Composition and methods related to modification of 5-hydroxymethylcytosine (5-hmC)

• US 8,741,567 B2
• Filed: 04/27/2011
• Issued: 06/03/2014
• Est. Priority Date: 04/06/2010
• Status: Active Grant

• Current Assignee: University of Chicago
• Original Assignee: University of Chicago
• Inventors: He, Chuan, Song, Chunxiao
• Primary Examiner(s): Sisson, Bradley L
• Application Number:
  US13/095,505
• Publication Number:
  US 20110301045A1
• Time in Patent Office: 1,133 Days
• Field of Search: None
• US Class Current: 435/6.11
• CPC Class Codes: C12P 19/34 Polynucleotides, e.g. nucleic acids, oligoribonucleotides
• CPC Class Codes: C12Q 1/48 involving transferase
• CPC Class Codes: C12Q 1/6827 for detection of mutation or polymorphism
• CPC Class Codes: C12Q 1/6883 for diseases caused by alterations of genetic material
• CPC Class Codes: C12Q 2521/531 Glycosylase
• 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: C12Y 204/01028 Glucosyl-DNA beta-glucosyltransferase (2.4.1.28)

Abstract

The present invention relates generally to the field of molecular biology. More particularly, it concerns methods and compositions for detecting, evaluating, and/or mapping 5-hydroxymethyl-modified cytosine bases within a nucleic acid molecule.

Claims

1. A method for distinguishing by glycosylation with a modified glucose molecule 5-hydroxymethylcytosine from 5-methylcytosine in a nucleic acid molecule of an isolated sample comprising:

placing in a reaction mixture the nucleic acid molecule with β

-glucosyltransferase and a synthetically modified uridine diphosphoglucose (UDP-Glu) molecule comprising a modification moiety to glycosylate 5-hydroxymethyl-cytosines in the nucleic acid molecule with a modified glucose molecule, wherein the modification moiety comprises a linker, a detectable moiety, an isolation tag, a blocking moiety, or a functional moiety.

2. The method of claim 1, wherein the nucleic acid molecule is a nucleic acid molecule further purified from the isolated sample.

3. The method of claim 2, wherein the purified nucleic acid molecule is located on an array.

4. The method of claim 1, wherein the modification moiety comprises a linker.

5. The method of claim 4, wherein the modification moiety comprises an azide linker.

6. The method of claim 4, wherein the modification moiety comprises a thiol linker.

7. The method of claim 1, further comprising isolating the nucleic acid molecule from a cell of the isolated sample prior to glycosylation of the nucleic acid molecule.

8. The method of claim 1, further comprising reacting the modified glucose in the glycosylated nucleic acid molecule with a detectable or functional moiety.

9. The method of claim 8, wherein the glucose is modified with an azide or thiol linker.

10. The method of claim 1, further comprising placing the glycosylated nucleic acid molecule in a reaction mixture with an enzyme that is not β

-glucosyltransferase.

11. The method of claim 10, wherein the enzyme is a restriction enzyme or a polymerase.

12. The method of claim 11, wherein the restriction enzyme is methylation-insensitive.

13. The method of claim 11, wherein the enzyme is polymerase.

14. The method of claim 13, wherein the glycosylated nucleic acid molecule is placed in a reaction mixture with polymerase, at least one primer, and one or more nucleotides under conditions to allow polymerization of a transcript of the glycosylated nucleic acid molecule.

15. The method of claim 14, further comprising sequencing the polymerized transcript of the glycosylated nucleic acid molecule.

16. The method of claim 1, further comprising isolating the glycosylated nucleic acid molecule from the reaction mixture.

19. The method of claim 14, wherein sequencing further comprises a primer extension assay.

17. A method for mapping by glycosylation with a modified glucose molecule 5-hydroxymethylcytosine in a nucleic acid molecule of an isolated sample comprising:

placing in a reaction mixture the nucleic acid molecule with β

-glucosyltransferase and a synthetically modified uridine diphosphoglucose (UDP-Glu) molecule comprising a modification moiety to glycosylate 5-hydroxymethylcytosines in the nucleic acid molecule with the modified Glu molecule, wherein the modification moiety comprises a linker, a detectable moiety, an isolation tag, a blocking moiety, or a functional moiety; and

mapping the glycosylated 5-hydroxymethylcytosines in the glycosylated nucleic acid molecule by sequencing the glycosylated nucleic acid molecule.

18. The method of claim 17, wherein sequencing further comprises sequencing a control nucleic acid molecule.

20. The method of claim 17, further comprising placing the glycosylated nucleic acid molecule in a reaction mixture with an enzyme that is not β

-glucosyltransferase before sequencing the glycosylated nucleic acid molecule.

21. The method of claim 20, wherein the enzyme is a restriction enzyme or a polymerase.

22. The method of claim 21, wherein the restriction enzyme is methylation-insensitive.

23. The method of claim 21, wherein the enzyme is polymerase.

24. The method of claim 23, wherein the glycosylated nucleic acid molecule is placed in a reaction mixture with polymerase, at least one primer, and one or more nucleotides under conditions to allow polymerization of a transcript of the glycosylated nucleic acid molecule.

25. The method of claim 24, further comprising sequencing the polymerized transcript of the glycosylated nucleic acid molecule.