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Greater Amplification Specificity with New Hot Start PCR Enzyme

sequencing of specific PCR products. Moreover, synthesis of misprimed products diverts reaction components (dNTPs, primers) away from the production of desired products, which leads to poor amplicon yield.

Various techniques have been developed to reduce nonspecific primer extension by Taq DNA polymerase at low temperatures. One method involves replacing Taq with Stratagenes Pfu DNA polymerase, which is less prone to zero-cycle artifacts due to lower activity at nonrestrictive temperatures (e.g., 8% maximum activity at 50C).1 Another method for reducing nonspecific amplification employs Stratagenes Perfect Match PCR enhancer, which decreases background by destabilizing mismatched primer-template complexes.2,3 In addition, various hot start procedures have been developed to prevent nonspecific primer extension at low temperatures. In these procedures, a critical PCR component (e.g., dNTPs, polymerase, or Mg2+) is withheld until stringent primer annealing temperatures are reached.4-7 The missing component can be added manually at the initial denaturation step4,5 or withheld by a physical wax barrier until temperatures sufficient to melt wax are achieved.6

More recently, reversibly inactivated DNA polymerases have been used to provide hot start. Heat-reversible inhibition is accomplished using neutralizing antibodies,8 oligonucleotide inhibitors,9 or enzyme modification.10 DNA polymerases reversibly inactivated by antibodies regain activity upon denaturation of the antibody (1 minute at 80C 8) durin
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