Advertisement

Polymerase Chain Reaction

Basic Principles and Routine Practice
  • Lori A. Kolmodin
  • David E. Birch
Part of the Methods in Molecular Biology™ book series (MIMB, volume 192)

Abstract

The polymerase chain reaction (PCR) is a primer-mediated enzymatic amplification of specifically cloned or genomic DNA sequences (1). This PCR process, invented more than a decade ago, has been automated for routine use in laboratories worldwide. The template DNA contains the target sequence, which may be tens or tens of thousands of nucleotides in length. A thermostable DNA polymerase such as Taq DNA polymerse, catalyzes the buffered reaction in which an excess of an oligonucleotide primer pair and four deoxynucleoside triphosphates (dNTPs) are used to make millions of copies of the target sequence. Although the purpose of the PCR process is to amplify template DNA, a reverse transcription step allows the starting point to be RNA (2-5).

Keywords

Polymerase Chain Reaction Polymerase Chain Reaction Product Polymerase Chain Reaction Amplification Polymerase Chain Reaction Cycle Polymerase Chain Reaction Process 
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.

References

  1. 1.
    Innes, M. A., Gelfand, D. H., Sninsky, J. J. and White, T. J., eds. (1990) PCR Protocols, A Guide to Methods and Application, Academic, San Diego, CA.Google Scholar
  2. 2.
    Mullis, K. B. and Faloonam F. A. (1987) Specific synthesis of DNA in vitro via a polymerase chain reaction. Meth. Enzymol. 155, 335–350.PubMedCrossRefGoogle Scholar
  3. 3.
    Saiki, R. K., Gelfand, D. H., Stoffel, S., Scharf, S. J., Higuchi, R., Horn, G. T., et al. (1988) Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science 239, 487–491.PubMedCrossRefGoogle Scholar
  4. 4.
    Saiki, R. K., Scharf, S. J., Faloona, F., Mullis, K. B., Horn, G. T., 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.PubMedCrossRefGoogle Scholar
  5. 5.
    Scharf, S. J., Horn, G. T., and Erlich, H. A. (1986) Direct cloning and sequence analysis of enzymatically amplified genomic sequences. Science 233, 1076–1087.PubMedCrossRefGoogle Scholar
  6. 6.
    Wang, A. M., Doyle, M. V., and Mark, D. F. (1989) Quantitation of mRNA by the polymerase chain reaction. Proc. Na.tAcad. Sci. USA 86, 9717–9721. Nature 1.CrossRefGoogle Scholar
  7. 7.
    Kwok, S. and Higuchi, R. (1989) Avoiding false positives with PCR. Nature 339, 237, 238.PubMedCrossRefGoogle Scholar
  8. 8.
    Orrego, C. (1990) Organizing a laboratory for PCR work, in PCR Protocols. A Guide to Methods and Applications (Innis, M. A., Gelfand, D. H., Sninsky, J. J., and White, T. J., eds.), Academic, San Diego, CA, pp. 447–454.Google Scholar
  9. 9.
    Kitchin, P. A., Szotyori, Z., Fromholc, C., and Almond, N. (1990) Avoiding false positives. Nature 344, 201.PubMedCrossRefGoogle Scholar
  10. 10.
    Longo, N., Berninger, N.S., and Hartley, J. L. (1990) Use of uracil DNA glycosylase to control carry-over contamination in polymerase chain reactions. Gene 93, 125–128.PubMedCrossRefGoogle Scholar
  11. 11.
    Chou, Q., Russell, M., Birch, D. E., Raymond, J., and Bloch, W. (1992) Prevention of prePCR mis-priming and primer dimerization improves low-copy-number amplifications. Nucl. Acids Res. 20, 1717–1723.PubMedCrossRefGoogle Scholar
  12. 12.
    Birch, D. E., Kolmodin, L., Laird, W. J., McKinney, N., Wong, J., Young, K. K. Y., et al. (1996) Simplified Hot Start PCR. Nature 381, 445,446.PubMedCrossRefGoogle Scholar
  13. 13.
    Ailenberg, M. and Silverman, M. (2000) Controlled hot start and improved specificity in carrying out PCR utilizing touch-up and loop incorporated primers (TULIPS). Biotechniques 29, 1018–1020, 1022-1024.PubMedGoogle Scholar
  14. 14.
    Kaboev, O. K., Luchkina, L. A., Tret′iakov, A. N., and Bahrmand, A. R. (2000) PCR hot start using primers with the structure of molecular beacons (hairpin-like structure). Nucl. Acids Res. 28, E94.PubMedCrossRefGoogle Scholar
  15. 15.
    Kainz, P., Schmiedlechner, A., and Strack, H. B. (2000) Specificity-enhanced hot-start PCR: addition of double-stranded DNA fragments adapted to the annealing temperature. Biotechniques 28, 278–82.PubMedGoogle Scholar
  16. 16.
    Dang, C. and Jayasena, S. (1996) Oligonucleotide inhibitors of Taq DNA polymerase facilitate detection of low copy number targets by PCR. J. Molec. Biol. 264, 268–278.PubMedCrossRefGoogle Scholar
  17. 17.
    Innis, M. A., Myambo, K. B., Gelfand, D. H., and Brow, M. A. D. (1988) DNA sequencing with Thermus aquaticus DNA polymerase and direct sequencing of polymerase chain reaction-amplified DNA. Proc. Nat. Acad. Sci. USA 85, 9436–9440.PubMedCrossRefGoogle Scholar
  18. 18.
    Abramson, R. D. (1995) Thermostable DNA polymerases, in PCR Strategies (Innes, M. A., Gelfand, D. H., and Sninsky, J. J., eds.), Academic, San Diego, CA, pp. 39–57.Google Scholar
  19. 19.
    Holland, P. M., Abramson, R. D., Watson, R., and Gelfand, D. H. (1991) Detection of specific polymerase chain reaction product by utilizing the 5′-3′ exonuclease activity of Thermus aquaticus DNA polymerase. Proc. Nat. Acad. Sci. USA 88, 7276–7280.PubMedCrossRefGoogle Scholar
  20. 20.
    Sobral, B. W. S. and Honeycutt, R. J. (1993) High output genetic mapping of polyploids using PCR generated markers. Theor. Appl. Genet. 86, 105–112.CrossRefGoogle Scholar
  21. 21.
    Myers, T. W. and Gelfand, D. H. (1991) Reverse transcription and DNA amplification by a Thermus thermophilus DNA polymerase. Biochemistry 30, 7661–7666.PubMedCrossRefGoogle Scholar
  22. 22.
    Myers, T. W. and Sigua, C. L. (1995) Amplification of RNA, in PCR Strategies (Innes, M. A., Gelfand, D. H., and Sninsky, J. J., eds.), Academic, San Diego, CA, pp. 58–58.Google Scholar
  23. 23.
    Cheng, S., Fockler, C., Barnes, W. M., and Higuchi, R. (1994) Effective amplification of long targets from cloned inserts and human genomic DNA. Proc Nat Acad Sci USA 91, 5695–5699.PubMedCrossRefGoogle Scholar
  24. 24.
    Cheng, S., Chen, Y., Monforte, J. A., Higuchi, R., and Van Houten, B. (1995) Template integrity is essential for PCR amplification of 20-to 30-kb sequences from genomic DNA. PCR Meth. Amplificat. 4, 294–298.Google Scholar
  25. 25.
    Erlich, H. A., ed. (1989) PCR Technology, Principles and Application for DNA Amplification. Stockton, New York.Google Scholar
  26. 26.
    Landre, P. A., Gelfand, D. H., and Watson, R. H. (1995) The use of cosolvents to enhance amplification by the polymerase chain reaction, in PCR Strategies (Innes, M. A., Gelfand, D. H., and Sninsky, J. J., eds.), Academic, San Diego, CA, pp. 3–16.Google Scholar
  27. 27.
    Henke, W., Herdel, K., Jung, K. Schnorr, D., and Loening, S. (1997) Betaine improves the PCR amplification of GC-rich DNA sequences. Nucl. Acids Res. 25 (19), 3957–3958.PubMedCrossRefGoogle Scholar
  28. 28.
    Paabo, S., Gifford, J. A., and Wilson, A. C. (1988) Mitochondrial DNA sequences from a 7000-year old brain. Nucl. Acids Res. 16, 9775–9787.PubMedCrossRefGoogle Scholar
  29. 29.
    Sarker, G, Kapeiner, S., and Sommer, S. S. (1990) Formamide can drastically increase the specificity of PCR. Nucl. Acid Res. 18, 7465.CrossRefGoogle Scholar
  30. 30.
    Smith, K. T., Long, C. M., Bowman, B. and Manos, M. M. (1990) Using cosolvents to enhance PCR amplification. Amplifications 9/90 (5), 16,17.Google Scholar
  31. 31.
    Kreader, C. (1996) Relief of amplification inhibition in PCR with bovine serum albumin or T4 gene 32 protein. Appl. Environ. Microbiol. 62, 1102–1106.PubMedGoogle Scholar
  32. 32.
    Kovarova, M. and Draber, P. (2000) New specificity and yield enhancer of polymerase chain reactions. Nucl. Acids Res. 28, E70.PubMedCrossRefGoogle Scholar
  33. 33.
    AmpliTaq Gold. Package Insert. BIO-142, 54, 670–3/96. Applied Biosystems, Foster City, CA.Google Scholar
  34. 34.
    Sambrook, J., Fritsch, E. F., and Maniatis, T. eds. (1989) Molecular Cloning: A Laboratory Manual, 2nd ed. Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, NY, pp. 6.20, 6.21, B.23, B.24.Google Scholar
  35. 35.
    Saiki, R. K., Walsh, P. S., Levenson, C. H., and Erlich, H. A. (1989) Genetic analysis of amplified DNA with immobilized sequence-specific oligonucleotide probes. Proc. Nat. Acad. Sci. USA 86, 6230–6234.PubMedCrossRefGoogle Scholar
  36. 36.
    Kolmodin, L., Cheng, S., and Akers, J. (1995) GeneAmp XL PCR Kit. Amplifications: A Forum for PCR Users (The Perkin-Elmer Corporation) 13, 1–5.Google Scholar

Copyright information

© Humana Press Inc. 2002

Authors and Affiliations

  • Lori A. Kolmodin
    • 1
  • David E. Birch
    • 2
  1. 1.Roche Molecular SystemsPleasanton
  2. 2.Roche Molecular SystemsAlameda

Personalised recommendations