Enhancing multiplex PCR efficiency using Hot Start dNTPs

Abstract

Multiplex PCR is a widespread molecular biology technique for amplification of multiple targets in a single PCR experiment. This approach saves both time and money because several loci can be amplified simultaneously. Today, this technique is applied in numerous areas, including molecular diagnostics and forensics (1). Although its utility is unquestioned, multiplex PCR can be particularly difficult to optimize. Because a unique primer pair is included for each additional target, reactions become more prone to off-target amplifications, such as mis-priming and primer dimerization, than traditional PCR setups. These nonspecific amplifications can hinder the sensitivity and selectivity of multiplex PCR because key components in the reaction mix are consumed, resulting in diminished target amplicon yields. Furthermore, preferential amplification of certain targets in multiplex PCR experiments can result in an imbalance of target amplicon yield formation. Many attempts have been made to improve the efficiency and specificity of multiplex PCR (2,3). Commonly used Hot Start approaches hold a key PCR reagent inactive until higher thermocycling temperatures are reached. Hot Start polymerases have shown tremendous promise in enhancing multiplex PCR, but their use can significantly increase the cost of PCR experiments. An approach that has yet to be examined is the use of chemically modified dNTPs as a heat-activatable component in multiplex PCR (4). Because dNTPs are essential to PCR, modified components can easily take the place of standard dNTPs in the reaction mix without significantly altering existing protocols. Hot Start dNTPs or CleanAmpTM dNTPs contain a thermolabile 3′-tetrahydrofuranyl (THF) protecting group that is released at high PCR temperatures (4,5). During the lower nonstringent temperatures of PCR setup, primers can interact nonspecifically and form mis-priming and primer dimerization products. The use of CleanAmpTM dNTPs blocks primer extension at these lower temperatures, thereby reducing off-target artifact accumulation during PCR. At Hot Start temperatures (~95°C), the 3′-THF protecting group is released, yielding a standard dNTP substrate suitable for DNA polymerase incorporation. Herein, we investigate the incorporation of CleanAmpTM dNTPs and how they can mitigate or eliminate the difficulties of multiplex PCR.