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Methods for reducing the complexity of DNA sequences

  • US 6,287,825 B1
  • Filed: 09/17/1999
  • Issued: 09/11/2001
  • Est. Priority Date: 09/18/1998
  • Status: Expired due to Term
First Claim
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1. A method for reducing the complexity of DNA sequences in a DNA sample by generating non-overlapping subsets of sequences which comprises:

  • (a) digesting the DNA sample with a restriction enzyme to obtain DNA fragments;

    (b) ligating Y-shaped adapters to both ends of the fragments to obtain fragment-plus-adapter constructs;

    (c) removing excess adapters that are not ligated to the fragments;

    (d) amplifying the fragment-plus-adapter constructs by carrying out a PCR reaction with a first DNA primer that is complementary to one strand of the Y-shaped adapters and that, at the 3′

    -end of the primer, overlaps the DNA fragment by at least one base, and a second PCR primer that is complementary to at least a portion of the adapter region of the 3′

    -end of the DNA product formed by elongation of the first primer and which also anneals to the adapter region, and, at the 3′

    -end of the primer, overlaps the DNA fragment sequence by at least one base; and

    (e) identifying DNA products obtained in step (d) from each primer set as non-overlapping subsets, the primer sets differing by the 3′

    -nucleotides overlapping the fragment DNA, wherein each subset is less complex than the DNA sample.

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