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. 1997 Jun 15;25(12):2516–2521. doi: 10.1093/nar/25.12.2516

A closed tube format for amplification and detection of DNA based on energy transfer.

I A Nazarenko 1, S K Bhatnagar 1, R J Hohman 1
PMCID: PMC146748  PMID: 9171107

Abstract

A new method for the direct detection of PCR-amplified DNA in a closed system is described. The method is based on the incorporation of energy transfer-labeled primers into the amplification product. The PCR primers contain hairpin structures on their 5'ends with donor and acceptor moieties located in close proximity on the hairpin stem. The primers are designed in such a way that a fluorescent signal is generated only when the primers are incorporated into an amplification product. A signal to background ratio of 35:1 was obtained using the hairpin primers labeled with fluorescein as a donor and 4-(4'-dimethylaminophenylazo) benzoic acid (DABCYL) as a quencher. The modified hairpin-primers do not interfere with the activity of DNA polymerase, and both thermostable Pfu and Taq polymerase can be used. This method was applied to the detection of cDNA for prostate specific antigen. The results demonstrate that the fluorescent intensity of the amplified product correlates with the amount of incorporated primers, and as few as 10 molecules of the initial template can be detected. This technology eliminates the risk of carry-over contamination, simplifies the amplification assay and opens up new possibilities for the real-time quantification of the amplified DNA over an extremely wide dynamic range.

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Selected References

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  1. 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 U S A. 1988 Dec;85(23):8790–8794. doi: 10.1073/pnas.85.23.8790. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Cimino G. D., Metchette K. C., Tessman J. W., Hearst J. E., Isaacs S. T. Post-PCR sterilization: a method to control carryover contamination for the polymerase chain reaction. Nucleic Acids Res. 1991 Jan 11;19(1):99–107. doi: 10.1093/nar/19.1.99. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Clegg R. M. Fluorescence resonance energy transfer and nucleic acids. Methods Enzymol. 1992;211:353–388. doi: 10.1016/0076-6879(92)11020-j. [DOI] [PubMed] [Google Scholar]
  4. 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 U S A. 1991 Aug 15;88(16):7276–7280. doi: 10.1073/pnas.88.16.7276. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Ju J., Ruan C., Fuller C. W., Glazer A. N., Mathies R. A. Fluorescence energy transfer dye-labeled primers for DNA sequencing and analysis. Proc Natl Acad Sci U S A. 1995 May 9;92(10):4347–4351. doi: 10.1073/pnas.92.10.4347. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Landegren U. Molecular mechanics of nucleic acid sequence amplification. Trends Genet. 1993 Jun;9(6):199–204. doi: 10.1016/0168-9525(93)90119-3. [DOI] [PubMed] [Google Scholar]
  7. Lee L. G., Connell C. R., Bloch W. Allelic discrimination by nick-translation PCR with fluorogenic probes. Nucleic Acids Res. 1993 Aug 11;21(16):3761–3766. doi: 10.1093/nar/21.16.3761. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Longo M. C., Berninger M. S., Hartley J. L. Use of uracil DNA glycosylase to control carry-over contamination in polymerase chain reactions. Gene. 1990 Sep 1;93(1):125–128. doi: 10.1016/0378-1119(90)90145-h. [DOI] [PubMed] [Google Scholar]
  9. Lyamichev V., Brow M. A., Dahlberg J. E. Structure-specific endonucleolytic cleavage of nucleic acids by eubacterial DNA polymerases. Science. 1993 May 7;260(5109):778–783. doi: 10.1126/science.7683443. [DOI] [PubMed] [Google Scholar]
  10. Mullis K. B., Faloona F. A. Specific synthesis of DNA in vitro via a polymerase-catalyzed chain reaction. Methods Enzymol. 1987;155:335–350. doi: 10.1016/0076-6879(87)55023-6. [DOI] [PubMed] [Google Scholar]
  11. Rye H. S., Yue S., Wemmer D. E., Quesada M. A., Haugland R. P., Mathies R. A., Glazer A. N. Stable fluorescent complexes of double-stranded DNA with bis-intercalating asymmetric cyanine dyes: properties and applications. Nucleic Acids Res. 1992 Jun 11;20(11):2803–2812. doi: 10.1093/nar/20.11.2803. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Selvin P. R. Fluorescence resonance energy transfer. Methods Enzymol. 1995;246:300–334. doi: 10.1016/0076-6879(95)46015-2. [DOI] [PubMed] [Google Scholar]
  13. Tyagi S., Kramer F. R. Molecular beacons: probes that fluoresce upon hybridization. Nat Biotechnol. 1996 Mar;14(3):303–308. doi: 10.1038/nbt0396-303. [DOI] [PubMed] [Google Scholar]
  14. Varani G. Exceptionally stable nucleic acid hairpins. Annu Rev Biophys Biomol Struct. 1995;24:379–404. doi: 10.1146/annurev.bb.24.060195.002115. [DOI] [PubMed] [Google Scholar]
  15. Walder R. Y., Hayes J. R., Walder J. A. Use of PCR primers containing a 3'-terminal ribose residue to prevent cross-contamination of amplified sequences. Nucleic Acids Res. 1993 Sep 11;21(18):4339–4343. doi: 10.1093/nar/21.18.4339. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Yamamoto N., Okamoto T. A rapid detection of PCR amplification product using a new fluorescent intercalator; the pyrylium dye, P2. Nucleic Acids Res. 1995 Apr 25;23(8):1445–1446. doi: 10.1093/nar/23.8.1445. [DOI] [PMC free article] [PubMed] [Google Scholar]

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