Advertisement

PCR-Assisted Mutagenesis for Site-Directed Insertion/Deletion of Large DNA Segments

  • Daniel C. Tessier
  • David Y. Thomas
Part of the Methods In Molecular Medicine™ book series (MIMB, volume 57)

Abstract

We present in this chapter a polymerase chain reaction (PCR)-based method to simultaneously introduce and remove large fragments of DNA in a single mutagenesis reaction without the need for restriction sites. We have favored the use of long single-stranded DNA primers synthesized by asymmetric PCR (1,2), whereas others have used double-stranded PCR-amplified fragment directly (3, 4, 5) as primers in in vitro mutagenesis reactions.

Keywords

Helper Phage Initial Polymerase Chain Reaction Restriction Enzyme Cleavage Site Asymmetric Polymerase Chain Reaction Mutagenesis Reaction 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

References

  1. 1.
    Tessier, D. C. and Thomas, D. Y. (1993) PCR-assisted large insertion/deletion mutagenesis. BioTechniques 15, 498–501.PubMedGoogle Scholar
  2. 2.
    Wychowski, C., Emerson, S. U., Silver, J., and Feinstone, S. M. (1990) Construction of recombinant DNA molecules by the use of a single stranded DNA generated by the polymerase chain reaction: its application to chimeric hepatitis A virus/poliovirus subgenomic cDNA. Nucleic Acids Res. 18, 913–918.PubMedCrossRefGoogle Scholar
  3. 3.
    Clackson, T. and Winter, G. (1989) “Sticky feet”-directed mutagenesis and its application to swapping antibody domains. Nucleic Acids Res. 17, 10,163–10,170.PubMedCrossRefGoogle Scholar
  4. 4.
    Near, R. I. (1992) Gene conversion of immunoglobulin variable regions in mutagenesis cassettes by replacement PCR mutagenesis. BioTechniques 12, 88–97.PubMedGoogle Scholar
  5. 5.
    Zhong, D. and Bajaj, S. P. (1993) A PCR-based method for site-specific domain replacement that does not require restriction recognition sequences. BioTechniques 15, 874–878.PubMedGoogle Scholar
  6. 6.
    Zoller, M. J. and Smith, M. (1982) Oligonucleotide-directed mutagenesis using M13-derived vectors: an efficient and general procedure for the production of point mutations in any fragment of DNA. Nucleic Acids Res. 10, 6487–6500.PubMedCrossRefGoogle Scholar
  7. 7.
    Saiki, R. K., Gelfand, D. H., Stoffel, S., Scharf, S. F., Higuchi, R., Horn, R. T., Mullis, K. B., and Erlich, H. A. (1988) Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science 238, 487–491.CrossRefGoogle Scholar
  8. 8.
    Vieira, J. and Messing, J. (1987) Production of single-stranded plasmid DNA. Methods Enzymol. 153, 3–11.PubMedCrossRefGoogle Scholar
  9. 9.
    Casadaban, M. and Cohen, S. N. (1980) Analysis of gene control signals by DNA fusions and cloning in Escherichia coli. J. Mol. Biol. 138, 179–207.PubMedCrossRefGoogle Scholar
  10. 10.
    Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning. A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY.Google Scholar
  11. 11.
    Kunkel, T. A. (1985) Rapid and efficient site-specific mutagenesis without phenotypic selection. Proc. Natl. Acad. Sci. USA 82, 488–492.PubMedCrossRefGoogle Scholar
  12. 12.
    Kunkel, T. A., Roberts, J. D., and Zakour, R. A. (1987) Rapid and efficient site-specific mutagenesis without phenotypic selection. Methods Enzymol. 154, 367–382.PubMedCrossRefGoogle Scholar
  13. 13.
    Hanahan, D. (1983) Studies on transformation of Escherichia coli with plasmids. J. Mol. Biol. 166, 557–580.PubMedCrossRefGoogle Scholar
  14. 14.
    Sanchez-Pescador, R. and Urdea, M. S. (1984) Use of unpurified synthetic deoxynucleotide primers for rapid dideoxynucleotide chain termination sequencing. DNA 3, 339–343.PubMedGoogle Scholar
  15. 15.
    Suggs, S. V, Hirose, T., Miyake, Y., Kawashima, E. H., and Johnson, M. J. (1981) Use of synthetic oligonucleotides for the isolation of specific cloned DNA sequences, in Developmental Biology Using Purified Genes (Brown, D. D., ed.), Academic, New York, pp 683–693.Google Scholar
  16. 16.
    Mattila, P., Korpela, J., Tenkanen, T., and Pitkanen, K. (1991) Fidelity of DNA synthesis by the Thermococcus litoralis DNA polymerase—an extremely heat stable enzyme with proofreading activity. Nucleic Acids Res. 19, 4967–4973.PubMedCrossRefGoogle Scholar
  17. 17.
    Perrin, S. and Gilliland, G. (1990) Site-specific mutagenesis using asymmetric polymerase chain reaction and a single mutant primer. Nucleic Acids Res. 18, 7433–7438.PubMedCrossRefGoogle Scholar

Copyright information

© Humana Press Inc. 1996

Authors and Affiliations

  • Daniel C. Tessier
    • 1
  • David Y. Thomas
    • 1
  1. 1.National Research Council of CanadaBiotechnology Research InstituteMontrealCanada

Personalised recommendations