Patent ID: 7319022

Claim:
A method for continuous monitoring of polynucleotide amplification, comprising: (a) combining a sample containing a target sequence, with one or more oligonucleotide primers complementary to regions of the target sequence, a polymerizing enzyme, nucleotide substrates, and an oligonucleotide conjugate having a formula: wherein MB is a minor groove binder, n is 1, one of Fl A and Fl B is a fluorophore and the other of Fl A and Fl B is a quencher, W is a trivalent linking group, ODN is an oligonucleotide or modified oligonucleotide, K is a linking group, wherein the ODN sequence overlaps from 1 to 7 bases with a primer sequence and the ODN 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.