Evelyn McGown and Michael Su
Molecular beacons are fluorogenic oligonucleotide probes developed by Kramerand associates to detect specific nucleic acids in homogeneous solutions.1 4 They are single-stranded oligonucleotides that exist in a hairpin shape because of thecomplementary arms at the ends which bind together to form a stem. Thestrained loop portion contains the nucleotide that is complementary to theintended target. A fluorophore is covalently bound to one end of theoligonucleotide and a quencher to the other end. In the absence of target, thefluorophore and quencher are held in close proximity by the arms and thefluorescence is internally quenched. When the probe hybridizes to the targetmolecule, it undergoes a conformational change resulting in separation of thefluorophore and quencher, and restoration of fluorescence. Molecular beacons areappealing because they can be used in homogeneous solutions and obviate theneed to isolate probe/target hybrids from an excess of unbound hybridizationprobes. They have been used to monitor polymerase chain reactions.3
Many combinations of fluorophores and quenchers are possible. Kramers grouphas found that 4-(4-dimethylaminophenylazo) benzoic acid (DABCYL) can serveas a universal quencher for a number of fluorophores. Even if the fluorophoreemission spectrum does not overlap with the DABCYL absorption spectrum,quenching (with >95% efficiency) occurs by direct energy transfer fromfluorophore to quencher because of their close proximity.2
This application note describes measurement of probe/target complexes with theSPECTRAmax GEMINI microplate spectrofluorometer.
MATERIALS AND METHODS
Samples of molecular beacons and complementary targets were obtained fromResearch Genetics, Huntsville, Alabama. The oligonucleotide sequences are notimportant to this discussion. The microplates used w