PCR & Molecular Biology

Many different types of fluorescent probes are used in PCR reactions, most of which rely on some forms of covalently linked fluorescent quencher molecule. Fluorescent probes are specific and must be designed exclusively for each reaction. All current probe-based PCR is based on the phenomenon of fluorescence resonance energy transfer (FRET), that is, the changes of each group in fluorescence intensity or fluorescence species in the system are directly related to the type and quantity of PCR products. By detecting these changes, the PCR products can be qualitatively or quantitatively determined. Common probes include hydrolysis probes, molecular beacons, double hybridization probes, small groove binder probes (MGB), UniPrimer, scorpion probes, etc.

(1) Hydrolysis probes

Hydrolysis probes, also called TaqMan or 5' nucleases, are one of the most commonly used types of probes. The hydrolysis probe includes a sequence-specific fluorescently labeled oligonucleotide probe with a fluorophore at the 5' end and a quencher at the 3' end. Under normal circumstances, the spatial distance between the two groups is quite close, and the fluorophore cannot emit fluorescence due to quenching. During PCR amplification, some polymerases cleave the fluorescent molecule attached to the 5' end of the probe, causing the fluorescent group to move away from the quenching group, thereby generating a fluorescent signal. TaqMan probe technology has the advantages of low fluorescence background, high sensitivity and high hybridization stability, but it is usually expensive and difficult to design. It is suitable for detecting reactions with a PCR product length of less than 150bp.

(2) Molecular beacon

Molecular beacon is a new type of fluorescently labeled nucleic acid probe with a hairpin structure. Its structure generally includes three parts: a loop region, a beacon stem region, a 5'-end fluorescent group and a 3'-end quencher group. The molecular beacon in the free state has a hairpin structure. At this time, the fluorescent group is very close to the quenching group, and the fluorescence is quenched; when it is combined with the target sequence, the fluorescent group and the quencher are separated, and the clusters thus fluoresce. Molecular beacons have a wide range of applications in analysis of nucleic acid sequences, dynamic detection of nucleic acids in living cells, and interactions between proteins (enzymes) and nucleic acids. In recent years, by modifying the structure of classic molecular beacons, many new types of molecular beacons have emerged, such as RNA-DNA chimeric molecular beacons using ssDNA strands as loops and RNA-DNA double strands as stems, etc.

(3) Double hybridization probe

Double hybridization probes, also known as LightCycler or FRET probes, use a pair of probes that bind adjacent template sequences. Probe pairs are labeled with a donor fluorophore and an acceptor fluorophore, respectively, to exhibit FRET. When the probes bind to their target sequences, the two probes bind to the target in a head-to-tail orientation during annealing, FRET occurs, and the acceptor fluorophore fluoresces. Double hybridization probes are used in the medical and food industry, microbial identification and environmental science, for the detection of various nucleic acids present in any biological sample, etc.