Touchdown polymerase chain reaction
- Touchdown polymerase chain reaction
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 (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.
References
:cite journal |author=Don R, Cox P, Wainwright B, Baker K, Mattick J |title='Touchdown' PCR to circumvent spurious priming during gene amplification |journal=Nucleic Acids Res |volume=19 |issue=14 |pages=4008 |year=1991 |pmid=1861999 |doi=10.1093/nar/19.14.4008:cite journal |author=Roux K |title=Using mismatched primer-template pairs in touchdown PCR |journal=Biotechniques |volume=16 |issue=5 |pages=812–4 |year=1994 |pmid=8068332:cite journal |author=Hecker K, Roux K |title=High and low annealing temperatures increase both specificity and yield in touchdown and stepdown PCR |journal=Biotechniques |volume=20 |issue=3 |pages=478–85 |year=1996 |pmid=8679209
Wikimedia Foundation.
2010.
Look at other dictionaries:
Polymerase chain reaction — PCR redirects here. For other uses, see PCR (disambiguation). A strip of eight PCR tubes, each containing a 100 μl reaction mixture The polymerase chain reaction (PCR) is a scientific technique in molecular biology to amplify a single or a… … Wikipedia
Polymerase chain reaction optimization — The polymerase chain reaction (PCR) is a commonly used molecular biology tool for amplifying DNA, and various techniques for PCR optimization have been developed by molecular biologists to improve PCR performance and minimize failure. Contents 1… … Wikipedia
Polymerase Chain Reaction — Réaction en chaîne par polymérase Pour les articles homonymes, voir PCR. Diagramme des 4 premiers cycles de la PCR La réaction en chaîne par polymérase (PCR est l abréviation an … Wikipédia en Français
Nested polymerase chain reaction — A diagram illustrating the method of nested PCR. Nested polymerase chain reaction is a modification of polymerase chain reaction intended to reduce the contamination in products due to the amplification of unexpected primer binding sites.… … Wikipedia
Multiplex polymerase chain reaction — (Multiplex PCR) is a modification of polymerase chain reaction in order to rapidly detect deletions or duplications in a large gene. This process amplifies genomic DNA samples using multiple primers and a temperature mediated DNA polymerase in a… … Wikipedia
Overlap extension polymerase chain reaction — This page assumes familiarity with the terms and components used in polymerase chain reaction (PCR) process. The overlap extension polymerase chain reaction (or OE PCR) is a variant of PCR. It is also referred to as Splicing by overlap extension… … Wikipedia
Touchdown (disambiguation) — A touchdown is the primary method of scoring in American and Canadian football.Touchdown may also refer to: * A similar method of scoring in touch football (rugby league) * Touchdown (song), by T.I. featuring Eminem * Touchdown (Bob James album)… … Wikipedia
Reaction En Chaine Par Polymerase — Réaction en chaîne par polymérase Pour les articles homonymes, voir PCR. Diagramme des 4 premiers cycles de la PCR La réaction en chaîne par polymérase (PCR est l abréviation an … Wikipédia en Français
Reaction en chaine par polymerase — Réaction en chaîne par polymérase Pour les articles homonymes, voir PCR. Diagramme des 4 premiers cycles de la PCR La réaction en chaîne par polymérase (PCR est l abréviation an … Wikipédia en Français
Réaction de polymérisation en chaine — Réaction en chaîne par polymérase Pour les articles homonymes, voir PCR. Diagramme des 4 premiers cycles de la PCR La réaction en chaîne par polymérase (PCR est l abréviation an … Wikipédia en Français
