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(12) Patent Application Publication (10) Pub. No.: US 2012/0142004 A1 Battersby Et Al 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.
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