Fluorescence quantitative PCR (also known as TaqMan PCR, hereinafter referred to as FQ-PCR) is a new nucleic acid quantitative technology developed by PE (Perkin Elmer) in the United States in 1995. This technology is based on conventional PCR by adding fluorescent labeled probes. Compared with flexible PCR, FQ-PCR has many advantages to realize its quantitative function. This article intends to briefly describe the characteristics, principles, methods, and applications of the technology.

1 Features

FQ-PCR not only has the high sensitivity of ordinary PCR, but also because of the application of fluorescent probes, it can directly detect the change of fluorescent signal during PCR amplification through the photoelectric conduction system to obtain quantitative results, which overcomes many shortcomings of conventional PCR, so it also has the high specificity of DNA hybridization and the high accuracy of spectroscopy technology.

For example, general PCR products need to be observed by agarose gel electrophoresis and ethidium bromide staining with ultraviolet light or by polyacrylamide gel electrophoresis and silver staining. This not only requires multiple instruments, but also takes time and effort. The stains used Ethidium bromide is harmful to the human body, and these complicated experimental procedures provide opportunities for pollution and false positives. However, FQ-PCR only needs to open the lid once during sample loading, and the subsequent process is completely closed-tube operation, which does not require PCR post-processing, avoiding many drawbacks in conventional PCR operations. The experiment generally uses the ABI7100 PCR thermal cycler developed by PE company.

The instrument has the following characteristics: ① Wide application: It can be used for DNA and RNA PCR product quantification, gene expression research, pathogen detection, and optimization of PCR conditions. ② Unique quantitative principle: Using fluorescently labeled probes, the amount of fluorescence will accumulate with the PCR cycle after laser excitation, so as to achieve the purpose of quantification. ③ High working efficiency: Built-in 9600 PCR thermal cycler, computer controlled 1 to 2 hours to complete the amplification and quantification of 96 samples automatically and synchronously. ④ No need for gel electrophoresis: No need to dilute and electrophoresis the sample, just use a special probe to detect directly in the reaction tube. ⑤No pollution in the pipeline: The unique fully enclosed reaction tube and photoelectric conduction system are adopted, so there is no need to worry about pollution. ⑥The results are reproducible: the quantitative dynamic range is up to five orders of magnitude. Therefore, since this technology was successfully developed, it has been valued by many scientific researchers and has been applied in many fields.

2 Principles and methods

The working principle of FQ-PCR is to use the 5′→3′ exonuclease activity of Taq enzyme to add a fluorescently labeled probe to the PCR reaction system. The probe can specifically hybridize with the DNA template contained in the primer sequence. The 5′end of the probe is labeled with the fluorescence emission gene FAM (6-carboxyfluorescein, fluorescence emission peak at 518nm), and the 3′end is labeled with The fluorescence quenching group TAMRA (6-carboxytetramethylrhodamine, fluorescence emission peak at 582nm), the 3′beginning of the probe is phosphorylated to prevent the probe from being extended during PCR amplification. When the probe remains intact, the quencher group suppresses the fluorescence emission of the emitting group. Once the emitting group is separated from the quenching group, the inhibition is lifted, and the optical density at 518nm increases and is detected by the fluorescence detection system.In the renaturation phase, the probe hybridizes with the template DNA, and the Taq enzyme in the extension phase moves along the DNA template with the extension of the primer. When the probe is cut off, the quenching effect is released and the fluorescent signal is released. Every time the template is copied, a probe is cut off, accompanied by the release of a fluorescent signal. Since there is a one-to-one relationship between the number of released fluorophores and the number of PCR products, this technique can be used to accurately quantify the template. The experimental instrument generally uses the ABI7100 PCR thermal cycler developed by PE company, and other thermal cyclers can also be used. If the ABI7700 reaction type reaction system is used for the experiment, after the reaction is completed, the quantitative results can be directly given through computer analysis. If you use other thermal cyclers, you need to use a fluorescence detector to measure the fluorescence signal in the reaction tube at the same time to calculate RQ+, RQ-, △RQ. RQ+ represents the ratio of the luminescence intensity of the fluorescent emission group of the sample tube to the luminescence intensity of the quenching group, RQ- represents the ratio of the two in the blank tube, △RQ (△RQ=RQ+-RQ-) represents the amount of fluorescence signal change during PCR After data processing, quantitative results can be obtained. Due to the introduction of fluorescent probes, the specificity of the experiment is significantly improved. The probe design should generally meet the following conditions: ①The length of the probe should be about 20-40 bases to ensure the specificity of binding. ②The content of GC bases is between 40% and 60% to avoid duplication of single nucleotide sequences. ③ Avoid hybridization or overlap with primers. ④ The stability of the binding between the probe and the template is greater than the stability of the binding between the primer and the template, so the Tm value of the probe should be at least 5°C higher than the Tm value of the primer. In addition, the concentration of the probe, the homology between the probe and the template sequence, and the distance between the probe and the primer all have an impact on the experimental results.

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Post time: Oct-15-2021