Methods for Detection of Nucleotide Modification

Abstract

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 oxidising or reducing a first portion of polynucleotides which comprise the sample nucleotide sequence; treating the oxidised 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 .- 32 . (canceled) 
     
     
         33 . A method comprising:
 contacting a sample comprising a 5-hydroxymethylcytosine with a metal oxo complex, wherein said metal oxo complex converts said 5-hydroxymethylcytosine to 5-formylcytosine.   
     
     
         34 . The method of  claim 33 , wherein said sample comprises a nucleotide sequence. 
     
     
         35 . The method of  claim 33 , wherein said metal oxo complex is a perruthenate. 
     
     
         36 . The method of  claim 35 , wherein said perruthenate is KRuO4. 
     
     
         37 . The method of  claim 34 , wherein said nucleotide sequence comprises genomic DNA. 
     
     
         38 . The method of  claim 34 , wherein said nucleotide sequence comprises RNA. 
     
     
         39 . The method of  claim 34 , wherein said nucleotide sequence is immobilized. 
     
     
         40 . The method of  claim 34 , further comprising confirming a presence of said 5-hydroxymethylcytosine. 
     
     
         41 . The method of  claim 40 , wherein said confirming comprises amplifying said nucleotide sequence. 
     
     
         42 . The method of  claim 40  or  41 , wherein said confirming comprises sequencing said nucleotide sequence. 
     
     
         43 . The method of  claim 34 , further comprising reducing said nucleotide sequence. 
     
     
         44 . The method of  claim 33 , wherein said contacting occurs under aqueous conditions.