荧光定量PCR.ppt

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1、Real-TimeQuantitativePCRContentPrinciplesofquantificationMethodsofquantificationApplicationsPrinciplesofquantificationThepolymerasechainreaction(PCR)hasrevolutionizedthedetectionofDNAandRNA.Aslittleasasinglecopyofaparticularsequencecanbespecificallyamplifiedanddetected.Theoretically,the

2、reisaquantitativerelationshipbetweenamountofstartingtargetsequenceandamountofPCRproductatanygivencycle.Inpractice,though,itisacommonexperienceforreplicatereactionstoyielddifferentamountsofPCRproduct.Thedevelopmentofreal-timequantitativePCRhaseliminatedthevariabilitytraditionallyassociat

3、edwithquantitativePCR,thusallowingtheroutineandreliablequantitationofPCRproductsPCRReactionPhaseInitialtemplate,moresignificantPerfectreproducibility,lesserrorConstantamplificationefficiency,linearstandardcurveSeveraBasicConceptsThreshold:ThelowlimitwhenPCRreactionsignalgoesintologphase

4、,usually,howtosetit?threshold=10′SDcycle3-15Ctvalue:ThecyclenumberofPCRreactionwhenthefluorescencesignalreachestothresholdStandardcurveStandardCurveCT=-klgX0+bThereisalinearrelationshipbetweenCTandlgX0(X0standsforamountofinitialDNA)Usestandardsamplewhichwehaveknownitsinitialconcentratio

5、ntomakestandardcurve.AndthenwecanidentifytheinitialconcentrationofunknowntemplatethroughthestandardcurvewhenweobtainitsCTvaluehowwecangetthisandwhy?PrinciplesofMathematicsweknow,X=X0(1+Ex)nX:amountofDNAwhenthePCRisfinishedX0：amountofinitialDNAEx：efficiencyofPCRn:cycleswhenn=ct,XCt=X0(

6、1+Ex)Ct=M(1)logM=logX0(1+Ex)Ct(2)logX0=-log(1+Ex)*Ct+logM(3)CT=-klgX0+bCT=-klgX0+b–Slope=-1/log(1+E)•IfE=1,Slope=-3.3219280……•Normally,slopeisabout-3.5~-4.2–Intercept•Normally,interceptisabout30~40MethodsofquantificationMethodsofquantificationDyemethod:SYBRGreenIProbemethods:TaqManProbe

7、,Molecularbeacons.etc..SYBRGreenISYBRGreenIdyeisahighlyspecificdoublestrandedDNAbindingdye.Itsfluorescenceincreaseswhenitboundtodouble-strandedDNAwhiledisappearswhenDNAdenatured.Thereisadirectratiobetweenfluorescencesignalandtheamountofdouble-strandedDNA.DissociationcurveEasily