Improved PCR flexibility with Hot Start dNTPs

Considered one of the most powerful tools in molecular biology, PCR is a well-used technique for the amplification of DNA fragments of interest. While ubiquitous, PCR is not flawless. Reactions are often handicapped by nonspecific amplifications such as primer dimerization and mis-priming (1). These off-target amplifications result from primer interaction and extension at lower, less-stringent temperatures and can significantly reduce the efficiency of PCR by inhibiting the amplification of the desired target (2). There have been numerous advancements in the field of PCR that aim to overcome such off-target amplifications – one approach is termed Hot Start. In this technique, primer extension is blocked until higher thermocycling temperatures are reached. Many technologies have evolved since the inception of Hot Start including methods that physically separate reaction components, inhibit DNA polymerase, employ accessory proteins, and utilize chemically modified primers. While these advancements are innovative and useful, they can drive up the cost of PCR considerably. This presents a dilemma between sacrificing an efficient and robust result for a lower cost reaction. One approach that was not explored yet is chemically modifying the dNTP component of the PCR mix. Since dNTPs are an essential part of all PCR reactions, modified dNTPs could easily replace standard dNTPs without the need to significantly alter existing PCR protocols. CleanAmp dNTPs contain a thermolabile tetrahydrofuranyl (THF) protecting group that allows for Hot Start activation in PCR and results in improved PCR performance (3). At the lower temperatures of PCR setup, the 3′-THF dNTP blocks primer extension and prevents primer dimerization and mis-priming. When the temperature of the reaction is elevated to higher temperatures, the protecting group is released. This forms the standard dNTP, which now becomes an available DNA polymerase substrate. Herein, the versatility and utility of CleanAmp dNTPs as compared to standard dNTPs are investigated.