Constructing Recombinant DNA Molecules by PCR

Elaine A. Elion1, Pablo Marina1, Lu Yu1

1 Harvard Medical School, Boston, Massachusetts
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 3.17
DOI:  10.1002/0471142727.mb0317s78
Online Posting Date:  April, 2007
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Abstract

This unit describes the use of PCR to construct hybrid DNA molecules. The unit provides an overview of how PCR can be exploited to accomplish numerous cloning strategies. The Basic Protocol outlines the PCR amplification and cloning strategies. The Commentary includes a troubleshooting guide for problems most frequently encountered in PCR cloning, plus four specific examples of the application of this technique to create inā€frame fusion proteins, to create recombinant DNA products, to generate deletions and inversions by inverse PCR, and to introduce mutagenized PCR products or specific mutations or fusions by gap repair in yeast.

Keywords: PCR; recombinant DNA; insertions; deletions; fusion proteins

     
 
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Table of Contents

  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1:

  Materials
  • Template DNA (1 to 10 ng of plasmid or phage DNA; 20 to 300 ng of genomic or cDNA)
  • Oligonucleotide primers (0.2 to 1.0 µM; unit 8.5)
  • Mineral oil (optional)
  • TE‐buffered phenol (unit 2.1) and chloroform
  • 100% ethanol
  • TE buffer, pH 8.0 ( appendix 22)
  • Klenow fragment of E. coli DNA polymerase I (unit 3.5)
  • Vector DNA
  • Calf intestinal phosphatase (unit 3.10)
  • Thin‐walled plastic PCR tubes
  • Additional reagents and equipment for phosphorylating synthetic oligonucleotides (unit 3.10), amplification of DNA by PCR (unit 15.1), agarose and polyacrylamide gel electrophoresis (units 2.5& 2.7), DNA extraction and precipitation (unit 2.1), purification of DNA by glass beads, electroelution from agarose gels, or from low gelling/melting temperature agarose gels (unit 2.6), repair of 3′ overhangs (unit 3.5), restriction endonuclease digestion (unit 3.1), dephosphorylating oligonucleotides (unit 3.10), ligation of DNA fragments (unit 3.16), transformation of E. coli (unit 1.8), plasmid DNA minipreps (unit 1.6), and DNA sequence analysis (unit 7.4)
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Figures

Videos

Literature Cited

   Arnheim, N. and Erlich, H. 1992. Polymerase chain reaction strategy. Annu. Rev. Biochem. 61:131‐156.
   Cariello, N.F., Swenberg, J.A., and Skopek, T.R. 1991. Fidelity of Thermococcus litoralis DNA polymerase (Vent) in PCR determined by denaturing gradient gel electrophoresis. Nucl. Acids Res. 19:4193‐4198.
   Clark, J.M. 1988. Novel nontemplated nucleotide addition reactions catalyzed by procaryotic and eucaryotic DNA polymerases. Nucl. Acids Res. 16:9677‐9689.
   D'Aquila, R.T., Bechtel, L.J., Videler, J.A., Eron, J.J., Gorczyca, P., and Kaplan, J.C. 1991. Maximizing sensitivity and specificity of PCR by pre‐amplification heating. Nucl. Acids Res. 19:3749.
   Fucharoen, S., Fucharoen, G., Fucharoen, P., and Fukamaki, Y. 1989. A novel ochre mutation in the beta‐thalassemia gene of a Thai identified by direct cloning of the entire beta‐globin gene amplified using polymerase chain reactions. J. Biol. Chem. 264:7780‐7783.
   Goodenow, M., Huet, T., Saurin, W., Kwok, S., Sninsky, J., and Wain‐Hobson, S. 1989. HIV‐1 isolates are rapidly evolving quasi‐species: Evidence for viral mixtures and preferred nucleotide substitutions. J. Acquired Immunol. Defic. Syndr. 2:344‐352.
   Innis, M.A., Gelfand, D.H., Sninsky, J.J., and White, T.J. (eds.) 1990. PCR Protocols. Academic Press, San Diego.
   Jung, V., Pestka, S.B., and Pestka, S. 1990. Efficient cloning of PCR‐generated DNA containing terminal restriction endonuclease sites. Nucl. Acids Res. 18:6156.
   Kohler, S.W., Provost, G.S., Fieck, A., Kretz, P.L., Bullock, W.O., Sorge, J.A., Putman, D.L., and Short, J.M. 1991. Spectra of spontaneous and mutagen‐induced mutations in the lacI gene in transgenic mice. Proc. Natl. Acad. Sci. U.S.A. 88:7958‐7962.
   Kornberg, A. and Baker, T.A. 1992. DNA Replication 2nd ed. W.H. Freeman, New York.
   Ma, H., Kunes, S., Schatz, P.J., and Botstein, D. 1987. Plasmid construction by homologous recombination in yeast. Gene 55:201‐216.
   Muhlrad, D., Hunter, R., and Parker, R. 1992. A rapid method for localized mutagenesis of yeast genes. Yeast 8:79‐82.
   Mullis, K.B. and Faloona, F.A. 1987. Specific synthesis of DNA in vitro via a polymerase‐catalyzed chain reaction. Methods Enzymol. 155:335‐350.
   Myers, R.M., Fischer, S.G., Maniatis, T., and Lerman, L.S. 1985. Modification of the melting properties of duplex DNA by attachment of a GC‐rich DNA sequence as determined by denaturing gradient gel electrophoresis. Nucl. Acids Res. 13:3111‐3129.
   Ochman, H., Medhora, M.M., Garza, D., and Hartl, D.L. 1990. Amplification of flanking sequences by inverse PCR. In PCR Protocols. (M.A. Innis, D.H. Gelfand, J.J. Sninsky, and T.J. White, eds.) pp. 219‐227. Academic Press, San Diego, Calif.
   Sheffield, V.C., Cox, D.R., Lerman, L.S., and Myers, R.M. 1989. Attachment of a 40 base pair G+C‐rich sequence (GC clamp) to genomic DNA fragments by the polymerase chain reaction results in improved detection of single‐base changes. Proc. Natl. Acad. Sci. U.S.A. 86:232‐236.
Key Reference
   Innis et al., 1990. See above
  Provides an in‐depth analysis of PCR methods and techniques.
Internet Resources
   http://www.bio.com/protocolstools/protocol.jhtml?id=p213
  Rine, J. High‐fidelity DNA amplification by PCR using Vent, Pwo, and Pfu DNA polymerases. bioProtocol, a Bio Online Site.
   http://www.zbiomed.com/
  Z‐BioMed welcome page, which includes a program for calculating the expected rate of PCR‐induced mutations using various DNA polymerases, depending on target size and cycle number (as shown in Table ).
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