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

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 t...