SELECTIVE OXIDATION OF 5-METHYLCYTOSINE BY TET-FAMILY PROTEINS
First Claim
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1. A method for detecting 5-methylcytosine in a nucleic acid comprising:
- a) converting 5-methylcytosine to an acid beyond hydroxymethylation in the nucleic acid using enzymatic modification by a dioxygenase;
b) bisulfite sequencing the converted nucleic acid; and
c) detecting the 5-methylcytosine in the nucleic acid at positions where the acid is identified by reading a thymine in the converted nucleic acid where a cytosine is read when the nucleic acid is bisulfite sequenced but not converted.
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Abstract
The present invention provides for novel methods for regulating and detecting the cytosine methylation status of DNA. The invention is based upon identification of a novel and surprising catalytic activity for the family of TET proteins, namely TET1, TET2, TET3, and CXXC4. The novel activity is related to the enzymes being capable of converting the cytosine nucleotide 5-methylcytosine into 5-hydroxymethylcytosine by hydroxylation.
22 Citations
20 Claims
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1. A method for detecting 5-methylcytosine in a nucleic acid comprising:
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a) converting 5-methylcytosine to an acid beyond hydroxymethylation in the nucleic acid using enzymatic modification by a dioxygenase; b) bisulfite sequencing the converted nucleic acid; and c) detecting the 5-methylcytosine in the nucleic acid at positions where the acid is identified by reading a thymine in the converted nucleic acid where a cytosine is read when the nucleic acid is bisulfite sequenced but not converted. - View Dependent Claims (2, 3, 4, 5, 6)
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7. A method of detecting 5-hydroxymethylcytosine from 5-methylctosine from a nucleic acid comprising:
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a) modifying 5-hydroxymethycytosine in the nucleic acid with a glucose or a modified glucose; b) treating the modified nucleic acid with dioxygenase to convert 5-methylcytosine to an acid beyond hydroxymethylation; c) sequencing the treated nucleic acid using bisulfite sequencing; and d) detecting 5-hydroxymethylcytosine from 5-methylcytosine by reading a cytosine for 5-hydroxymethylcytosine and a thymine for 5-methylcytosine in the bisulfite sequenced nucleic acid. - View Dependent Claims (8, 9, 10)
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11. A method for detecting 5-methylcytosine in a nucleic acid comprising:
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a) converting 5-methylcytosine to an acid beyond hydroxymethylation in the nucleic acid using enzymatic modification by a dioxygenase; b) bisulfite sequencing the converted nucleic acid; and c) detecting the 5-methylcytosine in the nucleic acid at positions where the acid is identified by reading a thymine in the converted nucleic acid. - View Dependent Claims (12, 13, 14, 15, 16)
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17. A method of detecting 5-hydroxymethylcytosine from 5-methylctosine from a nucleic acid comprising:
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a) modifying 5-hydroxymethycytosine in the nucleic acid with a glucose or a modified glucose; b) treating the modified nucleic acid with dioxygenase to convert 5-methylcytosine to an acid beyond hydroxymethylation; c) sequencing the treated nucleic acid using bisulfite sequencing; and d) detecting 5-hydroxymethylcytosine from 5-methylcytosine based on the bisulfite sequenced nucleic acid. - View Dependent Claims (18, 19, 20)
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Specification