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.