FAQ

KASP™ chemistry utilises a two-step touchdown PCR method, with the elongation and annealing steps incorporated into a single step. The temperature used for the annealing stage determines the specificity of the reaction and hence the ability of the primers to anneal to the DNA template. A touchdown PCR involves starting with a high annealing temperature and incrementally decreasing the annealing temperature each PCR cycle. The higher annealing temperatures in the early cycles of a touchdown ensure that only very specific base pairing will occur between the DNA and the primer, hence the first sequence to be amplified is most likely to be the sequence of interest.  The annealing temperature is gradually decreased to increase the efficiency of the reaction. The regions that were originally amplified during the highly specific early touchdown cycles will be further amplified and outcompete any non-specific amplification that may occur at the lower temperatures. The standard KASP thermal cycling protocol has 10 cycles of touchdown PCR (annealing 61°C to 55°C, decreasing 0.6°C per cycle), then 26 cycles of standard 2-step PCR at the lower annealing temperature (55°C).