References >> Molecular Beacons

Introduction to Molecular Beacons

Molecular beacons are single stranded hairpin shaped oligonucleotide probes. In the presence of the target sequence, they unfold, bind and fluoresce. The molecular beacon chemistry is one of the chemistries used to carry out a real time experiment.

Molecular Beacon

Structure A molecular beacon consists of 4 parts, namely 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 5' 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.

Molecular Beacons Functioning

Molecular beacons can report the presence of specific nucleic acids from a homogeneous solution. In the presence of a complementary target, the "stem" portion of the beacon separates out resulting in the probe hybridizing to the target. As explained in the illustration above, one end of the molecular beacon is tagged with a fluorophore, and the other one is tagged with a quencher. In the absence of a complimentary target sequence, the beacon remains closed and there is no appreciable fluorescence. When the beacon unfolds in the presence of the complementary target sequence, the fluorophore is no longer quenched, and the molecular beacon fluoresces. The fluorescence is easily detected in a thermal cycler.

The amount of fluorescence at any given cycle, or following cycling, depends on the amount of specific product. For quantitative PCR, molecular beacons bind to the amplified target following each cycle of amplification and the resulting signal is proportional to the amount of template. Fluorescence is monitored and reported during each annealing step when the beacon is bound to its complementary target. This information is then used during PCR or RT-PCR (reverse transcriptase PCR) experiments to quantify initial copy number.

For endpoint analysis, PCR reactions containing molecular beacons can be run on any 96-well thermal cycler, then read in a fluorescence reader.

Molecular Beacons Versus Linear Probes

While other systems use fluorescence to detect the accumulation of PCR product, molecular beacons add another level of specificity due to the presence of a distinct molecular probe apart from the primers. In addition, the stem probe structure of a molecular beacon makes it better able to discriminate single base-pair mismatches (compared to linear probes) because the hairpin makes mismatched hybrids thermally less stable than hybrids between the corresponding linear probes and their mismatched target.

Furthermore, unlike linear hydrolysis probes, quenching of molecular beacons has been shown to occur through a direct transfer of energy from the fluorophore to quencher. Consequently, a common quencher molecule can be used, increasing the number of possible fluorophores that can easily be used as reporters. This is an important advantage when designing Real time PCR experiments in which several molecular beacons with different colored fluorophores are used to detect multiple targets in the same tube (multiplexing).

The use of molecular beacons in diagnostic assays has thus been ever increasing. Diagnostic assays that aim at detecting single nucleotide polymorphisms, screening genetically diverse species and developing drugs through pharmacogenetic applications are now using molecular beacons based real time PCR assays. The ability of molecular beacons to fluoresce when bound specifically to a double stranded target and their accuracy to discriminate alleles, make them a powerful tool in the research lab armory. Molecular beacons can be successfully used to ascertain not only the presence or absence of a particular causative agent, but also in screening which antibodies will be effective against a particular strain of the causative organism.

In-Silico Molecular Beacons Design with Beacon Designer™

Beacon Designer™; for designing real time PCR assays, was developed in consultation with one of the inventors of molecular beacon technique, Dr. Sanjay Tyagi. Beacon Designer™ finds the best possible primer pair, TaqMan® probes and molecular beacons for single or multiplex real time PCR assays. For designing primers, Beacon Designer™ avoids cross homologies identified by automatically interpreting BLAST search results and template secondary structures. The resultant primers are highly specific and efficient. Beacon Designer can be used to design primers and probes for allele discrimination in multiplex experiments and to evaluate pre-designed assays as well. Verification BLAST for primers and probes is also made available.

Other Techniques Performed by Beacon Designer™

Other than the design of molecular beacons, Beacon Designer™ also supports the following real time PCR chemistries.

TaqMan® Probes: Beacon Designer™ makes it easy to design TaqMan® probe based real time PCR assays. The TaqMan®probes that the program designs are specific and efficient.

SYBR® Green Assays: Perhaps the most commonly used real time PCR assays. Beacon Designer™ can design and evaluate SYBR® Green primers in minutes.

FRET Probes: With a single click, you can design FRET probes that are free of dimers and other secondary structures.

Scorpions®: Scorpions® primers and probes can be designed using Beacon Designer™ for specific detection of the target.