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  1. 1. ASSIGNMENT: 1 APPLICATIONS RECOMBINANT DNA TECHNOLOGY (rDNA): Recombinant DNA technology-refers to the creation of new combinations of DNA segments that are not found together in nature. The isolation and manipulation of genes allows for more precise genetic analysis as well as practical applications in medicine, agriculture, and industry. Recombinant DNA molecules- which sometimes called chimeric DNA are DNA molecules formed by laboratory methods of genetic recombination (such as molecular cloning) to bring together genetic material from multiple sources, creating sequences that would not otherwise be found in biological organisms. Recombinant DNA is possible because DNA molecules from all organisms share the same chemical structure. They differ only in the nucleotide sequence within that identical overall structure. Photograph above is of GloFish, the first genetically modified animal to be sold as a pet. The GloFish is a patented and trademarked brand of genetically modified (GM) fluorescent fish. GloFish are a type of transgenic zebrafish (Danio rerio) that have been modified through the insertion of a green fluorescent protein (gfp) gene. Not all GloFish are green, however. Rather, there are several gfp gene constructs, each encoding a different colored phenotype, from fluorescent yellow to fluorescent red
  2. 2. APPLICATIONS OF RECOMBINANT DNA TECHNOLOGY: The most common application of recombinant DNA is in basic research, in which the technology is important to most current work in the biological and biomedical sciences. Recombinant DNA is used to identify, map and sequence genes, and to determine their function. rDNA probes are employed in analyzing gene expression within individual cells, and throughout the tissues of whole organisms. Recombinant proteins are widely used as reagents in laboratory experiments and to generate antibody probes for examining protein synthesis within cells and organisms. Many additional practical applications of recombinant DNA are found in industry, food production, human and veterinary medicine, agriculture, and bioengineering. Some specific examples are identified below; A) IN MEDICINE Recombinants DNA technology or Genetic engineering has resulted in a series of medical products. Multiple varieties of proteins are created from recombinant DNA technology and it is used for medications. Some can be extracts from humans, such as human growth hormone (rHGH), human insulin, folliclestimulating hormone (FSH) and factor VIII. Other proteins, when used as medication, only have recombinant DNA as a source, such as with erythropoietin. It has brought many revolutionary changes in the field of medicine and introduced such methods of treating diseases and delivering the drug. I) Recombinant human growth hormone (HGH, somatotropin) Administered to patients whose pituitary glands generate insufficient quantities to support normal growth and development. Before recombinant HGH became available, HGH for therapeutic use was obtained from pituitary glands of cadavers. This unsafe practice led to some patients developing Creutzfeldt-Jacob disease. Recombinant HGH eliminated this problem, and is now used therapeutically. It has also been misused as a performance enhancing drug by athletes and others. II) Recombinant human insulin Almost completely replaced insulin obtained from animal sources (e.g. pigs and cattle) for the treatment of insulin-dependent diabetes. A variety of different recombinant insulin preparations are in widespread use. Recombinant insulin is synthesized by inserting the human insulin gene into E. coli, which then produces insulin for human use. III) Recombinant blood clotting factor VIII A blood-clotting protein that is administered to patients with forms of the bleeding disorder hemophilia, who are unable to produce factor VIII in quantities sufficient
  3. 3. to support normal blood coagulation. Before the development of recombinant factor VIII, the protein was obtained by processing large quantities of human blood from multiple donors, which carried a very high risk of transmission of blood borne infectious diseases, for example HIV and hepatitis B. IV) Recombinant hepatitis B vaccine Hepatitis B infection is controlled through the use of a recombinant hepatitis B vaccine, which contains a form of the hepatitis B virus surface antigen that is produced in yeast cells. The development of the recombinant subunit vaccine was an important and necessary development because hepatitis B virus, unlike other common viruses such as polio virus, cannot be grown in vitro. V) Diagnosis of infection with HIV Each of the three widely used methods for diagnosing HIV infection has been developed using recombinant DNA. The antibody test (ELISA or western blot) uses a recombinant HIV protein to test for the presence of antibodies that the body has produced in response to an HIV infection. The DNA test looks for the presence of HIV genetic material using reverse transcriptase polymerase chain reaction (RTPCR). Development of the RT-PCR test was made possible by the molecular cloning and sequence analysis of HIV genomes. VI) Xenotransplantation Accordingly, the development of genetically engineered animals has overcome the hyper acute rejection barrier, with acute humoral xenograft rejection (AHXR) currently remaining the most important immunological obstacle. At this stage, a better control of the elicited anti-pig humoral immune response and avoidance of coagulation disorders are the two primary research fronts being pursued in order to overcome AHXR. Nonetheless, it is encouraging that porcine xenografts can sustain the life of non-human primates for several months. VII) Human gene therapy Human gene therapy involves adding a normal copy of a gene (transgene) to the genome of a person carrying defective copies of the gene. The potential for treatments for the 5,000 named genetic diseases is huge and transgenic animals
  4. 4. could play a role. For example, the A. I. Virtanen Institute in Finland produced a calf with a gene that makes the substance that promotes the growth of red cells in humans. Hence a transgenic cow exists that produces a substance to help human red cells grow. VIII) Control of Mosquito transmitted infections In 2010, scientists created "malaria-resistant mosquitoes" in the laboratory. The World Health Organization estimated that Malaria killed almost one million people in 2008.Genetically modified male mosquitoes containing a lethal gene have been developed in order to combat the spread of Dengue fever. Aedes aegypti mosquitoes, the single most important carrier of dengue fever, were reduced by 80% in a 2010 trial of these genetic modified (GM) mosquitoes in the Cayman Islands. Between 50 and 100 million people are affected by Dengue fever every year and 40,000 people die from it. Other applications of recombinant DNA technology in medicine are productions of following useful products;      Tumor necrosis factor. Treatment for certain tumor cells Interleukin-2 (IL-2). Cancer treatment, immune deficiency, and HIV infection treatment Prourokinase. Treatment for heart attacks Taxol. Treatment for ovarian cancer Interferon. Treatment for cancer and viral infections. B) IN AGRICULTURE Crop plants have been and continue to be the focus of biotechnology as efforts are made to improve yield and profitability by improving crop resistance to insects and certain herbicides and delaying ripening (for better transport and spoilage resistance). The creation of a transgenic plant, one that has received genes from another organism, proved more difficult than animals. Unlike animals, finding a vector for plants proved to be difficult until the isolation of the Ti plasmid, harvested from a tumor-inducing (Ti) bacteria found in the soil. The plasmid is “shot” into a cell, where the plasmid readily attaches to the plant's DNA. Although successful in fruits and vegetables, the Ti plasmid has generated limited success in grain crops.
  5. 5. I) Golden rice A recombinant variety of rice that has been engineered to express the enzymes responsible for β-carotene biosynthesis. This variety of rice holds substantial promise for reducing the incidence of vitamin A deficiency in the world's population. Golden rice is not currently in use, pending the resolution of regulatory issues. Photograph above show Golden rice (right) compared to white rice (left) II) Herbicide-resistant crops Commercial varieties of important agricultural crops (including soy, maize/corn, sorghum, canola, alfalfa and cotton) have been developed that incorporate a recombinant gene that results in resistance to the herbicide glyphosate, and simplifies weed control by glyphosate application. These crops are in common commercial use in several countries. III) Insect-resistant crops Bacillus thuringeiensis is a bacterium that naturally produces a protein (Bt toxin) with insecticidal properties. The bacterium has been applied to crops as an insectcontrol strategy for many years, and this practice has been widely adopted in agriculture and gardening. Recently, plants have been developed that express a
  6. 6. recombinant form of the bacterial protein, which may effectively control some insect predators. Environmental issues associated with the use of these transgenic crops have not been fully resolved. Photograph above show Kenyans examining insect-resistant transgenic Bt corn C) IN ANIMAL HUSBANDRY Neither the use of animal vaccines nor adding bovine growth hormones to cows to dramatically increase milk production can match the real excitement in animal husbandry: transgenic animals and clones. I) Transgenesis will allow larger herds with specific traits Farmers have always used selective breeding to produce animals that exhibit desired traits (e.g., increased milk production, high growth rate). Traditional breeding is a time-consuming, difficult task. When technology using molecular biology was developed, it became possible to develop traits in animals in a shorter time and with more precision. In addition, it offers the farmer an easy way to increase yields.
  7. 7. II) Improve the size and quality of livestock genetically. Transgenic cows exist that produce more milk or milk with less lactose or cholesterol, pigs and cattle that have more meat on them, and sheep that grow more wool. In the past, farmers used growth hormones to spur the development of animals but this technique was problematic, especially since residue of the hormones remained in the animal product. III) Disease-resistant livestock Scientists are attempting to produce disease-resistant animals, such as influenzaresistant pigs, but a very limited number of genes are currently known to be responsible for resistance to diseases in farm animals. Photograph above show transgenic casein cows D) IN FOOD INDUSTRY Recombinant chymosin Found in rennet, is an enzyme required to manufacture cheese. It was the first genetically engineered food additive used commercially. Traditionally, processors obtained chymosin from rennet, a preparation derived from the fourth stomach of milk-fed calves. Scientists engineered a non-pathogenic strain (K-12) of E. coli bacteria for large-scale laboratory production of the enzyme. This
  8. 8. microbiologically produced recombinant enzyme, identical structurally to the calf derived enzyme, costs less and is produced in abundant quantities. Today about 60% of U.S. hard cheese is made with genetically engineered chymosin. In 1990, FDA granted chymosin "generally-recognized-as-safe" (GRAS) status based on data showing that the enzyme was safe. E) INDUSTRY APPLICATION I) Uses in industry include material fabrication In 2001, two scientists at Nexia Biotechnologies in Canada spliced spider genes into the cells of lactating goats. The goats began to manufacture silk along with their milk and secrete tiny silk strands from their body by the bucketful. By extracting polymer strands from the milk and weaving them into thread, the scientists can create a light, tough, flexible material that could be used in such applications as military uniforms, medical microsutures, and tennis racket strings. II) Uses in determining safety tests of chemicals Toxicity-sensitive transgenic animals have been produced for chemical safety testing. Microorganisms have been engineered to produce a wide variety of proteins, which in turn can produce enzymes that can speed up industrial chemical reactions. REFERENCES: 1) 2) 3) 4) 5)