HYPERMUTAGENIC PCR INVOLVING ALL 4 TRANSITIONS AND A SIZABLE PROPORTION OF TRANSVERSIONS

Very complex mutant libraries of the dihydrofolate reductase (DHFR) ge ne encoded by the Escherichia coli plasmid R67 were created using hype rmutagenic PCR with biased deoxynucleotide triphosphate (dNTP) concent rations, 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 fractio n of hypermutants were functional, A combination of [dTTP] > [dCTP] an d [dGTP] > [dATP] biases generated mutations at unexpectedly low frequ encies, This could be overcome by the addition of Mn2+ cations, Overal l mutation frequencies of 10% per amplification (range 4-18% per clone ) could be attained, All four transitions and a smaller number of tran sversions were produced throughout the gene, PCR mutagenesis could be so extensive as to inactivate all amplified versions of the gene.