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Hot-start PCR is a technique that improves PCR performance by reducing nonspecific amplification during the initial setup stages of the PCR. This unit describes hot-start PCR protocols which utilize primers containing temperature-sensitive modifications. The introduction of 4-oxo-tetradecyl (OXT) phosphotriester groups onto the 3' end of the primer allows for primer-based hot-start PCR that is amenable for use in a number of PCR-based applications. The protocols described in this unit utilize OXT-modified primers in applications such as standard thermal cycling PCR, fast thermal cycling PCR, multiplex PCR, and one-step reverse-transcription PCR. This method is also advantageous for instances where improved PCR specificity is desired and a hot-start polymerase suitable for your application is not available.

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Multiplex PCR is an advantageous technique used in PCR applications to amplify multiple targets in a single reaction. As useful as it is, this technique presents a new set of challenges that further complicates PCR setup. For example, reactions are more prone to off‐target amplifications such as mis‐priming and primer dimer due to the increased number of primer pairs. Furthermore, preferential amplification of certain targets leads to an unequal distribution of amplicon products, making quantification and detection of problematic targets extremely difficult. To improve upon the problems specific to multiplex PCR, we evaluated Hot Start modified primers which contain either one or two thermolabile 4‐oxo‐tetradecyl (OXT) modifications to prevent DNA polymerase extension at low‐stringent temperatures, and that are released after a Hot Start activation step. Herein, we find that the singly‐modified primers provide greater amplification efficiency, specificity, and yield in the multiplex amplification of DNA targets. In reverse transcriptase PCR (RT‐PCR), the doubly‐modified primers have been proven to be the optimal choice. The presence of two thermolabile protecting groups allows for an efficient one‐step RT‐PCR reaction that provides high specificity for multiple targets. TriLink's innovative technology represents a convenient tool for multiplex PCR amplification of DNA and RNA samples.

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BackgroundMultiplex RT-PCR is a valuable technique used for pathogen identification, disease detection and relative quantification of gene expression. The simplification of this protocol into a one-step procedure saves time and reagents. However, intensive PCR optimization is often required to overcome competing undesired PCR primer extension during the RT step.ResultsHerein, we report multiplex one-step RT-PCR experiments in which the PCR primers contain thermolabile phosphotriester modification groups. The presence of these groups minimizes PCR primer extension during the RT step and allows for control of PCR primer extension until the more stringent, elevated temperatures of PCR are reached. Results reveal that the use of primers whose extension can be controlled in a temperature-mediated way provides improved one-step RT-PCR specificity in both singleplex and multiplex reaction formats.ConclusionsThe need for an accurate and sensitive technique to quantify mRNA expression levels makes the described modified primer technology a promising tool for use in multiplex one-step RT-PCR. A more accurate representation of the abundances in initial template sample is feasible with modified primers, as artifacts of biased PCR are reduced because of greater improvements in reaction specificity.

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.

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 Start system, 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 CleanAmpTM 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.