Patent ID: 7435541

Claim:
A method for determining the nucleotide present at a selected polymorphic site in a target nucleic acid molecule, comprising: (a) amplifying a portion of the target nucleic acid molecule comprising the selected polymorphic site using a first primer and a second primer, the second primer containing a recognition site for a first restriction enzyme and a recognition site for a second restriction enzyme, to generate amplification product containing a recognition site for the first restriction enzyme and a recognition site for the second restriction enzyme such that digestion of the amplification product with the first restriction enzyme and the second restriction enzyme generates a nucleic acid fragment containing the selected polymorphic site; (b) digesting the amplification product with the first restriction enzyme and the second restriction enzyme to generate a nucleic acid fragment having the selected polymorphic site within an overhanging end; (c) treating the nucleic acid fragment generated in step (b) to convert the overhanging end to a blunt end by extension of the recessed portion of the overhanging end; and (d) analyzing one or both strands of the nucleic acid fragment generated in step (c) to identify the nucleotide present at the selected polymorphic site.