应用DHPLC技术进行诊断性分析的质量保证体系

1、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分析的要要求临床分子子遗传学学分析的的复杂性性临床分子子检测结结果的一一致性和和精确性性变性高效效液相色色谱(DHPLC)作为一种种高效和和敏感的的基因突突变检测测技术DHPLC技术质量量控制AMERICANCOLLEGE OF MEDICALGENETICSStandardsand Guidelinesfor Clinical Genetics Laboratories2005 EditionG:CLINICALMOLECULARGENETICSTheseStandardsand Guidelinesspe

2、cificallyrefertothe useofmoleculartechniquestoexamine heritableorsomatic changesinthe human genome.G18Denaturing HighPerformanceLiquid Chromatography (dHPLC)(SectionAddedNovember2003)CMGSBest Practice GuidelinesUseoftheWAVESysteminDiagnosticServicePreparedandedited by JohnHarvey,NationalGeneticsRefe

3、renceLaboratory(Wessex),Salisbury,UKandEls Schollen,Centrefor Human Genetics,Leuven, Belgiumlast update:12March2004IntroductionLaboratoryprocessDHPLCsystemDataqualityChecking&reportingguidelinesReferencesDHPLCSOPsInstrument or maintenanceSOPTechniqueGeneral DHPLC SOPWAVE 3500, 3500HTMethodDisease-sp

4、ecific SOPsRett,BRCA,HNPCCMarfan, Applicationcompany +usersgeneral users +companyspecificusersSupplementary Appendix 1STANDARDOPERATINGPROCEDURE WAVE SystemOperationand MaintenanceSOP- O&MWAVESystemOperation andMaintenanceForWAVESystemModels 3500, 3500A and3500HTWAVESystemOperation andMaintenanceAna

5、lysisoftheWAVELow &HighRangeMutationStandardsThemaintenanceprocedureDNASep andDNASep HT cartridgemaintenanceRole of Mutation standards:checkingofcorrect functioning of theWAVESystem, includingoven calibration,cartridgeperformance, buffercompositionandstability,toensurereproducibility andaccuracyofth

6、echromatographic analysis.Mutationstandardsberun when:lTheroutinepre-run,lWeeklyand monthlymaintenanceprocedure,lAfterreplacementofanycomponent,lValidation fora newbatch,lAsanassaycontrol,atthebeginning andendofeveryrun,preferablyalso after every 100injections forlong runs.AnalysisoftheWAVELow &High

7、RangeMutationStandardsNormalrangesofthemutationstandardsThemaintenanceprocedure3.1Filterandflush3.2Pre-runmaintenance3.3Weeklymaintenance3.4Quarterlymaintenance3.5Other maintenance operations3.6Preventativemaintenanceprocedure andsystemvalidationFilterand flushTheprinciple of filtrationinvolvespreve

8、ntingunwantedcontaminantsfromenteringthesystem.Filtration appliestotwo specific areas:solvent filtrationand in-linefiltration.Thesystem flushing is to removemobilephasesaltcomponents thatcan precipitate under strongsolvent conditions.Pre-run maintenance1.Buffercheck2.Injectionsystem washing3.Pressur

9、echeck4.Checktheabsorbanceonthedetector5.PurgethelinesWeeklymaintenanceInlinefilter replacementCheckthesyringeQuarterlymaintenanceCheckUVlampUVlamp replacementCleaningthesystem (Isopropanolcleaning)DNASep andDNASep HT cartridgemaintenance1.Regular maintenance scheduleEvery96-192injections:Extendedho

10、t washEvery1000 injections:Reverse hotwashDNASep wash(if reversehot washfailstoresolvemutationstandards)2.Short-termcartridgestorage3.Long-Term cartridgestorage4.New cartridgeinstallationDailyMaintenanceEquilibratethecartridge50%A 50%Bfor 15 minutesRun1-2 blanksVerifysystem performance (pre-analysis

11、)RunastandardRunSamplesVerifysystem performance (post analysis)WeeklyMaintenanceAnExtendedActiveCleanWashisrecommendedevery100 injections.(Usuallydone after each96wellplate)OvenSet to:80 CPump Set to:100% D15-30minutesWASHOvenSet to:56 CPump Set to:50%A-50%BEQUILIBRATE45-90minutesRunStandards to Ver

12、ifySystemPerformance!1000InjectionMaintenanceA ReverseHot Washisrecommendedevery1000injectionsUV/FLDetectorTurn offthepumpReverse thecartridgedirection.Setthe ovento80C.Setthe pumpto100%D.60-90minutesStoring theCartridgeFlushthecartridge with100%DBuffer.Removethe cartridgefrom theWAVE System.Capthe

13、cartridgewith endplugs.Storethecartridge at roomtemperature.Installing aNew CartridgeStop thepump flowand removetheold cartridge.Removethe plugs fromthe newcartridge.Install thenewcartridge withthe arrow pointing towardtherearoftheoven.UV/FLDetectorMake surethe ovenheatsuptoatleast40C.Setthe pumpto1

14、00%D0.500mL/min.Ensurethe pressure is stableandgradually increase theflow (0.9mL/minor1.5mL/min).Flushthecartridge for15minutes.Setthe pumpto50% BufferA,50%Buffer Band equilibrate for30minutes.Equilibrating aNew Cartridge100%50% 50%VerifyNew CartridgePerformanceLow-RangeMutationStandardDNASizing Con

15、trolStandardSupplementary Appendix 2STANDARDOPERATINGPROCEDURE DHPLCSOP-DHPLCDHPLCmutationdetectiononTransgenomic WAVESystem3500Wavemaker 4.1.44& HSM3.0-2.1 (build2)Navigator 1.5.4 (build19)PCRrequirementsPrimerdesignTemplatepurityand concentrationDNAPolymerasesPCRbuffer mixPCRplatesPCRqualityandPro

16、ductmixingPost-PCR,filmuseControlsPrimerDesignUseaprimer-pickingprogramPrimers shouldideally be no closerthan 30 -50bpfromtheend of thesequencetobeanalyzedformutationsPrimers shouldbe18- 30 bp in lengthTheTmdifference betweenprimersina pairshould ideallybelessthan 2C.Size of PCRfragmentTheoptimalsiz

17、e range fordetectingmutation/SNPsbyDHPLCwith 100%accuracyis150 -500bp.Fragments500bpcanbegenerated butsensitivitydecreasedand timeofelutionincreased.Forfragments 150bp, differenceofmeltingpointbetween fragmentstoonarrow (thefragments meltovertoonarrow atemperaturerange).QuantityofPCRfragmentThePCR p

18、roductshould be sufficientlyconcentratedthat2 l runonanagarose gelproducesa clearlyvisibleband ( 20 ng/l)Dilutesamples(verylowyields)produce poorqualityresults (poor signal:noiseratio).Very highyields canlead to large proportions of misincorporations andhenceincreaseddifficultyincalling mutations.Us

19、ually 3-10l(50-200ng)ofunpurifiedPCRproductwouldbeinjectedonto thecolumn(peronetemperature).A 8lminimumaliquot of PCRproduct shouldbesuppliedinPCRtubesinstrips of 8(peronetemperature).SamplePreparationfor DHPLCDNAmustbeclean,all cellular debrisandorganiccompoundsmustberemoved.Salting outmethodispref

20、erred.DNAextracted withsomecommercial systemsmay be dilutedto10ngtoreduce effectsofimpuritiestoPCR.DNAoflow qualitywillresultinsub-optimalPCR results(hence DHPLC profiles).DNAquality& concentrarionTable1.Recommendedcleaningprocedures forDNAextraction.Isolation MethodRecommended Additional Cleaning:O

21、rganic Extraction(e.g. phenol/chloroform)Chloroform/isoamyl back-extraction followed by ethanol precipitation and washChaotropic Salts(e.g. guanidinium isothiocyanate)Ethanol precipitation and washSpin ColumnEthanol precipitation and washTable2.RecommendedDNAquantitiesused forPCR(50L reaction).Templ

22、ateRecommended QuantityHuman genomic DNA50-200 ng Phage DNA 1-10 pg Plasmid DNA 0.1-1.0 pgTheImportanceofPolymerase Fidelity forMutationDetectionImportance of highfidelityindHPLC500bpWild TypeFragmentRedTrace Optimase PolymeraseGreenTrace 9:1MixAmplitaqGoldandPfu TurboHeteroduplexdue to misincorpora

23、tionPolymerase Fidelity ComparisonMaximum recommended concentrationsofacceptable PCRadditivesAcceptable additives (maximum final concentration)Additives where final concentrationmust be 2mVatA260.Peaksofintensity30%ofthe averagepeakintensity.Weak peaks aremore likelytolead to false-negative/positive

24、 results.Minimum peakintensityIdentificationofsequencevariantsThepresenceofheteroduplexesisoftendetectedasachangeinthe numberofpeaks(may be 2, 3or4peak pattern).Twopeakpatternsaccount forthemajorityofmutations.Completeresolution of the2 heteroduplexes is notalwaysnecessary.Mutationsmay appearonly as

25、 aslight broadeningofthe singlepeak,orasa subtlechangetoashoulderonthepeak.Allsamplesidentified as heteroduplexes by DHPLC analysis mustbesequenced in bothdirectionstoconfirm anddeterminethe natureofthesequencechange.Thehomoduplexwild-typepatternistypically1peak,butmay be 2peaks, dependingupon theme

26、lting profile.Elution profiles thatdiffer fromthe wild-typeindicatethepresenceofDNA sequence changes. Butthemutationtypecannotbepredicted fromthe heteroduplexpattern.Each mutation in agivenPCR fragment is predictedtohave aunique heteroduplexpattern (highlyspecificelutionprofile).Thisisusefulfor quic

27、k genotypingofunknownsamples by comparisonwithpositivecontrol samples.However,traceprofilesarenot alwaysuniquefor aspecificmutation, i.e.different DNAvariantscangiveidenticalprofiles.Changes in retentiontime do notaccurately predictthe presence of asequencechange.TracespecificityData checking,report

28、ingand storageData checkingPositiveresultsFalsepositiveresultsNegativeresultsFalsenegativeresultsSensitivityDetectionofmosaicsArchivingSupplementary Appendix 3STANDARDOPERATINGPROCEDUREMECP2SOP-MECP2DHPLCscreeningofMECP2InthecontextofRett SyndromeRett syndromeChildhoodneurodevelopmentdisorderwith ap

29、revalenceof1/10.000to1/15.000infemalebirthsMutationsintheMECP2gene,codingfor MethylCpGBindingProtein 2, aretheprimarycauseofRTTEightmutationsare recurrently found in differentpopulations.Thefirstpartofthemolecular diagnosisofRTTisthe DHPLC-screeningofexons2,3 and4 ofMECP2. Thisallows theidentificati

30、onofmore than90% of alldescribedmutations.MaterialsWorksheet:-LotNo. of allproducts-Equipmentidentifiers-Patient-identifier-Performing technician(s)-Date of experimentsMaterialsPCR-StandardPCRequipment(Locationxxx)-OptimaseDNA polymerase(2.5U/l)(Locationxxx)-OptimasePCR bufferwith Mg2+(Locationxxx)-

31、Primers (Eurogentec)(stock)as250 pmol/l. (appendixA)(Locationxxx)-Primerworksolutionscontain2.5pmol/lofeach primer(Locationxxx)-Pure dNTPs (without dUTP) (2mM)(Locationxxx)-PCRsystemMaterialsDHPLCsystem-StandardDHPLCmaterial(for partnumbersseeappendixCinSOP-O&M)-WAVESystem3500HT,WAVEMAKER4.1.44& HSM

32、3.0-2.1build2.Patient material-Patient DNA-Positivecontrols-Negativecontrols-NormalcontrolsUses of PlasmidControlsasReference Reagents no ethical problems renewable resourceCan use same reference reagent as control for PCR, heteroduplex and mutation detection analysis Universal reagents which can be

33、 incorporated in QC procedures and SOPsAdvantages: Validation of new protocols Exon specific wild type and mutated controls for existing assays Validation of transfer of protocols between machines/labsUses:MethodPre-PCRPCRCompositionPCRConditionsPost PCRHeteroduplexformationAgarose gelelectrophoresi

34、sDHPLCInterpretationoftheresultsThemutationstandards at thebeginningand endoftherun areevaluatedAllpositivecontrolsshould be visibleattheirspecifictemperatureIfoneofthe controls doesnot fulfillthe criteria,negativeresults arenotvalidand havetoberepeated. Positive resultscan be processedasusual.Theel

35、utionprofilesofa specific fragment fromthe differentpatientsarecomparedwitheach other andscoredaccording to thegeneral DHPLC criteria.Theminimumpeak heightmust be 2mV.Any particularobservationshould be noted on worksheetsortechnical reports.Ampliconswithanaberrantelution patternare re-analysed by di

36、rectsequencing on an independent ampliconInterpretationoftheresultsAllpremature truncationmutations areimmediatelyreportable.Thepathogenicityofthemissensemutations willdepend on thepositionandthe type. Interpretation is thensubjecttogood practice andliterature review.Mutations in thetwohighly conser

37、vedMeCP2domains,themethyl bindingdomain andthetranscriptionrepression domain,are likelytobecausative.Ifa mutation is of unknownsignificance,samples shouldbeobtainedfrom thepatientsparents.Ifthe mutation is found to bedenovo, it is likelytobecausative. If themutationispresent in themother, X-inactiva

38、tion studiesneedtobecarried outonthemother of thepatient If boththe motherandthe daughter haverandom X-inactivation themutationisunlikelytobecausative.ReportingproceduresNEGATIVERESULTINAFEMALENEGATIVERESULTINAMALENORMALPARENTPOSITIVERESULTINAFEMALENEGATIVERESULTINAFEMALERett syndrome is causedbymut

39、ationsintheMECP2gene.Molecularanalysisofthisgene hasbeen carriedout on patient*,however no causativemutationhasbeenfound.DHPLCanalysiswasusedtoscreenfor mutationsintheMECP2gene.This techniquehasasensitivityof95% forthedetection of point mutations,microdeletionsand microinsertionsbutwillnotdetect gross deletionsofentireexons. Only80%ofRett syndrome patients haveadetectable mutation withintheMECP2gene.NEGATIVERESULTINAMALEMolecularanalysisoftheMECP2gene hasbeen carriedout