應(yīng)用DHPLC技術(shù)進(jìn)行診斷性分析的質(zhì)量保證體系ppt課件

1、運(yùn)用DHPLC技術(shù)進(jìn)展診斷性分析的質(zhì)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(xué)分析的復(fù)雜性臨床分子檢測結(jié)果的一致性和準(zhǔn)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(shù)DHPLC技術(shù)質(zhì)量控制AMERICAN COLLEGE OF MEDICAL GENETICSStandards and Guidelines for Clinical Genetics Laboratories2005 EditionG: CLINICAL MOLECULAR GENETICS These Standards and Guidelines specifical

2、ly refer to the use of molecular techniques to examine heritable or somatic changes in the human genome. G18Denaturing High Performance Liquid Chromatography (dHPLC) (Section Added November 2003)CMGS Best Practice Guidelines Use of the WAVE System in Diagnostic ServicePrepared and edited by John Har

3、vey, 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 Checking & reportingguidelines References DHPLC SOPsInstrument or maintenanceSOPTechniqu

4、eGeneral DHPLC SOPWAVE 3500, 3500 HTMethodDisease-specific SOPsRett, BRCA, HNPCCMarfan, Applicationcompany + usersgeneral users + companyspecific usersSupplementary Appendix 1STANDARD OPERATING PROCEDURE WAVE System Operation and MaintenanceSOP- O&MWAVE System Operation and MaintenanceFor WAVE Syste

5、m Models 3500, 3500A and 3500HTWAVE System Operation and Maintenance Analysis of the WAVE Low & High Range Mutation StandardsThe maintenance procedureDNASep and DNASep HT cartridge maintenance Role of Mutation standards:checking of correct functioning of the WAVE System, including oven calibration,

6、cartridge performance, buffer composition and stability, to ensure reproducibility and accuracy of the chromatographic analysis.Mutation standards be run when:lThe routine pre-run, lWeekly and monthly maintenance procedure, lAfter replacement of any component,lValidation for a new batch, lAs an assa

7、y control, at the beginning and end of every run, preferably also after every 100 injections for long runs. Analysis of the WAVE Low & High Range Mutation Standards Normal ranges of the mutation standardsThe maintenance procedure3.1 Filter and flush3.2 Pre-run maintenance3.3 Weekly maintenance3.4 Qu

8、arterly maintenance3.5 Other maintenance operations3.6 Preventative maintenance procedure and system validation Filter and flush The principle of filtration involves preventing unwanted contaminants from entering the system. Filtration applies to two specific areas: solvent filtration and in-line fi

9、ltration. The system flushing is to remove mobile phase salt components that can precipitate under strong solvent conditions. Pre-run maintenance1. Buffer check2. Injection system washing3. Pressure check4. Check the absorbance on the detector5. Purge the lines Weekly maintenanceInline filter replac

10、ement Check the syringe Quarterly maintenance Check UV lamp UV lamp replacement Cleaning the system (Isopropanol cleaning) DNASep and DNASep HT cartridge maintenance 1. Regular maintenance schedule Every 96-192 injections: Extended hot wash Every 1000 injections: Reverse hot wash DNASep wash (if rev

11、erse hot wash fails to resolve mutation standards) 2. Short-term cartridge storage 3. Long-Term cartridge storage 4. New cartridge installationDaily MaintenanceEquilibrate the cartridge50%A 50%B for 15 minutesRun 1-2 blanksVerify system performance (pre-analysis)Run a standardRun SamplesVerify syste

12、m performance (post analysis)Weekly MaintenanceAn Extended Active Clean Wash is recommended every 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 Perfor

13、mance!1000 Injection Maintenance A Reverse Hot Wash is recommended every 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 minutesStoring the CartridgeFlush the cartridge with 100%D Buffer.Remove the cartridge from the W

14、AVE System.Cap the cartridge with end plugs.Store the cartridge at room temperature.Installing a New CartridgeStop the pump flow and remove the old cartridge.Remove the plugs from the new cartridge.Install the new cartridge with the arrow pointing toward the rear of the oven.UV/FLDetectorMake sure t

15、he oven heats up to at least 40 C. Set the pump to 100%D 0.500mL/min.Ensure the pressure is stable and gradually increase the flow (0.9mL/min or 1.5mL/min).Flush the cartridge for 15 minutes.Set the pump to 50% Buffer A, 50% Buffer B and equilibrate for 30 minutes.Equilibrating a New Cartridge100%50

16、% 50%Verify New Cartridge PerformanceLow-Range Mutation StandardDNA Sizing Control StandardSupplementary Appendix 2STANDARD OPERATING PROCEDURE DHPLCSOP-DHPLCDHPLC mutation detection on Transgenomic WAVE System 3500Wavemaker 4.1.44 & HSM 3.0-2.1 (build 2)Navigator 1.5.4 (build 19) PCR requirementsPr

17、imer design Template purity and concentrationDNA PolymerasesPCR buffer mixPCR platesPCR quality and Product mixingPost-PCR, film useControlsPrimer DesignUse a primer-picking programPrimers should ideally be no closer than 30 - 50 bp from the end of the sequence to be analyzed for mutationsPrimers sh

18、ould be 18 - 30 bp in lengthThe Tm difference between primers in a pair should ideally be less than 2 C.Transgenomic WAVE System Customers can use the Advanced Features of Amplicon Design at mutationdiscoverySize of PCR fragment The optimal size range for detecting mutation/SNPs by DHPLC with 100% a

19、ccuracy is 150 - 500bp.Fragments 500 bp can be generated but sensitivity decreased and time of elution increased.For fragments 150 bp, difference of melting point between fragments too narrow (the fragments melt over too narrow a temperature range).Quantity of PCR fragment The PCR product should be

20、sufficiently concentrated that 2 l run on an agarose gel produces a clearly visible band ( 20 ng/l)Dilute samples (very low yields) produce poor quality results (poor signal:noise ratio).Very high yields can lead to large proportions of misincorporations and hence increased difficulty in calling mut

21、ations.Usually 3-10 l (50-200ng) of unpurified PCR product would be injected onto the column (per one temperature).A 8 l minimum aliquot of PCR product should be supplied in PCR tubes in strips of 8 (per one temperature).Sample Preparation for DHPLCDNA must be clean, all cellular debris and organic

22、compounds must be removed.Salting out method is preferred.DNA extracted with some commercial systems may be diluted to 10 ng to reduce effects of impurities to PCR.DNA of low quality will result in sub-optimal PCR results (hence DHPLC profiles). DNA quality & concentrarionTable 1. Recommended cleani

23、ng procedures for DNA extraction.Isolation MethodRecommended Additional Cleaning:Organic 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 pr

24、ecipitation and washTable 2. Recommended DNA quantities used for PCR (50 L reaction).TemplateRecommended QuantityHuman genomic DNA50-200 ng Phage DNA 1-10 pg Plasmid DNA 0.1-1.0 pgThe Importance of Polymerase Fidelity for Mutation DetectionImportance of high fidelity in dHPLC500 bp Wild Type Fragmen

25、tRed Trace Optimase PolymeraseGreen Trace 9:1 Mix Amplitaq Gold and Pfu TurboHeteroduplex due to misincorporationPolymerase Fidelity ComparisonMaximum recommended concentrationsof acceptable PCR additivesAcceptable additives (maximum final concentration)Additives where final concentrationmust be 2 m

26、V at A260.Peaks of intensity 30% of the average peak intensity. Weak peaks are more likely to lead to false-negative/positive results.Minimum peak intensityIdentification of sequence variantsThe presence of heteroduplexes is often detected as a change in the number of peaks (may be 2, 3 or 4 peak pa

27、ttern). Two peak patterns account for the majority of mutations.Complete resolution of the 2 heteroduplexes is not always necessary.Mutations may appear only as a slight broadening of the single peak, or as a subtle change to a shoulder on the peak. All samples identified as heteroduplexes by DHPLC

28、analysis must be sequenced in both directions to confirm and determine the nature of the sequence change.The homoduplex wild-type pattern is typically 1 peak, but may be 2 peaks, depending upon the melting profile.Elution profiles that differ from the wild-type indicate the presence of DNA sequence

29、changes. But the mutation type cannot be predicted from the heteroduplex pattern.Each mutation in a given PCR fragment is predicted to have a unique heteroduplex pattern (highly specific elution profile). This is useful for quick genotyping of unknown samples by comparison with positive control samp

30、les.However, trace profiles are not always unique for a specific mutation, i.e. different DNA variants can give identical profiles.Changes in retention time do not accurately predict the presence of a sequence change.Trace specificityData checking, reporting and storage Data checkingPositive results

31、False positive resultsNegative resultsFalse negative resultsSensitivityDetection of mosaicsArchiving Supplementary Appendix 3STANDARD OPERATING PROCEDURE MECP2SOP-MECP2DHPLC screening of MECP2In the context of Rett SyndromeRett syndrome Childhood neurodevelopment disorder with a prevalence of 1/10.0

32、00 to 1/15.000 in female births Mutations in the MECP2 gene, coding for Methyl CpG Binding Protein 2, are the primary cause of RTT Eight mutations are recurrently found in different populations. The first part of the molecular diagnosis of RTT is the DHPLC-screening of exons 2, 3 and 4 of MECP2. Thi

33、s allows the identification of more than 90% of all described mutations. Materials Worksheet :-Lot No. of all products - Equipment identifiers - Patient-identifier - Performing technician(s)- Date of experimentsMaterialsPCR - Standard PCR equipment (Location xxx) - Optimase DNA polymerase (2.5 U/l)

34、(Location 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 systemMaterials DHPLC system - Standa

35、rd 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. Patient material - Patient DNA - Positive controls - Negative controls - Normal controls Uses of Plasmid Controls as Reference Reagents no ethical problems renewable resourceCan

36、 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 of transfer of protoc

37、ols between machines/labsUses:Method Pre-PCR PCR Composition PCR Conditions Post PCR Heteroduplex formation Agarose gel electrophoresis DHPLC Interpretation of the results The mutation standards at the beginning and end of the run are evaluated All positive controls should be visible at their specif

38、ic temperature If 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. The elution profiles of a specific fragment from the different patients are compared with each other and scored according to the ge

39、neral DHPLC criteria. The minimum peak height must be 2mV. Any particular observation should be noted on worksheets or technical reports. Amplicons with an aberrant elution pattern are re-analysed by direct sequencing on an independent amplicon Interpretation of the results All premature truncation

40、mutations are immediately reportable. The pathogenicity of the missense mutations will depend on the position and the type. Interpretation is then subject to good practice and literature review. Mutations in the two highly conserved MeCP2 domains, the methyl binding domain and the transcription repr

41、ession domain, are likely to be causative. If 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 to be causative. If the mutation is present in the mother, X-inactivation studies need to be carried out on

42、the mother of the patient If both the mother and the daughter have random X-inactivation the mutation is unlikely to be causative. Reporting procedures NEGATIVE RESULT IN A FEMALE NEGATIVE RESULT IN A MALE NORMAL PARENT POSITIVE RESULT IN A FEMALE NEGATIVE RESULT IN A FEMALE Rett syndrome is caused

43、by mutations in the MECP2 gene. Molecular analysis of this 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 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 within the MECP2 gene. NEGATIVE RESULT IN A