Methods for detection of nucleotide modification
This invention relates to the identification of modified cytosine residues, such as 5-methylcytosine (5mC), 5-hydroxymethylcytosine (5hmC) and 5-formylcytosine (5fC) to be distinguished from cytosine (C) in a sample nucleotide sequence. Methods may comprise oxidizing or reducing a first portion of polynucleotides which comprise the sample nucleotide sequence; treating the oxidized or reduced first portion and a second portion of polynucleotides with bisulfite; sequencing the polynucleotides in the first and second portions of the population following steps ii) and iii) to produce first and second nucleotide sequences, respectively and; identifying the residue in the first and second nucleotide sequences which corresponds to a cytosine residue in the sample nucleotide sequence. These methods may be useful, for example in the analysis of genomic DNA and/or of RNA.
1. A method comprising:
contacting a sample comprising a 5-hydroxymethylcytosine with a perruthenate oxidizing agent, wherein said perruthenate oxidizing agent selectively converts said 5-hydroxymethylcytosine to 5-formylcytosine.
2. The method of claim 1 , wherein said sample comprises a nucleotide sequence.
3. The method of claim 1 , wherein said perruthenate oxidizing agent is KRuO 4 .
4. The method of claim 2 , wherein said nucleotide sequence comprises genomic DNA.
5. The method of claim 2 , wherein said nucleotide sequence comprises RNA.
6. The method of claim 2 , wherein said nucleotide sequence is immobilized.
7. The method of claim 2 , further comprising confirming a presence of said 5-hydroxymethylcyto sine.
8. The method of claim 7 , wherein said confirming comprises amplifying said nucleotide sequence.
9. The method of claim 7 or 8 , wherein said confirming comprises sequencing said nucleotide sequence.
10. The method of claim 2 , further comprising reducing said nucleotide sequence.
11. The method of claim 1 , wherein said contacting occurs under aqueous conditions.