Patent ID: 6887664

Claim:
A method of nucleic acid amplification comprising the steps of: (a) providing a mixture comprising (i) a target nucleic acid comprising complementary first and second target strands in denatured form, and (ii) a first primer and a second primer that are complementary to sequences in the first and second target strands, respectively, that flank a target sequence of interest, such that the first primer has a Tm that is higher than the Tm of the second primer; (b) annealing the first primer to the first target strand of the denatured target nucleic acid at a first annealing temperature to form an annealed first primer, (c) extending the annealed first primer with primer extension reagents at an extension temperature or the first annealing temperature to generate a first extension product, wherein the primer extension reagents comprise a polymerase, nucleotide 5â€²-triphosphates, and a buffer, under conditions such that the second primer is not extended; and said second target strand remains single stranded, (d) after said extending, annealing a detectable probe to the single stranded second target strand at a probe hybridization temperature; (e) after said annealing of the detectable probe, annealing the second primer to the second strand of the denatured target nucleic acid at a second annealing temperature wherein the second annealing temperature is lower than the first annealing temperature; to form an annealed second primer, (f) extending the annealed second primer with primer extension reagents at the extension temperature to generate a second extension product, (g) denaturing the first and second extension products from the first and second target strands; and (h) repeating steps (b)-(g) for from 2-50 cycles.