Real-Time PCR

Human genetic diseases are a group of diseases caused by abnormalities in genes that bring great suffering to patients and their families. To effectively prevent, diagnose, and treat these diseases, it is necessary to detect and analyze genes accurately and sensitively. Real-time PCR is a molecular biology experimental method based on PCR technology that can monitor the amplification of target DNA molecules in real time during the PCR process and perform qualitative or quantitative analysis. This technique has the characteristics of high sensitivity, high specificity, high efficiency, high throughput, and quantitative accuracy and does not require gel electrophoresis or other post-processing steps. Real-time PCR can be used to detect genetic variations such as gene mutations, polymorphisms, copy number variations, etc., as well as monitor gene expression levels, gene knockout or knock-in effects, etc., providing a powerful tool for the diagnosis and gene therapy of human genetic diseases.

Principles of Real-Time PCR

The working principle of real-time PCR is based on PCR technology, that is, using DNA templates, primers, enzymes, and dNTPs as reactants and performing repeated denaturation, annealing, and extension steps in a thermal cycler to exponentially amplify the target DNA molecule. Unlike conventional PCR, real-time PCR can monitor the amount of amplification product in real time during the PCR process and perform quantitative analysis. This is achieved by adding a fluorescent reporter molecule to each reaction well, which emits fluorescence as the amplification product DNA increases. The fluorescence detector can measure the fluorescence intensity in each reaction well at the end of each cycle and correlate it with the cycle number and the initial template DNA copy number, thereby calculating the relative or absolute amount of target DNA.

There are two common methods of fluorescence detection in real-time PCR: one is to use DNA-binding dyes, such as SYBR Green I, which can bind to any double-stranded DNA and emit fluorescence, so no specific probes are required, but it is also prone to interference from non-specific amplification or primer dimers; the other is to use fluorescently labeled sequence-specific probes, such as TaqMan probes, Molecular Beacon probes, etc., which only emit fluorescence after hybridizing with the target sequence, so they have high specificity and sensitivity, but they also require designing and synthesizing more primers and probes. Different types of fluorescence detection methods have their own advantages and disadvantages and application scopes and need to be selected and optimized according to the experimental purpose and conditions.

The Experimental Steps of Real-Time PCR

1. Sample preparation: First, RNA needs to be extracted from cells or tissues and purified and quantified. If the target is gene expression level, RNA also needs to be transcribed into cDNA, which is called reverse transcription.
2. Reaction system: According to the selected fluorescence detection method, the corresponding reactants need to be prepared, including DNA template, primers, enzymes, dNTPs, buffer, fluorescent dye or probe, etc., and mixed together in a certain proportion and loaded into special reaction tubes or plates.
3. Instrument setup: The reaction tubes or plates are placed in a thermal cycler, and the temperature, time, and cycle number parameters are set. At the same time, the wavelength, threshold, and baseline parameters of the fluorescence detector are also set so that the fluorescence intensity in each reaction well can be measured at the end of each cycle.
4. Amplification program: The thermal cycler starts to run and performs repeated denaturation, annealing, and extension steps according to the set program to exponentially amplify the target DNA molecule. At the same time, the fluorescence detector monitors the fluorescence signal changes in each reaction in real time and records them.
5. Data analysis: After the thermal cycler runs, the collected data are analyzed using special software, including drawing fluorescence curves, calculating threshold cycle numbers (Ct), performing the standard curve method or relative quantification method, etc., to obtain the relative or absolute amount of target DNA.

Characteristics of Real-Time PCR

Real-time PCR is a technique that can monitor and quantify the amplification of target DNA molecules in real time during the PCR process, and it has the characteristics of high sensitivity, high specificity, high efficiency, high throughput, and quantitative accuracy. Compared with conventional PCR, it can not only accurately measure the copy number of the initial template DNA but also does not require gel electrophoresis and other post-processing steps, saving time and increasing throughput. Real-time PCR achieves real-time detection of PCR products by adding a fluorescent reporter molecule to each reaction well, and the fluorescence detector can measure the fluorescence intensity in each reaction well at the end of each cycle and correlate it with the cycle number and the initial template DNA copy number, thereby calculating the relative or absolute amount of target DNA.

Applications of Real-Time PCR in Human Genetic Diseases

Real-time PCR has a wide range of applications in the diagnosis and gene therapy of human genetic diseases, and it can be used to detect genetic variations such as gene mutations, polymorphisms, copy number variations, etc., as well as monitor gene expression levels, gene knockout or knock-in effects, etc. For example, real-time PCR can be used to detect factor V Leiden and prothrombin gene mutations related to thrombosis, as well as hemochromatosis gene mutations related to iron overload, thus providing accurate diagnosis and personalized treatment options for patients. Real-time PCR can also be used to detect other genetic diseases, such as β-thalassemia, cystic fibrosis, and hereditary breast cancer. In addition, real-time PCR can also be used to monitor the effect of gene therapy, such as by measuring gene expression levels or copy numbers to evaluate the efficiency and stability of gene transfection or transduction.

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