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strains. When experimental conditions are strictly controlled,
molecular beacons can be used to sense DNA sequence with single-
base accuracy. Later, the quencher was replaced with an acceptor
dye, typically emitting at a lower energy than the donor dye. Thus,
two-dye molecular beacons [44] manifest molecular recognition in
dual fluorescence channels. In the absence of the target sequence,
the molecular beacon adopts a hairpin structure containing a six-
base-pair stem. The stem brings the two dyes in close proximity, thus
exciting the donor dye results in the emission of the acceptor dye
because of strong FRET. In principle, the two-dye molecular beacons
should have the same single-base accuracy of the dye
quencher
molecular beacons.
non-fluorescent
fluorescent
+
fluorescent
dye
quencher
+
FRET off
donor fluorescent
acceptor
fluorescent
donor dye
Figure 5.13
The first generation of molecular beacons uses a fluorescent
donor and a quencher. The second generation of molecular
beacons replaces the quencher with a fluorescent acceptor,
thus a FRET pair formed at the end of stem loop. Right, an
innovative approach uses conductive metal as the fluorescent
quencher.
A new type of molecular beacons illustrated in Fig. 5.13 uses
metal (Au) substrate as the quencher [45,46]. The hairpin secondary
structure and surface distribution of DNA hairpins on the substrate
affect the hybridization efficiency. Hybridizing with complementary
targets can increase the fluorescence signal more than 100-fold. The
bases used in the hairpin stem and the overall loop length significantly
influence sensitivity and selectivity. Surface-immobilized hairpins
can discriminate between two sequences with a single base-pair
mismatch with selectivity over an order of magnitude difference in
signal under identical assay conditions.
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