Basic principles of fluorescence and energy transfer applied to real-time PCR.

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.