Fig. 17.2.

Principle of how molecular beacons function. (a) When the probe sequence (loop portion) encounters a target that is perfectly complementary to it, a conformational reorganization of the molecule occurs, resulting in a separation of the stem and the generation of a fluorescence signal. (b) Thermal denaturation profiles of molecular beacons when they are with wild-type or mutant targets. The wild-type target is represented by solid lines and the mutant target is represented by dashed lines. The absence of target is indicated by a dotted line. The conformational state of the molecular beacon is shown directly above the line. By careful design of molecular beacons, mismatched targets can be easily discriminated from perfectly matched targets with “windows of discrimination” as high as 10°C. The optimal temperature for the annealing step from this thermal denaturation profile is found to be 50°C and therefore is used in real-time PCR. (c) An example of how each molecular beacon, the “red”-labeled or the “green”-labeled, competes to hybridize to the same region depending on whether it is perfectly complementary to the region.