A simple method for randomly mutating cloned DNA fragments by using chemical mutagens and the polymerase chain reaction

Abstract

Random mutagenesis of cloned DNA fragments can be an important tool for understanding genes and/or gene products. I report here a simple procedure for generating random mutations in any cloned DNA fragment in vitro. This method utilizes the polymerase chain reaction (PCR) to amplify chemically damaged single-stranded DNA containing the DNA fragment of interest. A mutagenized pool of plasmid DNA is produced by cloning the PCR product (containing chemically induced sequence heterogeneity) into a new vector. Therefore, extreme chemical treatments can be used that biologically inactivate the original vector. In this report, 10% of the plasmids contained a mutation in a 97-nucleotide DNA insert, giving a mutagenesis frequency of 10 −3/base pair. Of 3000 yeast transformants screened, seven intron mutations were isolated that activate a cryptic 3′ splice site, suggesting that 2% of the mutations could give rise to the desired phenotype. The seven mutations included transitions, transversions and single nucleotide deletions, demonstrating the well-balanced repertoire of nucleotide changes derived from this procedure.