Patent ID: 6977162

Claim:
A method for determining a sequence of a locus of interest, said method comprising: (a) amplifying a locus of interest on a template DNA using a first and second primers, each of the first and second primers comprising a 3′ annealing region that anneals to the template DNA, wherein the second primer contains a recognition site for a restriction enzyme such that digestion with the restriction enzyme generates a 5′ overhang containing the locus of interest, and wherein the melting temperature of the 3′ annealing region of the first primer is greater than the melting temperature of the 3′ annealing region of the second primer, and further wherein the annealing temperature for cycle 1 of the amplification is at about the melting temperature of the 3′ annealing region of the second primer, the annealing temperature for cycle 2 of the amplification is at about the melting temperature of the 3′ annealing region of the first primer, and the annealing temperature for cycle 3 is at about the melting temperature of the entire second primer, and wherein the annealing temperatures for cycle 2 and cycle 3 are greater than the annealing temperature for cycle 1; (b) digesting the amplified DNA with the restriction enzyme that recognizes the recognition site on the second primer; (c) incorporating a nucleotide into the digested DNA of (b) by using the 5′ overhang containing the locus of interest as a template; and (d) determining the sequence of the locus of interest by determining the sequence of the DNA of (c) containing the incorporated nucleotide.