Touchdown PCR

Touchdown PCR or Touchdown style PCR is a method of PCR (polymerase chain reaction) by which degenerate primers will avoid amplifying nonspecific sequence. The temperature at which primers anneal during a cycle of PCR determines the specificity of annealing. The melting point of the primer sets the upper limit on annealing temperature. At temperatures just below this point, only very specific base pairing between the primer and the template will occur. At lower temperatures, the primers bind less specifically. Nonspecific primer binding obscures PCR results, as the nonspecific sequences to which primers anneal in early steps of amplification will "swamp out" any specific sequences because of the exponential nature of PCR amplification.

The earliest steps of a Touchdown PCR cycle have high annealing temperatures. For every subsequent cycle, the annealing temperature is decreased by 1 degree Celsius. The primer will anneal at the highest (and therefore, least-permissive of nonspecific binding) temperature at which it is able. Thus, the first sequence amplified is the one between the regions of greatest primer specificity; it is most likely that this is the sequence of interest. These fragments will be further amplified during subsequent rounds at lower temperatures, and will swamp out the nonspecific sequences to which the primers will bind at those lower temperatures. If the primer initially binds to the sequence of interest at a low temperature, subsequent rounds of PCR can be performed upon the product to further amplify those fragments.

Touchdown PCR was first reported in Nucleic Acids Research.

Its original description can be found in the following article:

RH Don, PT Cox, BJ Wainwright, K Baker, and JS Mattick. "Touchdown' PCR to circumvent spurious priming during gene amplification." Nucleic Acids Res. 1991 19: 4008