![Polymerase Chain Reaction
• “Amplify” large quantities of DNA (μg quantities)
from small quantities (fg quantities) [billion fold
amplification]
• Analyze single DNA fragments out of large
complex mixture. [ Human genome mixture of 12
million 300bp fragments]
• Alter DNA sequence – directed mutagenesis.](https://image.slidesharecdn.com/pcrbasics-250113190405-909798a9/85/PCR-Basics-ppttttttttttttttttttttttttttt-2-320.jpg)
PCR Basics.ppttttttttttttttttttttttttttt
- 1. PCR Basics 1. Purpose of PCR 2. Overview 3. Components of PCR Reaction 4. Variables • Temperature • Cycle Times and Numbers • Primer • Buffer • Polymerase 5. Experimental Notes
- 2. Polymerase Chain Reaction • “Amplify” large quantities of DNA (μg quantities) from small quantities (fg quantities) [billion 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 1. Temperature Cycling Denaturation 94° Annealing 55° Extension 72° 2. Every cycle DNA between primers is duplicated
- 5. Exponential Amplification 30 cycles --- 1 billion 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 1. Temperature 2. Cycle Times and Temps 3. Primer 4. Buffer 5. 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 1 3 min at 95° 2 30 sec at 95° 3 1 min at 55° 4 2 min at 72° 5 Go to step 2 - 29 times 6 8 min 72° 7 0 min 4° 8 End
- 11. 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
- 12. 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)
- 13. 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
- 14. Notes • Typical Reaction • 32.5 μl dH2O • 5 μl 10 X PCR buffer + mg • 1 μl 200 μM dNTP • 0.5 μl 50 μM Left Primer • 0.5 μl 50 μM Right Primer • 10 μl Worm or Fly Lysate • 0.5 μl Taq Pol (5 Units/ μl) 50 μl Total Vol
- 15. Master Mix • 1 volume master mix – 32.5 μl dH2O – 5 μl 10 X PCR buffer – 1 μl 200 μM dNTP – 0.5 μl Taq Pol (5 Units/ μl) 39 μl Total Volume • To set up 4 reactions prepare 4.4 volumes of reaction master mix – 143 μl dH2O – 22 μl 10 X PCR buffer – 4.4 μl 200 μM dNTP – 2.2 μl Taq Pol (5 Units/ μl) • Individual reactions - 39 μl master mix - 0.5 μl Left primer - 0.5 μl Right primer - 10 μl worm lysate