The kinetic requirements of extreme qPCR.

The kinetic requirements of quantitative PCR were experimentally dissected into the stages of DNA denaturation, primer annealing, and polymerase extension. The temperature/time conditions for 2 stages were kept optimal, while the other was limited until the amplification efficiency decreased as measured by an increase in quantification cycle (Cq). Extension was studied in a commercial capillary LightCycler®.