Carl T Wittwer
University of Utah, United States of America
We experimentally dissected the kinetic requirements of PCR into the stages of DNA denaturation, primer annealing, and polymerase extension. A commercial capillary LightCycler™ was used to correlate PCR product length to required extension times. More accurate results for all stages were obtained by using a custom “extreme” temperature cycling instrument. Increased primer and polymerase concentrations were allowed to increase speed, and actual temperatures were measured in real time rather than programmed or predicted. Two stages were left long and not limiting, while the other was varied until Cqs increased. Under the conditions used, polymerase extension times depended on product length with about 1 s needed for every 100 bps. Cqs started to increase after 200-500 ms above the denaturation threshold, and after 300-1,000 ms below the annealing threshold. Temperature thresholds were set by including >98% of the derivative melting curve areas as experimentally determined by melting curves under PCR conditions. Progressing from rapid cycle PCR to extreme PCR has decreased cycling times by 10-60 fold. If temperatures are controlled accurately and flexibility in reagents is allowed, PCR of short products can be performed in less than 15 s. We also put PCR in context to other emerging methods and consider its relevance to the evolution of molecular diagnostics.
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