Touchdown polymerase chain reaction
The touchdown polymerase chain reaction or touchdown style polymerase chain reaction is a method of polymerase chain reaction by which primers will avoid amplifying nonspecific sequences. The annealing temperature during a polymerase chain reaction determines the specificity of primer 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. The annealing temperature is decreased in increments for every subsequent set of cycles (the number of individual cycles and increments of temperature decrease is chosen by the experimenter). The primer will anneal at the highest temperature which is least-permissive of nonspecific binding 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 out compete the nonspecific sequences to which the primers may bind at those lower temperatures. If the primer initially (during the higher-temperature phases) binds to the sequence of interest, subsequent rounds of polymerase chain reaction can be performed upon the product to further amplify those fragments. Touchdown increases specificity of the reaction at higher temperatures and increases the efficiency towards the end by lowering the annealing temperature.
Notes and references
- Don R, Cox P, Wainwright B, Baker K, Mattick J (1991). "'Touchdown' PCR to circumvent spurious priming during gene amplification". Nucleic Acids Res 19 (14): 4008. doi:10.1093/nar/19.14.4008. PMC 328507. PMID 1861999.
- Roux K (1994). "Using mismatched primer-template pairs in touchdown PCR". Biotechniques 16 (5): 812–4. PMID 8068332.
- Hecker K, Roux K (1996). "High and low annealing temperatures increase both specificity and yield in touchdown and stepdown PCR". Biotechniques 20 (3): 478–85. PMID 8679209.