The principle and process of polymerase chain reaction

Polymerase chain reaction (PCR) is a technology widely used in molecular biology. Its core principle is to rapidly amplify DNA fragments in vitro through specific enzymes. Since its advent in the 1980s, PCR technology has become an important tool in the fields of gene cloning, disease diagnosis, and forensic identification.

Polymerase chain reaction

1. Principle The principle of PCR is based on the semi-conservative replication of DNA, that is, during the DNA replication process, each newly synthesized strand retains a part of the original strand. Through PCR technology, target genes can be amplified in large quantities in a short time without having to rely on organisms such as E. coli or yeast.

2. Process The PCR process mainly consists of three basic reaction steps: denaturation, annealing and extension. These steps are realized through precise temperature control in the thermal cycler. 1. Denaturation: In the first stage of PCR, after the template DNA is heated to about 93°C for a certain period of time, the hydrogen bonds of the double-stranded DNA are broken and the double-stranded DNA is dissociated into single strands. This process is called denaturation of DNA. The denatured single-stranded DNA becomes the template for primer binding to prepare for the next round of reaction. 2. Annealing: When the temperature drops to about 55°C, the primer pairs with the complementary sequence of the template DNA single strand. Primers are short DNA fragments (oligonucleotides) designed to bind complementary to specific sites on target DNA. During the annealing process, the primer binds specifically to the template DNA, which ensures the accuracy and specificity of PCR amplification. 3. Extension: Under the action of DNA polymerase, using dNTP (deoxynucleoside triphosphate) as the reaction raw material and the target sequence as the template, a new strand complementary to the template DNA strand is synthesized according to the principles of base pairing and semi-conservative replication. Semi-conserved replication strand. During this process, the DNA polymerase extends along the template strand, and the 3' end of the primer gradually synthesizes a new DNA strand. The extension temperature is usually set around 72°C, which is the optimal activity temperature of most DNA polymerases. By repeating the three processes of cyclic denaturation, annealing and extension, more "semi-retained replication strands" can be obtained, and this new strand can become a template for the next cycle. It takes 24 minutes to complete each cycle, and the target gene to be amplified can be amplified millions of times in 23 hours.

3. Key factors 1. Efficient DNA polymerase: such as Taq DNA polymerase, which can maintain activity at high temperatures and synthesize new DNA strands along the template strand. 2. Specific primer design: The length, GC content and sequence specificity of primers have an important impact on the specificity and efficiency of the reaction. 3. Precise temperature control: The temperature and time of the three steps of denaturation, annealing and extension need to be precisely controlled to ensure the success of the PCR reaction. 4. Appropriate reaction conditions: including buffer composition, Mg²⁺ concentration, etc., all need to be optimized to obtain the best amplification effect.

4. Application PCR technology is widely used in gene cloning, gene expression analysis, mutation detection, genotyping, pathogen detection and other fields. Its high sensitivity and specificity make it an important tool in molecular biology research and clinical diagnosis.

Polymerase chain reaction is a simple, specific, sensitive and rapid method for amplifying specific DNA fragments. By precisely controlling reaction conditions and optimizing experimental design, efficient and specific DNA amplification can be achieved, providing a powerful tool for molecular biology research and applications.

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