PCR- and ligation-mediated synthesis of split-marker cassettes with long flanking homology regions for gene disruption in Candida albicans

Because Candida albicans is a diploid organism, two consecutive steps of ge ne disruption are required to generate a gene knock-out. The same marker (U RA3) is often used for disruption of both copies of the gene. This is possi ble because, after the first round of disruption, homologous recombination between direct repeats flanking the URA3 marker and the subsequent counters election allow for the efficient recovery of Ura(-) revertants. Unfortunate ly, the URA-blaster disruption cassette cannot be used in a PCR-based disru ption approach. The hisG repeats flanking the URA3 gene in the disruption c assette anneal to one another during PCR and thereby prevent amplification of the complete cassette. We explored the use of transformation based on sp lit-marker recombination to circumvent this problem. To avoid any cloning s teps and to retain the advantage of long flanking regions for disruption, w e combined this with a PCR- and ligation-mediated approach for generating m arker cassettes. We used this approach to disrupt the C. albicans FAL1 (ATP -dependent RNA helicase) gene. Long 5' and 3' FAL1-specific regions were am plified by PCR and individually ligated to a URA-blaster cassette. The resu lting ligation reactions were used separately as templates to generate two FAL1 disruption cassettes with overlapping URA3 marker regions. Simultaneou s transformation with both overlapping disruption cassettes yielded efficie nt disruption of one FAL1 allele.