The touchdown polymerase chain reaction or touchdown style polymerase chain reaction is a method of

polymerase chain reaction The polymerase chain reaction (PCR) is a method widely used to rapidly make millions to billions of copies (complete or partial) of a specific DNA sample, allowing scientists to take a very small sample of DNA and amplify it (or a part of it) ...

by which primers avoid amplifying nonspecific sequences. The annealing

temperature Temperature is a physical quantity that expresses quantitatively the perceptions of hotness and coldness. Temperature is measurement, measured with a thermometer. Thermometers are calibrated in various Conversion of units of temperature, temp ...

during a polymerase chain reaction determines the specificity of primer annealing. The

melting point The melting point (or, rarely, liquefaction point) of a substance is the temperature at which it changes state from solid to liquid. At the melting point the solid and liquid phase exist in equilibrium. The melting point of a substance depen ...

of the primer sets the upper limit on annealing temperature. At temperatures just above 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 Exponential may refer to any of several mathematical topics related to exponentiation, including: *Exponential function, also: **Matrix exponential, the matrix analogue to the above *Exponential decay, decrease at a rate proportional to value *Expo ...

nature of polymerase amplification.

Method

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 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 outcompete 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. From a mathematical point of view products of annealing at smaller temperatures are disadvantaged by

2^

for the first annealing in cycle

i

and the second one in cycle

j

for

i,j\in \mathbb N , i\geq j

References

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