Molecular Beacons for Single Nucleotide Polymorphism Detection

Molecular beacons are short oligonucleotide hybridization probes which can report the presence of specific nucleic acid present. Molecular Beacons are mostly used in real time PCR, which can even detect single nucleotide polymorphism. This makes it very advantageous to use where detection of antibiotic resistance, allelic discrimination, diagnostic assays etc. The sequence of each molecular beacon must be customized to detect the PCR product of interest.

Molecular Beacon Basic Structure

A typical molecular beacon probe is generally >25-30 nucleotide long and will have

1. Loop

2. Stem

3. 5' Flourophore

4. 3' Quencher

Hybridization of the molecular probe to the target nucleic acid occurs if the sequence of the probe exactly matches with the target nucleic acid. Once the hybridization occurs, quencher and flourophore moves apart and results in fluorescent emission. The presence of the emission reports that the event of hybridization has occurred and hence the target nucleic acid sequence is present in the test sample.

Attached to opposite ends of the beacon are a fluorescent reporter dye and a quencher dye. When the molecular beacon is in the hairpin conformation, any fluorescence emitted by the reporter is absorbed by the quencher dye and no fluorescence is detected.

Diagram of molecular beacon: This beacon is 33 nucleotides long with a reporter dye attached to the 5' end and a quencher attached to the 3' end. The nine 5' bases are able to form base pairs with the nine 3' bases which brings the reporter and quencher in very close proximity. Therefore, when the reporter is excited by the appropriate light, its emission is absorbed by the quencher and no fluorescence is detected. The pink lines represent nucleotides that can form base pairs with the PCR product under investigation.

Applications of Molecular Beacon

• SNP detection
• Real-time nucleic acid detection
• Real-time PCR quantification
• Allelic discrimination and identification
• Multiplex PCR assays
• Diagnostic clinical assays