Chemical Sciences: A Manual for CSIR-UGC National Eligibility Test for Lectureship and JRF/Molecular beacon

Molecular beacons are oligonucleotide hybridization probes that can report the presence of specific nucleic acids in homogenous solutions. The terms more often used is molecular beacon probes. Molecular beacons are hairpin shaped molecules with an internally quenched fluorophore whose fluorescence is restored when they bind to a target nucleic acid sequence. This is a novel nonradioactive method for detecting specific sequences of nucleic acids. They are useful in situations where it is either not possible or desirable to isolate the probe-target hybrids from an excess of the hybridization probes.

Molecular Beacon Probes
A typical molecular beacon probe is 25 nucleotides long. The middle 15 nucleotides are complementary to the target DNA and do not base pair with one another, and the five nucleotides at each end are complementary to each other and not to the target DNA. A typical molecular Beacon Structure can be divided in 4 parts :

Loop: This is the 18-30 base pair region of the molecular beacon which is complementary to the target sequence.

Stem: The beacon stem sequence lies on both the ends of the loop. It is typically 5-7 bp long at the sequences at both the ends are complementary to each other.

5' fluorophore: Towards the 3' end of the molecular beacon, is attached a dye that fluoresces in presence of a complementary target.

3' quencher (non fluorescent): The quencher dye is covalently attached to the 3' end of the molecular beacon and when the beacon is in closed loop shape, prevents the fluorophore from emitting light.

If the mRNA detected is complementary to the strand in the loop, then the mRNA goes into the loop causing the separation of the fluorophore and the quencher; therefore resulting in fluorescence.

Software
Beacon Designer - Software to design or evaluate standard molecular beacons or molecular beacons for NASBA® assays.