What is touchdown PCR and when is it used?

Touchdown PCR starts with annealing temperature above primer Tm, then decreases incrementally (0.5-1C per cycle) until reaching optimal Tm for remaining cycles. Early high-stringency cycles favor perfectly matched primer-template binding, enriching specific products before non-specific amplification can occur. This approach improves specificity without extensive optimization, is useful for primers with different Tm values, helps with GC-rich or difficult templates, and can reduce primer-dimer formation. It is particularly valuable for multiplex PCR, degenerate primers, and when specificity problems persist despite primer redesign. Cycle parameters might be: 68C to 58C over 10 cycles, then 30 cycles at 58C.