Megaprimer Method for Polymerase Chain Reaction-Mediated Generation of Specific Mutations in DNA

  • Jesper Brøs-Poulsen
  • Jane Nøhr
  • Leif Kongskov Larsen
Part of the Methods in Molecular Biology™ book series (MIMB, volume 182)

Abstract

During the past decade, a number of methods using polymerase chain reaction (PCR) for the generation of specific mutations in any nucleotide sequence have been described, these methods include such drawbacks as the possibility of generating undesired secondary mutations, because of the low fidelity of some of the thermostable DNA polymerases, the need for four or more specific oligonucleotide primers, and the use of sophisticated, individually optimized protocols. Finally, the selection of correctly mutated clones may also prove to be laborious (1, 2, 3). In contrast, the megaprimer principle described here is costefficient, fast, reliable, and convenient.

Keywords

Magnesium Glycerol Albumin Agarose Electrophoresis 

References

  1. 1.
    Picard, V., Ersdal-Badju, E., Lu, A., and Bock, S. C. (1994) A rapid and efficient one-tube PCR-based mutagenesis technique using Pfu DNA polymerase. Nucleic Acids Res. 22, 2587–2591.PubMedCrossRefGoogle Scholar
  2. 2.
    Weiner, M. P., Felts, K. A., Simcox, T. G., and Braman, J.C. (1993) A method for the site-directed mono-and multi-mutagenesis of double stranded DNA. Gene 126, 35–41.PubMedCrossRefGoogle Scholar
  3. 3.
    Jones, D. H. and Winistorfer, S. C. (1992) Recombinant circle PCR and recombination PCR for site-specific mutagenesis without PCR product purification. BioTechniques 12, 528–535.PubMedGoogle Scholar
  4. 4.
    Sambrook, J., Fritsch, E., and Maniatis, T. (1989) Molecular Cloning. A Laboratory Manual. 2nd ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY.Google Scholar
  5. 5.
    Br/Øs-Poulsen, J., Petersen, N. E., Horder, M., and Kristiansen, K. (1999) An improved PCR-based method for site directed mutagenesis using megaprimers. Mol. Cell. Probes. Google Scholar
  6. 6.
    Barettino, D., Feigenbutz, M., Valcárcel, R., and Stunnenberg, H. D. (1994) Improved method for PCR-mediated sitedirected mutagenesis. Nucleic Acids Res. 22,541,542.PubMedCrossRefGoogle Scholar
  7. 7.
    Good, L. and Nazar, R. N. (1992) An improved thermal cycle for two-step PCR-based targeted mutagenesis. Nucleic Acids Res. 20, 4934.PubMedCrossRefGoogle Scholar

Copyright information

© Humana Press Inc. 2002

Authors and Affiliations

  • Jesper Brøs-Poulsen
  • Jane Nøhr
  • Leif Kongskov Larsen
    • 1
  1. 1.CCBR A/SBallerupDenmark

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