HYPERMUTAGENIC PCR INVOLVING ALL FOUR TRANSITIONS AND A SIZEABLE PROPORTION OF TRANSVERSIONS

摘要

Very complex mutant libraries of the dihydrofolate reductase (DHFR) gene encoded by the Escherichia coli plasmid R67 were created using hypermutagenic PCR with biased deoxynucleotide triphosphate (dNTP) concentrations, Exploiting the particular stability of the G:T mismatch, the DHFR gene could be enriched in A+T by employing biased deoxypyrimidine triphosphate concentrations, i.e. [dTTP] > [dCTP], A sizeable fraction of hypermutants were functional, A combination of [dTTP] > [dCTP] and [dGTP] > [dATP] biases generated mutations at unexpectedly low frequencies, This could be overcome by the addition of Mn2+ cations, Overall mutation frequencies of 10% per amplification (range 4-18% per clone) could be attained, All four transitions and a smaller number of transversions were produced throughout the gene, PCR mutagenesis could be so extensive as to inactivate all amplified versions of the gene.