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Genetic Engineering (and other cool molecular biology techniques)

Genetic Engineering (and other cool molecular biology techniques). DNA Cloning. DNA cloning permits production of multiple copies of one particular gene

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Genetic Engineering (and other cool molecular biology techniques)

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  1. Genetic Engineering (and other cool molecular biology techniques)

  2. DNA Cloning • DNA cloning permits production of multiple copies of one particular gene • This gene copy will be used to produce a usable protein product (e.g. insulin) or for genetic engineering (e.g. gene for pest control inserted into plant)

  3. For Fun: • We want to clone a gene that produces a protein that will help you focus while studying.

  4. 1. We need the gene • This gene is located on chromosome 16

  5. 2. We need a vector • Vector – a DNA molecule (usually a bacterial plasmid) that is specifically designed to carry foreign DNA • Selectable marker • MCS

  6. 3. Restriction Enzymes • Restriction Enzymes – cut DNA at particular sequences • Ex: EcoR1 – recognizes GAATTC • 100’s of RE’s

  7. Transformation • Get “competent” bacteria (usually E.colithat is capable of being transformed) • Treat those cells to calcium chloride • Heat shock to force bacterial cell to take up plasmid DNA

  8. Now… • Select for bacteria that have taken up your plasmid (usually use antibiotic) • Grow bacteria in large quantities, purify out protein, and…

  9. PCR (polymerase chain reaction) • Specific sequence of DNA is amplified (copied many times) • Requires: • DNA template (contains your gene of interest) • Tac polymerase (a DNA polymerase that can work at high temperatures) • Nucleotides (to synthesize new DNA) • Primers (specific to the gene of interest)

  10. PCR is a cyclic process: • Heat to denature (open up) DNA • Cool to allow primers to anneal (stick to) single stranded DNA • Warm to allow DNA to be replicated by polymerase • REPEAT

  11. Gel Electrophoresis • Uses a gel as a molecular sieve to separate pieces of DNA based on size • Smaller pieces travel further than larger pieces

  12. Let’s discuss some examples • Genetically modified food • Transgenic animals • Pharmaceuticals

  13. 1. Genetically Modified Food • Insert particular genes within a type of food to make it better tasting, longer lasting, or more healthy. • Long term effects of consumption of this food are unknown.

  14. 2. Transgenic Animals • Research purposes • Knock outs • Overexpression • Use of GFP to select for animals that are transgenic • Consumer purposes (food or medication)

  15. 3. Pharmaceuticals • Use plants or other GMO to produce pharmaceuticals • Ex: Gaucher’s disease (lysosomal storage disease – buildup of fat in cells); Pfizer received patent to grow drug to treat this disease in tobacco plant • Ex: Use of a transgenic goat to produce an anticoagulant (in the goat milk) to reduce probability of blood clots • Use bacteria to produce insulin, human growth hormone, clotting factors for hemophaelics • GMO mammals (mice) used in pharmaceutical research labs to study disease and treatment

  16. Your Homework: • Read the entire laboratory handout. You must carefully read the background information AND the laboratory procedure as well. • Answer the pre-lab questions.

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