What is the working principle of a fluorescence quantitative PCR instrument?

The working principle of a fluorescence quantitative PCR instrument is based on polymerase chain reaction (PCR) technology and the detection and analysis of fluorescent signals. Here is the detailed principle:

Basic principle of PCR

PCR is a technique used for in vitro amplification of specific DNA fragments. Its process includes three basic steps: denaturation, annealing, and extension. Through multiple cycles, the target DNA fragment is exponentially amplified. In the denaturation stage, double-stranded DNA unwinds into single strands at high temperature. During annealing, primers specifically bind to single-stranded DNA templates. In the extension stage, under the action of DNA polymerase, using primers as starting points and dNTPs as raw materials, new DNA strands are synthesized.

Generation and detection of fluorescent signals

• Fluorescent dye method: Commonly used fluorescent dyes such as SYBR Green I can specifically embed in the minor groove of double-stranded DNA. When the DNA polymerization reaction proceeds, as the amount of double-stranded DNA products increases, the amount of bound fluorescent dye increases, and the intensity of the fluorescent signal also increases accordingly. The fluorescence quantitative PCR instrument detects the change of fluorescent signal in real time through a specific optical system, thus reflecting the situation of DNA amplification.
• Fluorescent probe method: For example, TaqMan probe, its 5′ end is labeled with a fluorescent reporter group and its 3′ end is labeled with a fluorescent quenching group. In the free state, the fluorescence emitted by the reporter group is absorbed by the quenching group, and the fluorescent signal is suppressed. When the PCR reaction proceeds to the extension stage, the 5’→3′ exonuclease activity of Taq enzyme cuts the probe, separating the reporter group from the quenching group, and releasing the fluorescent signal. As the amount of amplification products increases, the number of cut probes increases, and the fluorescent signal also continuously increases.

Principle of quantitative analysis

• Determination of Ct value: During the fluorescence quantitative PCR process, the instrument will monitor the change of fluorescent signal in real time and automatically set a threshold. When the fluorescent signal reaches the threshold, the corresponding number of PCR cycles is called the Ct value. There is a logarithmic linear relationship between the Ct value and the copy number of the initial template. That is, the more copies of the initial template, the smaller the Ct value; conversely, the larger the Ct value.
• Standard curve method: By performing fluorescence quantitative PCR reactions on standards with known initial copy numbers, a series of corresponding Ct values are obtained. Taking the logarithm of the initial copy number of the standard as the abscissa and the Ct value as the ordinate, a standard curve is drawn. When detecting an unknown sample, according to its Ct value, find the corresponding initial copy number on the standard curve, thus realizing the quantitative analysis of the target gene in the unknown sample.