PCRDerived In Vitro Nucleic Acid Amplification Techniques Hot Start PCR

Hot-start PCR was first described in the literature in 1991 by Kary Mullis (Mullis, 1991), and practical applications were demonstrated in 1992 (Chou et al., 1992). Hot-start PCR techniques focus on the inhibition of DNA polymerase activity during reaction setup. By limiting polymerase activity prior to the elevated temperatures of PCR, nonspecific amplification is reduced and target yield is increased. This is accomplished by physically separating or chemically inactivating one or more of the reaction components until high temperature triggers mixing or reactivation to give a complete reaction mixture.

In manual hot-start PCR, reactions lacking one essential component (usually DNA polymerase) are prepared and held at a temperature above the threshold of nonspecific binding of primer to template. Just prior to cycling, the missing component is added to allow the reaction to take place at higher temperature. This procedure limits nonspecific annealing of the primers and generally improves yield of the desired amplicon. This manual method is tedious and ungainly, as the tubes must be kept at 95-100°C. At this temperature, tubes are uncomfortable to handle. The additional opening of tubes to add the final reagent increases the chances of introducing contamination or cross-contaminating tubes. To simplify the process, tubes can be placed in the prewarmed thermal cycler just before adding the last component.

Hot-start PCR is also accomplished by creating a physical barrier between the essential components, such as primers and template or enzyme and magnesium chloride. This barrier can be created by adding wax over an incomplete PCR reaction mixture in a tube (Bassam and Caetano-Anolles, 1993; Horton et al., 1994; Riol et al., 1994). The wax can be preformulated for PCR reactions or can be in bulk form, such as paraffin. The remaining PCR component(s) is placed on top of the wax layer. During the first denaturation step, the wax barrier melts and convection currents mix the essential PCR components.

Additional hot-start methods include chemically modified Taq DNA polymerase (Birch, 1996; Kebelmann-Betzing et al., 1998) and an antibody-inhibited Taq DNA polymerase. The antibody is directed against the active site of the enzyme, preventing DNA replication until the high temperature of the denaturation step disassociates the antibody (Kellog, 1994). These modified enzyme preparations require a longer initial denaturation step than standard Taq DNA polymerase. Wax preparations and modified Taq DNA polymerase are commercially available.

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