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Molecular Biology / PCR / Variants of PCR / Touchdown PCR
Background: 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. 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 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.
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. 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
Berglund:Touchdown PCR
OpenWetWare
'Touchdown' PCR to circumvent spurious priming during gene amplification
Don RH et al. Nucleic Acids Res. 1991 Jul 25;19(14):4008.
Bio.net
Protocols for PCR and Touchdown PCR:
www.jcsg.org/prod/scripts/primer/pcr_protocol.doc
Combining Multiplex and
touchdown PCR for Microsatellite Analysis.
Springer Protocols
Touchdown' PCR to circumvent spurious priming during gene amplification.
Nucleic
Acid Res