Choosing Reporter-Quencher Pairs for Efficient Quenching Through Formation of Intramolecular Dimers

  • Mary Katherine Johansson
Part of the Methods in Molecular Biology™ book series (MIMB, volume 335)

Abstract

Fluorescent energy transfer within dual-labeled oligonucleotide probes is widely used in assays for genetic analysis. Nucleic acid detection/amplification methods, such as real-time polymerase chain reaction, use dual-labeled probes to measure the presence and copy number of specific genes or expressed messenger RNA. Fluorogenic probes are labeled with both a reporter and a quencher dye. Fluorescence from the reporter is only released when the two dyes are physically separated via hybridization or nuclease activity. Fluorescence resonance energy transfer (FRET) is the physical mechanism that is most often cited to describe how quenching occurs.

We have found that many dual-labeled probes have enhanced quenching through a nonFRET mechanism called static quenching. Static quenching, which is also referred to as contact quenching, can occur even in “linear” oligonucleotide probes that have no defined secondary structure to bring the reporter and quencher pair into proximity. When static quenching accompanies FRET quenching, the background fluorescence of probes is suppressed. This chapter describes how to pair reporter and quencher dyes for duallabeled probes to maximize both FRET and static quenching. Data comparing various reporter-quencher pairs is presented as well as protocols for evaluation and optimization of the probes.

Key Words

FRET intramolecular dimer stacking interactions fluorescent probes biosensors 

References

  1. 1.
    Didenko, V. V. (2001) DNA probes using fluorescence resonance energy transfer (FRET) designs and applications. Biotechniques 31, 1106–1121.PubMedGoogle Scholar
  2. 2.
    Johansson, M. K., Fidder H., Dick D., and Cook R. M. (2002) Intramolecular dimers a new strategy to fluorescence quenching in dual-labeled oligonucleotide probes. J. Am Chem. Soc. 124, 6950–6956.PubMedCrossRefGoogle Scholar
  3. 3.
    Johansson, M. K. and Cook, R. M. (2003) Intramolecular dimers a new design strategy for fluorescence-quenched probes. Chem. Eur. J. 9, 3466–3471.CrossRefGoogle Scholar
  4. 4.
    Rudert, W. A., Braun, E. R., Faas, S. J., Menon, R., Jaquins-Gerstl, A., and Trucco, M. (1997) Double labeled fluorescent probes for 5′ nuclease assays purification and performance evaluation. Biotechniques 22, 1140–1145.PubMedGoogle Scholar
  5. 5.
    Marras, S. A. E., Russell, F. R., and Tyagi, S. (2002) Efficiencies of fluorescence resonance energy transfer and contact-mediated quenching in oligonucleotide probes. Nucliec Acids Res. 30, e122.CrossRefGoogle Scholar
  6. 6.
    Seidel, C. A. M., Schulz, A., and Sauer, M. H. H. (1996) Nucleobase-specific quenching of fluorescent dyes. 1. Nucleobase one-electron redox potentials and their correlation with static and dynamic quenching efficiences. J. Phys. Chem. 100, 5541–5553.CrossRefGoogle Scholar
  7. 7.
    Haugland, R. P. (2002) Handbook of Fluorescent Probes and Research Products, Molecular Probes, Eugene, OR.Google Scholar
  8. 8.
    Lakowicz, J. (1999) Principles of Fluorescence Spectroscopy, Plenum, New York.Google Scholar
  9. 9.
    Andrus A., and Kuimelis, R. G. (2000) Current Protocols in Nucleic Acid Chemistry. Wiley and Sons, NY.Google Scholar
  10. 10.
    Bernacchi, S. and Mély, Y. (2001) Excitation interaction in molecular beacons: a sensitive sensor for short range modifications of the nucleic acid structure. Nucleic Acids Res. 29, e62.PubMedCrossRefGoogle Scholar
  11. 11.
    Crockett, A. O. and Wittwer, C. T. (2001) Fluorescein-labeled oligonucleotides for real-time PCR: using the inherent quenching of deoxyguanosine nucleotides. Anal. Biochem. 290, 89–97.PubMedCrossRefGoogle Scholar
  12. 12.
    Sjüback, R., Nygren, J., and Kubista, M. (1998) Characterization of fluoresceinoligonucleotide conjugates and measurements of local electrostatic potential. Biopolymers 46, 445–453.CrossRefGoogle Scholar

Copyright information

© Humana Press Inc., Totowa, NJ 2006

Authors and Affiliations

  • Mary Katherine Johansson

There are no affiliations available

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