Just as a lighthouse emits a guiding beam to safely navigate ships through treacherous waters, a Molecular Beacon casts a fluorescent glow, signaling the precise location of its target nucleic acids amidst the ocean of the genome.
WHAT ARE MOLECULAR BEACONS?
Molecular Beacons are a type of oligonucleotide probe (short singled-stranded nucleic acid sequences between 30–50 bases) used in molecular biology and genetics research. They are designed to have a unique sequence flanked by indirect repeats so that a stem-loop structure is formed, allowing the 5′ and 3′ ends to be maintained in proximity. Molecular Beacon probes fluoresce upon hybridization with a complementary target sequence. These structured probes are highly sensitive, sequence specific, and are used for sequence detection in qPCR for in vitro studies. Beacons are useful for real-time PCR (rtPCR), detecting specific nucleic acid sequences and monitoring gene expression due to their high specificity and sensitivity.1,2
HOW DO MOLECULAR BEACONS WORK?
A Molecular Beacon is a single-stranded bi-labeled fluorescent probe held in a hairpin-loop conformation (20 to 25 nt is most common) by complementary stem sequences (around 4 to 6 nt) at both ends of the probe. The 5’ and 3’ ends of the probe contain a reporter and a quencher molecule, respectively. The loop is a single-stranded DNA sequence complementary to the target sequence. The proximity of the reporter (fluorophore, or fluorescent dye) and quencher causes the quenching of the natural fluorescence emission of the reporter by bringing about energy transfer. The structure and mechanism of a molecular beacon is shown below (Figure 1).
Molecular Beacon Design and Function
- Molecular Beacon probes are designed with four essential parts: loop, stem, quencher, and reporter.
- The stem and loop regions of the Molecular Beacon form a stable hairpin structure, bringing the fluorophore and quencher in close proximity. This close proximity results in the quencher absorbing the fluorescence emitted by the fluorophore, effectively suppressing the signal.
- When Molecular Beacons encounter the complementary target sequence, the hairpin-loop structure opens and separates the 5’-end reporter from the 3’-end quencher. As the quencher is no longer in proximity to the reporter, the fluorescence of the reporter is no longer quenched, thereby allowing fluorescence emission.
- The intensity of the fluorescence signal is directly proportional to the amount of target nucleic acid present in the sample.
- In real-time PCR, when added prior to amplification, Molecular Beacons undergo disruption of base-pairing in the stem. This allows the probe to hybridize to the complementary target upon denaturation, which separates the reporter dye and quencher, thereby inducing reporter dye fluorescence.
Figure 1.How Molecular Beacons Work. The unbound beacon is in a hairpin configuration with quenched fluorescence (left graphic). Once the beacon binds to the target stretch of DNA the quencher and reporter are separated spatially and the signal is released (right graphic).
Applications of Molecular Beacons
- SNP detection
- Allele discrimination
- Pathogen detection
- Multiplexing
- Viral load quantification
- Gene expression analysis
- Gene copy determination
- Endpoint genotyping
- In vitro quantification or detection
Benefits of Using Molecular Beacons
High specificity and sensitivity
Molecular beacons can distinguish between sequences that differ by a single nucleotide, allowing for precise detection of specific nucleic acid sequences.
Real-time detection capabilities
Molecular Beacons enable real-time monitoring of PCR amplification, allowing researchers to track the progress of a reaction as it occurs.
Reduced background signal
The stem-loop structure keeps the fluorophore and quencher in close proximity, reducing background fluorescence and increasing signal-to-noise ratio when the target sequence is absent.
Versatility
Molecular beacons can be designed to target various sequences, making them useful in a wide range of applications, including gene expression studies, mutation detection, and viral load monitoring.
Non-invasive detection
Molecular beacons can be used in living cells to detect and visualize the presence of specific RNA molecules without disrupting cellular processes.
Multiplexing capability
Different Molecular Beacons can be labeled with different fluorophores, allowing simultaneous detection of multiple targets in a single reaction.
SNP Profiling
Molecular Beacons are ideal for single nucleotide polymorphism (SNP) profiling due to their high specificity and ability to distinguish sequences differing by a single nucleotide. Their unique design enables the simultaneous detection of multiple SNPs in a single reaction, enhancing efficiency and throughput. Additionally, Molecular Beacons can function within living cells, allowing non-invasive, real-time monitoring of gene expression and mutations.3
ADD LOCKED NUCLEIC ACIDS TO YOUR BEACON PROBE
Locked Nucleic Acids offer several advantages when used in Molecular Beacons, enhancing their performance and utility in various research, diagnostic, and preclinical applications:
- Increase thermal stability, binding affinity, and hybridization specificity.
- Obtain greater accuracy in SNP detection, allele discrimination and in vitro quantification or detection.
- Reduced background signal due to increased stability and reduced non-specific interactions.
- Achieve easier and more sensitive probe designs for problematic target sequences.
- Resistance to nucleases for long-term studies.
SIGMA-ALDRICH MOLECULAR BEACON Product Features
- Amounts: 1, 3, 5, and 10 OD
- Purification: HPLC
- Sequence Lengths: 15 - 40 bases
- Quality Control: 100% mass spectrometry
- Format: Supplied dry in amber tubes
- Custom formats available (normalizations, special plates, etc.)
Our probes are provided in a format to simplify your experimental planning.
Guaranteed Yields |
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*Estimate is based on 3 nmol or 32 µg for 1 OD and 200 nM in 25 µL reaction (5.0 pmol/reaction). Estimate is based on an average sequence length of 30 bases.
The most common fluorophore and quencher combinations are listed below:
Spectral Properties Table |
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Shipping Schedule |
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*Delivery times may be longer due to international transit, customs clearance delays, etc. Large projects will be placed on a delivery schedule based upon project needs.
References
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