Oxidising Agent for Modified Nucleotides

Abstract

This invention relates to the use of metal (VI) oxo complexes to catalyse the selective oxidation of 5hmC residues in polynucleotides to 5fC residues. This may be useful in the identification of modified cytosine residues in a population of polynucleotides comprising a sample nucleotide sequence. A first portion of the population is oxidised with a metal (VI) oxo complex and then the first portion and a second portion of said population are both treated with bisulfite. The residues in the first and second portions that correspond to a cytosine residue in the sample nucleotide sequence are identified following treatment and the identities of these residues are used to determine the modification of the cytosine residue in the sample nucleotide sequence. Methods, reagents and kits are provided.

What is claimed is: 
     
         1 .- 45 . (canceled) 
     
     
         46 . A method comprising: selectively converting 5-hydroxymethylcytosine (5-hmC) to 5-formylcytosine (5-fC) in a nucleotide sequence by contacting at least a portion of said nucleotide sequence with a metal (VI) oxo complex. 
     
     
         47 . The method of  claim 46 , further comprising treating said nucleotide sequence with bisulfite. 
     
     
         48 . The method of  claim 47 , further comprising sequencing said nucleotide sequence. 
     
     
         49 . The method of  claim 47 , further comprising amplifying said nucleotide sequence. 
     
     
         50 . The method of  claim 47 , wherein said method identifies a presence of 5-methylated cytosine (5-mC) or 5-hmC in said nucleotide sequence. 
     
     
         51 . The method of  claim 46 , wherein said metal (VI) oxo complex is manganate (Mn(VI)O 4   2− ). 
     
     
         52 . The method of  claim 46 , wherein said metal (VI) oxo complex is ruthenate (Ru(VI)O 4   2− ). 
     
     
         53 . The method of  claim 52 , wherein said metal (VI) oxo complex is K 2 RuO 4 . 
     
     
         54 . The method of  claim 46 , wherein said contacting is repeated. 
     
     
         55 . The method of  claim 46 , wherein said nucleotide sequence comprises DNA. 
     
     
         56 . The method of  claim 55 , wherein said DNA is genomic DNA. 
     
     
         57 . The method of  claim 46 , wherein said nucleotide sequence comprises RNA. 
     
     
         58 . The method of  claim 46 , wherein said nucleotide sequence or said portion of said nucleotide sequence is immobilized. 
     
     
         59 . The method of  claim 46 , further comprising contacting said nucleotide sequence with a detection oligonucleotide. 
     
     
         60 . The method of  claim 59 , wherein said detection oligonucleotide hybridizes to portions of said nucleotide sequence having a cytosine residue or a uracil residue. 
     
     
         61 . The method of  claim 59 , wherein said detection oligonucleotide is immobilized. 
     
     
         62 . The method of  claim 46 , wherein said nucleotide sequence is a mammalian nucleotide sequence. 
     
     
         63 . The method of  claim 46 , wherein said nucleotide sequence is from a tissue sample. 
     
     
         64 . The method of  claim 46 , further comprising converting a portion of said 5-hydroxymethylcytosine (5-hmC) to 5-carboxymethylcytosine (5-caC).