METHODS FOR DETECTION OF NUCLEOTIDE MODIFICATION
First Claim
1. A method of identifying a 5-formylcytosine residue in a sample nucleotide sequence comprising;
- (i) providing a population of single stranded polynucleotides which comprise the sample nucleotide sequence,(ii) reducing a first portion of said population by adding an alkaline borohydride solution,(iii) treating the reduced first portion of said population and a second portion of said population with bisulfite,(iv) 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;
(v) identifying the residues in the first and second nucleotide sequences which correspond to a 5-formylcytosine residue in the sample nucleotide sequence.
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Abstract
This invention relates to improved methods and kits for identification of 5-formylcytosine (5fC) to be distinguished from cytosine (C) in a sample nucleotide sequence. Methods comprise reducing a first portion of polynucleotides which comprise the sample nucleotide sequence; treating the reduced first portion and a second portion of polynucleotides with bisulfite; sequencing the polynucleotides in the first and second portions of the population to produce first and second nucleotide sequences respectively and; identifying the residues in the first and second nucleotide sequences which correspond 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.
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19 Claims
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1. A method of identifying a 5-formylcytosine residue in a sample nucleotide sequence comprising;
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(i) providing a population of single stranded polynucleotides which comprise the sample nucleotide sequence, (ii) reducing a first portion of said population by adding an alkaline borohydride solution, (iii) treating the reduced first portion of said population and a second portion of said population with bisulfite, (iv) 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; (v) identifying the residues in the first and second nucleotide sequences which correspond to a 5-formylcytosine residue in the sample nucleotide sequence. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19)
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Specification