Real-time PCR is widely welcomed by everyone because of its fully enclosed amplification, simple, rapid, reproducible, no post-amplification processing, and easy automation. In the field of molecular diagnosis, the real-time fluorescent PCR method involves many aspects such as infectious diseases, tumors, and genetic diseases.
When it comes to real-time PCR technology, is it the TaqMan probe method and dye method that first come to your mind? In fact, real-time PCR technology is more abundant than you think. Now let the editor take you into the world of real-time PCR and appreciate the richness of this technology.
Basic principles of real-time PCR
The current real-time PCR technology is mainly based on the principle of fluorescence resonance energy transfer (FRET). A pair of suitable fluorescent substances can form an energy donor (donor) and an energy acceptor (acceptor) pair. The emission spectrum of the body overlaps with the absorption spectrum of the acceptor. When they are close to a certain distance (1-10 nm) in space, the fluorescence energy generated by exciting the donor is just absorbed by the nearby acceptor, making the donor emit The fluorescence intensity decreases, and the fluorescence intensity of the acceptor fluorescent molecules increases.
Photo 1: Schematic diagram of fluorescence resonance energy transfer
Real-Time PCR Technology Type
SYBR Green I is a relatively common fluorescent dye that can bind to the minor grooves of double-stranded DNA non-specifically. It can be chimeric into the double-stranded DNA, but does not bind to the single-stranded DNA. When SYBR Green I does not bind double-stranded DNA on the surface of the solution, only very little fluorescence is produced. When it binds to double-stranded DNA, it emits a strong fluorescent signal. Since SYBR Green I can bind to any double-stranded DNA, it cannot distinguish primer dimers or non-specific amplification products. Generally, melting curve analysis is required to eliminate false positives.
Photo 2: Principle of SYBR Green I
The TaqMan probe is a fluorescent probe that specifically binds to the template. Its 5’end is labeled with a fluorescent reporter (Reporter, R), and its 3’end is labeled with a fluorescent quencher (Quencher, Q). When the probe is intact, the fluorophore and the quenching group are very close, so that the fluorescence emitted by the fluorophore is quenched. As PCR progresses, Taq enzyme encounters the probe bound to the template during the chain extension process, and its 5′-3′ exonuclease activity will cleave the probe, and the released 5’end reporter group will be free in the reaction system. , It is not affected by the 3’end fluorescence quenching group, the fluorescence signal can be detected by the instrument, and the fluorescence is accumulated. It can also be said that every time a DNA strand is amplified, a fluorescent molecule is formed, and the accumulation of fluorescent signal is synchronized with the formation of PCR products. The technology is currently widely used, including human or animal pathogen detection, biological product identification and other fields.
Photo 3: Principle of TaqMan Probe
Dual Hybridization probe
The dual hybridization probe real-time PCR is to label the fluorescent donor group and the fluorescent acceptor group on two oligonucleotide hybridization probes respectively, and the excitation light spectra of the two groups overlap to a certain extent. . The requirement for the two probes is that their hybridization positions with the target nucleic acid should be adjacent to each other. When the two probes hybridize to the target gene at the same time, the two fluorescent groups are very close, and the distance between them is 1~10 nm (usually 1~5 bases). According to the principle of FRET, in the donor group Under the action of a certain wavelength of excitation light, energy transfer occurs, thereby exciting the acceptor group to emit another kind of fluorescence, and the fluorescence intensity is proportional to the amount of DNA synthesis in the PCR reaction. Since two different probes must hybridize to the correct target sequence before fluorescence can be detected, the specificity of this method is enhanced.
Photo 4: Dual hybridization probe
The molecular beacon (MB) probe is composed of a single-stranded DNA molecule covalently labeled with a fluorescent group and a quenching group at both ends. The loop part of the molecular beacon is complementary to the target nucleic acid, and the two ends of the probe are Complementary to become a stem. When the molecular beacon has a stem-loop structure, the quenching group and the fluorescent group are very close, and the fluorescent signal of the reporter group is absorbed by the quenching group, thereby inhibiting the reporter group from producing fluorescence. In the PCR denaturation stage, the stem of the probe opens to form a single strand. When a specific template is present in the solution, the probe can hybridize to the template during the renaturation stage, making the 5’end fluorophore and the 3’end The quenching group is separated and the fluorescent signal is released. This method can be used for gene quantitative analysis, disease gene detection and diagnosis.
Photo 5: Principle of molecular beacon
Scorpions Probes are composed of probes, primers and PCR terminator. The probe part is similar to the molecular beacon. It also has a fluorescent group at the 5’end and a quenching group at the 3’end, and presents a stem-loop structure . The difference from the molecular beacon is that the scorpion probe has A specific primer can be combined with the corresponding target nucleic acid and polymerized and extended under the action of Taq DNA polymerase to obtain an amplified product, and the probe part with it can hybridize and bind to the complementary sequence in the specific product of the extended end. Therefore, such as Amplification exists. In the process of constant denaturation and renaturation, the scorpion probe can continuously hybridize with the amplicon, the stem-loop structure is opened, and the fluorescent group is no longer quenched and emitted. The fluorescence intensity and the amplified product The content is proportional. The specific probe sequence in the scorpion pro be has a circular structure, and a non-amplifiable monomer, the PCR terminator, is connected to the 5’end of the PCR primer. The PCR terminator can prevent amplification of the stem-loop part of the scorpion probe.