HotMasterAn innovative Hot Start/Cold Stop technology for better ,,, PCR* results
George Halley a...Hot Start DNA polymerases have become very popular because of their co... Introduction ...Hot Start PCR was developed as a method to minimize the deleterious ef...
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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