4 A Guided Tour to PCR-based Genomic Manipulations of S.cerevisiae (PCR-targeting)

Publisher Summary Polymerase chain reaction (PCR)-based targeting is a method to introduce foreign DNA or specific alterations into the yeast genome using homologous recombination between the targeted locus and homologous sequence information provided by PCR primers. It enables various genomic manipulations: gene deletions, gene truncations, C- and N-terminal gene tagging, promoter substitutions, and in vivo site-directed mutagenesis. Carrying out a particular manipulation relies on the availability of specific templates for PCR, called “modules” or “cassettes,” which provide characteristic features. PCR-based targeting has greatly accelerated directed gene studies and has been a key technique for genome-wide functional analysis. This chapter discusses the different applications of this method and the available cassettes and lists critical methods in a user-friendly manner. It also describes a number of considerations that has to be made while planning and conducting functional studies that rely on PCR-based targeting. When analyzing a new gene, one of the first things a researcher usually needs is a rapid and reliable method to detect the expression of the gene. The researcher also needs to detect the corresponding protein within the cell, in cell extracts obtained under different growth conditions, or in association with other proteins upon protein complex purification.