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TripleMaster PCR SystemThe Ideal Choice for Long-Range PCR

Scott Tarpinian
Eppendorf 5 Prime, Boulder, CO, USA Introduction

PCR* technology has been widely used in molecular biology research, providing scientists with an efficient tool for studies such as genome analysis, sequencing, and PCR cloning for gene expression. However, under standard PCR conditions, amplification of DNA fragments greater than 5 kb has been problematic. One major factor contributing to the inability of standard PCR to amplify long segments of DNA is template degradation during exposure to high temperatures in buffers that are unable to maintain pH. This is due to depurination, the hydrolysis of the N-glycosidic bond between the base and the sugar of a DNA molecule. Although depurination is a rare event, occurring approximately once every 25 kb of single-stranded DNA per minute, its effect on long-range PCR is more profound because long templates simply have more sites that are susceptible to depurination. In addition, depurination is more prevalent at the higher temperatures and lower pHs necessary for amplification of long targets. And once the N-glycosidic bond is cleaved, Taq Polymerase is unable to extend past the site of depurination and amplification of the desired target is not possible. Another crucial factor in the limitation of standard PCR to amplify long target sequences is the high rate of incorporation of incorrect bases by thermostable polymerases, such as Taq DNA Polymerase, that lack an editing function. The Eppendorf TripleMaster PCR System solves both of these problems with its innovative buffer system and unique enzyme mix, making it the ideal choice
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