Detection of hydroxymethylcytosine bases

• US 10,351,898 B2
• Filed: 01/05/2014
• Issued: 07/16/2019
• 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
• Primary Examiner(s): Dauner, Joseph G.
• Application Number:
  US14/894,350
• Publication Number:
  US 20160115525A1
• Time in Patent Office: 2,018 Days
• Field of Search: None
• 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 along a DNA molecule, the method comprising:

(a) attaching to the DNA molecule a 5-hydroxymethyl-cytosine specific fluorescent labeling agent, wherein said 5-hydroxymethyl-cytosine specific fluorescent labeling agent comprises a fluorescent moiety covalently attached to a chemical moiety that binds said 5-hydroxymethyl-cytosine; and

(b) extending the DNA molecule,

wherein attaching said 5-hydroxymethyl-cytosine specific fluorescent labeling agent comprises;

enzymatically synthesizing UDP-6-N₃-Glucose;

incubating the DNA molecule with β

-glucosyltransferase and said UDP-6-N₃-Glucose, to thereby obtain a 5-hydroxymethyl-cytosine glycosylated by a 6-azidoglucose residue in the DNA molecule;

reacting said DNA molecule comprising said 5-hydroxymethyl-cytosine glycosylated by a 6-azidoglucose residue with a labeling agent derivatized by a reactive group which is chemically compatible with the azide group of said 6-azidoglucose residue.

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

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

4. The method of claim 1, further comprising attaching to the DNA molecule an additional labeling agent distinct of said 5-hydroxymethyl-cytosine 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 along said DNA molecule.

11. The method of claim 1, wherein said labeling agent is derivatized by an alkyne, such that attaching said labeling agent to said DNA molecule is effected by a click chemistry.

12. The method of claim 1, wherein said reacting is free of a copper catalyst.

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

14. The method of claim 13, wherein the DNA molecule is longer than 20 Kb.

15. The method of claim 13, wherein the DNA molecule is longer than 30 Kb.

16. The method of claim 13, wherein the DNA molecule is longer than 40 Kb.

17. The method of claim 1, wherein said enzymatically synthesizing UDP-6-N₃-Glucose comprises subjecting phosphorylated 6-azidoglucose to enzymatic catalysis by uridyltransferase in the presence of UTP.

18. The method of claim 17, wherein said enzymatically synthesizing UDP-6-N₃-Glucose further comprises subjecting 6-azidoglucose to enzymatic catalysis by kinase N-acetylhexoseamine-1-kinase in the presence of ATP, to thereby obtain said phosphorylated 6-azidoglucose.

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

(a) attaching a 5-hydroxymethyl-cytosine specific fluorescent labeling agent to the DNA molecule according to the method of claim 1; and

(b) subjecting the DNA molecule to an imaging method suitable for detecting said 5-hydroxymethyl-cytosine specific fluorescent labeling agent.

20. The method of claim 19, wherein said imaging method is a fluorescence imaging.

21. The method of claim 19, further comprising generating an optical image of the DNA molecule following said imaging.