PCR Basics -Power Point

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Transcript of PCR Basics -Power Point

  • PCR BasicsPurpose of PCROverviewComponents of PCR ReactionVariablesTemperatureCycle Times and NumbersPrimerBufferPolymeraseExperimental Notes

  • Polymerase Chain ReactionAmplify large quantities of DNA (g 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.

  • Overview of PCRTemperature CyclingDenaturation 94Annealing 55Extension 72

    2. Every cycle DNA between primers is duplicated

  • PCR Amplification

  • Exponential Amplification30 cycles --- 1 billion copies in theory

  • Components of PCR ReactionTemplate DNAFlanking PrimersThermo-stable polymeraseTaq Polymerase dNTP (dATP, dTTP, dCTP, dGTP)PCR Buffer (mg++)ThermocylerThermus aquaticus

  • PCR VariablesTemperatureCycle Times and TempsPrimerBufferPolymerase

  • TemperatureDenaturationTrade off between denaturing DNA and not denaturing Taq PolymeraseTaq half-life 40min at 95 , 10min at 97.595AnnealingTrade off between efficient annealling and specificity2-5 below TmExtensionTemperature optimum for Taq Polymerase72

  • Cycle Times and TempsTypical PCR Run Step Time/Temp3 min at 9530 sec at 951 min at 552 min at 72Go to step 2 - 29 times8 min 720 min 4End

  • PrimersPaired flanking primersLength (17-28bp)GC content 50-60%GC ClampTms between 55-80Avoid simple sequences e.g. strings of GsAvoid primer self complementarye.g. hairpins, homodimers, heterodimers

  • PCR BufferBasic Components20mM Tris-HCL pH 8.450mM KCl1.5 mM MgCl2Magnesium 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 AdditivesHelix 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)

  • PCR PolymerasesTaq, Vent, Pfu, othersNative or ClonedHalf-lifee.g. Taq 40 min half-life, Vent 7 hour half-life3-5 Exo nuclease proofreadingFidelity (Error Rate)Taq 1/10,000nt, Pfu 1/1,000,000ProcessivityExtra bases at end

  • NotesTypical Reaction32.5 l dH2O5 l 10 X PCR buffer + mg1 l 200 M dNTP0.5 l 50 M Left Primer0.5 l 50 M Right Primer10 l Worm or Fly Lysate0.5 l Taq Pol (5 Units/ l)50 l Total Vol

  • Master Mix1 volume master mix 32.5 l dH2O5 l 10 X PCR buffer 1 l 200 M dNTP0.5 l Taq Pol (5 Units/ l) 39 l Total Volume

    To set up 4 reactions prepare 4.4 volumes of reaction master mix143 l dH2O22 l 10 X PCR buffer 4.4 l 200 M dNTP2.2 l Taq Pol (5 Units/ l)

    Individual reactions39 l master mix0.5 l Left primer0.5 l Right primer10 l worm lysate