Patent ID: 7919244
Filing Date: 2011-04-05
Classification: C12Q

Abstract:
1. A method for detecting a target nucleic acid in a sample, comprising the following steps: (1) contacting a sample with a first oligonucleotide primer which hybridizes with said target nucleic acid to leave one or more non-complementary nucleotides unpaired at the 3′ end of said first oligonucleotide primer, wherein said first oligonucleotide primer contains a fluorophore quencher at its 5′-end and a fluorophore at its 3′-end, or a fluorophore quencher at its 3′-end and a fluorophore at its 5′-end, and wherein the 3′-nucleotide of said first oligonucleotide primer contains a free hydroxyl group; (2) contacting said sample with at least one additional primer, a mixture of nucleotides, and a polymerase enzyme having 3′-5′ nuclease activity; (3) subjecting the sample containing said first oligonucleotide primer, said mixture of nucleotides, said at least one additional primer, and said enzyme, to real-time PCR; and (4) determining whether a measurable signal is produced by said fluorophore during said real-time PCR; wherein when said target nucleic acid is present in said sample, said first oligonucleotide primer hybridizes with the target nucleic acid so that nucleic acid extension occurs simultaneously with cleavage of one or more of said non-complementary nucleotides by said enzyme, producing a measurable signal by said fluorophore.