Lecture10BIO155 - BIO 155 DNA Technology Dr. Jessica...

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Unformatted text preview: BIO 155 DNA Technology Dr. Jessica Pamment Definition of Biotechnology Definition of Biotechnology • The use of microorganisms, such as bacteria or yeasts, or biological substances, such as enzymes, to perform specific industrial or manufacturing processes • Applications include the production of certain drugs, synthetic hormones, and bulk foodstuffs as well as the bioconversion of organic waste and the use of genetically altered bacteria in the cleanup of oil spills Overview Overview • Biotechnology history and applications • Recombinant DNA technology • Applications of DNA technology in our lives • Social and ethical issues History History • Biotechnology started with the agricultural revolution around 10,000 B.C. • Beer making started around 6,000 B.C. • Modern biotech started with the discovery of DNA structure and sequencing of genomes genomes to obtain desired results • Present day biotech involves manipulating Recombinant DNA Techniques Recombinant DNA Techniques • DNA cloning • Expressing cloned genes • The Polymerase Chain Reaction (PCR) • DNA sequencing DNA Cloning DNA Cloning • • • • i. ii. Provides a way of working directly with gene of interest Method used to make multiple copies of well­ defined DNA segments Commonly an E. coli plasmid is used to clone gene of interest Outcomes are : Make many copies of gene of interest Make protein product of interest DNA Cloning DNA Biostoning Biostoning * Waste, pollution, quality variability, and imperfections are all reduced. Unlike pumice or acid, which get used up during the wash, enzymes can be recycled. • A small dose of enzymes can replace several dozen pounds of pumice stones. So productivity can be increased by 30­50% because the room formerly taken up by the pumice stones in the washing machines can now be filled with more jeans. * There is no need for the time­consuming and expensive task of removing stone fragments from the jeans once the wash is done. * There is also no pumice dust to endanger employee health or gritty sediment to clog drains. Restriction Enzymes Restriction Enzymes • One of main tools used for cloning • Made by bacteria to digest foreign DNA • Specific enzymes that cut both DNA strands at precise points within restriction site • Restriction sites usually 4 to 8 nucleotides • Enzymes will cut to result in restriction fragments Cutting and Pasting DNA with restriction enzymes enzymes Cutting and Pasting DNA with restriction enzymes Cutting and Pasting DNA with restriction enzymes Bacterial Plasmids Bacterial Plasmids • A type of cloning vector: a DNA molecule that can carry foreign DNA into a host cell and replicate there • Easily isolated • Easily manipulated to form recombinant plasmids in vitro bacteria • Multiply rapidly due to high reproductive rate of A plasmid is separate and replicates independently A plasmid is separate and replicates independently of chromosomal DNA DNA Cloning DNA DNA Cloning DNA Cloning DNA DNA Cloning DNA Applications of DNA Technology Applications of DNA Technology • Production of insulin • Genetically modified foods • Farm and “Pharm” animals Production of Humulin Production of Humulin • World’s first genetically engineered pharm product • Produced by bacteria that express the human insulin gene • Produced in large vats • Used by people with diabetes who can’t produce their own insulin Vats with insulin producing bacteria Vats Glowing Fish Glowing Genetically Modified Crops Genetically Modified Crops • DNA technology is replacing traditional breeding programs • Genetic engineering can create plants with exact desired characteristics very rapidly • In US, approx. 50% of corn crop and 75% of soybean and cotton crops are GM’d Genetically Modified Corn Genetically Genetically Modified Rice Genetically “Pharm” Animals • Transgenic farm animals engineered to produce proteins to benefit humans • Transgenic animals are harder to make than transgenic plants • Transgenic animals used only to make useful proteins, not for food Genetically Modified Sheep Sheep A Genetically Modified Swine Genetically Transgenic Rubber Trees yields human Transgenic Rubber Trees yields human protein in sap • Albumin is a vital protein needed in huge quantities to drip into the bloodstreams of accident and surgery victims • Great new use for the rubber. You can get continual production just by tapping rubber trees • Plants require little more than sunlight, water and basic horticultural input to survive. As protein manufacturing factories, plants are solar­ powered and ecologically friendly • The albumin gene has been introduced into the tree's DNA sequence. 70% of the sticky liquid tapped from the tree consists of the protein DNA Fingerprinting and Forensic DNA Fingerprinting and Forensic Science • The analysis of DNA fragments to determine who they come from • Applications include identification of murderer in crime scene; paternity; evolution research • Techniques include: PCR; STR analysis; gel electrophoresis Cheddar Man and his descendant Cheddar DNA fingerprinting in solving a crime case case The Polymerase Chain Reaction (PCR) The Polymerase Chain Reaction (PCR) • Method for amplifying DNA found in small amounts • Can make billions of copies of a target segment in a few hours • PCR is a three step cycle that produces an exponential increase in identical DNA molecules • Involves cycles of heating and cooling DNA Amplification by PCR by Need: DNA of interest Nucleotides DNA polymerase Short Tandem Repeat Analysis Short Tandem Repeat Analysis • STRs are short, noncoding DNA segments that are repeated in tandem • The number of repeats varies between individuals • STRs are used as genetic markers • STR analysis usually compares 13 STRs Short Tandem Repeat (STR) Sites Short Visualizing STR Fragment Patterns Visualizing Gel Electrophoresis of DNA Molecules Gel Visualization of DNA in an EB gel under UV light Visualization Genomics and Proteomics Genomics and Proteomics • Genomics is the science of studying whole genomes • Proteomics is the study of proteins encoded by genomes • Genomics and proteomics enable scientists to approach study of life from a more holistic perspective than ever before The Human Genome Project The Human Genome Project • HGP started in 1990 and was completed in 2004 • Human genome consists of 3.2 billion nucleotide pairs and about 20,000 genes mostly unknown • Approx. 98% is noncoding DNA, function of which is • Huge potential benefits of having a complete map of human genome, e.g. mapping of disease­associated genes Human Gene Therapy Human Gene Therapy • This procedure involves inserting ‘healthy’ genes into an individual for therapeutic purposes • Holds great potential for treating conditions caused by single gene • Still little evidence of safe and effective gene therapy Gene Therapy Gene Therapy • Is a technique for correcting defective genes responsible for disease development • • • • Several approaches: Normal gene inserted randomly into genome Abnormal gene swapped for normalgene Regulation of particular gene can be altered Methods of Gene Delivery Methods of Gene Delivery • Viruses • Liposomes as delivery vectors • Introducing a 47th chromosome Current status of Gene Therapy Current status of Gene Therapy • • FDA has not approved any gene therapy product for sale Trials bean in 1990. Setbacks: failure due to severe immune response to adenovirus carrier one of them died • 1999­Death of 18yr old Jesse Gelsinger. Multiple organ • 2000­2006­3 kids treated for SCID developed leukemia, Problems with Gene Therapy Problems with Gene Therapy • Short­lived nature. Patients need to undergo multiple rounds of gene therapy • Immune response to introduction of foreign object • Viral vector could recover ability to cause disease • Many of the most common disorders are result of mutations in several genes. Best candidates are diseases caused by single gene Gene Therapy Gene SCID SCID • Severe combined immunodeficiency, ‘bubble boy’ disease • Rare, 1/200,000 births • It was fatal until recent years • Several underlying genetic mutations but all have the same effect, an individual who lacks almost all immune defenses SCID SCID • Treatments: • Bone marrow and cord blood transplants, only known cure • Gene therapy, has been successful in treating X­SCID but still experimental stages Bubble Boy Bubble Boy Bubble Boy Bubble Boy Gene Therapy to treat SCID Gene Therapy to treat SCID Safety and Ethical Issues Safety and Ethical Issues • Strict lab safety guidelines developed for working with recombinant DNA technology survive outside the lab • Microorganisms used are made so they won’t • Applications of biotech evaluated for potential risks by several regulatory agencies • Concern about GM foods (mainly plants) Genetically Modified Foods Genetically Modified Foods Comes from plants that have had their DNA modified in the lab to enhance desired traits Faster and more accurate than traditional breeding programs In US 60% of all processed foods contains GM ingredients Controversial: introduction of allergens, creation of ‘superweeds’ No convincing evidence that GM foods pose a real health threat Figure 12.25 Figure Ethical Questions Ethical Questions • Do we have the right to examine somebody else’s genetic information? • How should the information be used? • Should DNA ‘defects’ be corrected? Summary Summary • Recombinant DNA technology enables scientists to isolate and clone DNA of interest in order to study its function enzymes and cloning vector • DNA cloning involves use of restriction • Cloning a gene is a method of making many copies of gene of interest Summary • PCR as an alternative way to make many copies of gene of interest when starting material is scarce • Gel electrophoresis to separate nucleic acids or proteins dependent on size Summary Summary • Practical applications of DNA technology affect our every day lives agriculture, environment • Applications in the fields of medicine, • Safety and ethical concerns Recombinant DNA Techniques Techniques ...
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This note was uploaded on 04/25/2011 for the course BIO 155 taught by Professor Skoubis during the Fall '10 term at DePaul.

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