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How PCR works - PowerPoint PPT Presentation

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How PCR works

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  1. How PCR works • Cold Spring Harbor Animation • Animated .GIF files • Blackboard

  2. Review: The structure of DNA Unzipping Antiparallel Strands

  3. How PCR works • Cold Spring Harbor Animation PCR.EXE

  4. How PCR works • Animated .GIF #1

  5. How PCR works • Animated .GIF #2

  6. How PCR works • Blackboard (Dave attempts to draw this out!)

  7. PCR Reaction Components • Water • Buffer • DNA template • Primers • Nucleotides • Mg++ ions • DNA Polymerase

  8. PCR Reaction: Water • Water • The medium for all other components.

  9. PCR Reaction: Buffer • Water • Buffer • Stabilizes the DNA polymerase, DNA, and nucleotides • 500 mM KCl • 100 mM Tris-HCl, pH 8.3 • Triton X-100 or Tween

  10. PCR Reaction: Template DNA • Water • Buffer • DNA template • Contains region to be amplified • Any DNA desired • Purity not required • Should be free of polymerase inhibitors

  11. PCR Reaction: Primers • Water • Buffer • DNA template • Primers • Specific for ends of amplified region • Forward and Reverse • Annealing temps should be known • Depends on primer length, GC content, etc. • Length 15-30 nt • Conc 0.1 – 1.0 uM (pMol/ul)

  12. PCR Reaction: Nucleotides • Water • Buffer • DNA template • Primers • Nucleotides • Added to the growing chain • Activated NTP’s • dATP, dGTP, dCTP, dTTP • Stored at 10mM, pH 7.0 • Add to 20-200 uM in assay

  13. PCR Reaction: Magnesium • Water • Buffer • DNA template • Primers • Nucleotides • Mg++ ions • Essential co-factor of DNA polymerase • Too little: Enzyme won’t work. • Stabilizes the DNA double-helix • Too much: DNA extra stable, non-specific priming, band smearing • Used at 0.5 to 3.5 uM in the assay

  14. PCR Reaction: Polymerase • Water • Buffer • DNA template • Primers • Nucleotides • Mg++ ions • DNA Polymerase • The enzyme that does the extension • TAQ or similar • Heat-stable • Approx 1 U / rxn

  15. PCR Reaction Components • Water • Buffer • DNA template • Primers • Nucleotides • Mg++ ions • DNA Polymerase

  16. A Typical PCR Reaction Sterile Water 38.0 ul 10X PCR Buffer 5.0 ul MgCl2 (50mM) 2.5 ul dNTP’s (10mM each) 1.0 ul PrimerFWD (25 pmol/ul) 1.0 ul PrimerREV 1.0 ul DNA Polymerase 0.5 ul DNA Template 1.0 ul Total Volume 50.0 ul

  17. A Simpler PCR Reaction Sterile Water 38.0 ul 10X PCR Buffer 5.0 ul MgCl2 (50mM) 2.5 ul dNTP’s (10mM each) 1.0 ul PrimerFWD (25 pmol/ul) 1.0 ul PrimerREV 1.0 ul DNA Polymerase 0.5 ul DNA Template 1.0 ul Total Volume 50.0 ul PREMIX 24.0 ul Buffer MgCl2 dNTP’s DNA Polymerase “Enhancers” Sterile Water Primers FWD+Rev 1.0 ul DNA Template 25.0 ul Total Volume 50.0 ul PREMIXES CAN REDUCE THE NUMBER OF ITEMS ADDED TO THE MIX

  18. Using a PCR Mastermix Component 1X 20X Sterile Water 38.0 ul 760 ul 10X PCR Buffer 5.0 ul 100 ul MgCl2 (50mM) 2.5 ul 50 ul dNTP’s (10mM each) 1.0 ul 20 ul PrimerFWD (25 pmol/ul) 1.0 ul 20 ul PrimerREV 1.0 ul 20 ul DNA Polymerase 0.5 ul 10 ul DNA Template 1.0 ul -- Total Volume 50.0 ul 980 ul Aliquot 49 ul Add DNA as last step

  19. Typical Thermal Cycler Conditions 1. Initial Denaturation 95 C 4 min 2. DNA Denaturation 95 C 1 min 3. Primer Annealing 65 C 1 min 4. Primer Extension 72 C 1 min 5. Go to step #2, repeat 29 more times 6. Hold at 4 C 7. End

  20. “End” of PCR Introduction Let’s plan out the GMO kit reactions and set them up!