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Download III. Biotechnology
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Biotechnology Altering an organism's genetic code so that it produces desired protein A. A Brief History Lesson 1. Artificial Selection The breeding of plants and animals to produce offspring with desired traits has been practiced for thousands of years. For example, faster horses, cows that produce more milk and plants with higher yield. 2. Contributions of Salvador Luria -1950 a) Early biochemistry work was conducted on organisms with small genomes like E. coli and viruses that prey upon them b) A plate with nutrient agar would be inoculated with bacteria and they would be allowed to grow until they covered the plate c) Later, phages were added and they would attack and kill the bacteria leaving empty spots on the plate called plaques Salvador Luria’s Observation and Hypothesis d) In 1950, Luria observed some bacteria that were unaffected when exposed to phages e) Luria hypothesized that these bacteria had some type of primitive immune system that restricted phage growth 3. Bacteria Evolved Restriction Enzymes a) In order to reproduce, viruses must attach to a host cell Phage Viral DNA Host Cell Restriction Enzyme Host Cell DNA b) The virus then injects it’s DNA into the host cell How Restriction Enzymes Protect Bacteria c) Restriction enzymes bind with the viral DNA at specific base sequences called recognition sites d) The viral DNA is cut at specific sites called restriction sites which destroys it and protects the bacteria from infection 4. Contributions of Werner Arber, Daniel Nathans and Hamilton Smith In 1978 Arber, Nathans and Smith won the Nobel prize for isolating the first restriction enzyme 5. Restriction Enzymes- enzymes that cut DNA at a specific base sequence 6. Naming Restriction Enzymes EcoR I E BamH I genus Echericia B genus Co species coli am species amyloliquefaciens R Strain R H Strain H I Order found 1st I Order found 1st Hind III H genus Haemophilous in species influenzea d d Strain III Order found 3rd Bacillus B. Contributions Daniel Nathans and Joseph Sambrook 1. Nathans realized that DNA has a negative charge and therefore restriction fragments could be separated using an electric current 2. In 1970, Nathans used a polyacrylamide gel to separate DNA restriction fragments by size 3. Sambrook introduced the use of agarose gel and ethidium bromide staining used today 4. Gel Electrophoresis- separating cut DNA fragments by size using an electric current C. Bacterial Transformation 1. Experiments of Frederick Griffith 2. Griffith was a British army doctor trying to find a vaccine against Streptococcus pneumonie, a bacterium that causes pneumonia in mammals. He knew 3 things: a) There are two strains of the bacteria: one produces smooth colonies, S, and one that produces rough colonies, R. b) Cells of the smooth strain are covered with a polysaccharide coat and the rough strains are not. c) The alternative phenotypes (S&R) are inherited 3. Griffith preformed 4 experiments Griffith injected live S strain into mice. Results: Conclusion: The S strain is pathogenic Griffith injected mice with live R strain. Results: Conclusions: R strain is non pathogenic Mice were injected with heat killed S strain Results: Conclusions: Heat killed bacteria are non pathogenic Then for some reason known only to Griffith, he decided to inject Heat killed S and live R were injected into mice Results: Conclusion: The R strain acquired instructions to make the polysaccharide coat from the dead S strain Not only did the mouse die, live S strain was extracted from its body. Griffith could not explain what had happened, but he said the bacteria had been transformed We now call the assimilation of external genetic material by another cell Transformation 4. Morton Mandel and Akiko Higa a) Discovered a method in 1970 to make bacterial cell competent by treating them with calcium ions. b) Used a method involving a rapid change in temperature called heat shock D. Herbert Boyer & Stanley Cohen collaboration occurred in spring 1973 1. In 1973 Herbert Boyer, and Stanley Cohen constructed the first functional organism that combined and replicated genetic information from H. Boyer different species. 2. The science of genetic engineering was born S. Cohen E. Other Techniques 1. Dr. Kary Mullis won the 1993 Nobel Prize for the invention of PCR- Polymerase Chain Reactionallows specific DNA fragments to be copied millions of times 2. RFLPs- Restriction Fragment Length Polymorphism a) Used to identify DNA when a mutation adds or deletes a restriction site b) Gel electrophoresis separates the DNA fragments and mutations are identified by an abnormal number of fragments 3. VNTRs & STRPs- similar to RFLP analysis, but uses highly variable, noncoding sequences of DNA 4. DNA Sequencing a) A piece of DNA with an unknown sequence is placed in 4 Eppendorf tube containing primase, nucleotides, and DNA polymerase. Dideoxynucleotides (ddA, ddT, ddC & ddG) are added to different tubes. b) In the tube that contain ddT, sometimes deoxythymine will be added, but sometimes ddT will be added. Because dideoxynucleotides prevents the addition of the next base, the chain will be terminated c) The result will be fragments of DNA of various lengths. One corresponding to each base in the chain d) The tubes are incubated and the products are run through a gel which results in fragments which can be read dideoxynucleotides The resulting nucleotide sequence is complimentary to the unknown DNA sequence 5. Microinjection- thin needles insert DNA into cells 6. Synthetic Organisms Self replicating organisms whose genome is entirely constructed by man Craig Venter’s Institute manufactured the 1st entirely synthetic organisms on the 21st of May of 2010 F. Uses of Recombinant DNA Technology 1) Pharmaceuticals- Humilin, TPA, interferon, TNF, Artificial hemoglobin, human growth hormone 2) Agriculture- incide, Flavr-saver tomatoes, frost resistance, salt resistance, insect resistance, herbicide resistance, nitrogen fixation 3) Forensics- DNA finger printing 4) Medical- gene therapy G.. Dangers of Genetic Engineering 1. Pathogens- disease causing organisms 2. Eugenics- should we control or alter our own genome? 3. Stem Cells- growing new human tissues from cell derived from fertilized eggs 4. Legal Questions- can/should we patent life? 5. Genetic Screening- who would get the results of the tests and how could test results be used? 6. GMOs- Genetically modified organisms