15a cloning pcr-2011-successful cloning

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15a cloning pcr-2011-successful cloning

  1. 2. Cloning <ul><li>cloning – introducing a foreign DNA segment into an organism </li></ul><ul><ul><li>when the host organism replicates, copies of the DNA are also made </li></ul></ul><ul><ul><li>in vivo </li></ul></ul><ul><li>Plasmids are used to transport DNA into bacteria. </li></ul>
  2. 3. Why Organisms to Clone? <ul><li>All enzymes available to replicate DNA. </li></ul><ul><ul><li>plasmid has an origin of replication </li></ul></ul><ul><li>All enzymes available for transcription. </li></ul><ul><ul><li>plasmid has signals for starting and stopping transcription </li></ul></ul><ul><li>All enzymes available for protein synthesis. </li></ul><ul><ul><li>scientists usually interested in examining protein function </li></ul></ul>
  3. 4. Steps to Cloning <ul><li>Creation of recombinant DNA plasmid. </li></ul><ul><li>Bacterial transformation. </li></ul><ul><li>Screening for successful clones. </li></ul>
  4. 5. 1. Recombinant DNA Insert new sequence into Eco RI site.
  5. 6. 2. Bacterial Transformation <ul><li>Two main methods to help bacteria pick up plasmid: </li></ul><ul><ul><li>cold CaCl 2 treatment followed by heat-shocking </li></ul></ul><ul><ul><ul><li>makes cell membrane “leaky” and more permeable </li></ul></ul></ul><ul><ul><li>electroporation </li></ul></ul><ul><ul><ul><li>electric current to increase cell permeability </li></ul></ul></ul>
  6. 7. 3. Screening <ul><li>Screening is necessary as success is never 100% for all bacteria available. </li></ul><ul><li>Two main methods of screening: </li></ul><ul><ul><li>antibiotic resistance </li></ul></ul><ul><ul><li> -galactosidase activity </li></ul></ul>
  7. 8. a) Antibiotic Resistance
  8. 9. a) Antibiotic Resistance <ul><li>Bacteria are grown on Petri plate containing a specific antibiotic. </li></ul><ul><li>Only bacteria which accepted the new plasmid will grow. </li></ul>
  9. 10. b)  -galactosidase Screening <ul><li>plasmids contain  -lactamase gene </li></ul><ul><li> -lactamase acts on X-Gal (a chemical) to produce a blue precipitate </li></ul>
  10. 11. b)  -galactosidase Screening <ul><li>Bacteria are grown on Petri plates containing X-Gal. </li></ul><ul><li>Bacteria which accepted the new plasmid will be blue in colour. </li></ul>
  11. 12. Cloning Applications <ul><li>Transgenic plants </li></ul><ul><li>Plants cells can also take in bacterial plasmids. </li></ul>Flavr Savr Tomatoes Bt toxin – natural herbicide
  12. 14. Kary Mullis <ul><li>developed the PCR process in 1986 </li></ul><ul><li>Nobel Laureate, 1993 </li></ul>
  13. 15. Polymerase Chain Reaction <ul><li>PCR – a synthetic method (no living cells) to produce millions of copies of DNA </li></ul><ul><ul><li>use enzymes in a tube - in vitro </li></ul></ul><ul><li>Minimum requirements for DNA polymerase: </li></ul><ul><ul><li>template strand </li></ul></ul><ul><ul><li>primers </li></ul></ul><ul><ul><li>dNTPs </li></ul></ul>
  14. 16. Three Steps for each PCR Cycle <ul><li>DNA strand denaturation (95 °C) </li></ul><ul><ul><li>separate double stranded DNA </li></ul></ul><ul><ul><li>each strand becomes template strand </li></ul></ul><ul><li>Primer annealing (50 °C - 65°C) </li></ul><ul><ul><li>bind short DNA pieces to template strands </li></ul></ul><ul><li>DNA strand synthesis (72 °C) </li></ul><ul><ul><li>produce new DNA strands </li></ul></ul>
  15. 17. Problem <ul><li>Where do you find enzymes that don’t break down at 95 °C? </li></ul><ul><li>Thermus aquaticus bacterium live in hot springs and their enzymes are designed to withstand extreme temperatures. </li></ul><ul><li>Isolated Taq polymerase from these bacteria. </li></ul>
  16. 20. PCR Efficiency <ul><li>After 30 cycles, 2 30 (more than a billion) copies of DNA can be produced. </li></ul><ul><li>30 cycles of PCR can take anywhere from 1 – 2 hours to complete. </li></ul>
  17. 21. PCR Applications <ul><li>Genetic Screening </li></ul><ul><li>Early detection for certain diseases. </li></ul><ul><li>Forensic Analysis </li></ul><ul><li>Small amounts of DNA collected to identify individuals. </li></ul>

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