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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 nonsubstrates 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 mispriming products can form. At the initial heat-denaturing step and during the 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 an 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 "mispriming" products. Overall, PCR with 3'-THF-protected dNTPs demonstrated substantially improved performance and was more efficient and specific compared to PCR with standard dNTPs.

SUBMITTER: Koukhareva I

PROVIDER: S-EPMC2712722 | biostudies-literature | 2009 Jun

REPOSITORIES: biostudies-literature

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3'-Protected 2'-deoxynucleoside 5'-triphosphates as a tool for heat-triggered activation of polymerase chain reaction.

Koukhareva Inna I Lebedev Alexandre A

Analytical chemistry 20090601 12

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 nonsubstrates or terminating substrates for Taq DNA polymerase, they do not support primer extension/elongation at low stringency conditions during PCR sample prepar ...[more]

PMID: 19438248

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Dataset Information

3'-Protected 2'-deoxynucleoside 5'-triphosphates as a tool for heat-triggered activation of polymerase chain reaction.

Publications

3'-Protected 2'-deoxynucleoside 5'-triphosphates as a tool for heat-triggered activation of polymerase chain reaction.

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