DNA Technology

Abstract

Fluorescence resonance energy transfer (FRET) has become widely used in all applications of fluorescence, including medical diagnostics, DNA analysis, and optical imaging. The widespread use of FRET is due to the favorable distances for energy transfer, which are typically the size of a protein or the thickness of a membrane. Additionally, the extent of FRET is readily predictable from the spectral properties of the fluorophores. If the spectral properties of the fluorophores allow FRET, it will occur and will not be significantly affected by the biomolecules in the sample. These favorable properties allow for the design of experiments based on the known sizes and structural features of the sample. FRET is an electrodynamic phenomenon that can be explained using classical physics. FRET occurs between a donor (D) molecule in the excited state and an acceptor (A) molecule in the ground state. The donor molecules typically emit at shorter wavelengths that overlap with the absorption spectrum of the acceptor. Energy transfer occurs without the appearance of a photon and is the result of longrange dipole—dipole interactions between the donor and acceptor. The term resonance energy transfer (RET) is preferred because the process does not involve the appearance of a photon. The rate of energy transfer depends upon the extent of spectral overlap of the emission spectrum of the donor with the absorption spectrum of the acceptor, the quantum yield of the donor, the relative orientation of the donor and acceptor transition dipoles, and the distance between the donor and acceptor molecules. The distance dependence of RET allows measurement of the distances between donors and acceptors.