Taqman technology, as the gold standard for real-time fluorescence PCR detection, not only possesses outstanding specificity, sensitivity, and user-friendliness but also constitutes one of the fundamental principles of real-time PCR detection. This article will delve into the working principles of Taqman technology, aiming to provide readers with a comprehensive understanding of its operational mechanisms.

Similar to traditional PCR reactions, Taqman-based reactions require a double-stranded template and two specific primers. However, unlike conventional PCR, Taqman detection also requires a third specific sequence, namely the probe. The probe differs significantly from the primers in structure and function. Firstly, the probe lacks a free hydroxyl group, thus cannot be amplified by DNA polymerase. Secondly, the probe is covalently linked to two other molecules, with a fluorescent moiety called the reporter group at the 5' end and a quenching group at the 3' end. This design enables the probe to play a crucial role in PCR reactions.

During the PCR reaction, the probe binds to the target DNA. When the DNA polymerase encounters the probe during the extension phase and possesses exonuclease activity, it cleaves the probe into fragments. This process physically separates the reporter group and the quenching group, thereby triggering the emission of fluorescence from the reporter group. This emission signal is directly proportional to the generation of PCR products, enabling real-time monitoring of PCR reactions.

The design of Taqman technology ingeniously utilizes the principle of fluorescence resonance energy transfer (FRET). In the structure of the probe, the reporter group and the quenching group are very close in distance. When the quenching group receives energy, it transfers it to the reporter group, leading to quenching of the fluorescence signal. However, when the probe binds to the target DNA and is cleaved by DNA polymerase, the reporter group and the quenching group are permanently separated, thereby triggering an increase in the fluorescence signal. This process provides reliable signals for real-time PCR, enabling precise detection and quantification of PCR products.

Real-time PCR instruments can monitor and record the increase in fluorescence signal after each PCR cycle and generate amplification plots. These data can be further analyzed for diagnosis, research, and other applications.

In addition to the basic principles, we also make innovations at SBS Genetech. We have introduced double-quenched probes, solving the issue of fluorescence "leakage" in traditional probes as the length increases, thus improving detection sensitivity. We also utilize high-throughput DNA synthesis technology to achieve individual synthesis and purification of each sequence, controlling the synthesis yield at the pmol level, known as pmol-scale DNA Capture Probe Synthesis Service. Compared to traditional column-based synthesis methods, this approach saves raw materials, reduces costs, and is more suitable for downstream applications.

These innovations have enabled Taqman technology to play a crucial role in the field of molecular diagnostics, providing powerful tools for research and clinical diagnosis in medicine, biology, and other fields. Through a deep understanding of its working principles and the introduction of innovative technologies, we can better apply them in practice and drive the development and innovation of molecular diagnostic technologies. At SBS Genetech, we are committed to providing more advanced technologies and solutions to help our customers maintain a competitive edge in the market.