应用DHPLC技术进行诊断性分析的质量保证体系.ppt

Click to edit Master title style,Click to edit Master text styles,Second level,Third level,Fourth level,Fifth level,*,*,资料仅供参考,不当之处，请联系改正。,Click to Edit Master Title Style,Click to Edit Master Text Styles,Second Level,Third Level,Fourth Level,Fifth Level,资料仅供参考,不当之处，请联系改正。,Click to Edit Master Title Style,Click to Edit Master Text Styles,Second Level,Third Level,Fourth Level,Fifth Level,资料仅供参考,不当之处，请联系改正。,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,AMERICAN COLLEGE OF MEDICAL GENETICS,Standards and Guidelines for Clinical Genetics Laboratories,2005 Edition,G:CLINICAL MOLECULAR GENETICS,These,Standards and Guidelines,specifically refer to the use of molecular techniques to examine heritable or somatic changes in the human genome.,G18,Denaturing High Performance Liquid Chromatography(dHPLC),(Section Added November 2003),CMGS,Best Practice Guidelines,U,se of the WAVE System in Diagnostic Service,Prepared and edited by John Harvey,National Genetics Reference Laboratory(Wessex),Salisbury,UK and Els Schollen,Centre for Human Genetics,Leuven,Belgium,last update:12 March 2004,Introduction,Laboratory process,DHPLC system,Data quality,Checking&reportingguidelines,References,DHPLC SOPs,Instrument or maintenanceSOP,Technique,General DHPLC SOP,WAVE 3500,3500 HT,Method,Disease-specific SOPs,Rett,BRCA,HNPCCMarfan,Application,company+users,general users+company,specific users,Supplementary Appendix 1,STANDARD OPERATING PROCEDURE WAVE System Operation and Maintenance,SOP-O&M,WAVE,System Operation and Maintenance,For WAVE,System Models 3500,3500A and 3500HT,WAVE,System Operation and Maintenance,Analysis of the WAVE Low&High Range Mutation Standards,The maintenance procedure,DNASep and DNASep HT cartridge maintenance,Role of Mutation standards:checking of correct functioning of the WAVE System,including oven calibration,cartridge performance,buffer composition and stability,to ensure reproducibility 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 every 100 injections for long runs.,Analysis of the WAVE Low&High Range Mutation Standards,Normal ranges,of the mutation standards,The maintenance procedure,3.1 Filter and flush,3.2 Pre-run maintenance,3.3 Weekly maintenance,3.4 Quarterly maintenance,3.5 Other maintenance operations,3.6,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 filtration.,The system flushing is to remove mobile phase salt components that can precipitate under strong solvent conditions.,Pre-run maintenance,1.Buffer check,2.Injection system washing,3.Pressure check,4.Check the absorbance on the detector,5.Purge the lines,Weekly maintenance,Inline filter replacement,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 reverse hot wash fails to resolve mutation standards),2.Short-term cartridge storage,3.Long-Term cartridge storage,4.New cartridge installation,Daily Maintenance,Equilibrate the cartridge,50%A 50%B for 15 minutes,Run 1-2 blanks,Verify system performance(pre-analysis),Run a standard,Run Samples,Verify system performance(post analysis),Weekly Maintenance,An Extended Active Clean Wash is recommended every 100 injections.,(Usually done after each 96 well plate),Oven,Set to:,80 C,Pump,Set to:,100%D,15-30,minutes,WASH,Oven,Set to:,56 C,Pump,Set to:,50%A-50%B,EQUILIBRATE,45-90,minutes,Run Standards to Verify System Performance!,1000,Injection Maintenance,A Reverse Hot Wash is recommended every 1000 injections,UV/FL,Detector,Turn off the pump,Reverse the cartridge direction.,Set the oven to 80 C.,Set the pump to 100%D.,60-90,minutes,Storing the Cartridge,Flush the cartridge with 100%D Buffer.,Remove the cartridge from the WAVE System.,Cap the cartridge with end plugs.,Store the cartridge at room temperature.,Installing a New Cartridge,Stop 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/FL,Detector,Make sure the 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 Cartridge,100%,50%50%,Verify New Cartridge Performance,Low-Range Mutation Standard,DNA Sizing Control Standard,Supplementary Appendix 2,STANDARD OPERATING PROCEDURE DHPLC,SOP-DHPLC,DHPLC mutation detection on Transgenomic WAVE,System 3500,Wavemaker 4.1.44&HSM 3.0-2.1(build 2),Navigator 1.5.4(build 19),PCR requirements,Primer design,Template purity and concentration,DNA Polymerases,PCR buffer mix,PCR plates,PCR quality and Product mixing,Post-PCR,film use,Controls,Primer Design,Use a primer-picking program,Primers should ideally be no closer than 30-50 bp from the end of the sequence to be analyzed for mutations,Primers should be 18-30 bp in length,The T,m,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,Size of PCR fragment,The optimal size range for detecting mutation/SNPs by DHPLC with 100%accuracy 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 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 mutations.,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 DHPLC,DNA must be clean,all cellular debris and organic 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&concentrarion,Table 1.Recommended cleaning procedures for DNA extraction.,Isolation Method,Recommended Additional Cleaning:,Organic Extraction(e.g.phenol/chloroform),Chloroform/isoamyl back-extraction followed by ethanol precipitation and wash,Chaotropic Salts(e.g.guanidinium isothiocyanate),Ethanol precipitation and wash,Spin Column,Ethanol precipitation and wash,Table 2.Recommended DNA quantities used for PCR(50,L reaction).,Template,Recommended Quantity,Human genomic DNA,50-200,ng,Phage DNA,1-10,pg,Plasmid DNA,0.1-1.0,pg,The Importance of Polymerase Fidelity for Mutation Detection,Importance of high fidelity in dHPLC,500,bp Wild Type Fragment,Red Trace,Optimase Polymerase,Green Trace,9:1 Mix Amplitaq Gold and Pfu Turbo,Heteroduplex due to misincorporation,Polymerase Fidelity Comparison,Maximum recommended concentrationsof acceptable PCR additives,Acceptable additives,(maximum final concentration),Additives where final concentration,must be 2 mV 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 intensity,Identification of sequence variants,The presence of heteroduplexes is often detected as a change in the number of peaks(may be 2,3 or 4 peak pattern).,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 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 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 samples.,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 specificity,Data checking,reporting and storage,Data checking,Positive results,False positive results,Negative results,False negative results,Sensitivity,Detection of mosaics,Archiving,Supplementary Appendix 3,STANDARD OPERATING PROCEDURE,MECP2,SOP-,MECP2,DHPLC screening of,MECP2,In the context of Rett Syndrome,Rett syndrome,Childhood neurodevelopment disorder with a prevalence of 1/10.000 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,.This 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 experiments,Materials,PCR,-,Standard PCR equipment,(Location xxx),-,Optimase DNA polymerase(2.5 U/l),(Location xxx),-,Optimase PCR buffer with Mg,2+,(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,Materials,DHPLC system,-,Standard 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 resource,Can use same reference reagent as control for PCR,heteroduplex and mutation detection analysis,Universal reagents which can be incorporated in QC procedures and SOPs,Advantages:,Validation of new protocols,Exon specific wild type and mutated controls for existing assays,Validation of transfer of protocols between machines/labs,Uses:,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 specific 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 general 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 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 repression 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 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 FEMALENEGATIVE RESULT IN A MALE,NORMAL PARENT,POSITIVE RESULT IN A FEMALE,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 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 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 the detection of point mutations,microdeletions and microinsertions but will not detect gross deletions of entire exons.The frequency of,MECP2,mutations in mentally retarded males is estimated at 0.2%,NORMAL PARENT,*,is the parent of *,a patient with Rett syndrome who has the*mutation in the,MECP2,gene.,The parents DNA has been analysed for this specific mutation by,There is no evidence of the*mutation in the peripheral blood sample of*.However,we cannot rule out germline mosaicism for this mutation.Prenatal testing for this mutation is a possibility.,POSITIVE RESULT IN A FEMALE,*,has been found to carry*at base*of the,MECP2,gene(*).This result confirms the diagnosis of Rett syndrome in*.,More than 95%of Rett syndrome cases are sporadic,however*s mother may be a carrier of this mutation.Also,one of the parents may have germline mosaicism for this mutation.,诊断性,DHPLC,质量保证（,DDQA）,WAVE,系统的操作和维护,SOP；,DHPLC,技术分析基因突变的通用,SOP；,DHPLC,检测特定疾病基因的,SOP：Rett,综合征相关的,MECP2,基因筛查中,SOPs,。,DHPLC,技术性能发挥,首先，只有经过充分的实验设计，条件优化，和实际验证后才能达到这种高灵敏度；,第二，严格的质量控制，持续的系统验证，和严格的实验程序也是达到这种高灵敏度必不可少的；,第三，操作者的经验也不可低估；,第四，即便在最理想的条件下，也不能保证对所有的扩增产物达到100%的灵敏度；,最后，虽然不是直接由该方法引起，但必需意识到,DHPLC,分析的是小片段的,PCR,产物，因而只能检测单碱基的突变，小片段的插入和缺失，大片段的插入和缺失不能检测。,Thanks!,