Abstract
Gene Splicing by Overlap Extension (gene SOEing) provides a powerful method of recombining sequences without depending on restriction sites or ligase, and a simple, generally applicable way of using polymerase chain reaction (PCR) to perform site-directed mutagenesis in vitro. This technique allows even those with minimal molecular biology expertise to generate quickly genetic constructs that might otherwise have been impractical using only the older (restriction enzymebased) technology.
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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: Tailor-made 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, in Developmental 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. BioTechniques 8, 404–407.
Horton, R. M. and Pease, L. R. (1991) Recombination and mutagenesis of DNA sequences using PCR, in Directed 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.
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© 1993 Humana Press Inc., Totowa, NJ
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Horton, R.M. (1993). In Vitro Recombination and Mutagenesis of DNA. In: White, B.A. (eds) PCR Protocols. Methods in Molecular Biology, vol 15. Humana Press, Totowa, NJ. https://doi.org/10.1385/0-89603-244-2:251
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DOI: https://doi.org/10.1385/0-89603-244-2:251
Publisher Name: Humana Press, Totowa, NJ
Print ISBN: 978-0-89603-244-6
Online ISBN: 978-1-59259-502-0
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