Production of a vector to facilitate DNA mutagenesis and recombination

Most methods for the generation of site-specific mutants and recombinant constructs require primer extension in vitro. These methods include the Kunkel method and PCR-based methods. Such methods to manipulate DNA are prone to sequence error because they take place outside the complex in vivo mechanisms that increase sequence fidelity during plasmid replication in Escherichia coli. Sequence errors are of particular concern when using PCR-based methods. We have constructed two new plasmids that facilitate the generation of site-specific mutants and recombinant constructs. The plasmids we have constructed are designed to maximize the number of unique restriction enzyme recognition sites in inserts that have been cloned into either plasmid. This was accomplished by eliminating extraneous sequence and many restriction enzyme recognition sites. New recombinant circle and recombination PCR protocols for multiplex site-specific plasmid mutagenesis were used to make these plasmids. These plasmids permit small portions of an insert sequence to be readily removed by restriction enzyme digestion. A small DNA segment, containing the targeted sequence alteration, can subsequently be ligated into a plasmid construct that has not been subjected to primer extension in vitro, diminishing the probability of encountering a sequence error and reducing the amount of DNA sequencing necessary to assess for errors.