OXIDIZING AGENT FOR 5-HYDROXYMETHYLCYTOSINE AND METHOD FOR ANALYZING 5-HYDROXYMETHYLCYTOSINE

• US 20180251815A1
• Filed: 09/02/2016
• Published: 09/06/2018
• Est. Priority Date: 09/04/2015
• Status: Abandoned Application

• Current Assignee: University of Tokyo
• Original Assignee: University of Tokyo
• Inventors: OKAMOTO, Akimitsu, MATSUSHITA, Taku
• Application Number:
  US15/756,843
• Publication Number:
  US 20180251815A1
• Time in Patent Office: Days
• CPC Class Codes: C07D 207/46 with hetero atoms directly attached to the ring nitrogen atom
• CPC Class Codes: C07D 211/94 Oxygen atom, e.g. piperidine N-oxide
• CPC Class Codes: C07D 451/14 containing 9-azabicyclo [3.3.1] nonane ring systems, e.g. granatane, 2-aza-adamantane; Cyclic acetals thereof
• CPC Class Codes: C12N 15/09 Recombinant DNA-technology
• 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 1/6858 Allele-specific amplification
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2523/113 Denaturating agents
• CPC Class Codes: C12Q 2523/115 oxidising agents
• CPC Class Codes: C12Q 2523/125 Bisulfite(s)
• CPC Class Codes: C12Q 2537/164 Methylation detection other then bisulfite or methylation sensitive restriction endonucleases

Abstract

The purpose of the present invention is to develop and provide a less expensive and novel oxidizing agent for 5-hydroxymethylcytosine, which can selectively oxidize 5-hydroxymethylcytosine on DNA into 5-formylcytosine while preventing the DNA structure from destabilization and suppressing side reactions, and a method for detecting a demethylation site at a high accuracy in demethylation of DNA with the use of the aforesaid oxidizing agent.

Provided is an oxidizing agent for 5-hydroxymethylcytosine that comprises a nitroxyl radical molecule and a copper salt or a copper complex, and/or a nitroxyl radical molecule-copper complex.

Claims

1. An oxidizing agent for 5-hydroxymethylcytosine, selected from the group consisting of:

(a) a nitroxyl radical molecule and a copper salt or a copper complex;

(b) a nitroxyl radical molecule-copper complex;

and combinations (a) and (b).

2. The oxidizing agent for 5-hydroxymethylcytosine according to claim 1, further comprising (c):

(c) one or more reaction promoters selected from the group consisting of pyridine, bipyridine, phenanthroline, ethylenediamine, propanediamine, imidazole, and derivatives thereof.

3. The oxidizing agent for 5-hydroxymethylcytosine according to claim 1, wherein the nitroxyl radical molecule is 2,2,6,6-tetramethylpiperidine-1-oxyl, 3-carbamoyl-2,2,5,5-tetramethyl-3-pyrrolin-1-yloxy, 2-azaadamantane-N-oxyl, 9-azabicyclo[3.3.1]nonane-N′

-oxyl, or a derivative thereof.

4. A DNA demethylation analysis reagent, comprising the oxidizing agent for 5-hydroxymethylcytosine according to claim 1.

5. The DNA demethylation analysis reagent according to claim 4, further comprising a bisulfite.

6. A DNA demethylation analysis kit, comprising the DNA demethylation analysis reagent according to claim 5.

7. A method of oxidizing 5-hydroxymethylcytosine, comprising

mixing a test substance that may contain 5-hydroxymethylcytosine with the oxidizing agent for 5-hydroxymethylcytosine according to claim 1 in a reaction solution, and

incubating the reaction solution at a temperature of 4°

C. to 90°

C. for 1 to 100 hours to oxidize a hydroxy group in an allyl alcohol structure contained in 5-hydroxymethylcytosine.

8. A method of analyzing 5-hydroxymethylcytosine, comprising

mixing DNA with the oxidizing agent for 5-hydroxymethylcytosine according to claim 1 in a reaction solution,

incubating the reaction solution at a temperature of 4°

C. to 90°

C. for 1 to 100 hours to oxidize a hydroxy group in an allyl alcohol structure constituting 5-hydroxymethylcytosine, and

detecting 5-formylcytosine generated in the oxidation step.

9. The method according to claim 8, comprising cleaving a hydrogen bond contained in DNA, before performing the mixing step.

10. The method according to claim 9, comprising extracting DNA from a biological sample, before performing the hydrogen bond cleavage step.

11. The method according to claim 9, wherein in the hydrogen bond cleavage step, DNA is dissolved in a basic solution to cleave a hydrogen bond.

12. The method according to claim 8, wherein, in the detection step, 5-formylcytosine is detected by a bisulfite sequencing method.