonucleotide primer according to claim 1, ditions, thereby producing a polynucleotide product that is wherein at least one non-natural nucleobase is isocytosine. complementary to the polynucleotide target and that further 12. The oligonucleotide primer according to claim 1, comprises a 5' universal sequencing tag comprising at least wherein at least one non-natural nucleobase is isoguanine. one non-natural nucleobase which is capable of being repli 13. The oligonucleotide primer according to claim 1, cated during amplification. wherein at least one non-natural nucleobase is 5-methyliso 68. A method according to claim 67, further comprising the cytosine. step of sequencing the polynucleotide sequencing template 14. The oligonucleotide primer according to claim 1, com using a universal sequencing primer complementary to the prising at least four non-natural nucleobases. universal sequencing tag incorporated into the polynucle 15. The oligonucleotide primer according to claim 1, otide product, wherein the universal sequencing primer com wherein at least three of the four non-natural nucleobases are prises a non-natural nucleobase complementary to a non contiguous. natural nucleobase of polynucleotide product. US 2012/0142004 A1 Jun. 7, 2012 15

69. A method for sequencing a target polynucleotide, com- 70-85. (canceled) pr1S1ng: 86. A kit for sequencing a polynucleotide target, compris (a) providing an amplified polynucleotide sequencing tem- ing: plate comprising (a) an amplification primer comprising (i) a 3' universal sequencing tag comprising one or more t non-natural nucleobase capable of being replicated (i) a 3' target-specific nucleotide sequence and during amplification, and (ii) a 5' non-target-specific universal sequencing tag (ii) a 5' target polynucleotide; and comprising one or more non-natural nucleobases (b) hybridizing the polynucleotide sequencing template capable of complementary binding to a correspond with a universal sequencing primer at least a portion of ing second non-natural nucleobase, wherein the one which is complementary to the 3' universal sequencing or more non-natural nucleobases and the correspond tag of the polynucleotide sequencing template, wherein ing second non-natural nucleobases are capable of the universal sequencing primer comprises at least one being replicated during amplification; non-natural nucleobase capable of being replicated dur- (b) a universal sequencing primer comprising an oligo ing amplification and complementary to a non-natural nucleotide of the sequence comprising the 5' non-target nucleobase present in the 3' universal sequencing tag, sequencing tag. (c) generating primer extension products under conditions 87-102. (canceled) Sufficient to generate sequencing fragments correspond ing to a portion of the polynucleotide target. ck c. c. : :