Heat Activatable 3'-modified dNTPs: Synthesis and Application for Hot Start PCR

Abstract

Several 3'-ether and 3'-ester derivatives of 2'-deoxyribonucleoside 5'-triphosphates (dNTPs) were prepared. These dNTP derivatives were not substrates for DNA polymerase and did not support primer extension at room temperature. However, by short pre-heating to 95 degrees C in PCR buffer, these 3'-modified dNTPs can be converted to corresponding unmodified natural dNTPs that efficiently support PCR amplification. The analysis of PCR products obtained with 3'-modified dNTPs revealed a significant improvement in PCR performance resulting in higher amplicon yield and reduced formation of off-target products (mis-priming and primer dimer). Among the studied 3'-modified dNTPs, the 3'-tetrahydrofuranyl derivatives showed the best results.

3′-Protected 2′-Deoxynucleoside 5′-Triphosphates as a Tool for Heat-Triggered Activation of Polymerase Chain Reaction

“…The 3′-protected dNTPs are either nonsubstrates or terminating substrates for thermostable DNA polymerases, such as Thermus aquaticus ( Taq ) DNA polymerase. Regardless of the mechanism of action of 3′-protected dNTPs, ,, further primer extension or elongation is halted at ambient temperature, where the primer−template hybridization is not stringent. The PCR amplification can start only after heat-triggered activation when the 3′-protecting group is removed to produce a corresponding unprotected dNTP with a free 3′-hydroxyl group that is the normal substrate for Taq DNA polymerase.…”

“…Preliminary results on the synthesis and use of 3′protected dNTPs in PCR have been published recently. 38,39 Although modified dNTPs were used to improve PCR, [40][41][42][43] they were never previously proposed as a tool for hot start PCR applications. The report provides initial PCR results that should be of general interest to users of PCR and serve as a prelude for our continued investigations aimed at demonstrating utility of 3′protected dNTPs in heat-triggered PCR applications.…”

“…In this work, we describe several types of 3′-protected dNTPs and proof-of-principal experiments demonstrating that use of these compounds reduces the formation and accumulation of the off-target artifacts that interfere with the efficiency and specificity of PCR and leads to a substantial improvement in PCR performance. Preliminary results on the synthesis and use of 3′-protected dNTPs in PCR have been published recently. , Although modified dNTPs were used to improve PCR, − they were never previously proposed as a tool for hot start PCR applications. The report provides initial PCR results that should be of general interest to users of PCR and serve as a prelude for our continued investigations aimed at demonstrating utility of 3′-protected dNTPs in heat-triggered PCR applications.…”

Heat‐Activatable Primers for Hot‐Start PCR and Hot‐Start One‐Step RT‐PCR: Endpoint and Real‐Time Experiments

“…Moreover, not every commercially available DNA polymerase has been converted to the corresponding modified hot-start DNA polymerase, limiting users to a much smaller subset of DNA polymerases for their experiments. Much like the chemically modified DNA polymerases, chemically modified primers (Kaboev et al, 2000;Ullman et al, 2002;Ankenbauer et al, 2003;Bonner, 2003;Laird and Niemiec, 2004) and chemically modified dNTPs (Koukhareva et al, 2008) present a hot-start activation mechanism in which a heat-activation step provides a controlled release of unmodified primers and dNTPs, respectively, into the reaction. For each of these last two types of hot-start approaches, users can employ their DNA polymerase of choice, using modified primers or modified dNTPs to convert the DNA polymerase into its hot-start version.…”

Development of a high-throughput real time PCR based on a hot-start alternative for Pfu mediated by quantum dots

“…The third approach is to employ modified primers [13][14][15][16][17] or dNTP, 18,19 and largely depends on the efficiency of the modification. [20][21][22] Recently, we presented a new QD-based nano-engineering strategy to dynamically regulate the activity of DNA polymerases and achieved a HS-like effect in conventional PCR with a high-fidelity Pfu DNA polymerase, 23,24 which was a simple and low cost HS approach without the modification in However, most of these experiments were made on an endpoint PCR assay and PCR products were analyzed by electrophoresis in agarose gels. Evidently, there was no systematic evaluation of the HS effect of QDs modulated in real time PCR.…”