What is “Touchdown” PCR?
It is a method for increasing specificity of PCR reactions. Touchdown PCR uses a cycling program where the annealing temperature is gradually reduced e.g. 1-2 °C/every second cycle. The initial annealing temperature should be several degrees above the estimated Tm of the primers. Annealing temperature is then gradually decreased until it reaches the calculated annealing temperature of the primers or some degrees below. Amplification is then continued using this annealing temperature.
Touchdown PCR involves decreasing the annealling temperature by 1 degree C every second cycle to a ‘touchdown’ annealing temp which is then used for 10 or so cycles. It was originally intended to bypass more complicated optimization processes for determining optimal annealing temperatures. The idea is that any differences in Tm between correct and incorrect annealing gives a 2-fold difference in product amount per cycle (4-fold per degree C). You therefore enrich for the correct product over any incorrect products. Another use for this procedure is in determining DNA sequence for a known peptide sequence. The strategy here is to use two sets of degenerate primers that match potential coding sequences at the two ends of a peptide of known sequence. In practice, this requires that you know a stretch of peptide sequence of only 13 amino acids, with left and right primers of 18 nt (6 a.a.) and a space in between of one or more nt. Using these degenerate primers, you do a touchdown PCR. You