Abstract
Gene Splicing by Overlap Extension (gene SOEing) is a sequence-independent method for site-directed mutagenesis and/or recombination of DNA molecules. It is based on the idea that a PCR product can be engineered by adding or changing sequences at its ends so that the product can itself be used to prime DNA synthesis in a subsequent overlap-extension reaction to create mutant or recombinant molecules. As the engineered genes are created in vitro without reliance on host organisms or restriction sites, gene SOEing provides a powerful and versatile tool for genetic investigation and engineering.
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References
Mullis, K. B. and Faloona, F. A. (1987) Specific synthesis of DNA in vitro via a polymerase-catalysed chain reaction.Methods Enzymol. 155, 335–350.
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.
Kadowaki, H., Kadowaki, T., Wondisford, F. E., and Taylor, S. I. (1989) Use of polymerase chain reaction catalysed by Taq DNA polymerase for site-specific mutagenesis.Gene 76, 161–166.
Vallette, F., Mege, E., Reiss, A., and Milton, A. (1989) Constuction of mutant and chimeric genes using the polymerase chain reaction.Nucleic Acids Res. 17, 723–733.
Higuchi, R., Krummel, B., and Saiki, R.K. (1988) A general method of in vitro preparation and specific mutagenesis of DNA fragments: study of protein and DNA interactions.Nucleic Acids Res. 16, 7351–7367.
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.
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.
Horton, R. M., Cai, Z., Ho, S. N., and Pease, L. R. (1990) Gene splicing by overlap extension: tailormade genes using the polymerase chain reaction.BioTechniques 8, 528–535.
Kain, K. C., Orlandi, P. A., and Lanar, D. E. (1991) Universal promoter for gene expression without cloning: expression-PCR.BioTechniques 10, 366.
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, 165–169.
Daughtery, B. L., DeMartino, J. A., Law, M.-F., Kawka, D. W., Singer, I.I., and Mark, G. E. (1991) Polymerase chain reaction facilitates the cloning, CDR-grafting, and rapid expression of a murine monoclonal antibody directed against the CD18 component of leukocyte integrins.Nucleic Acids Res. 19, 2471–2476.
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, inDevelopmental Biology Using Purified Genes ( Brown, D. D. and Fow, C. F., eds.), Academic, New York, pp. 683–693.
Yon, J. and Fried, M. (1989) Precise gene fusion by PCR.Nucleic Acids Res. 17, 4895.
Sarkar, G. and Sommer, S. S. (1990) The “megaprimer” method of site-directed mutagenesis.Bio-Techniques 8, 404–407.
Horton, R. M. and Pease, L. R. (1991) Recombination and mutagenesis of DNA sequences using PCR, inDirected Mutagenesis: A Practical Approach. (McPherson, M. J., ed.) IRL, Oxford, pp. 217–247.
Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989)Molecular Cloning: A Laboratory Manual (2nd ed.) Cold Spring Harbor Laboratory, Cold Spring Harbor, NY.
Yolov, A. A. and Shaborova, Z. A. (1990) Constructing DNA by polymerase recombination.Nucleic Acids Res. 18, 3983–3986.
Eckert, K. A. and Kunkel, T. A. (1990) High fidelity DNA synthesis by the Thermus aquaticus DNA polymerase.Nucleic Acids Res. 18, 3739–3744.
Hanes, S. D. and Brent, R. (1991) A genetic model for interaction of the homeodomain recognition helix with DNA.Science 251, 426–430.
Rudert, R. A. and Trucco, M. (1990) DNA polymers of protein binding sequences generated by PCR.Nucleic Acids Res. 18, 6460.
Shuldiner, A. R., Scott, L. A., and Roth, J. (1990) PCR-induced (ligase-free) subcloning: a rapid reliable method to subclone polymerase chain reaction (PCR) products.Nucleic Acids Res. 18, 1920.
Jones, D. H. and Howard, B. H. (1990) A rapid method for site-specific mutagenesis and directional subcloning by using the polymerase chain reaction to generate recombinant circles.BioTechniques 8, 178–183.
White, B. A. (ed.) (1993)PCR Protocols: Current Methods and Applications, Humana, Totowa, NJ.
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Horton, R.M. PCR-mediated recombination and mutagenesis. Mol Biotechnol 3, 93–99 (1995). https://doi.org/10.1007/BF02789105
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DOI: https://doi.org/10.1007/BF02789105