Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

Multiplex PCR by multicolor fluorimetry and fluorescence melting curve analysis

Nature Medicine volume 7, pages 249–253 (2001)Cite this article

Sequence-specific fluorescent probe-based systems for the detection of nucleic acid sequences include those using linear hybridization probes1,2, TaqMan probes3, molecular beacons4,5,6 and peptide nucleic acid probes7. The linear hybridization probe format involves a fluorescence resonance energy transfer interaction8,9 between two fluorescently labeled oligonucleotide probes that hybridize adjacent to one another such that one fluorophore functions as the donor, while the other, the acceptor, emits light when juxtaposed to the donor1. This system is advantageous in that one probe may distinguish between products based on their distinct melting temperatures (Tms), which reflect the differences in the thermodynamic stabilities of the perfectly complementary and mismatched probe–target duplexes. Unfortunately, the number of products identifiable using this format is limited to those whose Tms can be distinguished10.

This is a preview of subscription content, access via your institution

Access options

Rent or buy this article

Prices vary by article type

from$1.95

to$39.95

Learn more

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Probe orientation, spectral overlap and color compensation for multicolor Nras mutation detection.
Figure 2: Detection of homozygous point mutations in the Nras gene using a probe complementary to the wild-type Nras sequence.
Figure 3: Fluorescence melting peak analysis of homozygous codon 13 mutants.
Figure 4: Fluorescence melting peak analysis showing heterozygous Nras mutations.

References

  1. Wittwer, C.T., Herrmann, M.G., Moss, A.A. & Rasmussen, R.P. Continuous fluorescence monitoring of rapid cycle DNA amplification. Biotechniques 22, 130–131, 134–138 (1997).

    Article  CAS  Google Scholar 

  2. Bernard, P.S., Lay, M.J. & Wittwer, C.T. Integrated amplification and detection of the C677T point mutation in the methylenetetrahydrofolate reductase gene by fluorescence resonance energy transfer and probe melting curves. Anal. Biochem. 255, 101–107 (1998).

    Article  CAS  Google Scholar 

  3. Heid, C.A., Stevens, J., Livak, K.J. & Williams, P.M. Real-time quantitative PCR. Genome Res. 6, 986–994 (1996).

    Article  CAS  Google Scholar 

  4. Kostrikis, L.G., Tyagi, S., Mhlanga, M.M., Ho, D.D. & Kramer, F.R. Spectral genotyping of human alleles. Science 279, 1228–1229 (1998).

    Article  CAS  Google Scholar 

  5. Tyagi, S. & Kramer, F.R. Molecular beacons: Probes that fluoresce upon hybridization. Nature Biotechnol. 14, 303–308 (1996).

    Article  CAS  Google Scholar 

  6. Vogelstein, B. & Kinzler, K.W. Digital PCR. Proc. Natl. Acad. Sci. USA 96, 9236–9241 (1999).

    Article  CAS  Google Scholar 

  7. Kyger, E. M., Krevolin, M. D. & Powell, M.J. Detection of the hereditary hemochromatosis gene mutation by real-time fluorescence polymerase chain reaction and peptide nucleic acid clamping. Anal. Biochem. 260, 142–148 (1998).

    Article  CAS  Google Scholar 

  8. Heller, M. J. & Morrison, L. E. Chemiluminescent and fluorescent probes for DNA hybridization systems. in Rapid Detection and Identification of Infectious Agents (eds. Kingsbury, D.T. & Falkow, S.) 245–256 (Academic Press, New York, 1985).

    Google Scholar 

  9. Cardullo, R.A., Agrawal, S., Flores, C., Zamecnik, P.C. & Wolf, D.E. Detection of nucleic acid hybridization by nonradiative fluorescence resonance energy transfer. Proc. Natl. Acad. Sci. USA 85, 8790–8794 (1988).

    Article  CAS  Google Scholar 

  10. Bernard, P.S., Ajioka, R.S., Kushner, J.P. & Wittwer, C.T. Homogeneous multiplex genotyping of hemochromatosis mutations with fluorescent hybridization probes. Am. J. Pathol. 153, 1055–1061 (1998).

    Article  CAS  Google Scholar 

  11. Lee, L.G. et al. Seven-color, homogeneous detection of six PCR products. Biotechniques 27, 342–349 (1999).

    Article  CAS  Google Scholar 

  12. Tyagi, S., Bratu, D.P. & Kramer, F.R. Multicolor molecular beacons for allele discrimination. Nature Biotechnol. 16, 49–53 (1998).

    Article  CAS  Google Scholar 

  13. Nelson, R.M. & Long, G.L. A general method of site-specific mutagenesis using a modification of the Thermus aquaticus polymerase chain reaction. Anal. Biochem. 180, 147–151 (1989).

    Article  CAS  Google Scholar 

  14. Lay, M.J. & Wittwer, C.T. Real-time fluorescence genotyping of factor V Leiden during rapid-cycle PCR. Clin. Chem. 43, 2262–2267 (1997).

    CAS  PubMed  Google Scholar 

  15. Bernard, P.S., Pritham, G.H. & Wittwer, C.T. Color multiplexing hybridization probes using the apolipoprotein E locus as a model system for genotyping. Anal. Biochem. 273, 221–228 (1999).

    Article  CAS  Google Scholar 

  16. Bagwell, C.B. & Adams, E.G. Fluorescence spectral overlap compensation for any number of flow cytometry parameters. Ann. NY Acad. Sci. 677, 167–184 (1993).

    Article  CAS  Google Scholar 

  17. Ririe, K.M., Rasmussen, R.P. & Wittwer, C.T. Product differentiation by analysis of DNA melting curves during the polymerase chain reaction. Anal. Biochem. 245, 154–160 (1997).

    Article  CAS  Google Scholar 

  18. Wetmur, J. Nucleic acid hybrids, formation and structure. in Molecular Biology and Biotechnology: A Comprehensive Desk Reference (ed. Myers, R.) 605–608 (VCH Publishers, New York, 1995).

    Google Scholar 

  19. Ikuta, S., Takagi, K., Wallace, R.B. & Itakura, K. Dissociation kinetics of 19 base paired oligonucleotide-DNA duplexes containing different single mismatched base pairs. Nucleic Acids Res. 15, 797–811 (1987).

    Article  CAS  Google Scholar 

  20. SantaLucia, J., Allawi, H.T. & Seneviratne, P.A. Improved nearest-neighbor parameters for predicting DNA duplex stability. Biochemistry 35, 3555–3562 (1996).

    Article  CAS  Google Scholar 

  21. Peyret, N., Seneviratne, P.A., Allawi, H.T. & SantaLucia, J., Jr. Nearest-neighbor thermodynamics and NMR of DNA sequences with internal A.A, C.C, G.G, and T.T mismatches. Biochemistry 38, 3468–3477 (1999).

    Article  CAS  Google Scholar 

  22. SantaLucia, J., Jr. A unified view of polymer, dumbbell, and oligonucleotide DNA nearest- neighbor thermodynamics. Proc. Natl. Acad. Sci. USA 95, 1460–1465 (1998).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

This work was supported by the ARUP Institute for Clinical and Experimental Pathology and by a Leukemia Research Foundation Grant to KSJE-J and National Institutes of Health Grants (CA83984) to KSJE-J and (GM58983) to CTW.

Author information

Authors and Affiliations

  1. Department of Pathology, University of Utah School of Medicine, Salt Lake City, Utah, USA

    Kojo S.J. Elenitoba-Johnson & Carl T. Wittwer

  2. ARUP Institute for Clinical and Experimental Pathology, Salt Lake City, Utah, USA

    Kojo S.J. Elenitoba-Johnson, Sandra D. Bohling & Carl T. Wittwer

  3. Lifespan Medical Center, and Brown University School of Medicine, Providence, Rhode Island, USA

    Thomas C. King

Authors
  1. Kojo S.J. Elenitoba-Johnson
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Sandra D. Bohling
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Carl T. Wittwer
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Thomas C. King
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Kojo S.J. Elenitoba-Johnson.

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Elenitoba-Johnson, K., Bohling, S., Wittwer, C. et al. Multiplex PCR by multicolor fluorimetry and fluorescence melting curve analysis. Nat Med 7, 249–253 (2001). https://doi.org/10.1038/84708

Download citation

  • Issue Date:

  • DOI: https://doi.org/10.1038/84708

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing