Multiple Site-directed Mutagenesis Research Articles

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.

Multiple site-directed mutagenesis.

Multiple site-directed mutagenesis.

Generation of a plasmid vector for deletion cloning by rapid multiple site-directed mutagenesis

The construction of a new plasmid vector, devoid of all MboI (GATC) and TspEI (AATT) restriction sites, is described. The lack of these two frequent-cutting restriction sites is a unique feature among plasmids. This new plasmid, pBRkanf1 −, allows selective fragmentation of a cloned insert. As a result, the vector offers an alternative strategy to create overlapping and sequentially deleted subclones. In addition, the construction of the new plasmid required the development of a rapid and accurate multiple site-directed mutagenesis procedure. The mutagenesis method uses a combination of DNA amplification and chain extension by DNA polymerase. By this method, mutations are created progressively from one end of a DNA molecule to the other.