Hot Start activation approaches are increasingly being used to improve the performance of PCR. Since the inception of Hot Start as a means of blocking DNA polymerase extension at lower temperatures, a number of approaches have been developed that target the essential reaction components such as magnesium ion, DNA polymerase, oligonucleotide primers, and dNTPs. Herein, five different Hot Start activation protocols are presented. The first method presents the use of barriers as a means of segregating key reaction components until a Hot Start activation step. The second and third protocols demonstrate Hot Start approaches to block DNA polymerase activity through the use of anti-DNA polymerase antibodies and accessory proteins, respectively. The fourth and fifth protocols utilize thermolabile chemical modifications to the oligonucleotide primers and dNTPs. The results presented demonstrate that all protocols significantly improve the specificity of traditional thermal cycling protocols.
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.
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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