Single-Step PCR Optimization Using Touchdown and Stepdown PCR Programming

Polymerase chain reaction (PCR) optimization and troubleshooting can consume considerable energy and resources because of the finicky and often unpredictable nature of the reactions. Small variations in any of the many variables in a given reaction can have a pronounced effect on the resultant amplicon profile. Reactions that are too stringent yield negligible product, and reactions that are not stringent enough yield artifactual amplicons. Variables include concentrations of Mg²⁺ , H⁺ , dNTPs, primers, and template, as well as cycling parameters. Regarding the latter, the value selected for the annealing temperature is most critical. Unfortunately, even with the most sophisticated algorithms (i.e., OLIGO) it is often difficult to predict the amplification optima a priori leaving no other choice but to employ empirical determination.