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 Cell Biology
Unit Number:  Appendix 3F
DOI:  10.1002/0471143030.cba03fs10
Online Posting Date:  May, 2001
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Abstract

This appendix 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.

     
 
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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 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
  • Additional reagents and equipment for DNA preparation ( appendix 3A), agarose gel electrophoresis ( appendix 3A), nondenaturing PAGE (unit 6.5), or sieving agarose gel electrophoresis ( appendix 3A), restriction endonuclease digestion ( appendix 3A), 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 mispriming 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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