A new approach to improved hot start PCR: nucleoside 5'-triphosphates with thermolabile 3'-protecting group

Koukhareva, Inna; Huang, H. T.; Yee, Joyclyn; Paul, Natasha; Hogrefe, Richard I.; Lebedev, A.

doi:10.1135/css200810259

Collection Symposium Series 2008

DOI: 10.1135/css200810259

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A new approach to improved hot start PCR: nucleoside 5'-triphosphates with thermolabile 3'-protecting group

Inna Koukhareva¹,

H. T. Huang²,

Joyclyn Yee³

et al.

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

Koukhareva

Lebedev

2009

Anal. Chem.

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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.

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

Koukhareva

Lebedev

2009

Anal. Chem.

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A new approach to improved hot start PCR: nucleoside 5'-triphosphates with thermolabile 3'-protecting group

Cited by 1 publication

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

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

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