Methods and kits for detection of methylation status
The present invention relates to methods and kits for the detection of 5-hydroxymethylcytosine (5hmC) and/or 5-methylcytosine (5meC). In some embodiments, the present invention relates to methods and kits for detection of 5hmC and/or 5meC in nucleic acid (e.g., DNA, RNA). In some embodiments, the present invention relates to detection of 5hmC in genomic DNA, e.g., mammalian genomic DNA.
1. A method for detecting 5-methylated cytosine and 5-hydroxymethylated cytosine of a nucleic acid sequence, wherein said method comprising:
(a) contacting a nucleic acid methyltransferase with said nucleic acid sequence and a daughter sequence of said nucleic acid sequence, which daughter sequence comprises a complementary sequence of said nucleic acid sequence, such that a pattern of 5-methylated cytosine residues of said nucleic acid sequence is maintained;
(b) after (a), oxidizing unmodified cytosine of said nucleic acid sequence and of said daughter sequence to yield a modified nucleic acid sequence and a modified daughter sequence;
(c) sequencing said modified nucleic acid sequence or derivative thereof and said modified daughter sequence or derivative thereof to obtain a first sequence corresponding to said modified nucleic acid sequence and a second sequence corresponding to said modified daughter sequence; and
(d) comparing said first sequence and said second sequence to identify and distinguish said 5-methylated cytosine and said 5-hydroxymethylated cytosine of said nucleic acid sequence.
2. The method of claim 1 , wherein a hemimethylated double stranded nucleic acid sequence comprises said nucleic acid sequence and said daughter sequence.
3. The method of claim 1 , wherein said nucleic acid sequence comprises genomic DNA.
4. The method of claim 1 , wherein said nucleic acid methyltransferase is a DNA methyltransferase.
5. The method of claim 4 , wherein said DNA methyltransferase is DNMT1.
6. The method of claim 5 , wherein said DNMT1 comprises a sequence corresponding to SEQ ID NO: 1 or SEQ ID NO: 2.
7. The method of claim 4 , wherein said DNA methyltransferase is M.SssI DNMT.
8. The method of claim 7 , wherein said M.SssI DNMT comprises a sequence corresponding to SEQ ID NO: 3.
9. The method of claim 1 , wherein said oxidizing comprises treating unmodified cytosine on said nucleic acid sequence and on said daughter sequence with bisulfite.
10. The method of claim 1 , wherein said 5-hydroxymethylated cytosine comprises 5-hydroxymethyl cytosine or beta-glu-5-hydroxymethyl cytosine.
11. The method of claim 10 , wherein said 5-hydroxymethylated cytosine comprises 5-hydroxymethyl cytosine.
12. The method of claim 1 , wherein said sequencing comprises a next generation sequencing method.
13. The method of claim 1 , wherein said nucleic acid sequence is obtained from a subject having or suspected of having a disease or condition.
14. The method of claim 13 , wherein said disease or condition comprises cancer.
15. The method of claim 1 , wherein said 5-methylated cytosine and 5-hydroxymethylated cytosine are identified at base specific resolution.