Construction of Mutant Alleles in Saccharomyces cerevisiae without Cloning: Overview and the Delitto Perfetto Method

Zarmik Moqtaderi1, Joseph V. Geisberg1

1 Harvard Medical School, Boston, Massachusetts
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 13.10C
DOI:  10.1002/0471142727.mb1310cs104
Online Posting Date:  October, 2013
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Traditionally, methods for introducing specific new mutations at target loci in the yeast genome have involved the preparation of disruption or gene‐replacement cassettes via multiple cloning steps. Sequences used for targeting these cassettes or integrating vectors are typically several hundred base pairs long. A variety of newer methods rely on the design of custom PCR oligonucleotides containing shorter sequence tails (∼50 nt) for targeting the locus of interest. These techniques obviate the need for cloning steps and allow construction of mutagenesis cassettes by PCR amplification. Such cassettes may be used for gene deletion, epitope tagging, or site‐specific mutagenesis. The strategies differ in several ways, most notably with respect to whether they allow reuse of the selection marker and whether extra sequences are left behind near the target locus. This unit presents a summary of methods for targeted mutagenesis of Saccharomyces cerevisiae loci without cloning, including PCR‐based allele replacement, delitto perfetto, and MIRAGE. Next, a protocol is provided for the delitto perfetto PCR‐ and oligonucleotide‐based mutagenesis method, which offers particular advantages for generating several different mutant alleles of the same gene. Curr. Protoc. Mol. Biol. 104:13.10C.1‐13.10C.17. © 2013 by John Wiley & Sons, Inc.

Keywords: allele replacement; delitto perfetto; mutagenesis; MIRAGE

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Table of Contents

  • Introduction
  • Basic Protocol 1: Construction of Mutant Alleles by Delitto Perfetto
  • Support Protocol 1: Design and Preparation of Oligonucleotides for Delitto Perfetto
  • Support Protocol 2: Colony PCR of Candidate Clones
  • Commentary
  • Literature Cited
  • Figures
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Basic Protocol 1: Construction of Mutant Alleles by Delitto Perfetto

  • Delitto perfetto CORE plasmid (e.g., pGSHU; Storici et al., )
  • 50 µM PCR primers for amplification of CORE cassette (see protocol 2)
  • 5 U/µl ExTaq polymerase with 10× buffer (TaKaRa)
  • 2.5 mM dNTP mix (2.5 mM each nucleotide)
  • PCR purification kit (Qiagen)
  • Yeast strain to be transformed
  • Yeast media and plates (unit 13.1):
    • YPD medium
    • SD or CAA plates lacking uracil
    • YP medium with 2% raffinose
    • YP medium with 2% galactose
    • SD plates with 5‐fluoroorotic acid (5‐FOA)
    • YPD plates with 300 µg/ml hygromycin or 200 µg/ml G418 (depending on CORE module)
  • 0.1 M LiAc/TE buffer, pH 7.5 (see appendix 22 for TE buffer)
  • 10 mg/ml salmon sperm DNA
  • URA3 CEN plasmid (for positive control)
  • LiAc/TE/PEG solution: 0.1 M LiAc/TE buffer containing 40% (w/v) PEG‐3350
  • 50 µM annealed mutagenic oligonucleotides (see protocol 2)
  • Hygromycin‐ or kanamycin‐resistant yeast strain (as positive control)
  • 0.5‐ml PCR tubes
  • Thermal cycler
  • 30°C incubator with tube roller
  • 15‐ml conical centrifuge tubes (e.g., Falcon)
  • 1.5‐ml microcentrifuge tubes
  • 30° and 42°C heat blocks
  • Snap‐cap tubes
  • Additional reagents and equipment for colony PCR (see protocol 3)

Support Protocol 1: Design and Preparation of Oligonucleotides for Delitto Perfetto

  • 10× PCR reaction buffer
  • Thermal cycler

Support Protocol 2: Colony PCR of Candidate Clones

  • Easily visible yeast colonies to be tested (see Basic Protocol)
  • Parental wild‐type strain (negative control)
  • Strain possessing desired mutation, if available (positive control)
  • 10× PCR buffer
  • 2.5 mM dNTP mix (2.5 mM each nucleotide)
  • Hot‐start Taq polymerase
  • 100 µM custom‐designed PCR primers flanking site of integration
  • Thin‐wall PCR tubes
  • Microwave oven
  • Thermal cycler
  • Additional reagents and equipment for agarose gel electrophoresis (unit 2.5) and sequencing
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Literature Cited

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