HIGH AND LOW ANNEALING TEMPERATURES INCREASE BOTH SPECIFICITY AND YIELD IN TOUCHDOWN AND STEPDOWN PCR

Touchdown (TD) PCR represents a versatile one-step procedure Sor optim izing PCRs even if the degree of primer-template complementary is not Sully known. The protocol relies on incremental annealing temperature decreases in progressive cycles designed to bracket the melting temper ature (T-m) of the reaction. Here we investigate the characteristics o f TD PCR that serve to minimize the need to optimize annealing tempera ture or buffer conditions and yet produce single strong target amplico ns. We demonstrate that priming initiates above the optimum annealing temperature; this helps to ensure a competitive advantage for the targ et amplicon. On the other hand as the cycling program progresses, anne aling temperatures well below the T-m can serve to significantly incre ase yields in reactions that would otherwise be marginal due to subopt imal buffer composition and yet do not promote spurious amplification. Modified forms of TD PCR, termed stepdown PCR, consisting of fewer bu t steeper incremental declines in annealing temperature, are also show n to be effective and can simplify thermal cycler programming.