Method for quantifying 5-hydroxymethylcytosine

A method for detecting or determining the presence or amount of 5-methylcytosine residues (5-mC) in a DNA, the method comprising:

• (a) splitting the DNA into a first sample and a second sample;
• (b) contacting the first sample with a 5-mC hydroxylase to form a first reaction mixture, wherein all 5-mC residues are hydroxylated to form 5-hmC;
• (c) contacting the first reaction mixture with a β-Glucosyltransferase (β-GT) and uridine diphospho-glucose (UDP-glucose) to form a second reaction mixture, wherein all 5-hmC residues are glucosylated;
• (d) contacting the second reaction mixture with ADP, a uridine/cytidine monophosphate kinase (CMK), and a buffer to form a second reaction mixture, wherein the buffer comprises a bioluminescent enzyme and a luciferin substrate;
• (e) detecting luminescence in the reaction mixture, thereby detecting or determining the presence or amount of 5-hydroxymethylcytosine (5-hmC) residues in the DNA in the first sample;
• (f) contacting the second sample with a β-Glucosyltransferase (β-GT) and uridine diphospho-glucose (UDP-glucose) to form a first reaction mixture, wherein 5-hmC residues are glucosylated;
• (g) contacting the first reaction mixture with ADP, a uridine/cytidine monophosphate kinase (CMK), and a buffer to form a second reaction mixture, wherein the buffer comprises a bioluminescent enzyme, and a luciferin substrate;
• (h) detecting luminescence in the reaction mixture, thereby detecting or determining the presence or amount of 5-hydroxymethylcytosine (5-hmC) residues in the DNA in the second sample;
• subtracting the number of relative light units in the second sample from the number of relative light units in the first sample, wherein the difference in luminescence between the second sample and the first sample corresponds to the presence or an amount of 5-methylcytosine (5-mC) residues in the DNA.

A method for determining whether a compound modulates the hydroxylation of 5-mC in a DNA, the method comprising:

• (a) splitting the DNA into a first sample and a second sample;
• (b) contacting the first sample with a 5-mC hydroxylase and a vehicle to form a first reaction mixture;
• (c) contacting the first reaction mixture with a β-GT and UDP-glucose to form a second reaction mixture, wherein all 5-hmC residues are glucosylated;
• (d) contacting the second reaction mixture with ADP, a CMK, and a buffer to form a second reaction mixture, wherein the buffer comprises a bioluminescent enzyme and a luciferin substrate;
• (e) detecting luminescence in the reaction mixture, thereby detecting or determining the presence or amount of 5-hmC residues in the DNA in the first sample;
• (f) contacting the second sample with a 5-mC hydroxylase and a compound to form a first reaction mixture;
• (g) contacting the first reaction mixture with a β-GT and a UDP-glucose to form a second reaction mixture, wherein all 5-hmC residues are glucosylated;
• (h) contacting the second reaction mixture with ADP, a CMK, and a buffer to form a second reaction mixture, wherein the buffer comprises a bioluminescent enzyme and a luciferin substrate;
• (i) detecting luminescence in the reaction mixture, thereby detecting or determining the presence or amount of 5-hmC residues in the DNA in the second sample;
• (j) wherein, if there is a difference in luminescence between (i) and (e), then the compound modulates the hydroxylation of 5-mC in DNA.