3′-Protected 2′-Deoxynucleoside 5′-Triphosphates as a Tool for Heat-Triggered Activation of Polymerase Chain Reaction
Year: 2009
Type: article
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.
Source: Analytical Chemistry
DOI: 10.1021/ac8026977
Authors: Inna Koukhareva, Alexandre Lebedev
Institution: TriLink BioTechnologies (United States)
Language: English
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: green
OpenAlex ID: W2029688215