Detection of hydroxymethylcytosine bases
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.
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-N 3 -Glucose;
incubating the DNA molecule with β-glucosyltransferase and said UDP-6-N 3 -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-N 3 -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-N 3 -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.