PCR

PolymeraseChainReaction

1)Addthefollowingtoamicrofugetube:10ulreactionbuffer1ul15uMforwardprimer1ul15uMreverseprimer1ultemplateDNA5ul2mMdNTP8ul25mMMgCl2orMgSO4(volumevariable)water(tomakeupto100ul)

2)PlacetubeinaThermocycler.Heatsampleto95°C,thenadd0.5-1ulofenzyme(Taq,Tli,Pfuetc.).Addafewdropsofmineraloil.

3)StartthePCRcyclesaccordingthefollowingschemes:

a)denaturation-94°C,30-90sec.b)annealing-55°C(or-5°Tm),0.5-2min.c)extension-72°C,1min.(timedependsonlengthofPCRproductandenzymeused)repeatcycles29times

4)Addafinalextensionstepof5min.tofillinanyuncompletedpolymerisation.Thencooleddownto4-25°C.

Note:MostoftheparameterscanbevariedtooptimisethePCR(moreatTavi"sPCRguide):a)Mg⁺⁺-oneofthemainvariables-changetheamountaddedifthePCRresultispoor.Mg⁺⁺affectstheannealingoftheoligotothetemplateDNAbystABIlisingtheoligo-templateinteraction,italsostabilisesthereplicationcomplexofpolymerasewithtemplate-primer.Itcanthereforealsoincreasesnon-specificannealingandproducedundesirablePCRproducts(givesmultiplebandsingel).EDTAwhichchelateMg⁺⁺canchangetheMg⁺⁺concentration.b)TemplateDNAconcentration-PCRisverypowerfultoolforDNAamplificationthereforeverylittleDNAisneeded.ButtoreducethelikelihoodoferrorbyTaqDNApolymerase,ahigherDNAconcentrationcanbeused,thoughtoomuchtemplatemayincreasetheamountofcontaminantsandreduceefficiency.c)Enzymesused-TaqDNApolymerasehasahighererrorrate(noproof-reADIng3"to5"exonucleaseactivity)thanTliorPfu.UseTli,Pfuorotherpolymeraseswithgoodproof-readingcapabilityifhighfidelityisneeded.Taq,however,islessfussythanotherpolymerasesandlesslikelytofail.Itcanbeusedincombinationwithotherenzymestoincreaseitsfidelity.TaqalsotendstoaddextraA"satthe3"end(extraA"sareusefulforTAcloningbutneedstoberemovedifbluntendligationistobedone).Moreenzymescanalsobeaddedtoimproveefficiency(sinceTaqmaybedamagedinrepeatedcycling)butmayincreasenon-specificPCRproducts.Ventpolymerasemaydegradeprimerandthereforenotidealformutagenesis-by-PCRwork.d)dNTP-canuseupto1.5mMdNTP.dNTPchelateMg⁺⁺,thereforeamountofMg⁺⁺usedmayneedtobechanged.HoweverexcessivedNTPcanincreasetheerrorrateandpossIBLyinhibitsTaq.LoweringthedNTP(10-50uM)maythereforealsoreduceerrorrate.LargersizePCRfragmentneedmoredNTP.e)primers-upto3uMofprimersmaybeused,buthighprimertotemplateratiocanresultsinnon-specificamplificationandprimer-dimerformation(note:storeprimersinsmallaliquots).f)Primerdesign-checkprimersequencestoavoidprimer-dimerformation.AddaGC-clampatthe5"endifarestrictionsiteisintroducedthere.OneortwoGorCatthe3"endisfinebuttrytoavoidhavingtoomany(itcanresultinnon-specificPCRproducts).Perfectcomplementarityof18basesormoreisideal.SeeGuide.g)Thermalcycling-denaturationtimecanbeincreasediftemplateGCcontentishigh.HigherannealingtemperaturemaybeneededforprimerswithhighGCcontentorlongerprimers(calculateTm).Usingagradient(ifyourPCRmachinepermitsit)isausefulwayofdeterminingtheannealingtemperature.ExtensiontimeshouldbeextendedforlargerPCRproducts;butreduceditwheneverpossibletolimitdamagetoenzyme.Extensiontimeisalsoaffectedbytheenzymesusede.gforTaq-assume1000base/min(alsochecksuppliers"recommendations,actualrateismuchhigher).ThenumberofcyclecanbeincreasedifthenumberoftemplateDNAisverylow,anddecreasedifhighamountoftemplateDNAisused(highertemplateDNAispreferableforPCRcloning-lowererrorrateinthePCR).

h)Additives-

1. Glycerol(5-10%),formamide(1-5%)orDMSO(2-10%)canbeaddedinPCRfortemplateDNAwithhighGCcontent(theychangetheTmofprimer-templatehybridisationreactionandthethermostabilityofpolymeraseenzyme).GlycerolcanprotectsTaqagainstheatdamage,whileformamidemaylowerenzymeresistence.
2. 0.5-2MBetaine(stocksolution-5M)isalsousefulforPCRoverhighGCcontentandlongstretchesofDNA(LongPCR/LAPCR).Performatitrationtodeterminetooptimumconcentration(1.3Mrecommended).Reducemeltingtemperature(92-93°C)andannealingtemperature(1-2°Clower).Itmaybeusefultousebetaineincombinationwithotherreagentslike5%DMSO.Betaineisoftenthesecret(andunnecessarilyexpensive)ingredientofmanycommercialkits.
3. >50mMTMAC(tetramethylammoniumchloride),TEAC(tetraethylammoniumchloride),andTMANO(trimethlamineN-oxide)canalsobeused.
4. BSA(upto0.8µg/µl)canalsoimproveefficiencyofPCRreaction.
5. SeealsoDanCruickshank"sPCRadditivesandAlkamiEnhancersformore.

1. HigherconcentrationofPCRbuffermaybeusedtoimproveefficiency.
2. Thisbuffermayworkbetterthanthebuffersuppliedfromcommercialsources.16.6mMammoniumsulfate67.7mMTRIS-HCl,pH8.8910mMbeta-mercaptoethanol170micrograms/mlBSA1.5-3mMMgCl2

k)TroubleshootingseeTavi"spage,MycoSite,AlkamiBiosystems,PromegaandSigma.

l)PCRmethods

1. Hot-startPCR-toreducenon-specificamplification.CanalsobedonebyseparatingtheDNAmixturesfromenzymebyalayerofwaxwhichmeltswhenheatedincyclingreaction.AnumberofcompaniesalsoproducehotstartPCRproducts,SeeAlkamiBiosystem.
2. "Touch-down"PCR-startathighannealingtemperature,thendecreaseannealingtemperatureinstepstoreducenon-specificPCRproduct.CanalsobeusedtodetermineDNAsequenceofknownproteinsequence.
3. NestedPCR-usetosynthesizemorereliableproduct-PCRusingaoutersetofprimersandtheproductofthisPCRisusedforfurtherPCRreactionusinganinnersetofprimers.
4. InversePCR-foramplificationofregionsflankingaknownsequence.DNAisdigested,thedesiredfragmentiscircularisebyligation,thenPCRusingprimercomplementarytotheknownsequenceextendingoutwards.
5. AP-PCR(arbitraryprimed)/RAPD(randomamplifiedpolymorphicDNA)-methodsforcreatinggenomicfingerprintsfromspecieswithlittle-knowntargetsequencesbyamplifyingusingarbitraryoligonucleotides.ItisnormallydoneatlowandthenhighstringencytodeterminetherelatednessofspeciesorforanalysisofRestrictionFragmentLengthPolymorphisms(RFLP).
6. RT-PCR(reversetranscriptase)-usingRNA-directedDNApolymerasetosynthesizeCDNAswhichisthenusedforPCRandisextremelysensitivefordetectingtheexpressionofaspecificsequenceinatissueorcells.ItmayalsobeusetoquantifymRNAtranscripts.SeealsoQuantiativeRT-PCR,CompetitiveQuantitativeRT-PCR,RTinsituPCR,NestedRT-PCR.
7. RACE(rapidamplificatonofcDNAends)-usedwhereinformationaboutDNA/proteinsequenceislimited.Amplify3"or5"endsofcDNAsgeneratingfragmentsofcDNAwithonlyonespecificprimereach(+oneadaptorprimer).OverlappingRACEproductscanthenbecombinedtoproducefullcDNA.SeealsoGibcomanual.
8. DD-PCR(differentialdisplay)-usedtoidentifydifferentiallyexpressedgenesindifferenttissues.FirststepinvolvesRT-PCR,thenamplificationusingshort,intentionallynonspecificprimers.Getseriesofbandinahigh-resolutiongelandcomparetothatfromothertissues,anybandsuniquetosinglesamplesareconsideredtobedifferentiallyexpressed.
9. Multiplex-PCR-2ormoreuniquetargetsofDNAsequencesinthesamespecimenareamplifiedsimultaneously.OnecanbeuseascontroltoverifytheintegrityofPCR.Canbeusedformutationalanalysisandidentificationofpathogens.
10. Q/C-PCR(Quantitativecomparative)-usesaninternalcontrolDNAsequence(butofdifferentsize)whichcompetewiththetargetDNA(competitivePCR)forthesamesetofprimers.Usedtodeterminttheamountoftargettemplateinthereaction.
11. RecusivePCR-Usedtosynthesisegenes.Oligosusedarecomplementarytostretchesofagene(>80bases),alternatelytothesenseandtotheantisensestrandswithendsoverlapping(~20bases).Designoftheoligoavoidinghomologoussequence(>8)iscrucialtothesuccessofthismethod.
12. AsymmetricPCR
13. InSituPCR
14. MutagenesisbyPCR
15. Fartoomanytolistproperly.

OtherPCRlinks-PCRlectures,radio-labelledprobes,Thermocyclersuppliers