Advertisement

Polymerase Chain Reaction

  • Yuri E. Nikiforov
  • Philip N. Howles

Abstract

The polymerase chain reaction (PCR) was introduced in 1985 by Saiki et al. (1), who recognized that sequence-specific oligonucleotides could be used to prime synthesis of complementary DNA strands by any of several DNA polymerases and that these primers could be chosen so as to replicate both strands of the DNA positioned between their cognate sequences in the template (1,2). Initial studies with the technique were laborious and thus limited in scope. Thermostable DNA polymerases were not yet commercially available, so the experimenter had to add fresh polymerase after the DNA denaturing step of each cycle (1,2). The process was not automated, so a student or technician was needed to transfer the reactions between water baths or heating blocks set for each temperature of the cycle. The original report was largely a “proof of principle” study in which a portion of the human ß-globin gene was amplified and then analyzed for the presence of normal versus sickle cell coding sequences (1,3). In 1988, the use of a thermostable DNA polymerase, isolated from Thermus aquaticus, was used in this procedure instead of the Escherichia coli Klenow fragment (4,5). This modification constituted the major advance needed to make the technique practical and commercially viable. Various preparations of heat-stable polymerases from T. aquaticus, as well as other thermophilic bacteria, were soon commercially available, as were several models of programmable instruments for rapid and reliable heating and cooling of samples.

Keywords

Polymerase Chain Reaction Product Thyroid Papillary Carcinoma Microcentrifuge Tube Reverse Transcription Reaction Upstream Primer 
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.

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

References

  1. 1.
    Saiki, R. K., Scharf, S. J., Faloona, F. A., et al. (1985) Enzymatic amplification of ß-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia. Science 230, 1350–1354.PubMedCrossRefGoogle Scholar
  2. 2.
    Mullis, K. B. and Faloona, F. A. (1987) Specific synthesis of DNA in vitro via a polymerase-catalyzed chain reaction. Methods Enzymol. 155, 335–350.PubMedCrossRefGoogle Scholar
  3. 3.
    Engelke, D. R., Hoener, P. A., and Collins, F. S. (1988) Direct sequencing of enzymatically amplified human genomic DNA. Proc. Natl. Acad. Sei. USA 85, 544–548.CrossRefGoogle Scholar
  4. 4.
    Wong, C, Dowling, C. E., Saiki, R. K., Higuchi, R. G., Erlich, H. A., and Kazazian, H. H. (1987) Characterization of ß-thalassemia mutations using direct genomic sequencing of amplified single copy DNA. Nature 330, 384–386.PubMedCrossRefGoogle Scholar
  5. 5.
    Saiki, R. K., Gelfand, D. H., Stoffel, S., et al. (1988) Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science 239, 487–491.PubMedCrossRefGoogle Scholar
  6. 6.
    Greer, C. E., Peterson, S. L., Kiviat, N. B., and Manos, M. M. (1991) PCR amplification from paraffin-embedded tissues: effects of fixative and fixation time. Am. J. Clin. Pathol. 95, 117–124.PubMedGoogle Scholar
  7. 7.
    Shibata, D. (1994) Extraction of DNA from paraffin-embedded tissue for analysis by polymerase chain reaction: new tricks from an old friend. Hum. Pathol. 25, 561–563.PubMedCrossRefGoogle Scholar
  8. 8.
    Ben-Ezra, J., Johnson, D. A., Rossi, J., Cook, N., and Wu, A. (1991) Effect of fixation on the amplification of nucleic acids from paraffin-embedded material by the polymerase chain reaction. J. Histochem. Cytochem. 39, 351–354.PubMedCrossRefGoogle Scholar
  9. 9.
    Greer, C. E., Lund, J. K., and Manos, M. M. (1991) PCR amplification from paraffin-embedded tissues: recommendations on fixatives for long-term storage and prospective studies. PCR Methods Appl. 1, 46–50.PubMedCrossRefGoogle Scholar
  10. 10.
    Shibata, D., Martin, W. J., and Arnheim, N. (1988) Analysis of DNA sequences in forty-year old paraffin-embedded thin-tissue sections: a bridge between molecular biology and classic histology. Cancer Res. 48, 4564–4566.PubMedGoogle Scholar
  11. 11.
    Kallio, P., Syrjanen, S., Tervahauta, A., and Syrjanen, K. (1991) A simple method for isolation of DNA from formalin-fixed paraffin-embedded samples for PCR. J. Virol. Meth. 35, 39–47.CrossRefGoogle Scholar
  12. 12.
    Heller, M. J., Robinson, R. A., Burgart, L. J., TenEyck, C. J., and Wilke, W. W. (1992) DNA extraction by sonication: a comparison of fresh, frozen, and paraffin-embedded tissue extracted for use in polymerase chain reaction assays. Mod. Pathol. 5, 203–206.PubMedGoogle Scholar
  13. 13.
    Sepp, R., Szabo, I., Uda, H., and Sakamoto, H. (1994) Rapid techniques for DNA extraction from routinely processed archival tissue for use in PCR. J. Clin. Pathol. 47, 318–323.PubMedCrossRefGoogle Scholar
  14. 14.
    Frank, T. S., Svoboda-Newman, S. M., and Hsi, E. D. (1996) Comparison of methods for extracting DNA from formalin-fixed paraffin sections for nonisotopic PCR. Diagn. Mol. Pathol. 5, 220–224.PubMedCrossRefGoogle Scholar
  15. 15.
    Birch, D. E. (1996) Simplified hot start PCR. Nature 381, 445–446.PubMedCrossRefGoogle Scholar
  16. 16.
    Bassam, B. J., Caetano-Anolles, G., Gresshoff, P. M. (1991) Fast and sensitive silver staining of DNA in Polyacrylamide gels. Anal. Biochem. 196, 80–83.PubMedCrossRefGoogle Scholar
  17. 17.
    Peats, S. (1984) Quantitation of protein and DNA in silver-stained agarose gels. Anal. Biochem. 140, 178–182.PubMedCrossRefGoogle Scholar
  18. 18.
    Liu, X. Y., Nelson, D., Grant, C, Morthland, V., Goodnight, S. H., Press, R. D. (1995) Molecular detection of a common mutation in coagulation factor V causing thrombosis via hereditary resistance to activated protein C. Diagn. Mol. Pathol. 4, 191–197.PubMedCrossRefGoogle Scholar
  19. 19.
    Sutcharitchan, P., Saiki, R., Huisman, T. H., et al. (1995) Reverse dot-blot detection of the African-American beta-thalassemia mutations. Blood 86, 1580–1585.PubMedGoogle Scholar
  20. 20.
    Kant, J. A., Mifflin, T. E., McGlennen, R., Pice, E., Naylor, E., and Cooper, D. L. (1995) Molecular diagnosis of cystis fibrosis. Clin. Lab. Med. 15, 877–898.PubMedGoogle Scholar
  21. 21.
    Orita, M., Suzuki, Y., Sekiya, T., and Hayashi, K. (1989) Rapid and sensitive detection of point mutations and DNA polymorphisms using the polymerase chain reaction. Genomics 5, 874–879.PubMedCrossRefGoogle Scholar
  22. 22.
    Ainsworth, P. J., Surh, L. C, and Coulter-Mackie, M. B. (1991) Diagnostic single strand conformation polymorphism (SSCP): a simplified non-radioisotopic method as applied to a Tay-Sachs Bl variant. Nucleic Acids Res., 19, 405–406.PubMedCrossRefGoogle Scholar
  23. 23.
    Lundberg, K. S., Shoemaker, D. D., Adams, M. W., Short, J. M., Sorge, J. A., Marthur, E. J. (1991) High-fidelity amplification using a thermostable DNA polymerase isolated from Pyrococcus furiosus. Gene 108, 1–6.Google Scholar
  24. 24.
    Cariello, N. F., Swenberg, J. A., Skopek, T. R. (1991) Fidelity of Thermococcus litoralis DNA polymerase (Vent) in PCR determined by denaturing gradient gel electrophoresis. Nucleic Acids Res. 19, 4193–4198.PubMedCrossRefGoogle Scholar
  25. 25.
    Chomczynski, P. and Sacchi, N. (1987) Single-step method of RNA isolation by acid guanidine thiocyanate-phenol-chloroform extraction. Anal. Biochem. 162, 156–159.PubMedCrossRefGoogle Scholar
  26. 26.
    Foss, R. D., Guha-Thakurta, N., Conran, R. M., Gutman, P. (1994) Effect of fixative time on the extraction and polymerase chain reaction amplification of RNA from paraffin-embedded tissue. Diagn. Mol. Pathol. 3, 148–155.PubMedCrossRefGoogle Scholar
  27. 27.
    Ladanyi, M. and Bridge, J. A. (2000) Contribution of molecular genetic data to the classification of sarcomas. Hum. Pathol. 31, 532–538.PubMedCrossRefGoogle Scholar
  28. 28.
    Nikiforov, Y. E., Rowland, J. M., Bove, K. E., Monforte-Munoz, H., and Fagin, J. A. (1997) Distinct pattern of ret oncogene rearrangements in morphologic variants of radiation-induced and sporadic thyroid papillary carcinomas in children. Cancer Res. 57, 1690–1694.PubMedGoogle Scholar
  29. 29.
    Argani, P., Żakowski, M. F., Klimstra, D. S., Rosai, J., and Ladanyi, M. (1998) Detection of the SYT-SSX chimeric RNA of synovial sarcoma in paraffin-embedded tissue and its application in problematic cases. Mod. Pathol. 11, 65–71.PubMedGoogle Scholar
  30. 30.
    Muthuchamy, M., Pajak, L., Howies, P., Doetschman, T., and Wieczorek, D. (1993) Developmental analysis of tropomyosin expression in embryonic stem cells and mouse embryos. Mol. Cell. Biol. 13, 3311–3323.PubMedGoogle Scholar
  31. 31.
    Becker-Andre, M. and Hahlbrock, K. (1989) Absolute mRNA quantification using the polymerase chain reaction (PCR). A novel approach by a PCR aided transcript titration assay (PATTY). Nucleic Acids Res. 17, 9438–9446.CrossRefGoogle Scholar
  32. 32.
    Gilliland, G., Perrin, S., Blanchard, K., and Bunn, F. (1990) Analysis of cytokine mRNA and DNA: detection and quantitation by competitive polymerase chain reaction. Proc. Natl. Acad. Sei. USA 87, 2725–2729.CrossRefGoogle Scholar
  33. 33.
    Wang, A. M., Doyle, M. V., and Mark, D. F. (1989) Quantitation of mRNA by the polymerase chain reaction. Proc. Natl. Acad. Sei. USA 86, 9717–9721.CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2001

Authors and Affiliations

  • Yuri E. Nikiforov
  • Philip N. Howles

There are no affiliations available

Personalised recommendations