关于数字PCR的报告 方昊ppt课件

关于数字PCR发展、原理、运用和前景调查汇报2017年3月,1,数字PCR(digitalPCR,dPCR)技术是近年来迅速发展起来的一种定量分析技术。与传统定量PCR技术不同的是数字PCR不依赖于扩增曲线的循环阈值(CT值)进行定量，不受扩增效率的影响，也不必采用看家基因和标准曲线，是对起始样的绝对定量。,2,主要内容,数字PCR的发展与原理,数字PCR的应用,3,1,2,数字PCR的前景展望,3,数字PCR的发展与原理20世纪末，Vogelstein等提出数字PCR的概念，通过将一个样本稀释后分成几十到几万份甚至上千万份，然后分配到不同的反应单元，每个单元不包含或包含一个或多个拷贝的目标分子(DNA模板)，在每个反应单元中分别对目标分子进行PCR扩增，扩增结束后对各个反应单元的荧光信号进行统计学分析。【1】,4,2006年美国Fluidigm公司率先推出第一款数字PCR产基于IFC（integratedfluidiccircuit）技术的BioMarkTM。2009年，BioTrove被LifeTechnologies(AppliedBiosystems)收购。QuantaLife公司开发出的微滴数字PCR技术。该产品还获得了2011年度Frost&Sullivan北美新产品创新奖。同年Bio-Rad收购QuantaLife的液滴数字PCR技术(ddPCR)，迅速推出QX100液滴数字PCR产品。,5,传统PCR或定量PCR反应都是在96/384孔板中进行的，因此早期的数字PCR技术也采用96/384孔板作为反应单元。这远远不能满足数字PCR对反应单元数的需求，于是开始制造微升和纳升级别的反应室，在提高灵敏度的同时减少了反应体系的总体积。,1.微反应室/孔板数字PCR,6,2.微流控数字PCR随着微流芯片技术的发展，数字PCR借助微流芯片，可以把样本准确并且快速的分成若干个独立的反应单元，各个单元之间互不影响，可以多步反应同时进行。提高反应通量的同时也降低了反应消耗成本。,7,3.微滴数字PCR微滴数字PCR借鉴了乳液PCR，即将DNA模板与连接引物的磁性微球以极低的浓度(比如单拷贝)包裹于油水两相形成的纳升至皮升级液滴中进行PCR扩增，扩增后的产物富集在磁性微球上，收集破乳后进行测序。通过油水两相间隔得到的以液滴为单位的PCR反应体系，比微孔板和IFC系统更容易实现小体积和高通量，而且系统简单，成本低，因此成为理想的数字PCR技术平台。【2】,8,数字PCR的应用,9,数字PCR的前景展望发展空间大数字PCR与传统PCR不同它有多种运作方式和原理，数字PCR技术还有很大的发展空间。在其运用方面，也可有有很大的拓展空间。因为他是对核酸的绝对定量，是一种更加直观、精确的PCR技术，在未来的研究中，数字PCR肯定会有更大的发挥空间。,10,数字PCR的前景展望高需求并有小众化趋势因为数字PCR在各项领域的突出表现，数字PCR的需求越来越迫切。例如：现在许多医疗机构提供DNA水平的疾病（主要为癌症）筛查服务，并为癌症患者提供靶向药品，虽然国内只有少数医疗机构提供该服务，但是需要这项服务的人却很多。,11,数字PCR的前景展望发展中的难点成本高通量低对引物有要求DNA聚合酶质量要求高严控污染,12,1林彩琴,姚波.数字PCR技术进展J.化学进展,2012,(12):2415-2423.2詹成,燕丽,王琳,金玉麟,陈力,时雨,王群.数字PCR技术的发展和应用J.复旦学报(医学版),2015,(06):786-789.3HolmbergRC,GindlespergerA,StokesT,etal.AkonniTruTip(R)andQiagen(R)methodsforextractionoffetalcirculatingDNA-evaluationbyreal-timeanddigitalPCR.PLoSOne,2013,8:e73068.4GuW,KohW,BlumenfeldYJ,etal.Noninvasiveprenataldiag-nosisinafetusatriskformethylmalonicacademia.GeneticsinMedicine,2014,16(7):564-567.5PornprasertS,PrasingW.Detectionofalpha(0)-thalassemiaSouth-EastAsian-typedeletionbydropletdigitalPCR.Euro-peanJournalofHaematology,2014,92:244-248.,13,6PodlesniyP,Figueiro-SilvaJ,LladoA,etal.LowcerebrospinalfluidconcentrationofmitochondrialDNAinpreclinicalAlzheimerdisease.AnnalsofNeurology,2013,74:655-668.7GevenslebenH,Garcia-MurillasI,GraeserMK,etal.NoninvasivedetectionofHER2amplificationwithplasmaDNAdigitalPCR.ClinicalCancerResearch,2013,19:3276-3284.8BeaverJA,JelovacD,BalukrishnaS,etal.DetectionofcancerDNAinplasmaofearlystagebreastcancerpatients.ClinicalCancerResearch,2014,20(10):2643-2650.9MaJ,LiN,GuarneraM,JiangF.QuantificationofplasmamiRNAsbydigitalPCRforcancerdiagnosis.BiomarkInsights,2013,8:127-136.,14,10PersaudD,GayH,ZiemniakC,etal.AbsenceofdetectableHIV-1viremiaaftertreatmentcessationinaninfant.NewEnglandJournalofMedicine,2013,369:1828-1835.11AubertM,BoyleNM,StoneD,etal.InvitroinactivationoflatentHSVbytargetedmutagenesisusinganHSV-specifichomingendonuclease,MolecularTherapy-NucleicAcids,2014,3:e146.12AbyzovA,MarianiJ,PalejevD,etal.Somaticcopynumbermosaicisminhumanskinrevealedbyinducedpluripotentstemcells.Nature,2012,492:438-442.13BoettgerLM,HandsakerRE,ZodyMC,etal.Structuralhaplotypesandrecentevolutionofthehuman17q21.31region.NatureGenetics,2012,44:881-885.,15,14HindsonBJ,NessKD,MasquelierDA,etal.High-throughputdrop-letdigitalPCRsystemforabsolutequantitationofDNAcopynumber.AnalyticalChemistry,2011,83:8604-8610.15ShlushLI,ZandiS,MitchellA,etal.Identificationofpre-leukaemichaematopoieticstemcellsinacuteleukaemia.Nature,2014,506:328-333.16JohnsonBE,MazorT,HongC,etal.Mutationalanalysisrevealstheoriginandtherapy-drivenevolutionofrecurrentglioma.Science,2014,343(6167):189-193.17MiyaokaY,ChanAH,JudgeLM,etal.Isolationofsingle-basegenome-editedhumaniPScellswithoutantibioticselection.NatureMethods,2014,11:291-293.,16,18Abdel-WahabO,KlimekVM,GaskellA,etal.Efficacyofinter-mittentcombinedRAFandMEKinhibitioninapatientwithconcurrentBRAFandNRASmutantmalignancies.Cancerdiscovery,2014,4(5):538-45.19NairVD,GeY,BalasubramaniyanN,etal.Involvementofhis-tonedemethylaseLSD1inshort-time-scalegeneexpressionchangesduringcellcycleprogressioninembryonicstemcells.MolecularandCellularBiology,2012,32:4861-4876.20GorbachevAY,FisunovGY,IzraelsonM,etal.DNArepairinMy-coplasmagallisepticum.BMCGenomics,2013,14:726.21JiangK,RenC,NairVD.MicroRNA-137repressesKlf4andTbx3duringdifferentiationofmouseembryonicstemcells.StemCellResearch,2013,11:1299-1313.,17,22GuoG,HussM,TongGQ,etal.Resolutionofcellfatedeci-sionsrevealedbysingle-cellgeneexpressionanalysisfromzygotetoblastocyst.DevelopmentalCell,2011,18(4):675-685.23WhiteAK,MichaelV,OlehIP,etal.High-throughputmicrofluidicsingle-cellRT-qPCR.ProcNatlAcadSciUSA,2011,108(34):13999-14004.24CangiMG,BiavascoR,CavalliG,etal.BRAFV600E-mutationisinvariablypresentandassociatedtooncogene-inducedsenes-cenceinErdheim-Chesterdisease.AnnalsoftheRheumatic,2014,doi:10.1136/annrheumdis-2013-204924.25ChongIY,CunninghamD,BarberLJ,etal.Thegenomicland-scapeofoesophagogastricjunctionaladenocarcinoma,JournalofPathology,2013,231:301-310.,18,26FresardL,LerouxS,ServinB,etal.Transcriptome-widein-vestigationofgenomicimprintinginchicken.NucleicAcidsResearch,2014,42(6):3768-37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