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Random Mutagenesis by Whole-Plasmid PCR Amplification

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PCR Cloning Protocols

Part of the book series: Methods in Molecular Biology™ ((MIMB,volume 192))

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Abstract

Mutagenesis is a popular tool used in the analysis of protein structure and function. Polymerase chain reaction (PCR)-based mutagenesis can be used to introduce mutations with the use of the appropriate primer. Although the majority of attention has been given to site-directed mutagenesis, random mutagenesis is actually an older approach and has considerable potential because of its limited bias, if an appropriate screening method is available. This approach has successfully been used to obtain “gain-of-function” mutants (1). The ability to target mutants to individual proteins and parts of proteins with modern molecular tools has considerable applicability.

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References

  1. Botstein, D. and Shortle, D. (1985) Strategies and application of in vitro mutagenesis. Science 229, 1193–1201.

    Article  CAS  PubMed  Google Scholar 

  2. Hill, D. E., Oliphant, A. R., and Struhl, K. (1997) Mutagenesis with degenerate oligonucleotides: an efficient method for saturating a defined DNA region with base pair substitutions. Meth. Enzymol. 155, 558–568.

    Article  Google Scholar 

  3. Fromant, M., Blanquet, S., and Plateau, P. (1995) Direct random mutagenesis of genesized DNA fragments using polymerase chain reaction. Analyt. Biochem. 224, 347–353.

    Article  CAS  PubMed  Google Scholar 

  4. Zhao, H., Giver, L., Shao, Z., Affholter, J. A., and Arnold, F. H. (1998) Molecular evolution by staggered extension process (StEP) in vitro recombination. Nature Biotechnol. 16, 258–261.

    Article  CAS  Google Scholar 

  5. Nakamaye, K. L. and Eckstein, F. (1986) Inhibition of restriction endonuclease NciI cleavage by phosphorothioate groups and its application to oligonucleotide-directed mutagenesis. Nucl. Acids Res. 14, 9679–9698.

    Article  CAS  PubMed  Google Scholar 

  6. Zaccolo, M., Williams, D. M., Brown, D. M., and Gherardi, E. (1996) An approach to random mutagenesis of DNA using mixtures of triphosphate derivatives of nucleotide analogues. J. Mol. Biol. 255, 589–603.

    Article  CAS  PubMed  Google Scholar 

  7. Tange, T., Taguchi, S., Kojima, S., Miura, K., and Momose, H. (1997) Improvement of a useful enzyme (substilisin BPN’) by an experimental evolution system. Appl. Microbiol. Biotechnol. 41, 239–244.

    Article  Google Scholar 

  8. Zoller, M. J. (1992) New recombinant DNA technology for protein engineering. Curr. Opin. Biotechnol. 3, 348–354.

    Article  CAS  PubMed  Google Scholar 

  9. Parikh, A. and Guengerich, F. P. (1998) Random mutagenesis by whole-plasmid PCR amplification. BioTechniques 24, 428–431.

    CAS  PubMed  Google Scholar 

  10. Lanio, T. and Jeltsch, A. (1998) PCR-Based random mutagenesis method using spiked oligonucleotides to randomize selected parts of a gene without any wild-type background. BioTechniques 25, 958–965.

    CAS  PubMed  Google Scholar 

  11. Sambrook, J., Fritsch, E. F., and Maniatis, T., eds. (1989) Molecular Cloning. A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.

    Google Scholar 

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© 2002 Humana Press Inc.

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Kim, D., Guengerich, F.P. (2002). Random Mutagenesis by Whole-Plasmid PCR Amplification. In: Chen, BY., Janes, H.W. (eds) PCR Cloning Protocols. Methods in Molecular Biology™, vol 192. Humana Press. https://doi.org/10.1385/1-59259-177-9:241

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  • DOI: https://doi.org/10.1385/1-59259-177-9:241

  • Publisher Name: Humana Press

  • Print ISBN: 978-0-89603-969-8

  • Online ISBN: 978-1-59259-177-0

  • eBook Packages: Springer Protocols

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