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- 1. MEWAR INSTITUTE OF MANAGEMENT Genetic engineering scopes and importance Priya yadav Msc. Biotech. Second sem
- 2. What is genetic engineering? • Genetic modification i.e. process of inserting new genetic information into existing cells in order to modify a specific organism for the purpose of changing its characteristics.
- 3. Restriction enzymes • Enzymes that cuts DNA at or near the specific recognition nucleotide sequence known as restriction sites. • These are of four types; type l, type ll , type lll and type lV based on their composition , enzyme cofactor requirements , nature of target sequence and the position of their DNA cleavage site relative to the target sequence.
- 4. • Type I enzymes are complex, multisubunit, combination restriction-and-modification enzymes that cut DNA at random far from their recognition sequences. • Type II enzymes cut DNA at defined positions close to or within their recognition sequences. • The most common Type II enzymes are those like HhaI (NEB #R0139), HindIII (NEB #R0104), and NotI ( NEB #R0189), that cleave DNA within their recognition sequences.
- 5. • Type III enzymes are also large combination restriction-and-modification enzymes. They cleave outside of their recognition sequences and require two such sequences in opposite orientations within the same DNA molecule to accomplish cleavage; they rarely give complete digests. • Type IV enzymes recognize modified, typically methylated DNA and are exemplified by the McrBC and Mrr systems of E. coli.
- 6. Action of restriction enzymes
- 7. Modifying enzymes • There are four types of modifying enzymes. These are: – Nucleases: DNase and RNase – DNA Ligase – Alkaline Phosphatase – Polynucleotide Kinase • The most widely used nucleases are DNase I and RNase A, both of which are purified from bovine pancreas: • Some of the common applications of DNase I are: 1.Eliminating DNA (e.g. plasmid) from preparations of RNA. 2.Analyzing DNA-protein interactions via DNase footprinting. 3.Nicking DNA prior to radiolabeling by nick translation.
- 8. Cont.. • major use of RNase A are: • Eliminating or reducing RNA contamination in preparations of plasmid DNA. • Mapping mutations in DNA or RNA by mismatch cleavage. RNase will cleave the RNA in RNA:DNA hybrids at sites of single base mismatches, and the cleavage products can be analyzed. • DNA ligases catalyze formation of a phosphodiester bond between the 5' phosphate of one strand of DNA and the 3' hydroxyl of the another. • This enzyme is used to covalently link or ligate fragments of DNA together
- 9. • This enzyme is used to covalently link or ligate fragments of DNA together. • The most widely used DNA ligase is derived from the T4 bacteriophage. T4 DNA ligase requires ATP as a cofactor • Alkaline phosphatase removes 5' phosphate groups from DNA and RNA. It will also remove phosphates from nucleotides and proteins. These enzymes are most active at alkaline pH - • Polynucleotide kinase (PNK) is an enzyme that catalyzes the transfer of a phosphate from ATP to the 5' end of either DNA or RNA.
- 10. Isoscizomers • Pairs of restriction enzymes specific to the same recognition sequence. • Eg., Sphl(CGTAC/G) and Bbul(CGTAC/G) are isoscizomers of each other. • First enzyme prototype recognizes such sequences. • Isolated from strains of bacteria. Neoscizomers • Enzyme that recognize the same sequence but cut it differently . • Smal(CCC/GGG) and Xmal(C/CCGGG) are neoscizomers of each other.
- 12. • Only one out of pair can recognizes both methylated and unmethylated forms of restriction sites. • In contrast other one just recognizes unmethylated recognition sites allowing identification Of methylation state of restriction site while isolating it from bacterial strain. • Hpall and Mspl are isoscizomers , as they both recognize the sequence 5’-CCGG-3’ when it is unmethylated but when the second C of the sequence is methylated , only Mspl can recognize it while Hpall can’t.
- 13. process of genetic engineering involes: Genetic engineering is accomplished in three basic steps. These are (1) The isolation of DNA fragments from a donor organism; (2) The insertion of an isolated donor DNA fragment into a vector genome and (3) The growth of a recombinant vector in an appropriate host.
- 14. Process
- 17. Concept and importance of RDT • Isolation and manipulation of DNA is the object of recombinant DNA research. This requires several techniques and reagents. • RESTRICTION ENZYMES : Some endonucleases that cut DNA at specific sites within molecule. More than 200 known defensive enzymes protecting host bacterial DNA from foreign organisms. • Preparation of chimerical DNA molecules .
- 18. Concept and importance of RDT • Molecular basis of no. of diseases(e.g., familial hypercholesterolemia, sickle cell  , thalassemia , cystic fibrosis , Huntington’s chorea). • Large quantity of human proteins production. • Proteins for vaccines and for diagnostic tests can be obtained • Diagnosis of existing disease and prediction of risk of its development. • Gene therapy.
- 19. Applications • Applications in Medicines... • Applications in Industries. • Applications for Environment. • Applications in Agriculture. • Applications for Humans.
- 20. Application in medicines •   Mass-production of insulin, human growth hormones, follistim (for treating infertility), human albumin, monoclonal antibodies, antihemophilic factors, vaccines, and many other drugs. • Production of interferons.
- 21. Genetic engineering in research • Organisms are genetically engineered to discover the functions of certain genes. • The production of a cloned embryo by transplanting the nucleus of a somatic cell into an ovum. • Gene replacement therapy in severe combined immune deficiency(SCID) caused by lack of adenine deaminase (AD). • Human genome project (1990-2003) to study human races and evolution ,genetic diseases and their cure etc.
- 22. Genetic engineering in industries •  Transforming microorganisms such as bacteria or yeast, or insect mammalian cells with a gene coding for a useful protein. •  Mass quantities of the protein can be produced by growing the transformed organism in bioreactors  using fermentation, then purifying the protein.
- 23. Genetic engineering in environment protection • Use of catalase enzyme for dying of jeans. • Oil eating bacteria patented by Chakrobarty to get rid of oil spilling in oceans. • Enzyme such as protease and lipase for washing  purposes. • Bio fuels.Â
- 24. Genetic engineering in agriculture • To create genetically-modified crops or genetically- modified organisms. • For e.g. , FlavrSavr (tomato) for ripening without softening or rotection against extreme weather conditions. • Virus resistant potatoes. • Pest resisted cotton ,peanuts etc. • Plants with extra vitamins and nutrients like golden rice.
- 25. Genetic engineering in animal husbandry • High milk yielding capacity of cows and buffalows. • High yield of egg , meat yielding capacity like  growth in salmon fish size. • For aqua culture like goldenfish. • High wool production (sheep with mouse keratin gene).
Editor's Notes
- Genetic engineering, also called genetic modification, is the direct manipulation of an organism's genome using biotechnology. New DNA may be inserted in the host genome by first isolating and copying the genetic material of interest, using molecular-cloning methods to generate a DNA sequence; or by synthesizing the DNA, and then inserting this construct into the host organism. Genes may be removed, or "knocked out" , using a nuclease. Genetically manipulated mice Laboratory mice are genetically manipulated by deleting a gene for use in biomedical research. Gene targeting is a different technique that uses homologous recombination to change an endogenous gene, and can be used to delete a gene, remove exons, add a gene, or introduce point mutations. Genetic engineering has applications in medicine, research, industry and agriculture and can be used on a wide range of plants, animals and microorganisms. Genetic engineering has produced a variety of drugs and hormones for medical use. For example, one of its earliest uses in pharmaceuticals was gene splicing to manufacture large amounts of insulin, made using cells of E. coli bacteria. Interferon, which is used to eliminate certain viruses and kill cancer cells, also is a product of genetic engineering, as are tissue plasminogen activator and urokinase, which are used to dissolve blood clots. Another byproduct is a type of human growth hormone; it's used to treat dwarfism and is produced through genetically-engineered bacteria and yeasts. The evolving field of gene therapy involves manipulating human genes to treat or cure genetic diseases and disorders. Modified plasmids or viruses often are the messengers to deliver genetic material to the body's cells, resulting in the production of substances that should correct the illness. Sometimes cells are genetically altered inside the body; other times scientists modify them in the laboratory and return them to the patient's body. Since the 1990s, gene therapy has been used in clinical trials to treat diseases and conditions such as AIDS, cystic fibrosis, cancer, and high cholesterol. Drawbacks of gene therapy are that sometimes the person's immune system destroys the cells that have been genetically altered, and also that it is hard to get the genetic material into enough cells to have the desired effect.
- 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. Creating a crop that is resistant to a specific herbicide proved to be a success because the herbicide eliminated weed competition from the crop plant. Researchers discovered herbicide-resistant bacteria, isolated the genes responsible for the condition, and “shot” them into a crop plant, which then proved to be resistant to that herbicide. Similarly, insect-resistant plants are becoming available as researchers discover bacterial enzymes that destroy or immobilize unwanted herbivores, and others that increase nitrogen fixation in the soil for use by plants. Geneticists are on the threshold of a major agricultural breakthrough. All plants need nitrogen to grow. In fact, nitrogen is one of the three most important nutrients a plant requires. Although the atmosphere is approximately 78 percent nitrogen, it is in a form that is unusable to plants. However, a naturally occurring rhizobium bacterium is found in the soil and converts atmospheric nitrogen into a form usable by plants. These nitrogen-fixing bacteria are also found naturally occurring in the legumes of certain plants such as soybeans and peanuts. Because they contain these unusual bacteria, they can grow in nitrogen-deficient soil that prohibits the growth of other crop plants. Researchers hope that by isolating these bacteria, they can identify the DNA segment that codes for nitrogen fixation, remove the segment, and insert it into the DNA of a profitable cash crop! In so doing, the new transgenic crop plants could live in new fringe territories, which are areas normally not suitable for their growth, and grow in current locations without the addition of costly fertilizers!
- 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. Transgenic animals model advancements in DNA technology in their development. The mechanism for creating one can be described in three steps: Healthy egg cells are removed from a female of the host animal and fertilized in the laboratory. The desired gene from another species is identified, isolated, and cloned. The cloned genes are injected directly into the eggs, which are then surgically implanted in the host female, where the embryo undergoes a normal development process. It is hoped that this process will provide a cheap and rapid means of generating desired enzymes, other proteins, and increased production of meat, wool, and other animal products through common, natural functions. Ever since 1997 when Dolly was cloned, research and experimentation to clone useful livestock has continued unceasingly. The attractiveness of cloning is the knowledge that the offspring will be genetically identical to the parent as in asexual reproduction. Four steps describe the general process: A differentiated cell, one that has become specialized during development, with its diploid nucleus is removed from an animal to provide the DNA source for the clone. An egg cell from a similar animal is recovered and the nucleus is removed, leaving only the cytoplasm and cytoplasm organelles. The two egg cells are fused with an electric current to form a single diploid cell, which then begins normal cell division. The developing embryo is placed in a surrogate mother, who then undergoes a normal pregnancy.