Touchdown Polymerase Chain Reaction (PCR)

“Touchdown polymerase chain reaction (PCR)” is a method to decrease off-target priming and hence to increase the specificity of PCRs. In touchdown PCR the temperature selected for the annealing step is initially set 5°C–10°C higher than the calculated T m of the primers. Annealing under conditions of high stringency favors the formation of perfect primer–template hybrids. In subsequent cycles, the annealing temperature is gradually decreased by a small amount so that by the end of the PCR, the annealing temperature is 2°C–5°C below the calculated T m of the primers. By then, the target sequence will have undergone several cycles of geometric amplification and therefore becomes the dominant product of the PCR. To minimize mispriming during the early stages of the PCR, touchdown PCR should always be performed in conjunction with a hot start protocol. The use of touchdown PCR is essential when the sequence of the primer might not match that of the target—for example, if the sequence of the primer has been deduced from amino acid sequences, when the template DNA may contain several closely related targets, or when the target DNA is of a different species from that used to design the primers.