Detection of hydroxymethylcytosine bases
Methodologies for labeling the epigenetic modification 5-hydroxymethylcytosine (5hmC) along a DNA molecule, and for determining a presence or a level of this epigenetic modification based on a ratio of fluorescence intensity of a labeled DNA sample to absorption intensity of the DNA sample at 260 nm are disclosed. Related compositions and reagents, and methods of preparing same are also disclosed.
1. A method of detecting 5-hydroxymethyl-cytosine in a DNA sample, the method comprising:
(a) reacting the DNA sample with a 5-hydroxymethyl-cytosine-specific fluorescent labeling agent under conditions which allow staining of the DNA sample with said 5-hydroxymethyl-cytosine-specific fluorescent labeling agent so as to obtain a 5-hydroxymethyl-cytosine-labeled DNA sample;
(b) measuring fluorescence intensity of said 5-hydroxymethyl-cytosine-labeled DNA sample (X) and absorption intensity of said 5-hydroxymethyl-cytosine-labeled DNA sample at 260 nm (Y), and (c) comparing a ratio between X to Y to a ratiometric calibration curve, to thereby determine a presence or a level of 5-hydroxymethyl-cytosine in the DNA sample.
2. The method of claim 1 , wherein said detecting is effected in a high throughput setting of at least 300 DNA samples.
3. The method of claim 1 , wherein said 5-hydroxymethyl-cytosine specific fluorescent labeling agent comprises a fluorescent moiety covalently attached to a chemical moiety that binds 5-hydroxymethyl-cytosine.
4. The method of claim 1 , not comprising subjecting DNA of said DNA sample or said 5-hydroxymethyl-cytosine-labeled DNA sample to fragmentation.
5. The method of claim 1 , wherein reacting the DNA sample with a 5-hydroxymethyl-cytosine-specific fluorescent labeling agent comprises:
reacting a fluorescent labeling agent derivatized by a second reactive group with a DNA molecule in which the 5-hydroxymethy-cytosines 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.
6. The method of claim 5 , further comprising glycosylating the 5-hydroxymethy-cytosines in the DNA molecule comprises incubating the DNA molecule with β-glucosyltransferase and a uridine diphosphoglucose derivatized by said first reactive group.
7. The method of claim 6 , wherein said uridine diphosphoglucose derivatized by said first reactive group is a UDP-6-N 3 -Glucose.
8. The method of claim 7 , wherein said UDP-6-N 3 -Glucose is synthesized chemically.
9. The method of claim 7 , wherein said UDP-6-N 3 -Glucose is synthesized enzymatically.
10. The method of claim 9 , wherein enzymatically synthesizing said UDP-6-N 3 -Glucose comprises subjecting phosphorylated 6-azidoglucose to enzymatic catalysis by uridyltransferase in the presence of UTP.
11. The method of claim 10 , wherein said enzymatically synthesizing said 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.
12. The method of claim 5 , wherein one of said first and second reactive groups is azide and the other is alkyne, such that said 5-hydroxymethyl-cytosine-labeled DNA sample is obtained by a click chemistry.
13. The method of claim 5 , wherein said reacting is free of a copper catalyst.
14. The method of claim 5 , wherein said first reactive group is azide.
15. The method of claim 1 , wherein said DNA sample comprises a genomic DNA molecule.
16. The method of claim 15 , wherein the genomic DNA molecule is longer than 20 Kb.
17. The method of claim 15 , wherein the genomic DNA molecule is longer than 30 Kb.
18. The method of claim 15 , wherein the genomic DNA molecule is longer than 40 Kb.
19. The method of claim 1 , wherein a concentration of DNA in said DNA sample and said 5-hydroxymethyl-cytosine-labeled DNA sample is in a range of from 1 to 350 ng/μl.