Detection of hydroxymethylcytosine bases
Provided herein is a method of labeling the epigenetic modification 5 -hydroxymethyl-cytosine along a DNA molecule that involves attaching a 5 -hydroxymethyl-cytosine specific labeling agent to the DNA molecule, which is effected by enzymatically synthesizing UDP- 6 -N 3 -Glucose by subjecting 6 -azidoglucose to an enzymatic catalysis by N-acetylhexoseamine 1 -kinase (NahK) in the presence of ATP to thereby obtain a phosphorylated 6 -azidoglucose, followed by subjecting the phosphorylated 6 -azidoglucose to enzymatic catalysis by uridyltransferase (GlmU) in the presence of UTP to thereby obtain UDP- 6 -N 3 -Glucose, followed by incubating the DNA molecule with UDP- 6 -N 3 -Glucose and ß-glucosyltransferase, to thereby obtain a 5 -hydroxymethyl-cytosin e glycosylated by a 6 -azidoglucose residue in the DNA molecule, and reacting the modified DNA molecule with a labeling agent which is chemically compatible with the azide group of the 6 -azidoglucose residue.
1. A method of labeling the epigenetic modification 5-hydroxymethyl-cytosine along a DNA molecule, the method comprising attaching to said DNA molecule a 5-hydroxymethyl-cytosine specific labeling agent, wherein said attaching comprises:
subjecting 6-azidoglucose to an enzymatic catalysis by N-acetylhexoseamine 1-kinase (NahK) in the presence of ATP to thereby obtain a phosphorylated 6-azidoglucose;
subjecting said phosphorylated 6-azidoglucose to enzymatic catalysis by uridyltransferase (GlmU) in the presence of UTP to thereby obtain UDP-6-N 3 -Glucose;
said UDP-6-N3-Glucose and β-glucosyltransferase, to thereby obtain a 5-hydroxymethyl-cytosine glycosylated by a 6-azidoglucose residue in said DNA molecule; and
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 labeling agent derivatized by said reactive group is a fluorescent labeling agent.
3. The method of claim 1 , further comprising attaching to said DNA molecule an additional labeling agent distinct of said 5-hydroxymethyl-cytosine specific labeling agent.
4. The method of claim 3 , wherein said additional labeling agent is a 5-methyl-cytosine specific labeling agent.
5. The method of claim 3 , wherein said additional labeling agent is an epigenetic modification specific labeling agent.
6. The method of claim 3 , wherein said additional labeling agent is a non-epigenetic modification specific labeling agent.
7. The method of claim 6 , wherein said non-epigenetic modification specific labeling agent is a fluorescent agent which binds to nucleic acids.
8. The method of claim 1 , not comprising subjecting said DNA molecule to fragmentation.
9. The method of claim 1 , wherein said reactive group is an alkyne.
10. The method of claim 9 , wherein attaching said labeling agent to said DNA molecule is effected by a click chemistry.
11. The method of claim 1 , wherein said reacting is free of a copper catalyst.
12. The method of claim 1 , wherein said DNA molecule is a genomic DNA molecule.
13. The method of claim 12 , wherein said DNA molecule is longer than 20 Kb.
14. The method of claim 12 , wherein said DNA molecule is longer than 40 Kb.
15. The method of claim 1 , further comprising detecting the 5-hydroxymethyl-cytosine specific labeling agent.
16. The method of claim 15 , wherein said detecting is effected in a high throughput setting of at least 300 DNA samples.
17. A method of in-situ imaging a DNA molecule, the method comprising:
(a) attaching a 5-hydroxymethyl-cytosine specific labeling agent to said DNA molecule according to the method of claim 2 ; and
(b) subjecting said DNA molecule to an imaging method suitable for detecting said 5-hydroxymethyl-cytosine specific labeling agent.
18. The method of claim 17 , wherein said labeling agent is a fluorescent labeling agent said imaging method is a fluorescent imaging.
19. The method of claim 17 , further comprising generating an optical image of said DNA molecule following said imaging.