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KaryMullis1983,POLYMERASECHAINREACTION,AlicencetodomolecularbiologyAkeycentraltechniquethathasrevolutionisedmolecularandconsequentlycellbiology,InthisLecture,PrincipleofPCRPCR,thebasicsOptimizethePCRandtroubleshootApplicationofPCRReverse-TranscriptionPCR（反转录PCRPCRcloning（PCR克隆技术Real-TimePCR（实时定量PCRHistoryofPCR,PolymeraseChainReaction,PCR是一种体外酶促合成特定DNA片断的技术，是根据人类的需要对复杂生命过程的一种简单化的模拟。PCR技术的原理是DNA半保留复制。,DNAReplicationinvivo,http:/www.accessexcellence.org/AB/GG/collaboration.html,StrandseparationSynthesisofshortRNAprimersSynthesisofnewDNAstrands,DENATURATION93C-95C,DNAreplicationinvitro,ThePCRProcessinvitro,TemplateDNA:ThesampletoamplifiedPrimersShort,specificsegementsofDNA,provideSPECIFICITYdATP,dTTP,dCTP,dGTPThermostableDNApolymerase(e.g.,Taq,Pfu)Bufferandsalts(KCl,MgCl2)Optional:BSA,DMSO,Formamide,TYPICALREACTIONMIXTURE,25or50lsinamicroEppendorf(0.5ml)tube,(1.0-4.5mM),ThePCRinvitro,http:/vector.cshl.org/resources/BiologyAnimationLibrary.htm,PCR,Agarosegelelectrophoresis,Thefinalproduct,UVvisualisation,3-4hours,Startingnucleicacid-DNA/RNATissue,cells,blood,hairroot,saliva,semenThermo-stableDNApolymerasee.g.TaqpolymeraseOligonucleotidesDesignthemwell!BufferTris-HCl(pH7.6-8.0)Mg2+dNTPs(dATP,dCTP,dGTP,dTTP),OPTIMISINGPCRTHEREACTIONCOMPONENTS,Startingnucleicacid-DNA/RNATissue,cells,blood,hairroot,saliva,semenThermo-stableDNApolymerasee.g.TaqpolymeraseOligonucleotidesDesignthemwell!BufferTris-HCl(pH7.6-8.0)Mg2+dNTPs(dATP,dCTP,dGTP,dTTP),OPTIMISINGPCRTHEREACTIONCOMPONENTS,Startingnucleicacid-DNA/RNATissue,cells,blood,hairroot,saliva,semenThermo-stableDNApolymerasee.g.TaqpolymeraseOligonucleotidesDesignthemwell!BufferTris-HCl(pH7.6-8.0)Mg2+dNTPs(dATP,dCTP,dGTP,dTTP),OPTIMISINGPCRTHEREACTIONCOMPONENTS,ThermastatDNApolymerase:Taq,Pfu,Vente.g.,Isolatedfromathermophilicbacterium(Thermusaquaticus)fromaculturederivedfromahotspringinYellowstone.CatalyzesDNApolymerizationatelevatedtemperature(72C)andisresistantto98C.Similarenzymeshavehavebeenidentifiedfromotherthermophilicbacteria,suchasthoselivingindeepseavents.,NumberofoptionsavailableTaqpolymerasePfupolymeraseTthpolymeraseHowbigistheproduct?100bp40-50kbWhatisendpurposeofPCR?Sequencing/mutationdetection-NeedhighfidelitypolymeraseCloning(TAcloning)-TaqDNAPolymerase,CHOOSEYOURPOLYMERASEWITHCARE,Startingnucleicacid-DNA/RNATissue,cells,blood,hairroot,saliva,semenThermo-stableDNApolymerasee.g.TaqpolymeraseOligonucleotidesDesignthemwell!BufferTris-HCl(pH7.6-8.0)Mg2+dNTPs(dATP,dCTP,dGTP,dTTP),OPTIMISINGPCRTHEREACTIONCOMPONENTS,Length18-30nt(21nt)Basecomposition;50-60%GCrichpairsshouldhaveequivalentTmsTm=(numberofA+Tresidues)x2C+(numberofG+Cresidues)x4CInitialuseTm5CAvoidinternalhairpinstructuresnosecondarystructureAvoidaTatthe3endAvoidoverlapping3endswillformprimerdimersCanmodify5endstoaddrestrictionsitesetc,PRIMERDESIGNISVITAL,optimiseforeachsetofprimers,OPTIMISEPCRCONDITIONS-Primer,X,DNAprimersforPCR,PRIMERDESIGN,Primer5fromPremierprimerCo.,Alsoavailableoninternethttp:/www.hgmp.mrc.ac.uk/GenomeWeb/nuc-primer.htmlOligoSys:,Startingnucleicacid-DNA/RNATissue,cells,blood,hairroot,saliva,semenThermo-stableDNApolymerasee.g.TaqpolymeraseOligonucleotidesDesignthemwell!BufferTris-HCl(pH7.6-8.0)Mg2+dNTPs(dATP,dCTP,dGTP,dTTP),OPTIMISINGPCRTHEREACTIONCOMPONENTS,TITRATEYOURMg2+CONCENTRATION!,43.532.521.51mM,Normally,1.5mMMgCl2isoptimalBestsuppliedasseparatetubeAlwaysvortexthawedMgCl2Mg2+concentrationwillbeaffectedbytheamountofDNA,primersandnucleotides,USEMASTERMIXESWHEREPOSSIBLE,Takenfrom-http:/info.med.yale.edu/genetics/ward/tavi/PCR.html,PCROptimization,FocusontemperatureandMgCl2,THEPERFECTRESULT,APPLICATIONSOFPCR,基因克隆：基因表达的定量：半定量RT-PCR和实时定量RT-PCT反转录PCR，RT-PCR快速扩增cDNA末端RACEDNA序列分析,已知序列基因的PCR研究,代表性差异显示PCR:RDA差异显示RT-PCR:：DDRT快速扩增多态性DNA：RAPD,未知序列基因的PCR研究,PCR技术在基因克隆基因表达差异分析中常用的方法,PCRcloningSemi-quantitativeRT-PCRReal-TimePCR,CloningofPCRproducts,ModifiedprimersbyaddingREsitesatthe5endofeachprimerT-Astrategy:becausesomethermostaticDNApolymerasecanaddanon-template-dependedAatthe3endofPCRproducts,socaninsertedthePCRproductsintoaT-vectordiractlyBlunt-endPCRproductscloning,manythermostaticDNApolymerasecanamplifytheDNAcorrectly,InsertedtheDNAproductsintoblunt-REdigestedplasmiddiractly.,DNA聚合酶的特性及在基因克隆中的应用,TaqDNA聚合酶：某些耐热的DNA聚合酶，在DNA链延伸到模板DNA的末端时，并不是立即终止新链的延伸，而是在新链的3末端加上一个A，形成突出的3末端，为基因克隆提供了一个天然的粘末端。,逆转录酶：来自于逆转录病毒的逆转录酶，在以mRNA为模板，反转录cDNA时，当新链延伸到模板的末端时，并不是立即终止新链的延伸，而是在新链的3末端加上几个CCCC，形成突出的3末端，为合成全长的cDNA提供了一个天然接头位点。,PCRT-Acloning,PCR,TT,PCRcloningT-Astrategy,NNNGGATCC,TCTAGAMMM,NNNGGATCCYYYCCTAGG,AGATCTMMMTCTAGAWWW,EcoRI和BglII,GATCCG,ATCTAG,相应酶切的克隆质粒,PCR克隆：通过引物对克隆片断添加酶切位点,T4DNA酶连接,NNNGGATCC,TCTAGAMMM,NNNGGATCCYYYCCTAGG,AGATCTMMMTCTAGAWWW,EcoRI和BglII,GATCCG,ATCTAG,相应酶切的克隆质粒,PCR克隆：通过引物对克隆片断添加酶切位点,T4DNA酶连接,RT-PCR,RTase:Avianmyeloblastosisvirus(AMV)andMoloneystrainofmurineleukemiavirus(MMLV).Primersforfirst-strandedcDNAsynthesis:Genespecificprimers,Oligo(dT)primersforbindingthepoly(A)mRNAandRandomhexamerprimers,RT-PCR,ToamplifycDNAcopiesofRNAToretrieveandclonethe5and3terminusofRNATogeneratecDNAlibraryfromaverysmallamountsofmRNAToidentifythemutationsandpolymorphismsTomeasurethestrengthofgeneexpression(semi-quantitativeRT-PCR),RT-PCR,FulllengthcDNAsynthesiswithPCR,(SwitchingMechanismAtthe5endoftheRNATranscript)technology,RT-PCR,RTase:Avianmyeloblastosisvirus(AMV)andMoloneystrainofmurineleukemiavirus(MMLV).Primersforfirst-strandedcDNAsynthesis:Genespecificprimers,Oligo(dT)primersforbindingthepoly(A)mRNAandRandomhexamerprimers,基因表达差异的分析技术,NorthernBlotWesternblotSemi-quantatitiveRT-PCRDNAChipProteinChip二维电泳质谱,NORTHERN,targetgene,internalcontrolgeneactin,GAPDH,RPLP0etc,10X,2X,control,expt,Correctedfoldincrease=10/2=5,Semi-quantitativeRT-PCR,Correctedfoldincrease=810问题:难以准确设定PCR扩增的平台期,Real-TimePCRInstruments,dilutionstargetDNA,dilutionsreferenceDNA,C,C,C,C,C,C,E,E,E,E,E,E,targetprimers,referenceprimers,triplicatescDNA,triplicatescDNA,Standardcurvemethod,DifferencesbetweenbasicandReal-TimePCRs,Semi-quantitative,notsensitivityPCRproductsvaryfrom100toseveralkbps,Real-timedetection:EachcycleproducesafluorescentsignalAbsolutequantitative,muchmoresensitivityPCRproductsarearound60-150bps,SemiquantatativePCR,Real-TimePCR,Real-TimePCR-Taqmanprobe,ABIPRISM7000and7700SequenceDetectionSystemReal-timedetection:EachcycleproducesafluorescentsignalproportionaltotheamountofPCRproductpresent,“Real-Time”PCR,F,Q,.,.,2.Laserexcitesfluorescenttagonprimer.,3.Fluorescenceisabsorbedbyquenchermolecule,4.Detectorregisters“noreaction”.,“Real-Time”PCR,F,Q,.,.,T,.,5.PrimerisextendedbyTaqPolymerase,“Real-Time”PCR,F,Q,.,.,T,.,.,6.ExtensioncontinuesuntilthePolymeraseMeetsthefluorescentmolecule.,7.Thepolymerasebreaksdownthetaggedprimer.Thefluorescenttagandquenchermoleculebecomeseparated.,“Real-Time”PCR,F,Q,.,T,.,.,.,Q,Q,F,F,8.AsPCRcontinues,thetaggedprimersaredegradedand“free”fluorescentmoleculesaccumulate.,9.Thedetectorrecordsfluorescenceasameasureofamplificationprogress.,Real-TimePCR-SYBRgreen,SERIESOF10-FOLDDILUTIONS,IL1-bcon,IL1-bvit,av=18.03,av=29.63,IL1-beta,Real-TimePCR,TaqmanprobeSpecificprimersFluorescentdyelabeledprobeExpensiveHighspecificity,SRBRgreenSpecificprimersNoprobesCheaperReliable,HistoryofPCR,PCR技术是由美国科学家KaryMullis于1983年发明的。由于当时没有耐热DNA聚合酶，因此PCR过程中每个循环都需要添加DNA聚合酶，PCR技术操作十分烦琐而且不实用。PCR技术的自动化归因另外2个重大发现：耐热DNA聚合酶的发现，和计算机控制的热循环仪（DNAthermalcycler)的出现。TaqDNA聚合酶被美国Science杂志评为1993年的明星分子。,HistoryofPCR,1987年PCR技术得到美国专利局的专利授权。1989年DuPont公司对该专利提出异议：PCR技术应当是公共产权。斯坦福大学的KornbergHG也对PCR技术专利提出异议。理由是认可具有生物学知识的人都可以从Kornberg的文章推知如何操作PCR。1993年MullisKary获得诺贝尔奖。,HistoryofPCR,MilestonesofPCR,DNApolymerase:KornbergA.(1957)Oligonucleotidesynthesis:KhoranaHG(1967-1968)ThePCRidea:KhoranaHG(1971,J.Mol.Biol)Theidea:KaryMullis(1983)ThermostableDNApolymerase(1989)AutomatedThermalcyclerInnovativeapplicationsReal-TimePCRRACEFlowChipPCR,HistoryofPCR,LouisPasteur:Onceremarkedthatchancefavorsthepreparedmind,andcertainlythehistoryofscientificprogresssupportshiscontention,suchasNewtonsdiscoveryofgravityfollowinghisencounterwithanapple,Flemingsdiscoveryofpenicillinonacontaminatedpetridish.KaryMullis:Scientiststodaycontinuetotakeunexpectedturnsontheirpathstodiscovery.Onesuchrecentdetouroccurredin1983onU.S.Route101innorthernCalifornia.,HistoryofPCR,KaryMulliswasbornin1944inNorthCarolina.HeobtainedhisBachelorsdegreeinChemistryin1966fromtheGeorgiaInstituteofTechnologyandin1972receivedaPhDinBiochemistryfromtheUniversityofCaliforniaatBerkeley.HewasthenofferedatechnicianspostattheCetusCorporationofEmeryvillein1978.In1983,whilstdrivingalongthehighwayfromSanFranciscotohishomeinLaJolla,California,MulliswasthinkingaboutasimplemethodofexponentiallyamplifyingaDNAsequenceinatesttube.MullisthentookhisconcepttohisassociatesatCetusandtogethertheytooktheideaandmadeitworkinanexperimentalsystem,Mulliswasawarded10,000US$forhisidea.DuetotheunprecedentedpopularityofthetechniqueanditsrevolutionaryimpactonMolecularBiology,KaryMulliswasawardedtheNobelPrizeforChemistryin1993.,HistoryofPCR,TheDNAcomplexwouldbedenaturedtoformsinglestrands,thisdenaturationstepwouldbecarriedoutinthepresenceofasufficientlyLargeexcessofthetwoappropriateprimers.DNApolymerasewillbeaddedtocompletetheprocessofrepair.thewholecyclewouldberepeated(H.G.Khorana,1971;JournalofMolecularBiology,56:341IstoppedthecaragainandstarteddrawinglinesoftheDNAmoleculeshybridizingandextending,theproductofonecyclebecomingthetemplatesforthenextinachainreaction.(K.B.Mullis,1990,ScientificAmerican,262.56,Continuous-FlowPCRonaChip,Science,280(5366),1046-1048,15May1998,PolymeraseChainReactionPCRProteinChainReactionPCR,WhowillbethenextKaryMullis?,KhoranaHG.1971;JournalofMolecularBiology,56:341:DNAPCRMullisK.1983;PCR,TellingGC.2001;Protein-basedPCR,NatureMedicine,2001Jul;7(7):778-9Who?ProteinPCR,
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