Illumina光纤微珠芯片技术.ppt

Illumina BeadArray Technology & Application Illumina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 * * * * * 基因芯片的制作 点样法 原位合成 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。 Date Peter J. Russell, iGenetics: Copyright Pearson Education, Inc., publishing as Benjamin Cummings. 原位合成 Date Peter J. Russell, iGenetics: Copyright Pearson Education, Inc., publishing as Benjamin Cummings. Date Peter 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25bp Date 点样法 Date Date Date 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 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. Date 1.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-技术原理 有效的反应表面积和体积比 低/无背景 大小均一 Date 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 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 Date 2. 寡聚核苷酸探针 3 End5 End 50 mer 探针 合成探针 23 mer 地址序列 光纤微珠芯片-技术原理 73mer 35合成方向 Date 23mer50mer 探针 3 End 5 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 Date Key Technology Elements: Bead Chemistry How do we make our Beads ? A three step process! Raw Beads Silanization produces a general coupling molecule an amine Trimethoxy amino propyl silane Very stable compared to other techniques Activation Prepares the bead to attach the Oligo Cyanuric Chloride Immobilization of DNA Complexity Discrete reactions 96 Well microtiter plate NH2 OH N N N NH Cl Cl Date 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 (1624 24,000+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 Date Key Technology Elements: Bead Production Synthesis of Oligos Activated Bead Plates + Oligo Plates Gilson 96 Tip Robot Oven Pooled Bead Set QC Date acid etch strand cladding strand core photo- resist silicon wafer plasma etching cleaning Optical fiber 3 m beads in wells 3.芯片的组装 光纤微珠芯片-技术原理 Date Key Elements: Random Bead Assembly Mixture of Different Bead Types 6x2 Whole Genome Bead Chip Bead Pool is pipetted on the Substrate No Information of Bead Identity and location on Array Date 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 Date Decoder 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-技术原理 Date Key Elements: Decoding Strategy Expands Exponentially指数级解码 XY = total number of codes X = total number of colors or states Y = total number of hybridization stages Single IllumiCode Strategy Example: 38 = 6,561 48= 65,536 24 mer ICode Date 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 Date 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 Types Decoding Intensities of each stage Focus Metrics All parameters monitored in real time Date Key Elements: Decoding Quality Metrics Date BeadArray Formats Sentrix Array Matrix Sentrix BeadChip Formats Human-6 HumanRef-8 Universal-16 Custom-16 Human-1 Date BeadArray Formats Sentrix Array Matrix Sentrix BeadChip Formats Human-6 HumanRef-8 Universal-16 Custom-16 Human-1 GoldenGateTM Genotyping and DASLTM “Universal Array” Date BeadArray Formats Sentrix Array Matrix Sentrix BeadChip Formats Human-6 HumanRef-8 Universal-16 Custom-16 Human-1 Focused GEX Sets (Standard Samplers, Custom BeadPools) Date BeadArray Formats Sentrix Array Matrix Sentrix BeadChip Formats Human-6 HumanRef-8 Universal-16 Custom-16 Human-1 Whole Genome Gene Expression Date BeadArray Formats Sentrix Array Matrix Sentrix BeadChip Formats Human-6 HumanRef-8 Universal-16 Custom-16 Infinium InfiniumTM Whole Genome Genotyping Date BeadStation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-技术原理 超高分辨率 全自动化定位,扫描 Date BeadArray Reader Confocal laser scanning system Sub-micron, 0.8 micron Two lasers 532, 635 nm Supports Cy3 & Cy5 imaging Auto-focusing, centering and tilting without photo bleaching Supports genotyping, gene expression applications Scans both Array Matrix and BeadChip microarrays Date Sentrix Bead Chip: Analytical Fluorescent Image Date User Supplied Dual-block 96-well thermal cycler w/heated lid 2 plate centrifuges (pre and post-PCR) pipette Illumina 提供 BeadArray Reader PC Hybridization chamber Raised bar magnet Heat blocks Oven capable of 45/70oC Plate vortex User documentation BeadStudio software Installation & training Illuminas BeadStation 500 System Date 灵活性 扩展性 重复性 均一性 ？ 特点 Date mVjSgOdLaI6F3B0y)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(u%rZoWlThQeNbJ8G4D1A-w*t$qYnVjSgPdLaI7F3C0z)v&s#pXmUiRfOcK9H5E2B+x(u%rZoWkThQeMbJ8G4D1z- 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