《应用DHPLC技术进行诊断性分析质量保证体系.ppt》由会员分享，可在线阅读，更多相关《应用DHPLC技术进行诊断性分析质量保证体系.ppt（65页珍藏版）》请在三一文库上搜索。
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 GENETICSStandards and Guidelines for Clinical Genetics Laboratories2005 EditionG: CLINICAL MOLECULAR GENETICS These Standards and Guidelines specif
2、ically 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 Joh
3、n 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 Checking & reportingguidelines References,DHPLC SOPs,Instrument or maintenanceSOPTe
4、chniqueGeneral DHPLC SOPWAVE 3500, 3500 HTMethodDisease-specific SOPsRett, BRCA, HNPCCMarfan, Application,company + users,general users + company,specific users,Supplementary Appendix 1STANDARD OPERATING PROCEDURE WAVE System Operation and MaintenanceSOP- O&MWAVE System Operation and MaintenanceFor
5、WAVE System Models 3500, 3500A and 3500HT,WAVE 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 ca
6、libration, 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,
7、lAs 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 flush3.2 Pre-run maintenance3.3 Weekly main
8、tenance3.4 Quarterly 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 a
9、nd 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 check2. Injection system washing3. Pressure check4. Check the absorbance on the detector5. Purge the lines,Weekly maintenance,Inlin
10、e 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 DNAS
11、ep 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 cartridge50%A 50%B for 15 minutesRun 1-2 blanksVerify system performance (pre-analysis)Run a standardRun Sa
12、mplesVerify system performance (post analysis),Weekly Maintenance,An Extended Active Clean Wash is recommended every 100 injections. (Usually done after each 96 well plate),15-30 minutes,WASH,EQUILIBRATE,45-90 minutes,Run Standards to Verify System Performance!,1000 Injection Maintenance,A Reverse H
13、ot Wash is recommended every 1000 injections,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
14、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.,Make sure the oven heats up to at least 40 C. Set the pump to 100%D 0.500mL
15、/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,Verify New Cartridge Performance,Low-Range Mutation Standard,DNA Siz
16、ing Control Standard,Supplementary 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 requirements,Primer design Template purity and concentrationDNA PolymerasesPCR buffe
17、r mixPCR platesPCR quality and Product mixingPost-PCR, film useControls,Primer Design,Use a primer-picking programPrimers should ideally be no closer than 30 - 50 bp from the end of the sequence to be analyzed for mutationsPrimers should be 18 - 30 bp in lengthThe Tm difference between primers in a
18、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
19、 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 visib
20、le 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
21、 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 so
22、me 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.,Table 2. Recommended DNA quantities used for
23、 PCR (50 L reaction).,The Importance of Polymerase Fidelity for Mutation Detection,Importance of high fidelity in dHPLC,500 bp Wild Type FragmentRed Trace Optimase PolymeraseGreen Trace 9:1 Mix Amplitaq Gold and Pfu Turbo,Heteroduplex due to misincorporation,Polymerase Fidelity Comparison,Maximum re
24、commended concentrationsof acceptable PCR additives,Use of these components requires the use of Active clean for each injection, and other additional cleaning.Fluorescent labels will not damage the column but are not recommended. Digoxigenin/biotin labelled primers have not been tested.,Unacceptable
25、 PCR components.,Template DNA extracted or purified in a manner not consistent with WAVEs recommendationGel purification cannot be used to clean up samples for DHPLC, as the reagents and residual agarose damage the column.Unidentified reagent:“proprietary”- “stabilizers”“enhancers”- “additives”Autoc
26、laved WaterMineral OilFormamideProteinase KBovine serum albumin (BSA)Loading dyes (cresol red),(Components causing irreversible DNASep cartridge damage),Analysis of PCR Products on the WAVE System,Run PCR Products at 50 C (non-denaturing conditions) to verify size, yield and purity Run PCR Products
27、under partially-denaturing conditions to verify fidelity.,Controls,PCR Positive controls PCR Negative controls Instrument controls: Low and High Range Mutation standards,Control samples in DHPLC,Wherever possible, a confirmed wild-type control should be run, and compared with each sample.A mutation
28、(positive) control should be included.When amplifying large numbers of samples for many different gene fragments with a low frequency mutation pickup rate, it is sometimes acceptable to omit a normal control.,Instrument Buffers,Commercial buffersPreparation of In-house Buffers,Heteroduplex formation
29、,All samples must be heteroduplexed ; Heteroduplex formation : 95 C for 5 min and cool slowly (minimum 10s/C) to room temperature. Faster cooling protocols used for heteroduplex formation can seriously impair heteroduplexing.Purification of PCR products can seriously impair heteroduplex formation.,S
30、oftware,WaveMaker 4.1.44 Navigator Project Set-upSelection of analysis temperatures based on melt profilesAdjustment of gradients with time shifts.,Results Interpretation,ChromatogramControlsMinimum peak intensityVisual assessment of chromatogramsTrace specifity,Assessment of chromatogram quality,A:
31、 Contains unincorporated nucleotides and primers which do not bind to the columB: Contains primer dimersC: May be caused either by Taq errors during PCR or by non-template A addition.D: The majority of wild type samples appear as a single peak.E: ACN absorbs at 260nm. Any high MW contaminants appear
32、 as spikes on this peak.,Typical homozygous wild-type chromatogram,Controls,Check the mutation standards and see if they fulfil the criteria described in SOP-O&M. Check the known sequence variants (positive controls). If they do not present an aberrant chromatogram at the optimal screening temperatu
33、re, the results should be rejected and the analysis repeated,Ideally the signal intensity of DHPLC profiles should 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 seq
34、uence 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 broade
35、ning 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 pea
36、ks, 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 (high
37、ly 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
38、predict the presence of a sequence change.,Trace specificity,Data checking, reporting and storage,Data checkingPositive resultsFalse positive resultsNegative resultsFalse negative resultsSensitivityDetection of mosaicsArchiving,Supplementary Appendix 3STANDARD OPERATING PROCEDURE MECP2SOP-MECP2DHPLC
39、 screening of MECP2In 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
40、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 - Perfor
41、ming 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 Mg2+ (Location xxx) - Primers (Eurogentec)(stock) as 250 pmol/l. (appendix A) (Location xxx) - Primer work solutions contain 2.5
42、 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 cont
43、rols - Negative controls - Normal controls,Uses of Plasmid Controls as Reference Reagents,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 evalu
44、ated 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
45、 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 ampl
46、icon,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 d
47、omains, 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 t
48、he 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 FEMALE NEGATIVE RESULT IN A MALE NORMAL PARENT POSITIVE RESULT IN A
49、 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 sensiti
50、vity 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 carri