Multipurpose Transposon Insertion Libraries for Large-Scale Analysis of Gene Function in Yeast

Abstract

Transposons have long been recognized as useful laboratory tools facilitating genome-scale studies of gene function. Relative to traditional methods, transposon mutagenesis offers a rapid and economical means of generating large numbers of independent insertions in target DNA through minimal experimental manipulation. In particular, the transposon insertion library described here is an excellent tool for the analysis of gene function on a large scale in the budding yeast Saccharomyces cerevisiae. The transposon utilized in this library is multifunctional, such that the library can be used to screen for disruption phenotypes while also providing a means to generate epitope-tagged alleles and, in many cases, conditional alleles. Provided here are complete protocols by which the transposon insertion library may be used to screen for mutant phenotypes in yeast as well as accompanying protocols describing a means of identifying transposon insertion sites within strains of interest. In total, this insertion library is a singularly useful tool for genome-wide functional analysis, and the general approach is applicable to other organisms in which transforming DNA tends to integrate by homologous recombination.