PCR principle and application

Polymerase Chain Reaction (PCR) is an in vitro gene amplification technology. In 1985, the human genetic research laboratory of PE Company of the United States invented this technology. Saiki et al first applied to the prenatal diagnosis of sickle cell anemia, but due to the operation. The cumbersome method has not been fully promoted and applied. Until 1988, the discovery and application of heat-resistant DNA polymerase (Taq enzyme) made PCR technology extremely simple, and it was quickly applied in the fields of molecular biology, bioengineering, medicine, forensic science and agronomy. PCR technology has been used as a molecule. A milestone on the road to biological development, forever in history.
(I) Basic Principles of PCR Technology Similar to the natural replication process of DNA, its specificity depends on oligonucleotide primers complementary to both ends of the target sequence. PCR consists of three basic reaction steps: denaturation-annealing-extension:
1 Denaturation of template DNA: After the template DNA is heated to about 93 ° C for a certain period of time, the double-stranded DNA of the template DNA double-stranded or amplified by PCR is dissociated to make it a single strand so that it binds to the primer. Prepare for a round reaction;
2 annealing of the template DNA and the primer (refolding): after the template DNA is denatured into a single strand by heating, the temperature is lowered to about 55 ° C, and the primer is paired with the complementary sequence of the single strand of the template DNA;
3 primer extension: DNA template--primer conjugate under the action of Taq DNA polymerase, dNTP as the reaction material, target sequence as template, according to the principle of base pairing and semi-reserved replication, synthesize a new complementary to the template DNA strand The semi-reserved replication chain repeats the cyclic denaturation-annealing-extension process to obtain more "semi-reserved replication chains", and this new chain can be used as a template for the next cycle. It takes 2 to 4 minutes to complete each cycle, and the gene to be amplified can be amplified several million times (Plateau) in 2 to 3 hours. The number of cycles required to reach the plateau depends on the copy of the template in the sample. At the same time, it is recognized that proteins are synthesized by receiving genetic information of RNA.

In the early 1950s, Zamecnik et al. found in the morphological and isolated subcellular component experiments that microsomes are the sites of intracellular protein synthesis; in 1957 Hoagland, Zamecnik and Stephenson isolated tRNAs and synthesized them in synthetic proteins. The function of transporting amino acids raises hypotheses; in 1961 Brenner and Gross observed the binding of mRNA to ribosomes during protein synthesis; in 1965, Holley first measured the primary structure of yeast alanine tRNA; especially in the 1960s. The joint efforts of several groups of scientists, such as Nirenberg, Ochoa, and Khorana, have deciphered the genetic code for encoding synthetic proteins on RNA. Subsequent studies have shown that this set of genetic codes is universal in the biological world, thus understanding the basic process of protein translation and synthesis.
From the development of molecular biology, it can be seen that in the past half century, it has been the most rapid development of the life sciences, driving the development of the entire life sciences. Molecular biology is still developing rapidly, new achievements and new technologies are emerging, but the history of molecular biology is still short, and the accumulated data is not enough. For example, the vast variety of life on earth carries a huge amount of life information. So far, human beings know only a very small part. They have not yet recognized many basic laws of nucleic acid and protein composition. For example, even if we have obtained it in 2005, The complete sequence of 3×109 bp of human genomic DNA determines the primary structure of 50,000-100,000 human genes, but it is necessary to thoroughly understand the function, regulation, inter-gene relationship and coordination of these gene products, and understand more than 80%. The role of sequences that are not encoded by proteins, etc., has to go through a long research path. It can be said that the development prospects of molecular biology are glorious, and the road will be difficult and tortuous.
4. The correctness of binding of the oligonucleotide with specificity as a primer to the template is the key to determining whether the reaction product is specific.
5. The crude product or total RNA of the target DNA with low trace amount (pg, ng) is required for the quality of the original material, and can be used as a reaction starting material to obtain the target product.
(2) Application of PCR technology
1. Life Science a. Human Genome Project With the improvement of PCR, the scientists have completed, classified, and arranged the human genome in 2003, which is more accurate, clear and complete. Genomic map. This is the first disclosure of the basic features of the human genome, indicating that scientists began to “read” the content of the human life “Tianshu”.
b. Post-genome planning After completion of the human genomic DNA sequence map, identification of genomic polymorphisms and their haplotypes and their search for important applications in biological and medical applications have become a hot topic. The “post-genome era” with research on gene function has come, and large-scale research projects on structural genomes, proteomics and pharmacogenomics have become new hotspots.
c. Classification, evolution and genetic relationship of species can be used for conservation analysis of species evolution, species polymorphism analysis, and species identification.
2, medicine a, disease diagnosis and treatment of genetic diseases: such as thalassemia, sickle cell anemia, clotting factor deficiency and other genetically predisposed diseases, especially senile diseases such as diabetes, hyperlipidemia, and even tumors Some can be predicted; oncogene detection and diagnosis: detection of markers of malignant tumors to diagnose diseases such as cancer; gene therapy to treat neoplastic diseases.
b. Detection and detection of pathogenic pathogens include bacteria, viruses (SARS and avian influenza virus H5N1, etc.), protozoa and parasites, mold, rickettsia, chlamydia and mycoplasma. The sensitivity and specificity of the assay are much higher than current immunological methods, and the time required has reached the clinical requirements for viruses that are difficult to culture (hepatitis B), bacteria (such as tuberculosis, anaerobic bacteria) and protozoa (such as Treponema pallidum). ) and so on.
c, DNA fingerprinting, individual identification (DNA identification), paternity identification and forensic evidence can be done with a hair, a cell, a sperm, this field has also developed into bone marrow or organ transplantation.
d. Bioengineering Pharmaceuticals Many drugs can be produced in large quantities by engineering bacteria and cells, such as interferon, interleukin, erythropoietin and other drugs.
e. Transgenic animal pharmaceuticals and disease models Transgenic animals are increasingly closely related to medical and biomedical research. In recent years, a variety of human disease transgenic animal models have been established. Such as the application of transgenic animals in genetic diseases, cardiovascular diseases, tumors, hypertension, viral diseases, xenotransplantation, transfusion medicine, pharmacology research; the use of transgenic animal-mammary gland bioreactor to produce drug protein, such as on animals The establishment of a "pharmaceutical factory" can continuously obtain genetic products with stable biological activity from animal milk. This is a brand-new drug production model with low investment costs, short drug development cycle and high economic efficiency. f. The authenticity of Chinese herbal medicines identifies the DNA molecular marker identification method using the characteristics of DNA molecules for species identification. Compared with the existing traditional Chinese medicine identification methods, it has the following main features: 1) high accuracy, good reproducibility, real and stable Reliable; 2) Unconstrained by the form of the sample, the original medicinal materials, decoction pieces, powders and even proprietary Chinese medicines (pills, powders, etc.) containing the prototype of the crude drug can be applied; 3) the required sample amount is small, for rare herbs and fossil specimens The identification of the application is more applicable; 4) The rapid and convenient PCR technology makes the DNA molecular marker identification method more suitable for popularization.
3. Agricultural Science a. Transgenic plants are mainly divided into higher yield, disease resistance, herbicide resistance, improved quality and developmental regulation according to their functions. Such as: soybeans, corn, potatoes, tomatoes, etc.
b. In the agricultural sector, genetically modified animals mainly improve the production traits of livestock and poultry, improve the disease resistance of livestock and poultry, and use non-conventional animal products to produce GM livestock.
4. Environmental Science a. Environmental Ecology Research PCR-FLP technology provides a new research method and a new way of thinking for environmental geochemistry. The method has the advantages of low sample volume, quick and simple, and strong characteristics, and can be widely applied to biogeochemical cycle of materials, biological action of environmental processes, biodiversity and judgment of organic matter sources. Looking forward to future research results, more and new microbial species will be discovered in natural environments such as marine and lake sediments; the mechanisms and processes of biological action and material circulation will be further clarified; and the changes of biological macromolecules in the natural environment will be further clarified. Mechanism and its environmental effects and find out the sediment organic matter.
b. Environmental monitoring The long-term detection of the external environment (Vibrio cholerae in water samples, Clostridium perfringens in the air, known pathogens in the ocean, and harmful organisms such as algae, etc.) are both traditional and isolated, which is cumbersome and time consuming. The detection rate is low, and the PCR method can quickly and accurately detect the pathogenic bacteria in the external environment.
5, archaeological and historical events interpretation Using human short tandem repeat STR-PCR technology to study the genetic polymorphism of human race, the effect is very stable. At present, this technology has been widely used in various fields such as biological archaeology, germline development, ethnology, anthropology and archaeology.

6, health and safety a, food microbial detection Traditional pathogen detection first after a long period of training, time-consuming and laborious, the application of PCR technology is very fast and accurate. Mainly used for: detection of food pathogenic bacteria, such as botulinum toxin; detection of lactic acid bacteria; determination of bacterial indicators in water.

b. Detection of genetically modified foods There are currently more than 100 species of genetically modified foods in the world, most of which have been used in food. The harm of genetically modified foods to human health and its impact on the ecology are also widely recognized in the world. Therefore, the detection of genetically modified foods has become a means of controlling their proliferation.
c. The sensitive, specific and rapid diagnostic test method for animal and plant quarantine is the guard at the port of import and export in China. Check whether the personnel, animals and plants (sows, seeds), etc. entering and leaving the country carry strong infectious diseases (AIDS, animal viruses, plants). Viruses, etc.) Pathogens, food, feed, etc., whether or not with Salmonella require genetic diagnosis to reject these bacteria outside the country, is a necessary guarantee to improve China's overall national strength.

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