SITE-SPECIFIC MUTAGENESIS USING SYNTHETIC OLIGODEOXYRIBONUCLEOTIDE PRIMERS .2. INVITRO SELECTION OF MUTANT-DNA

A method for the in vitro selection of mutant DNA was devised as an adjunct to the recently developed method for the use of short enzymatically-synthesized oligodeoxyribonucleotides of defined sequence as site-specific mutagens for circular DNA. The selection method uses the mutating oligodeoxyribonucleotide as a primer for Escherichia coli DNA polymerase I (large fragment) under conditions where there is preferential interaction with mutant DNA template. After ligation using T4 DNA ligase, endonuclease S1 is used to degrade single-stranded non-mutant DNA leaving the desired mutant as closed circular duplex DNA. The development of the method using mutants in .phi.X174 DNA as the model system is described. Studies on the changes A .fwdarw. G and G .fwdarw. A at position 587 of .phi.X174 viral DNA (am3 to wild-type and its reversal) show that 1 or 2 cycles of selection can lead to a population of phage consisting of close to 100% mutants.