Abstract
The capacity to recombine and modify DNA are underpinnings of the recombinant DNA revolution. The polymerase chain reaction (PCR) (1,2) provides a rapid means for the site-directed mutagenesis of DNA and for the recombination of DNA (1-9). Recently, two methods have been introduced that permit site-directed mutagenesis and DNA recombination without any enzymatic reaction in vitro apart from DNA amplification (5-9). The first method is accomplished by using separate PCR amplifications to generate products, such that when these products are combined, denatured, and reannealed, they form doublestranded DNA with single-stranded ends that are designed to anneal to each other to yield circles, an application termed recombinant circle PCR (RCPCR; see Chapter 27).
This is a preview of subscription content, log in via an institution.
Buying options
Tax calculation will be finalised at checkout
Purchases are for personal use only
Learn about institutional subscriptionsSpringer Nature is developing a new tool to find and evaluate Protocols. Learn more
References
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, in Cold Spring Harbor Symposia on Quantitative Biology, vol. 51, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, pp. 263–273.
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.
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.
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.
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
Jones, D. H., Sakamoto, K., Vorce, R. L., and Howard B. H. (1990) DNA mutagenesis and recombination. Nature 344, 793,794.
Jones, D. H. and Howard, B. H. (1991) A rapid method for recombination and site-specific mutagenesis by placing homologous ends on DNA using polymerase chain reaction. BioTechniques 10, 62–66.
Jones, D. H. and Winistorfer, S. C. (1990) Simultaneous site-specific mutagenesis of two distal sites by in vivo recombination of PCR products. Technique 2, 273–278.
Jones, D. H. and Winistorfer, S. C. (1991) Site-specific mutagenesis and DNA recombination by using PCR to generate recombinant circles in vitro or by recombination of linear PCR products in vivo. Methods-A Companion to Methods in Enzymology 2, 2–10.
Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual (2nd Ed.), Cold Spring Harbor Laboratory, Cold Spring Harbor, NY.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 1993 Humana Press Inc., Totowa, NJ
About this protocol
Cite this protocol
Jones, D.H., Winistorfer, S.C. (1993). Use of Polymerase Chain Reaction for Making Recombinant Constructs. In: White, B.A. (eds) PCR Protocols. Methods in Molecular Biology, vol 15. Humana Press, Totowa, NJ. https://doi.org/10.1385/0-89603-244-2:241
Download citation
DOI: https://doi.org/10.1385/0-89603-244-2:241
Publisher Name: Humana Press, Totowa, NJ
Print ISBN: 978-0-89603-244-6
Online ISBN: 978-1-59259-502-0
eBook Packages: Springer Protocols