Methods for Detection of Nucleotide Modification

• US 20140178881A1
• Filed: 07/27/2012
• Published: 06/26/2014
• Est. Priority Date: 07/29/2011
• Status: Active Grant

• Current Assignee: Biomodal Limited
• Original Assignee: Cambridge Epigenetix Limited
• Inventors: Balasubramanian, Shankar, Booth, Michael John
• Granted Patent: US 9,290,807 B2
• Time in Patent Office: Days
• US Class Current: 435/6.11
• CPC Class Codes: C01G 55/002 Compounds containing, besides ruthenium, rhodium, palladium, osmium, iridium, or platinum, two or more other elements, with the exception of oxygen or hydrogen C01G55/007 takes precedence
• CPC Class Codes: C07H 1/00 Processes for the preparation of sugar derivatives
• CPC Class Codes: C07H 21/02 with ribosyl as saccharide radical
• CPC Class Codes: C07H 21/04 with deoxyribosyl as saccharide radical
• CPC Class Codes: C12Q 1/6806 Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6827 for detection of mutation or polymorphism
• CPC Class Codes: C12Q 1/6876 Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
• CPC Class Codes: C12Q 2523/115 oxidising agents
• CPC Class Codes: C12Q 2523/125 Bisulfite(s)
• CPC Class Codes: C12Q 2600/154 Methylation markers

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.

Claims

1. A method of identifying a modified cytosine residue in a sample nucleotide sequence comprising;

(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 the residue in the first and second nucleotide sequences which corresponds to a cytosine residue in the sample nucleotide sequence.

2. A method according to claim 1 wherein the residues identified in the first and second nucleotide sequences are indicative of a modified cytosine at the corresponding position in the sample nucleotide sequence.

3. A method according to claim 1 wherein the modified cytosine is 5-methylcytosine (5mC), 5-hydroxymethylcytosine (5hmC) or 5-formylcytosine (5fC).

4. A method according to claim 1 wherein the first portion of said population is oxidised in step ii).

5. A method according to claim 4 wherein said oxidation selectively oxidises 5-hydroxymethylcytosine residues.

6. A method according to claim 4 wherein step ii) comprises multiple steps of oxidation and purification of the first portion of polynucleotides.

7. A method according to claim 4 wherein the first portion is oxidised using a chemical oxidising agent.

8. A method according to claim 4 wherein the first portion is oxidised using a perruthenate oxidising agent.

9. A method according to claim 4 wherein the first portion is oxidised using KRuO₄.

10. A method according to claim 4 wherein identification of uracil at a position in the first nucleotide sequence which corresponds to a cytosine residue in the sample nucleotide sequence and cytosine at the same position in the second nucleotide sequence is indicative that the cytosine residue in the sample nucleotide sequence is 5-hydroxylmethylcytosine (5hmC).

11. A method according to claim 4 wherein identification of cytosine at a position in both the first and second nucleotide sequences which corresponds to a cytosine residue in the sample nucleotide sequence is indicative that the cytosine residue in the sample nucleotide sequence is 5-methylcytosine (5mC).

12. A method according to claim 4 wherein identification of cytosine at a position in the first nucleotide sequence which corresponds to a cytosine residue in the sample nucleotide sequence is indicative that the cytosine residue in the sample nucleotide sequence is 5-methylcytosine (5mC).

13. A method according to claim 4 comprising;

vi) reducing a third portion of the population of polynucleotides comprising the sample nucleotide sequence,

vii) treating the reduced third portion of the population with bisulfite,

viii) sequencing the polynucleotides in the third portion following steps vi) and vii) to produce a third nucleotide sequence, and;

ix) identifying the residue in the third nucleotide sequence which corresponds to a cytosine residue in the sample nucleotide sequence.

14. A method according to claim 13 wherein the residues identified in the first, second and third nucleotide sequences are indicative of a modified cytosine at the corresponding position in the sample nucleotide sequence.

15. A method according to claim 13 wherein identification of uracil at a position in the first and second nucleotide sequences which corresponds to a cytosine residue in the sample nucleotide sequence and a cytosine at this position in the third nucleotide sequence is indicative that the cytosine residue in the sample nucleotide sequence is 5-formylcytosine.

16. A method according to claim 1 wherein the first portion of said population is reduced in step ii).

17. A method according to claim 16 wherein the first portion of said population is reduced using NaBH₄.

18. A method according to claim 16 wherein identification of cytosine at a position in the first nucleotide sequence which corresponds to a cytosine in the sample nucleotide sequence and uracil at the same position in the second nucleotide sequence is indicative that the cytosine residue in the sample nucleotide sequence is 5-formylcytosine.

19. A method according to claim 16 wherein identification of uracil at a position in both the first and the second nucleotide sequence which corresponds to a cytosine in the sample nucleotide sequence is indicative that the cytosine residue in the sample nucleotide sequence is unmodified cytosine.

20. A method according to claim 13 comprising;

providing a fourth portion of the population of polynucleotides comprising the sample nucleotide sequence; and

,

sequencing the polynucleotides in the fourth portion to produce the sample nucleotide sequence.

22. A method according to claim 1 wherein the polynucleotides are genomic DNA.

23. A method according to claim 22 wherein the genomic DNA is mammalian genomic DNA.

24. A method according to claim 1 wherein the polynucleotides are RNA.

25. A method according to claim 24 wherein the RNA is genomic RNA, mRNA, tRNA, rRNA or non-coding RNA.

26. A method according to claim 25 wherein the genomic RNA is mammalian, plant or viral genomic RNA.

27. A method according to claim 20 wherein the population of polynucleotides or one or more of the first, second, third and fourth portions of the population are immobilised.

28. A method according to claim 20 wherein one or more of the first, second, third and fourth portions of the population are amplified before sequencing.

29. A method according to claim 28 wherein one or more of the first, second, third portions of the population are amplified following treatment with bisulfite.

30. A kit for use in a method of identifying a modified cytosine residue according to claim 1 comprising;

(i) a oxidising agent and/or a reducing agent; and

,

(ii) a bisulfite reagent.

31. A kit according to claim 30 wherein the oxidising agent is a perruthenate oxidising agent.

32. A kit according to claim 31 wherein the oxidising agent is potassium perruthenate (KRuO₄).

21. A method of identifying 5-methylcytosine in a sample nucleotide sequence comprising;

(i) providing a population of polynucleotides which comprise the sample nucleotide sequence,

(ii) oxidising said population,

(iii) treating the oxidised population with bisulfite,

(iv) sequencing the polynucleotides in the population following steps ii) and iii) to produce a treated nucleotide sequence, and;

(v) identifying the 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 (5mC).