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 .- 10 . (canceled)
11 . A method comprising:
(a) obtaining a sample, wherein said sample is at least a portion of an extracellular fluid sample that comprises a nucleic acid sequence; and
(b) adding a control sample to said sample, wherein said control sample comprises a nucleic acid sequence having an epigenetically modified base.
12 . The method of claim 11 , wherein said epigenetically modified base is a non-natural epigenetically modified base.
13 . The method of claim 11 , wherein said epigenetically modified base is a hydroxymethylated cytosine or a methylated cytosine.
14 . The method of claim 11 , comprising denaturing said sample.
15 . The method of claim 11 , comprising preparing said sample for high-throughput sequencing.
16 . The method of claim 11 , wherein said sample is obtained from a subject having cancer or suspected of having cancer.
17 . The method of claim 11 , comprising contacting said sample with an antibody or antigen-binding portion thereof that specifically binds to an epigenetic modification of said sample.
18 . The method of claim 17 , comprising performing solid-phase purification, wherein said antibody or antigen-binding portion thereof is immobilized on a substrate.
19 . The method of claim 17 , wherein said antibody or antigen-binding portion thereof is a monoclonal antibody.
20 . The method of claim 17 , wherein said antibody or antigen-binding portion thereof is a hydroxymethyl cytosine-specific antibody or binding fragment thereof.
21 . The method of claim 17 , wherein said antibody or antigen-binding portion thereof binds cytosine-5-methylsulfonate.
22 . A method comprising:
(a) contacting a nucleic acid sequence with an antibody or antigen-binding portion thereof that specifically binds to an epigenetically modified base of said nucleic acid sequence; and
(b) detecting a presence or an absence of a binding of said antibody or said antigen-binding portion thereof to said nucleic acid sequence.
23 . The method of claim 22 , wherein said epigenetically modified base is a hydroxymethylated cytosine or a methylated cytosine.
24 . The method of claim 22 , wherein said epigenetically modified base is a non-natural base.
25 . The method of claim 22 , wherein said mammalian nucleic acid sequence is obtained from an extracellular fluid sample.
26 . The method of claim 22 , comprising denaturing said mammalian nucleic acid sequence.
27 . The method of claim 22 , comprising preparing said mammalian nucleic acid sequence for high-throughput sequencing.
28 . The method of claim 22 , wherein said mammalian nucleic acids sequence is of a sample obtained from a subject having cancer or suspected of having cancer.
29 . The method of claim 22 , comprising performing solid-phase purification, wherein said antibody or antigen-binding portion thereof is immobilized on a substrate.
30 . The method of claim 22 , wherein said antibody or antigen-binding portion thereof is a hydroxymethyl cytosine-specific antibody or binding fragment thereof.
31 . The method of claim 22 , wherein said antibody or antigen-binding portion thereof binds cytosine-5-methylsulfonate.
32 . The method of claim 22 , wherein said antibody or antigen binding portion thereof is a monoclonal antibody.
33 . The method of claim 22 , further comprising contacting said mammalian nucleic acid sequence with an antibody or antigen-binding portion thereof that specifically binds to a 5-methylcytosine.