Asymmetric pcr methods, primers and kits

A method for production of single stranded DNA amplicons via an asymmetric polymerase chain reaction (PCR), comprising the steps of:

• (a) subjecting an initial mixture to a first phase of thermal cycling under amplifying PCR conditions, said initial mixture comprising:
• a nucleic acid sample;
• (ii) an Asymmetric Primer Pair,
• (iii) a thermostable DNA polymerase, and
• (iv) PCR Reagents,
• wherein the first phase of thermal cycling comprises cycling through at least three temperatures, said three temperatures comprising:
• (1) a first temperature above the Tm of the target nucleic acid for denaturation, (2) a second temperature below the Tm of the Unextended Primer for primer annealing, and (3) a third temperature suitable for extension by the thermostable DNA polymerase,
• thereby producing an intermediate mixture, said intermediate mixture comprising double stranded DNA amplicons extended from both the Unextended Primer and the Extended Primer if the target nucleic acid is present in the sample;
• (b) subjecting the intermediate mixture to a second phase of thermal cycling, said second phase of thermal cycling comprising cycling through:
• (1) a fourth temperature above the Tm of the double stranded DNA amplicons for denaturation, (2) a fifth temperature that is (i) below the Tm of the Extended Primer, (ii) above the Tm of the Unextended Primer and (iii) suitable for extension by the thermostable DNA polymerase,
• thereby producing a final mixture, said final mixture comprising single stranded DNA amplicons if the target nucleic acid is present in the sample.