Illumina光纤微珠芯片技术

,IlluminaBeadArrayTechnology&ApplicationIllumina光纤微珠芯片技术及应用,邱亚静,May20,内容第一部分：光纤微珠芯片技术第二部分：基因表达研究第三部分：基因分型SNP研究第四部分：最新技术,May20,基因芯片的基本原理,基因芯片又称DNA微阵列（microarray），把大量已知序列探针集成在一个基片上，然后与标记的样品进行杂交，通过检测杂交信号来检测信息,May20,基因芯片的制作,点样法原位合成,May20,基因芯片制备,原位合成法，原位合成法是基于组合化学的合成原理,它通过一组定位模板来决定基片表面上不同化学单体的偶联位点和次序。原位合成法制备DNA芯片的关键是高空间分辨率的模板定位技术和固相合成化学技术的精巧结合。点样法。基因芯片点样法首先按常规方法制备cDNA（或寡核苷酸）探针库，然后通过特殊的针头和微喷头,分别把不同的探针溶液，逐点分配在玻璃、尼龙或者其它固相基底表面上不同位点,并通过物理和化学的结合使探针被固定于芯片的相应位点。,May20,PeterJ.Russell,iGenetics:CopyrightPearsonEducation,Inc.,publishingasBenjaminCummings.,原位合成,May20,PeterJ.Russell,iGenetics:CopyrightPearsonEducation,Inc.,publishingasBenjaminCummings.,May20,PeterJ.Russell,iGenetics:CopyrightPearsonEducation,Inc.,publishingasBenjaminCummings.,May20,原位合成技术,可达很高的密度合成过程中大量不完全片断残留在基片上探针序列不能太长，一般为25bp,May20,点样法,May20,May20,May20,点样技术,片断长度不受限制需要预先准备大量的核苷酸点的大小，间距较难控制,May20,新一代芯片技术光纤微珠芯片,ScreeningUnlabeledDNATargetswithRandomlyOrderedFiber-OpticGeneArrays,Steemers,F.J.,Ferguson,J.A.,Walt,D.R.,NatureBiotechnology,18,91-94,2000.Techview:MolecularBiology.Bead-BasedFiber-OpticArrays,Walt,D.R.,Science,287,451-452,2000DecodingRandomlyOrderedDNAArrays,KevinL.Gunderson,GenomeResearch,14:870-877,2004.,May20,1.微珠,3.1um无孔无荧光硅珠每个微珠单独质控,光纤微珠芯片-技术原理,有效的反应表面积和体积比低/无背景大小均一,May20,Oligator合成工厂,2.寡聚核苷酸探针,光纤微珠芯片-技术原理,96孔板全自动化合成LIMS控制所有过程年生产能力20million(20-mer)严格的QC系统OD260检测产量MALDI-TOF质谱检测UV毛细管电泳实时数码色彩监控,May20,2.寡聚核苷酸探针,光纤微珠芯片-技术原理,73mer35合成方向,May20,探针与微珠连接,光纤微珠芯片-技术原理,2.寡聚核苷酸探针,每个微珠上连接100万左右相同的探针,May20,KeyTechnologyElements:BeadChemistry,HowdowemakeourBeads?Athreestepprocess!,RawBeadsSilanizationproducesageneralcouplingmoleculeanamineTrimethoxyaminopropylsilaneVerystablecomparedtoothertechniquesActivationPreparesthebeadtoattachtheOligoCyanuricChlorideImmobilizationofDNAComplexityDiscretereactions96Wellmicrotiterplate,May20,每种(微珠+探针)单独合成并QC不同种微珠混合在一起形成微珠池(162424,000+种微珠),2.寡聚核苷酸探针,光纤微珠芯片-技术原理,高杂交特异性全长序列探针定制灵活100%质量检控QC,May20,KeyTechnologyElements:BeadProduction,SynthesisofOligos,ActivatedBeadPlates,+,OligoPlates,Gilson96TipRobot,Oven,PooledBeadSet,QC,May20,acidetch,strandcladding,strandcore,photo-resist,siliconwafer,plasmaetching,cleaning,Opticalfiber,3mbeadsinwells,3.芯片的组装,光纤微珠芯片-技术原理,May20,KeyElements:RandomBeadAssembly,MixtureofDifferentBeadTypes,6x2WholeGenomeBeadChip,BeadPoolispipettedontheSubstrate,NoInformationofBeadIdentityandlocationonArray,May20,将微珠池内微珠倒入蚀刻光纤，微珠无序自组装进入小孔，每根光纤上带一个微珠每种微珠大约有30个重复,3.芯片的组装,光纤微珠芯片-技术原理,消除系统随机误差高重复性微珠之间间距均一,May20,光纤束-微珠,3.芯片的组装,光纤微珠芯片-技术原理,传统芯片图,Decoderhybridization1,Decoderhybridization2,May20,4.芯片解码,光纤微珠芯片-技术原理,Gundersonetal.(2004)DecodingrandomlyorderedDNAarrays.GenomeResearch14,870-877.,4.芯片解码,光纤微珠芯片-技术原理,May20,KeyElements:Decoding,StrategyExpandsExponentially指数级解码,XY=totalnumberofcodes,X=totalnumberofcolorsorstatesY=totalnumberofhybridizationstages,SingleIllumiCodeStrategy,Example:38=6,56148=65,536,24merICode,May20,每张芯片上每个微珠每个特性都被质控(100%QC)位置和强度控制有多少重复小部分不合格地被捡出是Illumina出厂前的生产步骤每个芯片在出厂前已通过解码和质控解码CD随芯片送至用户处,4.芯片解码,光纤微珠芯片-技术原理,May20,KeyElements:DecodingQualityMetrics,%ofBeadsLoaded:98%ofBeadsDecoded:98%BeadTypeRepresentation:30ErrorRate:0.05%PresenceofallBeadTypesDecodingIntensitiesofeachstageFocusMetricsAllparametersmonitoredinrealtime,May20,KeyElements:DecodingQualityMetrics,May20,BeadArrayFormats,SentrixArrayMatrix,SentrixBeadChipFormats,Human-6HumanRef-8,Universal-16Custom-16,Human-1,May20,BeadArrayFormats,SentrixArrayMatrix,SentrixBeadChipFormats,Human-6HumanRef-8,Universal-16Custom-16,Human-1,GoldenGateTMGenotypingandDASLTM“UniversalArray”,May20,BeadArrayFormats,SentrixArrayMatrix,SentrixBeadChipFormats,Human-6HumanRef-8,Universal-16Custom-16,Human-1,FocusedGEXSets(StandardSamplers,CustomBeadPools),May20,BeadArrayFormats,SentrixArrayMatrix,SentrixBeadChipFormats,Human-6HumanRef-8,Universal-16Custom-16,Human-1,WholeGenomeGeneExpression,May20,BeadArrayFormats,SentrixArrayMatrix,SentrixBeadChipFormats,Human-6HumanRef-8,Universal-16Custom-16,Infinium,InfiniumTMWholeGenomeGenotyping,May20,BeadStation扫描系统,激光共聚焦扫描系统高分辨率,0.8micron双色激光at532&635nm,光纤微珠芯片-技术原理,超高分辨率全自动化定位,扫描,May20,BeadArrayReader,ConfocallaserscanningsystemSub-micron,0.8micronTwolasers532,635nmSupportsCy3&Cy5imagingAuto-focusing,centeringandtiltingwithoutphotobleachingSupportsgenotyping,geneexpressionapplicationsScansbothArrayMatrixandBeadChipmicroarrays,May20,SentrixBeadChip:AnalyticalFluorescentImage,May20,UserSuppliedDual-block96-wellthermalcyclerw/heatedlid2platecentrifuges(prea