RT Journal Article SR Electronic(1) A1 Davidson, Robert C. A1 Blankenship, Jill R. A1 Kraus, Peter R. A1 de Jesus Berrios, Marisol A1 Hull, Christina M. A1 D’Souza, Cletus A1 Wang, Ping A1 Heitman, JosephYR 2002 T1 A PCR-based strategy to generate integrative targeting alleles with large regions of homology JF Microbiology, VO 148 IS 8 SP 2607 OP 2615 DO https://doi.org/10.1099/00221287-148-8-2607 PB Microbiology Society, SN 1465-2080, AB Cryptococcus neoformans is an opportunistic fungal pathogen with a defined sexual cycle for which genetic and molecular techniques are well developed. The entire genome sequence of one C. neoformans strain is nearing completion. The efficient use of this sequence is dependent upon the development of methods to perform more rapid genetic analysis including gene-disruption techniques. A modified PCR overlap technique to generate targeting constructs for gene disruption that contain large regions of gene homology is described. This technique was used to disrupt or delete more than a dozen genes with efficiencies comparable to those previously reported using cloning technology to generate targeting constructs. Moreover, it is shown that disruptions can be made using this technique in a variety of strain backgrounds, including the pathogenic serotype A isolate H99 and recently characterized stable diploid strains. In combination with the availability of the complete genomic sequence, this gene-disruption technique should pave the way for higher throughput genetic analysis of this important pathogenic fungus., UL https://www.microbiologyresearch.org/content/journal/micro/10.1099/00221287-148-8-2607