Cloning <ul><li>cloning  – introducing a foreign DNA segment into an organism </li></ul><ul><ul><li>when the host organism...
Why Use Organisms to Clone? <ul><li>All enzymes available to replicate DNA. </li></ul><ul><ul><li>plasmid has an origin of...
Cloning Applications <ul><li>Transgenic plants </li></ul><ul><li>Plants cells can also take in bacterial plasmids. </li></...
 
Kary Mullis <ul><li>developed the PCR process in 1986 </li></ul><ul><li>Nobel Laureate, 1993 </li></ul>
Polymerase Chain Reaction <ul><li>PCR  – a synthetic method (no living cells) to produce millions of copies of DNA </li></...
in vitro  and  in vivo  DNA Amplification DNA amplification Advantage of system in vivo Definition in vitro System
in vitro  and  in vivo  DNA Amplification DNA amplification Advantage of system Inside natural environment (cellular) in v...
in vitro  and  in vivo  DNA Amplification Bacterial cell  transformed with DNA PCR DNA amplification Advantage of system I...
in vitro  and  in vivo  DNA Amplification Bacterial cell  transformed with DNA PCR DNA amplification few errors in replica...
Three Steps for each PCR Cycle <ul><li>DNA strand denaturation (95 °C) </li></ul><ul><ul><li>separate double stranded DNA ...
Problem <ul><li>Where do you find enzymes that don’t break down at 95 °C? </li></ul><ul><li>Thermus   aquaticus   bacteriu...
McGraw Video Clip <ul><li>http://highered.mcgraw-hill.com/olc/dl/120078/micro15.swf </li></ul>
 
 
PCR Efficiency <ul><li>After 30 cycles, 2 30  (more than a billion) copies of DNA can be produced. </li></ul><ul><li>30 cy...
PCR Applications <ul><li>Genetic Screening </li></ul><ul><li>Early detection for certain diseases. </li></ul><ul><li>Foren...
PCR clip <ul><li>http://www.sumanasinc.com/webcontent/animations/content/pcr.html </li></ul>
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14 cloning pcr-2010-stacy update

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14 cloning pcr-2010-stacy update

  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 Use 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. 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
  4. 6. Kary Mullis <ul><li>developed the PCR process in 1986 </li></ul><ul><li>Nobel Laureate, 1993 </li></ul>
  5. 7. 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>
  6. 8. in vitro and in vivo DNA Amplification DNA amplification Advantage of system in vivo Definition in vitro System
  7. 9. in vitro and in vivo DNA Amplification DNA amplification Advantage of system Inside natural environment (cellular) in vivo Outside of natural environment Definition in vitro System
  8. 10. in vitro and in vivo DNA Amplification Bacterial cell transformed with DNA PCR DNA amplification Advantage of system Inside natural environment (cellular) in vivo Outside of natural environment Definition in vitro System
  9. 11. in vitro and in vivo DNA Amplification Bacterial cell transformed with DNA PCR DNA amplification few errors in replication due to proofreading [thus limitation to the number of times PCR cycle can be repeated] DNA can be in a partially degraded state and still be amplified Advantage of system Inside natural environment (cellular) in vivo Outside of natural environment Definition in vitro System
  10. 12. 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>
  11. 13. 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>
  12. 14. McGraw Video Clip <ul><li>http://highered.mcgraw-hill.com/olc/dl/120078/micro15.swf </li></ul>
  13. 17. 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>
  14. 18. 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>
  15. 19. PCR clip <ul><li>http://www.sumanasinc.com/webcontent/animations/content/pcr.html </li></ul>

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