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Methods and compositions for generating and amplifying DNA libraries for sensitive detection and analysis of DNA methylation

  • US 20050202490A1
  • Filed: 03/03/2005
  • Published: 09/15/2005
  • Est. Priority Date: 03/08/2004
  • Status: Active Grant
First Claim
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1. A method of preparing a DNA molecule, comprising:

  • (a) providing a DNA molecule;

    (b) digesting the DNA molecule with at least one methylation-sensitive restriction enzyme and, optionally, an additional nuclease, to provide digested DNA molecules;

    (c) incorporating a nucleic acid molecule into at least some of the digested DNA molecules to provide first modified DNA molecules, by one of the following;

    (1) incorporating at least one primer from a plurality of primers, said primers comprising a 5′

    constant sequence and a 3′

    variable sequence that is substantially non-self-complementary and substantially non-complementary to other primers in the plurality;

    or (2) incorporating an oligonucleotide comprising an inverted repeat and a loop, under conditions wherein the oligonucleotide becomes blunt-end ligated to one strand of the molecule, thereby producing an oligonucleotide-linked molecule comprising a nick having a 3′

    hydroxyl group, wherein there is polymerization from the 3′

    hydroxyl group of at least part of the oligonucleotide-linked molecule; and

    (d) amplifying one or more of the first modified DNA molecules to provide amplified first modified DNA molecules.

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