Methods for detection of nucleotide modification
First Claim
1. A method of identifying a modified cytosine residue in a sample nucleotide sequence comprising the steps of:
- (i) providing a population of polynucleotides which comprise the sample nucleotide sequence,(ii) oxidising or reducing a first portion of said population,(iii) treating the oxidised or 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 a residue in the first and second nucleotide sequences which corresponds to a cytosine residue in the sample nucleotide sequence.
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Accused Products
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 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.
24 Citations
27 Claims
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1. A method of identifying a modified cytosine residue in a sample nucleotide sequence comprising the steps of:
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(i) providing a population of polynucleotides which comprise the sample nucleotide sequence, (ii) oxidising or reducing a first portion of said population, (iii) treating the oxidised or 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 a residue in the first and second nucleotide sequences which corresponds to a cytosine 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, 20, 21, 22, 23, 24, 25, 26, 27)
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19. A method of identifying 5-methylcytosine in a sample nucleotide sequence comprising the steps of:
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(i) providing a population of polynucleotides which comprise the sample nucleotide sequence, (ii) oxidising said population of polynucleotides, (iii) treating the oxidised population of polynucleotides with bisulfite to generate an oxidised, bisulfite treated population of polynucleotides, (iv) sequencing the polynucleotides in the oxidised, bisulfite treated population to produce a treated nucleotide sequence, and (v) identifying a residue in the treated nucleotide sequence which corresponds to a cytosine residue in the sample nucleotide sequence, wherein the presence of a cytosine in the treated nucleotide sequence is indicative that the cytosine residue in the sample nucleotide sequence is 5-methylcytosine.
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Specification