Parole chiave: Fluorescence, fluorophore, FRET, quencher
Definizione:Molecular beacons are single stranded hairpin-shaped oligonucleotide probes designed to report the presence of specific nucleic acids in a solution
TECHNOLOGY
A molecular beacon consists of four components; a stem, hairpin loop, 5' fluorophore and 3' quencher. When the hairpin-like beacon is not bound to a target, the fluorophore and quencher lie close together and fluorescence is suppressed. In the presence of a complementary target nucleotide sequence, the stem of the beacon opens to hybridize to the target. This separates the fluorophore and quencher allowing the fluorophore to fluoresce. 'Wavelength-shifting Molecular Beacons', incorporate an additional harvester fluorophore enabling the fluorophore to emit more strongly.
The basic mode of operation for molecular beacons has been modified for use in living cells, where enzymic degradation of the beacon can cause separation of fluorophore and quencher and the generation of an erroneous signal. One solution is a system where two beacons attach to the same target on adjacent binding sites. Fluorescence resonance energy transfer (FRET) between a donor beacon and acceptor beacon results in a red-shifted signal that can be distinguished from false positive signals. Cell-based beacons can be detected using confocal fluorescence microscopy.
APPLICATIONS:
Molecular beacons are widely used in genomic and pharmacogenomic applications (for the detection of single nucleotide polymorphisms (SNPs), realtime monitoring of PCR, detection of specific nucleotide sequences) and in cell-based diagnostic applications where they can be used, for example, to detect cancer cells or the presence of microorganisms such as bacteria and viruses.
MICROSCOPE CONFIGURATION:
Observation of molecular beacons in living cells is usually carried out using confocal microscopy techniques. Confocal imaging provides the high resolution and sensitivity required to accurately locate beacon fluorescence and enables 3-D reconstruction for spatial analysis.
RECOMMENDED SYSTEM:
Ti series inverted microscope with confocal system (EC1, C1+, C1si or LiveScan sweptfield confocal).
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