SELECTIVE OXIDATION OF 5-METHYLCYTOSINE BY TET-FAMILY PROTEINS
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.
1 .- 20 . (canceled)
21 . A method comprising:
(a) obtaining a sample comprising a nucleic acid sequence, wherein said nucleic acid sequence comprises a 5-hydroxymethylcytosine and a 5-methylcytosine;
(b) performing a sequencing; and
(c) distinguishing said 5-hydroxymethylcytosine from said 5-methylcytosine.
24 . The method of claim 1 , wherein said nucleic acid sequence is a mammalian nucleic acid sequence.
25 . The method of claim 1 , wherein said sample comprises genomic DNA.
26 . The method of claim 1 , wherein said sample comprises an extracellular fluid.
27 . The method of claim 1 , wherein said sequencing is high-throughput sequencing.
28 . The method of claim 1 , wherein said sample is from a subject having or suspected of having cancer.
29 . The method of claim 1 , wherein said distinguishing comprises detecting said 5-hydroxymethylcytosine.
30 . The method of claim 1 , wherein said distinguishing comprises detecting said 5-methylcytosine.
31 . The method of claim 1 , further comprising contacting said nucleic acid sequence with a dioxygenase or catalytically active fragment thereof to convert a methylated cytosine in said nucleic acid sequence to a modified base, wherein said dioxygenase or said catalytically active fragment thereof comprises TET1, TET2, TET3, CXXC4, a catalytically active fragment of any of these, or any combination thereof.
32 . The method of claim 31 , further comprising contacting said nucleic acid sequence with sodium bisulfite.