Spectroscopic features of dual fluorescence/luminescence resonance energy-transfer molecular beacons.
Molecular beacons have the potential to become a powerful tool in gene detection and quantification in living cells. Here we report a novel dual molecular beacons approach to reduce false-positive signals in detecting target nucleic acids in homogeneous assays. A pair of molecular beacons, each containing a fluorescence quencher and a reporter fluorophore, one with a donor and a second with an acceptor fluorophore, hybridize to adjacent regions on the same target resulting in fluorescence resonance energy transfer (FRET). The detection of a FRET signal leads to a substantially increased signal-to-background ratio compared with that seen in single molecular beacon assays and enables discrimination between fluorescence due to specific probe/target hybridization and a variety of possible false-positive events. Further, when a lanthanide chelate is used as a donor in a dual-probe assay, extremely high signal-to-background ratios can be achieved owing to the long lifetime and sharp emission peaks of the donor and the time-gated detection of acceptor fluorescence emission. These new approaches allow for the ultrasensitive detection of target molecules in a way that could be readily applied to real-time imaging of gene expression in living cells.
Tsourkas
Andrew
A
Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, Atlanta, Georgia 30332, USA.
Behlke
Mark A
MA
Xu
Yangqing
Y
Bao
Gang
G
D001483:Base Sequence; D005189:False Positive Reactions; D005453:Fluorescence; D031541:Fluorescence Resonance Energy Transfer / Q000295:instrumentation / Q000379:methods*; D005456:Fluorescent Dyes / Q000032:analysis; D020869:Gene Expression Profiling / Q000379:methods; D009693:Nucleic Acid Hybridization; D009696:Nucleic Acids / Q000032:analysis / Q000235:genetics; D015345:Oligonucleotide Probes / Q000032:analysis* / Q000235:genetics; D013997:Time Factors
D005456:Fluorescent Dyes; D009696:Nucleic Acids; D015345:Oligonucleotide Probes