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Rapid analysis of variations in a genome

  • US 7,208,274 B2
  • Filed: 02/28/2003
  • Issued: 04/24/2007
  • Est. Priority Date: 03/01/2002
  • Status: Active Grant
First Claim
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1. A method for determining a sequence of alleles of a locus of interest, said method comprising:

  • (a) amplifying a locus of interest on a template DNA using a first and second primers, wherein the second primer contains a recognition site for a restriction enzyme that cuts DNA at a distance from the recognition site and digestion with the restriction enzyme generates a 5′

    overhang containing the locus of interest, and wherein the first primer contains a recognition site for a restriction enzyme that is different from the recognition site for the restriction enzyme on the second primer and contains a tag at the 5′

    end;

    (b) digesting the amplified DNA with the restriction enzyme that recognizes the recognition site on the second primer;

    (c) incorporating nucleotides into the digested DNA of (b), wherein;

    (i) a labeled nucleotide that terminates elongation, and is complementary to the locus of interest of an allele, is incorporated into the 5′

    overhang of said allele, and(ii) a nucleotide complementary to the locus of interest of a different allele is incorporated into the 5′

    overhang of said different allele, and said terminating nucleotide, which is complementary to a nucleotide in the 5′

    overhang of said different allele, is incorporated into the 5′

    overhang of said different allele;

    (d) digesting the DNA of (c) with the restriction enzyme that recognizes the recognition site on the first primer; and

    (e) determining the sequence of the alleles of the locus of interest by determining the sequence of the digested DNA of (d) containing the labeled nucleotide.

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