Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Protocol
  • Published:

Gene splicing and mutagenesis by PCR-driven overlap extension

Abstract

Extension of overlapping gene segments by PCR is a simple, versatile technique for site-directed mutagenesis and gene splicing. Initial PCRs generate overlapping gene segments that are then used as template DNA for another PCR to create a full-length product. Internal primers generate overlapping, complementary 3′ ends on the intermediate segments and introduce nucleotide substitutions, insertions or deletions for site-directed mutagenesis, or for gene splicing, encode the nucleotides found at the junction of adjoining gene segments. Overlapping strands of these intermediate products hybridize at this 3′ region in a subsequent PCR and are extended to generate the full-length product amplified by flanking primers that can include restriction enzyme sites for inserting the product into an expression vector for cloning purposes. The highly efficient generation of mutant or chimeric genes by this method can easily be accomplished with standard laboratory reagents in approximately 1 week.

This is a preview of subscription content, access via your institution

Access options

Rent or buy this article

Prices vary by article type

from$1.95

to$39.95

Prices may be subject to local taxes which are calculated during checkout

Figure 1: PCR-mediated overlap extension can create specific nucleotide mutations or generate chimeric gene products.
Figure 2: Mutagenic primers b and c share complementary sequence.
Figure 3: Glycine to arginine mutation at position 83 of murine MHC Kb is created by overlap extension mutagenesis.

Similar content being viewed by others

References

  1. Hunt, H.D., Pullen, J.K., Dick, R.F., Bluestone, J.A. & Pease, L.R. Structural basis of Kbm8 alloreactivity. Amino acid substitutions on the beta-pleated floor of the antigen recognition site. J. Immunol. 145, 1456–1462 (1990).

    CAS  PubMed  Google Scholar 

  2. Pullen, J.K., Hunt, H.D. & Pease, L.R. Peptide interactions with the Kb antigen recognition site. J. Immunol. 146, 2145–2151 (1991).

    CAS  PubMed  Google Scholar 

  3. Lohi, J., Lehti, K., Valtanen, H., Parks, W.C. & Keski-Oja, J. Structural analysis and promoter characterization of the human membrane-type matrix metalloproteinase-1 (MT1-MMP) gene. Gene 242, 75–86 (2000).

    Article  CAS  Google Scholar 

  4. Seyboldt, C., Granzow, H. & Osterrieder, N. Equine herpesvirus 1 (EVH-1) glycoprotein M: effect of deletions of transmembrane domains. Virology 278, 477–489 (2000).

    Article  CAS  Google Scholar 

  5. Lemberg, M.K. & Martoglio, B. Requirements for signal peptide peptidase-catalyzed intramembrane proteolysis. Mol Cell 10, 735–744 (2002).

    Article  CAS  Google Scholar 

  6. Ho, S.N., Hunt, H.D., Horton, R.M., Pullen, J.K. & Pease, L.R. Site-directed mutagenesis by overlap extension using the polymerase chain reaction. Gene 77, 51–59 (1989).

    Article  CAS  Google Scholar 

  7. Horton, R.M., Hunt, H.D., Ho, S.N., Pullen, J.K. & Pease, L.R. Engineering hybrid genes without the use of restriction enzymes: gene splicing by overlap extension. Gene 77, 61–68 (1989).

    Article  CAS  Google Scholar 

  8. Zoller, M.J. & Smith, M. Oligonucleotide-directed mutagenesis using M13-derived vectors: an efficient and general procedure for the production of point mutations in any fragment of DNA. Nuc. Acids Res. 10, 6487–6500 (1982).

    Article  CAS  Google Scholar 

  9. Morinaga, Y., Franceschini, T., Inouye, S. & Inouye, M. Improvement of oligonucleotide-directed site-specific mutagenesis using double-stranded plasmid DNA. Biotechnology 2, 636–639 (1984).

    CAS  Google Scholar 

  10. Lai, D., Zhu, X. & Pestka, S. A simple and efficient method for site-directed mutagenesis with double-stranded plasmid DNA. Nucleic Acids Res. 21, 3977–3980 (1993).

    Article  CAS  Google Scholar 

  11. Brown, T. Hybridization analysis of DNA blots. in Current Protocols in Molecular Biology, Unit 2.10, January 1993 (ed. Harkins, B.) 2.10.1–2.10.16 (John Wiley & Sons Inc., Hoboken, NJ, 2003).

    Google Scholar 

  12. Duby, A., Jacobs, K.A. & Celeste, A. Using synthetic oligonucleotides as probes. in Current Protocols in Molecular Biology, Unit 6.4, January 1990 (ed. Harkins, B.) 6.4.1–6.4.10 (John Wiley & Sons Inc., Hoboken, NJ, 2003).

    Google Scholar 

  13. Engebrecht, J., Brent, R. & Kaderbhai, M.A. Minipreps of plasmid DNA. in Current Protocols in Molecular Biology, Unit 1.6, July 1991 (ed. Harkins, B.) 1.6.1–1.6.10 (John Wiley & Sons Inc., Hoboken, NJ, 2003).

    Google Scholar 

  14. Slatko, B.E., Eckert, R.L., Albright, L.M. & Ausubel, F.M. DNA sequencing strategies. in Current Protocols in Molecular Biology, Unit 7.1, April 1999 (ed. Harkins, B.) 7.1.1–7.1.7 (John Wiley & Sons Inc., Hoboken, NJ, 2003).

    Google Scholar 

  15. Kammann, M., Laufs, J., Schell, J. & Gronenborn, B. Rapid insertional mutagenesis of DNA by polymerase chain reaction (PCR). Nucleic Acids Res. 17, 5404 (1989).

    Article  CAS  Google Scholar 

  16. Sarkar, G. & Sommer, S.S. The “megaprimer” method of site-directed mutagenesis. Biotechniques 8, 404–407 (1990).

    CAS  PubMed  Google Scholar 

  17. Mikaelian, I. & Sergeant, A. Modification of the overlap extension method for extensive mutagenesis on the same template. Methods Mol. Biol. 57, 193–202 (1996).

    CAS  PubMed  Google Scholar 

  18. Urban, A., Neukirchen, S. & Jaeger, K. A rapid and efficient method for site-directed mutagenesis using one-step overlap extension PCR. Nucleic Acids Res. 25, 2227–2228 (1997).

    Article  CAS  Google Scholar 

  19. Kramer, M.F. & Coen, D.M. Enzymatic amplification of DNA by PCR: standard procedures and optimization. in Current Protocols in Molecular Biology, Unit 15.1, October 2001 (ed. Harkins, B.) 15.1.1–15.1.14 (John Wiley & Sons Inc., Hoboken, NJ, 2003).

    Google Scholar 

  20. Rohren, E.M., Pease, L.R., Ploegh, H.L. & Schumacher, T.N.M. Polymorphisms in pockets of major histocompatibility complex class I molecules influence peptide preference. J. Exp. Med. 177, 1713–1721 (1993).

    Article  CAS  Google Scholar 

  21. Weiss, E. et al. The DNA sequence of the H-2Kb gene: evidence for gene conversion as a mechanism for the generation of polymorphism in histocompatibility antigens. EMBO J. 2, 453–462 (1983).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank Rudy Hanson for his technical contributions to this paper and Virginia Van Keulen and Michael Bell for helpful discussions.

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Larry R Pease.

Ethics declarations

Competing interests

Mayo Clinic is assigned intellectual property rights related to the described technology that have been and continue to be licensed in a non-exclusive manner to a number of non-academic private interests. Mayo Clinic and I (author) have received royalty payments in excess of $10,000. K. Heckman has no competing financial interest.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Heckman, K., Pease, L. Gene splicing and mutagenesis by PCR-driven overlap extension. Nat Protoc 2, 924–932 (2007). https://doi.org/10.1038/nprot.2007.132

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nprot.2007.132

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing