Overview of PCR

Christine D. Kuslich1, Buena Chui2, Carl T. Yamashiro3

1 Molecular Profiling Institute, Phoenix, Arizona, 2 GE Healthcare, Piscataway, New Jersey, 3 Arizona State University, Tempe, Arizona
Publication Name:  Current Protocols Essential Laboratory Techniques
Unit Number:  Unit 10.2
DOI:  10.1002/9780470089941.et1002s00
Online Posting Date:  October, 2008
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Abstract

As a means of rapidly copying and amplifying a selected template sequence from a pool of DNA in vitro, the polymerase chain reaction (PCR) as a stand‐alone technique and in combinations with other methods has a vast range of applications. This chapter provides an overview of the theory and applications for this powerful and versatile laboratory method. A generic protocol for the broadest application is described in this unit along with the basic theory underpinning PCR to foster an understanding of how to make modifications to the protocol that can be applied to specific applications of the PCR technique. There is a troubleshooting table provided to describe and resolve the most common problems associated with PCR, as well as a table for suppliers and manufacturers of thermal cycling instruments. A detailed discussion of various DNA polymerases and suppliers is also provided.

Keywords: polymerase chain reaction (PCR); dNTPs; thermal cycler; DNA polymerase; DNA template; denaturation; annealment; elongation; melting temperature (Tm); hot start; primer design; contamination

     
 
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Table of Contents

  • Overview and Principles
  • Strategic Planning
  • Safety Considerations
  • Protocols
  • Basic Protocol 1: Routine PCR
  • Support Protocol 1: Using Temperature Gradients for Rapid Optimization of PCR Cycling Conditions
  • Support Protocol 2: Titration of MgCl2 Concentration
  • Reagents and Solutions
  • Understanding Results
  • Troubleshooting
  • Variations
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Routine PCR

  Materials
  • 10% (w/v) bleach solution
  • Master mix components (see Table 10.2.5):
    • 5 U/µl DNA polymerase (e.g., Taq)
    • 10× PCR buffer with MgCl 2 (e.g., Promega, no. M8295; also see recipe) or without MgCl 2 (e.g., Promega, no. M3005; also see recipe); typically optimized for DNA polymerase of choice and provided by the manufacturer of the enzyme
    • 25 mM MgCl 2 (if not already included in PCR buffer)
    • 40 mM dNTPs (dATP, dTTP, dCTP, and dGTP; e.g., Promega, no. C1141; also see recipe)
    • 10 µm forward (upstream) and 10 µM reverse (downstream) primer (see recipe for Primers; custom synthesis available from Invitrogen)
    • Molecular‐biology‐grade, sterile, nuclease‐free ddH 2O (e.g., Invitrogen, no. 10977‐015)
  • DNA template (unit 5.2)
  • Sterile, nuclease‐free mineral oil (e.g., Sigma, no. M5904; only necessary if thermal cycler does not have heated lid)
  • Gel loading buffer/dye
  • Agarose gel
  • 100‐bp PCR ladder (e.g., Sigma, no. D3687)
  • Ethidium bromide
  • 0.2‐ml (e.g., VWR cat. no. 10011‐802) or 0.5‐ml (e.g., VWR, no. 10011836) thin‐walled reaction tubes (size dependent on thermal cycler block and manufacturer specifications)
  • Dedicated pipets used only for setting up PCR reactions (2 µl, 20 µl, 200 µl, and 1000 µl)
  • Sterile micropipettor tips (made for the pipets used for PCR reaction set up) with aerosol barrier (e.g., Rainin Instruments)
  • Vortex
  • Thermal cycler (see Table 10.2.2 for a list of manufacturers)
  • UV transilluminator
  • Additional reagents and equipment for preparing the DNA template (unit 5.2) and agarose gel electrophoresis including staining with ethidium bromide (unit 7.2)
NOTE: The time to complete reaction preparation will vary depending on the number of samples to be run. Typically setting up ten samples will take 30 to 45 min.
Table 0.2.5   MaterialsStandard PCR Reaction Mixture

Components Final concentration Per tube volume Master mix for 10 tubes (prepare for 12 tubes)
10× PCR buffer MgCl 2‐free 5 µl 60 µl
25 mM MgCl 2 a 1.5 mM 3 µl 36 µl
40 mM dNTP mix b 0.2 mM each dNTP 1 µl 12 µl
10 µM forward primer 1 µM c 5 µl 60 µl
10 µM reverse primer 1 µM c 5 µl 60 µl
Sterile, nuclease‐free H 2O 30.75 µl (to a final volume of 50 µl) 369 µl
5 U/µl hot‐start Taq DNA polymerase d 0.025 U/µl 0.25 µl 3 µl
>100 ng/µl template e 2 ng/µl 1 µl

 aMgCl 2 concentration can be titrated to optimize the PCR reaction. MgCl 2 concentration ranges between 1.5 and 4.0 mM for most PCR.
 bThere are numerous commercially available dNTP mixes. Most are 40 mM (10 mM of each of the 4 dNTPs).
 cPrimer concentration can also be titrated to optimize the PCR reaction. Final primer concentrations generally can be adjusted within the range of 0.2 to 1 µM.
 dConcentrations of Taq will vary. When using a non‐hot‐start polymerase, the enzyme should not be added to the reaction vessel until the tubes are at 95°C in the thermal cycler to avoid the formation of nonspecific products.
 eTemplate DNA concentration can vary, but generally the final concentration of the template DNA will not exceed 10 ng/µl. The technique is sensitive enough to detect pmol quantities of template.

Support Protocol 1: Using Temperature Gradients for Rapid Optimization of PCR Cycling Conditions

  • Thermal cycler with temperature gradient function
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Figures

Videos

Literature Cited

   Ballabio, A., Ranier, J.E., Chamberlain, J.S., Zollo, M., and Caskey, C.T. 1990. Screening for steroid sulfatase (STS) gene deletions by multiplex DNA amplification. Hum. Genet. 84:228‐232.
   Baskaran, N., Kandpal, R.P., Bhargava, A.K., Glynn, M.W., Bale, A., and Weissman, S.M. 1996. Uniform amplification of a mixture of deoxyribonucleic acids with varying GC content. Genome Res. 6:633‐638.
   Bassam, B.J. and Caetano‐Anolles, G. 1993. Automated “hot start” PCR using mineral oil and paraffin wax. BioTechniques 14:30‐34.
   Birch, D.E. 1996. Simplified Hot Start PCR. Nature 381:445‐446.
   Breslauer, K.J., Frank, R., Blocker, H., and Marky, L.A. 1986. Predicting DNA duplex stability from the base sequence. Proc. Nat. Acad. Sci. U.S.A. 83:3746‐3750.
   Brooks‐Wilson, A.R., Goodfellow, P.N., Povey, S., Nevanlinna, H.A., deJong, P.J., and Goodfellow, P.J. 1990. Rapid cloning and characterization of new chromosome 10 DNA markers by Alu element‐mediated PCR. Genomics 7:614‐620.
   Chakrabarti, R. and Schutt, C.E. 2001a. The enhancement of PCR amplification by low molecular‐weight sulfones. Gene. 274:293‐298.
   Chakrabarti, R. and Schutt, C.E. 2001b. The enhancement of PCR amplification by low molecular weight amides. Nucl. Acids Res. 29:2377‐2381.
   Cheng, S., Chang, S.Y., Gravitt, P., and Respess, R. 1994. Long PCR. Nature 369:684‐685.
   Cheng, S., Fockler, C., Barnes, W.M., and Higuchi, R. 1994. Effective amplification of long targets from cloned inserts and human genomic DNA. Proc. Natl. Acad. Sci. U.S.A.. 91:5695‐5699.
   Cimino, G.D., Metchette, J.W., Tessman, J.W., Hearst, J.E., and Isaacs, S.T. 1991. Post‐PCR sterilization: A method to control carryover contamination for the polymerase chain reaction. Nucl. Acids Res. 19:99‐107.
   Dang, C. and Jayasena, S.D. 1996. Oligonucleotide inhibitors of Taq DNA polymerase facilitate detection of low copy number targets by PCR. J. Mol. Biol. 264:268‐278.
   D'Aquila, R.T., Bechtel, L.J., Videler, J.A., Eron, J.J., Gorczyca, P., and Kaplin, J.A. 1991. Maximizing sensitivity and specificity of PCR by pre‐amplification heating. Nucl. Acids Res. 19:3749.
   Don, R.H., Cox, P.T., Wainwright, B.J., Baker, K., and Mattick, J.S. 1991. ‘Touchdown’ PCR to circumvent spurious priming during gene amplification. Nucl. Acids Res. 19:4008.
   Ehrlich, H.A., Gelfand, D.H., and Sninsky, J.J. 1991. Recent advances in the polymerase chain reaction. Science 252:1643‐1651.
   Eldadah, Z.A., Asher, D.M., Godec, M.S., Pomeroy, K.L., Goldfarb, L.G., Feinstone, S.M., Levitan, H., Gibbs, C.J. Jr., and Gajdusek, D.C. 1991. Detection of flaviviruses by reverse‐transcriptase polymerase chain reaction. J. Med. Virol. 33:260‐267.
   GE Healthcare. 2006. Increased polymerase chain reaction (PCR) amplification specificity using illustra Hot Start Master Mix. Application Note 28‐4073‐38 AB.
   Hebert, J.M., Basilico, C., Goldfarb, M., Haub, O., and Martin, G.R. 1990. Isolation of cDNAs encoding four mouse FGF family members and characterization of their expression patterns during embryogenesis. Dev. Biol. 138:454‐463.
   Henke, W., Herdel, K., Jung, K., Schnorr, D., and Loening, S.A. 1997. Betaine improves the PCR amplification of GC‐rich DNA sequences. Nucl. Acids Res. 25:3957‐3958.
   Herman, J.G., Graff, J.R., Myohanen, S., Nelkin, B.D., and Baylin, S.B. 1996. Methylation‐specific PCR: A novel PCR assay for methylation status of CpG islands. Proc. Natl. Acad. Sci. U.S.A. 93:9821‐9826.
   Higuchi, R., Fockler, C., Dollinger, G., and Watson, R. 1993. Kinetic PCR analysis: Real‐time monitoring of DNA amplification reactions. Biotechnology 11:1026‐1030.
   Innis, M., Gelfand, D., and Sninsky, J. 1999. PCR Applications, Protocols for Functional Genomics. Academic Press, San Diego, California.
   Kellogg, D.E., Rybalkin, I., Chen, S., Mukhamedova., N., Vlasik, T., Siebert, P.D., and Chenchik, A. 1994. TaqStart Antibody: “hot start” PCR facilitated by a neutralizing monoclonal antibody directed against Taq DNA polymerase. BioTechniques 16:1134‐1137.
   Korn, S.H., Moerkerk, P.T., and de Goeij, A.F. 1993. K‐ras point mutations in routinely processed tissues: non‐radioactive screening by single strand conformational polymorphism analysis. J. Clin. Pathol. 46:621‐623.
   Kreader, C.A. 1996. Relief of amplification inhibition in PCR with bovine serum albumin or T4 gene 32 protein. Appl. Environ. Microbiol. 62:1102‐1106.
   Kwok, S. and Higuchi, R. 1989. Avoiding false positives with PCR. Nature 339:237‐238.
   Lawyer, F.C., Stoffel, S., Saiki, R.K., Myambo, K., Drummond, R., and Gelfand, D.H. 1989. Isolation, characterization, and expression in Escherichia coli of the DNA polymerase gene from Thermus aquaticus. J. Biol. Chem. 264:6427‐6437.
   Liang, P. and Pardee, A.B. 1992. Differential display of eukaryotic messenger RNA by means of the polymerase chain reaction. Science 257:967‐971.
   Lin, Y. and Jayasena, S.D. 1997. Inhibition of multiple thermostable DNA polymerases by a heterodimeric aptamer. J. Mol. Biol. 271:100‐111.
   Lin‐Goerke, J.L., Robbins, D.J., and Burczak, J.D. 1997. PCR‐based random mutagenesis using manganese and reduced dNTP concentration. Biotechniques 23:409‐412.
   Lundberg, K.S., Shoemaker, D.D., Adams, M.W., Short, J.M., Sorge, J.A., and Mathur, E.J. 1991. High‐fidelity amplification using a thermostable DNA polymerase isolated from Pyrococcus furiosus. Gene 108:1‐6.
   Mattila, P., Korpela, J., Tenkanen, T., and Pitkanen, K. 1991. Fidelity of DNA synthesis by the Thermococcus litoralis DNA polymerase–an extremely heat stable enzyme with proofreading activity. Nucl. Acids Res. 19:4967‐4973.
   Melissis, S., Labrou, N.E., and Clonis, Y.D. 2006. Nucleotide‐mimetic synthetic ligands for DNA‐recognizing enzymes One‐step purification of Pfu DNA polymerase. J. Chromatogr. A. 1122:63‐75.
   Melissis, S., Labrou, N.E., and Clonis, Y.D. 2007. One‐step purification of Taq DNA polymerase using nucleotide‐mimetic affinity chromatography. Biotechnol. J. 2:121‐132.
   Mifflin, T.E. 1995. Setting up a PCR laboratory. In PCR Primer: A Laboratory Manual. (C. Dieffenbach and G. Dveksler, eds.) pp. 5‐14. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
   Mifflin, T.E. 1997. Control of contamination associated with PCR and other amplification reactions. http://www.mbpinc.com/html/pdf/techreport/MifflinReport.pdf.
   Morin, P.A. and Smith, D.G. 1995. Nonradioactive detection of hypervariable simple sequence repeats in short polyacrylamide gels. Biotechniques 19:223‐228.
   Mullis, K.B., Faloona, F.A., Scharf, S., Saiki, R.K., Horn, G., and Erlich, H.A. 1986. Specific enzymatic amplification of DNA in vitro: The polymerase chain reaction. Cold Spring Harb. Symp. Quant. Biol. 51 Pt 1:263‐273.
   Musso, M., Bocciardi, R., Parodi, S., Ravazzolo, R., and Ceccherini, I. 2006. Betaine, dimethyl sulfoxide, and 7‐deaza‐dGTP, a powerful mixture for amplification of GC‐rich DNA sequences. J. Mol. Diagn. 8:544‐550.
   Ochman, H., Gerber, A.S., and Hartl, D.L. 1988. Genetic applications of an inverse polymerase chain reaction. Genetics 120:621‐623.
   Parker, L.T., Zakeri, H., Deng, Q., Spurgeon, S., Kwok, P.‐Y., and Nickerson, D.A. 1996. AmpliTaq DNA Polymerase, FS dye‐terminator sequencing: Analysis of peak height patterns. BioTechniques 21:694‐699.
   Rychlik, W., Spencer, W.J., and Rhoads, R.E. 1990. Optimization of the annealing temperature for DNA amplification in vitro. Nucl. Acids Res. 18:6409‐6412.
   Saiki, R.K., Scharf, S., Faloona, F., Mullis, K.B., Horn, G.T., Erlich, H.A., and Arnheim, N. 1985. Enzymatic amplification of beta‐globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia. Science 230:1350‐1354.
   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.
   Scharf, S.J., Horn, G.T., and Erlich, H.A. 1986. Direct cloning and sequence analysis of enzymatically amplified genomic sequences. Science 233:1076‐1078.
   Sharkey, D.J., Scalice, E.R., Christy, K.G. Jr., Atwood, S.M., and Daiss, J.L. 1994. Antibodies as thermolabile switches: High temperature triggering for the polymerase chain reaction. Biotechnology 12:506‐509.
   Shyamala, V. and Ames, G.F. 1989. Amplification of bacterial genomic DNA by the polymerase chain reaction and direct sequencing after asymmetric amplification: application to the study of periplasmic permeases. J. Bacteriol. 171:1602‐1608.
   Singh, B., Cox‐Singh, J., Miller, A.O., Abdullah, M.S., Snounou, G., and Rahman, H.A. 1996. Detection of malaria in Malaysia by nested polymerase chain reaction amplification of dried blood spots on filter‐paper. Trans. R. Soc. Trop. Med. Hyg. 90:519‐521.
   Schneeberger, C., Speiser, P., Kury, F., and Zeillinger, R. 1995. Quantitative detection of reverse transcriptase‐PCR products by means of a novel and sensitive DNA stain. P.C.R. Methods Appl. 4:234‐238.
   Sugimoto, N., Nakano, S., Yoneyama, M., and Honda, K. 1996. Improved thermodynamic parameters and helix initiation factor to predict stability of DNA duplexes. Nucl. Acids Res. 24:4501‐4505.
   Tabor, S. and Richardson, C.C. 1995. A single residue in DNA polymerases of the Escherichia coli DNA polymerase I family is critical for distinguishing between deoxy‐ and dideoxyribonucleotides. Proc. Natl. Acad. Sci. U.S.A. 92:6339‐6343.
   Telenius, H., Carter, N.P., Bebb, C.E., Nordenskjold, M., Ponder, B.A., and Tunnacliffe, A. 1992. Degenerate oligonucleotide‐primed PCR: General amplification of target DNA by a single degenerate primer. Genomics 13:718‐725.
   Thein, S.L. and Wallace, R.B. 1986. Human genetic diseases: A practical approach (K.E. Davis, ed.) pp. 33‐50. IRL Press, Oxford, U.K.
   Thornton, C.G., Hartley, J.L., and Rashtakian, A. 1992. Use of uracil DNA glycosylase to control carryover contamination in the polymerase chain reaction. BioTechniques 13:80‐82.
   Vos, P., Hogers, R., Bleeker, M., Reijans, M., van der Lee, T., Hornes, M., Frijters, A., Pot, J., Peleman, J., Kuiper, M., and Zabeau, M. 1995. AFLP: A new technique for DNA fingerprinting. Nucl. Acids Res. 23:4407‐4414.
   Wainwright, L.A. and Seifert, H.S. 1993. Paraffin beads can replace mineral oil as an evaporation barrier in PCR. BioTechniques 14:34‐36.
   Williams, J.G., Kubelik, A.R., Livak, K.J., Rafalski, J.A., and Tingey, S.V. 1990. DNA polymorphisms amplified by arbitrary primers are useful as genetic markers. Nucl. Acids Res. 18:6531‐6535.
   Zon, L.I., Dorfman, D.M., and Orkin, S.H. 1989. The polymerase chain reaction colony miniprep. BioTechniques 7:696‐698.
Key References
   Mullis et al., 1986. See above.
  The three key references provided above are historical in nature and illustrate some of the noteworthy efforts made early on in the development of this prevalent technique.
   Saiki et al., 1985. See above.
   Saiki et al., 1988. See above.
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