Advertisement

PCR-Directed In Vivo Plasmid Construction Using Homologous Recombination in Baker’s Yeast

  • Erik C. Andersen
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 772)

Abstract

A variety of applications require the creation of custom-designed plasmids, including transgenic reporters, heterologous gene fusions, and phenotypic rescue plasmids. These plasmids are created traditionally using restriction digests and in vitro ligation reactions, but these techniques are dependent on available restriction sites and can be laborious given the size and number of fragments to be ligated. The baker’s yeast Saccharomyces cerevisiae provides a powerful platform to create nearly any plasmid through PCR-directed yeast-mediated ligation. This technique can ligate complex plasmids of up to 50 kilobasepairs (kb) in vivo to produce plasmids with precisely defined sequences.

Key words

Yeast Homologous recombination Tagging Plasmid Construct creation Ligation cloning Custom vectors 

Notes

Acknowledgments

The author would like to thank Dr. Leonid Kruglyak for financial support and laboratory space. Additionally, Amy Caudy, Justin Gerke, and Robyn Tanny for many helpful comments. This protocol was adapted from one used by the laboratory of Dr. Jay Dunlap (Dartmouth Medical School) and communicated to the author by Dr. Allan Froelich. Many others have contributed to protocols and studies using yeast to create custom vectors and some of whom are referenced below. The author would like to apologize for any omissions of published works or protocols relevant to these studies. E.C.A. is supported by a Ruth L. Kirschstein National Research Service Award from the National Institutes of Health.

References

  1. 1.
    Orr-Weaver TL, Szostak JW, Rothstein RJ (1981) Yeast transformation: a model system for the study of recombination. Proc Natl Acad Sci USA 78:6354–6358PubMedCrossRefGoogle Scholar
  2. 2.
    Rothstein R (1991) Targeting, disruption, replacement, and allele rescue: integrative DNA transformation in yeast. Methods Enzymol 194:281–301PubMedCrossRefGoogle Scholar
  3. 3.
    Raymond CK, Pownder TA, Sexson SL (1999) General method for plasmid construction using homologous recombination. Biotechniques 26:134–138, 140–141Google Scholar
  4. 4.
    Oldenburg KR, Vo KT, Michaelis S et al (1997) Recombination-mediated PCR-directed plasmid construction in vivo in yeast. Nucleic Acids Res 25:451–452PubMedCrossRefGoogle Scholar
  5. 5.
    Nikawa J, Kawabata M (1998) PCR- and ligation-mediated synthesis of marker cassettes with long flanking homology regions for gene disruption in Saccharomyces cerevisiae. Nucleic Acids Res 26:860–861PubMedCrossRefGoogle Scholar
  6. 6.
    Ma H, Kunes S, Schatz PJ et al (1987) Plasmid construction by homologous recombination in yeast. Gene 58:201–216PubMedCrossRefGoogle Scholar
  7. 7.
    Manivasakam P, Weber SC, McElver J et al (1995) Micro-homology mediated PCR targeting in Saccharomyces cerevisiae. Nucleic Acids Res 23:2799–2800PubMedCrossRefGoogle Scholar
  8. 8.
    Baudin A, Ozier-Kalogeropoulos O, Denouel A et al (1993) A simple and efficient method for direct gene deletion in Saccharomyces cerevisiae. Nucleic Acids Res 21:3329–3330PubMedCrossRefGoogle Scholar
  9. 9.
    Collopy PD, Colot HV, Park G et al (2010) High-throughput construction of gene deletion cassettes for generation of Neurospora crassa knockout strains. Methods Mol Biol 638:33–40PubMedCrossRefGoogle Scholar
  10. 10.
    Winston F, Dollard C, Ricupero-Hovasse SL (1995) Construction of a set of convenient Saccharomyces cerevisiae strains that are isogenic to S288C. Yeast 11:53–55PubMedCrossRefGoogle Scholar
  11. 11.
    Sikorski RS, Hieter P (1989) A system of shuttle vectors and yeast host strains designed for efficient manipulation of DNA in Saccharomyces cerevisiae. Genetics 122:19–27PubMedGoogle Scholar
  12. 12.
    Ling M, Merante F, Robinson BH (1995) A rapid and reliable DNA preparation method for screening a large number of yeast clones by polymerase chain reaction. Nucleic Acids Res 23:4924–4925PubMedCrossRefGoogle Scholar
  13. 13.
    Woodman ME (2008) Direct PCR of intact bacteria (colony PCR). Curr Protoc Microbiol 9:A.3D.1-A.3D.6Google Scholar

Copyright information

© Springer Science+Business Media, LLC 2012

Authors and Affiliations

  1. 1.Lewis-Sigler Institute for Integrative GenomicsPrinceton UniversityPrincetonUSA

Personalised recommendations