Illumina BeadArray® Technology & Application Illumina光纤微珠芯片技术光纤微珠芯片技术及应用及应用 邱亚静邱亚静 基因芯片的基本原理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 *****基因芯片的制作基因芯片的制作•点样法•原位合成 基因芯片制备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 Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.原位合成原位合成 Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings. Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings. 原位合成技术原位合成技术•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 点样法点样法 点样技术点样技术 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 新一代芯片技术新一代芯片技术 光纤微珠芯片光纤微珠芯片 Screening Unlabeled DNA Targets with Randomly Ordered Fiber-Optic Gene Arrays, Steemers, F.J., Ferguson, J.A., Walt, D.R., Nature Biotechnology,18, 91-94, 2000. Techview: Molecular Biology. Bead-Based Fiber-Optic Arrays, Walt, D.R., Science, 287, 451-452, 2000 Decoding Randomly Ordered DNA Arrays, Kevin L. Gunderson, Genome Research, 14: 870-877, 2004. 1.微珠微珠3.1 um无孔无荧光硅珠每个微珠单独质控光纤微珠芯片光纤微珠芯片---技术原理技术原理•有效的反应表面积和体积比有效的反应表面积和体积比•低低/无背景无背景•大小均一大小均一 Oligator® 合成工厂合成工厂2. 寡聚核苷酸探针寡聚核苷酸探针光纤微珠芯片光纤微珠芯片---技术原理技术原理96 孔板全自动化合成LIMS 控制所有过程年生产能力 >20 million (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 2. 寡聚核苷酸探针寡聚核苷酸探针3’ End5’ End50 mer 探针探针合成探针合成探针23 mer 地址序列地址序列光纤微珠芯片光纤微珠芯片---技术原理技术原理73mer3’5’合成方向合成方向 23mer50mer 探针探针3’ End5’ End探针与微珠连接探针与微珠连接光纤微珠芯片光纤微珠芯片---技术原理技术原理2. 寡聚核苷酸探针寡聚核苷酸探针每个微珠上连接每个微珠上连接100万左右万左右相同相同的探针的探针 Key Technology Elements: Bead ChemistryHow do we make our Beads ? A three step process! Raw BeadsSilanization – produces a general coupling molecule – an amineTrimethoxy amino propyl silaneVery stable compared to other techniquesActivationPrepares the bead to attach the OligoCyanuric ChlorideImmobilization of DNAComplexityDiscrete reactions96 Well microtiter plateNH2OHNNNNHClCl 每种(微珠+探针)单独合成并 QC不同种微珠混合在一起形成 微珠池微珠池 (1624 － 24,000+种微珠)2. 寡聚核苷酸探针寡聚核苷酸探针光纤微珠芯片光纤微珠芯片---技术原理技术原理•高杂交特异性高杂交特异性•全长序列探针全长序列探针•定制灵活定制灵活•100%质量检控质量检控QC Key Technology Elements: Bead ProductionSynthesis of OligosActivated Bead Plates+Oligo Plates Gilson 96 Tip RobotOvenPooled Bead SetQC acid etchstrandcladdingstrandcorephoto-resistsiliconwaferplasmaetchingcleaningOptical fiber3 µm beads in wells3.芯片的组装芯片的组装光纤微珠芯片光纤微珠芯片---技术原理技术原理 Key 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 Array •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.芯片的组装芯片的组装光纤微珠芯片光纤微珠芯片---技术原理技术原理•消除系统随机误差消除系统随机误差•高重复性高重复性•微珠之间间距均一微珠之间间距均一 光纤束光纤束-微珠微珠3.芯片的组装芯片的组装光纤微珠芯片光纤微珠芯片---技术原理技术原理传统芯片图 Decoder hybridization 1Decoder hybridization 28/31/20244.芯片解码芯片解码光纤微珠芯片光纤微珠芯片---技术原理技术原理 • Gunderson et al. (2004) Decoding randomly ordered DNA arrays.Genome Research 14, 870-877.4.芯片解码芯片解码光纤微珠芯片光纤微珠芯片---技术原理技术原理 Key Elements: DecodingStrategy Expands Exponentially指数级解码指数级解码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 ICode 每张每张芯片上每个每个微珠每个每个特性都被质控(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.芯片解码芯片解码光纤微珠芯片光纤微珠芯片---技术原理技术原理 Key Elements: Decoding Quality Metrics% of Beads Loaded: ~ 98%% of Beads Decoded: ~98%Bead Type Representation: ~ 30 Error Rate: 0.05%Presence of all Bead TypesDecoding Intensities of each stageFocus MetricsAll parameters monitored in ~ real time Key Elements: Decoding Quality Metrics BeadArray FormatsSentrix Array MatrixSentrix BeadChip Formats Human-6HumanRef-8Universal-16Custom-16Human-1 BeadArray FormatsSentrix Array MatrixSentrix BeadChip Formats Human-6HumanRef-8Universal-16Custom-16Human-1GoldenGateTM Genotyping and DASLTM “Universal Array” BeadArray FormatsSentrix Array MatrixSentrix BeadChip Formats Human-6HumanRef-8Universal-16Custom-16Human-1Focused GEX Sets(Standard Samplers, Custom BeadPools) BeadArray FormatsSentrix Array MatrixSentrix BeadChip Formats Human-6HumanRef-8Universal-16Custom-16Human-1Whole GenomeGene Expression BeadArray FormatsSentrix Array MatrixSentrix BeadChip Formats Human-6HumanRef-8Universal-16Custom-16InfiniumInfiniumTM Whole Genome Genotyping BeadStation®扫描系统扫描系统 激光共聚焦扫描系统 高分辨率, 0.8 micron 双色激光 at 532 & 635 nm光纤微珠芯片光纤微珠芯片---技术原理技术原理•超高分辨率超高分辨率•全自动化定位全自动化定位,扫描扫描 BeadArray ReaderConfocal laser scanning systemSub-micron, 0.8 micronTwo lasers 532, 635 nmSupports Cy3 & Cy5 imagingAuto-focusing, centering and tilting without photo bleachingSupports genotyping, gene expression applicationsScans both Array Matrix and BeadChip microarrays Sentrix Bead Chip: Analytical Fluorescent Image User SuppliedDual-block 96-well thermal cycler w/heated lid2 plate centrifuges (pre and post-PCR)pipette Illumina 提供提供BeadArray ReaderPCHybridization chamber Raised bar magnetHeat blocksOven capable of 45/70oCPlate vortexUser documentationBeadStudio softwareInstallation & training Illumina’s BeadStation 500 System 灵活性扩展性重复性均一性？？？特点特点 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