Patent ID: 7192700

Claim:
A method of performing a primer extension reaction, comprising: obtaining an amplicon having a sequence generated from a target nucleic acid and a sequence generated from a first strand amplification primer, by amplifying a target nucleic acid having a nucleotide sequence containing a variant nucleotide flanked by an invariant nucleotide, wherein a first strand amplification primer is employed that comprises a 5′ tag substantially incapable of hybridizing to the target nucleic acid under amplification conditions, and wherein the 5′ tag contains the same nucleotide bases in sequence as the variant nucleotide and the invariant nucleotide of the target nucleic acid, and employing a second strand amplification primer; employing the amplicon in a primer extension reaction wherein the identity of the variant nucleotide in the sequence generated from the target nucleic acid is determined by hybridizing a first identification primer immediately adjacent to the variant nucleotide in the sequence generated from the target nucleic acid; hybridizing a second identification primer immediately adjacent to the variant nucleotide in the sequence generated from the 5′ tag of the first strand amplification primer; extending the first and the second identification primers in the presence of one or more nucleotides and a polymerizing agent; determining the identity of the variant nucleotide generated from the target nucleic acid; and comparing extension product of the first identification primer and extension product of the second identification primer, thereby performing the primer extension reaction.