Recombinant DNA PDF

ASSIGNMENT In a 1 whole sheet of paper/bond paper, list down at least 5 products of Genetic Engineering in the field of Medicine, Agriculture, Industry, Environment, etc. and choose 1 product then explain why and how it was engineered and it’s Pros and Cons. Glow-in-the-dark cats Scientist used a...

ASSIGNMENT In a 1 whole sheet of paper/bond paper, list down at least 5 products of Genetic Engineering in the field of Medicine, Agriculture, Industry, Environment, etc. and choose 1 product then explain why and how it was engineered and it’s Pros and Cons. Glow-in-the-dark cats Scientist used a virus to insert DNA from jellyfish The gene made the cat produce a fluorescent protein in its fur. Activity 1: Am I Good Enough? Example of plants/animals Desirable or enhanced trait 1. Grapes Seedless 2. Guapple 3. Corn 4. Rice 5. Coconut 6. Banana 7. Ampalaya 8. Cow 9.Chicken 10. Pig  GENERAL BIOLOGY 2 Recombinant DNA Presented by: JOAN A. RIPARIP Learning Objectives At the end of the lesson, you should be able to: Outline the processes involved in genetic engineering Discuss the applications of recombinant DNA Introduction to Genetic Engineering The term “genetic engineering” (recombinant DNA technology and biotechnology ) was coined in 1919 by Karl Ereky, a Hungarian engineer. Introduction to Genetic Engineering 8000 and 1000 B.C. - Man has used artificial selection to exploit and manipulate organisms for thousands of years; horses, camels, oxen, and many other species were already domesticated; 6000 B.C. - yeast was used to make beer; 5000 B.C. - plants such as maize, wheat, and rice were bred 420 B.C. - Socrates speculated on why children do not necessarily resemble their parents; Introduction to Genetic Engineering 400 B.C. - Hippocrates would propose that males contribute to a child’s character through semen: the idea of heredity was thus established. It was not just Greeks or Romans who were in a constant quest for an answer to how life originates. 100–300 A.D.- Hindu philosophers were giving much thought to the same questions of reproduction and inheritance. By the first millennium, they had already established the foundations of genetics; they observed that certain diseases might run in the family. They also came to believe, almost correctly, that children inherit all the characteristics of their parents. Introduction to Genetic Engineering 19th century With the exponential increase in the number of biochemical studies during the 19th century, such as those on nucleic acids and amino acids, and the speeding up of the fermentation industry, biology took on a whole new direction. 1864-1865 - Mendel presented his work on peas and published the results in 1865 in. The work was largely neglected for quite some time, and the term gene or genetics was not yet coined. Introduction to Genetic Engineering 1882 - first biotechnology products, including the use of agar described by the Koch lab 1884 - the development of the autoclave in 1884 by a French company (Chamberland’s Autoclaves), 1895 - the discovery of X-rays by W. Roentgen 1913 - the application of this information to X-ray crystallography by physicist Sir William Henry Bragg and his son William Lawrence Bragg and many others Introduction to Genetic Engineering 1945 - William Astbury, a leading biophysicist in the field of X-ray diffraction analysis of structures of biological macromolecules, devised the term molecular biology. early 1950s - Rosalind Franklin and Maurice Wilkins obtained the X-ray diffraction data for DNA, which would prove crucial for Watson and Crick to establish their model of two helically intertwined chains tied together by hydrogen bonds between the purines and pyrimidines in 1953. 1951 - Hershey performed his famous ―blender experiment‖ with his assistant Martha Chase, showing that the hereditary material is DNA and not protein. Luria and Hershey also demonstrated that bacteriophages mutated, and introduced criteria for distinguishing mutations from other modifications.  Introduction to Genetic Engineering At around the same time, bacterial plasmids were defined as autonomously replicating material, late 1960s - Werner Arber identified the restriction enzymes in bacteria that were designed to cleave DNA 1970 - Temin and Baltimore independently identified the viral enzyme reverse transcriptase, which would result in the birth of recombinant DNA technology 1971 - Paul Berg (Stanford) succeeded in proving the possibility to splice and to recombine genetic material 1972 - the first recombinant DNA was produced in Boyer Laboratory 1976 - first biotechnology company Genentech was born. Introduction to Genetic Engineering The big biotech boom would be seen in the 1980s, 1983 - the invention of the polymerase chain reaction (PCR) by Karen Mullis 1982 - Genentech’s recombinant interferon gamma and Eli Lilly’s recombinant human insulin appeared on the market 1986 - The Human Genome Initiative, later to be renamed the Human Genome Project, was launched in and its completion was announced nearly two decades later.  Introduction to Genetic Engineering 1990s - Another biotech company, GenPharm International, created the first transgenic dairy cow to produce human milk proteins for infant formula in the, and in the same period the first authorized gene therapy began on a four-year-old girl with an immune disorder known as ADA, or adenosine deaminase deficiency. 1997 - This hype was perhaps at its peak in 1997, when flash news came from Scotland’s Roslin Institute that the first mammalian clone, Dolly the sheep, was born, through a procedure known as somatic cell nuclear transfer.  GENERAL BIOLOGY 2 Recombinant DNA Presented by: JOAN A. RIPARIP What is RECOMBINANT DNA TECHNOLOGY? This involves using enzymes and various laboratory techniques to manipulate and isolate DNA segments of interest. This method can be used to combine (or splice) DNA from different species or to create genes with new functions. The resulting copies are often referred to as recombinant DNA. Such work typically involves propagating the recombinant DNA in a bacterial or yeast cell, whose cellular machinery copies the engineered DNA along with its own.  What is Recombinant DNA Technology? “the joining together of DNA molecules from different organisms and inserting it into a host organism to produce new genetic combinations that are of value to science, medicine, agriculture and industry.” (Encyclopedia Britannica)  What is Recombinant DNA Technology? It is a biotechnology approach that has multidisciplinary applications and the potential to deal with important aspects of life, from health issues (e.g. by the means of recombinant antibodies) to food resources, and resistance to divergent adverse environmental effects (Eberle C. 2022) Basic Principle of rDNA Recombinant DNA is made from combining DNA from different sources History of Recombinant DNA technology Recombinant DNA in the Lab In a series of experiments, between 1972 and 1974, Stanley Cohen, Herbert Boyer, and their colleagues, at Stanford University and the University of California, San Francisco built on the work of recombinant DNA pioneers such as Paul Berg to develop techniques that would form the basis of recombinant DNA technology. These experiments helped spur the birth of the biotechnology industry. Recombinant DNA in the Lab Since 1959, scientists knew that bacteria contain extra loops of DNA called “plasmids” in addition to their chromosome. In nature, bacteria can swap these plasmids with one another, quickly transferring beneficial genes like those that code for antibiotic resistance. By the early 1970s, investigators had isolated several plasmids as well as special enzymes known as “restriction endonucleases” that work like scissors to cut open the loops of plasmids.  Recombinant DNA in the Lab Herbert Boyer had expertise with restriction endonucleases and Stanley Cohen studied plasmids, and after meeting at a conference in 1972, the two decided to combine their research efforts. After preliminary experiments in 1973, the Cohen- Boyer team was able to cut open a plasmid loop from one species of bacteria, insert a gene from different bacterial species and close the plasmid. This created a recombinant DNA molecule-- a plasmid containing recombined DNA from two different sources. Next, they inserted the plasmid into bacteria and demonstrated that the recombinant DNA could be used by bacteria. The team had created the first genetically modified organisms. Recombinant DNA in the Lab A year later, they would use this technique to insert a gene from a frog into bacteria, proving that it was possible to transfer genes between two very different organisms. The technology for creating these “molecular chimeras” was patented on December 2, 1980 (US Patent 4,237,224.) Tools of Recombinant DNA Technology  Plasmids Plasmids are physically separate from chromosomal DNA and replicate independently. They typically have a small number of genes — notably, some associated with antibiotic resistance — and can be passed from one cell to another. Scientists use recombinant DNA methods to splice genes that they want to study into a plasmid. When the plasmid copies itself, it also makes copies of the inserted gene. A plasmid is a small circular DNA molecule found in bacteria and some other microscopic organisms. Restriction enzymes These are molecular scissors used in molecular biology for cutting DNA sequences at a specific site. It plays an important role in gene manipulation. Restriction enzymes Although there are several different types of restriction enzymes, those most useful for rDNA technology recognize specific short sequences in DNA and cleave the DNA at that site to produce cohesive (sticky) or blunt-ended fragments DNA Ligase It attaches 2 pieces of DNA together Process of Recombinant DNA Technology The complete process of recombinant DNA technology includes multiple steps, maintained in a specific sequence to generate the desired product. Step-1. Isolation of Genetic Material. The first and the initial step in Recombinant DNA technology is to isolate the desired DNA in its pure form i.e. free from other macromolecules. Step-2. Cutting the gene at the recognition sites. The restriction enzymes play a major role in determining the location at which the desired gene is inserted into the vector genome. These reactions are called ‘restriction enzyme digestions’. Step-3. Amplifying the gene copies through Polymerase chain reaction (PCR). It is a process to amplify a single copy of DNA into thousands to millions of copies once the proper gene of interest has been cut using restriction enzymes. Step-4. Ligation of DNA Molecules. In this step of Ligation, the joining of the two pieces – a cut fragment of DNA and the vector together with the help of the enzyme DNA ligase. Step-5. Insertion of Recombinant DNA Into Host. In this step, the recombinant DNA is introduced into a recipient host cell. This process is termed as TRANSFORMATION. Once the recombinant DNA is inserted into the host cell, it gets multiplied and is expressed in the form of the manufactured protein under optimal conditions. Production of an Insulin Transformation Vectorless Gene Transfer Transduction  Vectorless Gene Transfer 1. Electroporation Electroporation, also called electropermeabilization, is an efficient, non- viral delivery system that allows genetic material (DNA and RNA), proteins, drugs or other molecules to enter cells. It uses an accurately pulsed electrical current to create temporary pores in the cell membrane through which the molecules can then pass. This process can be used on a wide variety of cells including mammalian, insect, yeast, plant and bacterial cells.  Vectorless Gene Transfer 2. Protoplast fusion The protoplasm of the living plant cell excluding the cell wall is called protoplast. Fusion of the protoplast derived from somatic cells of plants belonging to different species produces a hybrid protoplast, this process is called protoplast fusion. Fusion of protoplasts can be obtained - 1. Either chemically with the help of a mixture of polyethylene glycol (PEG) and a high concentration of calcium at pH 8. 2. Or physically by electrofusion. 3. The pomato is a transgenic crop produced by protoplast fusion of a tomato plant and a potato plant.  Vectorless Gene Transfer 3. Microinjection The transformation method of microinjection is particularly efficient when inserting DNA into giant cells. The microinjection technique introduces DNA into animal cells (eggs, oocytes, and embryos) or plant protoplasts using a micropipette (fine- tipped glass needle). This technique is more appropriate for producing transgenic mice. This process involves incorporating DNA straight into the cytoplasm or nucleus.  Transduction Transduction is the process wherein genetically engineered bacteriophages-viruses that parasitize bacteria are introduced into the cell to create the desired recombinant DNA.  Applications of Recombinant DNA Technology  Industry Cyanobacteria have been modified to produce plastic (polyethylene) and fuel (butanol) as byproducts of photosynthesis. E. coli bacteria have been modified to produce diesel fuel. Health and Medicine Treatment of genetic diseases (gene therapy) Gene Therapy: Removal and replacement of defective genes with normal healthy functional genes is known as gene therapy e.g. Sickle cell anemia, Severe Combined Immuno-Deficiency (SCID). SCID is due to a defect in the gene for the enzyme adenosine deaminase (ADA) in 25 per cent of the cases.  Health and Medicine Production of medically useful biologicals (e.g. insulin) Recombinant Human Growth Hormone Recombinant insulin (Humulin)  Health and Medicine Vaccines production Pharmacogenomics - the study of how genes affect a person’s response to drugs. This field combines pharmacology (the science of drugs) and genomics (the study of genes and their functions) to develop effective, safe medications that can be prescribed based on a person’s genetic makeup.  Environment Development and usage of alternative fuels that burn cleaner and improve air quality through reduced pollution of the environment is possible by genetic engineering means. Micro-organisms are used to decompose wastes and clean up contaminated sites by the technology of bioremediation. The use of disease resistant cultivars can make crop production less environmentally intrusive by reducing the use of agrochemicals  Agriculture Reasons to Genetically Modify Crops, Insect resistant, Herbicide resistant, Drought/freeze resistant, Disease resistant, Higher yield, Faster growth, Improved nutrition & Longer shelf life