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

Journal Article (Journal Article)

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.

Full Text

Duke Authors

Cited Authors

  • Davidson, RC; Blankenship, JR; Kraus, PR; de Jesus Berrios, M; Hull, CM; D'Souza, C; Wang, P; Heitman, J

Published Date

  • August 2002

Published In

Volume / Issue

  • 148 / Pt 8

Start / End Page

  • 2607 - 2615

PubMed ID

  • 12177355

International Standard Serial Number (ISSN)

  • 1350-0872

Digital Object Identifier (DOI)

  • 10.1099/00221287-148-8-2607


  • eng

Conference Location

  • England