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HotMasterAn innovative Hot Start/Cold Stop technology for better ,,, PCR* results

George Halley and Vincent Prezioso, PhD
George Halley, Eppendorf 5 Prime, Inc., Boulder, CO, USA
Vincent Prezioso, Brinkmann Instruments, BioSytems Application Lab, Westbury, NY

Abstract

Hot Start DNA polymerases have become very popular because of their convenience and their ability to reduce nonspecific amplification. Typical Hot Start enzymes utilize chemical or antibody mediated inhibition, which impart disadvantages such as a long initial denaturing step or excessive protein contamination. In contrast, Eppendorfs HotMaster uses an innovative temperature-dependent inhibitor to achieve Hot Start activity. In addition to increasing specificity and sensitivity in PCR reactions, HotMaster offers several advantages not available in other Hot Start DNA polymerases: the Mg++ concentration is pre-optimized and self adjusting; no heat activation step is required, the polymerase is inhibited in a temperature-dependent manner during each annealing step of the PCR (at temperatures below ~60C); and larger target sizes can be amplified.

Introduction

Hot Start PCR was developed as a method to minimize the deleterious effects of mispriming at lower temperatures during PCR. In a PCR reaction, even short incubations at temperatures below the optimum annealing temperature for a particular set of primers can result in mispriming, elongation and the subsequent formation of spurious bands. The Hot Start technique involves inactivating (or leaving out) one critical component of the PCR reaction until the temperature has risen above this optimal annealing temperature. Most Hot Start kits on the market today
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