Patent ID: 7348146

Claim:
A method for continuous monitoring of polynucleotide amplification of a target nucleic acid sequence having at least two single nucleotide polymorphisms wherein a first single nucleotide polymorphism is to be distinguished and a second single nucleotide polymorphism is not distinguished, each of said polymorphisms being in a probe region of said target nucleic acid, the method comprising: (a) combining a sample containing said target nucleic acid with one or more oligonucleotide primers adjacent to or overlapping with said probe region of the target sequence, a polymerizing enzyme, nucleotide substrates, and an oligonucleotide conjugate having a 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 [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, and a peptidic backbone; 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 (indiscriminative) base and the oligonucleotide portion has a sequence complementary to a portion of the target sequence being amplified, to provide a mixture; (b) incubating the mixture under conditions favorable for polymerization; and (c) continuously monitoring the amplification by monitoring the fluorescence produced upon conjugate hybridization to the amplified target.