Standard vs. Touchdown PCR

Polymerase chain reaction (PCR) has become one of the most standard tools throughout biological, medical, and clinical sciences, and advances in technology have increased its scope and usability in many fields. Some frustrations of the technique lie in regard to length of the process, consumption of resources, and in refining and troubleshooting the procedure when many off-target products are generated. The development of off-target products may often be caused by poor melting temperature (Tm) estimations along with improper annealing conditions. Because of which, touchdown PCR was developed to reduce mispriming and the production of nonspecific products.

In touchdown PCR, the setpoint temperature of the annealing step is initially set 5-10°C above the calculated Tm of the primers. These more stringent conditions allow the primers to favor the formation of perfect primer-template hybrids and reduce off-target binding, providing an empirical and simple solution to mispriming in traditional PCR. In following cycles, the annealing temperature gradually decreased in small increments, generally between 0.5-1°C per cycle, so that in the finishing stages of PCR, the annealing temperature is only 2-5°C below the Tm of the primers. At this point the template will have undergone several cycles of amplification and has become the dominant product in the PCR reaction.

To minimize mispriming it is recommended that touchdown PCR be performed in conjunction with a hot start protocol, where either an essential reaction component is withheld from the reaction mix prior to denaturing step or a reversible polymerase inhibitor is included, to allow for amplification of trickier templates.

Tools:

Temperature of Annealing Calculator

One of the largest benefits of touchdown PCR is that it offers a simple and rapid means to optimize PCR procedures, and increase specificity, sensitivity and yield, without the need for lengthy experimental optimization or the need to redesign primers. Touchdown PCR, and its sister technique stepdown PCR, have found wide applicability in reverse transcriptase-dependent PCR, as well as in the generation of cDNA libraries and single nucleotide polymorphism screening.

Touchdown PCR has been widely used for primer and template combinations that inherently require high annealing temperatures. It is also particularly useful for templates that are difficult to amplify, for example templates with extensive secondary structures, and ones that contain a high cytosine (C) and guanine (G) content or CpG islands. Touchdown PCR is essential when the sequence of the primer may not match the target exceptionally well or when the degree of identity between the primer and template is unknown. For example, if a DNA template contains several closely related targets, when members of a multigene family are amplified, or when the target DNA is of a different species than what was used in the primers (evolutionary PCR).

An inherent limitation of touchdown PCR is that it is not suitable for quantitative assessment of target concentration, which is ideally performed by optimized real-time PCR. It is also important to note that temperature is a key component in any successful PCR experiment, and temperatures that are too high can result in low product yields.

More Reading

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Comparing Hot Start vs. Standard PCR
Thermal Cycling Optimization

Products

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References

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Touchdown PCR for increased specificity and sensitivity in PCR amplification
Touchdown Polymerase Chain Reaction (PCR)
Optimization and troubleshooting in PCR