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Molecular Biotechnology

, Volume 29, Issue 3, pp 225–232 | Cite as

A single-stage polymerase-based protocol for the introduction of deletions and insertions without subcloning

  • John C. Salerno
  • Rachel J. Jones
  • Eda Erdogan
  • Susan M. E. Smith
Research Protocol

Abstract

A single-stage polymerase-based procedure is described that allows extensive modifications of DNA. The version described here uses the QuikChange Site-Directed Mutagenesis System kit supplied by Stratagene. The original protocol is replaced by a single-stage method in which linear production of complementary strands is accomplished in separate single primer reactions. This has proved effective in introducting insertions and deletions into large gene/vector combinations without subcloning.

Index Entries

Linear amplification site-directed mutagenesis 

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References

  1. 1.
    Mullis, K., Faloona, F., Scharf, S., Saiki, R., Horn, G., and Ehrlich, H. (1986) Specific enzymatic amplification of DNA in vitro: the polymerase chain reaction. Cold Spring Harbor Symp. Quant. Biol. 51, 262–273.Google Scholar
  2. 2.
    Cormack, B. (1994) Mutagenesis of cloned DNA. In: Current Protocols in Molecular Biology (Ausubel, F. M., Brent, R. E., Kingston, D. D., et al., eds.), John Wiley and Sons, New York, pp. 8.5.1–8.5.12.Google Scholar
  3. 3.
    Sarkar, G. and Sommer, S. S. (1990) The “megaprimer” method of site-directed mutagenesis. Biotechniques 8, 404–407.PubMedGoogle Scholar
  4. 4.
    Aiyar, A. and Leis, J. (1993) Modification of the megaprimer method of PCR mutagenesis: improved amplification of the final product. Biotechniques 15, 366–369.Google Scholar
  5. 5.
    Barik, S. (1996) Site-directed mutagenesis in virto by megaprimer PCR. Methods Mol. Biol. 57, 203–215.PubMedGoogle Scholar
  6. 6.
    Wang, W. Y. and Malcolm, B. A. (1999) Two-stage PCR protocol allowing introduction of multiple mutations, deletions and insertions using QuikChange Site-Directed Mutagenesis. Biotechniques 26, 680–682.PubMedGoogle Scholar
  7. 7.
    Wang, W. Y. and Malcolm, B. A. (2002) Two-stage polymerase chain reaction protocol allowing introduction of multiple mutations, deletions, and insertions, using QuikChange site-directed mutagenesis. Methods Mol. Biol. 182, 37–43.PubMedGoogle Scholar
  8. 8.
    Makarova, O., Kamberov, E., and Margolis, B. (2000) Generation of deletion and point mutations with one primer in a single cloning. Biotechniques 29, 970–972.PubMedGoogle Scholar
  9. 9.
    Geiser, M., Gee, R., Drewello, D., and Schmitz, R. (2001) Integration of PCR fragments at any specific site within cloning vectors without the use of restriction enzymes and DNA ligase Biotechniques 31, 88–92.Google Scholar

Copyright information

© Humana Press Inc 2005

Authors and Affiliations

  • John C. Salerno
    • 1
  • Rachel J. Jones
    • 1
  • Eda Erdogan
    • 1
  • Susan M. E. Smith
    • 1
  1. 1.Biology DepartmentRensselaer Polytechnic Institute, Science CenterTroy

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