Additional modules for versatile and economical PCR-based gene deletion and modification in Saccharomyces cerevisiae

  • Longtine M
  • McKenzie A
  • Demarini D
 et al. 
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Abstract

An important recent advance in the functional analysis of Saccharomyces cerevisiae genes is the development of the one-step PCR-mediated technique for deletion and modification of chromosomal genes. This method allows very rapid gene manipulations without requiring plasmid clones of the gene of interest. We describe here a new set of plasmids that serve as templates for the PCR synthesis of fragments that allow a variety of gene modifications. Using as selectable marker the S. cerevisiae TRP1 gene or modules containing the heterologous Schizosaccharomyces pombe his5+ or Escherichia coli kan(r) gene, these plasmids allow gene deletion, gene overexpression (using the regulatable GAL1 promoter), C- or N-terminal protein tagging [with GFP(S65T), GST, or the 3HA or 13Myc epitope], and partial N- or C-terminal deletions (with or without concomitant protein tagging). Because of the modular nature of the plasmids, they allow efficient and economical use of a small number of PCR primers for a wide variety of gene manipulations. Thus, these plasmids should further facilitate the rapid analysis of gene function in S. cerevisiae.

Author-supplied keywords

  • Epitope tagging
  • Functional analysis
  • Gene deletion
  • Gene truncation
  • Green fluorescent protein
  • Overexpression studies
  • Polymerase chain reaction

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Authors

  • Mark S. Longtine

  • Amos McKenzie

  • Douglas J. Demarini

  • Nirav G. Shah

  • Achim Wach

  • Arndt Brachat

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