Skip to main content

PCR Methods for Identification of Point Mutations and Gene Rearrangements

  • Protocol
Book cover Protocols in Molecular Parasitology

Part of the book series: Methods in Molecular Biology™ ((MIMB,volume 21))

  • 1414 Accesses

Abstract

The polymerase chain reaction (PCR) allows the specific amplifica tion of either RNA or DNA nucleotide sequences (1,2). The hallmarks of this technique are specificity, sensitivity, and speed. The specific ity of the reaction is a result of the requirement of DNA polymerases for a primer that is extended only when annealed to its complemen tary sequence. The primers in the PCR are synthetic oligonucleotides designed to be complementary to the intended target sequence. The sensitivity of PCR is shown by the ability to amplify a target sequence from a single cell under appropriate conditions (3). Finally, PCR analy sis is normally rapid; a typical 30 cycle reaction is complete in about 3 h. In addition, the introduction of a thermostable DNA polymerase isolated from Thermus aquaticus (Taq) has allowed the reaction to be automated, greatly increasing the number of samples that can be con veniently processed at once.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 149.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 199.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Mullis, K., Faloona, F, Scharf, S., Saiki, R., Horn, G., and Erlich, H. (1986) Specific amplification of DNA in vitro: the polymerase chain reaction. Cold Spring Harbor Symp. Quant. Biol. 51, 263–273.

    PubMed  CAS  Google Scholar 

  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

    Article  PubMed  CAS  Google Scholar 

  3. Li, H. H., Gyllensten, U. B., Cui, X. F., Saiki, R. K., Erlich, H. A, and Arnheim, N. (1988) Amplification and analysis of DNA sequences in single human sperm and diploid cells. Nature 335, 414–417.

    Article  PubMed  CAS  Google Scholar 

  4. Wright, P. A. and Wynford-Thomas, D. (1990) The polymerase chain reac tion Miracle or Mirage? A critical review of its uses and limitations in diag nosis and research J. Pathol. 162, 99–117.

    Article  PubMed  CAS  Google Scholar 

  5. Bruce-Chwatt, L. J., Black, R. H., Canfield, C J., Clyde, D. F., Peters, W., and Wernsdorfer, W. H. (1986) Chemotherapy of malaria. World Health Organi zation, Geneva, Switzerland

    Google Scholar 

  6. Peterson, D. S., Walliker, D, and Wellems, T. E. (1988) Evidence that a point mutation in dihydrofolate reductase-thymidylate synthase confers resistance to pyrimethamine in falciparum malaria. Proc Natl. Acad Sci. USA 85,9114–9118

    Article  PubMed  CAS  Google Scholar 

  7. Cowman, A. F, Morry, M. J., Biggs, B. A., Cross, G. A., and Foote, S. J. (1988) Amino acid changes linked to pyrimethamine resistance in the dihydro folate reductase-thymidylate synthase gene of Plasmodium falciparum. Proc. Natl. Acad Sci. USA 85, 9109–9113.

    Article  PubMed  CAS  Google Scholar 

  8. Zolg, J, W, Plitt, J R., Chen, G. X., and Palmer, S (1989) Point mutations in the dihydrofolate reductase-thymidylate synthase gene as the molecular basis for pyrimethamine resistance in Plasmodium falciparum Mol. Biochem Parasitol. 36, 253–262

    Article  CAS  Google Scholar 

  9. Snewin, V A., England, S. M, Sims, P F G., and Hyde, J E. (1989) Charac terization of the dihydrofolate reductase-thymidylate synthetase gene from human malaria parasites highly resistant to pyrimethamine. Gene 76, 41–52.

    Article  PubMed  CAS  Google Scholar 

  10. Zolg, J.W, Chen, G X., and Plitt, J. R. (1990) Detection of pyrimethamine resistance in Plasmodium falciparum by mutation-specific polymerase chain reaction Mol. Biochem. Parasitol. 39,257–265.

    Article  PubMed  CAS  Google Scholar 

  11. Peterson, D. S., Di Santi, S. M., Povoa, M., Calvosa, V. S., Do Rosario, V. E, and Wellems, T. E. (1991) High incidence of Asn-108 mutations in dihydro folate reductase as the basis for pyrimethamine resistant falciparum malaria in the Brazilian Amazon. Am J. Trop. Med. Hyg. 45, 492–497.

    PubMed  CAS  Google Scholar 

  12. Wu, D. Y., Ugozzoli, L., Pal, B. K., and Wallace, R B. (1989) Allele-specific enzymatic amplification of beta-globin genomic DNA for diagnosis of sickle cell anemia Proc. Natl Acad. Sci. USA 86, 2757–2760.

    Article  PubMed  CAS  Google Scholar 

  13. Newton, C. R., Graham, A., Heptinstall, L. E., Powell, S. J., Summers, C, Kalsheker, N., Smith, J. C, and Markham, A. F. (1989) Analysis of any point mutation in DNA The amplification refractory mutation system (ARMS). Nucl. Acids Res 17, 2503–2516

    Article  PubMed  CAS  Google Scholar 

  14. Ehlen, T. and Dubeau, L. (1989) Detection of ras point mutations by poly merase chain reaction using mutation-specific, inosine-containing oligonucle otide primers. Biochem. Biophys. Res. Commun 160, 441–447.

    Article  PubMed  CAS  Google Scholar 

  15. Kwok, S., Kellogg, D. E., McKinney, N., Spasic, D., Goda, L., Levenson, C, and Sninsky, J. J. (1990) Effects of primer-template mismatches on the poly merase chain reaction: human immunodeficiency virus type 1 model studies. Nucl. Acids Res 18, 999–1005.

    Article  PubMed  CAS  Google Scholar 

  16. Okayama, H., Curiel, D. T., Brantly, M. L., Holmes, M. D., and Crystal, R. G (1989) Rapid, nonradioactive detection of mutations in the human genome by allele-specific amplification. J. Lab. Clin. Med. 114, 105–113.

    PubMed  CAS  Google Scholar 

  17. Roos, M. H. (1990) The molecular nature of benzimidazole resistance in hel minths. Parasitol Today 6,125–127

    Article  PubMed  CAS  Google Scholar 

  18. Driscoll, M, Dean, E., Reilly, E, Bergholz, E., and Chalfie, M. (1989) Genetic and Molecular Analysis of a Caenorhabditis elegans β-tubulin that conveys benzimidazole sensitivity. J Cell Biol. 109, 2992–3003

    Article  Google Scholar 

  19. Brindley, P. J, Heath, S., Waters, A. P., McCutchan, T F., and Sher, A. (1991) Characterization of a programmed alteration on an 18S ribosomal gene that accompanies the experimental induction of drug resistance in Schistosoma mansoni. Proc Natl. Acad. Sci. USA 88, 7754–7758

    Article  CAS  Google Scholar 

  20. Sambrook, J, Fritsch, E F, and Maniatis, T. (1989) Molecular Cloning. A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY

    Google Scholar 

  21. Sommer, R. and Tautz, D. (1989) Minimal homology requirements for PCR primers Nucl Acids Res. 17, 6749.

    Article  PubMed  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

Authors

Editor information

Editors and Affiliations

Rights and permissions

Reprints and permissions

Copyright information

© 1993 Humana Press Inc, Totowa, NJ

About this protocol

Cite this protocol

Peterson, D.S. (1993). PCR Methods for Identification of Point Mutations and Gene Rearrangements. In: Hyde, J.E. (eds) Protocols in Molecular Parasitology. Methods in Molecular Biology™, vol 21. Humana Press. https://doi.org/10.1385/0-89603-239-6:213

Download citation

  • DOI: https://doi.org/10.1385/0-89603-239-6:213

  • Publisher Name: Humana Press

  • Print ISBN: 978-0-89603-239-2

  • Online ISBN: 978-1-59259-508-2

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics