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Basic Principles of Fluorescence and Energy Transfer Applied to Real-Time PCR

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Abstract

Fluorescence is highly sensitive to environment, and the distance separating fluorophores and quencher molecules can provide the basis for effective homogeneous nucleic acid hybridization assays. Molecular interactions leading to fluorescence quenching include collisions, ground state and excited state complex formation, and long-range dipole-coupled energy transfer. These processes are well understood and equations are provided for estimating the effects of each process on fluorescence intensity. Estimates for the fluorescein-tetramethylrhodamine donor–acceptor pair reveal the relative contributions of dipole-coupled energy transfer, collisional quenching, and static quenching in several common assay formats, and illustrate that the degree of quenching is dependent upon the hybridization complex formed and the manner of label attachment.

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Notes

  1. This separation distance assumes the stem regions are included in the rigid helix. However, since the stem portions are not complementary to the target they would have some random coil nature and a somewhat smaller separation distance would result.

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  1. Abbott Molecular Inc. , Des Plaines, IL, USA

    Larry E. Morrison

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  1. Larry E. Morrison
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Correspondence to Larry E. Morrison.

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Morrison, L.E. Basic Principles of Fluorescence and Energy Transfer Applied to Real-Time PCR. Mol Biotechnol 44, 168–176 (2010). https://doi.org/10.1007/s12033-009-9225-1

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