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Improve Amplification Specificity with Hot Start PCR Enzyme


SureStart Taq DNA polymerase utilizes ultra-pure Taq2000 DNA polymerase

SureStart Taq DNA polymerase features a modified version of Taq2000 DNA polymerase with hot start capability. Using SureStart Taq, hot start is in- corporated into PCR protocols previously optimized with Taq DNA polymerase, with little or no modification of cycling parameters or reaction conditions. SureStart Taq can be used in a variety of amplification systems to improve specificity, yield, and amplification of difficult targets.

A 105 bp fragment of the glucocerebrosidase gene was amplified from human genomic DNA using SureStart Taq (lane 1), unmodified Taq (lane 2), a competitors antibody-based hot start Taq (lane 3), or a competitors modified Taq (lane 4) DNA polymerase preparation. PCR reactions were assembled at room temperature and employed 2.5 U of each enzyme and the recommended buffer. Identical temperature cycling parameters were used, except that a heat step 95C, 10 minute heat step was added to the beginning of the thermal cycling program for lanes 1 and 4.

Combining PCR reactants at low temperatures can lead to high background and low product yields in some amplification systems. Certain PCR enzymes exhibit significant polymerase activity at temperatures encountered during reaction set-up (4 to 25C) or while ramping up to stringent primer annealing temperatures. For example, Taq DNA polymerase exhibits 2 to 3% maximum activity at 25C (room temperature PCR set up) and 70% maximum activity at 50C (generally at or below the Tm of PCR primers)1. Non-specific primer annea
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