A recyclable Candida albicans URA3 cassette for PCR product-directed gene disruptions

For some time, gene disruptions in Candida albicans have been made with the hisG-URA3-hisG ('Ura-blaster') cassette, which can be re-used in successiv e transformations of a single strain after homologous excision of URA3. How ever, the hisG repeats are too large for efficient PCR amplification of the entire cassette, so it cannot be used for PCR product-directed gene disrup tions. We describe here a gene disruption cassette, URA3-dpl200, with 200 b p flanking repeats that permit efficient PCR amplification. After transform ation and integration to produce both arg5::URA3-dpl200 and rim101::URA3-dp l200 alleles, we find that arg5::dpl200 and rim101::dpl200 segregants, resp ectively, can be obtained. We have used the cassette to create rim101::dpl2 00/rim101::URA3-dpl200 mutants exclusively through PCR product-directed dis ruption. Copyright (C) 2000 John Wiley & Sons, Ltd.