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

Abstract

A new approach that improves the efficiency and specificity of Polymerase Chain Reaction (PCR) has been developed. Heat sensitive 3’-protected derivatives of 2’-deoxyribonucleoside 5’-triphosphates (dNTPs) have been synthesized and used as substitutes for natural dTTP, dCTP, dATP and dGTP in PCR. Since 3’-protected dNTPs are either non-substrates or terminating substrates for Taq DNA polymerase they do not support primer extension/elongation at low stringency conditions during PCR sample preparation when PCR artifacts such as primer dimers and mis-priming products can form. At initial heat-denaturing step and during PCR sequence the 3’-protecting group is cleaved releasing 3’-unprotected dNTP that is a natural substrate for DNA polymerase. As a result, the primer extension/elongation proceeds only at elevated temperature of PCR, when the interaction of primers and template is highly stringent and specific. Several 3’-protecting groups covering a wide range of deprotection kinetics have been tested. The 3’-O-tetrahydrofuranyl derivatives of dNTPs have demonstrated the best properties leading to a drastically reduced accumulation of PCR artifacts such as “primer dimers” and “mis-priming” products. Overall, PCR with 3’-THF protected dNTPs demonstrated substantially improved performance and was more efficient and specific compared to PCR with standard dNTPs.

Chemoenzymatic Synthesis of 3′‐O‐Acetal‐Protected 2′‐Deoxynucleosides as Building Blocks for Nucleic Acid Chemistry

“…The easy access to 3Ј-O-acetal-protected nucleosides will lead to the expeditious synthesis of 5Ј-triphosphates (dNTPs) required for PCR related applications. [5,22] The chemoenzymatic methodology detailed herein is a significant improvement over reported methods. A key step in the synthesis is the regioselective 5Ј-hydroxy protection of the 2Ј-deoxynucleosides catalyzed by Candida antarctica lipase B. Enzyme-catalyzed reactions are environmentally attractive due to their safe and cost-effective features relative to conventional chemistry-based methods using silyl or dimethoxytrityl reagents.…”

“…A recent report on the thermolabile deprotection of tetrahydropyranyl (THP) and tetrahydrofuranyl (THF) ethers during the polymerase chain reaction (PCR) has attracted our attention. [5] The merits of acetal groups like THP and THF lie in their stability in the presence of various reagents such as basic media, alkyllithiums, metal hydrides, Grignard reagents, oxidative reagents, and alkylating or acylating reagents and under mild acidic conditions or heating, which promote cleavage. [6] Numerous methods have been reported for the tetrahydropyranylation of alcohols.…”

A hot start alternative for high-fidelity DNA polymerase amplification mediated by quantum dots

“…Although the use of HS polymerase results in high-specificity amplification, it has greatly increased the cost of PCR amplification. The third approach is to employ modified primers [20][21][22][23] or dNTP [24], and largely depends on the efficiency of the modification [25][26][27].…”

Quantum dots trigger hot-start effects for pfu-based polymerase chain reaction

“…Although capable of significantly suppressing non-specific amplification, these polymerase-based approaches have significantly added the cost for PCR amplification. The third approach achieves HS effects by the modifications of the primers [29][30][31][32] or dNTP [33], and such methods largely depend on the efficiency of the modification of primers or dNTP.…”