Patent ID: 8735067

Claim:
An asymmetric PCR amplification method to produce single-stranded amplification products comprising amplification of a target nucleic acid sequence from a sample using primers and a DNA polymerase, wherein the method comprises the steps of: 1) pre-denaturing the target nucleic acid sequence; and 2) asymmetrically amplifying the target nucleic acid sequence by a PCR amplification reaction comprising i) a first phase of PCR amplification comprising one or more cycles of denaturation, primer annealing, and primer extension; and; ii) a second phase of PCR amplification comprising one or more cycles of denaturation, primer annealing, and primer extension, wherein the temperature for primer annealing and the temperature for primer extension in the second phase are the same, wherein the temperature for primer annealing in the second phase of PCR is higher than the temperature for primer annealing in the first phase of PCR, wherein the primers comprise a primer pair comprising a first primer and a second primer, wherein the first primer in the primer pair has at its 5′ end an oligonucleotide tail having a sequence that is unrelated to the target sequence to be amplified, and wherein the T m of the first primer is greater than the T m of the second primer, thereby producing single-stranded amplification products.