Oligonucleotide design and optimized protocol for site-directed mutagenesis.

We have optimized conditions for the successful execution of site-directed mutagenesis (SDM) in systems that utilize mutagenic oligonucleotide primers to direct the synthesis of mutant plasmids. In this report, we describe strategies for the design of single-strand DNA templates for SDM, mutagenic oligonucleotide primers, as well as conditions for the annealing, synthesis and propagation of mutant plasmids. The primary focus of the report details the technical aspects of computer-assisted mutagenic oligonucleotide design. Important features include oligonucleotide length, number of matched bases flanking the point(s) of mutation(s), melting temperature, internal stability of the 5' and 3' ends, hairpin and dimer formation, and potential false-priming sites. Largely through a retrospective analysis of our successes and failures, we describe features of the mutagenic oligonucleotide primer that appear critical in this mutagenesis system. Specific examples of efficient and inefficient oligonucleotides are discussed. These characteristics and guidelines should be applicable for SDM of a broad range of target sequences of varying composition, complexity and secondary structure.