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.
Wilson
R B
RB
Department of Microbiology, Institute of Cancer Research, Columbia University, New York, NY 10032, USA.
Davis
D
D
Enloe
B M
BM
Mitchell
A P
AP
D002176:Candida albicans / Q000235:genetics*; D005656:Fungal Proteins / Q000235:genetics*; D016133:Polymerase Chain Reaction; D014170:Transformation, Genetic
D005656:Fungal Proteins