Composition and Methods Related to Modification of 5-Hydroxymethylcytosine (5-hmC)

• US 20110301045A1
• Filed: 04/27/2011
• Published: 12/08/2011
• Est. Priority Date: 04/06/2010
• Status: Active Grant

• Current Assignee: University of Chicago
• Original Assignee: University of Chicago Uchicago Tech
• Inventors: He, Chuan, Zhang, Liang, Song, Chunxiao
• Granted Patent: US 8,741,567 B2
• Time in Patent Office: Days
• US Class Current: 506/7
• 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 5-hydroxymethylcytosine from 5-methylcytosine in a nucleic acid molecule comprising incubating the nucleic acid molecule with β

-glucosyltransferase and a modified uridine diphosphoglucose (UDP-Glu) molecule to glycosylate 5-hydroxymethylcytosines in the nucleic acid molecule with a modified glucose molecule.

2. The method of claim 1, wherein the nucleic acid molecule is an isolated nucleic acid molecule.

8. The method of claim 2, wherein the isolated nucleic acid molecule is on an array.

10. The method of claim 1, wherein the modified UDP-Glu comprises a modification moiety.

11. The method of claim 10, wherein the modification moiety comprises an azide linker.

12. The method of claim 10, wherein the modification moiety comprises a thiol linker.

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

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

26. The method of claim 25, wherein the glucose is modified with an azide or thiol linker.

29. The method of claim 1, further comprising incubating the glycosylated nucleic acid molecule with an enzyme that is not β

-glucosyltransferase.

30. The method of claim 29, wherein the enzyme is a restriction enzyme or a polymerase.

31. The method of claim 30, wherein the restriction enzyme is methylation-insensitive.

32. The method of claim 30, wherein the enzyme is polymerase.

33. The method of claim 32, wherein the glycosylated nucleic acid molecule is incubated with polymerase, at least one primer, and one or more nucleotides under conditions to allow polymerization of the glycosylated nucleic acid.

34. The method of claim 33, further comprising sequencing the glycosylated nucleic acid molecule.

35. The method of claim 33, wherein the glycosylated nucleic acid is used in a primer extension assay.

36. The method of claim 1, further comprising isolating the glycosylated nucleic acid molecule.

3-7. -7. (canceled)

9. (canceled)

13-19. -19. (canceled)

21-24. -24. (canceled)

27. (canceled)

28. (canceled)

37-153. -153. (canceled)

154. A method for mapping 5-hydroxymethylcytosine in a nucleic acid molecule comprising incubating the nucleic acid molecule with β

-glucosyltransferase and a modified uridine diphosphoglucose (UDP-Glu) molecule to glycosylate 5-hydroxymethylcytosines in the nucleic acid molecule with the modified Glu molecule; and

mapping the 5-hydroxymethylcytosines in the nucleic acid molecule.

155. The method of claim 154, wherein the 5-hydroxymethylcytosines in the nucleic acid are mapped by sequencing the glycosylated nucleic acid and comparing the results to a control nucleic acid.

156. The method of claim 154, wherein the 5-hydroxymethylcytosines in the nucleic acid are mapped by subjecting the glycosylated nucleic acid to a primer extension assay and comparing the results to a control nucleic acid.

157-162. -162. (canceled)

163. A kit comprising purified β

-glucosyltransferase and a modified uridine diphosphoglucose (UDP-Glu) molecule.

164. The kit of claim 163, further comprising one or more nucleic acid buffers.

165. The kit of claim 163, further comprising a polymerase.

166. The kit of claim 165, further comprising a composition comprising nucleotides for use with the polymerase.

167. The kit of claim 165, further comprising a restriction enzyme.

168-171. -171. (canceled)