A molecular beacon is the hairpin-shaped single-stranded structured probe which is highly sequence-specific and sensitive.
The Molecular Beacons are used in the real-time PCR assay for the quantification of RNA or DNA present into the sample. It is used in place of the linear TaqMan probe.
The real-time PCR method is one of the excellent modification of the conventional PCR method. Quantification of the sample is only possible in the qPCR only.
In the conventional PCR, we can only amplify the DNA however, in the qPCR or rt PCR we can even quantify it by using the fluorochrome chemistry.
The real-time PCR method is based on the two types of chemistry,
- Probe-based hybridization method
- Dye-based method
We have covered an in-depth article on the when to use dye-based method and when to use Probe-based method. Read the article here: Real-time PCR: Principle, Procedure, Advantages, Limitations and Applications
In the present article, we will discuss the basic principle behind the use of molecular beacons and why it is applied instead of the linear probe.
- A brief introduction to real-time PCR
- What is the molecular beacon?
- Structure of molecular beacons
- Designing of molecular beacons
- Importance of molecular beacons
- Application in genomics
A brief introduction to real-time PCR:
The real-time PCR or the quantitative real-time PCR is a molecular method in which we can quantify the nucleic acid (DNA or RNA) efficiently.
Even the expression of a gene in a particular tissue can also be measured using the qPCR method.
The method is based on the use of fluorochromes. Once the fluoro-labelled oligo binds to its complementary sequence on the target DNA, it emits fluorescence. The level of fluorescence emitted is measured by the real-time PCR instrument.
The amount of the fluorescence emitted is directly proportional to the amount of target DNA amplified.
Two methods are used for quantitative analysis.
- Dye-based method
- Probe-based method
In the dye-based method, the dye binds to the double-stranded DNA emits fluorescent which is measured for the quantification.
In the probe-based method, the fluoro-labelled probe directly binds to the complementary sequence on the target DNA.
The fluorescence released by the probe is directly proportional to the amplified target.
This is a brief and general idea behind the real-time PCR. Read our article on more detail of real-time PCR: Real-time PCR: Principle, Procedure, Advantages, Limitations and Applications
Three types of probes are used in the probe-based hybridization method:
- Linear probes
- Molecular beacons
- Scorpion probes
Here we are going to discuss only about the molecular beacons.
What is the molecular beacon?
The molecular beacons are the TaqMan probe used in the real-time PCR, mainly for increasing the specificity of the reaction.
It is a single-stranded oligonucleotide hairpin structure made up of 25 to 30 nucleotides. In which the 15 to 20 nucleotide present into the middle-loop is complementary to the sequence of our interest.
Whereas the sequences present on both arm are complementary to each other.
Hence it remains bounded with each other.
The molecular beacons consist of 4 parts.
A stem, loop, 3′ end and the 5′ end.
The step consists of 7 to 9 nucleotides which are complementary to each other. The nucleotides in the step remain bounded with each other if the beacon is in the relaxed state.
The loop is the central part of the molecular beacons which is very much important as it is complementary to the target sequence.
The central loop part contains 15 to 20 nucleotides, complementary to the sequence of our interest.
The 3′ end of the molecular beacons contains the quencher dye. This quencher dye on the 3′ end is in the close proximity with the 5′ end, prevent the emission of fluorescence.
The 5′ OH end of the probe contains the fluorescent dye. Once the beacon binds to the complementary sequence, the fluorescent dye is unquenched, released and emits the fluorescence.
The linear TaqMan probe can work efficiently than question arise in mind why to use the molecular beacons?
What is the importance of molecular beacons?
Non-specific binding is one of the major problems in any PCR reaction even in the real-time PCR too.
The real-time PCR quantification assay is too sensitive, it detects some of the sensitive templates and quantifies it. It is applicable mainly in the diagnostic field so imagine how critical it is to avoid any mistakes during the PCR.
What is the problem with the linear TaqMan probe?
The linear TaqMan probes are 18 to 20 nucleotide long sequences on which one end contains the fluorescent dye while another end contains the quencher dye.
The problem with it is if it finds some of the complementary bases, it hybridized, the fluorochrome unquenched and release the fluorescence.
So using a linear probe in the real-time endpoint or real-time PCR assay is not a good choice always.
The molecular beacons are specially designed probes on which if it is not bound to any sequences, the majority of its energy is saved (remains in the hairpin state- relaxed state).
But once it bound to its complementary sequence or in the presence of the complementary sequence the beacons unfolds.
The fluorochrome no longer quenched. It is released and emits the fluorescence.
The detailed stepwise explanation of the mechanism of molecular beacons action is shown in the figure below.
As each probe binds to its complementary sequence, the amount of the fluorescence emitted is measured in a real-time.
Depending upon that, the template DNA is measured.
After each PCR cycle, more probes are hybridized more fluorescence is emitted and more templates are quantified.
The entire mechanism of the molecular beacons is based on the stability of the hybrid formed during the amplification (DNA: DNA hybrid or RNR: RNR hybrid).
The stability of the perfectly matched probe and DNA is higher as compared with the stability of the mismatch. The hairpin makes the mismatch hybrid thermally less stable than the exact match.
Therefore, the hairpin is open up only in the presence of the more stable hybrid viz, exact base pairing.
Further, the stability of the complementary base pairing is more as compared with the stability of the stem base pairing therefore once the exact match is found the stem unfolds.
Designing the molecular beacons:
Designing the molecular beacons are quite a tedious job but it will give a good output.
There are several points you have to understand if you want to design your own beacon.
Designing the primer:
The primer used for the real-time quantification must follow all the standard rules of primer designing. Read our article on primer designing: PCR primer design guidelines
Broadly, the primer has the following characteristics:
- 18- 2o nucleotides long
- 50 to 55% of GC content
- No secondary structure formation
- No complementary bases
- Annealing temperature between 55°C to 65°C
The annealing temperature of primers and beacons must be same or nearer.
Designing the target:
Target designing is as important as the primer designing.
The amplified product of the target DNA must be between 100 to 300 base pairs. A fragment larger than this will create unnecessary problem in the PCR amplification.
The target region must be less GC rich and can be secondary structure free therefore the molecular beacon can bind efficiently.
Designing the molecular beacon:
Design beacon in two different parts, first design the loop part and then the stem part.
The loop must have a controlled GC content and melting temperature 7 to 9°C higher than the annealing temperature.
The annealing temperature of the beacon must match the annealing temperature of the primers or we can say with the target region.
In simple words, we can say that at the same annealing temperature the primer and probe bind to the complementary region.
The maximum nucleotides in a loop are 30, do not use more than this.
The stem contains 5 to 7 complementary bases.
Keep one thing in mind: The stability of the stem must not be more than the stability of the probe and DNA hybrid.
If the stem base pairing is more stable than the loop nucleotides cannot bind to its complementary sequence.
Nonetheless, the stem base pairing must be stable enough that it can not unfold in the presence of mismatch base pairing of the loop.
Check the melting temperature of the molecular beacon using the melting curve analysis.
Adding the fluorochrome and quencher dye:
A single fluorochrome added at the 5′ OH end of the beacon while the quencher molecule is added to the 3′ end in close proximity of the fluorochrome.
Advantages of molecular beacons:
It increases the specificity and the sensitivity of the reaction.
It increases the thermal stability and efficiency of hybridization.
Probes can be preserved during the reaction.
The molecular beacons facilitate detection of multiple targets using multiple fluorochromes in a single reaction, efficiently.
Applications of molecular beacons:
The diagnostic value of the beacon chemistry is more as compared to the dye method.
It is used in the determination or separation of different alleles.
In microbial genetics, it is used in the detection of the pathogen. Furthermore, the accurate quantification of an infectious pathogen can be done.
Multiplexing is more easy using the molecular beacons probe-based method. By using the differently fluoro-labelled molecular beacons different alleles or templates in a sample can be amplified and quantified.
Genotyping using the endpoint real-time method is more accurately done by utilising the beacons.
Gene expression, gene quantification and copy number variation can be done efficiently using this probe.
SNP detection is also possible by “real-time endpoint molecular beacon probe-based method.”
Viral load quantification can be done preciously.
Interestingly, it is used as an intercellular probe for the detection of DNA or mRNA.
- Real-time PCR: Principle, Procedure, Advantages, Limitations and Applications
- Inverse PCR: Principle, Procedure, Protocol and Applications
External resources related to this article:
The molecular beacons provide the next level of sensitivity in the diagnostic practises. Ready to use molecular beacon kits for different templates are available nowadays. The probe-based method is more accurately quantify the nucleic acid than the dye-based method.