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DNA Manipulation 2

DNA Manipulation 2. The nucleus. contains DNA. DNA. Restriction Digestion. Restriction enzymes are proteins that cut DNA at specific sequences. Have optimal condition of pH, temperature, ionic strength etc... (enzymes) Scan DNA until they find the sequence.

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DNA Manipulation 2

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  1. DNA Manipulation 2

  2. The nucleus contains DNA DNA

  3. Restriction Digestion • Restriction enzymes are proteins that cut DNA at specific sequences. • Have optimal condition of pH, temperature, ionic strength etc... (enzymes) • Scan DNA until they find the sequence. • http://www.youtube.com/watch?v=-sI5vy-cD2g

  4. DNA Hybridisation • DNA denaturation: Two DNA strands can be separated by heat without breaking phosphodiester bonds • DNA renaturation = hybridization: Two single strands that are complementary or nearly complementary in sequence can come together to form a different double helix • Single strands of DNA can also hybridize complementary sequences of RNA

  5. DNA Hybridisation

  6. Gene Cloning • Inserting new DNA into another piece of DNA. • Most commonly used with bacterial plasmids which are easily manipulated. • Usually used antibiotic resistance genes to select for cloned DNA. • Recombination: • http://www.youtube.com/watch?v=8rXizmLjegI&feature=relmfu

  7. Example of a cloning vector • What is a vector? • A piece of DNA that can be replicated (usually a plasmid) that cloned DNA can be inserted into.

  8. Cloning steps

  9. Gene Delivery • Gene delivery depends on the organism... • Bacteria: Plasmids, electroporation, conjugation or bacteria phage. • Yeast: Yeast artificial chromosomes • Plants: Agrobacterium or gene gun • Animal:Electroporation, gene gun, transfection reagents (lipid balls) or viral vectors.

  10. Delivering genes to Plants

  11. The RoundUp Ready Story • Glyphosate is a broad-spectrum herbicide • Active ingredient in RoundUp herbicide • Kills all plants it come in contact with • Inhibits a key enzyme (EPSP synthase) in an amino acid pathway • Plants die because they lack the key amino acids • A resistant EPSP synthase gene allows crops • to survive spraying

  12. Shikimic acid + Phosphoenol pyruvate Plant EPSP synthase 3-Enolpyruvyl shikimic acid-5-phosphate (EPSP) Aromatic amino acids RoundUp Sensitive Plants + Glyphosate X X Without amino acids, plant dies X X

  13. RoundUp Resistant Plants Shikimic acid + Phosphoenol pyruvate + Glyphosate RoundUp has no effect; enzyme is resistant to herbicide Bacterial EPSP synthase 3-enolpyruvyl shikimic acid-5-phosphate (EPSP) With amino acids, plant lives Aromatic amino acids

  14. Delivering genes to Animals Lentivirus Liposomes (Lipids) Adenovirus Gene Gun

  15. Transgenic animals

  16. Cloned Genes to make therapies Human insulin produced in bacteria....

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