應用DHPLC技術進行診斷性分析的質量保證體系張澤云美國環(huán)球基因公司中國代表處診斷性分析的要求臨床分子遺傳學分析的復雜性臨床分子檢測結果的一致性和精確性

變性高效液相色譜(DHPLC)作為一種高效和敏感的基因突變檢測技術DHPLC技術質量控制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