Abstract
It is difficult to underestimate the impact of the polymerase chain reaction (PCR) and related DNA amplification techniques on modern molecular biology and applied molecular medicine. PCR represents a rapid, sensitive, and specific method for amplification of nucleic acid sequences and is the basis for numerous molecular techniques that have become the mainstay of the basic research laboratory, as well as the clinical diagnostics laboratory. The concept of PCR was first described in 1985 (1), and the modern technique emerged a few years later (2). Since that time, the technology has evolved into a reliable and affordable method that is performed in laboratories worldwide. When reduced to its essence, PCR is a molecular technology that facilitates the amplification of rare copies of specific nucleic acid sequences to produce a quantity of amplified product that can be analyzed. In early descriptions of PCR (1,3,4), the Klenow fragment of Escherichia coli DNA polymerase I was used for DNA synthesis during each amplification cycle. However, Klenow fragment is not thermal-stable. Therefore, after each denaturation step the samples were quickly cooled before the addition of enzyme to avoid heat denaturation of the polymerase enzyme and it was necessary to add a fresh aliquot of Klenow fragment enzyme after each denaturation cycle. In addition, the primer hybridization and DNA synthesis steps were carried out at 30°C to preserve the activity of the poly-merase enzyme, resulting in hybridization of primers to nontarget sequences and considerable nonspecific amplification (4). Even with these drawbacks, the original PCR methodology was successfully applied to gene cloning and molecular diagnostic experiments (1,3,4). The major technological breakthrough in development of PCR came with the introduction of a thermostable polymerase to PCR (2). Thermus aquaticus is a bacterium that lives in hot springs and is adapted to the variations in ambient temperature that accompany its environment. The DNA polymerase enzyme expressed by T. aquaticus (known as Taq polymerase) exhibits robust polymerase activity that is relatively unaffected by rapid fluctuations in temperature over a wide range (5). Introduction of Taq to PCR improved the practicality of this methodology. Because Taq polymerase can survive extended incubation at the elevated temperatures required for DNA denaturation (93–95°C), there is no need to add a new enzyme after each cycle. In addition, by using a heat block that automatically changes temperatures (a thermocy-cler), the PCR cycles becomes automated. Incredibly, the basic PCR technique has not changed that much since 1988 (2), although new developments in commercially available molecular reagents have made the technique easier to perform.
Keywords
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Preview
Unable to display preview. Download preview PDF.
References
Saiki, R. K., Scharf, S., Faloona, F., Mullis, K. B., Horn, G. T., Erlich, H. A., et al. Enzymatic amplification of beta-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia. Science 230:1350–1354, 1985.
Saiki, R. K., Gelfand, D. H., Stoffel, S., Scharf, S. J., Higuchi, R., Horn, G. T., et al. Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science 239: 487–491, 1988.
Mullis, K., Faloona, F., Scharf, S., Saiki, R., Horn, G., and Erlich, H. Specific enzymatic amplification of DNA in vitro: the poly-merase chain reaction. Cold Spring Harbor Symp. Quant. Biol. 51 (Pt. 1):263–273, 1986.
Mullis, K. B. and Faloona, F. A. Specific synthesis of DNA in vitro via a polymerase-catalyzed chain reaction. Methods Enzymol. 155:335–350. 1987.
Lawyer, F. C., Stoffel, S., Saiki, R. K., Myambo, K., Drummond, R., and Gelfand, D. H. Isolation, characterization, and expression in Escherichia coli of the DNA polymerase gene from Thermus aquaticus. J. Biol. Chem. 264:6427–6437, 1989.
Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., Seidman, J. G., Smith, J. A., et al., eds. Short Protocols in Molecular Biology, 5th ed., Wiley, New York, 2002.
O’Leary, J. J., Engels, K., and Dada, M. A. The polymerase chain reaction in pathology. J. Clin. Pathol. 50:805–810, 1997.
Brinkmann, B. Overview of PCR-based systems in identity testing. Methods Mol. Biol. 98:105–119, 1998.
Rogers, B. B. Application of the polymerase chain reaction to archival material. Perspect. Pediatr. Pathol. 16:99–119, 1992.
Cano, R. J., Poinar, H. N., Pieniazek, N. J.,Acra, A., and Poinar, G. O., Jr. Amplification and sequencing of DNA from a 120–135-million-year-old weevil. Nature 363:536–538, 1993.
Vieille, C. and Zeikus, G. J. Hyperthermophilic enzymes: sources, uses, and molecular mechanisms for thermostability. Microbiol. Mol. Biol. Rev. 65:1–43, 2001.
Lawyer, F. C., Stoffel, S., Saiki, R. K., Chang, S. Y., Landre, P. A., Abramson, R. D., et al. High-level expression, purification, and enzymatic characterization of full-length Thermus aquaticus DNA polymerase and a truncated form deficient in 5′ to 3′ exonuclease activity. PCR Methods Appl. 2:275–287, 1993.
Chien, A., Edgar, D. B., and Trela, J. M. Deoxyribonucleic acid polymerase from the extreme thermophile Thermus aquaticus. J. Bacteriol. 127:1550–1557, 1976.
Kaledin, A. S., Sliusarenko, A. G., and Gorodetskii, S. I. [Isolation and properties of DNA polymerase from extreme thermophylic bacteria Thermus aquaticus YT-1]. Biokhimiia 45:644–651, 1980.
Fanning, S. and Gibbs, R. A. PCR in genome analysis, in Genome Analysis. Volume 1: Analyzing DNA, Birren, B., Green, E. D., Klapholz, S., Myers, R. M., and Roskams, J., eds., Cold Spring Harbor Laboratory Press, Plainview, NY, pp. 249–299, 1997.
Perler, F. B., Kumar, S., and Kong, H. Thermostable DNA poly-merases. Adv. Protein Chem. 48:377–435, 1996.
Eckert, K. A. and Kunkel, T. A. DNA polymerase fidelity and the polymerase chain reaction. PCR Methods Appl. 1:17–24, 1991.
Tindall, K. R. and Kunkel, T. A. Fidelity of DNA synthesis by the Thermus aquaticus DNA polymerase. Biochemistry 27:6008–6013, 1988.
Mattila, P., Korpela, J., Tenkanen, T., and Pitkanen, K. Fidelity of DNA synthesis by the Thermococcus litoralis DNA polymerase— an extremely heat stable enzyme with proofreading activity. Nucleic Acids Res. 19:4967–4973, 1991.
Cariello, N. F., Swenberg, J. A., and Skopek, T. R. Fidelity of Thermococcus litoralis DNA polymerase (Vent) in PCR determined by denaturing gradient gel electrophoresis. Nucleic Acids Res. 19:4193–4198, 1991.
Lundberg, K. S., Shoemaker, D. D., Adams, M. W., Short, J. M., Sorge, J. A., and Mathur, E. J. High-fidelity amplification using a thermostable DNA polymerase isolated from Pyrococcus furiosus. Gene 108:1–6, 1991.
Bloch, W. A biochemical perspective of the polymerase chain reaction. Biochemistry 30:2735–2747, 1991.
Wu, D. Y., Ugozzoli, L., Pal, B. K., Qian, J., and Wallace, R. B. The effect of temperature and oligonucleotide primer length on the specificity and efficiency of amplification by the polymerase chain reaction. DNA Cell Biol. 10:233–238, 1991.
Frohman, M. A., Dush, M. K., and Martin, G. R. Rapid production of full-length cDNAs from rare transcripts: amplification using a single gene-specific oligonucleotide primer. Proc. Natl. Acad. Sci. USA 85:8998–9002, 1988.
Pallansch, L., Beswick, H., Talian, J., and Zelenka, P. Use of an RNA folding algorithm to choose regions for amplification by the polymerase chain reaction. Anal. Biochem. 185:57–62, 1990.
Wallace, R. B., Shaffer, J., Murphy, R. F., Bonner, J., Hirose, T., and Itakura, K. Hybridization of synthetic oligodeoxyribonucleotides to phi chi 174 DNA: the effect of single base pair mismatch. Nucleic Acids Res. 6:3543–3557, 1979.
Sarkar, G., Kapelner, S., and Sommer, S. S. Formamide can dramatically improve the specificity of PCR. Nucleic Acids Res. 18:7465, 1990.
Kaijalainen, S., Karhunen, P. J., Lalu, K., and Lindstrom, K. An alternative hot start technique for PCR in small volumes using beads of wax-embedded reaction components dried in trehalose. Nucleic Acids Res. 21:2959–2960, 1993.
Bassam, B. J. and Caetano-Anolles, G. Automated “hot start” PCR using mineral oil and paraffin wax. Biotechniques 14:30–34, 1993.
Roux, K. H. Using mismatched primer-template pairs in touchdown PCR. Biotechniques 16:812–814, 1994.
Don, R. H., Cox, P. T., Wainwright, B. J., Baker, K., and Mattick, J. S. ‘Touchdown’ PCR to circumvent spurious priming during gene amplification. Nucleic Acids Res. 19:4008, 1991.
Bachmann, B., Luke, W., and Hunsmann, G. Improvement of PCR amplified DNA sequencing with the aid of detergents. Nucleic Acids Res. 18:1309, 1990.
Casanova, J. L., Pannetier, C., Jaulin, C., and Kourilsky, P. Optimal conditions for directly sequencing double-stranded PCR products with sequenase. Nucleic Acids Res. 18:4028, 1990.
Chirgwin, J. M., Przybyla, A. E., MacDonald, R. J., and Rutter, W. J. Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease. Biochemistry 18:5294–5299, 1979.
Chomczynski, P. and Sacchi, N. Single-step method of RNA isolation by acid guanidinium thiocyanate–phenol–chloroform extraction. Anal. Biochem. 162:156–159, 1987.
Liang, P. and Pardee, A. B. Differential display of eukaryotic messenger RNA by means of the polymerase chain reaction. Science 257:967–971, 1992.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2006 Humana Press, a part of Springer Science+Business Media, LLC
About this chapter
Cite this chapter
Coleman, W.B., Tsongalis, G.J. (2006). The Polymerase Chain Reaction. In: Coleman, W.B., Tsongalis, G.J. (eds) Molecular Diagnostics. Humana Press. https://doi.org/10.1385/1-59259-928-1:047
Download citation
DOI: https://doi.org/10.1385/1-59259-928-1:047
Publisher Name: Humana Press
Print ISBN: 978-1-58829-356-5
Online ISBN: 978-1-59259-928-8
eBook Packages: MedicineMedicine (R0)