DETECTING 5-HYDROXYMETHYLATED CYTOSINE

• US 20180119225A1
• Filed: 02/23/2017
• Published: 05/03/2018
• Est. Priority Date: 09/26/2008
• Status: Abandoned Application

• Current Assignee: Children's Medical Center Corporation, United States Department Of Health And Human Services
• Original Assignee: Children's Medical Center Corporation, The United States of America As Represented By The Secretary of Agriculture
• Inventors: Rao, Anjana, Tahiliani, Mamta, Koh, Kian Peng, Agarwal, Suneet, Iyer, Aravind
• Application Number:
  US15/440,822
• Publication Number:
  US 20180119225A1
• Time in Patent Office: Days
• CPC Class Codes: C12N 2501/70 Enzymes
• CPC Class Codes: C12N 2501/71 Oxidoreductases (EC 1.)
• CPC Class Codes: C12N 2501/724 Glycosyltransferases (EC 2.4.)
• CPC Class Codes: C12N 9/0071 acting on paired donors with incorporation of molecular oxygen (1.14)
• CPC Class Codes: C12N 9/1007 Methyltransferases (general) (2.1.1.)
• CPC Class Codes: C12Q 1/26 involving oxidoreductase
• 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 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2521/531 Glycosylase
• CPC Class Codes: C12Q 2522/10 Nucleic acid binding proteins
• CPC Class Codes: C12Q 2537/164 Methylation detection other then bisulfite or methylation sensitive restriction endonucleases
• CPC Class Codes: C12Q 2600/154 Methylation markers
• CPC Class Codes: G01N 2500/00 Screening for compounds of potential therapeutic value
• CPC Class Codes: G01N 33/5011 for testing antineoplastic activity
• CPC Class Codes: G01N 33/5308 for analytes not provided for elsewhere, e.g. nucleic acids, uric acid, worms, mites

Abstract

Provided herein are methods and kits for detecting 5-hydroxymethylated cytosine.

Claims

1. A process for detecting a 5-hydroxymethylated cytosine residue in a sample comprising a nucleic acid, the process comprising:

a) replicating said nucleic acid to produce a hemimethylated double stranded nucleic acid, wherein said hemimethylated double stranded nucleic acid comprises a daughter strand and a parent strand;

b) treating said hemimethylated double stranded nucleic acid with DNA methyltransferase (DNMT) 1 to generate a methyltransferase treated sample;

c) generating a bisulfite treated sample by contacting said methyltransferase treated sample with a composition comprising bisulfite;

d) sequencing said bisulfite treated sample; and

e) detecting uracil or thymine in said daughter strand and cytosine in said parent strand, wherein said detecting is indicative of a presence of the 5-hydroxymethylated cytosine residue in said sample.

3. The process of claim 1, wherein said replicating comprises amplifying said nucleic acid with a polymerase.

4. The process of claim 3, wherein a) and b) are performed multiple times.

5. The process of claim 1, wherein said replicating comprises employing a polymerase chain reaction.

9. The process of claim 1, further comprising comparing the presence of the 5-hydroxymethylated cytosine residue to a control.

13. The process of claim 1, wherein said nucleic acid comprises DNA.

15. The process of claim 1, wherein said nucleic acid is isolated.

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