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PCR Thermocyclers.Real Time Fluorescence PCR
PCR thermal cyclers, despite their relatively simple design, play an important role in modern medicine and research. As we continue to face new medical challenges, the value of accurate, rapid DNA replication is only going to increase, cementing the thermal cycler's place in the history of medical equipment.
The cost of purchasing and maintaining a PCR thermal cycler can be quite high, but their invaluable contribution to medical diagnostics and research is worth the investment. Efforts are underway to develop more affordable and portable versions to improve accessibility, especially in resource-poor settings.
The development of PCR thermocyclers has left an indelible mark on the medical field, greatly improving diagnostic accuracy and research capabilities. Their continued advancement bodes well for the future as fast, accurate and convenient DNA analysis continues to support medical diagnostic and therapeutic strategies.
Are you looking for a high quality PCR thermal cycler product for your research or clinical practice? We offer a range of high quality thermal cyclers, from compact to high throughput systems, designed to meet your needs. Our PCR thermal cyclers amplify nucleic acid sequences quickly and accurately, enabling you to achieve reliable and reproducible results in your PCR process. Take the next step in advancing your research or clinical practice by browsing our portfolio today.
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The basic principle of real-time quantitative fluorescence PCR is to take advantage of the fact that DNA polymerase synthesizes new DNA strands during the PCR process, and in combination with a fluorescently labeled probe or dye, measure the extent of the PCR reaction by monitoring the increase in fluorescence signal in real time [2]. Since there is a linear relationship between the Ct value of a template and the starting copy number of that template during the exponential period of PCR amplification, it becomes the basis for quantification. Real-time fluorescence quantitative PCR has several advantages, including high sensitivity, high specificity, wide linear range, rapidity, high automation, and low requirements for sample quantity and quality. It is widely used in gene expression analysis, pathogen detection, genotype analysis, and environmental microbiology research.
At the beginning of the PCR reaction, two specific primers (a pair of primers) bind to the ends of the DNA template sequence to be tested. These primers are designed by the experimenter to be complementary to specific regions of the sequence to be tested. In real-time fluorescent quantitative PCR, a fluorescently labeled probe is also added. This probe is also complementary to specific regions of the DNA sequence to be tested and binds to the target DNA during PCR. The fluorescent molecules on the probe are usually masked by a bursting apparatus, so the fluorescence is burst. However, when the probe binds to the target DNA, it is cleaved by DNA polymerase, releasing fluorescent molecules that result in an enhanced fluorescent signal. The intensity of this fluorescent signal is proportional to the amount of target DNA in the PCR reaction.
After mixing the Taqman probe labeled with fluorescein with the template DNA, completing the thermal cycle of high temperature denaturation, low temperature denaturation, and moderate temperature extension, and obeying the law of polymerase chain reaction, the Taqman probe that is complementary paired with the template DNA is cut [3], and the fluorescein is freed from the reaction system, emitting fluorescence under specific light excitation, and with the increase of the number of cycles, the amplified target gene fragments show an As the number of cycles increases, the amplified fragments of the target gene grow exponentially, and the Ct value is obtained by real-time detection of the fluorescence signal intensity corresponding to the amplification [4], and the copy number of the target gene of the specimen to be tested is obtained by utilizing a number of standards with known template concentration as a control.
Detection method
1. SYBRGreen Ⅰ method:
In the PCR reaction system, an excess of SYBR fluorescent dye is added. The SYBR fluorescent dye specifically doped into the DNA double strand emits fluorescent signals, while the SYBR dye molecules not doped into the strand do not emit any fluorescent signals, thus ensuring that the increase in fluorescent signals is completely synchronized with the increase in PCR products.
SYBR quantitative PCR amplification fluorescence curve graphs
PCR product melting curve (a single peak indicates the singularity of the PCR amplification product)
2. TaqMan probe method:
When the probe is intact, the fluorescent signal emitted by the reporter group is absorbed by the quenching group; during PCR amplification, the 5'-3' exonuclease activity of Taq enzyme degrades the probe by enzymatic cleavage, which separates the reporter fluorescent group and the quenching fluorescent group, so that the fluorescence signal can be received by the fluorescence monitoring system, i.e., for every DNA strand amplified, a fluorescent molecule is formed, realizing the accumulation of fluorescent signals is completely synchronized with the formation of PCR products.
Instrumentation
Thermal cycling device
One of the core components of a real-time PCR instrument is a thermal cycler, which controls the temperature of the PCR reaction system. The thermal cycling device usually consists of a Peltier temperature control module, which can quickly and accurately regulate the temperature of the reaction system, usually including heating and cooling functions, and can quickly switch the temperature during different PCR reaction steps (e.g. denaturation, annealing, extension) to meet the requirements of the PCR reaction.
Optical Detection System
Real-time fluorescence PCR instruments are equipped with a highly sensitive optical detection system for real-time monitoring of fluorescence signal changes during the PCR reaction process, usually including lasers, filters, photodiodes and other components, which can accurately and stably detect fluorescence signals.
Fluorescence Detection Channel
Real-time PCR instruments are usually equipped with multiple fluorescence detection channels for simultaneous detection of multiple target sequences or multiple fluorescent markers. Each fluorescence detection channel has a specific wavelength range for detecting the fluorescence signals generated by different fluorescent markers.
Automation
Some real-time PCR instruments are equipped with automation features, which can automate the processes of sample loading, PCR reaction, data analysis, etc. These automation features can improve the efficiency of the experiments. These automation functions can improve experimental efficiency and reduce operational errors, especially for large-scale sample processing and high-throughput experiments.
