《Illumina光纤微珠芯片技术11教学材料》由会员分享，可在线阅读，更多相关《Illumina光纤微珠芯片技术11教学材料（42页珍藏版）》请在金锄头文库上搜索。
1、IlluminaBeadArrayTechnology&ApplicationIllumina光纤微珠芯片技术及应用邱亚静Date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Date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Date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
2、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。DatePeter J. Russell, iGenetics: Copyright Pearson Education, Inc., publishing as Benjamin Cummings.原位合成 DatePeter J. Russell, iGenetics: Copyright Pearson Education, Inc., publishin
3、g as Benjamin Cummings.DatePeter J. Russell, iGenetics: Copyright Pearson Education, Inc., publishing as Benjamin Cummings.Date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25bpDate点样法DateDateDate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Date新一代芯片技术 光纤微珠芯片ScreeningUnlabeledDNATargetswithRandomlyOrderedFiber-
4、OpticGeneArrays,Steemers,F.J.,Ferguson,J.A.,Walt,D.R.,Nature Biotechnology,18,91-94,2000.Techview:MolecularBiology.Bead-BasedFiber-OpticArrays,Walt,D.R.,Science,287,451-452,2000DecodingRandomlyOrderedDNAArrays,KevinL.Gunderson,Genome Research,14:870-877,2004.Date1.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
5、的反应表面积和体积比低/无背景大小均一DateOligator 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Date2. 寡聚核苷酸探针3 End5 End50 mer 探针合成探针23 mer 地址序列光纤微珠芯片-技术原理73mer35合成方向Date23mer50mer 探针3 End5 End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DateKey Techno
6、logy Elements: Bead ChemistryHowdowemakeourBeads?Athreestepprocess!RawBeadsSilanizationproduces a general coupling molecule an amineTrimethoxy amino propyl silaneVery stable compared to other techniquesActivationPrepares the bead to attach the OligoCyanuric ChlorideImmobilizationofDNAComplexityDiscr
7、ete reactions96 Well microtiter plateNH2OHNNNNHClClDate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QCDateKey Technology Elements: Bead ProductionSynthesis of OligosActivated Bead Plates+Oligo Plates Gilson 96 Tip RobotOvenPooled Bead SetQCDateacidetchs
8、trandcladdingstrandcorephoto-resistsiliconwaferplasmaetchingcleaningOptical fiber3mbeadsinwells3.芯片的组装光纤微珠芯片-技术原理DateKey Elements: Random Bead AssemblyMixture of Different Bead Types6x2 Whole Genome Bead ChipBead Pool is pipetted on the SubstrateNo Information of Bead Identity and location on ArrayD
9、ate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Date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DateDecoder hybridization 1Decoder hybridization 2*4.芯片解码光纤微珠芯片-技术原理Date Gunderson et al. (2004) Decoding randomly ordered DNA arrays.Genome Research 14, 870-877.4.芯片解码光纤微珠芯片-技术原
10、理DateKey Elements: DecodingStrategyExpandsExponentially指数级解码XY = total number of codesX = total number of colors or statesY = total number of hybridization stagesSingle IllumiCode StrategyExample: 38 = 6,561 48= 65,53624 mer ICodeDate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
11、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DateKey Elements: Decoding Quality Metrics%ofBeadsLoaded:98%ofBeadsDecoded:98%BeadTypeRepresentation:30ErrorRate:0.05%PresenceofallBeadTypesDecodingIntensitiesofeachstageFocusMetricsAllparametersmonitoredinrealtimeDateKey Elements: Decoding Quality MetricsD
12、ateBeadArray FormatsSentrix Array MatrixSentrix BeadChip Formats Human-6HumanRef-8Universal-16Custom-16Human-1DateBeadArray FormatsSentrix Array MatrixSentrix BeadChip Formats Human-6HumanRef-8Universal-16Custom-16Human-1GoldenGateTM Genotyping and DASLTM “Universal Array”DateBeadArray FormatsSentri
13、x Array MatrixSentrix BeadChip Formats Human-6HumanRef-8Universal-16Custom-16Human-1Focused GEX Sets(Standard Samplers, Custom BeadPools)DateBeadArray FormatsSentrix Array MatrixSentrix BeadChip Formats Human-6HumanRef-8Universal-16Custom-16Human-1Whole GenomeGene ExpressionDateBeadArray FormatsSent
14、rix Array MatrixSentrix BeadChip Formats Human-6HumanRef-8Universal-16Custom-16InfiniumInfiniumTM Whole Genome GenotypingDateBeadStation扫描系统激光共聚焦扫描系统高分辨率,0.8 micron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DateBeadArray ReaderConfocallaserscanningsystemSub-micron,0.8micronTwolasers532,635nmSupportsCy3
15、&Cy5imagingAuto-focusing,centeringandtiltingwithoutphotobleachingSupportsgenotyping,geneexpressionapplicationsScansbothArrayMatrixandBeadChipmicroarraysDateSentrix Bead Chip: Analytical Fluorescent ImageDateUser SuppliedDual-block96-wellthermalcyclerw/heatedlid2platecentrifuges(preandpost-PCR)pipett
16、eIllumina 提供BeadArrayReaderPCHybridizationchamberRaisedbarmagnetHeatblocksOvencapableof45/70oCPlatevortexUserdocumentationBeadStudiosoftwareInstallation&trainingIlluminas BeadStation 500 SystemDate灵活性扩展性重复性均一性？特点DatemVjSgOdLaI6F3B0y)v%s#pXlUiRfNcK8H5E2A+x(u$rZnWkThPeMbJ7G4C1z-w&t!qYmVjRgOdL9I6F3B0y(v%s#oXlUiQfNbK8H5D2A+x*u$qZnWkShPeMaJ7G4C1z)w&t!pYmVjRgOcL9I6E3B0y(v%r#oXlTiQfNbK8G5D2A-x*u$qZnVkShPdMaJ7F4C0z)w&s!pYmUjRfOcL9H6E3B+y(u%r#oWlTiQeNbK8G5D1A-x*t$qZnVkSgPdMaI7F4C0z)v&s!pXmUjRfOcK9H6E2B+y
