应用DHPLC技术进行诊断性分析的质量保证体系.ppt

DHPLC技术进行诊断性分析的质量保证体系 诊断性分析的要求 临床分子遗传学分析的复杂性临床分子检测结果的一致性和精确性变性高效液相色谱 DHPLC 作为一种高效和敏感的基因突变检测技术DHPLC技术质量控制 AMERICANCOLLEGEOFMEDICALGENETICSStandardsandGuidelinesforClinicalGeneticsLaboratories2005EditionG CLINICALMOLECULARGENETICSTheseStandardsandGuidelinesspecificallyrefertotheuseofmoleculartechniquestoexamineheritableorsomaticchangesinthehumangenome G18DenaturingHighPerformanceLiquidChromatography dHPLC SectionAddedNovember2003 CMGSBestPracticeGuidelinesUseoftheWAVESysteminDiagnosticServicePreparedandeditedbyJohnHarvey NationalGeneticsReferenceLaboratory Wessex Salisbury UKandElsSchollen CentreforHumanGenetics Leuven Belgiumlastupdate 12March2004 Introduction Laboratoryprocess DHPLCsystem Dataquality Checking reportingguidelines References DHPLCSOPs InstrumentormaintenanceSOPTechniqueGeneralDHPLCSOPWAVE3500 3500HTMethodDisease specificSOPsRett BRCA HNPCCMarfan Application company users generalusers company specificusers SupplementaryAppendix1STANDARDOPERATINGPROCEDUREWAVE SystemOperationandMaintenanceSOP O MWAVE SystemOperationandMaintenanceForWAVE SystemModels3500 3500Aand3500HT WAVE SystemOperationandMaintenance AnalysisoftheWAVE Low HighRangeMutationStandardsThemaintenanceprocedureDNASep andDNASep HTcartridgemaintenance RoleofMutationstandards checkingofcorrectfunctioningoftheWAVE System includingovencalibration cartridgeperformance buffercompositionandstability toensurereproducibilityandaccuracyofthechromatographicanalysis Mutationstandardsberunwhen lTheroutinepre run lWeeklyandmonthlymaintenanceprocedure lAfterreplacementofanycomponent lValidationforanewbatch lAsanassaycontrol atthebeginningandendofeveryrun preferablyalsoafterevery100injectionsforlongruns AnalysisoftheWAVE Low HighRangeMutationStandards Normalrangesofthemutationstandards Themaintenanceprocedure 3 1Filterandflush3 2Pre runmaintenance3 3Weeklymaintenance3 4Quarterlymaintenance3 5Othermaintenanceoperations3 6Preventativemaintenanceprocedureandsystemvalidation Filterandflush Theprincipleoffiltrationinvolvespreventingunwantedcontaminantsfromenteringthesystem Filtrationappliestotwospecificareas solventfiltrationandin linefiltration Thesystemflushingistoremovemobilephasesaltcomponentsthatcanprecipitateunderstrongsolventconditions Pre runmaintenance 1 Buffercheck2 Injectionsystemwashing3 Pressurecheck4 Checktheabsorbanceonthedetector5 Purgethelines Weeklymaintenance InlinefilterreplacementCheckthesyringe Quarterlymaintenance CheckUVlampUVlampreplacementCleaningthesystem Isopropanolcleaning DNASep andDNASep HTcartridgemaintenance 1 RegularmaintenancescheduleEvery96 192injections ExtendedhotwashEvery1000injections ReversehotwashDNASep wash ifreversehotwashfailstoresolvemutationstandards 2 Short termcartridgestorage3 Long Termcartridgestorage4 Newcartridgeinstallation DailyMaintenance Equilibratethecartridge50 A50 Bfor15minutesRun1 2blanksVerifysystemperformance pre analysis RunastandardRunSamplesVerifysystemperformance postanalysis WeeklyMaintenance AnExtendedActiveCleanWashisrecommendedevery 100injections Usuallydoneaftereach96wellplate 15 30minutes WASH EQUILIBRATE 45 90minutes RunStandardstoVerifySystemPerformance 1000InjectionMaintenance AReverseHotWashisrecommendedevery 1000injections TurnoffthepumpReversethecartridgedirection Settheovento80 C Setthepumpto100 D 60 90minutes StoringtheCartridge Flushthecartridgewith100 DBuffer RemovethecartridgefromtheWAVESystem Capthecartridgewithendplugs Storethecartridgeatroomtemperature InstallingaNewCartridge Stopthepumpflowandremovetheoldcartridge Removetheplugsfromthenewcartridge Installthenewcartridgewiththearrowpointingtowardtherearoftheoven Makesuretheovenheatsuptoatleast40 C Setthepumpto100 D 0 500mL min Ensurethepressureisstableandgraduallyincreasetheflow 0 9mL minor1 5mL min Flushthecartridgefor15minutes Setthepumpto50 BufferA 50 BufferBandequilibratefor30minutes EquilibratingaNewCartridge VerifyNewCartridgePerformance Low RangeMutationStandard DNASizingControlStandard SupplementaryAppendix2STANDARDOPERATINGPROCEDUREDHPLCSOP DHPLCDHPLCmutationdetectiononTransgenomicWAVE System3500Wavemaker 4 1 44 HSM3 0 2 1 build2 Navigator 1 5 4 build19 PCRrequirements PrimerdesignTemplatepurityandconcentrationDNAPolymerasesPCRbuffermixPCRplatesPCRqualityandProductmixingPost PCR filmuseControls PrimerDesign Useaprimer pickingprogramPrimersshouldideallybenocloserthan30 50bpfromtheendofthesequencetobeanalyzedformutationsPrimersshouldbe18 30bpinlengthTheTmdifferencebetweenprimersinapairshouldideallybelessthan2 C TransgenomicWAVESystemCustomerscanusetheAdvancedFeaturesofAmpliconD SizeofPCRfragment Theoptimalsizerangefordetectingmutation SNPsbyDHPLCwith100 accuracyis150 500bp Fragments 500bpcanbegeneratedbutsensitivitydecreasedandtimeofelutionincreased Forfragments 150bp differenceofmeltingpointbetweenfragmentstoonarrow thefragmentsmeltovertoonarrowatemperaturerange QuantityofPCRfragment ThePCRproductshouldbesufficientlyconcentratedthat2 lrunonanagarosegelproducesaclearlyvisibleband 20ng l Dilutesamples verylowyields producepoorqualityresults poorsignal noiseratio Veryhighyieldscanleadtolargeproportionsofmisincorporationsandhenceincreaseddifficultyincallingmutations Usually3 10 l 50 200ng ofunpurifiedPCRproductwouldbeinjectedontothecolumn peronetemperature A8 lminimumaliquotofPCRproductshouldbesuppliedinPCRtubesinstripsof8 peronetemperature SamplePreparationforDHPLC DNAmustbeclean allcellulardebrisandorganiccompoundsmustberemoved Saltingoutmethodispreferred DNAextractedwithsomecommercialsystemsmaybedilutedto10ngtoreduceeffectsofimpuritiestoPCR DNAoflowqualitywillresultinsub optimalPCRresults henceDHPLCprofiles DNAquality concentrarion Table1 RecommendedcleaningproceduresforDNAextraction Table2 RecommendedDNAquantitiesusedforPCR 50 Lreaction TheImportanceofPolymeraseFidelityforMutationDetection ImportanceofhighfidelityindHPLC 500bpWildTypeFragmentRedTrace OptimasePolymeraseGreenTrace 9 1MixAmplitaqGoldandPfuTurbo Heteroduplexduetomisincorporation PolymeraseFidelityComparison MaximumrecommendedconcentrationsofacceptablePCRadditives Useofthesecomponentsrequirestheuseof Activeclean foreachinjection andotheradditionalcleaning Fluorescentlabelswillnotdamagethecolumnbutarenotrecommended Digoxigenin biotinlabelledprimershavenotbeentested UnacceptablePCRcomponents TemplateDNAextractedorpurifiedinamannernotconsistentwithWAVE srecommendationGelpurificationcannotbeusedtocleanupsamplesforDHPLC asthereagentsandresidualagarosedamagethecolumn Unidentifiedreagent proprietary stabilizers enhancers additives AutoclavedWaterMineralOilFormamideProteinaseKBovineserumalbumin BSA Loadingdyes cresolred ComponentscausingirreversibleDNASepcartridgedamage AnalysisofPCRProductsontheWAVESystem RunPCRProductsat50 C non denaturingconditions toverifysize yieldandpurityRunPCRProductsunderpartially denaturingconditionstoverifyfidelity Controls PCRPositivecontrolsPCRNegativecontrolsInstrumentcontrols LowandHighRangeMutationstandards ControlsamplesinDHPLC Whereverpossible aconfirmedwild typecontrolshouldberun andcomparedwitheachsample Amutation positive controlshouldbeincluded Whenamplifyinglargenumbersofsamplesformanydifferentgenefragmentswithalowfrequencymutationpickuprate itissometimesacceptabletoomitanormalcontrol InstrumentBuffers CommercialbuffersPreparationof In house Buffers Heteroduplexformation Allsamplesmustbeheteroduplexed Heteroduplexformation 95 Cfor5minandcoolslowly minimum10s C toroomtemperature Fastercoolingprotocolsusedforheteroduplexformationcanseriouslyimpairheteroduplexing PurificationofPCRproductscanseriouslyimpairheteroduplexformation Software WaveMaker 4 1 44Navigator ProjectSet upSelectionofanalysistemperaturesbasedonmeltprofilesAdjustmentofgradientswithtimeshifts ResultsInterpretation ChromatogramControlsMinimumpeakintensityVisualassessmentofchromatogramsTracespecifity Assessmentofchromatogramquality A ContainsunincorporatednucleotidesandprimerswhichdonotbindtothecolumB ContainsprimerdimersC MaybecausedeitherbyTaqerrorsduringPCRorbynon templateAaddition D Themajorityofwildtypesamplesappearasasinglepeak E ACNabsorbsat260nm AnyhighMWcontaminantsappearasspikesonthispeak Typicalhomozygouswild typechromatogram Controls CheckthemutationstandardsandseeiftheyfulfilthecriteriadescribedinSOP O M Checktheknownsequencevariants positivecontrols Iftheydonotpresentanaberrantchromatogramattheoptimalscreeningtemperature theresultsshouldberejectedandtheanalysisrepeated IdeallythesignalintensityofDHPLCprofilesshouldbe 2mVatA260 Peaksofintensity 30 oftheaveragepeakintensity Weakpeaksaremorelikelytoleadtofalse negative positiveresults Minimumpeakintensity Identificationofsequencevariants Thepresenceofheteroduplexesisoftendetectedasachangeinthenumberofpeaks maybe2 3or4peakpattern Twopeakpatternsaccountforthemajorityofmutations Completeresolutionofthe2heteroduplexesisnotalwaysnecessary Mutationsmayappearonlyasaslightbroadeningofthesinglepeak orasasubtlechangetoashoulderonthepeak AllsamplesidentifiedasheteroduplexesbyDHPLCanalysismustbesequencedinbothdirectionstoconfirmanddeterminethenatureofthesequencechange Thehomoduplexwild typepatternistypically1peak butmaybe2peaks dependinguponthemeltingprofile Elutionprofilesthatdifferfromthewild typeindicatethepresenceofDNAsequencechanges Butthemutationtypecannotbepredictedfromtheheteroduplexpattern EachmutationinagivenPCRfragmentispredictedtohaveauniqueheteroduplexpattern highlyspecificelutionprofile Thisisusefulforquickgenotypingofunknownsamplesbycomparisonwithpositivecontrolsamples However traceprofilesarenotalwaysuniqueforaspecificmutation i e differentDNAvariantscangiveidenticalprofiles Changesinretentiontimedonotaccuratelypredictthepresenceofasequencechange Tracespecificity Datachecking reportingandstorage DatacheckingPositiveresultsFalsepositiveresultsNegativeresultsFalsenegativeresultsSensitivityDetectionofmosaicsArchiving SupplementaryAppendix3STANDARDOPERATINGPROCEDUREMECP2SOP MECP2DHPLCscreeningofMECP2InthecontextofRettSyndrome Rettsyndrome Childhoodneurodevelopmentdisorderwithaprevalenceof1 10 000to1 15 000infemalebirthsMutationsintheMECP2gene codingforMethylCpGBindingProtein2 aretheprimarycauseofRTTEightmutationsarerecurrentlyfoundindifferentpopulations ThefirstpartofthemoleculardiagnosisofRTTistheDHPLC screeningofexons2 3and4ofMECP2 Thisallowstheidentificationofmorethan90 ofalldescribedmutations Materials Worksheet LotNo ofallproducts Equipmentidentifiers Patient identifier Performingtechnician s Dateofexperiments Materials PCR StandardPCRequipment Locationxxx Optimase DNApolymerase 2 5U l Locationxxx Optimase PCRbufferwithMg2 Locationxxx Primers Eurogentec stock as250pmol l appendixA Locationxxx Primerworksolutionscontain2 5pmol lofeachprimer Locationxxx PuredNTPs withoutdUTP 2mM Locationxxx PCRsystem Materials DHPLCsystem StandardDHPLCmaterial forpartnumbersseeappendixCinSOP O M WAVE System3500HT WAVEMAKER 4 1 44 HSM3 0 2 1build2 Patientmaterial PatientDNA Positivecontrols Negativecontrols Normalcontrols UsesofPlasmidControlsasReferenceReagents Method Pre PCRPCRCompositionPCRConditionsPostPCRHeteroduplexformationAgarosegelelectrophoresisDHPLC Interpretationoftheresults ThemutationstandardsatthebeginningandendoftherunareevaluatedAllpositivecontrolsshouldbevisibleattheirspecifictemperatureIfoneofthecontrolsdoesnotfulfillthecriteria negativeresultsarenotvalidandhavetoberepeated Positiveresultscanbeprocessedasusual TheelutionprofilesofaspecificfragmentfromthedifferentpatientsarecomparedwitheachotherandscoredaccordingtothegeneralDHPLCcriteria Theminimumpeakheightmustbe2mV Anyparticularobservationshouldbenotedonworksheetsortechnicalreports Ampliconswithanaberrantelutionpatternarere analysedbydirectsequencingonanindependentamplicon Interpretationoftheresults Allprematuretruncationmutationsareimmediatelyreportable Thepathogenicityofthemissensemutationswilldependonthepositionandthetype Interpretationisthensubjecttogoodpracticeandliteraturereview MutationsinthetwohighlyconservedMeCP2domains themethylbindingdomainandthetranscriptionrepressiondomain arelikelytobecausative Ifamutationisofunknownsignificance samplesshouldbeobtainedfromthepatient sparents Ifthemutationisfoundtobedenovo itislikelytobecausative Ifthemutationispresentinthemother X inactivationstudiesneedtobecarriedoutonthemotherofthepatientIfboththemotherandthedaughterhaverandomX inactivationthemutationisunlikelytobecausative Reportingprocedures NEGATIVERESULTINAFEMALENEGATIVERESULTINAMALENORMALPARENTPOSITIVERESULTINAFEMALE NEGATIVERESULTINAFEMALE RettsyndromeiscausedbymutationsintheMECP2gene Molecularanalysisofthisgenehasbeencarriedoutonpatient howevernocausativemutationhasbeenfound DHPLCanalysiswasusedtoscreenformutationsintheMECP2gene Thistechniquehasasensitivityof 95 forthedetectionofpointmutations micr