Two-step method for constructing unmarked insertions, deletions and allele substitutions in the yeast genome

Authors

  • Misa Gray,

    1. Division of Cell Biology and Biophysics, School of Biological Sciences, University of Missouri-Kansas City, 5100 Rockhill Road, Kansas City, MO 64110, United States
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  • Sarah Piccirillo,

    1. Division of Cell Biology and Biophysics, School of Biological Sciences, University of Missouri-Kansas City, 5100 Rockhill Road, Kansas City, MO 64110, United States
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  • Saul M. Honigberg

    Corresponding author
    1. Division of Cell Biology and Biophysics, School of Biological Sciences, University of Missouri-Kansas City, 5100 Rockhill Road, Kansas City, MO 64110, United States
    • Corresponding author. Tel.: +1 816 235 2578; fax: +1 816 235 6553., E-mail address: honigbergs@umkc.edu

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  • Edited by M. Jacquet

Abstract

We describe three extensions of the method of site-specific genomic (SSG) mutagenesis. These three extensions of SSG mutagenesis were used to generate precise insertion, deletion, and allele substitution mutations in the genome of the budding yeast, Saccharomyces cerevisiae. These mutations are termed precise because no attached sequences (e.g., marker genes or recombination sites) are retained once the method is complete. Because the method is PCR-based, neither DNA cloning nor synthesis of long oligonucleotides is required. We demonstrated the efficacy of these methods by deleting an ORF, inserting the tandem affinity purification (TAP) tag, and replacing a wild-type allele with a mutant allele.

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