Hot start PCR

Hot Start PCR is a modified form of Polymerase chain reaction (PCR) which avoids non-specific amplification of DNA by inactivating the taq polymerase at lower temperature. In this dsDNA is denatured by heating the sample at its denaturing temperature and then the temperature is suddenly reduced to 55 degree C at which primer and Taq-polymerase is added, but here the difference arises i.e. specific antibodies are used to block this Taq-polymerase at annealing temperature. Now when the temperature raises for amplification to 72 degrees, the specific antibody detaches from Taq-polymerase and the amplification with greater specificity starts. In conventional PCR, the Taq DNA polymerase is active at room temperature and to a lesser degree, even on ice. In some instances, when all the reaction components are put together, nonspecific primer annealing can occur due to these low temperatures. This nonspecific annealed primer can then be extended by the Taq DNA polymerase, generating nonspecific products and lowering product yields. Hot Start PCR significantly reduces nonspecific priming, the formation of primer dimers, and often, increases product yields. In Hot Start Long and Accurate PCR, the impact on yield can be dramatic. Classic methods, while effective, involve additional handling and increased risk of contamination.

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a technique that reduces non-specific amplification during the initial set up stages of the PCR. It may be performed manually by heating the reaction components to the denaturation temperature (e.g., 95°C) before adding the polymerase.[28] Specialized enzyme systems have been developed that inhibit the polymerase's activity at ambient temperature, either by the binding of an antibody[11][29] or by the presence of covalently bound inhibitors that dissociate only after a high-temperature activation step. Hot-start/cold-finish PCR is achieved with new hybrid polymerases that are inactive at ambient temperature and are instantly activated at elongation temperature.