PRESERVATION OF INFORMATION RELATED TO GENOMIC DNA METHYLATION
First Claim
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1. A method of sequencing nucleic acid comprising deaminated cytosine, said method comprising:
- providing a sample comprising a template nucleic acid;
generating a complementary copy of said template nucleic acid, said generating being directed by an oligonucleotide primer using a nucleic acid polymerase in the presence of a bisulfite-resistant cytosine analog, wherein said generating produces a complementary copy of said template nucleic acid such that cytosine residues in said complementary copy are bisulfite-resistant;
subjecting said template nucleic acid and said complementary copy to bisulfite treatment to convert unmethylated cytosine residues in said template nucleic acid into uracil residues, resulting in a bisulfite-converted template nucleic acid and a non-converted complementary copy; and
determining the nucleotide sequence of said bisulfite-converted template nucleic acid and said non-converted complementary copy.
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Abstract
The present invention relates to compositions, methods and systems for analyzing the methylation state of nucleic acids. Some embodiments relate to a compositions, methods and systems for analyzing the methylation state of DNA with a gene array.
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Citations
141 Claims
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1. A method of sequencing nucleic acid comprising deaminated cytosine, said method comprising:
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providing a sample comprising a template nucleic acid; generating a complementary copy of said template nucleic acid, said generating being directed by an oligonucleotide primer using a nucleic acid polymerase in the presence of a bisulfite-resistant cytosine analog, wherein said generating produces a complementary copy of said template nucleic acid such that cytosine residues in said complementary copy are bisulfite-resistant; subjecting said template nucleic acid and said complementary copy to bisulfite treatment to convert unmethylated cytosine residues in said template nucleic acid into uracil residues, resulting in a bisulfite-converted template nucleic acid and a non-converted complementary copy; and determining the nucleotide sequence of said bisulfite-converted template nucleic acid and said non-converted complementary copy. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21)
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22. A method of identifying methylated cytosines in a plurality of nucleic acids, said method comprising:
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providing a sample comprising a plurality of template nucleic acids; generating complementary copies of said template nucleic acids, said generating being directed by an oligonucleotide primer using a nucleic acid polymerase in the presence of a bisulfite-resistant cytosine analog, wherein said generating produces a complementary copy of each of said template nucleic acids such that cytosine residues in each of said complementary copy are bisulfite-resistant, and wherein each complementary copy is coupled to one of said template nucleic acids; subjecting said template nucleic acids and said complementary copies to bisulfite treatment to convert unmethylated cytosine residues in said template nucleic acids into uracil residues, resulting in each bisulfite-converted template nucleic acid being coupled to a non-converted complementary copy; determining the nucleotide sequence of said bisulfite-converted template nucleic acids and said non-converted complementary copies; and comparing said nucleotide sequence of said bisulfite-converted template nucleic acids to the nucleotide sequence of said non-converted complementary copies for each of said bisulfite-converted template nucleic acids coupled to non-converted complementary copies, thereby determining the methylation status of the template nucleic acids prior to bisulfite conversion. - View Dependent Claims (23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48)
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49. A method of identifying methylated cytosines in a nucleic acid, said method comprising:
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obtaining bisulfite-converted template nucleic acid, obtaining a non-converted complementary copy of said template nucleic acid, determining the nucleotide sequence of said bisulfite-converted template nucleic acid; determining the nucleotide sequence of said non-converted complementary copy of said template nucleic acid; comparing said nucleotide sequence of said non-converted complementary copy of said template nucleic acid to said bisulfite-converted template nucleic acid, thereby determining the nucleotide sequence and the methylation status of the template nucleic acid prior to bisulfite conversion. - View Dependent Claims (50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71)
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72. A nucleic acid pair comprising:
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a template nucleic acid comprising a cytosine residue; a complementary copy of said template nucleic acid having every cytosine replaced by a bisulfite-resistant cytosine analog; and a tag capable of identifying said template nucleic acid and said complementary copy of said template nucleic acid as members of said nucleic acid pair, wherein said template nucleic acid and said complementary copy of said template nucleic acid are coupled to said tag. - View Dependent Claims (73, 74, 75, 76, 77, 78, 79, 80, 81, 82)
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83. A population of nucleic acid pairs, comprising:
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template nucleic acids and complementary copies of said template nucleic acids attached to each other as covalent pairs via a nucleic acid loop, wherein said template nucleic acids each comprise at least one cytosine residue, wherein every cytosine of said complementary copies is replaced by a bisulfite-resistant cytosine analog; and wherein different nucleic acid pairs in said population have different sequences for said template nucleic acids and the same sequence for said nucleic acid loop. - View Dependent Claims (84, 85, 86, 87, 88, 89)
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90. A method of making an array comprising:
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providing a solid support with a plurality of sites; providing a sample comprising a template nucleic acid; generating a complementary copy of said template nucleic acid, said generating being directed by an oligonucleotide primer using a nucleic acid polymerase in the presence of a bisulfite-resistant cytosine analog, wherein said generating produces a complementary copy of said template nucleic acid such that each cytosine residue in said complementary copy is bisulfite-resistant; subjecting said template nucleic acid and said complementary copy to bisulfite treatment to convert unmethylated cytosine residues in said template nucleic acid into uracil residues, resulting in a bisulfite-converted template nucleic acid and a non-converted complementary copy; and coupling said template and said complementary copy of said template to said solid support. - View Dependent Claims (91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109)
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110. An array comprising:
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a solid support with a plurality of sites, a bisulfite-converted template nucleic acid, and a non-converted complementary copy of said template nucleic acid, wherein said template nucleic acid is coupled to at least one of said plurality of sites and said non-converted complementary copy is coupled to at least one of said plurality of sites. - View Dependent Claims (111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128)
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129. A method of identifying methylated cytosines in a nucleic acid, said method comprising:
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obtaining a template nucleic acid comprising at least a first methyl CpG dinucleotide, obtaining a complementary copy of said template nucleic acid, wherein said complementary copy comprises a complementary methyl CpG dinucleotide in a position opposite said first methyl CpG dinucleotide, subjecting said template nucleic acid and said complementary copy to bisulfite treatment to convert unmethylated cytosine residues in said template nucleic acid into uracil residues, resulting in a bisulfite-converted template nucleic acid and a bisulfite-converted converted complementary copy; determining the nucleotide sequence of said bisulfite-converted template nucleic acid; determining the nucleotide sequence of said bisulfite-converted complementary copy; and comparing said nucleotide sequence of said bisulfite-converted complementary copy to said nucleotide sequence of said bisulfite-converted template nucleic acid, thereby determining the nucleotide sequence of said template nucleic acid prior to bisulfite conversion and the methylation status of the template nucleic acid prior to bisulfite conversion. - View Dependent Claims (130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141)
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Specification