Principles and methods of in vitro DNA amplification

DNA is the carrier of genetic information, and its structure and function have always been the focus of life science research. In vitro DNA amplification technology provides a powerful tool for us to unveil the mystery of DNA. This technology allows scientists to quickly replicate DNA in the laboratory for in-depth analysis and research. Through the mastery of DNA amplification, we can better understand the role of genes and the transmission of genetic information.

Principles of in vitro DNA amplification

1. Basic Principles The basic principle of in vitro DNA amplification is based on the mechanism of semi-retaining replication of DNA in cells, as well as the properties that double-stranded and single-stranded DNA molecules can transform into each other at different temperatures. By artificially controlling the temperature of the in vitro synthesis system, the double-stranded DNA is turned into single-stranded, and the single-stranded DNA is then bound to artificially synthesized primers. Then, under the action of DNA polymerase, dNTP (deoxyribonucleoside triphosphate) is used as raw material to extend the primer along the single-stranded template into double-stranded DNA.

2. Main methods At present, the most commonly used method for in vitro DNA amplification is polymerase chain reaction (PCR). PCR technology was first created by scholars such as Karray in 1985 and developed by Cetus, the United States. The basic steps of PCR technology include: 1. High temperature denaturation: unspin the double strand of DNA to be amplified at high temperature (such as 94-98°C) into a single strand for binding to primers. The time of this step depends on the polymerase used. 2. Low temperature annealing: When the reaction temperature drops to a lower temperature (such as 50-65°C), artificially synthesized primers form hydrogen bonds with complementary DNA single-stranded sequences, thereby binding to the DNA template. The annealing temperature depends on the Tm value (melting temperature) of the primer used. 3. Medium temperature extension: Under the action of DNA polymerase (such as Taq DNA polymerase), using dNTP as raw material, the primer synthesizes a new strand complementary to the template DNA strand along the 5’→3’ direction of the DNA template. The temperature during extension is usually around 72°C. The above steps form a cycle, and by repeating this cycle continuously (usually 30-35 times), the DNA fragment of interest can be amplified efficiently and rapidly.

3. Application and Features 1. Application: In vitro DNA amplification technology has been widely used in many fields. In medical diagnosis, it can be used to detect pathogens, genetic disease genes, etc.; in forensics, it can be used for DNA fingerprinting, paternity testing, etc.; in molecular biology research, it can be used for gene cloning, gene expression analysis, etc. 2. Features: In vitro DNA amplification technology has high specificity and sensitivity, and can amplify extremely small amounts of DNA samples. At the same time, this technology also has the advantages of simple operation, fast reaction and accurate results.

In vitro DNA amplification technology, as a key technology in biological research and application, has made great progress. Through precise DNA amplification, scientific researchers can achieve in-depth exploration and precise operation of genes, which has promoted the development of many fields such as genomics, medicine, and agriculture.

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