Detection of Fluorescence in Real-Time PCR Using Molecular Beacons and Taqman Probes
Molecular beacons (MB) and Taqman (TQ) are used as probes in Real time PCR experiments. Both these probes are based on the principle FRET and employ a fluorophor (F) and a quencher (Q). However the mechanisms by which they function are different as illustrated below
At what stage of the PCR we would be able to detect fluorescence?
A. Annealing step for both.
B. Extension step for both
C. Annealing for A and Extension for B.
D. Extension for A and Annealing for B.
Real-time PCR (qPCR) is a widely used technique in molecular biology to quantify nucleic acids in real-time. It involves monitoring the accumulation of PCR products during the reaction cycles using fluorescence-based probes. Among the most commonly used probes in qPCR are Molecular Beacons (MB) and Taqman Probes (TQ). Both of these probes rely on the principle of FRET (Fluorescence Resonance Energy Transfer) to generate fluorescence signals, but they function through distinct mechanisms at different stages of the PCR cycle. Understanding when fluorescence is detected for each probe type is crucial for interpreting qPCR results accurately.
Correct Answer:
The correct answer is (C) Annealing for A and Extension for B.
- Molecular Beacons – Fluorescence is detected during the annealing step.
- Taqman Probes – Fluorescence is detected during the extension step.
What are Molecular Beacons and Taqman Probes?
1. Molecular Beacons (MB):
Molecular beacons are hairpin-shaped oligonucleotide probes labeled with a fluorophore (F) at one end and a quencher (Q) at the other.
Mechanism of Molecular Beacons:
- In the absence of a complementary target sequence, the hairpin structure keeps the fluorophore and quencher in close proximity, leading to FRET-based quenching of fluorescence.
- During the annealing step of PCR, the molecular beacon binds to the target DNA, separating the fluorophore from the quencher.
- This separation prevents quenching and allows fluorescence emission, which is detected in real-time.
✅ Fluorescence detection occurs during the annealing step.
2. Taqman Probes (TQ):
Taqman probes are linear oligonucleotide probes labeled with a fluorophore (F) at one end and a quencher (Q) at the other.
Mechanism of Taqman Probes:
- During the annealing step, the Taqman probe hybridizes to the target DNA.
- During the extension step, the 5′ to 3′ exonuclease activity of Taq DNA polymerase cleaves the probe.
- This cleavage separates the fluorophore from the quencher, leading to fluorescence emission.
✅ Fluorescence detection occurs during the extension step.
Why Fluorescence is Detected at Different Stages
- Molecular Beacons: The fluorophore-quencher separation occurs upon hybridization with the target sequence, which happens during the annealing phase of PCR.
- Taqman Probes: The fluorophore-quencher separation requires exonuclease activity, which occurs during the extension phase when DNA polymerase cleaves the probe.
Comparison of Molecular Beacons and Taqman Probes
| Feature | Molecular Beacons | Taqman Probes |
|---|---|---|
| Structure | Hairpin-shaped | Linear |
| Fluorophore-Quencher Positioning | Close in hairpin loop | Linear and adjacent |
| Mechanism of Fluorescence Generation | Hybridization with target | Probe cleavage by DNA polymerase |
| Stage of Fluorescence Detection | Annealing | Extension |
| Specificity | High | High |
| Sensitivity | Moderate | High |
| Reusability | Reused until denatured | Single-use per cycle |
Why Other Options Are Incorrect:
- (A) Annealing step for both – Incorrect because fluorescence detection for Taqman probes occurs during the extension step, not annealing.
- (B) Extension step for both – Incorrect because Molecular Beacons emit fluorescence during the annealing step.
- (D) Extension for A and Annealing for B – Incorrect as the detection stages are reversed.
Applications of Molecular Beacons and Taqman Probes
- Genotyping:
- Taqman probes are widely used in SNP (Single Nucleotide Polymorphism) analysis and mutation detection.
- Pathogen Detection:
- Both probes are used in real-time PCR assays for detecting bacterial and viral infections (e.g., COVID-19).
- Gene Expression Analysis:
- Taqman probes provide high sensitivity and accuracy for quantitative gene expression analysis.
- Allelic Discrimination:
- Molecular beacons offer high specificity for detecting single nucleotide mismatches.
Advantages and Disadvantages of Molecular Beacons and Taqman Probes
✅ Advantages of Molecular Beacons:
- High specificity for distinguishing similar sequences.
- Reusable until denatured.
- No degradation by DNA polymerase.
❌ Disadvantages of Molecular Beacons:
- More complex to design due to hairpin structure.
- Lower sensitivity compared to Taqman probes.
✅ Advantages of Taqman Probes:
- High sensitivity due to probe cleavage and signal amplification.
- Simplified design and high efficiency.
❌ Disadvantages of Taqman Probes:
- Single-use per cycle due to probe degradation.
- May produce false positives if the probe binds non-specifically.
Challenges in Using Molecular Beacons and Taqman Probes
- Non-Specific Binding: Can lead to false-positive signals.
- Temperature Sensitivity: Incorrect annealing temperatures can reduce specificity.
- Fluorophore Bleaching: Prolonged exposure to light can reduce fluorescence signal.
- Probe Degradation: Taqman probes are degraded after each cycle, increasing costs.
Conclusion
In real-time PCR, Molecular Beacons generate fluorescence during the annealing step, while Taqman Probes produce fluorescence during the extension step due to polymerase-mediated probe cleavage. Both techniques offer high specificity and sensitivity for nucleic acid detection, but their distinct mechanisms make them suitable for different applications. Understanding these differences is crucial for designing accurate and efficient real-time PCR assays.
71 Comments
Akshay mahawar
March 16, 2025Done 👍
Suman bhakar
March 17, 2025👍
pallavi gautam
March 18, 2025doneee
Parul
March 21, 2025Done sir.
Abhilasha
March 25, 2025Done
Nisha
March 27, 2025Done
Mohd juber Ali
August 24, 2025Annealing (MB) and extention (taq man)
During annealing step of PCR molecular beacons binds with target dna and fluorochrome separate from quencher and produce fluoroscenc
In extention step taq man probe also produce fluoroscenc
Sakshi yadav
August 24, 2025Extension for B taq.poly.breaks the fluorescent from quencher and than emit the fluorescent
karishma don
August 24, 2025for taqman the fluorescence detects at extension step while for molecular beacon it detects in annealing step
Mansukh Kapoor
August 24, 2025The correct answer is option 3rd
Annealing for A and Extension for B
Mol baecons detects flourescent during annealing and Taq Man detects flourescent during extension
Anurag Giri
August 24, 2025Ans 3
Molecular Beacons – Fluorescence is detected during the annealing step.
Taqman Probes – Fluorescence is detected during the extension step
Soniya Shekhawat
August 24, 2025Answer is 3rd Annealing of A and EXTENSION of B, molecular beacons show flurosence during the annealing in PCR when MB hybridised with DNA and tagman probe Fluroscence show during extension of PCR than taq polymers are degrade the taqman probe.
Manisha gujar
August 24, 2025Option c is correct
Khushi Agarwal
August 24, 2025Option c is correct here
Annealing for A and Extension for B
Avni
August 24, 2025The correct answer is (C) Annealing for A and Extension for B.
Khushi Pareek
August 24, 2025Annealing: molecular beacon
Extension : Taqmann probe
Roopal Sharma
August 24, 2025Anealing for a and extension for b .option c is correct.
Priyanka Choudhary
August 24, 2025Option c is correct answer,
Annealing for A and Extension for B
Divya rani
August 24, 2025C is the answer
Molecular beacons….fluorescent in annealing step because here the fluorescent is separated from quencher.
Taqman probe…..fluorescent in extension step because here the fluorescent is separated from quancher.
Dipti Sharma
August 24, 2025(C) Annealing for A and Extension for B.
AKANKSHA RAJPUT
August 24, 2025The correct answer is (C) Annealing for A and Extension for B.
Neelam Sharma
August 24, 2025Annealing for A and Extension for B
anjani sharma
August 24, 2025c is answer
Annealing for A and Extension for B
Neha Yadav
August 24, 2025Fluorescence is detected during Annealing in Molecular beacons and in Taqman fluorescence during extension
Tanvi Panwar
August 24, 2025Annealing for A and extension for B.
MOHIT AKHAND
August 24, 2025Done ✅
Heena Mahlawat
August 24, 2025Annealing for A and extension for B
Aakanksha Sharma
August 24, 2025Extension for taqman probe
Annealing for molecular beacon
Arushi Saini
August 24, 2025Anneling for A and extention for B
Bharti Yadav
August 24, 2025molecular beacon detect flourescent during annealing while Taqmann probe detect flourescent during extension.
Santosh Saini
August 24, 2025In real time PCR, molecular beacon binds with target DNA during annealing, while Taq probe produce fluorescence during the extension step and in extension step fluorochrome separated from quencher
Dharmpal Swami
August 25, 2025Molecular beacon in which flurosence detect in anneling step
Taq man in which flurosence detect in extention step
Ayush Dubey
August 25, 2025Annealing for A and Extension for B.
Surbhi Rajawat
August 25, 2025To detect fluorescent in real time PCR we nust free the fluorochrome of the quencher and that is in option C
Palak Sharma
August 25, 2025molecular beacon binds with target DNA during annealing, while Taq probe produce fluorescence during the extension step
shruti sharma
August 25, 2025Annealing for A and Extension for B
Varsha Tatla
August 25, 2025In real time PCR,mol. Beacon binds with target during annealing nd during extension in taqman
Payal Gaur
August 25, 2025C. Annealing for A and extension for B
Pallavi Ghangas
August 25, 2025Annealing for molecular beacon an extension for tackman
Monika jangid
August 25, 2025Annealing for a and extension for b bcoz mb detect the fluorescent in annealing nd taaman during extension
Vanshika Sharma
August 26, 2025Molecular beacon bind with target during annealing and in taqman during extension
Aafreen Khan
August 26, 2025Annealing for A and Extension for B because Molecular Beacons detects fluorescence during the annealing step, while Taqman Probes detects fluorescence during the extension step
Minal Sethi
August 26, 2025In case of Molecular beacon Amplification is detected at Annealing step and for Taqman Probe amplification is detected at Extension step
Mitali saini
August 26, 2025The correct answer is (C) Annealing for A and Extension for B.
Molecular Beacons – Fluorescence is detected during the annealing step.
Taqman Probes – Fluorescence is detected during the extension step.
Aman Choudhary
August 26, 2025Option C is correct
In annealing step fluorescence is detected by molecular beacon and in extension step Taqman probe is responsible to detect fluorescence
Shivani
August 26, 2025Annealing for A and Extension for B.
Rishita
August 26, 2025Correct answer is c
Surendra Doodi
August 26, 2025Annealing for molecular beacon and Extension for Taq.man
Devika
August 27, 2025Annealing for A extension for B
Muskan singodiya
August 27, 2025Anneleing for a and extension for b
Bhawna Choudhary
August 27, 2025Correct answer is C
Sakshi Kanwar
August 27, 2025For molecular beacon due to high temperature of denaturation the the distance between flourochrome quencher increases and it bind to DNA during annealing
Where as in TAQMAN due to taq polymerase activity in extension flourochrome dissociate from quencher
Annealing for A and Extension for B
Nilofar Khan
August 28, 2025correct answer is (C)
Annealing for moleculer beacon and Extension for taqman
Mohini
August 28, 2025Option C: Annealing for A and Extension for B.
Kanica Sunwalka
August 28, 2025done
Deepika Sheoran
August 28, 2025Option C is correct
Annealing – Molecular Beacon
Extension – Taqman Probe.
Khushi Vaishnav
August 28, 2025Annealing for A and Extension for B.
Parul Yadav
August 29, 2025Fluorescence is observed in Annealing step of Molecular Beacon PCR and Extension step of Taqman Probe PCR.
Simran Saini
August 29, 2025Annealing for A and Extension for B.
Molecular Beacons – fluorescence is detected during the annealing step.
Taqman Probes – fluorescence is detected during the extension step.
Priya dhakad
August 29, 2025Option c is correct
Molecular Beacons – Fluorescence is detected during the annealing step.
Taqman Probes – Fluorescence is detected during the extension step.
Anisha Beniwal
August 29, 2025Annealing for molecular beacon and extension for taq man
Asha Gurzzar
August 30, 2025Annealing for a and extension for b
Khushi Singh
August 30, 2025C is correct
Sonam Saini
August 30, 2025Option A for analying and B for extinction
Kirti Agarwal
August 31, 2025Annealing for molecular beakers
Extention for taqman
Kajal
August 31, 2025correct answer is (C) Annealing for A and Extension for B
Kajal
August 31, 2025Option c is correct as in molecular beacon annealing step or in taqman extension step are used for detection of florosence
Konika Naval
August 31, 2025Annealing for A and Extension for B.
Khushi Mehra
September 8, 2025Molecular beacons detect fluorescent during annealing step and taqman detect fluorescent during extension step.
Muskan Yadav
September 17, 2025Annealing for A and Extension for B.
Molecular Beacons – Fluorescence is detected during the annealing step.
Taqman Probes – Fluorescence is detected during the extension step.
Meenakshi Choudhary
November 11, 2025Annealing for A and Extension for B.