Myeloid Leukemia

Real-Time RT-PCR Strategies 41
Fig. 8. Specific fragment detection with molecular beacon probes. (A) Structure of
a molecular beacon probe, which consists of a hairpin loop structure where the loop is
a single-stranded probe that is complementary to the amplicon. The arm sequences
flanking either side of the probe are complementary to one another. A fluorophore
(circle) and a quencher (pentagon) nonfluorescent chromophore are attached to
the termini. (B) During the denaturation step, the hairpin is opened out and the
fluorophore and quencher are separated. During annealing step, the probe binds to
target sequence and fluorescence can be monitored. During primer extension, the
molecular beacons dissociate from their targets, and fluorescence is again quenched.
fluorophore at the 5� end, (3) a quencher at the 3� end, and (4) a target-specific
PCR primer linked to the 3� end of the hairpin loop structure via (5) a PCR
stopper (26,27). The PCR stopper prevents read-through of the probe element.
After PCR extension of the scorpion primer, the resultant amplicon contains
a sequence that is complementary to the probe, which is rendered single stranded