Hot Start

Regular DNA polymerases exhibit some activity below their optimal temperature, which for Taq polymerase is 72°C. Thus, primers can anneal non-specifically to the template DNA at room temperature when PCR reactions are being set-up and non-specific products may result. It is also possible at a low temperature for the primers to bind to each other creating products called 'primer dimers.' These are a particular problem because their small size relative to the PCR products means that they will be preferentially amplified.

Once low-temperature non-specific priming occurs, these undesirable products will be efficiently amplified throughout the remaining PCR cycles. Because the polymerase is busy amplifying these competing products, the target DNA region will be amplified less efficiently. If this happens, you will get less of what you are looking for and you may not have enough specific DNA to run your other tests.

Low-temperature mispriming can be avoided by initiating PCR at an elevated temperature, a process usually referred to as 'hot start' PCR. Hot start PCR may be performed by introducing a critical reaction component, such as the poly-merase, after the temperature of the sample has been raised above the desired annealing temperature (e.g., 60°C). This minimizes the possibility of misprim-ing and misextension events by not having the polymerase present during reaction setup. However, this approach is cumbersome and time-consuming when working with large numbers of samples. Perhaps a more important disadvantage is the fact that the sample tubes must be opened at the thermal cycler to introduce the essential component, which gives rise to a greater opportunity for cross-contamination between samples. As will be discussed in the next section, a modified form of Taq DNA polymerase has been developed that requires thermal activation and thus enables a closed-tube hot start PCR.

This enzyme, named AmpliTaq Gold, has greatly benefited the specificity of PCR amplifications.

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