Hot Start dNTPs‐ Novel chemistries for use in advanced PCR applications

Abstract

PCR is a widely used scientific tool whose specificity can be increased by the use of Hot Start technologies. Although many Hot Start technologies exist, recently developed CleanAmpTM dNTPs are a distinct approach that employs modified nucleoside triphosphates with a thermolabile protecting group at the 3′‐hydroxyl. The presence of the protecting group blocks low temperature primer extension, which can often be a significant problem in PCR. At higher temperatures, the protecting group is released to allow for incorporation by the DNA polymerase and more specific amplification of the intended target. These modified dNTPs provide comparable performance to other Hot Start technologies and can be used with a variety of thermostable DNA polymerases to turn a reaction into a Hot Start version. This thermolabile chemistry can be applied to dNTP analogs such as dUTP, which is used in UNG decontamination methods, and 7‐deaza‐dGTP, which is used to amplify difficult GC‐rich targets. In addition, further studies have led to the development of 3′‐protecting groups that deprotect more quickly than the current 3′‐modification group, allowing these modified dNTPs to be used in fast PCR. With the evolving chemistry of these Hot Start dNTPs, the areas of application benefiting from the versatility and flexibility of this technology continue to grow.