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Recombinant DNA

Recombinant DNA. Purpose: To ___________of one organism into another organism. Recombinant DNA Applications. Green-glowing aquarium fish (jellyfish genes) Fast-growing fish (Salmon with Pout genes) Herbicide-_____________ crops (Round-Up Ready). _____-resistant crops (__ toxin).

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Recombinant DNA

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  1. Recombinant DNA • Purpose: To ___________of one organism into another organism

  2. Recombinant DNA Applications • Green-glowing aquarium fish (jellyfish genes) • Fast-growing fish (Salmon with Pout genes) • Herbicide-_____________ crops (Round-Up Ready)

  3. _____-resistant crops (__ toxin)

  4. Recombinant DNA Applications • _________ production (bacteria can make insulin, hGH, etc)

  5. Making Recombinant Bacteria • Important players: • ________ of interest (i.e. insulin, hGH, etc) • ______________ enzymes – cut the DNA • ___________ – circular DNA found in bacteria • ________________

  6. What are Restriction Enzymes? • Restriction enzymes: • Target very ___________ ______ sequences • Are found in more than 100 different varieties • Are used in nature to ______________ bacteria from foreign invaders

  7. What are Restriction Enzymes? • Each restriction enzyme recognizes a very specific nucleotide _______ EcoR1 recognizes: GAATTC CTTAAG The enzyme cuts it:G AATTC CTTAA G

  8. Making Recombinant DNA • Plasmids - self-replicating rings of _______ containing 2-30 genes, found in bacterial cells

  9. Making Recombinant DNA • __________ the gene of interest using a restriction enzyme • Cut the __________ (using the same enzyme) • __________ gene into the plasmid • Insert the plasmid into _________ • Grow bacteria and harvest the ____________

  10. Making a paper bracelet • You will need: • A small circle of paper (this is your plasmid) • A strip of paper in a different color about 5 inches long. In the center is a 1-2 inch piece that is your gene of interest. • Cut out the gene with your scissors. • Cut the circle once and using a glue stick or tape, add your gene to the open circle to form a closed circle.

  11. 1. Isolating the gene • Use restriction enzymes to cut the DNA strand at specific places that surround the gene

  12. 2. Cut the plasmid • Cut the plasmid using the __________ restriction enzyme

  13. 3. Put the gene in the plasmid • Since the gene and the plasmid have the same ______________ ________, the gene will stick to the plasmid

  14. 5/6. Put plasmid in bacteria • Insert plasmid into bacteria • Let bacteria make the protein

  15. Cloning a gene: • http://highered.mcgraw-hill.com/sites/0072437316/student_view0/chapter16/animations.html#

  16. DNA Fingerprinting • Definition: Creating a __________ _______ ______________ for someone • Purpose: • Forensic science • Determining paternity • Research • Diagnosing Disease

  17. Gel Electrophoresis Analogy(DO NOT COPY THIS DOWN) "Imagine a huge swimming pool full of water and many entangled nets (this is the agarose gel). A dump truck comes along and dumps its load into one end of the pool. That load contains an extensive collection of long, skinny things of varying lengths and sizes (analogous to DNA of varying sizes), e.g. from boa constrictors to smaller snakes to worms to microscopic bacteria. There is a vacuum at the other end of the pool (analogous to the electric current), which can be turned on and off. When on, the vacuum pulls all of the creatures across the pool. But because of the netting, movement of the skinny things is impeded."

  18. Gel Electrophoresis • Separates DNA fragments by _________ using ______________ current

  19. Gel Electrophoresis • Larger fragments move more _________ • Bands of fragments result

  20. DNA Fingerprinting Method • Use restriction enzymes to cut the DNA • Load the DNA onto agarose gel for gel electrophoresis • Analyze the banding pattern

  21. Gel Electrophoresis

  22. Gel Electrophoresis

  23. RFLP Analysis RFLP – Restriction Fragment Length Polymorphism: for related DNA molecules, a difference in DNA fragment sizes after restriction enzyme digestion • Difference results from presence of different DNA sequences • Certain regions of genome are highly variable

  24. A single nucleotide change can make a difference Wild-type allele AGATCT TCTAGA Restriction site Mutant allele AGAGCT TCTCGA Not a restriction site

  25. No cut site = 1 long DNA fragment RFLP Cut site present = 2 shorter DNA fragments

  26. NOVA: Cracking the Code - #9: Finding Cures is Hard http://www.pbs.org/wgbh/nova/genome/program.html

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