Patent ID: 7718374

Claim:
A method for distinguishing in a sample between wild-type, mutant and heterozygous target polynucleotides in a target polynucleotide sequence having at least two single nucleotide polymorphisms wherein a first polymorphism is to be distinguished and a second single nucleotide polymorphism is not distinguished, each of said first and second polymorphisms being in a probe region of said target polynucleotides, the method comprising: (a) contacting said sample containing said target polynucleotide sequence with a first probe and a second probe that each distinguish a first polymorphism of interest, wherein the first probe preferentially hybridizes to the wild-type target polynucleotide and the second probe preferentially hybridizes to the mutant target polynucleotide, each of the first and second probes having an independently selected formula: wherein M is a minor groove binder; the subscript t is 0 or 1; Q is a quencher; W is a linking group; K is a bond or a linking group; Fl is a fluorophore and each probe has a different fluorophore; and [A-B] n represents a nucleic acid oligomer having n units, wherein n is an integer of from 5 to 50; each A independently represents a nucleic acid backbone component selected from the group consisting of a sugar phosphate backbone, a modified sugar phosphate backbone, a locked nucleic acid backbone, a peptidic backbone or a variant thereof used in nucleic acid preparation; and each B independently represents a nucleic acid base, a modified base or a base analog and wherein the base at the site complementary to said second single nucleotide polymorphism, is a universal or promiscuous base, and the oligonucleotide portion has a sequence complementary to a portion of the target sequence being amplified, to provide a mixture; and (b) measuring the fluorescence produced on hybrid formation, whereby hybrid formation indicates the presence or absence of each of the wild-type, mutant and heterozygous target polynucleotides.