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Fluorescence in Nucleic Acid Hybridization Assays

  • Chapter
Topics in Fluorescence Spectroscopy

Part of the book series: Topics in Fluorescence Spectroscopy ((TIFS,volume 7))

Conclusions

Fluorescence has provided a large variety of DNA hybridization assay formats, both heterogeneous and homogeneous. Heterogeneous formats offer high sensitivity, with time-resolved lanthanide fluorescence providing detection down to 0.01 amol, or about 6000 molecules of target nucleic acid. With amplification, the detection limit drops to the tens or hundreds of molecules, or even the single molecule level. For many applications amplification would not be necessary. The variety of environment-sensitive fluorescence properties available makes fluorescence particularly valuable to homogeneous assays where measurable properties must change in response to hybridization. Homogeneous assays conserve time and labor, but suffer in sensitivity due to fundamental constraints on probe concentration. Homogeneous assay detection levels are closer to the fmol or 0.1 fmol level. Since homogeneous assay detection levels are limited by probe concentration, sensitivity would increase tremendously if sample analytes could be concentrated into microvolumes, thereby providing amol or lower detection levels. Combining hybridization assays with amplification, however, provides 10 molecule or lower detection levels to even homogeneous assays. Since target amplification methods tend to be homogeneous, combination with a homogeneous detection system provides a completely homogeneous high sensitivity assay that can be performed in sealed vessels, thereby reducing the risk of sample cross contamination. Given the wide range of assay formats already demonstrated in fluorescence approaches to hybridization assays, it should be relatively easy to tailor either a heterogeneous or homogeneous assay to any particular need.

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  1. Vysis, Inc., Downers Grove, Illinois, 60515-5400

    Larry E. Morrison

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  1. Center for Fluorescence Spectroscopy and Department of Biochemistry and Molecular Biology, University of Maryland School of Medicine, Baltimore, Maryland

    Joseph R. Lakowicz

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Morrison, L.E. (2003). Fluorescence in Nucleic Acid Hybridization Assays. In: Lakowicz, J.R. (eds) Topics in Fluorescence Spectroscopy. Topics in Fluorescence Spectroscopy, vol 7. Springer, Boston, MA. https://doi.org/10.1007/0-306-47947-8_2

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  • DOI: https://doi.org/10.1007/0-306-47947-8_2

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