Effect of heat denaturation of target DNA on the PCR amplification

Abstract

The polymerase chain reaction (PCR) and amplification of specific regions of DNA in vitro is a widely used and powerful technique, and the optimization of conditions used to maximize PCR product yield has received much attention. We have shown that lengthy denaturation times of template DNA ranging from 1 to 7 min at pH 7.0–8.0, that are often employed prior to the start of a PCR reaction, result in marked degradation of the template. This can result in a significant reduction in the yield of PCR products larger than 500 bp, by up to 99%. This effect was demonstrated for both complex genomic template DNA, and also for a 2691-bp linear piece of template DNA using both a rapid hot-air thermocycler and a conventional block thermocycler. This decrease in product yield is likely due to the increased degradation of the template or target DNA as a result of pre-amplification denaturation (PAD). We therefore recommend that when amplifying larger pieces of DNA, the template DNA should not be exposed to PAD prior to a PCR reaction, irrespective of the starting pH of the template solution.