13 biotech techniques-stacy-2010 update

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13 biotech techniques-stacy-2010 update

  1. 2. Biotechnology <ul><li>biotechnology – manipulation of biological organisms (usually with DNA itself) </li></ul><ul><li>To study the functions of individual genes, molecular biologists will cut them out of a genome and place them into bacteria. </li></ul>
  2. 4. Isolating DNA <ul><li>Before DNA can be manipulated, it needs to be isolated from the cells. </li></ul><ul><li>Cell membranes are disrupted </li></ul><ul><ul><li>use a detergent </li></ul></ul><ul><li>DNA precipitation </li></ul><ul><ul><li>ethanol used to dehydrate and aggregate DNA </li></ul></ul><ul><li>DNA isolation / storage </li></ul>
  3. 6. Manipulating DNA <ul><li>restriction endonucleases – enzymes that cut DNA backbones at specific sequences through hydrolysis </li></ul><ul><li>recognition site – the DNA sequence which restriction enzymes bind to </li></ul><ul><ul><li>unsure if enzymes scan DNA to find sequences </li></ul></ul>
  4. 7. Why Restriction Enzymes? <ul><li>These enzymes are naturally found in bacteria. </li></ul><ul><li>Bacteria found to be resistant to some bacteriophage. </li></ul><ul><li>Restriction enzymes would cut viral DNA, not its own genome. </li></ul>
  5. 8. Recognition Sites <ul><li>Recognition sites are typically 4 to 8 bp in length. They are always palindromic. </li></ul><ul><li>Nucleic acid is the same whether you read it from the 5’-3’ or 3’-5’ end. </li></ul><ul><li>This sequence is specific for the Eco RI enzyme. </li></ul>5’ G A A T T C 3’ 3’ C T T A A G 5’
  6. 9. Restriction Enzymes <ul><li>Restriction enzymes are named according to the organism from which it was identified. </li></ul><ul><li>Ex. Eco RI </li></ul><ul><li>E - E scherichia </li></ul><ul><li>co - co li </li></ul><ul><li>R - strain R Y13 </li></ul><ul><li>I - 1 st enzyme in this strain </li></ul>
  7. 10. Restriction Enzymes <ul><li>Bacillus amyloliquefaciens , strain H, 5 th endonuclease identified </li></ul><ul><li>Nocardia otitidis , 3 rd endonuclease identified </li></ul>BamHV NotIII
  8. 11. Restriction Enzyme Cutting <ul><li>sticky ends – enzyme cuts to make overhangs </li></ul><ul><li>Eco RI 5’ G A A T T C 3’ 3’ C T T A A G 5’ </li></ul><ul><li>5’ G 3’ 5’ A A T T C 3’ </li></ul><ul><li>3’ C T T A A 5’ 3’ G 5’ </li></ul><ul><li>5’ overhang </li></ul>
  9. 13. Restriction Enzyme Cutting <ul><li>Pst I 5’ C T G C A G 3’ </li></ul><ul><li>3’ G A C G T C 5’ </li></ul><ul><li>5’ C T G C A 3’ 5’ G 3’ </li></ul><ul><li>3’ G 5’ 3’ A C G T C 5’ </li></ul><ul><li>3’ overhang </li></ul>
  10. 14. Restriction Enzyme Cutting <ul><li>blunt ends – enzyme cuts to make straight ends </li></ul><ul><li>Sma I 5’ C C C G G G 3’ </li></ul><ul><li>3’ G G G C C C 5’ </li></ul><ul><li>5’ C C C 3’ 5’ G G G 3’ </li></ul><ul><li>3’ G G G 5’ 3’ C C C 5’ </li></ul>
  11. 15. Recombinant DNA <ul><li>Complementary sticky ends from different pieces of DNA can be joined together – recombinant DNA </li></ul>
  12. 16. DNA Ligase <ul><li>T4 DNA ligase – used to chemically join two pieces of DNA together </li></ul>
  13. 18. DNA Methylation <ul><li>Inevitably, the organism which creates the restriction enzyme will also have, in its genome, the sequence which will be cut. </li></ul><ul><li>methylases (methyltransferases) – a methyl functional group is added to the nitrogen base of a nucleotide </li></ul><ul><ul><li>REs do not recognize methylated bases </li></ul></ul><ul><ul><li>methyl groups can be used by the scientist to protect DNA regions from being cut </li></ul></ul>
  14. 19. Restriction Enzymes <ul><li>http://highered.mcgraw-hill.com/olc/dl/120078/bio37.swf </li></ul><ul><li>http://highered.mcgraw-hill.com/olc/dl/120078/micro10.swf </li></ul>

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