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 successive transformations of a single strain after homologous excision of URA3. However, the hisG repeats are too large for efficient PCR amplification of the entire cassette, so it cannot be used for PCR product‐directed gene disruptions. We describe here a gene disruption cassette, URA3‐dpl200, with 200 bp flanking repeats that permit efficient PCR amplification. After transformation and integration to produce both arg5::URA3‐dpl200 and rim101::URA3‐dpl200 alleles, we find that arg5::dpl200 and rim101::dpl200 segregants, respectively, can be obtained. We have used the cassette to create rim101::dpl200/rim101::URA3‐dpl200 mutants exclusively through PCR product‐directed disruption. Copyright © 2000 John Wiley & Sons, Ltd.