References >> FRET Probe

Introduction to FRET Probes

Fluorescence Resonance Energy Transfer (FRET) occurs due to the interaction between the electronic excited states of two dye molecules. The excitation is transferred from one (the donor) dye molecule to the other (the acceptor) dye molecule without emission of a photon. This is distance-dependent, that is the donor and the acceptor dye must be in close proximity. FRET has been used for investigating a variety of biological phenomena that produce changes in molecular proximity.

FRET Probes

FRET probes are a pair of fluorescent probes placed in close proximity. Fluorophores are so chosen that the emission spectrum of one overlaps significantly with the excitation spectrum of the other. During FRET, the donor fluorophore excited by a light source, transfers its energy to an acceptor fluorophore when positioned in the direct vicinity of the former.

The acceptor fluorophore emits light of a longer wavelength, which is detected in specific channels. The light source cannot excite the acceptor dye.

FRET Probes Functioning

The hybridization probe system consists of two oligonucleotides labeled with fluorescent dyes. The hybridization probe pair is designed to hybridize to adjacent regions on the target DNA.

Each probe is labeled with a different marker dye. Interaction of the two dyes can only occur when both are bound to their target. The donor probe is labeled with fluorophore at the 3' end and the acceptor probe at 5' end. During PCR, the two different oligonucleotides hybridize to adjacent regions of the target DNA such that the fluorophores, which are coupled to the oligonucleotides, are in close proximity in the hybrid structure. The donor fluorophore (F1) is excited by an external light source, then passes part of its excitation energy to the adjacent acceptor fluorophore (F2). The excited acceptor fluorophore (F2) emits light at a different wavelength which can then be detected and measured.