应用DHPLC技术进行诊断性分析的质量保证体系课件.ppt

1、路漫漫其修远兮路漫漫其修远兮,吾将上下而求索吾将上下而求索2022年10月30日星期日应用应用DHPLC技术进行诊技术进行诊断性分析的质量保证体断性分析的质量保证体系系路漫漫其修远兮路漫漫其修远兮,吾将上下而求索吾将上下而求索诊断性分析的要求诊断性分析的要求n临床分子遗传学分析的复杂性临床分子遗传学分析的复杂性n临床分子检测结果的一致性和精确性临床分子检测结果的一致性和精确性 n变性高效液相色谱变性高效液相色谱(DHPLC)(DHPLC)作为一种高效和敏作为一种高效和敏感的基因突变检测技术感的基因突变检测技术nDHPLCDHPLC技术质量控制技术质量控制路漫漫其修远兮路漫漫其修远兮,吾将上下而

2、求索吾将上下而求索AMERICAN COLLEGE OF MEDICAL GENETICSStandards and Guidelines for Clinical Genetics Standards and Guidelines for Clinical Genetics LaboratoriesLaboratories2005 Edition G:CLINICAL MOLECULAR GENETICSG:CLINICAL MOLECULAR GENETICS These Standards and Guidelines specifically refer to the use of m

3、olecular techniques to examine heritable or somatic changes in the human genome.G18G18Denaturing High Performance Liquid Chromatography(dHPLC)Denaturing High Performance Liquid Chromatography(dHPLC)(Section Added November 2003)(Section Added November 2003)路漫漫其修远兮路漫漫其修远兮,吾将上下而求索吾将上下而求索CMGS Best Pract

4、ice Guidelines Use of the WAVE System in Diagnostic ServicePrepared and edited by John Harvey,National Genetics Reference Laboratory(Wessex),Salisbury,UK and Els Schollen,Centre for Human Genetics,Leuven,Belgiumlast update:12 March 2004Introduction Laboratory process DHPLC system Data quality Checki

5、ng&reporting guidelines References 路漫漫其修远兮路漫漫其修远兮,吾将上下而求索吾将上下而求索路漫漫其修远兮路漫漫其修远兮,吾将上下而求索吾将上下而求索DHPLC SOPsnInstrument or maintenance SOPuTechniquenGeneral DHPLC SOPWAVE 3500,3500 HTuMethodnDisease-specific SOPsRett,BRCA,HNPCCMarfan,uApplication company+users general users+company specific users路漫漫其修远兮路

6、漫漫其修远兮,吾将上下而求索吾将上下而求索 Supplementary Appendix 1 STANDARD OPERATING PROCEDURE WAVE System Operation and Maintenance SOP-O&M WAVE System Operation and MaintenanceFor WAVE System Models 3500,3500A and 3500HT路漫漫其修远兮路漫漫其修远兮,吾将上下而求索吾将上下而求索WAVE System Operation and Maintenance Analysis of the WAVE Low&High

7、Range Mutation StandardsThe maintenance procedureDNASep and DNASep HT cartridge maintenance 路漫漫其修远兮路漫漫其修远兮,吾将上下而求索吾将上下而求索 Role of Mutation standards:checking of correct functioning of the WAVE System,including oven calibration,cartridge performance,buffer composition and stability,to ensure reproduc

8、ibility and accuracy of the chromatographic analysis.Mutation standards be run when:l The routine pre-run,l Weekly and monthly maintenance procedure,l After replacement of any component,l Validation for a new batch,l As an assay control,at the beginning and end of every run,preferably also after eve

9、ry 100 injections for long runs.Analysis of the WAVE Low&High Range Mutation Standards 路漫漫其修远兮路漫漫其修远兮,吾将上下而求索吾将上下而求索路漫漫其修远兮路漫漫其修远兮,吾将上下而求索吾将上下而求索Normal ranges of the mutation standards路漫漫其修远兮路漫漫其修远兮,吾将上下而求索吾将上下而求索The maintenance procedure3.1 Filter and flush3.2 Pre-run maintenance3.3 Weekly maintena

10、nce3.4 Quarterly maintenance3.5 Other maintenance operations3.6 Preventative maintenance procedure and system validation 路漫漫其修远兮路漫漫其修远兮,吾将上下而求索吾将上下而求索Filter and flush nThe principle of filtration involves preventing unwanted contaminants from entering the system.nFiltration applies to two specific a

11、reas:solvent filtration and in-line filtration.nThe system flushing is to remove mobile phase salt components that can precipitate under strong solvent conditions.路漫漫其修远兮路漫漫其修远兮,吾将上下而求索吾将上下而求索Pre-run maintenance1.Buffer check2.Injection system washing3.Pressure check4.Check the absorbance on the det

12、ector5.Purge the lines 路漫漫其修远兮路漫漫其修远兮,吾将上下而求索吾将上下而求索Weekly maintenancenInline filter replacement nCheck the syringe 路漫漫其修远兮路漫漫其修远兮,吾将上下而求索吾将上下而求索Quarterly maintenance nCheck UV lamp nUV lamp replacement nCleaning the system(Isopropanol cleaning)路漫漫其修远兮路漫漫其修远兮,吾将上下而求索吾将上下而求索DNASep and DNASep HT cartr

13、idge maintenance 1.Regular maintenance schedule Every 96-192 injections:Extended hot wash Every 1000 injections:Reverse hot wash DNASep wash(if reverse hot wash fails to resolve mutation standards)2.Short-term cartridge storage 3.Long-Term cartridge storage 4.New cartridge installation路漫漫其修远兮路漫漫其修远兮

14、,吾将上下而求索吾将上下而求索Daily MaintenancenEquilibrate the cartridge50%A 50%B for 15 minutesRun 1-2 blanksnVerify system performance(pre-analysis)Run a standardnRun SamplesnVerify system performance(post analysis)路漫漫其修远兮路漫漫其修远兮,吾将上下而求索吾将上下而求索Weekly MaintenanceAn Extended Active Clean Wash is recommended every

15、 100 injections.(Usually done after each 96 well plate)OvenSet to:80 CPump Set to:100%D15-30 minutesWASHOvenSet to:56 CPump Set to:50%A-50%BEQUILIBRATE45-90 minutesRun Standards to Verify System Performance!路漫漫其修远兮路漫漫其修远兮,吾将上下而求索吾将上下而求索1000 Injection Maintenance A Reverse Hot Wash is recommended eve

16、ry 1000 injectionsUV/FLDetector Turn off the pump Reverse the cartridge direction.Set the oven to 80 C.Set the pump to 100%D.60-90 minutes路漫漫其修远兮路漫漫其修远兮,吾将上下而求索吾将上下而求索Storing the CartridgenFlush the cartridge with 100%D Buffer.nRemove the cartridge from the WAVE System.nCap the cartridge with end pl

17、ugs.nStore the cartridge at room temperature.路漫漫其修远兮路漫漫其修远兮,吾将上下而求索吾将上下而求索Installing a New Cartridge1.Stop the pump flow and remove the old cartridge.2.Remove the plugs from the new cartridge.3.Install the new cartridge with the arrow pointing toward the rear of the oven.UV/FLDetector路漫漫其修远兮路漫漫其修远兮,

18、吾将上下而求索吾将上下而求索1.Make sure the oven heats up to at least 40 C.Set the pump to 100%D 0.500mL/min.2.Ensure the pressure is stable and gradually increase the flow(0.9mL/min or 1.5mL/min).3.Flush the cartridge for 15 minutes.4.Set the pump to 50%Buffer A,50%Buffer B and equilibrate for 30 minutes.Equilib

19、rating a New Cartridge100%50%50%路漫漫其修远兮路漫漫其修远兮,吾将上下而求索吾将上下而求索Verify New Cartridge PerformanceLow-Range Mutation StandardDNA Sizing Control Standard路漫漫其修远兮路漫漫其修远兮,吾将上下而求索吾将上下而求索 Supplementary Appendix 2 STANDARD OPERATING PROCEDURE DHPLC SOP-DHPLC DHPLC mutation detection on Transgenomic WAVE System

20、3500 Wavemaker 4.1.44&HSM 3.0-2.1(build 2)Navigator 1.5.4(build 19)路漫漫其修远兮路漫漫其修远兮,吾将上下而求索吾将上下而求索 nPrimer design nTemplate purity and concentrationnDNA PolymerasesnPCR buffer mixnPCR platesnPCR quality and Product mixingnPost-PCR,film usenControls路漫漫其修远兮路漫漫其修远兮,吾将上下而求索吾将上下而求索Primer DesignnUse a prime

21、r-picking programnPrimers should ideally be no closer than 30-50 bp from the end of the sequence to be analyzed for mutationsnPrimers should be 18-30 bp in lengthnThe Tm difference between primers in a pair should ideally be less than 2 C.路漫漫其修远兮路漫漫其修远兮,吾将上下而求索吾将上下而求索Size of PCR fragment Size of PCR

22、 fragment nThe optimal size range for detecting mutation/SNPs by DHPLC with 100%accuracy is 150-500bp.nFragments 500 bp can be generated but sensitivity decreased and time of elution increased.nFor fragments 150 bp,difference of melting point between fragments too narrow(the fragments melt over too

23、narrow a temperature range).路漫漫其修远兮路漫漫其修远兮,吾将上下而求索吾将上下而求索Quantity of PCR fragment Quantity of PCR fragment nThe PCR product should be sufficiently concentrated that 2 l run on an agarose gel produces a clearly visible band(20 ng/l)nDilute samples(very low yields)produce poor quality results(poor sig

24、nal:noise ratio).nVery high yields can lead to large proportions of misincorporations and hence increased difficulty in calling mutations.nUsually 3-10 l(50-200ng)of unpurified PCR product would be injected onto the column(per one temperature).nA 8 l minimum aliquot of PCR product should be supplied

25、 in PCR tubes in strips of 8(per one temperature).路漫漫其修远兮路漫漫其修远兮,吾将上下而求索吾将上下而求索Sample Preparation for DHPLCSample Preparation for DHPLCnDNA must be clean,all cellular debris and organic compounds must be removed.nSalting out method is preferred.nDNA extracted with some commercial systems may be dilu

26、ted to 10 ng to reduce effects of impurities to PCR.nDNA of low quality will result in sub-optimal PCR results(hence DHPLC profiles).DNA quality&concentrarion路漫漫其修远兮路漫漫其修远兮,吾将上下而求索吾将上下而求索Table 1.Recommended cleaning procedures for DNA extraction.Isolation MethodRecommended Additional Cleaning:Organi

27、c 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 washTable 2.Recommended DNA quantities used for PCR(50 L reaction).Templa

28、teRecommended QuantityHuman genomic DNA50-200 ng Phage DNA 1-10 pg Plasmid DNA 0.1-1.0 pg路漫漫其修远兮路漫漫其修远兮,吾将上下而求索吾将上下而求索The Importance of Polymerase Fidelity for Mutation DetectionnImportance of high fidelity in dHPLC500 bp Wild Type FragmentRed Trace Optimase PolymeraseGreen Trace 9:1 Mix Amplitaq Go

29、ld and Pfu TurboHeteroduplex due to misincorporation路漫漫其修远兮路漫漫其修远兮,吾将上下而求索吾将上下而求索Polymerase Fidelity Comparison路漫漫其修远兮路漫漫其修远兮,吾将上下而求索吾将上下而求索Maximum recommended concentrationsMaximum recommended concentrationsof acceptable PCR additivesof acceptable PCR additivesAcceptable additives(maximum final con

30、centration)Additives where final concentrationmust be 2 mV at A260.nPeaks of intensity 30%of the average peak intensity.nWeak peaks are more likely to lead to false-negative/positive results.Minimum peak intensityMinimum peak intensity路漫漫其修远兮路漫漫其修远兮,吾将上下而求索吾将上下而求索Identification of sequence variantsI

31、dentification of sequence variantsnThe presence of heteroduplexes is often detected as a change in the number of peaks(may be 2,3 or 4 peak pattern).nTwo peak patterns account for the majority of mutations.nComplete resolution of the 2 heteroduplexes is not always necessary.nMutations may appear onl

32、y as a slight broadening of the single peak,or as a subtle change to a shoulder on the peak.nAll samples identified as heteroduplexes by DHPLC analysis must be sequenced in both directions to confirm and determine the nature of the sequence change.路漫漫其修远兮路漫漫其修远兮,吾将上下而求索吾将上下而求索nThe homoduplex wild-ty

33、pe pattern is typically 1 peak,but may be 2 peaks,depending upon the melting profile.nElution profiles that differ from the wild-type indicate the presence of DNA sequence changes.But the mutation type cannot be predicted from the heteroduplex pattern.nEach mutation in a given PCR fragment is predic

34、ted to have a unique heteroduplex pattern(highly specific elution profile).This is useful for quick genotyping of unknown samples by comparison with positive control samples.nHowever,trace profiles are not always unique for a specific mutation,i.e.different DNA variants can give identical profiles.n

35、Changes in retention time do not accurately predict the presence of a sequence change.Trace specificityTrace specificity路漫漫其修远兮路漫漫其修远兮,吾将上下而求索吾将上下而求索 nData checkingnPositive resultsnFalse positive resultsnNegative resultsnFalse negative resultsnSensitivitynDetection of mosaicsnArchiving 路漫漫其修远兮路漫漫其修

36、远兮,吾将上下而求索吾将上下而求索Supplementary Appendix 3STANDARD OPERATING PROCEDURE MECP2SOP-MECP2 DHPLC screening of MECP2In the context of Rett Syndrome路漫漫其修远兮路漫漫其修远兮,吾将上下而求索吾将上下而求索Rett syndrome n Childhood neurodevelopment disorder with a prevalence of 1/10.000 to 1/15.000 in female births nMutations in the ME

37、CP2 gene,coding for Methyl CpG Binding Protein 2,are the primary cause of RTT n Eight mutations are recurrently found in different populations.nThe first part of the molecular diagnosis of RTT is the DHPLC-screening of exons 2,3 and 4 of MECP2.This allows the identification of more than 90%of all de

38、scribed mutations.路漫漫其修远兮路漫漫其修远兮,吾将上下而求索吾将上下而求索Materials nWorksheet:-Lot No.of all products -Equipment identifiers -Patient-identifier -Performing technician(s)-Date of experiments路漫漫其修远兮路漫漫其修远兮,吾将上下而求索吾将上下而求索MaterialsnPCR -Standard PCR equipment(Location xxx)-Optimase DNA polymerase(2.5 U/l)(Locati

39、on xxx)-Optimase PCR buffer with Mg2+(Location xxx)-Primers(Eurogentec)(stock)as 250 pmol/l.(appendix A)(Location xxx)-Primer work solutions contain 2.5 pmol/l of each primer(Location xxx)-Pure dNTPs(without dUTP)(2mM)(Location xxx)-PCR system路漫漫其修远兮路漫漫其修远兮,吾将上下而求索吾将上下而求索Materials nDHPLC system -Sta

40、ndard DHPLC material(for part numbers see appendix C in SOP-O&M)-WAVE System 3500 HT,WAVEMAKER 4.1.44&HSM3.0-2.1 build2.n Patient material -Patient DNA -Positive controls -Negative controls -Normal controls 路漫漫其修远兮路漫漫其修远兮,吾将上下而求索吾将上下而求索Uses of Plasmid Controls as Reference Reagents no ethical proble

41、ms renewable resourceCan use same reference reagent as control for PCR,heteroduplex and mutation detection analysis Universal reagents which can be incorporated in QC procedures and SOPsAdvantages:Validation of new protocols Exon specific wild type and mutated controls for existing assays Validation

42、 of transfer of protocols between machines/labsUses:路漫漫其修远兮路漫漫其修远兮,吾将上下而求索吾将上下而求索Method n Pre-PCR PCR Composition PCR Conditions nPost PCR Heteroduplex formation Agarose gel electrophoresis n DHPLC 路漫漫其修远兮路漫漫其修远兮,吾将上下而求索吾将上下而求索Interpretation of the results nThe mutation standards at the beginning an

43、d end of the run are evaluated nAll positive controls should be visible at their specific temperature nIf one of the controls does not fulfill the criteria,negative results are not valid and have to be repeated.Positive results can be processed as usual.nThe elution profiles of a specific fragment f

44、rom the different patients are compared with each other and scored according to the general DHPLC criteria.The minimum peak height must be 2mV.Any particular observation should be noted on worksheets or technical reports.nAmplicons with an aberrant elution pattern are re-analysed by direct sequencin

45、g on an independent amplicon 路漫漫其修远兮路漫漫其修远兮,吾将上下而求索吾将上下而求索Interpretation of the results nAll premature truncation mutations are immediately reportable.nThe pathogenicity of the missense mutations will depend on the position and the type.Interpretation is then subject to good practice and literature

46、review.Mutations in the two highly conserved MeCP2 domains,the methyl binding domain and the transcription repression domain,are likely to be causative.nIf a mutation is of unknown significance,samples should be obtained from the patients parents.If the mutation is found to be de novo,it is likely t

47、o be causative.If the mutation is present in the mother,X-inactivation studies need to be carried out on the mother of the patient If both the mother and the daughter have random X-inactivation the mutation is unlikely to be causative.路漫漫其修远兮路漫漫其修远兮,吾将上下而求索吾将上下而求索Reporting procedures nNEGATIVE RESUL

48、T IN A FEMALE nNEGATIVE RESULT IN A MALE nNORMAL PARENT nPOSITIVE RESULT IN A FEMALE 路漫漫其修远兮路漫漫其修远兮,吾将上下而求索吾将上下而求索NEGATIVE RESULT IN A FEMALE Rett syndrome is caused by mutations in the MECP2 gene.Molecular analysis of this gene has been carried out on patient*,however no causative mutation has been

49、 found.DHPLC analysis was used to screen for mutations in the MECP2 gene.This technique has a sensitivity of 95%for the detection of point mutations,microdeletions and microinsertions but will not detect gross deletions of entire exons.Only 80%of Rett syndrome patients have a detectable mutation wit

50、hin the MECP2 gene.路漫漫其修远兮路漫漫其修远兮,吾将上下而求索吾将上下而求索NEGATIVE RESULT IN A MALE Molecular analysis of the MECP2 gene has been carried out on patient*.However no causative mutation has been found.DHPLC analysis was used to screen for mutations in the MECP2 gene.This technique has a sensitivity of 95%for th