METHODS FOR DETECTION OF NUCLEOTIDE MODIFICATION

• US 20160258014A1
• Filed: 03/21/2016
• Published: 09/08/2016
• Est. Priority Date: 07/29/2011
• Status: Active Grant

• Current Assignee: Biomodal Limited
• Original Assignee: Cambridge Epigenetix Limited
• Inventors: Booth, Michael John, Balasubramanian, Shankar
• Granted Patent: US 10,428,381 B2
• Time in Patent Office: Days
• US Class Current: 1/1
• 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

1-32. -32. (Canceled)

33. A method of modifying 5-hydroxymethylcytosine residues in a sample nucleotide sequence comprising the step of oxidizing said sample nucleotide sequence, thereby selectively oxidising said 5-hydroxymethylcytosine to 5-formylcytosine residues, wherein the oxidation is carried out using a metal oxide.

34. The method according to claim 33, further comprising multiple steps of oxidation of the sample nucleotide sequence.

35. The method according to claim 33, wherein the metal oxide is a perruthenate oxidising agent.

36. The method according to claim 35 wherein the metal oxide is KRuO₄.

37. The method according to claim 33, wherein the sample nucleotide sequence is genomic DNA.

38. The method according to claim 37, wherein the genomic DNA is mammalian genomic DNA.

39. The method according to claim 33, wherein the sample nucleotide sequence is RNA.

40. The method according to claim 39, wherein the RNA is genomic RNA, mRNA, tRNA, rRNA or non-coding RNA.

41. The method according to claim 40, wherein the genomic RNA is mammalian, plant or viral genomic RNA.

42. The method according to claim 33, wherein the sample nucleotide sequence is immobilised.

43. The method according to claim 33, wherein the location of the 5-hydroxymethylcytosine residues is identified.

44. The method according to claim 43, wherein the identification is sequencing the sample.

45. The method according to claim 44, wherein the sample is amplified before sequencing.

46. A kit for use in a method of modifying a hydroxymethylcytosine residue according to claim 33 comprising a metal oxide.

47. The kit according to claim 46, wherein the metal oxide is a perruthenate oxidising agent.

48. The kit according to claim 47, wherein the perruthenate oxidising agent is selected from potassium perruthenate (KRuO₄), tetraalkylammonium perruthenates, polymer-supported perruthenate (PSP), and tetraphenylphosphonium ruthenate.

49. The kit according to claim 48, wherein the metal oxide is potassium perruthenate (KRuO₄).

50. The kit according to claim 46, further comprising instructions for use in the method of identifying a hydroxymethylcytosine residue.