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Microbiology 148 (2002), 2607-2615
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Microbiology (2002), 148, 2607-2615.
© 2002 Society for General Microbiology

Research Paper

A PCR-based strategy to generate integrative targeting alleles with large regions of homology

Robert C. Davidson1, Jill R. Blankenship1, Peter R. Kraus1, Marisol de Jesus Berrios1, Christina M. Hull1, Cletus D’Souza1, Ping Wang1 and Joseph Heitman1

Departments of Molecular Genetics and Microbiology, Pharmacology and Cancer Biology, and Medicine, and the Howard Hughes Medical Institute, Duke University Medical Center, Research Drive, Durham, NC 27710, USA1

Author for correspondence: Joseph Heitman. Tel: +1 919 684 2824. Fax: +1 919 684 5458. e-mail: heitm001{at}duke.edu

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.

Keywords: gene disruption, PCR overlap, Cryptococcus neoformans

Abbreviations: MAP, mitogen-acitvated protein




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