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Detection of hydroxymethylcytosine bases

  • US 10,351,898 B2
  • Filed: 01/05/2014
  • Issued: 07/16/2019
  • Est. Priority Date: 05/28/2013
  • Status: Active Grant
First Claim
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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-N3-Glucose;

    incubating the DNA molecule with β

    -glucosyltransferase and said UDP-6-N3-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.

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