Detection of PCR products using self-probing amplicons and fluorescence (original) (raw)

Nature Biotechnology volume 17, pages 804–807 (1999)Cite this article

Abstract

Molecular diagnostics is progressing from low-throughput, heterogeneous, mostly manual technologies to higher throughput, closed-tube, and automated methods. Fluorescence is the favored signaling technology for such assays, and a number of techniques rely on energy transfer between a fluorophore and a proximal quencher molecule. In these methods, dual-labeled probes hybridize to an amplicon and changes in the quenching of the fluorophore are detected. We describe a new technology that is simple to use, gives highly specific information, and avoids the major difficulties of the alternative methods. It uses a primer with an integral tail that is used to probe an extension product of the primer. The probing of a target sequence is thereby converted into a unimolecular event, which has substantial benefits in terms of kinetics, thermodynamics, assay design, and probe reliability.

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References

  1. Ishiguro, T. et al. Homogeneous quantitative assay of hepatitis C virus RNA by polymerase chain reaction in the presence of a fluorescent intercalater. Anal. Biochem. 229, 207–213 (1995).
    Article CAS Google Scholar
  2. Tseng, S.Y. et al. An homogeneous fluorescence polymerase chain reaction assay to identify Salmonella. Anal. Biochem. 245, 207–212 (1997).
    Article CAS Google Scholar
  3. Wittwer, C.T. et al. The LightcyclerTM a microvolume multisample fluorimeter with rapid temperature control. Biotechniques 22, 176–181 (1997).
    Article CAS Google Scholar
  4. Wittwer, C.T., Herrmann, M.G., Moss, A.A. & Rasmussen, R.P. Continuous fluorescence monitoring of rapid cycle DNA amplification. Biotechniques 22, 130. (1997).
    Article Google Scholar
  5. Nazarenko, I.A., Bhatnagar, S.K. & Hohman, R.J. A closed tube format for amplification and detection of DNA based on energy transfer. Nucleic Acids Res. 25, 2516–2521 (1997).
    Article CAS Google Scholar
  6. Brownie, J. et al. The elimination of primer-dimer accumulation in PCR. Nucleic Acids Res. 25, 3235–3241 (1997).
    Article CAS Google Scholar
  7. Gibson, N. J. et al. A homogeneous method for genotyping with fluorescence polarization. Clin. Chem. 43, 1336–1341 (1997).
    CAS PubMed Google Scholar
  8. Holland, P.M., Abramson, R.D., Watson, R. & Gelfand, D.H. Detection of specific polymerase chain reaction product by utilizing the 5'–3' exonuclease activity of Thermus aquaticus DNA polymerase. Proc. Natl. Acad. Sci. USA 88, 7276– 7280 (1991).
    Article CAS Google Scholar
  9. Gelmini, S. et al. Quantitative polymerase chain reaction-based homogeneous assay with fluorogenic probes to measure C-Erbb-2 oncogene amplification. Clin. Chem. 43, 752–758 (1997).
    CAS PubMed Google Scholar
  10. Livak, K.J., Marmaro, J. & Todd, J.A. Towards fully automated genome-wide polymorphism screening. Nat. Genet. 9, 341–342 (1995).
    Article CAS Google Scholar
  11. Tyagi, S. & Kramer, F.R. Molecular beacons—probes that fluoresce upon hybridization. Nat. Biotechnol. 14, 303–308 (1996).
    Article CAS Google Scholar
  12. Tyagi, S., Bratu, D.P. & Kramer, F.R. Multicolor molecular beacons for allele discrimination. Nat. Biotechnol. 16, 49– 53 (1998).
    Article CAS Google Scholar
  13. Newton, C.R. et al. The production of PCR products with 5´ single-stranded tails using primers that incorporate novel phosphoramidite intermediates. Nucleic Acids Res. 21, 1155– 1162 (1993).
    Article CAS Google Scholar
  14. Ibrahim, M.S., Esposito, J.J., Jahrling, P.B. & Lofts, R.S. The potential of 5´ nuclease PCR for detecting a single-base polymorphism in orthopoxvirus. Mol. Cell. Probes 11, 143–147 (1997).
    Article CAS Google Scholar
  15. Ibrahim, M.S. et al. Real-time microchip PCR for detecting single-base differences in viral and human DNA. Anal. Chem. 70, 2013–2017 (1998).
    Article CAS Google Scholar
  16. Couch, F. J. et al. BRCA2 germline mutations in male breast cancer cases and breast cancer families. Nat. Genet. 13, 123– 125 (1996).
    Article CAS Google Scholar
  17. Ferrie, R.M., Ellison, G., Callaghan, K. & Fox, J.C. UK Patent Application, number 9815224.2 ( 1998).
    Google Scholar
  18. van den Velden, P.A. & Reitsma, P.H. Amino acid dimorphism in IL1A is detectable by PCR amplification; Hum. Mol. Genet. 2, 1753 (1993).
    Article CAS Google Scholar
  19. Zuker, M. mfold Program website. http://www.ibc.wustl.edu/\~zuker.
  20. Santalucia, J. A unified view of polymer, dumbbell, & oligonucleotide DNA nearest-neighbor thermodynamics. Proc. Natl. Acad. Sci. USA 95, 1460–1465 (1998).
    Article CAS Google Scholar
  21. Newton, C.R. et al. Analysis of any point mutation in DNA. The amplification refractory mutation system (ARMS). Nucleic Acids Res. 17, 2503–2516 (1989).
    Article CAS Google Scholar
  22. Ferrie, R.M. et al. Development, multiplexing, and application of ARMS tests for common mutations in the CFTR gene. Am. J. Hum. Genet. 51, 251–262 (1992).
    CAS PubMed PubMed Central Google Scholar

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Acknowledgements

We thank Dr. Neil Gibson for helpful discussions, Kay Callaghan and Kemal Haque for sharing of unpublished K-ras data, and Dr. Julian Sorrell for information concerning IL-1A polymorphisms. The methods described in this paper form part of worldwide patent applications. ARMS is the subject of European patent number 0332435 (ZENECA Ltd.) and corresponding US patent #5595890. The use of nonamplifiable tails and blocking monomers are the subjects of European patent 0416817 (ZENECA Ltd.) and corresponding US patent #5525494.

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Authors and Affiliations

  1. AstraZeneca Diagnostics, Gadbrook Park , Rudheath, Northwich, CW9 7RA , Cheshire, UK
    David Whitcombe, Jane Theaker, Simon P. Guy & Steve Little
  2. Department of Chemistry, University of Southampton, Highfield, SO17 1BJ, Southampton, UK
    Tom Brown

Authors

  1. David Whitcombe
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  2. Jane Theaker
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  3. Simon P. Guy
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  4. Tom Brown
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  5. Steve Little
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Correspondence toDavid Whitcombe.

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Whitcombe, D., Theaker, J., Guy, S. et al. Detection of PCR products using self-probing amplicons and fluorescence .Nat Biotechnol 17, 804–807 (1999). https://doi.org/10.1038/11751

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