DETECTION OF HYDROXYMETHYLCYTOSINE BASES

• US 20160115525A1
• Filed: 01/05/2014
• Published: 04/28/2016
• Est. Priority Date: 05/28/2013
• Status: Active Grant

• Current Assignee: Ramot at Tel Aviv University Limited (Tel Aviv University)
• Original Assignee: Ramot at Tel Aviv University Limited (Tel Aviv University)
• Inventors: EBENSTEIN, Yuval, FRIDMAN, Micha
• Granted Patent: US 10,351,898 B2
• Time in Patent Office: Days
• US Class Current: 1/1
• CPC Class Codes: C12P 19/305 Pyrimidine nucleotides
• 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 2537/164 Methylation detection other then bisulfite or methylation sensitive restriction endonucleases
• CPC Class Codes: G01N 21/6428 Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" in vivo A61B5/00; immunoassay G01N33/53
• CPC Class Codes: G01N 21/6458 Fluorescence microscopy fluorescence microscopes per se G02B21/0076 and G02B21/16

Abstract

Methodologies for labeling the epigenetic modification 5-hydroxymethyl-cytosine (5hmC) along a DNA molecule, and for imaging this epigenetic modification along a DNA molecule are disclosed. Related compositions and reagents, and methods of preparing same are also disclosed.

Claims

1. A method of labeling the epigenetic modification 5-hydroxymethyl-cytosine (5hmC) along a DNA molecule:

(a) attaching to the DNA molecule a 5hmc specific labeling agent; and

(b) extending the DNA molecule.

2. The method of claim 1, wherein said extending is linearly extending.

3. The method of claim 1, wherein step (b) is effected following step (a).

4. The method of claim 1, further comprising attaching to the DNA molecule an additional labeling agent distinct of said 5hmc specific labeling agent.

5. The method of claim 4, wherein said additional labeling agent is a 5mc specific labeling agent.

6. The method of claim 4, wherein said additional labeling agent is an epigenetic modification specific labeling agent.

7. The method of claim 4, wherein said additional labeling agent is a non-epigenetic modification specific labeling agent.

8. The method of claim 1 not comprising subjecting the DNA molecule to fragmentation.

9. The method of claim 1, wherein said extending is effected by depositing the DNA molecule on a surface or extending the DNA molecule in a nanochannel.

10. The method of claim 1 further comprising identifying a position of said 5-hydroxymethyl-cytosine (5hmC) along said DNA molecule.

11. The method of claim 1, wherein attaching said labeling agent comprises:

reacting a labeling agent derivatized by a second reactive group with a DNA molecule in which the 5-hydroxymethylcytosines are glycosylated by a glucose molecule derivatized by a first reactive group,

wherein said first and second reactive groups are chemically compatible to one another.

12. The method of claim 11, wherein glycosylating the 5-hydroxymethylcytosines in the DNA molecule comprises incubating the DNA molecule with β

-glucosyltransferase and a uridine diphosphoglucose (UDP-Glu) derivatized by said first reactive group.

13. The method of claim 11, wherein one of said first and second reactive groups is azide and the other is alkyne, such that attaching said labeling agent to said DNA molecule is effected by a click chemistry.

14. The method of claim 11, wherein said reacting is free of a copper catalyst.

15. The method of claim 11, wherein said first reactive group is azide.

16. The method of claim 15, wherein said uridine diphosphoglucose (UDP-Glu) derivatized by said first reactive group is a UDP-6-N₃-Glucose.

17. The method of claim 16, wherein said UDP-6-N₃-Glucose is synthesized chemically.

18. The method of claim 16, wherein said UDP-6-N₃-Glucose is synthesized enzymatically.

19. The method of claim 1, wherein said labeling agent is a fluorescent labeling agent.

20. A method of in-situ imaging a DNA molecule, the method comprising:

(a) attaching a labeling agent to the DNA molecule according to the method of claim 11; and

(b) subjecting the DNA molecule to an imaging method suitable for detecting said labeling agent.

21. The method of claim 20, wherein said labeling agent is a fluorescent agent and said imaging method is a fluorescence imaging.

22. The method of claim 20, further comprising generating an optical image of the DNA molecule following said imaging.

34. The method of claim 1, wherein the DNA molecule is a genomic DNA molecule.

35. The method of claim 34, wherein the DNA molecule is longer than 20 Kb.

36. The method of claim 34, wherein the DNA molecule is longer than 30 Kb.

37. The method of claim 34, wherein the DNA molecule is longer than 40 Kb.

23. An extended DNA molecule comprising at least one 5hmc-specific labeling agent.

24. A DNA molecule comprising at least two different labeling agents, wherein a first labeling agent of said at least two different labels is a 5hmc-specific labeling agent.

41. A composition-of-matter comprising the DNA molecule of claim 24, wherein the DNA molecule is surface deposited or extended in a microchannel.

25-33. -33. (canceled)

38. A method of detecting 5-hydroxymethyl-cytosine (5hmC) in a DNA sample the method comprising:

(a) reacting the DNA sample with a 5hmc-specific fluorescent agent under conditions which allow staining of the DNA sample with said 5hmc-specific labeling agent so as to obtain a 5hmC-labeled DNA sample; and

(b) measuring fluorescence intensity of said 5hmC-labeled DNA sample (X) and adsorption intensity of the DNA, at 260 nm (Y), wherein a ratio between X to Y is indicative of presence or level of 5hmC in the DNA sample.

39. The method of claim 38, wherein said ratio is compared to a ratiometric calibration curve.

40. The method of claim 38, wherein said detecting is effected in a high throughput setting of at least 300 DNA samples.

42-45. -45. (canceled)