Exploring the Principles of Probing in QPCR in 2023

Probe-based quantitative PCR (qPCR) probe is an extension of non-quantitative PCR technology in which specific DNA ‘probe’ sequences replace the PCR primers.

It allows for measuring gene expression levels by measuring Tm (melting temperature) using a thermal cycler. The principle behind probing in QPCR is that it uses fluorescently labelled primers and probes to detect the presence of a specific sequence and then amplifies the signal using a thermostable DNA polymerase.

Here are some key principles of probe-based qPCR;

Fluorescent Probe

The principle of the probe in QPCR is that the fluorescent probes have a sequence complementary to the target sequence, and the probe can be bound to the target DNA by complementary base pairing. The fluorescence quenching effect is caused by the non-specific binding of the probe to non-target DNA strands, which increases background fluorescence and decreases target gene amplification efficiency. Therefore, it is necessary to select appropriate fluorescent probes with high specificity for target genes to not interfere with non-specific binding.

Probe Hybridization

Hybridization is the reaction of two complementary single-stranded DNA or RNA molecules to form a double-stranded molecule. In QPCR, hybridization is used to detect the presence of target DNA/RNA. The probe is labelled with a fluorescent dye that emits light when excited by a laser. The fluorescence emitted from the probe will be captured by a CCD camera and analyzed by software. A threshold can be set to determine if there is enough signal fluorescence.

Quantification

Quantification is the most important principle of probes in QPCR. The probe will be designed to have a fluorescent reporter and a quencher. Usually, the reporter is attached to the 5′ end of the probe, and the quencher is attached to its 3′ end. When these two groups are close together, they can form a base pair called an oligonucleotide duplex (OD). In this situation, it would be impossible for the reporter to fluoresce because fluorescence requires a certain amount of energy which would be absorbed by the quencher group instead. However, when no DNA is present in the reaction mixture, no OD will be formed so that energy can pass through and cause fluorescence emission.

Fluorescence detection

QPCR probes are fluorescently labelled oligonucleotides that bind directly to the target sequence. QPCR probes are designed to be complementary to a segment of a target gene. The polymerase chain reaction (PCR) is then used to amplify the fragment of interest, which allows for the detection of the amplified fragment by the probe. The probe hybridizes with its target sequence and emits fluorescence when excited at the appropriate wavelength, allowing real-time detection.

Probe cleavage

Probes are designed to be cleaved by the target DNA or RNA. The cleavage reaction is catalyzed by a thermostable endonuclease (Taq polymerase). In some cases, two probes are used, one each for the target and reference gene. The second probe is a control to ensure the reaction works properly. The principle of probe cleavage is based on a sequence-specific interaction between the probe and its target DNA or RNA. This interaction is usually between a part of the probe sequence and a complementary base pair in the target molecule.

Conclusion

The probe is the key component of QPCR. The probe should be bound to the target DNA (or RNA) with a specific base sequence. Such a probe will then trigger a fluorescence reporter attached to the QPCR machine and can be detected during PCR amplification. This process can quantitatively measure the target DNA (or RNA) amount under certain conditions.