Transplacement Mutagenesis: A Recombination-Based In Situ Mutagenesis Protocol

The introduction of subtle mutations into genomic or cDNA regions of interest provides a powerful approach to the study of gene expression and function. Such manipulations of DNA permit numerous small physical changes, and their effects, to be examined. Unfortunately, most mutagenesis procedures require either tedious subcloning schemes to generate gene constructs, or the use of mutagenic polymerase chain reaction (PCR) methods that are not easily applicable to large DNA clones. Meanwhile, recombination-based procedures have readily become adapted for the introduction of mutations, ranging from single base-pair alterations to the introduction of large disruption cassettes or reporter genes. This chapter describes a recombination-based method using genetic positive-negative selection procedures for the deposition of subtle mutations from plasmids into phage clones.