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1、Downloadedfromgenome.cshlp.orgonAugust13,2018-PublishedbyColdSpringHarborLaboratoryPressImprovedQuantitativePCRUsingNestedPrimersLawrenceA.HaftAppliedBiosystemsDivision,ThePerkin-ElmerCorporation,Norwalk,Connecticut06859QuantitativePCRcanoftenbeim-Higuchietal.~1~des

2、cribedapowerfulsinglesetofprimers,evenwithhotprovedbyconductingtheamplifica-andconvenientmethodtodetectandstarts.tionwithnestedprimers.First,fewerquantifyPCRamplificationproductsbyAwidelyacceptedandgenerallyeffec-nonspeciflcamplificationproducts,fluorescenceenhancem

3、entinethidiumtivesolutiontoPCRnonspecificityiswhichcouldotherwiseinterferewithbromide.ThesedatacanbetreatedinatnestedprimerPCR.(3)NestedprimerPCRquantitation,areproduced.Often,leasttwowaystoestimatetheinitialcon-involvesaninitialsetofthermalcyclesnonspeciflcproducts

4、canbeelimi-centrationofatargetsequence.First,withapairofprimersspacedrelativelynated.Inthesecases,relativelysim-fromdatatakenintheexponentialam-farapart.Then,anadditionalprimerorplenonspeciflcdetectiontechniquesplificationphase,thestepcycleeffi-pairofprimers,located

5、betweenthearesuitableforquantitation.Inad-ciencycanbecalculatedandoriginaltar-outerprimers,isadded,followedbyadition,nestedprimerPCRprovidesgetconcentrationeasilycalculated.(2~secondroundofthermalcycling.ThesetintrinsicPCRproductcarryoverpro-Higuchietal.~1~alsodemon

6、stratedthatofinnerprimersisaddedatahighercon-tectionandgenerallyimprovesthetheinitialtargetsequenceconcentrationcentrationthantheouterprimers.Afterrobustnessandlowerlimitofdetec-couldbecalculatedfromdataobtainedafewthermalcycles,innerprimerPCRtionofPCR.ForanestedPCR

7、topro-inthenonexponentialphase(orplateauproductpredominates.videusefulquantitativeinformation,phase),basedonthecalculatednumberBecausethediscriminatorypowerofitisImportantthattheinitialphaseofPCRcyclesrequiredforPCRproductnestedprimerPCRissohigh,asingleofamplificati

8、on,performedwiththetoreachanarbitrarilyselectedthresholdPCRproductcanoftenbeobtainedwithouterpairofprimers,takesplaceen-concentration.Amaj