Recombinant DNA PDF

RECOMBINANT DNA Michael Angelo B. Palomar Where is DNA found? DNA is in every cell of every living thing. It is found within the chromosomes of the cell. Chromosomes work to build proteins and assist in duplication or division of the cells.  The unique structure of DNA allows it to be...

RECOMBINANT DNA Michael Angelo B. Palomar Where is DNA found? DNA is in every cell of every living thing. It is found within the chromosomes of the cell. Chromosomes work to build proteins and assist in duplication or division of the cells.  The unique structure of DNA allows it to be the hereditary molecule and allows it to store instructions for directing cell activities. DNA DNA stands for deoxyribonucleic acid. It is a long molecule made up of monomers called nucleotides. PHOSPHATE BASE Deoxyribonucleic Acid Sugar macro-molecule [ stores the genetic material ] DEOXYRIBOSE SUGAR The twisted ladder shape is called a DOUBLE HELIX.  NITROGEN BASES Adenine Thymine Cytosine Guanine What does DNA look like? The bases of DNA pair with each other in a Adenine Thymine predictable way. A ALWAYS PAIRS WITH T C ALWAYS PAIRS WITH G Cytosine Guanine  NUCLEOTIDE The backbone of DNA is formed by alternating sugar and phosphates held together by a strong bond. The rungs of the ladder are formed by the four nitrogen bases and are held together by weak HYDROGEN BONDS. How does DNA work? The 4 letters of DNA make up codons. These chemicals are repeated in various orders over and over. These codons make up genes. These genes tell cells how to make a protein that controls everything in the cell.  Let's Practice! One strand of DNA has the base sequence TACGATTGA What is the complementary strand of DNA? Answer! TACGATTGA ATGCTAACT  RECOMBINANT DNA Molecules of DNA from two different species that are inserted into host organism to produce new genetic combinations that are of value to science, medicine, agriculture, and industry. It is often shortened to rDNA. It is an artificially made DNA strand that is formed by the combination of two or more gene sequences. This new combination may or may not occur naturally but is engineered specifically for a purpose to be used in one of the many applications of recombinant DNA. What are the important applications of recombinant DNA? The three important applications are (1) agricultural applications (applications in crop improvement), (2) medicinal applications (applications in medicines), and (3) industrial applications.  I. AGRICULTURAL APPLICATIONS 1. DISTANT HYBRIDIZATION 2. DEVELOPMENT OF TRANSGENIC PLANTS 3. DEVELOPMENT OF ROOT NODULES IN CEREAL CROPS 4. DEVELOPMENT OF C4 PLANTS 1. DISTANT HYBRIDIZATION Transfer genes between distantly related species 2. DEVELOPMENT OF TRANSGENIC PLANTS Genetically transformed plants which contain foreign genes are called transgenic plants. 3. DEVELOPMENT OF ROOT NODULES IN CEREAL CROPS Leguminous plants have root nodules which contain nitrogen-fixing bacteria Rhizobium. 4. DEVELOPMENT OF C4 PLANTS The photosynthetic rate can be increased by conversion of C3 plants into C4 plants, which can be achieved either through protoplasm fusion or recombinant DNA technology.  II. MEDICINAL APPLICATIONS (APPLICATIONS IN MEDICINES) 1. Production of antibiotics 2. Production of hormone insulin 3. Production of vaccines 4. Production of interferon 5. Production of enzymes 6. Gene therapy 7. Solution of disputed parentage 8. Diagnosis of disease 9. Production of transgenic animals 1. PRODUCTION OF ANTIBIOTICS Penicillium and Streptomyces fungi are used for mass production of famous antibiotics penicillin and streptomycin. Genetically efficient strains of these fungi have been developed to greatly increase the yield of these antibiotics. 2. PRODUCTION OF HORMONE INSULIN Insulin, a hormone used by diabetics, is usually extracted from the pancreas of cows and pigs. This insulin is slightly different in structure from human insulin. As a result, it leads to allergic reactions in about 5% patients. Human gene for insulin production has been incorporated into bacterial DNA and such genetically engineered bacteria are used for large-scale production of insulin. 3. PRODUCTION OF VACCINES Vaccines are now produced by transfer of antigen-coding genes to disease- causing bacteria. Such antibodies provide protection against the infection by the same bacteria or virus. 4. PRODUCTION OF INTERFERON Interferons are virus-induced proteins produced by virus- infected cells. Interferons are antiviral in action and act as first line of defense against viruses causing serious infections, including breast cancer and lymph node malignancy. Natural interferon is produced in very small quality from human blood cells. It is thus very costly also. It is now possible to produce interferon by recombinant DNA technology at a much cheaper rate. 5. PRODUCTION OF ENZYMES Some useful enzymes can also be produced by recombinant DNA technique. For instance, enzyme urokinase, which is used to dissolve blood clots, has been produced by genetically engineered microorganisms. 6. GENE THERAPY Genetic engineering may one day enable the medical scientists to replace the defective genes responsible for hereditary diseases (e.g., hemophilia, phenylketonuria, alkaptonuria) with normal genes. This new system of therapy is called gene therapy. 7. SOLUTION OF DISPUTED PARENTAGE Disputed cases of parentage can now be solved most accurately by recombinant technology than by blood tests. 8. DIAGNOSIS OF DISEASE Recombinant DNA technology has provided a broad range of tools to help physicians in the diagnosis of diseases. Most of these involve the construction of probes: short segments of single- stranded DNA attached to a radioactive or fluorescent marker. Such probes are now used for the identification of infectious agents, for instance, food poisoning Salmonella, pus-forming Staphylococcus, hepatitis virus, HIV, etc. By testing the DNA ofprospective genetic disorder carrier parents, their genotype can be determined and their chances of producing an afflicted child can be predicted. 9. PRODUCTION OF TRANSGENIC ANIMALS Animals which carry foreign genes are called transgenic animals. Examples: Cow, sheep, goat – therapeutic human proteins in their milk. Fish like common carp, catfish, salmon and goldfish contain human growth hormone (hGH).  III. INDUSTRIAL APPLICATIONS In industries, recombinant DNA technique will help in the production of chemical compounds of commercial importance, improvement of existing fermentation processes, and production of proteins from wastes. This can be achieved by developing more efficient strains of microorganisms. Specially developed microorganisms may be used even to clean up the pollutants. Thus, biotechnology, especially recombinant DNA technology, has many useful applications in crop improvement, medicines, and industry. What is genetic engineering?  It is the process of using rDNA technology to alter the genetic makeup of an organism. Traditionally, humans have manipulated genomes indirectly by controlling breeding and selecting offspring with desired traits.  It involves the direct manipulation of one or more genes. Most often, a gene from another species is added to an organism's genome to give it a desired phenotype.  It is the artificial modification of an organism’s genetic composition. Genetic engineering typically involves transferring genes from one organism into another organism of a different species to give the latter specific traits of the former. The resulting organism is called a transgenic or genetically modified organism (GMO). 5 Basic Processes in Genetic Engineering Gene Cloning Gene Design DNA Extraction Transformation Backcross Breeding  STEP 1: DNA EXTRACTION The process of genetic engineering requires the successful completion of a series of five steps. DNA extraction is the first step in the genetic engineering process. In order to work with DNA, scientists must extract it from the desired organism. A sample of an organism containing the gene of interest is taken through a series of steps to remove the DNA.  STEP 2: GENE CLONING The second step of the genetic engineering process is gene cloning. During DNA extraction, all of the DNA from the organism is extracted at once. Scientists use gene cloning to separate the single gene of interest from the rest of the genes extracted and make thousands of copies of it.  STEP 3: GENE DESIGN Once a gene has been cloned, genetic engineers begin the third step, designing the gene to work once inside a different organism. This is done in a test tube by cutting the gene apart with enzymes and replacing gene regions that have been separated. STEP 4: TRANSFORMATION (E.G., PLANTS) The modified gene is now ready for the fourth step in the process, transformation, or gene insertion. Since plants have millions of cells, it would be impossible to insert a copy of the transgene into every cell. Therefore, tissue culture is used to propagate masses of undifferentiated plant cells called callus. These are the cells to which the new transgene will be added. The new gene is inserted into some of the cells using various techniques. Some of the more common methods include the gene gun, agrobacterium, microfibers, and electroporation. The main goal of each of these methods is to transport the new gene(s) and deliver them into the nucleus of a cell without killing it. Transformed plant cells are then regenerated into transgenic plants. The transgenic plants are grown to maturity in greenhouses, and the seed they produce, which has inherited the transgene, is collected. The genetic engineer's job is now complete. He/she will hand the transgenic seeds over to a plant breeder who is responsible for the final step. STEP 5 : BACKCROSS BREEDING (E.G., ENGINEERED CROP) Transgenic plants are crossed with elite breeding lines using traditional plant breeding methods to combine the desired traits of elite parents and the transgene into a single line. The offspring are repeatedly crossed back to the elite line to obtain a high-yielding transgenic line. The result will be a plant with a yield potential close to current hybrids that expresses the trait encoded by the new transgene.