By using a higher annealing temperature in the initial PCR cycles, touchdown PCR favors accumulation of amplicons for sequences with the highest primer-template complementarity, thereby enriching for the most specific amplicons. In subsequent cycles, the annealing temperature is slowly decreased until it reaches the calculated annealing temperature of the primers (Don 1991). The initial annealing temperature is set to several degrees above the estimated T m of the primers. Touchdown PCR increases specificity by using reaction conditions that gradually reduce the annealing temperature. To achieve higher specificity, heteroduplex formation should be minimized by increasing stringency (i.e., increasing the temperature) during the initial PCR cycles.
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