qPCR experiments are always disturbed by background fluorescence? "Quenching codes" of 7 types of probes and a guide to avoid pitfalls

The qPCR amplification curve is abnormally elevated?
The negative control (NTC) is strangely fluorescent?
Repeating the experiment but getting jumping Ct values?
A scientific researcher quietly broke down again...

The root cause is often hidden in the quantum world of probe design - the energy transfer efficiency between the quencher group and the reporter group, which directly determines the signal-to-noise ratio of the fluorescence signal. This article will interpret the fluorescence quenching code of 7 types of probes and help you crack the experimental traps hidden behind the light signal.


1. TaqMan probe: classic exonuclease activity probe

Molecular architecture

• Linear oligonucleotides (15-30 bp)
• 5' end labeled with FAM/VIC/HEX and other reporter groups (Reporter)
• The 3' end is bound to a quencher group such as TAMRA/MGB (Quencher)

Mechanism of action

During the annealing phase, the probe binds specifically to the target sequence. Taq DNA polymerase exhibits 5'→3' exonuclease activity during the extension process, hydrolyzing the probe to release the reporter group (Figure 1). The fluorescence intensity is positively correlated with the accumulation of the amplified product, achieving a "hydrolysis-dequenching" signal transduction mode.

Technical advantages

• Compatible with multiplex detection (multi-color fluorescent labeling)
• Simple probe design and cost-effective
• Applies to<150 bp短片段扩增（如SNP分型） 

2. Molecular beacon probes (MB): dynamic conformational switching probes

Molecular architecture

• Stem-loop structure design: loop region (15-30 nt targeting sequence)
• Stem region (5-7 bp reverse complementary sequence)
• The 5'/3' ends are labeled with fluorescent pairs such as Cy3/BHQ1

Mechanism of action

When the target sequence is not bound, the stem region forms a close proximity between the fluorescent group and the quenching group (<10 Å）。当靶序列扩增后，环区与靶序列杂交导致茎环结构打开，淬灭效应解除（图2）。该构象转换可实现单核苷酸多态性（SNP）的高分辨率检测。 

Technical advantages

• Low background signal (background quenching efficiency >95%)
• Suitable for high GC content templates
• Real-time monitoring of hybridization kinetics

Molecular architecture

• Two probes separated by 1-5 nt (donor probe: FAM; acceptor probe: LC Red 640)
• The donor probe is labeled at the 3' end and the acceptor probe is labeled at the 5' end

Mechanism of action

Based on the principle of fluorescence resonance energy transfer (FRET), when two probes bind to the target sequence at the same time, the donor group (Donor) transfers energy to the acceptor group (Acceptor), and the detection channel switches to the acceptor emission wavelength (Figure 3). This design achieves the decoupling of the excitation/emission spectrum and significantly improves the signal-to-noise ratio.

Technical advantages

• Eliminate primer dimer interference
• Suitable for long fragment (>300 bp) detection
• Requires FRET detection module (such as LightCycler system)

4. Scorpion: intramolecular self-hybridization probe

Molecular architecture

• 3' extension primer (18-25 nt)
• The 5' probe region (15-20 nt) is connected by a spacer arm
• The probe ends form an intramolecular quenching structure

Mechanism of action

During the extension process, the newly synthesized complementary strand causes the probe region to form intramolecular hybridization with the product strand, without the need for diffusion between the probe and target molecules (Figure 4). The intramolecular reaction kinetics are 100 times faster than traditional probes, making it particularly suitable for rapid cycling experiments.

Technical advantages

• Improved reaction efficiency (kcat≈10^7 M^-1s^-1)
• Suitable for fast qPCR (<40分钟全程）  
• Able to detect low-abundance targets (LOD up to 10 copies/μL)

5. MGB probe: double-stranded stability enhancement probe

Molecular architecture

• The 3' end is covalently linked to a tripeptide minor groove binder (MGB)
• Use of non-fluorescent quencher (NFQ)
• Probe length shortened to 13-18 bp

Mechanism of action

MGB molecules bind to the minor groove of the DNA double strand through van der Waals forces, stabilizing the probe-target complex. The increase in Tm value is positively correlated with the GC content (ΔTm=0.5-1.5℃/bp), and combined with the NFQ quenching group, the background signal is reduced to 1/5 of the TAMRA system (Figure 5).

Technical advantages

• High specificity (distinguishing single base mismatches)
• Adapt to complex templates (such as direct amplification of genomic DNA)
• Mg²+ concentration needs to be optimized (2.5-4 mM recommended)

6. Dual quenching probes: optimized probes for multiplex detection

Molecular architecture

• 3' end primary quencher (such as BHQ1)
• Internal secondary quencher (such as Iowa Black RQ)
• Probe length up to 40 bp

Mechanism of action

The dual quenching system enhances the quenching efficiency (QY>99.5%) through the steric hindrance effect, and can cover more mutation sites with the long probe design. In the six-fold detection system, the cross-interference rate is <0.3% (traditional probes>5%).

Technical advantages

• Supports high-density multiplex detection (≥6 plex)
• Reduce cross-quenching between probes
• A staged annealing procedure is required

7. LNA probe: high affinity modified probe

Molecular architecture

• 2'-O,4'-C Methylene Bridge Locked Nucleic Acid Modification
• Each additional LNA monomer increases ΔTm by 2-8℃
• Conventional mixing ratio 20-40%

Mechanism of action

The rigid double-ring structure enhances the base stacking force and significantly improves the stability of the double strand. In miRNA detection, the Tm value of the 8-mer LNA probe can reach 65°C (the traditional DNA probe is only 35°C), achieving high-sensitivity detection of short sequences.

Technical advantages

• Detection of short RNA/DNA (such as miRNA)
• Improve mismatch discrimination ability (ΔTm>10℃)
• Higher crafting cost (+30-50%)