METHYLATION ANALYSIS OF MATE PAIRS
First Claim
1. A method of analyzing the methylation state of genomic DNA, comprising:
- fragmenting a genomic DNA sample, whereby genomic DNA fragments are produced;
circularizing a genomic DNA fragment to produce a double-stranded circular DNA comprising a nick on at least one strand of the double-stranded circular DNA;
linearizing the circular DNA;
adding a nick translation enzyme in the presence of methylation conversion agent resistant nucleotide triphosphates, whereby a partially methylation conversion agent resistant polynucleotide is generated, wherein the partially methylation conversion agent resistant polynucleotide has a tag region that is methylation conversion agent resistant and a tag region that is not methylation conversion agent resistant.
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Abstract
Various embodiments of the present teachings relate to methods for the methylation analysis of nucleic acids. The subject methods include methods that result in the preparation of mate-pair libraries suitable for highly multiplexed DNA sequencing. Embodiments include methods of preparing mate-pair libraries comprising a first tag sequence and a second tag sequence, wherein one of the tag sequences has been converted by a methylation conversion agent and the other tag sequence has not been converted by the methylation conversion agent. Other embodiments provided include intermediates for making the mate-pair library and kits for making the mate-pair libraries. Also provided is software and computer systems for analyzing the methylation levels of genomic DNA from which the tag sequences were derived.
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Citations
50 Claims
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1. A method of analyzing the methylation state of genomic DNA, comprising:
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fragmenting a genomic DNA sample, whereby genomic DNA fragments are produced; circularizing a genomic DNA fragment to produce a double-stranded circular DNA comprising a nick on at least one strand of the double-stranded circular DNA; linearizing the circular DNA; adding a nick translation enzyme in the presence of methylation conversion agent resistant nucleotide triphosphates, whereby a partially methylation conversion agent resistant polynucleotide is generated, wherein the partially methylation conversion agent resistant polynucleotide has a tag region that is methylation conversion agent resistant and a tag region that is not methylation conversion agent resistant. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8)
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- 9. The method of 8, further comprising exposing the adapter modified polynucleotide to a nick translation enzyme and a set of dNTPS.
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19. A method of analyzing the methylation state of genomic DNA, comprising:
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fragmenting a genomic DNA sample, whereby genomic DNA fragments are produced; forming a first tag sequence and a second tag sequence, wherein the first tag sequence and the second tag sequence are derived from a single genomic DNA fragment; wherein the first tag sequence has been converted by a methylation conversion agent and the second tag sequence has not been converted by a methylation conversion agent. - View Dependent Claims (20, 21, 22, 23, 24)
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- 25. A polynucleotide construction comprising a first tag sequence and a second tag sequence, wherein the first tag sequence and the second tag sequence are derived from a single fragment of genomic DNA, wherein the first tag comprises methylation conversion resistant nucleotide that have been incorporated into the construction by an in vitro reaction and the second tag does not comprise methylation conversion resistant nucleotide that have been incorporated into the construction by an in vitro reaction.
- 29. An adapter comprising a first strand having methylation conversion resistant nucleotides and a second strand complementary to the first strand, wherein the second strand optionally contains methylation conversion resistant nucleotides.
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31. A method of matching a DNA sequence to a genomic sequence database, said method comprising:
comparing a data record comprising (1) a first tag sequence that corresponds to a DNA sequence that has not been modified by a methylation conversion agent and (2) a second tag sequence that corresponds to a DNA sequence that may have been modified by a methylation conversion agent, with DNA sequence information in the genomic database. - View Dependent Claims (32, 33, 34, 35)
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36. A method of amplifying polynucleotides converted by a methylation conversion agent, comprising;
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providing a polynucleotide fragment having two termini; ligating a primer-adapter to both of the termini, wherein the primer-adapter is a double-stranded polynucleotide having a first stand and second strand complementary to the first strand, wherein the first strand comprises methylation conversion resistant nucleotides and the second strand optionally comprises methylation conversion resistant nucleotides, whereby an adapter modified polynucleotide is produced; exposing the adapter-modified polynucleotide to a methylation conversion reagent, whereby a converted adapter modified polynucleotide is produced; and amplifying the converted adapter modified polynucleotide, wherein amplifying the converted adapter modified polynucleotide uses primers specific for sequences in the second strand of the adapter. - View Dependent Claims (37)
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38. A method of analyzing the methylation state of a genomic DNA sample, said method comprising:
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mixing a DNA sample with formamide, whereby a sample mixture is formed; heating the sample mixture at temperature sufficient to denature the genomic DNA; and adding a bisulfite salt to the sample mixture. - View Dependent Claims (39, 40, 41, 42, 43, 44, 45, 46)
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47. A method of analyzing the methylation state of a polynucleotide, comprising:
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providing a polynucleotide fragment having two termini; ligating a primer-adapter to both of the termini; circularizing the adapter-modified polynucleotide with an internal adapter to produce a double-stranded circular polynucleotide comprising a nick on one strand of the circular polynucleotide, wherein the internal adapter comprises a specific binding moiety; nick-translating the circular polynucleotide; capturing the strand comprising the specific binding moiety with a cognate specific binding moiety on a solid support; separating the captured strand and the non-captured strand; and exposing at least one of the captured strand and the non-captured strand to a methylation conversion reagent, whereby at least one converted strand is produced; and sequencing the at least one converted strand. - View Dependent Claims (48, 49, 50)
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Specification