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 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.
Trilink BioTechnologies, Inc., 9955 Mesa Rim Road, San Diego, California 92121, USA.
Inna
I
Koukhareva
Alexandre
A
Lebedev
D003854:Deoxyribonucleotides / Q000737:chemistry*; D016133:Polymerase Chain Reaction / Q000379:methods*; D013696:Temperature
D003854:Deoxyribonucleotides