Four steps to build a PCR system

PCR (polymerase chain reaction) is a widely used technology in molecular biology for amplifying specific DNA sequences. Whether it is gene cloning, mutation analysis, or gene expression detection, PCR plays an irreplaceable role. Building an efficient and reliable PCR system is the key to ensuring the success of the experiment, and this process usually includes four important steps. This article will introduce in detail the four steps of PCR system construction to help everyone better understand and apply this technology.

Construction of PCR System

1. Preparation of template DNA1. Purpose: Extract DNA from the sample as a template for PCR amplification. 2. Methods: Common extraction methods include CTAB method, saline solution method, and the use of commercial DNA extraction kits. The integrity and purity of DNA should be ensured during the extraction process. 3. Notes: According to experimental needs, select the appropriate DNA concentration as the template for PCR. Too high concentrations may inhibit the PCR reaction, while too low concentrations may lead to poor amplification.

2. Preparation of PCR reaction system1. Components: Template DNA: Provide the DNA sequence to be amplified. Primers: Short-chain DNA molecules complement each end of the DNA fragment to be amplified, which are used to guide DNA polymerase to synthesize new DNA strands. dNTPs: Deoxynucleotides, including A, T, C, and G, are the raw materials for DNA synthesis. Buffer: Provides the appropriate pH and ionic environment to maintain the normal progress of the PCR reaction. Polymerase: For example, Taq DNA polymerase is heat-resistant, can maintain activity at high temperatures, and catalyze the synthesis of DNA strands. 2. Preparation method: According to experimental requirements, mix the above ingredients in a certain proportion to form a PCR reaction system.

3. Settings of PCR cycle reaction1. Basic steps: Denaturation: Heat the PCR reaction mixture to about 95°C, untie the double-stranded DNA of the template DNA to obtain single-stranded DNA. Annealing (primer binding): Cool the reaction system to an appropriate temperature (usually 45~65°C) to bind the primer to the complementary region of single-stranded DNA. Extension: The reaction system is warmed to the optimal temperature of the polymerase (usually 65~75°C), and the polymerase synthesizes new DNA strands based on primers. 2. Number of cycles: The number of cycles of PCR depends on the initial concentration of the DNA fragment to be amplified and the amplification efficiency of PCR. Usually, 25 to 35 PCR cycles are performed.

IV. Analysis of PCR products1. Method: Gel electrophoresis: Commonly used gels include agarose gel and polyacrylamide gel. PCR products can be separated and detected by gel electrophoresis, and the length and amplification efficiency of the PCR products can be judged based on the product's migration distance and band diagram. Other methods: such as fluorescence quantitative PCR, sequencing, etc., can also be used to analyze the specificity and concentration of PCR products. 2. Results interpretation: Amplification efficiency: The quality of the amplification efficiency can be judged based on the band diagram of the PCR product. Amplification specificity: The amplified PCR product should have only a single target sequence, nothing more than specific amplification. Growth degree: Gel electrophoresis can be used to determine whether the length of the PCR product meets expectations. Purity to concentration: The purity of the PCR product can be evaluated by determining the ratio of A260/A280, and its concentration can be estimated by comparing the density of the PCR product to the DNA size standard.

After deeply analyzing and elaborating in detail the four core steps of PCR system construction, it is not difficult to find that every step of this process embodies the rigor of scientific research and the exquisiteness of technology. From the precise extraction of template DNA, to the careful formulation of reaction systems, to the precise regulation of cycle parameters, to the strict identification of the final product, every step of the construction of the PCR system is like a precision gear, interlocking each other and jointly driving the operation of this powerful biotechnology.

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