应用DHPLC技术进行诊断性分析的质量保证体系课件

路漫漫其修远兮路漫漫其修远兮,吾将上下而求索吾将上下而求索26六月2024应用应用DHPLC技术进行诊技术进行诊断性分析的质量保证体断性分析的质量保证体系系11八月2023应用DHPLC技术进行诊断性分析的质量保1路漫漫其修远兮路漫漫其修远兮,吾将上下而求索吾将上下而求索诊断性分析的要求诊断性分析的要求n临床分子遗传学分析的复杂性临床分子遗传学分析的复杂性n临床分子检测结果的一致性和精确性临床分子检测结果的一致性和精确性n变性高效液相色谱变性高效液相色谱(DHPLC)(DHPLC)作为一种高效和敏作为一种高效和敏感的基因突变检测技术感的基因突变检测技术nDHPLCDHPLC技术质量控制技术质量控制诊断性分析的要求临床分子遗传学分析的复杂性2路漫漫其修远兮路漫漫其修远兮,吾将上下而求索吾将上下而求索AMERICAN COLLEGE OF MEDICAL GENETICSStandards and Guidelines for Clinical Genetics Standards and Guidelines for Clinical Genetics LaboratoriesLaboratories2005 EditionG:CLINICAL MOLECULAR GENETICSG:CLINICAL MOLECULAR GENETICS These Standards and Guidelines specifically refer to the use of molecular techniques to examine heritable or somatic changes in the human genome.G18G18Denaturing High Performance Liquid Chromatography(dHPLC)Denaturing High Performance Liquid Chromatography(dHPLC)(Section Added November 2003)(Section Added November 2003)AMERICANCOLLEGEOFMEDICALGE3路漫漫其修远兮路漫漫其修远兮,吾将上下而求索吾将上下而求索CMGSBestPracticeGuidelinesUseoftheWAVESysteminDiagnosticServicePreparedandeditedbyJohnHarvey,NationalGeneticsReferenceLaboratory(Wessex),Salisbury,UKandElsSchollen,CentreforHumanGenetics,Leuven,Belgiumlastupdate:12March2004IntroductionLaboratoryprocessDHPLCsystemDataqualityChecking&reportingguidelinesReferencesCMGSBestPracticeGuidelines4路漫漫其修远兮路漫漫其修远兮,吾将上下而求索吾将上下而求索应用DHPLC技术进行诊断性分析的质量保证体系课件5路漫漫其修远兮路漫漫其修远兮,吾将上下而求索吾将上下而求索DHPLC SOPsnInstrumentormaintenanceSOPuTechniquenGeneralDHPLCSOPWAVE3500,3500HTuMethodnDisease-specificSOPsRett,BRCA,HNPCCMarfan,uApplicationcompany+usersgeneral users+companyspecific usersDHPLCSOPsInstrumentormainte6路漫漫其修远兮路漫漫其修远兮,吾将上下而求索吾将上下而求索SupplementaryAppendix1STANDARDOPERATINGPROCEDUREWAVESystemOperationandMaintenanceSOP-O&MWAVESystemOperationandMaintenanceForWAVESystemModels3500,3500Aand3500HTSupplementaryAppendix1ST7路漫漫其修远兮路漫漫其修远兮,吾将上下而求索吾将上下而求索WAVESystemOperationandMaintenance AnalysisoftheWAVELow&HighRangeMutationStandardsThemaintenanceprocedureDNASepandDNASepHTcartridgemaintenanceWAVESystemOperationandMai8路漫漫其修远兮路漫漫其修远兮,吾将上下而求索吾将上下而求索RoleofMutationstandards:checkingofcorrectfunctioningoftheWAVESystem,includingovencalibration,cartridgeperformance,buffercompositionandstability,toensurereproducibilityandaccuracyofthechromatographicanalysis.Mutationstandardsberunwhen:lTheroutinepre-run,lWeeklyandmonthlymaintenanceprocedure,lAfterreplacementofanycomponent,lValidationforanewbatch,lAsanassaycontrol,atthebeginningandendofeveryrun,preferablyalsoafterevery100injectionsforlongruns.AnalysisoftheWAVELow&HighAnalysisoftheWAVELow&HighRangeMutationStandardsRangeMutationStandards RoleofMutations9路漫漫其修远兮路漫漫其修远兮,吾将上下而求索吾将上下而求索应用DHPLC技术进行诊断性分析的质量保证体系课件10路漫漫其修远兮路漫漫其修远兮,吾将上下而求索吾将上下而求索NormalrangesofthemutationstandardsNormalrangesofthemutation11路漫漫其修远兮路漫漫其修远兮,吾将上下而求索吾将上下而求索The maintenance procedure3.1Filterandflush3.2Pre-runmaintenance3.3Weeklymaintenance3.4Quarterlymaintenance3.5Othermaintenanceoperations3.6PreventativemaintenanceprocedureandsystemvalidationThemaintenanceprocedure3.112路漫漫其修远兮路漫漫其修远兮,吾将上下而求索吾将上下而求索Filterandflush nTheprincipleoffiltrationinvolvespreventingunwantedcontaminantsfromenteringthesystem.nFiltrationappliestotwospecificareas:solventfiltrationandin-linefiltration.nThesystemflushingistoremovemobilephasesaltcomponentsthatcanprecipitateunderstrongsolventconditions.FilterandflushTheprinciple13路漫漫其修远兮路漫漫其修远兮,吾将上下而求索吾将上下而求索Pre-run maintenance1.Buffercheck2.Injectionsystemwashing3.Pressurecheck4.Checktheabsorbanceonthedetector5.PurgethelinesPre-runmaintenance1.Bufferc14路漫漫其修远兮路漫漫其修远兮,吾将上下而求索吾将上下而求索Weekly maintenancenInlinefilterreplacementnCheckthesyringeWeeklymaintenanceInlinefilte15路漫漫其修远兮路漫漫其修远兮,吾将上下而求索吾将上下而求索Quarterlymaintenance nCheckUVlampnUVlampreplacementnCleaningthesystem(Isopropanolcleaning)QuarterlymaintenanceCheckUV16路漫漫其修远兮路漫漫其修远兮,吾将上下而求索吾将上下而求索DNASepandDNASepHTcartridgemaintenance 1.RegularmaintenancescheduleEvery96-192injections:ExtendedhotwashEvery1000injections:ReversehotwashDNASepwash(ifreversehotwashfailstoresolvemutationstandards)2.Short-termcartridgestorage3.Long-Termcartridgestorage4.NewcartridgeinstallationDNASepandDNASepHTcartrid17路漫漫其修远兮路漫漫其修远兮,吾将上下而求索吾将上下而求索Daily MaintenancenEquilibratethecartridge50%A50%Bfor15minutesRun1-2blanksnVerifysystemperformance(pre-analysis)RunastandardnRunSamplesnVerifysystemperformance(postanalysis)DailyMaintenanceEquilibratet18路漫漫其修远兮路漫漫其修远兮,吾将上下而求索吾将上下而求索Weekly MaintenanceAnExtendedActiveCleanWashisrecommendedevery100injections.(Usuallydoneaftereach96wellplate)OvenOvenSetto:Setto:80C80CPumpPumpSetto:Setto:100%D100%D15-30minutes15-30minutesWASHWASHOvenOvenSetto:Setto:56C56CPumpPumpSetto:Setto:50%A-50%B50%A-50%BEQUILIBRATEEQUILIBRATE45-90minutes45-90minutesRunStandardstoVerifySystemPerformance!RunStandardstoVerifySystemPerformance!WeeklyMaintenanceAnExtended19路漫漫其修远兮路漫漫其修远兮,吾将上下而求索吾将上下而求索1000 Injection Maintenance AReverseHotWashisrecommendedevery1000injectionsUV/FLDetectorTurnofftheTurnoffthepumppumpReversetheReversethecartridgecartridgedirection.direction.SettheovenSettheovento80C.to80C.SetthepumpSetthepumpto100%D.to100%D.60-90minutes60-90minutes1000InjectionMaintenanceAR20路漫漫其修远兮路漫漫其修远兮,吾将上下而求索吾将上下而求索Storing the CartridgenFlushthecartridgewith100%DBuffer.nRemovethecartridgefromtheWAVESystem.nCapthecartridgewithendplugs.nStorethecartridgeatroomtemperature.StoringtheCartridgeFlushthe21路漫漫其修远兮路漫漫其修远兮,吾将上下而求索吾将上下而求索Installing a New Cartridge1.1.StopthepumpflowStopthepumpflowandremovetheoldandremovetheoldcartridge.cartridge.2.2.RemovetheplugsRemovetheplugsfromthenewfromthenewcartridge.cartridge.3.3.InstallthenewInstallthenewcartridgewiththecartridgewiththearrowpointingarrowpointingtowardtherearoftowardtherearoftheoven.theoven.UV/FLDetectorInstallingaNewCartridgeStop22路漫漫其修远兮路漫漫其修远兮,吾将上下而求索吾将上下而求索1.1.Makesuretheovenheatsuptoatleast40C.Setthepumpto100%DMakesuretheovenheatsuptoatleast40C.Setthepumpto100%D0.500mL/min.0.500mL/min.2.2.Ensurethepressureisstableandgraduallyincreasetheflow(0.9mL/minorEnsurethepressureisstableandgraduallyincreasetheflow(0.9mL/minor1.5mL/min).1.5mL/min).3.3.Flushthecartridgefor15minutes.Flushthecartridgefor15minutes.4.4.Setthepumpto50%BufferA,50%BufferBandequilibratefor30minutes.Setthepumpto50%BufferA,50%BufferBandequilibratefor30minutes.Equilibrating a New Cartridge100%100%50%50%50%50%Makesuretheovenheatsupto23路漫漫其修远兮路漫漫其修远兮,吾将上下而求索吾将上下而求索Verify New Cartridge PerformanceLow-RangeMutationLow-RangeMutationStandardStandardDNASizingControlDNASizingControlStandardStandardVerifyNewCartridgePerforman24路漫漫其修远兮路漫漫其修远兮,吾将上下而求索吾将上下而求索SupplementaryAppendix2STANDARDOPERATINGPROCEDUREDHPLCSOP-DHPLCDHPLCmutationdetectiononTransgenomicWAVESystem3500Wavemaker4.1.44&HSM3.0-2.1(build2)Navigator1.5.4(build19)SupplementaryAppendix225路漫漫其修远兮路漫漫其修远兮,吾将上下而求索吾将上下而求索PCRrequirementsnPrimerdesignnTemplatepurityandconcentrationnDNAPolymerasesnPCRbuffermixnPCRplatesnPCRqualityandProductmixingnPost-PCR,filmusenControlsPCRrequirementsPrimerdesign26路漫漫其修远兮路漫漫其修远兮,吾将上下而求索吾将上下而求索Primer DesignnUseaprimer-pickingprogramnPrimersshouldideallybenocloserthan30-50bpfromtheendofthesequencetobeanalyzedformutationsnPrimersshouldbe18-30bpinlengthnTheTmdifferencebetweenprimersinapairshouldideallybelessthan2C.TransgenomicWAVESystemCustomerscanuseTransgenomicWAVESystemCustomerscanusetheAdvancedFeaturesofAmpliconDesignattheAdvancedFeaturesofAmpliconDPrimerDesignTransgenomicWAVE27路漫漫其修远兮路漫漫其修远兮,吾将上下而求索吾将上下而求索Size of PCR fragment nTheoptimalsizerangefordetectingmutation/SNPsbyDHPLCwith100%accuracyis150-500bp.nFragments500bpcanbegeneratedbutsensitivitydecreasedandtimeofelutionincreased.nForfragments150bp,differenceofmeltingpointbetweenfragmentstoonarrow(thefragmentsmeltovertoonarrowatemperaturerange).SizeofPCRfragmentTheoptim28路漫漫其修远兮路漫漫其修远兮,吾将上下而求索吾将上下而求索Quantity of PCR fragment nThePCRproductshouldbesufficientlyconcentratedthat2lrunonanagarosegelproducesaclearlyvisibleband(20ng/l)nDilutesamples(verylowyields)producepoorqualityresults(poorsignal:noiseratio).nVeryhighyieldscanleadtolargeproportionsofmisincorporationsandhenceincreaseddifficultyincallingmutations.nUsually3-10l(50-200ng)ofunpurifiedPCRproductwouldbeinjectedontothecolumn(peronetemperature).nA8lminimumaliquotofPCRproductshouldbesuppliedinPCRtubesinstripsof8(peronetemperature).QuantityofPCRfragmentTheP29路漫漫其修远兮路漫漫其修远兮,吾将上下而求索吾将上下而求索Sample Preparation for DHPLCnDNAmustbeclean,allcellulardebrisandorganiccompoundsmustberemoved.nSaltingoutmethodispreferred.nDNAextractedwithsomecommercialsystemsmaybedilutedto10ngtoreduceeffectsofimpuritiestoPCR.nDNAoflowqualitywillresultinsub-optimalPCRresults(henceDHPLCprofiles).DNAquality&concentrarionSamplePreparationforDHPLCDN30路漫漫其修远兮路漫漫其修远兮,吾将上下而求索吾将上下而求索Table1.RecommendedcleaningproceduresforDNAextraction.IsolationMethodRecommendedAdditionalCleaning:OrganicExtraction(e.g.phenol/chloroform)Chloroform/isoamylback-extractionfollowedbyethanolprecipitationandwashChaotropicSalts(e.g.guanidiniumisothiocyanate)EthanolprecipitationandwashSpinColumnEthanolprecipitationandwashTable2.RecommendedDNAquantitiesusedforPCR(50Lreaction).TemplateRecommendedQuantityHumangenomicDNA50-200ngPhageDNA1-10pgPlasmidDNA0.1-1.0pgTable1.Recommendedcleaning31路漫漫其修远兮路漫漫其修远兮,吾将上下而求索吾将上下而求索The Importance of Polymerase Fidelity for Mutation DetectionnImportanceofhighfidelityindHPLC500bpWildTypeFragmentRedTraceOptimasePolymeraseGreenTrace9:1MixAmplitaqGoldandPfuTurboHeteroduplexduetomisincorporationTheImportanceofPolymeraseF32路漫漫其修远兮路漫漫其修远兮,吾将上下而求索吾将上下而求索Polymerase Fidelity ComparisonPolymeraseFidelityComparison33路漫漫其修远兮路漫漫其修远兮,吾将上下而求索吾将上下而求索Maximum recommended concentrationsof acceptable PCR additivesAcceptableadditives(maximumfinalconcentration)Additiveswherefinalconcentrationmustbe2mVatA260.nPeaksofintensity30%oftheaveragepeakintensity.nWeakpeaksaremorelikelytoleadtofalse-negative/positiveresults.Minimum peak intensityIdeallythesignalintensityo45路漫漫其修远兮路漫漫其修远兮,吾将上下而求索吾将上下而求索Identification of sequence variantsnThepresenceofheteroduplexesisoftendetectedasachangeinthenumberofpeaks(maybe2,3or4peakpattern).nTwopeakpatternsaccountforthemajorityofmutations.nCompleteresolutionofthe2heteroduplexesisnotalwaysnecessary.nMutationsmayappearonlyasaslightbroadeningofthesinglepeak,orasasubtlechangetoashoulderonthepeak.nAllsamplesidentifiedasheteroduplexesbyDHPLCanalysismustbesequencedinbothdirectionstoconfirmanddeterminethenatureofthesequencechange.Identificationofsequencevar46路漫漫其修远兮路漫漫其修远兮,吾将上下而求索吾将上下而求索nThehomoduplexwild-typepatternistypically1peak,butmaybe2peaks,dependinguponthemeltingprofile.nElutionprofilesthatdifferfromthewild-typeindicatethepresenceofDNAsequencechanges.Butthemutationtypecannotbepredictedfromtheheteroduplexpattern.nEachmutationinagivenPCRfragmentispredictedtohaveauniqueheteroduplexpattern(highlyspecificelutionprofile).Thisisusefulforquickgenotypingofunknownsamplesbycomparisonwithpositivecontrolsamples.nHowever,traceprofilesarenotalwaysuniqueforaspecificmutation,i.e.differentDNAvariantscangiveidenticalprofiles.nChangesinretentiontimedonotaccuratelypredictthepresenceofasequencechange.Trace specificityThehomoduplexwild-typepatte47路漫漫其修远兮路漫漫其修远兮,吾将上下而求索吾将上下而求索Datachecking,reportingandstorage nDatacheckingnPositiveresultsnFalsepositiveresultsnNegativeresultsnFalsenegativeresultsnSensitivitynDetectionofmosaicsnArchivingDatachecking,reportingands48路漫漫其修远兮路漫漫其修远兮,吾将上下而求索吾将上下而求索SupplementaryAppendix3STANDARDOPERATINGPROCEDUREMECP2SOP-MECP2DHPLCscreeningofMECP2InthecontextofRettSyndromeSupplementaryAppendix349路漫漫其修远兮路漫漫其修远兮,吾将上下而求索吾将上下而求索RettsyndromenChildhoodneurodevelopmentdisorderwithaprevalenceof1/10.000to1/15.000infemalebirthsnMutationsintheMECP2gene,codingforMethylCpGBindingProtein2,aretheprimarycauseofRTTnEightmutationsarerecurrentlyfoundindifferentpopulations.nThefirstpartofthemoleculardiagnosisofRTTistheDHPLC-screeningofexons2,3and4ofMECP2.Thisallowstheidentificationofmorethan90%ofalldescribedmutations.RettsyndromeChildhoodneuro50路漫漫其修远兮路漫漫其修远兮,吾将上下而求索吾将上下而求索Materials nWorksheet:-LotNo.ofallproducts-Equipmentidentifiers-Patient-identifier-Performingtechnician(s)-DateofexperimentsMaterialsWorksheet:51路漫漫其修远兮路漫漫其修远兮,吾将上下而求索吾将上下而求索MaterialsnPCR-StandardPCRequipment(Location xxx)-OptimaseDNApolymerase(2.5U/l)(Location xxx)-OptimasePCRbufferwithMg2+(Location xxx)-Primers(Eurogentec)(stock)as250pmol/l.(appendixA)(Location xxx)-Primerworksolutionscontain2.5pmol/lofeachprimer(Location xxx)-PuredNTPs(withoutdUTP)(2mM)(Location xxx)-PCRsystemMaterialsPCR52路漫漫其修远兮路漫漫其修远兮,吾将上下而求索吾将上下而求索Materials nDHPLCsystem-StandardDHPLCmaterial(forpartnumbersseeappendixCinSOP-O&M)-WAVESystem3500HT,WAVEMAKER4.1.44&HSM3.0-2.1build2.nPatientmaterial-Patient DNA-Positive controls-Negative controls-Normal controlsMaterialsDHPLCsystem53路漫漫其修远兮路漫漫其修远兮,吾将上下而求索吾将上下而求索UsesofPlasmidControlsasReferenceReagentsnoethicalproblemsrenewableresourceCanusesamereferencereagentascontrolforPCR,heteroduplexandmutationdetectionanalysisUniversalreagentswhichcanbeincorporatedinQCproceduresandSOPsAdvantages:ValidationofnewprotocolsExonspecificwildtypeandmutatedcontrolsforexistingassaysValidationoftransferofprotocolsbetweenmachines/labsUses:UsesofPlasmidControlsasRe54路漫漫其修远兮路漫漫其修远兮,吾将上下而求索吾将上下而求索Method nPre-PCRPCRCompositionPCRConditionsnPostPCRHeteroduplexformationAgarosegelelectrophoresisnDHPLCMethodPre-PCR55路漫漫其修远兮路漫漫其修远兮,吾将上下而求索吾将上下而求索Interpretationoftheresults nThemutationstandardsatthebeginningandendoftherunareevaluatednAllpositivecontrolsshouldbevisibleattheirspecifictemperaturenIfoneofthecontrolsdoesnotfulfillthecriteria,negativeresultsarenotvalidandhavetoberepeated.Positiveresultscanbeprocessedasusual.nTheelutionprofilesofaspecificfragmentfromthedifferentpatientsarecomparedwitheachotherandscoredaccordingtothegeneralDHPLCcriteria.Theminimumpeakheightmustbe2mV.Anyparticularobservationshouldbenotedonworksheetsortechnicalreports.nAmpliconswithanaberrantelutionpatternarere-analysedbydirectsequencingonanindependentampliconInterpretationoftheresults56路漫漫其修远兮路漫漫其修远兮,吾将上下而求索吾将上下而求索Interpretationoftheresults nAllprematuretruncationmutationsareimmediatelyreportable.nThepathogenicityofthemissensemutationswilldependonthepositionandthetype.Interpretationisthensubjecttogoodpracticeandliteraturereview.MutationsinthetwohighlyconservedMeCP2domains,themethylbindingdomainandthetranscriptionrepressiondomain,arelikelytobecausative.nIfamutationisofunknownsignificance,samplesshouldbeobtainedfromthepatientsparents.Ifthemutationisfoundtobede novo,itislikelytobecausative.Ifthemutationispresentinthemother,X-inactivationstudiesneedtobecarriedoutonthemotherofthepatientIfboththemotherandthedaughterhaverandomX-inactivationthemutationisunlikelytobecausative.Interpretationoftheresults57路漫漫其修远兮路漫漫其修远兮,吾将上下而求索吾将上下而求索Reportingprocedures nNEGATIVERESULTINAFEMALEnNEGATIVERESULTINAMALEnNORMALPARENTnPOSITIVERESULTINAFEMALEReportingproceduresNEGATIVE58路漫漫其修远兮路漫漫其修远兮,吾将上下而求索吾将上下而求索NEGATIVERESULTINAFEMALE Rett syndrome is caused by mutations in theMECP2gene.Molecularanalysisofthisgenehasbeen carried out on patient*,however nocausativemutationhasbeenfound.DHPLCanalysiswasusedtoscreenformutationsintheMECP2gene.Thistechniquehasasensitivityof95%forthedetectionofpointmutations,microdeletionsandmicroinsertionsbutwillnotdetectgrossdeletionsofentireexons.Only80%ofRettsyndromepatientshaveadetectablemutationwithintheMECP2gene.NEGATIVERESULTINAFEMALE59路漫漫其修远兮路漫漫其修远兮,吾将上下而求索吾将上下而求索NEGATIVERESULTINAMALE Molecular analysis of the MECP2 gene has beencarriedoutonpatient*.Howevernocausativemutationhasbeenfound.DHPLCanalysiswasusedtoscreenformutationsintheMECP2gene.Thistechniquehasasensitivityof95%forthedetectionofpointmutations,microdeletionsandmicroinsertionsbutwillnotdetectgrossdeletionsofentireexons.ThefrequencyofMECP2mutationsinmentallyretardedmalesisestimatedat0.2%NEGATIVERESULTINAMALEM60路漫漫其修远兮路漫漫其修远兮,吾将上下而求索吾将上下而求索NORMALPARENT*istheparentof*,apatientwithRettsyndromewhohasthe*mutationintheMECP2gene.TheparentsDNAhasbeenanalysedforthisspecificmutationbyThereisnoevidenceofthe*mutationintheperipheralbloodsampleof*.However,wecannotruleoutgermlinemosaicismforthismutation.Prenataltestingforthismutationisapossibility.NORMALPARENT*isth61路漫漫其修远兮路漫漫其修远兮,吾将上下而求索吾将上下而求索POSITIVERESULTINAFEMALE*hasbeenfoundtocarry*atbase*oftheMECP2gene(*).ThisresultconfirmsthediagnosisofRettsyndromein*.Morethan95%ofRettsyndromecasesaresporadic,however*smothermaybeacarrierofthismutation.Also,oneoftheparentsmayhavegermlinemosaicismforthismutation.POSITIVERESULTINAFEMALE62路漫漫其修远兮路漫漫其修远兮,吾将上下而求索吾将上下而求索诊断性诊断性DHPLCDHPLC质量保证（质量保证（DDQADDQA）nWAVEWAVE系统的操作和维护系统的操作和维护SOPSOP；nDHPLCDHPLC技术分析基因突变的通用技术分析基因突变的通用SOPSOP；nDHPLCDHPLC检测特定疾病基因的检测特定疾病基因的SOPSOP：RettRett综合征综合征相关的相关的MECP2MECP2基因筛查中基因筛查中SOPsSOPs。诊断性DHPLC质量保证（DDQA）WAVE系统的操作和维63路漫漫其修远兮路漫漫其修远兮,吾将上下而求索吾将上下而求索DHPLC技术性能发挥技术性能发挥1.1.首先，只有经过充分的实验设计，条件优化，和实首先，只有经过充分的实验设计，条件优化，和实际验证后才能达到这种高灵敏度；际验证后才能达到这种高灵敏度；2.2.第二，严格的质量控制，持续的系统验证，和严格第二，严格的质量控制，持续的系统验证，和严格的实验程序也是达到这种高灵敏度必不可少的；的实验程序也是达到这种高灵敏度必不可少的；3.3.第三，操作者的经验也不可低估；第三，操作者的经验也不可低估；4.4.第四，即便在最理想的条件下，也不能保证对所有第四，即便在最理想的条件下，也不能保证对所有的扩增产物达到的扩增产物达到100%100%的灵敏度；的灵敏度；5.5.最后，虽然不是直接由该方法引起，但必需意识到最后，虽然不是直接由该方法引起，但必需意识到DHPLCDHPLC分析的是小片段的分析的是小片段的PCRPCR产物，因而只能检测单产物，因而只能检测单碱基的突变，小片段的插入和缺失，大片段的插入碱基的突变，小片段的插入和缺失，大片段的插入和缺失不能检测。和缺失不能检测。DHPLC技术性能发挥首先，只有经过充分的实验设计，条件优化64