In vitro DNA amplification technology

In vitro DNA amplification technology, as the name suggests, refers to the quantitative amplification of the target DNA molecule in vitro in a laboratory environment through specific methods and tools. This technology has been widely used in molecular biology research, genetic engineering, disease diagnosis, forensic medicine and other fields, and has become one of the indispensable technologies in modern biological research and practice.

In vitro DNA amplification

1. Basic Principles The basic principle of in vitro DNA amplification technology is that under the catalysis of DNA polymerase, the parent strand DNA is used as the template and specific primers as the starting point for extension, and through denaturation, annealing, extension and other steps, the child strand DNA complementary to the parent strand template DNA is replicated in vitro. During this process, dNTP (deoxynucleotide), Mg²⁺, elongation factor and amplification enhancement factor are added to the system. 1. Denaturation: Use high temperature to separate DNA double-strands. The hydrogen bond between the double-stranded DNA is interrupted at high temperatures (such as 93~98℃) to form single-stranded DNA. 2. Annealing: After the DNA double-stranded separation, the temperature is reduced so that the primer can bind to the single-stranded DNA. Primers are oligonucleotide sequences complementary to both ends of the target DNA fragments, which are able to specifically recognize and bind to the target DNA. 3. Extension: Under the action of DNA polymerase, using dNTP as raw material, a new DNA strand is synthesized from the 3' end of the primer. During this process, DNA polymerase moves along the template chain and continuously adds dNTP until a complete complementary chain is synthesized. After completing one cycle, the number of DNA fragments doubles. Through multiple cycles (usually 25 to 35 times), the number of DNA fragments will increase exponentially.

2. Main technology - PCR technology The most representative of in vitro DNA amplification technology is polymerase chain reaction (PCR) technology. PCR technology was invented by American scientist Mullis in 1985 and published a groundbreaking paper on this technology in the journal Science. This invention not only won Mullis the Nobel Prize in Chemistry in 1993, but also completely changed the research method of DNA engineering and promoted the development of the field of life sciences. PCR technology has the characteristics of high efficiency, specificity, and sensitivity, and can amplify a large number of target DNA fragments in a short time. Through PCR technology, researchers can easily amplify any specific DNA sequence, which has a wide range of applications in gene cloning, disease diagnosis, forensics and other fields.

3. Classification of PCR technology According to different applications and needs, PCR technology can be divided into many types: 1. Ordinary PCR: Amplification is used by ordinary DNA polymerase, and then the product is detected by agarose gel electrophoresis. Ordinary PCR can only perform qualitative analysis, that is, to determine whether the target DNA exists, but quantitative detection cannot be performed. 2. Fluorescence quantitative PCR (Real-Time PCR or qPCR): Add a fluorescent probe that can indicate the reaction process to monitor the accumulation of amplified products by accumulating fluorescence signals. Fluorescence quantitative PCR can achieve quantitative detection, i.e., determine the concentration or copy number of the target DNA. Fluorescent substances can be divided into TaqMan fluorescent probes, molecular beacons and fluorescent dyes. 3. Digital PCR (Digital PCR): Calculate the copy number of the target sequence through endpoint detection, and accurately and absolutely quantitative detection can be performed without using internal parameters and standard curves. Digital PCR has the characteristics of high sensitivity and high accuracy, and is not easily interfered by PCR reaction inhibitors, and can achieve true absolute quantification without the need for standards. According to the different forms of the reaction unit, digital PCR can be divided into three categories: microfluidic, chip and microdroplet systems.

4. Application and Prospects In vitro DNA amplification technology has broad application prospects in the field of life sciences: 1. Gene cloning: PCR technology can amplify specific gene fragments, and then insert them into the vector to construct recombinant DNA molecules, thereby achieving gene cloning. 2. Disease diagnosis: PCR technology can be used to amplify specific DNA fragments related to the disease, such as pathogen genes, mutant genes, etc., thereby providing a strong basis for the diagnosis and treatment of the disease. 3. Forensic Science: In the field of forensic science, PCR technology is used to extract and analyze trace DNA samples, such as blood stains, hair, tissue fragments, etc., providing key evidence for the investigation of cases. 4. Biodiversity research: PCR technology can amplify specific DNA fragments of different species, and then study their genetic diversity and evolutionary relationship.

In vitro DNA amplification technology, especially PCR technology, plays an irreplaceable role in modern life science research and medical diagnosis. With the continuous advancement of technology, in vitro DNA amplification technology will bring changes in more fields. Whether in the fields of gene cloning, disease diagnosis, or forensics, this technology has provided scientists with powerful tools and has promoted human health, life sciences and social development to new heights.

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