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 Toxicology
Unit Number:  Appendix 3C
DOI:  10.1002/0471140856.txa03cs03
Online Posting Date:  May, 2001
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Abstract

The polymerase chain reaction (PCR) is used to enzymatically amplify small quantities of specific DNA sequences. The reaction must optimized to specifically amplify the sequences and primers of interest; this includes titration of magnesium chloride and selection of enhancing agents, if appropriate, to minimize nonspecific primer target interactions and maximize the specificity, sensitivity, and yield.

     
 
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Table of Contents

  • Reagents and Solutions
  • Commentary
  • 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 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.
  • 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 ( appendix 3A)
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

Literature Cited
   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 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 .
   Demonstrates the ease and power of PCR using Taq DNA polymerase.
Internet Resources
   http://www.pebio.com
   Most companies can be accessed at “http://company‐name.com”. Many such company Web sites provide protocols, information on primer design, and other PCR assistance.
   http://www.promega.com
   http://www.stratagene.com
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