Sanjay Tyagi, Salvatore A.E. Marras
& Fred Russell Kramer
Department of Molecular Genetics, Public Health Research
Institute, 455 First Avenue, New York,
NY 10016.
Correspondence should be addressed to Sanjay Tyagi sanjay@phri.nyu.eduhybridization probesFRETreal-time PCRhomogeneous assayshaplotype determination
We describe wavelength-shifting molecular beacons, which are nucleic acid
hybridization probes that fluoresce in a variety of different colors, yet
are excited by a common monochromatic light source. The twin functions of
absorption of energy from the excitation light and emission of that energy
in the form of fluorescent light are assigned to two separate fluorophores
in the same probe. These probes contain a harvester fluorophore that absorbs
strongly in the wavelength range of the monochromatic light source, an emitter
fluorophore of the desired emission color, and a nonfluorescent quencher.
In the absence of complementary nucleic acid targets, the probes are dark,
whereas in the presence of targets, they fluoresce—not in the emission
range of the harvester fluorophore that absorbs the light, but rather in the
emission range of the emitter fluorophore. This shift in emission spectrum
is due to the transfer of the absorbed energy from the harvester fluorophore
to the emitter fluorophore by fluorescence resonance energy transfer, and
it only takes place in probes that are bound to targets. Wavelength-shifting
molecular beacons are substantially brighter than conventional molecular beacons
that contain a fluorophore that cannot efficiently absorb energy from the
available monochromatic light source. We describe the spectral characteristics
of wavelength-shifting molecular beacons, and we demonstrate how their use
improves and simplifies multiplex genetic analyses.
