New modules for PCR‐based gene targeting in Candida albicans: rapid and efficient gene targeting using 100 bp of flanking homology region

The use of PCR‐based techniques for directed gene alterations has become a standard tool in Saccharomyces cerevisiae. In our efforts to increase the speed of functional analysis of Candida albicans genes, we constructed a modular system of plasmid vectors and successfully applied PCR‐amplified functional analysis (FA)‐cassettes in the transformation of C. albicans. These cassettes facilitate: (a) gene disruptions; (b) tagging of 3′‐ends of genes with green fluorescent protein (GFP); and (c) replacements of endogenous promoters to achieve regulated expression. The modules consists of a core of three selectable marker genes, CaURA3, CaHIS1 and CaARG4. Modules for C‐terminal GFP‐tagging were generated by adding GFP‐sequences flanked at the 5′‐end by a (Gly‐Ala)3‐linker and at the 3′‐end by the S. cerevisiae URA3‐terminator to these selection markers. Promoter exchange modules consist of the respective marker genes followed by the regulatable CaMAL2 or CaMET3 promoters at their 3′‐ends. In order to ensure a reliably high rate of homologous gene targeting, the flanking homology regions required a size of 100 bp of gene‐specific sequences, which were provided with the oligonucleotide primers. The use of shorter flanking homology regions produced unsatisfactory results with C. albicans strain BWP17. With these new modules only a minimal set of primers is required to achieve the functional analysis of C. albicans genes and, therefore, provides a basic tool to increase the number of functionally characterized C. albicans genes of this human pathogen in the near future. Copyright © 2003 John Wiley & Sons, Ltd.