应用DHPLC技术进行诊断性分析的质量保证体系

应用DHPLC技术进行诊断性分析的质量保证体系张泽云美国环球基因公司中国代表处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AMERICANCOLLEGEOFMEDICALGENETICS

StandardsandGuidelinesforClinicalGeneticsLaboratories

2005Edition

G:CLINICALMOLECULARGENETICS

TheseStandardsandGuidelinesspecificallyrefertotheuseofmoleculartechniquestoexamineheritableorsomaticchangesinthehumangenome.

G18

DenaturingHighPerformanceLiquidChromatography(dHPLC)

(SectionAddedNovember2003)CMGSBestPracticeGuidelinesUseoftheWAVESysteminDiagnosticService

PreparedandeditedbyJohnHarvey,NationalGeneticsReferenceLaboratory(Wessex),Salisbury,UKandElsSchollen,CentreforHumanGenetics,Leuven,Belgium

lastupdate:12March2004

•Introduction

•Laboratoryprocess

•DHPLCsystem

•Dataquality

•Checking&reporting

guidelines

•References

DHPLCSOPsInstrumentormaintenance

SOPTechnique

GeneralDHPLCSOPWAVE3500,3500HTMethod

Disease-specificSOPsRett,BRCA,HNPCC

Marfan,…Applicationcompany+usersgeneralusers+companyspecificusers

SupplementaryAppendix1

STANDARDOPERATINGPROCEDUREWAVE®SystemOperationandMaintenance

SOP-O&M

WAVE®SystemOperationandMaintenance

ForWAVE®SystemModels3500,3500Aand3500HTWAVE®SystemOperationandMaintenance

AnalysisoftheWAVE®Low&HighRangeMutationStandardsThemaintenanceprocedureDNASep®andDNASep®HTcartridgemaintenance

RoleofMutationstandards:

checkingofcorrectfunctioningoftheWAVE®System,includingovencalibration,cartridgeperformance,buffercompositionandstability,toensurereproducibilityandaccuracyofthechromatographicanalysis.

Mutationstandardsberunwhen:

l

Theroutinepre-run,

l

Weeklyandmonthlymaintenanceprocedure,

l

Afterreplacementofanycomponent,

l

Validationforanewbatch,

l

Asanassaycontrol,atthebeginningandendofeveryrun,preferablyalsoafterevery100injectionsforlongruns.

AnalysisoftheWAVE®Low&HighRangeMutationStandards

NormalrangesofthemutationstandardsThemaintenanceprocedure3.1Filterandflush3.2Pre-runmaintenance3.3Weeklymaintenance3.4Quarterlymaintenance3.5Othermaintenanceoperations3.6Preventativemaintenanceprocedureandsystemvalidation

Filterandflush

Theprincipleoffiltrationinvolvespreventingunwantedcontaminantsfromenteringthesystem.Filtrationappliestotwospecificareas:solventfiltrationandin-linefiltration.Thesystemflushingistoremovemobilephasesaltcomponentsthatcanprecipitateunderstrongsolventconditions.

Pre-runmaintenance1.Buffercheck2.Injectionsystemwashing3.Pressurecheck4.Checktheabsorbanceonthedetector5.Purgethelines

WeeklymaintenanceInlinefilterreplacement

Checkthesyringe

Quarterlymaintenance

CheckUVlamp

UVlampreplacement

Cleaningthesystem(Isopropanolcleaning)

DNASep®andDNASep®HTcartridgemaintenance

1.

Regularmaintenanceschedule

Every96-192injections:Extendedhotwash

Every1000injections:Reversehotwash

DNASep®wash(ifreversehotwashfailstoresolvemutationstandards)2.Short-termcartridgestorage3.Long-Termcartridgestorage4.NewcartridgeinstallationDailyMaintenanceEquilibratethecartridge50%A50%Bfor15minutesRun1-2blanksVerifysystemperformance(pre-analysis)RunastandardRunSamplesVerifysystemperformance(postanalysis)WeeklyMaintenanceAnExtendedActiveCleanWashisrecommendedevery~100injections.(Usuallydoneaftereach96wellplate)OvenSetto:80°CPumpSetto:100%D15-30minutesWASHOvenSetto:56°CPumpSetto:50%A-50%BEQUILIBRATE45-90minutesRunStandardstoVerifySystemPerformance!1000InjectionMaintenanceAReverseHotWashisrecommendedevery~1000injectionsUV/FLDetector

TurnoffthepumpReversethecartridgedirection.Settheovento80°C.Setthepumpto100%D.60-90minutesStoringtheCartridgeFlushthecartridgewith100%DBuffer.RemovethecartridgefromtheWAVESystem.Capthecartridgewithendplugs.Storethecartridgeatroomtemperature.InstallingaNewCartridgeStopthepumpflowandremovetheoldcartridge.Removetheplugsfromthenewcartridge.Installthenewcartridgewiththearrowpointingtowardtherearoftheoven.UV/FLDetectorMakesuretheovenheatsuptoatleast40°C.Setthepumpto100%D@0.500mL/min.Ensurethepressureisstableandgraduallyincreasetheflow(0.9mL/minor1.5mL/min).Flushthecartridgefor15minutes.Setthepumpto50%BufferA,50%BufferBandequilibratefor30minutes.EquilibratingaNewCartridge100%50%50%VerifyNewCartridgePerformanceLow-RangeMutationStandardDNASizingControlStandard

SupplementaryAppendix2STANDARDOPERATINGPROCEDUREDHPLCSOP-DHPLC

DHPLCmutationdetectiononTransgenomicWAVE®System3500Wavemaker™4.1.44&HSM3.0-2.1(build2)Navigator™1.5.4(build19)

PCRrequirementsPrimerdesignTemplatepurityandconcentrationDNAPolymerasesPCRbuffermixPCRplatesPCRqualityandProductmixingPost-PCR,filmuseControlsPrimerDesignUseaprimer-pickingprogramPrimersshouldideallybenocloserthan30-50bpfromtheendofthesequencetobeanalyzedformutationsPrimersshouldbe18-30bpinlengthTheTmdifferencebetweenprimersinapairshouldideallybelessthan2°C.SizeofPCRfragmentTheoptimalsizerangefordetectingmutation/SNPsbyDHPLCwith100%accuracyis150-500bp.Fragments>500bpcanbegeneratedbutsensitivitydecreasedandtimeofelutionincreased.Forfragments<150bp,differenceofmeltingpointbetweenfragmentstoonarrow(thefragmentsmeltovertoonarrowatemperaturerange).QuantityofPCRfragmentThePCRproductshouldbesufficientlyconcentratedthat2µlrunonanagarosegelproducesaclearlyvisibleband(~20ng/µl)Dilutesamples(verylowyields)producepoorqualityresults(poorsignal:noiseratio).Veryhighyieldscanleadtolargeproportionsofmisincorporationsandhenceincreaseddifficultyincallingmutations.Usually3-10µl(~50-200ng)ofunpurifiedPCRproductwouldbeinjectedontothecolumn(peronetemperature).A8µlminimumaliquotofPCRproductshouldbesuppliedinPCRtubesinstripsof8(peronetemperature).SamplePreparationforDHPLCDNAmustbeclean,allcellulardebrisandorganiccompoundsmustberemoved.Saltingoutmethodispreferred.DNAextractedwithsomecommercialsystemsmaybedilutedto10ngtoreduceeffectsofimpuritiestoPCR.DNAoflowqualitywillresultinsub-optimalPCRresults(henceDHPLCprofiles).DNAquality&concentrarionTable1.RecommendedcleaningproceduresforDNAextraction.IsolationMethodRecommendedAdditionalCleaning:OrganicExtraction

(e.g.phenol/chloroform)Chloroform/isoamylback-extractionfollowedbyethanolprecipitationandwashChaotropicSalts

(e.g.guanidiniumisothiocyanate)EthanolprecipitationandwashSpinColumnEthanolprecipitationandwashTable2.RecommendedDNAquantitiesusedforPCR(50µLreaction).TemplateRecommendedQuantityHumangenomicDNA50-200ngPhageDNA1-10pgPlasmidDNA0.1-1.0pgTheImportanceofPolymeraseFidelityforMutationDetectionImportanceofhighfidelityindHPLC500bpWildTypeFragmentRedTrace–OptimasePolymeraseGreenTrace–9:1MixAmplitaqGoldandPfuTurboHeteroduplexduetomisincorporationPolymeraseFidelityComparisonMaximumrecommendedconcentrations

ofacceptablePCRadditivesAcceptableadditives(maximumfinalconcentration)Additiveswherefinalconcentrationmustbe<1%10%

DMSOHighMolecularweightstabilizers:-polyethyleneglycol(PEG)2%

GlycerolDetergentsincludingbutnotlimitedto:TritionX100-NP40Tween20-SDS/SLS1.25to2.5M

BetaineUseofthesecomponentsrequirestheuseof'Activeclean'foreachinjection,andotheradditionalcleaning.Fluorescentlabelswillnotdamagethecolumnbutarenotrecommended.Digoxigenin/biotinlabelledprimershavenotbeentested.UnacceptablePCRcomponents.TemplateDNAextractedorpurifiedinamannernotconsistentwithWAVE’srecommendationGelpurificationcannotbeusedtocleanupsamplesforDHPLC,asthereagentsandresidualagarosedamagethecolumn.Unidentifiedreagent:“proprietary” -“stabilizers”“enhancers” -“additives”AutoclavedWaterMineralOilFormamideProteinaseKBovineserumalbumin(BSA)Loadingdyes(cresolred)(ComponentscausingirreversibleDNASepcartridgedamage)AnalysisofPCRProductsontheWAVESystemRunPCRProductsat50°C(non-denaturingconditions)toverifysize,yieldandpurityRunPCRProductsunderpartially-denaturingconditionstoverifyfidelity.Controls

PCRPositivecontrols

PCRNegativecontrolsInstrumentcontrols:LowandHighRangeMutationstandardsControlsamplesinDHPLCWhereverpossible,aconfirmedwild-typecontrolshouldberun,andcomparedwitheachsample.Amutation(positive)controlshouldbeincluded.Whenamplifyinglargenumbersofsamplesformanydifferentgenefragmentswithalowfrequencymutationpickuprate,itissometimesacceptabletoomitanormalcontrol.InstrumentBuffersCommercialbuffersPreparationof‘In-house’Buffers

Heteroduplexformation

·

Allsamplesmustbeheteroduplexed;·Heteroduplexformation:95ºCfor5minandcoolslowly(minimum10s/°C)toroomtemperature.

·Fastercoolingprotocolsusedforheteroduplexformationcanseriouslyimpairheteroduplexing.·

PurificationofPCRproductscanseriouslyimpairheteroduplexformation.

SoftwareWaveMaker™4.1.44

Navigator™

ProjectSet-upSelectionofanalysistemperaturesbasedonmeltprofilesAdjustmentofgradientswithtimeshifts.

ResultsInterpretation

ChromatogramControlsMinimumpeakintensityVisualassessmentofchromatogramsTracespecifity

AssessmentofchromatogramqualityA:ContainsunincorporatednucleotidesandprimerswhichdonotbindtothecolumB:ContainsprimerdimersC:MaybecausedeitherbyTaqerrorsduringPCRorbynon-templateAaddition.D:Themajorityofwildtypesamplesappearasasinglepeak.E:ACNabsorbsat260nm.AnyhighMWcontaminantsappearasspikesonthispeak.Typicalhomozygouswild-typechromatogramACBDERetentiontime(minutes)A260nmInjectionpeakACNwashpeakSamplepeakControlsCheckthemutationstandardsandseeiftheyfulfilthecriteriadescribedinSOP-O&M.Checktheknownsequencevariants(positivecontrols).Iftheydonotpresentanaberrantchromatogramattheoptimalscreeningtemperature,theresultsshouldberejectedandtheanalysisrepeatedIdeallythesignalintensityofDHPLCprofilesshouldbe>2mVatA260.Peaksofintensity>30%oftheaveragepeakintensity.Weakpeaksaremorelikelytoleadtofalse-negative/positiveresults.MinimumpeakintensityIdentificationofsequencevariantsThepresenceofheteroduplexesisoftendetectedasachangeinthenumberofpeaks(maybe2,3or4peakpattern).Twopeakpatternsaccountforthemajorityofmutations.Completeresolutionofthe2heteroduplexesisnotalwaysnecessary.Mutationsmayappearonlyasaslightbroadeningofthesinglepeak,orasasubtlechangetoashoulderonthepeak.AllsamplesidentifiedasheteroduplexesbyDHPLCanalysismustbesequencedinbothdirectionstoconfirmanddeterminethenatureofthesequencechange.Thehomoduplexwild-typepatternistypically1peak,butmaybe2peaks,dependinguponthemeltingprofile.Elutionprofilesthatdifferfromthewild-typeindicatethepresenceofDNAsequencechanges.Butthemutationtypecannotbepredictedfromtheheteroduplexpattern.EachmutationinagivenPCRfragmentispredictedtohaveauniqueheteroduplexpattern(highlyspecificelutionprofile).Thisisusefulforquickgenotypingofunknownsamplesbycomparisonwithpositivecontrolsamples.However,traceprofilesarenotalwaysuniqueforaspecificmutation,i.e.differentDNAvariantscangiveidenticalprofiles.Changesinretentiontimedonotaccuratelypredictthepresenceofasequencechange.TracespecificityDatachecking,reportingandstorage

DatacheckingPositiveresultsFalsepositiveresultsNegativeresultsFalsenegativeresultsSensitivityDetectionofmosaicsArchiving

SupplementaryAppendix3STANDARDOPERATINGPROCEDUREMECP2SOP-MECP2

DHPLCscreeningofMECP2InthecontextofRettSyndromeRettsyndrome

Childhoodneurodevelopmentdisorderwithaprevalenceof1/10.000to1/15.000infemalebirthsMutationsintheMECP2gene,codingforMethylCpGBindingProtein2,aretheprimarycauseofRTT

Eightmutationsarerecurrentlyfoundindifferentpopulations.ThefirstpartofthemoleculardiagnosisofRTTistheDHPLC-screeningofexons2,3and4ofMECP2.Thisallowstheidentificationofmorethan90%ofalldescribedmutations.Materials

Worksheet:-

LotNo.ofallproducts-

Equipmentidentifiers-

Patient-identifier-

Performingtechnician(s)-

DateofexperimentsMaterialsPCR

-

StandardPCRequipment(Locationxxx)

-

Optimase™DNApolymerase(2.5U/µl)(Locationxxx)

-

Optimase™PCRbufferwithMg2+

(Locationxxx)

-

Primers(Eurogentec)(stock)as250pmol/µl.(appendixA)(Locationxxx)

-

Primerworksolutionscontain2.5pmol/µlofeachprimer(Locationxxx)

-

PuredNTPs(withoutdUTP)(2mM)(Locationxxx)

-

PCRsystemMaterials

DHPLCsystem

-

StandardDHPLCmaterial(forpartnumbersseeappendixCinSOP-O&M)-

WAVE®System3500HT,WAVEMAKER™4.1.44&HSM3.0-2.1build2.

Patientmaterial

-

PatientDNA

-

Positivecontrols

-

Negativecontrols

-

Normalcontrols

UsesofPlasmidControlsasReferenceReagents

noethicalproblems renewableresource CanusesamereferencereagentascontrolforPCR, heteroduplexandmutationdetectionanalysisUniversalreagentswhichcanbeincorporatedinQC proceduresandSOPsAdvantages:

Validationofnewprotocols

Exonspecificwildtypeandmutatedcontrolsforexistingassays

Validationoftransferofprotocolsbetweenmachines/labsUses:Method

Pre-PCR

PCRComposition

PCRConditions

PostPCR

Heteroduplexformation

Agarosegelelectrophoresis

DHPLC

Interpretationoftheresults

ThemutationstandardsatthebeginningandendoftherunareevaluatedAllpositivecontrolsshouldbevisibleattheirspecifictemperatureIfoneofthecontrolsdoesnotfulfillthecriteria,negativeresultsarenotvalidandhavetoberepeated.Positiveresultscanbeprocessedasusual.TheelutionprofilesofaspecificfragmentfromthedifferentpatientsarecomparedwitheachotherandscoredaccordingtothegeneralDHPLCcriteria.Theminimumpeakheightmustbe2mV.Anyparticularobservationshouldbenotedonworksheetsortechnicalreports.Ampliconswithanaberrantelutionpatternarere-analysedbydirectsequencingonanindependentampliconInterpretationoftheresults

Allprematuretruncationmutationsareimmediatelyreportable.Thepathogenicityofthemissensemutationswilldependonthepositionandthetype.Interpretationisthensubjecttogoodpracticeandliteraturereview.MutationsinthetwohighlyconservedMeCP2domains,themethylbindingdomainandthetranscriptionrepressiondomain,arelikelytobecausative.Ifamutationisofunknownsignificance,samplesshouldbeobtainedfromthepatient’sparents.Ifthemutationisfoundtobedenovo,itislikelytobecausative.Ifthemutationispresentinthemother,X-inactivationstudiesneedtobecarriedoutonthemotherofthepatientIfboththemotherandthedaughterhaverandomX-inactivationthemutationisunlikelytobecausative.Reportingprocedures

NEGATIVERESULTINAFEMALENEGATIVERESULTINAMALENORMALPARENTPOSITIVERESULTINAFEMALENEGATIVERESULTINAFEMALE

RettsyndromeiscausedbymutationsintheMECP2gene.Molecularanalysisofthisgenehasbeencarriedoutonpatient******,howevernocausativemutationhasbeenfound.

DHPLCanalysiswasusedtoscreenformutationsintheMECP2gene.Thistechniquehasasensitivityof>95%forthedetectionofpointmutations,microdeletionsandmicroinsertionsbutwillnotdetectgrossdeletionsofentireexons.Only~80%ofRettsyndromepatientshaveadetectablemutationwithintheMECP2gene.NEGATIVERESULTINAMALE

MolecularanalysisoftheMECP2genehasbeencarriedoutonpatient******.Howevernocausativemutationhasbeenfound.

DHPLCanalysiswasusedtoscreenformutationsintheMECP2gene.Thistechniquehasasensitivityof>95%forthedetectionofpointmutations,microdeletionsandmicroinsert