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.
Applications
of FRET probes:
1. Quantitative PCR.
2. DNA copy number measurements.
3. Pathogen detection assays.
4. SNP genotyping.
5. Verification of microarray results.
Beacon Designer designs optimal
FRET probes free of dimers, repeats and runs
to ensure signal fidelity. Using Beacon Designer
a pre-designed FRET probes can be evaluated
or a probe can be designed for a pre-designed
primer set. This facilitates using SYBR® Green
primers for FRET assays.
