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

YeastVolume 14, Issue 10 p. 953-961 Yeast Functional Analysis Report Additional modules for versatile and economical PCR-based gene deletion and modification in Saccharomyces cerevisiae Mark S. Longtine, Corresponding Author Mark S. Longtine mlunc@isis.unc.edu. Department of Biology, University of North Carolina, Chapel Hill, NC 27599–3280, U.S.A.Department of Biology, CB#3280, Coker Hall, University of North Carolina, Chapel Hill, NC 27599–3280, U.S.A. Tel: (+1) 919/962 2293; fax: (+1) 919/962 0320Search for more papers by this authorAmos Mckenzie III, Amos Mckenzie III Department of Biology, University of North Carolina, Chapel Hill, NC 27599–3280, U.S.A.Search for more papers by this authorDouglas J. Demarini, Douglas J. Demarini Department of Biology, University of North Carolina, Chapel Hill, NC 27599–3280, U.S.A.Search for more papers by this authorNirav G. Shah, Nirav G. Shah Department of Biology, University of North Carolina, Chapel Hill, NC 27599–3280, U.S.A.Search for more papers by this authorAchim Wach, Achim Wach Institut für Angewandte Mikrobiologie, Biozentrum, Universität Basel, CH-4056 Basel, SwitzerlandSearch for more papers by this authorArndt Brachat, Arndt Brachat Institut für Angewandte Mikrobiologie, Biozentrum, Universität Basel, CH-4056 Basel, SwitzerlandSearch for more papers by this authorPeter Philippsen, Peter Philippsen Institut für Angewandte Mikrobiologie, Biozentrum, Universität Basel, CH-4056 Basel, SwitzerlandSearch for more papers by this authorJohn R. Pringle, John R. Pringle Department of Biology, University of North Carolina, Chapel Hill, NC 27599–3280, U.S.A.Search for more papers by this author Mark S. Longtine, Corresponding Author Mark S. Longtine mlunc@isis.unc.edu. Department of Biology, University of North Carolina, Chapel Hill, NC 27599–3280, U.S.A.Department of Biology, CB#3280, Coker Hall, University of North Carolina, Chapel Hill, NC 27599–3280, U.S.A. Tel: (+1) 919/962 2293; fax: (+1) 919/962 0320Search for more papers by this authorAmos Mckenzie III, Amos Mckenzie III Department of Biology, University of North Carolina, Chapel Hill, NC 27599–3280, U.S.A.Search for more papers by this authorDouglas J. Demarini, Douglas J. Demarini Department of Biology, University of North Carolina, Chapel Hill, NC 27599–3280, U.S.A.Search for more papers by this authorNirav G. Shah, Nirav G. Shah Department of Biology, University of North Carolina, Chapel Hill, NC 27599–3280, U.S.A.Search for more papers by this authorAchim Wach, Achim Wach Institut für Angewandte Mikrobiologie, Biozentrum, Universität Basel, CH-4056 Basel, SwitzerlandSearch for more papers by this authorArndt Brachat, Arndt Brachat Institut für Angewandte Mikrobiologie, Biozentrum, Universität Basel, CH-4056 Basel, SwitzerlandSearch for more papers by this authorPeter Philippsen, Peter Philippsen Institut für Angewandte Mikrobiologie, Biozentrum, Universität Basel, CH-4056 Basel, SwitzerlandSearch for more papers by this authorJohn R. Pringle, John R. Pringle Department of Biology, University of North Carolina, Chapel Hill, NC 27599–3280, U.S.A.Search for more papers by this author First published: 04 December 1998 https://doi.org/10.1002/(SICI)1097-0061(199807)14:10<953::AID-YEA293>3.0.CO;2-UCitations: 3,843AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat 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 kanr 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. © 1998 John Wiley & Sons, Ltd. 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