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PCR Basics: Amplifying DNA Fragments and Altering Sequences

PCR (Polymerase Chain Reaction) is a powerful technique used to amplify DNA, analyze DNA fragments, and alter DNA sequences. Learn about the purpose, components, variables, and experimental notes of PCR. Discover the temperature cycling, reaction components, and polymerases crucial for PCR success.

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PCR Basics: Amplifying DNA Fragments and Altering Sequences

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  1. PCR Basics Purpose of PCR Overview Components of PCR Reaction Variables Temperature Cycle Times and Numbers Primer Buffer Polymerase Experimental Notes

  2. Polymerase Chain Reaction • “Amplify” large quantities of DNA (μg quantities) from small quantities (ag quantities) [Trillion fold amplification] • Analyze single DNA fragments out of large complex mixture. [ Human genome mixture of 12 million 300bp fragments] • Alter DNA sequence – directed mutagenesis.

  3. Overview of PCR • Temperature Cycling • Denaturation 94° • Annealing 55° • Extension 72° • 2. Every Cycle DNA between primers is duplicated

  4. PCR Amplification

  5. Exponential Amplification 30 cycles --- 2 Trillion copies in theory

  6. Components of PCR Reaction • Template DNA • Flanking Primers • Thermo-stable polymerase • Taq Polymerase • dNTP • (dATP, dTTP, dCTP, dGTP) • PCR Buffer (mg++) • Thermocyler Thermus aquaticus

  7. PCR Variables • Temperature • Cycle Times and Temps • Primer • Buffer • Polymerase

  8. Temperature • Denaturation • Trade off between denaturing DNA and not denaturing Taq Polymerase • Taq half-life 40min at 95 °, 10min at 97.5° • 95° • Annealing • Trade off between efficient annealling and specificity • 2-5 ° below Tm • Extension • Temperature optimum for Taq Polymerase • 72 °

  9. Cycle Times and Temps Typical PCR Run Step Time/Temp • 3 min at 95° • 30 sec at 95° • 1 min at 55° • 2 min at 72° • Go to step 2 - 29 times • 8 min 72° • 0 min 4° • End

  10. Primers • Paired flanking primers • Length (17-28bp) • GC content 50-60% • GC Clamp • Tm’s between 55-80 • Avoid simple sequences – e.g. strings of G’s • Avoid primer self complementary • e.g. hairpins, homodimers, heterodimers

  11. PCR Buffer • Basic Components • 20mM Tris-HCL pH 8.4 • 50mM KCl • 1.5 mM MgCl2 • Magnesium – Since Mg ions form complexes with dNTPs, primers and DNA templates, the optimal concentration of MgCl2 has to be selected for each experiment. Too few Mg2+ ions result in a low yield of PCR product, and too many increase the yield of non-specific products and promote misincorporation. • Potential Additives • Helix Destabilisers - useful when target DNA is high G/CWith NAs of high (G+C) content. • dimethyl sulphoxide (DMSO), • dimethyl formamide (DMF), • urea • formamide  • Long Targets >1kb. Formamide and glycerol   • Low concentration of template: Polyethylene glycol (PEG)

  12. PCR Polymerases • Taq, Vent, Pfu, others • Native or Cloned • Half-life • e.g. Taq 40 min half-life, Vent 7 hour half-life • 3’-5’ Exo nuclease – proofreading • Fidelity (Error Rate • Taq 1/10,000nt, Pfu 1/1,000,000 • Processivity • Extra bases at end

  13. Notes • Typical Reaction • 80 μl dH2O • 10 μl 10 X PCR buffer + mg • 2 μl 200 μM dNTP • 1 μl 50 μM Left Primer • 1 μl 50 μM Right Primer • 5 μl Worm Lysate • 1 μl Taq Pol (5 Units/ μl) 100 μl Total Vol

  14. Master Mix • 1 Reaction Common Comp • 80 μl dH2O • 10 μl 10 X PCR buffer • 2 μl 200 μM dNTP • 1 μl Taq Pol (5 Units/ μl) 93 μl Total Volume • 9.5 reaction master mix – add 93 μl to each reaction • 760 μl dH2O • 95 μl 10 X PCR buffer • 19 μl 200 μM dNTP • 9.5 μl Taq Pol (5 Units/ μl)

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