FRET Probes

Fluorescence resonance energy transfer (FRET) is a quantum phenomenon that occurs between two dye molecules. Excitation is transferred from one dye molecule to the other dye molecule via dipole-dipole interactions without photon emission. The fluorescence of the donor molecule is quenched and the acceptor molecule is excited. Energy is then lost by heating or fluorescence emission. The occurrence of FRET results in a decrease in the fluorescence intensity, lifetime, and quantum efficiency of the donor, and an increase in the fluorescence intensity of the acceptor. FRET technique is widely utilized in biological experiments such as molecular structure detection.

LightCycler probes or FRET probes are hybridization probes based on the FRET technique. They consist of a pair of single-stranded fluorescent-labeled oligonucleotides. The donor fluorophore is attached to the 3' end of the upstream probe, and the acceptor fluorophore is attached to the 5' end of the downstream probe. The two fluorescent labeled oligonucleotide probes hybridize in a head to tail arrangement to adjacent sequences on the target DNA. Interaction between the two fluorescent labels occurs only when both probes are bound. During FRET, the donor fluorophore excited by a light source, transfers its energy to the acceptor fluorophore. Since FRET is a distance-dependent process, the gap of 1-5 bases between neighboring sequences on the same strand is optimal.

The donor fluorophore emits light at a wavelength within the absorption range of the acceptor fluorophore. During annealing, when the donor fluorophore is excited by the instrument light source, the acceptor fluorophore absorbs the donor emission and emits light at the wavelength detected by the qPCR instrument. If the two probes are not simultaneously bound to the target, no or very little fluorescence will be detected. The hybridization probes measure the amount of product produced each PCR cycle by producing FRET fluorescence in proportion to the amount of accumulating target DNA. FRET probes are suitable for detecting substitutions and small deletion or insertion mutations by performing dissociation or melting curve analysis of the probe for the target.

Amerigo Scientific provides custom LightCycler/FRET probes for quantitative PCR (qPCR), DNA copy number measurements, SNP genotyping, pathogen detection assays and other applications. The free 3' hydroxyl group of the acceptor probe is blocked with a phosphate group (P) to prevent Taq DNA polymerase extension.

Specifications of FRET Probes

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Design and synthesize FRET probes with highly sensitivity and sequence-specificity.