Patent ID: 7723038

Claim:
A method for continuous monitoring of nucleic acid amplification, said method comprising: (a) providing a mixture comprising a sample containing a target sequence, with one or more than one 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 0 or 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 bond or a linking group, and wherein the ODN sequence has an overlap of from 1 to 7 bases with at least one of said primer sequences and the ODN has a sequence complementary to a portion of the target sequence being amplified; (b) incubating the mixture under conditions favorable for polymerization with a polymerase with 5′-nuclease activity; and (c) continuously monitoring the amplification by monitoring the fluorescence produced upon conjugate hybridization to the amplified target and cleavage by 5′-nuclease activity.