RECOMBINANT DNA TECHNOLOGY AND GENETIC ENGINEERING GENETIC ENGINEERING Genetic engineering – refers to the development and application of scientific methods, procedures, and technologies that permit direct manipulation of genetic material in order to alter the hereditary traits of a cell, organism, or population. BIOTECHNOLOGY Biotechnology– is the use of biological processes, organisms, or systems to manufacture products intended to improve the quality of human life. RECOMBINANT DNA Recombinant DNA– DNA in which one or more segments or genes have been inserted, either naturally or by laboratory manipulation, from a different molecule or from another part of the same molecule, resulting in a new genetic combination. Classical Breeding Technique and Modern Genetic Techniques CLASSICAL BREEDING TECHNIQUE Classing Breeding– Practices focus on the mating of organisms with desirable qualities. With this, a new population of organisms with favorable traits will remain. CLASSICAL BREEDING Classical breeding is widely applicable to food crops for the following objectives: 1. Improved food quality 2. Increased yield 3. Increased tolerance and resistance from environmental threats 4. Increased shelf life Disadvantage One disadvantage of classical breeding technique is that it takes time to produce the desired variety due to the randomness of meiosis. Through the years, scientists have found way to insert desirable traits to organisms by altering the genes themselves or their expression. MODERN GENETIC TECHNIQUES Modern genetic techniques involves the use of molecular techniques to modify the traits of a target organism. The Modification of traits may involve 1. Introducing new traits to an organism 2. Enhancement of a trait by increasing the expression of the gene 3. Enhancement of a trait by disrupting the inhibition of the gene expression General Outline of Recombinant DNA Technology Gene modification can be outlined as follows: 1. 2. 3. 4. 5. 6. Cutting the desired gene by restriction enzymes Selection of an appropriate vector which will propagate the gene of interest Ligation (joining together) of the desired gene and the vector’s gene Transferring the desired gene to the vector for it to be reproduced Select which vectors were able to contain the new gene Sequencing the new game to make sure it produced the proper product Gene modification can be outlined as follows: PLASMID DNA IN BACTERIA • Some bacteria have more DNA than their own bacterial DNA. These pieces of DNA are called plasmids • Plasmids are shorter, circular sequences of DNA that is also replicated by the bacteria prior to binary fission. • Plasmids are great targets for genetic modification. Making the desired plasmid A restriction enzyme is used for this purpose. Restriction enzyme – “scissors” of DNA; cuts a DNA along a specific sequence. EcoRI – a restriction enzyme that cuts along G|AATT Making the desired plasmid 2. The restriction enzyme cuts the vector’s plasmid and the desired gene from the original DNA. 3. Both DNA will have a sticky end of AATT on one direction and TTAA on the other. 4. AATT and TTAA are complementary. Thus, the vector plasmid and the desired gene can be ligated to form the new plasmid. Human Insulin Production Ways of introducing desired gene to host organism Heat Shock Treatment Heat shock treatment - is a process used to transfer plasmid DNA into bacteria. The target cells are pre-treated before the procedure to increase the pore sizes of their plasma membranes. This pretreatment (usually with CaCl2) is said to make the cells “competent” for accepting the plasmid DNA. After the cells are made competent, they are incubated with the desired plasmid at about 4°C for about 30min. Biolistics Biolistics – a “gene gun” is used to fire dna-coated pellets on plant tissues. Cells that survive the bombardment and are able to take up the expression plasmid coated pellets and acquire the ability to express the designed protein Electropolation Electroporation – this technique follows a similar methodology as heat shock treatment, but, the expansion of the membrane pores is done through an electric “shock”. This method is commonly used for insertion of genes into mammalian cells. Genetically Modified Organisms Genetically Modified Organisms With the ability to insert gene sequences, comes the possibility of providing new traits for these target organisms. This has allowed the development of GMOs. Some of these genetic modifications promise higher product yield for their targets. These include the flavr-savr tomato and bt-corn. Genetically Modified Organisms The flavr-savr (“flavor savor”) • Tomato was the first genetically modified organism that was licensed for human consumption. The trait modified in this tomato is its ripening process. • A gene for an enzyme that causes the degradation of pectin in the cell walls (i.E. Polygalacturonase) normally softens the fruit as it ripens. In flavr savr tomatoes, an inhibitor (i.E.Antisense RNA) disrupts the expression of this gene, thereby delaying the softening of the fruit and extending the time it may be kept in storage and transported to markets. Genetically Modified Organisms Bt-corn • was developed to incorporate the production of a toxin (i.E. Bt-endotoxin) from bacillus thuringensis in corn plants. This toxin results in the death of pests that feed on these plants like the corn borer larvae. • The toxin has been shown to be selective for lepidoptera larvae and is non-toxic to humans, mammals, fish and birds. The selective toxicity of the toxin allows its use in foodcrops. • The introduction of the toxin is believed to increase crop production due to decreased losses from pest infestation. The same technology has been applied in the Philippines for the development of bt-eggplant.
