Enzymatic Amplification of DNA by PCR: Standard Procedures and Optimization

Martha F. Kramer1, Donald M. Coen1

1 Harvard Medical School, Boston, Massachusetts
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 10.20
DOI:  10.1002/0471142735.im1020s24
Online Posting Date:  May, 2001
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Abstract

This unit describes a method for amplifying DNA enzymatically by the polymerase chain reaction (PCR) and for optimizing this reaction for the sequence and primer set of interest. Important variables that can influence the outcome of PCR include the MgCl2 concentration and the cycling temperatures. Additives that promote polymerase stability and processivity or increase hybridization stringency, and strategies that reduce nonspecific primer‐template interactions, especially prior to the critical first cycle, can greatly improve sensitivity, specificity, and yield. This protocol is designed to optimize the reaction components and conditions in one or two stages. The first stage determines the optimal MgCl2 concentration and screens several enhancing additives. To further improve specificity, sensitivity and yield, the second stage compares methods for optimizing initial specific hybridization to prevent polymerization of misprimed sequences prior to thermal cycling. For initial inhibition of polymerase activity, temperature (i.e., cooling reagents), physical separation (“hot start” method), and reversible antibody binding are compared.

     
 
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Table of Contents

  • Reagents and Solutions
  • Commentary
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1:

  Materials
  • Sterile H 2O
  • 15 mM (low), 30 mM (medium), and 45 mM (high) MgCl 2
  • recipe10× MgCl 2‐free PCR amplification buffer (see recipe)
  • recipe25 mM 4dNTP mix (see recipe)
  • 50 µM oligonucleotide primer 1:50 pmol/µl in sterile H 2O (store at −20°C)
  • 50 µM oligonucleotide primer 2:50 pmol/µl in sterile H 2O (store at −20°C)
  • Template DNA: 1 µg mammalian genomic DNA/10 µl or 0.1 ng plasmid DNA/10 µl
  • 5 U/µl Taq DNA polymerase
  • DMSO, cell culture grade
  • Glycerol
  • Perfect Match Polymerase Enhancer (PMPE, Stratagene)
  • Taq pol + TaqStart: 1:1 mixture of Taq DNA polymerase and TaqStart Antibody (Clontech; store at −20°C)
  • Mineral oil
  • Ficoll 400 (optional)
  • Tartrazine dye (optional)
  • Automated thermal cycler
  • Additional reagents and equipment for agarose gel electrophoresis (unit 10.4) or sieving agarose gel electrophoresis (unit 10.5) and restriction endonuclease digestion (unit 10.8)
NOTE: Do not use DEPC to treat water or reagents.
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Figures

Videos

Literature Cited

Literature Cited
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   Chou, Q., Russell, M., Birch, D.E., Raymond, J., and Bloch, W. 1992. Prevention of pre‐PCR mis‐priming and primer dimerization improves low‐copy‐number amplifications. Nucl. Acids Res. 20:1717‐1723.
   Eckert, K.A. and Kunkel, T.A. 1990. High fidelity DNA synthesis by the Thermus aquaticus DNA polymerase. Nucl. Acids Res. 18:3739‐3752.
   Embury, S.H., Scharf, S.J., Saiki, R.K., Gholson, M.A., Golbus, M., Arnheim, N., and Erlich, H.A. 1987. Rapid prenatal diagnosis of sickle cell anemia by a new method of DNA analysis. N. Engl. J. Med. 316:656‐661.
   Finney, M., Nisson, P.E., and Rashtchian, A. 1995. Molecular cloning of PCR products. In Current Protocols in Molecular Biology (F.A. Ausubel, R. Brent, R.E. Kingston, D.D. Moore, J.G. Seidman, J.A. Smith, and K. Struhl, eds.) pp. 15.7.1‐15.7.11. John Wiley & Sons, New York.
   Gelfand, D.H. 1989. Taq DNA polymerase. In PCR Technology: Principles and Applications for DNA Amplification (H.A. Erlich, ed.) pp. 17‐22. Stockton Press, New York.
   Gyllensten, U. 1989. Direct sequencing of in vitro amplified DNA. In PCR Technology: Principles and Applications for DNA Amplification (H.A. Erlich, ed.) pp. 45‐60. Stockton Press, New York.
   Higuchi, R. 1989. Simple and rapid preparation of samples for PCR. In PCR Technology: Principles and Applications for DNA Amplification (H.A. Erlich, ed.) pp. 31‐38. Stockton Press, New York.
   Jeffreys, A.J., Wilson, V., Neumann, R., and Keyte, J. 1988. Amplification of human minisatellites by the polymerase chain reaction: Towards DNA fingerprinting of single cells. Nucl. Acids Res. 16:10,953‐10,971.
   Kellogg, D.E., Rybalkin, I., Chen, S., Mukhamedova, N., Vlasik, T., Siebert, P.D., and Chencik, A. 1994. TaqStart antibody: “Hot start” PCR facilitated by a neutralizing monoclonal antibody directed against Taq DNA polymerase. BioTechniques 16:1134‐1137.
   Kleppe, K., Ohtsuka, E., Kleppe, R., Molineux, I., and Khorana, H.G. 1971. Studies on polynucleotides. XCVI. Repair replication of short synthetic DNA's as catalyzed by DNA polymerases. J. Mol. Biol. 56:341‐361.
   Martin, C.S. 1997. Dideoxy DNA sequencing with chemiluminescent detection. In Current Protocols in Molecular Biology (F.A. Ausubel, R. Brent, R.E. Kingston, D.D. Moore, J.G. Seidman, J.A. Smith, and K. Struhl, eds.) pp. 7.4.36‐7.4.50. John Wiley & Sons, New York.
   Mueller, P.R., Garrity, P.A., and Wold, B. 1992. Ligation‐mediated PCR for genomic sequencing and footprinting. In Current Protocols in Molecular Biology (F.A. Ausubel, R. Brent, R.E. Kingston, D.D. Moore, J.G. Seidman, J.A. Smith, and K. Struhl, eds.) pp.15.5.1‐15.5.26. John Wiley & Sons, New York.
   Mullis, K.B., Faloona, F., Scharf, S.J., Saiki, R.K., Horn, G.T., and Erlich, H.A. 1986. Specific enzymatic amplification of DNA in vitro: The polymerase chain reaction. Cold Spring Harbor Symp. Quant. Biol. 51:263‐273.
   Saiki, R.K. 1989. The design and optimization of the PCR. In PCR Technology: Principles and Applications for DNA Amplification (H.A. Erlich, ed.) pp.7‐16. Stockton Press, New York.
   Saiki, R.K., Scharf, S., Faloona, F., Mullis, K., Horn, G., Erlich, H.A., and Arnheim, N. 1985. Enzymatic amplification of β‐globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia. Science 230:1350‐1354.
   Saiki, R.K., Bugawan, T.L., Horn, G.T., Mullis, K.B., and Erlich, H.A. 1986. Analysis of enzymatically amplified β‐globin and HLA‐DQα DNA with allele‐specific oligonucleotide probes. Nature 324:163‐166.
   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.
   Wittwer, C.T. and Garling, D.J. 1991. Rapid cycle DNA amplification: Time and temperature optimization. BioTechniques 10:76‐83.
Key Reference
   Saiki et al., 1988. See above.
  Demonstrates the ease and power of PCR using Taq DNA polymerase.
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