Touchdown PCR

The touchdown PCR is a modification of PCR in which the initial annealing temperature is higher than the optimal melting temperature (Tm) of the primers, and is gradually reduced over subsequent cycles until the Tm temperature (also known as "touchdown temperature") is reached. This type of PCR avoids the amplification of non-specific sequences since a gradual lowering of temperature to a more permissive annealing temperature during the course of cycling favors the amplification of the desired amplicon.

Figure 1. Standard touchdown PCR protocol. It includes two phases: (A) PCR starts using an annealing temperature of Tm + Y °C and then it gradually decreases 1 °C each new cycle (Y is normally equal to 10-15); (B) when Tm is reached, ca. 20-25 cycles are performed until the protocol ends.