Rapid and economical multiple base site-directed mutagenesis of double stranded plasmid DNA

This method for the mutagenesis of ds-DNA, utilizing the best features of previously published protocols, incorporates a fragmentation procedure on the plasmid, thermostable enzymes and two transformations in E. coli. Screening of positive clones can begin after about two days. Insertions, deletions and substitutions of up to 50 bp are routinely obtained with 90-95% of clones positive as proven by sequencing. The cost is about one half to one third of equiv-alent commercial procedures.