A common problem with the polymerase chain reaction is the formation of non-specific products, especially primer-dimers. These unwanted products not only interfere with generating the desired amplicons but also obscure analyses following the reaction. A major cause of this lack of specificity occurs during reaction set-up and before the initial denaturation step. At the lower, less stringent temperatures before denaturation, primers may bind to targets that are not completely homologous, such as other primers ((1). These non-specific hybrids may be extended by the polymerase, even at lower temperatures, which creates competing targets (e.g. primer-dimers) during subsequent cycles. Hot start PCR methods provide a solution to this lack of specificity by reducing or eliminating non-specific product formation before high-temperature cycling((2).
Current hot start methods have targeted the polymerase by muting its activity before the initial denaturation step, most commonly with a blocking antibody or chemical modification. USB has developed an alternative strategy, called primer sequestration, which uses a recombinant protein to bind primers at lower temperatures making them unavailable for extension by the polymerase†. This novel primer-sequestration technique effectively blocks non-specific product formation before thermal-cycling. Following the initial denaturation step, the protein is inactivated and the primers are free to participate in the amplification reaction. This hot start method enhances many complex PCR reactions by increasing both specificity and yield (see figure).
USB currently offers two hot start PCR products using the primer sequestration method, HotStart-IT™ Taq DNA Polymerase and HotStart-IT™ Taq Master Mix. HotStart-IT™ Polymerase (PN 71195) is provided with a reaction buffer and a separate MgCl2 solution. HotStart-IT™ Master Mix (PN 71196) combines all of the necessary reaction components for PCR, except for primers and template, in a 2X concentrate. The master mix provides maximum convenience and reduced experimental variability. Prior to thermal-cycling, the reaction components for both products may be assembled at room temperature or on ice, whichever is most convenient. No special modifications to standard thermal-cycling protocols are necessary, such as an extensive initial denaturation step used with chemically-modified hot start polymerases.
USB HotStart-IT PCR products have been tested and validated in both end-point and real-time PCR with complex templates such as human genomic DNA. Real-time PCR reactions may be performed with probes or with dyes such as SYBR® Green I. TA-and TOPO®-based cloning procedures from PCR products have also been validated. Simply use USB HotStart-IT products as a seamless replacement for standard or hot start PCR protocols and procedures. The primer sequestration method is an effective technique for increasing PCR specificity and yield.