Touchdown polymerase chain reaction

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Touchdown polymerase chain reaction or touchdown style polymerase chain reaction is a method of polymerase chain reaction by which primers will avoid amplifying nonspecific sequence. The temperature at which primers anneal during a cycle of polymerase chain reaction 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 polymerase chain reaction 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 polymerase amplification.

The earliest steps of a touchdown polymerase chain reaction cycle have high annealing temperatures. For every subsequent 2 cycles, the annealing temperature is decreased by 1 degree Celsius. The primer will anneal at the highest temperature which is least-permissive of nonspecific that it is able to tolerate. 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 polymerase chain reaction can be performed upon the product to further amplify those fragments.

Touchdown polymerase chain reaction was first reported in Nucleic Acids Research [1] in "Touchdown' PCR to circumvent spurious priming during gene amplification" article[2].