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1、JForensicSci,January2010,Vol.55,No.1PAPERdoi:10.1111/j.1556-4029.2009.01245.xAvailableonlineat:interscience.wiley.comCRIMINALISTICS123KerryL.Opel,Ph.D.;DeniseChung,Ph.D.;andBruceR.McCord,Ph.D.AStudyofPCRInhibitionMechanisms,UsingRealTimePCR*ABSTRACT:Inthisproject,realtimepolymerasechainreaction(PC
2、R)wasutilizedtostudythemechanismofPCRinhibitionthroughexami-nationoftheeffectofampliconlength,meltingtemperature,andsequence.SpecificallydesignedprimerswiththreedifferentampliconlengthsandthreedifferentmeltingtemperatureswereusedtotargetasinglehomozygousalleleintheHUMTH01locus.Theeffectonamplificat
3、ionefficiencyforeachprimerpairwasdeterminedbyaddingdifferentconcentrationsofvariousPCRinhibitorstothereactionmixture.TheresultsshowthatavarietyofinhibitionmechanismscanoccurduringthePCRprocessdependingonthetypeofco-extractedinhibitor.TheseincludeTaqinhibition,DNAtemplatebinding,andeffectsonreaction
4、efficiency.Inaddition,someinhibitorsappeartoaffectthereactioninmorethanonemanner.Overallwefindthatampliconsizeandmeltingtemperatureareimportantinsomeinhibitionmechanismsandnotinothersandthekeyissueinunderstand-ingPCRinhibitionisdeterminingtheidentityoftheinterferingsubstance.KEYWORDS:forensicscienc
5、e,inhibition,DNAtyping,realtimepolymerasechainreaction,HUMTH01,humicacid,hematin,melanin,calciumDegradedandenvironmentallychallengedsamplescanproduceInpreviouswork(6)wehavedeterminedthatcertainprimersnumerousproblemsinforensicDNAtypingincludinglossofsig-withahighermeltingtemperaturearelessaffectedb
6、yinhibitionnal,peakimbalance,andalleledropout.However,DNAdegradation(Fig.1),andthatnotallinhibitorshavethesameeffectondifferentisnottheonlyissueencounteredwhenanalyzingchallengingsam-STRloci.Thissuggeststhatthesequenceoftheampliconorpri-ples.Manysuchsamplescontainsubstanceswhichareco-extractedmerma
7、yhaveanaffectonPCRinhibition.PrimerswithhigherwiththeDNAandinhibitthepolymerasechainreaction(PCR).meltingtemperaturesaremorestronglyboundtotheDNAandWhiletheeffectofthepresenceofinhibitorsiswellknown,themayp
