Hot Start dNTPs - A Novel Tool for Controlled Nucleotide Incorporation in PCR
Poster Jan 08, 2010
Tony Le, Elena Hidalgo Ashrafi, Sabrina Shore, Victor Timoshchuk, Natasha Paul, Richard Hogrefe, Inna Koukhareva, Alexandre Lebedev
PCR is a widely used scientific tool employed by a variety of applications. Various Hot Start technologies have already been developed using modified PCR components to increase specificity of a reaction. Recently developed CleanAmpTM dNTPs are modified nucleoside triphosphates with a thermolabile 3’-tetrahydrofuranyl protecting group that is released at higher temperatures. These modified dNTPs prevent low temperature primer extension, which can often be a significant problem in PCR. At higher temperatures, the modified dNTPs are deprotected, to allow for incorporation by the DNA polymerase and more specific amplification of the intended target. The use of CleanAmpTM dNTPs provides comparable performance to other Hot Start technologies and shows promise to provide a synergistic effect when used in conjunction with other Hot Start methods. This modified dNTP technology also has the ability to use any DNA polymerase in a Hot Startsystem, which can be very cost effective. Although the utility of CleanAmpTM dNTPs in traditional Hot Start PCR has been previously demonstrated, they can also be used in more advanced PCR applications that require temperature-controlled nucleotide incorporation. In these advanced applications, the CleanAmp TM dNTP modification blocks nucleotide incorporation during initial, lower temperature reactions, allowing for delayed nucleotide activation in a reaction. In summary, CleanAmpTM dNTPs have the potential to provide great versatility and flexibility in a vast number of applications including traditional Hot Start PCR.
When there is a need to quickly analyze samples using a number of different PCR assays, it is likely that optimal conditions for each assay will not be the same. First, different assays often will require different annealing temperatures for their primers. In addition, amplicons may be designed to be of different lengths and therefore require varying durations of the extension step.READ MORE