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Interactive Fluorophore and Quencher Pairs for Labeling Fluorescent Nucleic Acid Hybridization Probes

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Abstract

The use of fluorescent nucleic acid hybridization probes that generate a fluorescence signal only when they bind to their target enables real-time monitoring of nucleic acid amplification assays. Real-time nucleic acid amplification assays markedly improves the ability to obtain qualitative and quantitative results. Furthermore, these assays can be carried out in sealed tubes, eliminating carryover contamination. Fluorescent nucleic acid hybridization probes are available in a wide range of different fluorophore and quencher pairs. Multiple hybridization probes, each designed for the detection of a different nucleic acid sequence and each labeled with a differently colored fluorophore, can be added to the same nucleic acid amplification reaction, enabling the development of high-throughput multiplex assays. In order to develop robust, highly sensitive and specific real-time nucleic acid amplification assays it is important to carefully select the fluorophore and quencher labels of hybridization probes. Selection criteria are based on the type of hybridization probe used in the assay, the number of targets to be detected, and the type of apparatus available to perform the assay. This article provides an overview of different aspects of choosing appropriate labels for the different types of fluorescent hybridization probes used with different types of spectrofluorometric thermal cyclers currently available.

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Acknowledgments

The studies on the measurements of quenching efficiencies in fluorescent hybridization probes described in this article are the result of a long-time collaboration with Drs. Fred Russell Kramer and Sanjay Tyagi, and was supported by National Institutes of Health Grant EB-000277.

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  1. Public Health Research Institute and the Department of Microbiology and Molecular Genetics, UMDNJ-New Jersey Medical School, 225 Warren Street, Newark, NJ, 07103, USA

    Salvatore A. E. Marras

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  1. Salvatore A. E. Marras
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Correspondence to Salvatore A. E. Marras.

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Marras, S.A.E. Interactive Fluorophore and Quencher Pairs for Labeling Fluorescent Nucleic Acid Hybridization Probes. Mol Biotechnol 38, 247–255 (2008). https://doi.org/10.1007/s12033-007-9012-9

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