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 Pharmacology
Unit Number:  Unit 6.7
DOI:  10.1002/0471141755.ph0607s13
Online Posting Date:  August, 2001
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Abstract

This unit describes a method for amplifying DNA enzymatically by the polymerase chain reaction (PCR), including procedures to quickly determine conditions for successful amplification of the sequence and primer sets of interest, and to optimize for specificity, sensitivity, and yield. Hot‐start methods are described which can greatly improve specificity, sensitivity, and yield. This protocol suggests some relatively inexpensive methods to achieve hot start, and lists several commercial hot‐start options which may be more convenient, but of course more expensive. The unit has recently been updated to include new information on reagents to enhance the reaction, better cycling parameters, and innovations in robotics and high‐performance thermocyclers.

     
 
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Table of Contents

  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Tables
     
 
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Materials

Basic Protocol 1:

  Materials
  • Sterile H 2O
  • 15 mM (L), 30 mM (M), and 45 mM (H) MgCl 2
  • 10× MgCl 2‐free PCR amplification buffer (see recipe)
  • 25 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 or 1.0 to 100.0 pg of plasmid DNA
  • 5 U/µl Taq DNA polymerase (native or recombinant; many suppliers)
  • Enhancer agents (optional; see recipe)
  • TaqStart Antibody (Clontech)
  • Mineral oil
  • Ficoll 400 (optional)
  • Tartrazine dye (optional)
  • Thin‐walled PCR tubes
  • Automated thermal cycler
NOTE: See Ausubel et al. ( ) for additional reagents and equipment for DNA preparation, agarose gel electrophoresis, nondenaturing PAGE, or sieving agarose gel electrophoresis, restriction endonuclease digestion, and Southern blotting and hybridization.NOTE: Do not use DEPC to treat water, reagents, or glassware.NOTE: Reagents should be prepared in sterile, disposable labware, taken directly from its packaging, or in glassware that has been soaked in 10% bleach, thoroughly rinsed in tap water followed by distilled water, and if available, exposed to UV irradiation for ∼10 min. Multiple small volumes of each reagent should be stored in screw‐cap tubes. This will then serve as the user's own optimization “kit.” Thin‐walled PCR tubes are recommended.
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Figures

Videos

Literature Cited

   Ausubel, F.A., Brent, R., Kingston, R.E., Moore, D.D., Seidman, J.G., Smith, J.A., and Struhl, K. (eds.). 2001. Current Protocls in Molecular Biology. John Wiley and Sons, New York.
   Beck, S. 1998. How low can you go? Nineteen thermal cyclers priced under $5000. The Scientist 12:19‐20.
   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.
   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.
   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.
   Rees, W.A., Yager, T.D., Korte, J., and von Hippel, P.H. 1993. Betaine can eliminate the base pair composition dependence of DNA melting. Biochemistry 32:137‐144.
   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.
Key Reference
   Saiki et al., 1988. See above.
  Demonstrates the ease and power of PCR using Taq DNA polymerase.
Internet Resources
  http://www.promega.com/techserve/
   Offers Amplification Assistant, a PCR troubleshooting program.
  http://www.genome.wi.mit.edu/
  Provides access to www Primer Picking (Primer 3); select experimental web‐based software under Genome Center Software.
  http://www.alkami.com/primers/idxprmr.htm
  Contains primer design tools and tips.
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