Additional Modules for Versatile and Economical PCR-based Gene Deletion and Modification in Saccharomyces Cerevisiae

Yeast. 1998 Jul;14(10):953-61. doi: 10.1002/(SICI)1097-0061(199807)14:10<953::AID-YEA293>3.0.CO;2-U.

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.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • DNA Primers
  • Gene Deletion
  • Gene Expression
  • Genetic Vectors
  • Green Fluorescent Proteins
  • Luminescent Proteins
  • Molecular Biology / methods*
  • Plasmids*
  • Polymerase Chain Reaction*
  • Recombinant Fusion Proteins
  • Reproducibility of Results
  • Saccharomyces cerevisiae / genetics*
  • Transformation, Genetic

Substances

  • DNA Primers
  • Luminescent Proteins
  • Recombinant Fusion Proteins
  • Green Fluorescent Proteins