Linker Scanning Mutagenesis by Three-Step PCR

A number of mutagenesis methods allow the systematic survey of a region of transcriptional regulatory sequence for identifying functional elements. In these methods, clusters or blocks of point mutations are introduced at discrete locations that span a suspected regulatory region of a gene. The individual mutants are then examined for retention of transcriptional activity typically in a reporter gene assay. Traditional methods, including linker-scanning mutagenesis (1 ) and microscale “shot-gun” gene synthesis (2 ), are either tedious and time-consuming or relatively inefficient at producing the desired mutated sequence. A modification of the PCR-based mutagenesis method originally described by Li and Shapiro (3 ) is much simpler, faster, and more efficient than the traditional methods.