Molecular Beacons
Beacons are a refinement of the non-specific dyes used in RT-PCR for DNA detection. They are able to specifically recognise a particular DNA sequence and fluoresce upon binding to it. Essentially they consist of 3 regions.Regions 1 and 3 can bind to each other to form a double stranded DNA helix. Region 1 forma a loop between these regions and consists of a sequence that can recognise a target of choice. Regions 1 and 3 contain a fluorescent dye and a quencher molecule that stops the dye fluorescing. When regios 1 and 3 are double stranded the dye and quencher are close together and no fuorescence can be detected. In the presence of the target DNA th emolecule unfolds and the dye and quencher are separated so that the molecule fluoresces. Thus the eacon is a molecule that will only fuoresce in the presence of a specific target. This property can be used to add an extra level of specificity to the PCR detection system in RT-PCR. Non-specific products will not react with the beacon and no signal will be detected.
The animation below shows a beacon binding to its target and fluorescing:
Other types of specific probes are available. The most commonly used are Taqman and Scorpions. These rely on broadly similar principles to beacons and both contain sequences that are complementary to the target.