An in vitro screening technique for DNA polymerases that can incorporate modified nucleotides. Pseudothymidine as a substrate for thermostable polymerases

DNA polymerases are desired that incorporate modified nucleotides into DNA with diminished pausing, premature termination and infidelity. Reported her e is a simple in vitro assay to screen for DNA polymerases that accept modi fied nucleotides based on a set of primer extension reactions, In combinati on with the scintillation proximity assay (SPA(TM)), this allows rapid and simple screening of enzymes for their ability to elongate oligonucleotides in the presence of unnatural nucleotides, A proof of the concept is obtaine d using pseudo-thymidine (psi T), the C-nucleoside analog of thymidine, as the unnatural substrate. The conformational properties of psi T arising fro m the carbon-carbon bond between the sugar and the base make it an interest ing probe for the importance of conformational restraints in the active sit e of polymerases during primer elongation, From a pool of commercially avai lable thermostable polymerases, the assay identified Taq DNA polymerase as the most suitable enzyme for the PCR amplification of oligonucleotides cont aining psi T. Subsequent experiments analyzing PCR performance and fidelity of Taq DNA polymerase acting on psi T are presented. This is the first tim e that PCR has been performed with a C-nucleoside.