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 Cytometry
Unit Number:  Appendix 3K
DOI:  10.1002/0471142956.cya03ks37
Online Posting Date:  August, 2006
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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. The first step of PCR simply entails mixing template DNA, two appropriate oligonucleotide primers, Taq or other thermostable DNA polymerases, deoxyribonucleoside triphosphates (dNTPs), and a buffer. Once assembled, the mixture is cycled many times (usually 30) through temperatures that permit denaturation, annealing, and synthesis to exponentially amplify a product of specific size and sequence. The PCR products are then displayed on an appropriate gel and examined for yield and specificity. Recommended optimization conditions are included.

     
 
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Table of Contents

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

Basic Protocol 1:

  Materials
  • recipe10× MgCl 2‐free PCR buffer (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
  • recipe25 mM 4dNTP mix (see recipe)
  • 5 U/µl Taq DNA polymerase (native or recombinant)
  • recipeEnhancer agents (optional; see recipe)
  • 15 mM (L), 30 mM (M), and 45 mM (H) MgCl 2
  • Mineral oil
  • TaqStart Antibody (Clontech)
  • Ficoll 400 (optional): prepare as 10× stock; store indefinitely at room temperature
  • Tartrazine dye (optional): prepare as 10× stock; store indefinitely at room temperature
  • 0.5 ml 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 (Ausubel et al., )
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.M., Brent, R., Kingston, R.E., Moore, D.D., Seidman, J.G., Smith, J.A., and Struhl, K. (eds.) 2006. Current Protocols in Molecular Biology. John Wiley & Sons, Hoboken, N.J.
   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: Princi ples 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/amplification/amptech.html
  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/
  Contains free primer design tools and tips.
  http://bioinformatics.weizmann.ac.il/mb/bioguide/pcr/contents.html
  Contains useful tips and links.
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