Patent ID: 6475729
Filing Date: 2002-11-05
Classification: C12Q

Abstract:
A method for the amplification of a nucleic acid comprising the steps of:A. heating a targeted double-stranded nucleic acid at a first temperature of about 85 to about 100Â° C. for about 1 to about 40 seconds to denature the strands of said nucleic acid, B. cooling said denatured strands to a second temperature over a time period of about 5 to about 20 seconds, C. in the presence of 1) a thermostable DNA polymerase present in an amount of at least 5 units/100 microliters of solution, 2) deoxyribonucleoside-5â€²-triphosphates present in amounts effective for DNA polymerization, and 3) a set of primers specific for said denatured strands, said primers being present in amounts effective for DNA polymerization, forming hybridized primer extension products of said primers and denatured strands by incubating said denatured strands at said second temperature for about 1 to about 80 seconds, said second temperature being in the range of about (Tmâˆ’15)Â° C. to about (Tm+5)Â° C. wherein Tm is the melting temperature of said denatured strands and said primers, and the difference &Dgr;T between said first and second temperatures being from about 5 to about 45Â° C., D. heating said hybridized primer extension products to said first temperature over a period of time of about 5 to about 20 seconds and keeping said products at said temperature for about 1 to about 40 seconds, and E. repeating steps B through D sequentially as a cycle at least once wherein each cycle of steps B through D is carried out within about 20 to about 120 seconds, provided that if the concentration of each of said primers is less than about 0.075 micromolar the time for each cycle of Steps B through D is at least about 60 seconds.