Patent ID: 7208274

Claim:
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.