Detection of 5-hydroxymethylcytosine by glycosylation
Provided herein are methods and kits for detecting a modified cytosine.
1. A method for detecting 5-hydroxymethylcytosine in a mammalian nucleic acid, the method comprising:
contacting a sample comprising said mammalian nucleic acid with an enzyme that utilizes a labeled glucose or a labeled glucose-derivative donor substrate to add a labeled glucose molecule or a labeled glucose-derivative to a 5-hydroxymethylcytosine nucleic acid residue to generate a labeled glucosylated-5-hydroxymethylcytosine,
wherein the enzyme is a beta-glucosyltransferase and
wherein the labeled glucose or the labeled glucose-derivative donor substrate comprises a uridine diphosphate glucose (UDPG); and treating the contacted sample with a TET enzyme or a catalytically active fragment thereof comprising SEQ ID NO: 1.
2. The method of claim 1 , wherein the labeled glucose or the labeled glucose-derivative donor substrate is radioactively labeled.
3. The method of claim 1 , wherein the beta-glucosyltransferase comprises an Asp-Ile-Arg-Leu sequence.
4. The method of claim 1 , wherein the beta-glucosyltransferase is encoded by a T4 bacteriophage.
5. The method of claim 1 , wherein the mammalian nucleic acid comprises mammalian DNA.
6. The method of claim 1 , further comprising conducting a nucleic acid sequencing.
7. The method of claim 1 , wherein the mammalian nucleic acid comprises a 5-methylcytosine.
8. The method of claim 7 , further comprising distinguishing a 5-hydroxymethylcytosine from the 5-methylcytosine.