應用dhplc技術進行診斷性分析的質(zhì)量保證體系(編輯修改稿)
2025-02-24 01:19 本頁面
【文章內(nèi)容簡介】 lity ComparisonMaximum remended concentrationsof acceptable PCR additivesAcceptableadditives(maximumfinalconcentration)Additiveswherefinalconcentrationmustbe1%10%DMSO HighMolecularweightstabilizers:polyethyleneglycol(PEG)2%Glycerol Detergentsincludingbutnotlimitedto:TritionX100NP40Tween20SDS/SLStoBetainen Useoftheseponentsrequirestheuseof39。Activeclean39。foreachinjection,andotheradditionalcleaning.n Fluorescentlabelswillnotdamagethecolumnbutarenotremended.Digoxigenin/biotinlabelledprimershavenotbeentested.Unacceptable PCR ponents.n TemplateDNAextractedorpurifiedinamannernotconsistentwithWAVE’sremendation– GelpurificationcannotbeusedtocleanupsamplesforDHPLC,asthereagentsandresidualagarosedamagethecolumn.n Unidentifiedreagent:– “proprietary” “stabilizers”– “enhancers” “additives”n AutoclavedWatern MineralOiln Formamiden ProteinaseKn Bovineserumalbumin(BSA)n Loadingdyes(cresolred)(ComponentscausingirreversibleDNASepcartridgedamage)Analysis of PCR Products on the WAVE SystemnRunPCRProductsat50176。C(nondenaturingconditions)toverifysize,yieldandpuritynRunPCRProductsunderpartiallydenaturingconditionstoverifyfidelity.Controls n PCRPositivecontrolsn PCRNegativecontrolsn Instrumentcontrols:LowandHighRangeMutationstandardsControl samples in DHPLCn Whereverpossible,aconfirmedwildtypecontrolshouldberun,andparedwitheachsample.n Amutation(positive)controlshouldbeincluded.n Whenamplifyinglargenumbersofsamplesformanydifferentgenefragmentswithalowfrequencymutationpickuprate,itissometimesacceptabletoomitanormalcontrol.InstrumentBuffersn Commercialbuffersn Preparationof‘Inhouse’BuffersHeteroduplexformation AllsamplesmustbeheteroduplexedHeteroduplexformation:95186。Cfor5minandcoolslowly(minimum10s/176。C)toroomtemperature. Fastercoolingprotocolsusedforheteroduplexformationcanseriouslyimpairheteroduplexing.PurificationofPCRproductscanseriouslyimpairheteroduplexformation. SoftwareWaveMaker? Navigator? n ProjectSetupn Selectionofanalysistemperaturesbasedonmeltprofilesn Adjustmentofgradientswithtimeshifts.ResultsInterpretation n Chromatogramn Controlsn Minimumpeakintensityn Visualassessmentofchromatogramsn TracespecifityAssessment of chromatogram qualityA:ContainsunincorporatednucleotidesandprimerswhichdonotbindtothecolumB:ContainsprimerdimersC:MaybecausedeitherbyTaqerrorsduringPCRorbynontemplateAaddition.D:Themajorityofwildtypesamplesappearasasinglepeak.E:ACNabsorbsat260nm.AnyhighMWcontaminantsappearasspikesonthispeak.TypicalhomozygouswildtypechromatogramACBDERetentiontime(minutes)A260nmInjectionpeak ACNwashpeakSamplepeakControlsn CheckthemutationstandardsandseeiftheyfulfilthecriteriadescribedinSOPOM.n Checktheknownsequencevariants(positivecontrols).Iftheydonotpresentanaberrantchromatogramattheoptimalscreeningtemperature,theresultsshouldberejectedandtheanalysisrepeatedn IdeallythesignalintensityofDHPLCprofilesshouldbe2mVatA260.n Peaksofintensity30%oftheaveragepeakintensity.n Weakpeaksaremorelikelytoleadtofalsenegative/positiveresults.Minimum peak intensityIdentification of sequence variantsn Thepresenceofheteroduplexesisoftendetectedasachangeinthenumberofpeaks(maybe2,3or4peakpattern).n Twopeakpatternsaccountforthemajorityofmutations.n Completeresolutionofthe2heteroduplexesisnotalwaysnecessary.n Mutationsmayappearonlyasaslightbroadeningof160
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