OLIGONUCLEOTIDE DESIGN AND OPTIMIZED PROTOCOL FOR SITE-DIRECTED MUTAGENESIS

We have optimized conditions Sor the successful execution of site-dire cted mutagenesis (SDM) in systems that utilize mutagenic oligonucleoti de 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 prima ry focus of the report details the technical aspects of computer assis ted mutagenic oligonucleotide design. Important features include oligo nucleotide length, number of matched bases flanking the points(s) of m utation(s), melting temperature, internal stability of the 5' and 3' e nds, hairpin and dimer formation, and potential Salse-priming sites. L argely through a retrospective analysis of our successes and failures, we describe features of the mutagenic oligonucleotide primer that app ear critical in this mutagenesis system. Specific examples of efficien t and inefficient oligonucleotides are discussed. These characteristic s and guidelines should be applicable for SDM of a broad range of targ et sequences of varying composition, complexity and secondary structur e.