Recombinant dna technology by 269

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Recombinant dna technology by 269

  1. 1.  Deoxyribonucleic acid (DNA) is an informational molecule encoding the genetic instructions used in the development and functioning of all known living organisms and many viruses.  Recombinant DNA (rDNA) molecules are DNA sequences that result from the use of laboratory methods (molecular cloning) to bring together genetic material from multiple sources, creating sequences that would not otherwise be found in biological organisms.  Recombinant DNA technology is the technology of preparing recombinant DNA in vitro by cutting up DNA molecules and splicing together fragments from more than one organism.
  2. 2. How is Recombinant DNA made? There are three different methods by which Recombinant DNA is made. They are - 1. Transformation 2. Phage Introduction 3. Non-Bacterial Transformation.
  3. 3. Transformation  The first step in transformation is to select a piece of DNA to be inserted into a vector.  The second step is to cut that piece of DNA with a restriction enzyme and then ligate the DNA insert into the vector with DNA Ligase.  The insert contains a selectable marker which allows for identification of recombinant molecules.  The vector is inserted into a host cell, in a process called transformation. One example of a possible host cell is E. Coli. The host cells must be specially prepared to take up the foreign DNA.
  4. 4. Non-bacterial Transformation  This is a process very similar to Transformation, The only difference between the two is non-bacterial does not use bacteria such as E. Coli for the host.  In microinjection, the DNA is injected directly into the nucleus of the cell being transformed.  In biolistics, the host cells are bombarded with high velocity microprojectiles, such as particles of gold or tungsten that have been coated with DNA.
  5. 5. Phage Introduction  Phage introduction is the process of transfection, which is equivalent to transformation, except a phage is used instead of bacteria. In vitro packagings of a vector is used. This uses lambda or MI3 phages to produce phage plaques which contain recombinants. The recombinants that are created can be identified by differences in the recombinants and non-recombinants using various selection methods.
  6. 6. Why is rDNA important? Recombinant DNA has been gaining in importance over the last few years, and recombinant DNA will only become more important in the 21st century as genetic diseases become more prevalent and agricultural area is reduced. Below are some of the areas where Recombinant DNA will have an impact -  Better Crops (drought & heat resistance)  Recombinant Vaccines (i.e. Hepatitis B)  Prevention and cure of sickle cell anemia  Prevention and cure of cystic fibrosis  Production of clotting factors  Production of insulin  Production of recombinant pharmaceuticals  Plants that produce their own insecticides  Germ line and somatic gene therapy
  7. 7. Advantages of Recombinant DNA Technology Benefits include engineering organisms that have a desirable trait. For example, insulin for many decades was harvested from animal pancreases. Recombinant bacteria have been designed that produce human insulin in large quantities, so animal pancreases are no longer required. Gene therapy is another benefit, as it seeks to supplement mutant copies of genes with the correct copies. A success in gene therapy is the recent treatment of metastatic melanoma (cancer) with gene-therapy. Genetically modified crops with traits such as resistance to disease and insects or other desirable characteristics.  Provide substantial quantity  No need for natural or organic factors  Tailor made product that you can control  Unlimited utilizations  Cheap  Resistant to natural inhibitors
  8. 8. Disadvantages of Recombinant DNA Technology A drawback is that modified organisms can spread through nature and interbreed with natural organisms, thereby contaminating environments and future generations in an unforeseeable and uncontrollable way. Also, the interactions of the designer genes with the wild-type genes can be unpredictable.  Commercialized and became big source of income for businessmen  Effects natural immune system of the body  Can destroy natural ecosystem that relies on organic cycle  Prone to cause mutation that could have harmful effects  Major international concern: manufacturing of biological weapons such as botulism & anthrax to target humans with specific genotype  Concerns of creating super‐human race
  9. 9. Production of Insulin The first step in creating synthetic human insulin is to extract human DNA. The method to extracting human DNA is as follows:  First charge a sample of blood, normally between 300 and 2500 microlitres, and allow it to undergo a treatment of lysis by a cationic detergent, for example tetradecyltrimethilammoniumbromide (TTAB) or dodecyltrimethilammoniumbromide (DTAB) both with sodium chloride at a concentration higher than 0.5 M. Then mix up the solution and heat it up to 68 C and incubate it for five minutes. Add 1 volume of chloroform or another organic solvent. Allow the mixture to undergo centrifuging with a normal bench centrifuge for a few minutes to eliminate the protein portion which forms a clog with the organic base.  After centrifuging, to the aqueous phase add a quantity of water to decrease the ionic strength below 0.5 M NaCl, and a cationic detergent (for instance a solution of 5% of Cetyl-trimetil-ammonium bromide), so that precipitation of the cationic detergent micellar complex-DNA takes place after a short mixing operation. The solution now containing the micellar-DNA complex then undergoes filtration. The ultra filtration takes place with a filter (for instance, sintered borosilicate glass or an organic matrix like polypropilene or polyethylene) of known and tested porosity (pore size between 5 and 15 micron), which retains the DNA-cationic detergent micellar complex in a satisfactory way. The hydrophilic surface enables DNA to be recovered easily and speedily after the washing operations. The organic matrix filter allows a slower recovery of DNA so it is not commonly used at the moment. As genomic DNA is immobilized on the filter, it is then eluted. (Schneider, 1995).
  10. 10.  Once the human DNA has been extracted, it is necessary to isolate the exact gene for insulin. It is located in the top of the short arm of the eleventh chromosome. The DNA sequence for the A chain is compromised of sixty three nucleotides and the sequence for the B chain is compromised of ninety nucleotides. An extra codon must be placed at the end of both sequences to signal the termination of protein synthesis. Also, an anti-codon, consisting of the amino acid methionine, must be placed at the beginning of each chain in order to make the removal of the insulin possible. (Singh-Khaira, 1994).  In order to “cut” the genes for insulin production from the DNA you must mix the human DNA with the enzymes HincII and BamHI (Laub, Rall, Bell, Rutter, 1982).  Once the DNA has been cleaved and the insulin gene and been isolated, the mixture must be run through an agarose gel electrophoresis in order to be able to remove the insulin gene. First you must cast the agarose gel. To do this place the well-forming comb into the gel-casting tray and pour enough agarose solution to fill the tray to 6mm.  After the gel has set (15-20 minutes), place it into the gel box, fill the box with enough TBE buffer to cover the gel, and remove the comb. After these steps have been completed, the DNA must be loaded into the gel by inserting it into the wells with a pipet. After all of the DNA has been loaded, close the electrophoresis chamber and connect the electrical leads to a power supply and turn it on. Wait until the bands are almost to the end and then remove the gel and stain it (Owen, 2000). Once the gel has been stained, elute the DNA fragments by shaking the gel in a solution of 0.2 N NaCl, 1mm EDTA, and 10 mm Trk (Laub, 1982).
  11. 11.  Now that the insulin gene has been isolated and removed, it must be inserted into the plasmid of the vector cell, which is E. coli. The genes for the A and B chains of insulin are inserted into the gene for the bacterial enzyme, B- galactosidase. Once the genes have been inserted into the plasmid, the plasmid is reinserted into the E. coli cell.  When the cell replicates, the B-galactosidase is formed with either the A or B chain of insulin attached to it. The two chains are then extracted and purified. Then they are mixed together thus connecting to each other by forming disulfide cross bridges. The end result is Humulin, or synthetic human insulin (Singh-Khaira, 1994).
  12. 12. Scopes  Agriculture: Growing crops of your choice (GM food), pesticide resistant crops, fruits with attractive colors, all being grown in artificial conditions.  Pharmacology: Artificial insulin production, drug delivery to target sites.  Medicine: Gene therapy, antiviral therapy, vaccination, synthesizing clotting factors.  Other uses: Fluorescent fishes, glowing plants etc.
  13. 13. Environmental Conversation  The use of recombinant DNA technology to control feral pests such as the European rabbit (Oryctolagus cuniculus) is undergoing research.  Rabbits have developed a resistance to the myxomatosis virus. A naturally occurring rabbit gene (ZPC ) affects the zona pellucida and blocks fertilization in rabbits. This gene has been isolated and added to the myxomatosis virus. The resulting recombinant virus, acting as a vector for ZPC, has been very successful under laboratory conditions as a contraceptive. If released into the wild it may reduce the rabbit population and the rabbit's status as a major pest.  Another example of environmental clean-up and conservation using recombinant DNA technology is the use of bacteria in clearing areas containing landmines.
  14. 14. Use in Agriculture  One of the most important applications of biotechnology is in the production of food. Crop plants and livestock have been genetically modified using recombinant DNA techniques to increase yield, improve quality and develop new varieties that are resistant to disease, or can survive in arid or high salt conditions.
  15. 15. Recombinant Vaccines  A viral disease called infectious bursal disease (IBD) attacks the white blood cells that normally produce antibodies in chicken. This weakens the immune system and makes chickens susceptible to other diseases.  A protein from the virus causing IBD was found to produce a high immune response, i.e. the production of antibodies. The gene that produces the viral protein has been isolated and introduced into a bacterial plasmid. The plasmid, now containing recombinant DNA, is inserted into a bacterial cell and cloned. Large quantities of the viral protein are produced and form the basis of a vaccine for IBD in chickens.  Protein-based vaccines for dealing with footrot in sheep and cattle tick in cattle have also been developed in this way.
  16. 16. Use in Medicine  Human enzymes, like insulin or human growth hormone, are created in normally functioning bodies. Enzymes are proteins and proteins are made up of a specific sequence of amino acids. The amino acid sequence is determined by the person's DNA. Previously, diabetics used insulin from pigs but it was not accepted well by all diabetics as the amino acid sequence was slightly different. Now, scientists have developed bacteria which have had the human gene for insulin inserted into them using recombinant DNA techniques. Since the amino acid sequence is the same, diabetics readily accept it even though it was manufactured by bacteria. In a similar fashion, scientists have developed protocols for the production of clotting factors, hGH, virus fighting proteins, and many other medicines are in development.  Genetic engineering has resulted in a series of medical products. The first two commercially prepared products from recombinant DNA technology were insulin and human growth hormone, both of which were cultured in the E. coli bacteria.
  17. 17. Use in Bioplastics Regular plastics are a polymer created from petroleum, which is a nonrenewable fossil fuel. When we run out of petroleum, we could run out of plastic, not just gas for our cars. This possibility has spurred the science of creating bioplastics. Bioplastics are created from products produced by plants, often through means of recombinant DNA. Scientists have engineered a gene that will produce a compound nearly identical to commercial plastics and it's application is a hot area of research today. If successful, scientists will have ensured that we will never run out of plastic or have to worry about the pollution of creating plastic from old, nonrenewable technologies.
  18. 18. Use in Pharmaceutical Products  By recombinant DNA technology discussed previously, human genes for various proteins have been engineered into expression vectors and then into bacterial host cells that produce and secrete the protein. Examples include insulin, used to treat diabetes, and human growth hormone, used to treat hypopituitarism, which causes a form of dwarfism.  Another example is tissue plasminogen activator (TPA), which helps dissolve blood clots and reduces the risk of subsequent heart attacks if administered shortly after an initial attack. It is also possible to construct desired molecules.  Genetically engineered proteins can block or mimic surface receptors on cell membranes; an example is a molecule designed to mimic a receptor protein that HIV binds to in entering white blood cells (if HIV binds to the drug molecules instead of those on the cell surface, it would fail to enter the blood cell).  Recombinant DNA techniques can generate large amounts of proteins associated with the immune response against pathogens or be used to modify the genome of a pathogen to attenuate it, and thus lead to more specific and safer vaccines.

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