Recombination and Mutagenesis by Overlap Extension PCR

  • Robert J. Pogulis
  • Abbe N. Vallejo
  • Larry R. Pease
Part of the Springer Protocols Handbooks book series (SPH)


The polymerase chain reaction (PCR) (1,2) is now a fundamental tool of molecular biology. Although PCR provides the basis for a variety of sensitive analytical techniques, it can also be used in a synthetic capacity to generate large quantities of specific DNA fragments. The alteration of amplified DNA sequences is also possible, since synthetic oligonucleotide primers become incorporated into the final PCR product. Although the 3′ ends of these primers must match the target DNA sequence, the 5′ ends may contain modifications. Sequence modifications in the primers will therefore be present in the ends of the amplified DNA fragment, offering a straightforward, although limited, ability to introduce site-directed mutations during PCR.


Polymerase Chain Reaction Polymerase Chain Reaction Buffer Internal Primer Flank Primer Sensitive Analytical Technique 
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.


  1. 1.
    Mullis, K., Faloona, F., Scharf, S., Saiki, R., Horn, G., and Erlich, H. (1986) Specific enzymatic amplification of DNA in vitro: the polymerase chain reaction. Cold Spring Harbor Symp. Quant. Biol. 51, 263–273.PubMedCrossRefGoogle Scholar
  2. 2.
    Saiki, R. K., Gelfand, D. H., Stoffel, S., Scharf, S. J., Higuchi, R., Horn, G. T., Mullis, K. B., and Erlich, H. A. (1988) Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science 239, 487–491.PubMedCrossRefGoogle Scholar
  3. 3.
    Ho, S. N., Hunt, H. D., Horton, R. M., Pullen, J. K., and Pease, L. R. (1989) Site-directed mutagenesis by overlap extension using the polymerase chain reaction. Gene 77, 51–59.PubMedCrossRefGoogle Scholar
  4. 4.
    Horton, R. M., Hunt, H. D., Ho, S. N., Pullen, J. K., and Pease, L. R. (1989) Engineering hybrid genes without the use of restriction enzymes: gene splicing by overlap extension. Gene 77, 61–68.PubMedCrossRefGoogle Scholar
  5. 5.
    Horton, R. M., Cai, Z., Ho, S. N., and Pease, L. R. (1990) Gene splicing by overlap extension: tailor-made genes using the polymerase chain reaction. Biotechniques 8, 528–535.PubMedGoogle Scholar
  6. 6.
    Rychlik, W. (1993) Selection of primers for polymerase chain reaction, in PCR Protocols: Current Methods and Applications (White, B. A., ed), Humana, Totowa, NJ, pp. 31–40.CrossRefGoogle Scholar
  7. 7.
    Suggs, S. V., Hirose, T., Miyake, T., Kawashima, E. H., Johnson, M. J., Itakura, K., and Wallace, R. B. (1981) Use of synthetic oligo-deoxyribonucleotides for the isolation of cloned DNA sequences, in Developmental Biology Using Purified Genes (Brown, D. D. and Fow, C. F., eds.), Academic, New York, pp. 683–693.Google Scholar
  8. 8.
    Davis, G. T., Bedzyk, W. D., Voss, E. W., and Jacobs, T. W. (1991) Single chain antibody (SCA) encoding genes: one-step construction and expression in eukaryotic cells. Biotechnology 9, 165–179.PubMedCrossRefGoogle Scholar
  9. 9.
    Tautz, D. and Renz, M. (1983) An optimized freeze-squeeze method for the recovery of DNA fragments from agarose gels. Anal. Biochem. 132, 14–19.PubMedCrossRefGoogle Scholar
  10. 10.
    Sambrook, J., Fritsch, E. F., and Maniatis, T. (eds.) (1989) Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, NY.Google Scholar
  11. 11.
    Morrison, H. G. and Desrosiers, R. C. (1993) A PCR-based strategy for extensive mutagenesis of a target DNA sequence. Biotechniques 14, 454–457.PubMedGoogle Scholar
  12. 12.
    Kirchhoff, F. and Desrosiers, R. C. (1993) A PCR-derived library of random point mutations within the V3 region of simian immunodeficiency virus. PCR Methods Appl. 2, 301–304.PubMedCrossRefGoogle Scholar
  13. 13.
    Hanes, S. D. and Brent, R. (1991) A genetic model for interaction of the homeodomain recognition helix with DNA. Science 251, 426–430.PubMedCrossRefGoogle Scholar
  14. 14.
    Cease, K. B., Potcova, C. A., Lohoff, C. J., and Zeigler, M. E. (1994) Optimized PCR using Vent polymerase. PCR Methods Appl. 3, 298–300.PubMedCrossRefGoogle Scholar
  15. 15.
    Juncosa-Ginestra, M., Pons, J., Planas, A., and Querol, E. (1994) Improved efficiency in site-directed mutagenesis by PCR using a Pyrococcus sp. GB-D polymerase. Biotechniques 16, 820–823.Google Scholar
  16. 16.
    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
  17. 17.
    Clark, J. M. (1988) Novel non-templated nucleotide addition reactions catalyzed by prokaryotic and eukaryotic DNA polymerase. Nucleic Acids Res. 17, 3319.Google Scholar
  18. 18.
    Landt, O., Grunert, H. P., and Hahn, U. (1990) A general method for rapid site-directed mutagenesis using the polymerase chain reaction. Gene 96, 125–128.PubMedCrossRefGoogle Scholar
  19. 19.
    Kuipers, O. P., Boot, H. J., and de Vos, W. M. (1991) Improved site-directed mutagenesis method using PCR. Nucleic Acids Res. 19, 4558.PubMedCrossRefGoogle Scholar

Copyright information

© Humana Press Inc., Totowa, NJ 2000

Authors and Affiliations

  • Robert J. Pogulis
    • 1
  • Abbe N. Vallejo
    • 1
  • Larry R. Pease
    • 1
  1. 1.Department of ImmunologyMayo Clinic and Mayo FoundationRochester

Personalised recommendations