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Recombinant DNA technology -- the development of the 20th century the 1950s, the double helix structure of DNA was explained, opened a new chapter in the life sciences, creating a new scientific and technological era. Subsequently, the molecular mechanism of genetic -- DNA replication, the genetic code, genetic information transfer in the center rules As the basic unit of heredity and cell engineering and genetic blueprint for the Regulation of gene expression have been recognized. So far, it has been fully aware of have all the biological fate of the things is that it contains DNA and genes Biological evolutionary process and the process of life, because DNA and gene locus different operation. Aware of the important role of DNA and value, Life scientists first thought and can in certain human closely related to the interests of natural genetic Breaking the ironclad rule Let ill gene repented to achieve medical purpose different sources of gene fragments were "grafted" to produce new varieties and new quality ... So, one full of temptations of science fantasy, miraculously become a reality. This is the 20th century occurred in the early 1970s things. Realization of the scientific miracle of technology means of recombinant DNA technology. In 1972, American scientists Paul? Burg first successful reorganization of the world's first batch of DNA molecules, marked recombinant DNA technology -- genetic engineering as a modern biological engineering foundation modern biotechnology and life science base and core. Recombinant DNA technology is the specific content using artificial means to different sources of a specific gene with the DN A fragment reorganization, to change the biological gene type and availability of specific gene products that the purpose of a high science and technology. Of the 20th century, 70 years late Due to the emergence of bacteria and the realization of the project recombinant DNA and reprocessing is the nature of the project, genetic engineering or genetically engineered recombinant DNA technology as a synonym for wide use. Now, genetic engineering also includes the transformation of the genome, DNA sequence analysis and molecular evolution analysis, molecular immunology, gene cloning, genetic diagnosis and gene therapy, and other content. It can be said that DNA recombinant technology created nearly 30 years and was a tremendous success has been to put people into an incredible dream as the scientific world in which human life is a mystery and opened preventing and curing diseases "demons" golden key. Currently, recombinant DNA technology has achieved results in many ways. End of the 20th century, the largest recombinant DNA technology in the field of medicine, including active peptides, Protein and vaccine production, the mechanism of the disease, diagnosis and treatment, the new gene separation and purification and environmental monitoring. Many active peptides and proteins have the treatment and prevention of diseases, they are from the corresponding gene produced. However, because the cells within the production is minimal and the use of conventional methods is very difficult to obtain enough for clinical use. Gene breakthrough in the construction of this limitation, to such large-scale production of peptides and proteins, so far has been successful in the treatment of diabetes produce insulin and schizophrenia. right leukemia and certain solid tumors curative antivirals -- interferon, dwarfism treatment of human growth hormone, treatment of acromegaly and acute pancreatitis in the release of growth hormone inhibitor 100 kinds of products. Genetic engineering may be the antigen DNA into living microorganisms, Such microbes in by the immune stress vivo growth may have attenuated live vaccine, with large doses of antigen stimulation. lasted a long time and so on. Currently being developed genetically engineered vaccine alone dozens, in terms of dealing with bacteria are against leprosy bacillus. pertussis bacteria, and Neisseria gonorrhoeae, meningococcal vaccines, etc.; in dealing with a virus against hepatitis A, hepatitis B, cytomegalovirus, herpes simplex, influenza, human immunodeficiency virus vaccine .... Hepatitis B virus carriers in China and as many as hepatitis B patients Besides, This situation also prompted China's own scientists successfully developed hepatitis B vaccine made tremendous social and economic benefits. Antibodies are the body's immune system resistant disease prevention as one of the principal weapons, 1970s in the 20th century created the monoclonal antibody technology in the aspects of preventing resistance played an important role, However, as Human monoclonal antibodies is difficult to obtain, making monoclonal antibody in clinical application is restricted. To solve this problem, In recent years, scientists have used recombinant DNA technology has been a human antibody, This antibody can guarantee it with the antigen-binding specificity and affinity, but also to ensure the normal functioning of the play. At present, there are such a variety of antibodies for clinical trials, If anti - HER-2 human monoclonal antibody treatment of breast cancer has entered Phase III testing, IGE anti-human monoclonal antibody treatment of asthma has entered Phase II trial. Antibiotics in the treatment of disease has played an important role, along with the increase in the number of antibiotics, with the traditional method of discovering new antibiotics probability of getting lower and lower. To obtain more new antibiotics, using recombinant DNA technology has become an important tool. Currently people have been dozens of genetic engineering "hybrid" of antibiotics for clinical application opening a new therapeutic approach. What is worth noting is that the above-mentioned genetic engineering peptides, proteins, vaccines, antibiotics to combat drug is not only effective in controlling the disease. and the need to avoid drug side effects are often superior to the traditional method of production of similar medicines, which also favored by people. Human diseases are directly or indirectly associated with the gene, the gene level of disease diagnosis and treatment, it will not only meet the diagnostic accuracy and originality, will enable diagnosis and treatment to achieve high specificity, high sensitivity, Simple and Rapid purposes. At the level of gene diagnosis and treatment of the professional as genetic diagnosis and gene therapy. Currently genetic diagnosis as a fourth-generation clinical diagnosis technology has been widely used for genetic diseases, cancer, cardiovascular and cerebrovascular diseases, Bacterial viruses and parasitic diseases in the diagnosis of occupational diseases; and the goal of gene therapy is through recombinant DNA technology to create specific functions with the recombinant gene. to compensate for the loss of the gene function, or an increase in some functions for the benefit of the abnormal cells were corrected or eliminated. In theory, gene therapy cure is permanent cure without any toxic side effects of therapy. However, despite the international community so far has been more than 100 gene therapy program is in the clinical trial stage. But gene therapy in theory and some technical problems still make such treatment from the large-scale practical application is still a very long distance . Determine whether genes or genetic etiology diagnosis, gene therapy, study the mechanism of disease, The key prerequisite is to understand the specific disease-related genes. Along with the "Human Genome Project" is approaching completion, the scientists of all human genes will be comprehensive understanding, This use of gene recombination technology made forced to create human health conditions. However, although the human genome technology to display its mysterious "magician - like" charisma, But there are also a large number of scientists to the development of such technology to be human ethical and ecological evolution of the impact of natural law expressed very The big worry. Theoretically speaking, development of this technology is to make an extreme of human beings to create any life form, or never have the biological capacity. People can imagine how this will be the outcome? [DNA paternity test -- Identification of parent-child relations with the most DNA typing identification. Person's blood, hair, saliva, oral cells and so be used for paternity testing, it is very convenient. A person is 23 pairs (46) chromosome, on the same chromosome on the same location of a gene known as alleles, a general from the father and one from the mother. If detecting a DNA loci allele, the same one with the mother, the other on the same with his father, Otherwise there is a doubt. The use of DNA paternity testing, for as long as a dozen to several dozen sites for DNA testing, if all the same. will be able to define parent-child relationship, if any of the above three different loci can be excluded from parent-child relationship, a 12-point, Gene mutation, it should consider the possibility to carry out some testing site identification. DNA paternity testing and deny the parent-child relationship accuracy rate nearly 100%. definitely parent-child relationship accuracy can be achieved 99.99%. DNA paternity testing test FAQ What is DNA paternity test test? DNA (deoxy nuclear Sugar nucleic acid) is a physical body cells atomic material. Each atom has 46 chromosomes, while men's sperm cells and the woman's egg, have 23 chromosomes, When the sperm and egg of times. This chromosome 46 atoms on a manufacturing life, therefore, each inherited from the father half of the molecular material, and the other half from the mother receives. DNA paternity testing test with the traditional blood tests are quite different. It can be in different sample tests, including blood, gills cavity cells, tissue samples and semen samples. Because blood type, such as Type A, Type B, Type O or Type RH, the population is relatively common, for the resolution of each individual, DNA paternity would not be as effective identification tests. Apart from genuine twins, each person's DNA is unique. Because it is so unique, like fingerprints, for paternity testing, DNA is the most effective method. Our results is often requested by the court also accurate from 10 to 100 times. [DNA ultracentrifugation -- Modern purified plasmid DNA from E. coli to separation of representatives, In view of the overall situation bacilli (E. coli) in molecular biology research of the important position, from the E. coli DNA Purification quality control in recent years - centrifuge technology an important issue. And the rapid plasmid DNA Purification also right - centrifuge equipment (speed centrifuges, turn and ancillary equipment) put a higher demand. E. coli is a typical prokaryotic cell biology, Due to the lack of prokaryotic cells their cells have the kind of flat membrane can be composed of a variety of functional components of the separation of specificity regional and local independent of the endometriosis, thus its nuclear cells contained cells (nuclear, endoplasmic reticulum, Golgi apparatus, Many studies, lysosomes, etc.). Electron microscope micrographs showed that E. coli can be two different regions within the cytoplasmic and nuclear January 1 quality, They surrounded the outside of the thinner layer of the cell membrane and a very thick cell wall, the external wall attached to the free end some flagella. Plasmid DNA in the nuclear area, there is a fine filament, Such filaments membranes in a variety of circumstances it is an extremely long circular DNA fragments by the folding up of poly secret body. E.coli response to the microstructure, During super centrifugal separation and purification of plasmid DNA pretreatment before the order is : E.coli ¡ú ¡ú lysozyme to the cell wall using surfactants, such as SDS, Trit¨®n X-100 and other EE acid membrane ¡ú DNA pot, Most of RNA and protein precipitation (90%). Sediments can join in TE buffer (10m-MTris - HCL lmMEDTA. pH8.0) zeolites to live protein to RNA; can also be used to ultracentrifugation protein C to RNA, to rank-DNA or DNA fragments. ¡¾Ultracentrifugation Plasmid DNA Isolation Method -- the traditional separation methods : a few years ago, because of equipment limitations, Plasmid DNA Isolation general balance with CsCl density centrifugation, since the formation of gradient. 10 ~ TOCE single-tube capacity for example, with turn-rejection separation 36.000rpm ¡Á 60 hours, Angular separation of 45-turn, OOOrpm ¡Á 36 hours. The former include the slowdown, amounted to 130 million drive to the Ministry of life, the latter should also consume 100 million to the Department of Driver life, This right was speeding centrifuge total life of ~ 100 to 20 billion, undoubtedly each experiment costs are too high, CsCl with dosage, your prices and other factors, such as separation and purification work very expensive experiment. ¡¾Plasmid DNA ultracentrifugation separation of the latest developments -- (1) speeding vertical tube to put centrifugal separation (carbon fiber or titanium alloy Victoria Manufacturing) : From 1975 vertical tube turn to the world, In recent years, the major producers of centrifuge development of the vertical pipe to apply the old, single-tube capacity of 0.2 ml of 4Oml. The maximum speed from 50 rpm to 120,000 rpm, up to 700 RCFmax, OOOXg. 1990s development of the new models and turn so that others can plasmid DNA vertical tube centrifugal separation experiment done with high proficiency. (3) near vertical pipe to put centrifugal separation : In order to eliminate the vertical pipe to put centrifugal for plasmid DNA in the form of wall RNA precipitation have become the DNA bands pollution, but also to improve the general turn-angle (angle &S226; 25 -- 35 &S226;) due to the settlement longer distance thus separated for a longer period of time shortcomings, In recent years the development of a wide range of near-vertical tube to put (Near VerticalTube compost, referred to turn Linux or Neo Angle Rotor, small angle at the turn, listed NT). their centrifuge tube axis and the vertical section centrifuges driven angle between the axis of the &S226; 7.5 -- 10 &S226; between speed from 65, to 120 rpm, OOOrpm, RCFmax up to 646, 000 ¡Á g single-tube capacity from 2 ml to 13.5ml. Linux (or NT) to put the development is mainly for plasmid DNA isolation and design, Of course, it also applies to mitochondrial DNA, chromosomal DNA, RNA and serum lipoproteins &S226; Isolation and purification. (3) discontinuous gradient separation ladder : school quality DNA Purification is the traditional method used since the formation of CsCl balance of density gradient centrifugation. at the beginning of the centrifuge tube CsCl density uniform, uniform distribution of these samples. [Human Genome Project -- The Human Genome Project (human genome project, HGP) is a scientist in the United States in 1985, first proposed in 1990 the official launch. The United States, the United Kingdom, the Republic of France, the Federal Republic of Germany. Japan and Chinese scientists involved in the common value of up to 30 billion dollars in the Human Genome Project. This program aims to over 30 billion base pairs form of the Human Genome Sequencing precise, found that all human genes and determining their position on the chromosome and decipher all human genetic information. And the Manhattan atomic bomb project and the Apollo program and called the three major scientific project. June 26, 2000, participated in the Human Genome Project in the United States, the United Kingdom, the Republic of France, Federal Republic of Germany, Japan and China, the six scientists jointly announced, draft human genome mapping has been completed. Final completion of the map requested by the sequencing of clones can be faithfully represented often genomic structure, Sequence error rate of less than one ten thousandth. 95% regular chromatin regions were sequenced, Gap is less than 150kb each. Completed plans will be completed by 2003, is expected to more than two years in advance. American and British scientists May 18, 2006 in the United Kingdom "Nature" magazine published on the Internet version of a human final chromosome -- the chromosome gene sequencing. In all the 22 pairs of human chromosomes often, chromosome 1 gene contains the largest number, reaching 3,141. is twice the average, a total of more than 223 million base pairs on the deciphering of the greatest difficulty. 1 by 150 British and American scientists team took 10 years before he completed the sequencing of chromosome work. Scientists declared on more than one occasion the Human Genome Project completed, but have not launched all of this, This time fixing the "book of life" is more accurate, coverage of the human genome by 99.99%. Interpretation of the human genetic code of the "book of life" and declared completed. During the 16 years of the Human Genome Project finished writing the final chapter. DNA is a history of the development of DNA in 1944 by the Americans Avery discovered. Professor Crick in 1953 to map out the DNA double helix structure. In 1985 the University of Leicester, Professor Alex Jefferies also invented the use of DNA to identify the human approach. DNA from the 1988 applications starting in the administration of justice. July 29, 1994, French law provides for the use of gene markers conditions. According to scientific analysis, each individual has 400 trillion cells (skin, muscle, nerve, etc.) Apart from human red blood cells outside cells have a 46 by the chromosomal composition of the nucleus, Chromosomal DNA by itself constitute chromosome silk, silk chromosome in all cells are the same. DNA has been called by the A (adenine), T (Morus). G (guanine) and C (cytosine) nucleic acid composition, It is they constitute our human genes. DNA can be determined based on the intergenerational relationship, always as a child from his father and mother who receive half of the genetic material. Scientists also study DNA on the goal to determine the cause of illness origin of the gene, so that in the future a better understanding, treatment and prevention of hazards to human health diseases. DNA credibility? Chromosome 2 of whether similar? According to scientific experiments, such a possibility only Qianmobanzhiyi. However, in the course of all errors will be possible, mainly in the extraction and testing of specimens, specimens may also be another person's DNA contamination. To ensure the reliability of the DNA must be extracted specimens and laboratory analysis strictly checked. Now, due to the use of genome sequence project and the development of new equipment, not only to avoid possible errors, but also greatly accelerate the speed of the DNA test. ¡¾ "Junk DNA" -- yeast and worms such as the simple biological evolution is how birds and mammals such a complex biological ? One genome against the extensive comparative studies show that The answer may be hidden in the biological refuse deoxyribonucleic acid (DNA). American scientists found that the more complex biological, carrying garbage DNA will be, It is precisely those which have not "useless" Higher DNA to help out the biological evolution of complex organisms. Since the first eukaryote -- ranging from yeast to human cells are biological -- of the genome is deciphered, Scientists have long wondered why the majority of biological DNA and the lack of useful genes. From the protection of the chromosome mutation of the support structure for the so-called garbage DNA may explain why there are many species. But last year from the human, mouse and rat body in exactly the same DNA on refuse the findings show that In this region may contain an important regulatory mechanism so as to control the basis of biochemical reactions and the development process, This will help biological evolution is even more complex organism. And simple eukaryotes, the more complex biological gene mutation not doubt the fact that greatly strengthened this discovery. To right this issue will have a deeper understanding, from the University of California Santa Cruz branch (UCSC) calculated biologist David Hauss ler leadership of a research group of five species of invertebrates -- human, mouse, rat, chicken and globefish -- refuse DNA sequence with four types of insects, Two worms, and seven species of yeast DNA sequence of garbage were compared. Researchers compared results from the received a shocking pattern : The more complex biological refuse DNA seems more important. This implied that the possibility is, if different types of biological have the same DNA, Well, this DNA must be used to resolve some key issues. Yeast and vertebrates share a certain amount of DNA, after all, they all need to manufacture proteins, However, only 15% of the total DNA and gene irrelevant. Study Team in July 14 "Genome Research" magazine on the internet version of the report said, They will yeast and more complex worms were compared, the latter is a multi-cell biology, found that 40% of the total DNA was not coding. Subsequently, the researchers will invertebrates and insects were compared, these organisms more complex than worms and found that More than 66% of the total DNA contains no coding DNA. Participate in the study calculated the UCSC biologist Adam Siepel, the worm's findings must be carefully treated, This is because scientists only to the two genomes were analyzed. Nevertheless, Siepel is of the view that this was found to effectively support such a theory, invertebrates and insects that the biological complexity of the increase was mainly due to the precise gene regulation patterns. [DNA probe -- DNA probe is the most commonly used DNA probe refer to a few hundred base pairs in length on these double-stranded DNA or single stranded DNA probes. This has been the number of DNA probes that there are many bacteria, viruses, protozoa, fungi, animals and human cell DNA probe. Such probes for a number of gene sequences in whole or in part, or a non-coding sequences. These DNA fragments to be specific, such as bacterial virulence factor gene probes and human Alu probe. These DNA probe is dependent on molecular cloning technology development and application. To bacteria for example, Currently molecular hybridization technology for bacterial strain identification and classification than the G + C percentage of the value of accurate, Bacterial taxonomy is a development direction. Moreover, molecular hybridization of high sensitivity molecular hybridization in the diagnosis of clinical microbiology has broad prospects. Bacterial genome size of about 5 ¡Á 106bp, containing about 3,000 genes. Most types of germs between DNA is the same, to obtain a bacteria-specific nucleic acid probe usually taken to the establishment of bacterial genomic DNA library, about bacterial DNA into small fragments were cloned after the genome contains all the information for the cloning. Then use a variety of other bacteria's DNA as a probe to screen, hybridization signals have been removed from the cloning, The last remaining not linked to any other bacterial hybrid cloning may contain the bacteria-specific DNA fragment. This recombinant plasmid marker probe after further identification, but also by DNA sequence analysis identified its source and function of genes. Therefore, in order to obtain a specific DNA probes, often more cumbersome. Cloning DNA probe can be applied to the screening serological methods, different is built for DNA expression library, Cloning colony or plaque by cracking after the release of antigen expression, and then use the source of bacteria in the polyclonal antiserum screening positive clones received a number of positive clones via other bacteria in the anti-serum screening, Finally only with the anti-bacterial expression of the serum response cloning that contain this bacteria-specific gene fragment it is the protein encoded by the bacteria are endemic. Use this expression library screening of the apparently is a specific gene probe. [DNA repair -- DNA repair (repairing DNA) cells to DNA damage after a reaction that may enable the resumption of the structure of DNA as it is, can re-enforce its original function; But sometimes not able to completely eliminate the DNA damage, cells can only make this tolerance of DNA damage and can continue to survive. Perhaps this has not been fully restored and retained from the injury in suitable conditions are displayed (for example, cancer cells, etc.) But if these cells have restored function, it can not deal with the frequent occurrence of DNA damage, others. So studies of DNA repair is also exploring the lives of one important aspect, but also with military medicine, oncology and other closely related. Different pairs of DNA damage, cells may have different reactions repair. [DNA replication -- DNA replication is double-stranded DNA prior to cell division in the reproduction process Replication of results is a double-stranded two into the same double-stranded (if replication of normal), with each double-strand of the double-stranded, like the original. This process is known as semi-reservation mechanism to copy the successful completion. Replication can be divided into the following phases : initial stages : gyrase in local start the double helix structure of DNA as a single chain, primer on the identification initiation site to remove the section of DNA as a template, according to the 5 'to 3' direction of short-chain RNA synthesis. Formation of RNA primer. DNA fragments generated : a primer 3 '- OH ends of the foundation, Catalytic DNA polymerase chain two DNA replication process simultaneously, As the process of reproduction only by 5 '- "3' direction synthesis, it can be a continuous chain synthesis, Another one sub-chain synthesis, Each of the short chain become Okazaki fragment (Okazaki fragments). RNA hydrolysis primer : When a certain length of DNA synthesis, DNA polymerase hydrolysis of RNA primer, Buchen gap. DNA ligase DNA fragments will link up to form a DNA molecule. Finally the new synthetic DNA fragment in gyrase with the help of re-forming spiral. ¡¾-- Single strand of DNA single-strand DNA (single-stranded DNA) most of the DNA double Spiral structure, but one by heat or alkali treatment will be turned into single-chain state. Single strand DNA refers to the existence of such a state of the DNA. Single strand DNA molecules in the fluid dynamics nature, absorption spectra, the nature of the base response are different and double-stranded DNA. Certain phage particles containing single-strand DNA ring, This phage DNA in the cell proliferation when it is formed double-stranded DNA. ¡¾Closed loop of DNA -- DNA (closed circular DNA) without the double-stranded circular fracture DNA, which is also known as the super-helical DNA. Because of double-stranded helix structure of each closed, with the result that the entire DNA molecule further Rotary song structure and form three. Also, if one or two different parts of the chain generated a fracture, it will become Irrotational song of open-loop DNA molecules. From cells extracted from the plasmid or viral DNA contains the closed-loop and open-loop that two kinds of molecules. Under both pigment and the ability to integrate different, and the two are separated. [Link connecting DNA -- DNA (DNA Linker) : Nuclear body in addition to 146 bp DNA outside the core of all the DNA. [Template DNA -- DNA template can be single-chain molecule, it can also be a double-stranded molecule, it can be a linear elements, it can also be cyclic molecules (linear molecule cyclic molecules than the amplification effect was somewhat better). on the template DNA, The main impact of PCR template is the quantity and purity. [Complementary DNA -- complementary DNA (cDNA. complementary DNA) genes constitute the double-stranded DNA molecule with a single strand as a template. transcription sequence complementary to its messenger RNA molecules, and then reverse transcriptase role, to mRNA molecules as templates synthesized mRNA sequence with a complementary single strand DNA, Finally, a single-stranded DNA as a template synthesis with another complementary single strand DNA, two complementary single strand DNA molecules composed of a double-stranded cDNA molecule. Therefore, Double-stranded cDNA molecule with the sequence of the mRNA transcription of genetic elements are the same. Therefore, a cDNA - on behalf of a gene. But still different from the cDNA gene, because the gene mRNA transcription. some coding intron sequences that were deleted, retained only coding sequence, that the exon. So cDNA sequences than to be much shorter, because cDNA does not include the non-coding gene sequences --- introns. [DNA restriction fragment length polymorphism analysis -- in the human genome. average of about 200 pairs of nucleotide can occur one pair of variation (known as the neutral mutations), neutral mutations lead to individuals nucleotide sequence differences, known as DNA polymorphism. Many polymorphisms in DNA restriction enzyme recognition site, hydrolysis of the DNA fragments will result in the fragmentation of different length, known as restriction fragment length polymorphism (RFLP). RFLP approach by Mendelian genetics, in a particular clan, If a certain disease genes and specific fragment polymorphism closely linked, Polymorphism can use this footage as a "genetic marker" family members to determine whether a fetus or genes that cause carriers. Hemophilia A, cystic fibrosis and phenylketonuria, etc. can use this method to be diagnosed. [Gene therapy -- Gene therapy is normal integrated into the cell genome to correction and replacement of a specific gene therapy. Currently Broadly speaking, some of the genetic material transferred to the cell patients, so intimate a role to play, to achieve the purpose of treating disease, also known as gene therapy. At present, gene therapy, the method used can basically be divided into the following categories : 1 ª± gene corrected gene correction refers to the abnormal genes that cause the base corrected, and normal part be retained. 2. Gene replacement gene replacement is to use the normal gene in vivo gene by homologous recombination. Replacement situ lesions within the cell disease genes and the DNA within the cells fully restored to normal conditions. 3. Gene added gene refers to the addition of target gene into tumor cells or other cells, not remove abnormal gene, Rather, the purpose of gene-targeted integration, its expression products compensation defect gene function or to enable the original function has been strengthened. At present, the use of gene therapy in this manner. 4. Early gene inactivation generally refers antisense oligonucleotide technology. It is a specific antisense oligonucleotides, including antisense RNA, antisense and ribozymes DNA into cells. in the translation and transcription level of blocking certain abnormal gene expression. In recent years by anti-gene strategy, peptide nucleic acid, RNA genes and remove