Patent ID: 7915014
Filing Date: 2011-03-29
Classification: C12Q

Abstract:
1. A non-symmetric polymerase chain reaction (PCR) amplification and detection method comprising (a) thermally cycling a reaction mixture through repeated thermal cycles that include a primer-annealing temperature, said reaction mixture containing a first deoxyribonucleic acid (DNA) amplification target sequence; a first excess primer and a first limiting primer; dNTPs; a thermostable DNA polymerase that is capable of extending said primers when they are hybridized to said target sequence but that does not exhibit target-independent-probe-cleavage; and a low-temperature, fluorophore-labeled first hybridization probe that signals upon hybridization to a first amplicon strand that is the extension product of the first excess primer, that has a concentration-adjusted melting temperature at least 5° C. below the concentration-adjusted melting temperature of the first limiting primer, and that is a linear DNA oligonucleotide whose susceptibility to primer-independent 5′ exonuclease cleavage has been enhanced by a structural modification of said oligonucleotide; (b) hybridizing said first hybridization probe to said first amplicon strand at a first hybridization temperature after exhaustion of the limiting primer; and (c) detecting cleavage of said probe, wherein the structural modification of said first hybridization probe is selected from the group consisting of a 5′ terminal nucleotide that is not complementary to the corresponding nucleotide of said first amplicon strand and a 5′ terminal nucleotide having a label moiety linked to it by a chain comprised of at least three continuous methylene groups.