Composition and methods related to modification of 5-methylcytosine (5-mC)

• US 9,611,510 B2
• Filed: 04/06/2012
• Issued: 04/04/2017
• Est. Priority Date: 04/06/2011
• Status: Active Grant

• Current Assignee: University of Chicago
• Original Assignee: University of Chicago
• Inventors: Song, Chunxiao, Yu, Miao, He, Chuan, Zhang, Liang
• Primary Examiner(s): Dauner, Joseph G
• Application Number:
  US14/110,007
• Publication Number:
  US 20140322707A1
• Time in Patent Office: 1,824 Days
• Field of Search: None
• 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 2537/164 Methylation detection other then bisulfite or methylation sensitive restriction endonucleases

Abstract

The present invention relates to methods and compositions for detecting, evaluating, and/or mapping 5-methyl-modified and/or 5-hydroxylmethyl-modified cytosine bases within a nucleic acid molecule.

Claims

1. A method for detecting 5-methylcytosine in a nucleic acid comprising converting 5-methylcytosine to 5-carboxylcytosine in the nucleic acid using enzymatic modification by methylcytosine dioxygenase;

bisulfite sequencing the converted nucleic acid; and

detecting 5-methylcytosine in the nucleic acid at positions where 5-carboxylcytosine is identified by reading a thymine in the converted nucleic acid as compared to a cytosine in the positions of a control sequence, wherein the control sequence is the sequence of the nucleic acid when bisulfite sequenced but not converted.

2. The method of claim 1, wherein the methylcytosine dioxygenase is TET1, TET2, or TET3, or a homologue thereof.

3. The method of claim 1, wherein the methylcytosine dioxygenase comprises amino acids 1367-2039 of SEQ ID NO:

1.

4. The method of claim 1, further comprising modifying 5-hydroxymethylcytosine with a first detectable label or a first detectable functional group prior to converting 5-methylcytosine to 5-carboxylcytosine.

5. The method of claim 4, further comprising detecting 5-hydroxymethylcytosine in the nucleic acid.

6. The method of claim 1, wherein the nucleic acid is genomic DNA.

7. A method for distinguishing 5-hydroxymethylcytosine from 5-methylcytosine in a nucleic acid molecule comprising:

a) modifying 5-hydroxymethylcytosine in the nucleic acid molecule with a glucose or a modified glucose;

b) treating the modified nucleic acid molecule with methylcytosine dioxygenase under conditions to convert 5-methylcytosine to 5-carboxylcytosine;

c) then sequencing the treated nucleic acid using bisulfite; and

d) distinguishing 5-hydroxymethylcytosine from 5-methylcytosine in the bisulfite sequence by reading a cytosine for 5-hydroxymethylcytosine and a thymine for 5-methylcytosine.

8. The method of claim 7, wherein the methylcytosine dioxygenase is TET1, TET2, or TET3, or a homologue thereof.

9. The method of claim 7, wherein the 5-hydroxymethylcytosine is modified by a process comprising incubating the nucleic acid molecule with β

-glucosyltransferase and a glucose or modified glucose molecule.

10. The method of claim 7, wherein the nucleic acid molecule is genomic DNA.