13a biotech techniques

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13a biotech techniques

  1. 1. What can you do with DNA?
  2. 2. Biotechnology biotechnology – manipulation of biological organisms (usually with DNA itself) To study the functions of individual genes, molecular biologists will cut them out of a genome and place them into bacteria.
  3. 3. Basic Techniques Six basic techniques will be covered: 1. 2. 3. 4. 5. 6. DNA isolation restriction enzyme digest PCR gel electrophoresis sequencing gene cloning
  4. 4. Isolating DNA Before DNA can be manipulated, it needs to be isolated from the cells. 1. Cell membranes are disrupted  use a detergent 1. DNA precipitation  ethanol used to dehydrate and aggregate DNA 1. DNA isolation / storage
  5. 5. pp 377 - 378
  6. 6. Restriction Enzymes DNA must be cut into smaller pieces before they can be used in other techniques.
  7. 7. Restriction Enzymes restriction endonucleases – enzymes that cut DNA backbones at specific sequences through hydrolysis recognition site – the DNA sequence which restriction enzymes bind to unsure if enzymes scan DNA to find sequences
  8. 8. Restriction Enzymes Why are these enzymes used? These enzymes are naturally found in bacteria. Bacteria found to be resistant to some bacteriophage (bacterial virus). Restriction enzymes would cut viral DNA, not its own genome.
  9. 9. Restriction Enzymes Restriction enzymes cut at recognition sites. Recognition sites are typically 4 to 8 bp in length. They are always palindromic. 5’ G A A T T C 3’ 3’ C T T A A G 5’ This sequence is specific for the EcoRI enzyme.
  10. 10. Restriction Enzymes Restriction enzymes are named according to the organism from which it was identified. Ex. EcoRI E co R I - Escherichia - coli - strain RY13 - 1st enzyme in this strain
  11. 11. Restriction Enzymes Bacillus amyloliquefaciens, strain H, 5th endonuclease identified BamHV Nocardia otitidis, 3rd endonuclease identified NotIII
  12. 12. Restriction Enzymes sticky ends – enzyme digests (cuts) to make overhangs EcoRI 5’ G A A T T C 3’ 3’ C T T A A G 5’ 5’ G 3’ 5’ A A T T C 3’ 3’ C T T A A 5’ 3’ G 5’ 5’ overhang
  13. 13. Restriction Enzyme Cutting PstI 5’ C T G C A G 3’ 3’ G A C G T C 5’ 5’ C T G C A 3’ 5’ G 3’ 3’ G 5’ 3’ A C G T C 5’ 3’ overhang
  14. 14. Restriction Enzyme Cutting blunt ends – enzyme digests to make straight ends SmaI 5’ C C C G G G 3’ 3’ G G G C C C 5’ 5’ C C C 3’ 3’ G G G 5’ 5’ G G G 3’ 3’ C C C 5’
  15. 15. DNA Ligase T4 DNA ligase – used to chemically join two pieces of DNA together
  16. 16. Recombinant DNA Complementary sticky ends from different pieces of DNA can be joined together – recombinant DNA
  17. 17. Recombinant DNA Animation

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