Detection of 5-hydroxymethylcytosine by glycosylation

• US 9,816,986 B2
• Filed: 02/23/2017
• Issued: 11/14/2017
• Est. Priority Date: 09/26/2008
• Status: Active Grant

• Current Assignee: Children's Medical Center Corporation, United States Department Of Health And Human Services (Government of the United States of America)
• Original Assignee: Children's Medical Center Corporation
• Inventors: Rao, Anjana, Tahiliani, Mamta, Koh, Kian Peng, Agarwal, Suneet, Iyer, Aravind
• Primary Examiner(s): Dauner, Joseph G.
• Application Number:
  US15/440,408
• Publication Number:
  US 20170176421A1
• Time in Patent Office: 264 Days
• Field of Search: None
• 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 a modified cytosine.

Claims

1. A method for detecting 5-hydroxymethylcytosine in a mammalian nucleic acid, the method comprising:

contacting a sample comprising said mammalian nucleic acid with an enzyme that utilizes a labeled glucose or a labeled glucose-derivative donor substrate to add a labeled glucose molecule or a labeled glucose-derivative to a 5-hydroxymethylcytosine nucleic acid residue to generate a labeled glucosylated-5-hydroxymethylcytosine,

wherein the enzyme is a beta-glucosyltransferase and

wherein the labeled glucose or the labeled glucose-derivative donor substrate comprises a uridine diphosphate glucose (UDPG); and

treating the contacted sample with a TET enzyme or a catalytically active fragment thereof comprising SEQ ID NO;

1.

2. The method of claim 1, wherein the labeled glucose or the labeled glucose-derivative donor substrate is radioactively labeled.

3. The method of claim 1, wherein the beta-glucosyltransferase comprises an Asp-Ile-Arg-Leu sequence.

4. The method of claim 1, wherein the beta-glucosyltransferase is encoded by a T4 bacteriophage.

5. The method of claim 1, wherein the mammalian nucleic acid comprises mammalian DNA.

6. The method of claim 1, further comprising conducting a nucleic acid sequencing.

7. The method of claim 1, wherein the mammalian nucleic acid comprises a 5-methylcytosine.

8. The method of claim 7, further comprising distinguishing a 5-hydroxymethylcytosine from the 5-methylcytosine.