Transcript 生物芯片技术
生物芯片技术 生物芯片原理和应用 何为生物芯片? 生物芯片主要指通过平面微细加工技术在固体芯片 表面构建的微流体分析单元和系统，以实现对细胞 、蛋白质、核酸以及其他生物组分的准确、快速、 大信息量的检测。 他是继大规模集成电路之后的又一次具有深远意义 的科学技术革命。 生物芯片分类 Biochips DNA Chips Protein Chips Lab Chips 蛋白芯片(Protein Chips) a microarray-based high-throughput protein assay method Chemiluminescence or Fluorescence based detection methods can be used to visualize bound antibodies. 蛋白芯片应用 • Diagnostic immunoassay that allows the simultaneous high-throughput analysis of known autoantigens. In order to quantify antibodies in the sera of patients with autoimmune diseases, Recombinant antigens and control proteins were immobilized on slides with reactive aldehyde groups as replicas in serial dilutions of the various antigens thereby allowing accurate determination of autoantibody titer using minimal amounts of serum. Miniaturized and highly paralleled immunoassays like these will reduce cost by decreasing reagent consumption and improve performance by greatly increasing the number of assays that can be performed with a single serum sample. • Protein-Protein Interaction. • DNA- Protein Interaction 基因芯片（Gene chip） • 基因芯片(Gene chip)技术是指通过微阵列 (Microarray)技术将高密度DNA片段阵列通 过高速机器人或原位合成方式以一定的顺 序或排列方式使其附着在如玻璃片等固相 表面，以荧光标记的DNA探针，借助碱基互 补杂交原理，进行大量的基因表达及监测 等方面研究的最新革命性技术 基因芯片发展历史 Southern & Northern Blot Dot Blot Macroarray Microarray Types of DNA Chips DNA Chips Expression Chips Genomic Chips Sequencing Chips Comparison of DNA Chip Technologies Oligo-Chip cDNA-Chip 8 n or 20 n < 2,000 n sequencing expression expression Genomic Chip > 50,000 n genomic analysis Sensitivity of DNA chip based assays is a function of: – Probe and target DNA/RNA (Complexity) – Chip surface (autofluorescence & non-spec. bkg) – Attachment chemistry/methodology (hyb. efficiency & crosshyb.) – Hybridization efficiency (lots of factors) – Detection technology (signal type, efficiency, noise) Why Genomic Biochips? • Screen specimens to determine gene copy number changes • Establish correlations between gene copy number changes and disease biology • Determine the interaction of multiple genes on the initiation and progression of disease • Accelerate development of products for genomic disease management to guide therapeutic intervention • Combined with expression chips, gives full understanding of disease process DNA Chip Technology • Solid support (glass, plastic, metal, silicon) • Miniaturized array of DNA (genetic material) • Work on the biochemical principle of DNA/DNA hybridization • Hybridized probes (DNA molecules) are fluorescently labeled Assay Formats Genomic 1. Extract genomic DNA from tissue Expression Tumor Sample Normal Sampl e 1. Extract mRNA from tissue 2. Label 2. Produce cDNA by RT & Label 3. Mix with labeled reference DNA 3. Mix with labeled reference cDNA 4. Hybridize to Chip 4. Hybridize to Chip 5. Wash and Image 5. Wash and Image Areas of Biochip Applications • Academic research of genetic diseases – Cancer – Prenatal genetics – General genetic diseases • • • • • Infectious diseases Drug discovery Animal farming/veterinary Industrial (fermentation, corrosion) Environmental 应用之一 基因表达谱（gene expression pattern） • Research Use. Biological Sample Functional Information One Disease——One Gene Expression Pattern • Clinical Diagnostic Use. Research Use—— From Sequence to function • 计算Ratio 值 (= Cy3/Cy5) – 在 0.5-2.0 之外的定义为在两样本中有 明显差异表达。进而获取初步功能信息。 • Clustering Research Use—— From Sequence to function Challenges • 庞大的数据库 • 数学模型 Research Use —— From Sequence to function 可以大大推进包括人类（后）基因组计划在内 的各类基因组研究，通过比较不同个体或物种 之间以及同一个体在不同生长发育阶段、正常 和疾病状态下基因及其表达的差异，寻找和发 现新的基因，研究基因的功能以及生物体在进 化、发育、遗传等过程中的规律。 Research Use —— From Sequence to function 基因芯片可为研究不同层次多基因协 同作用的生命过程提供手段。 将在研究人类重大疾病如癌症、心血 管疾病等相关基因及作用机理方面发 挥巨大的作用。 Clinical Diagnostic Use One Disease=One Expression Pattern • Academic research of genetic diseases – Cancer – Prenatal genetics – General genetic diseases • Infectious diseases – HAV、HBV、HCV….. – HIV Clinical Diagnostic Use 生物芯片能为现代医学科学及医学诊断学 的发展提供强有力的手段，促进医学从 “系统、血管、组织和细胞层次”(通常称 之为“第二阶段医学”)向“DNA、RNA、 蛋白质及其相互作用层次”(第三阶段医学) 过渡，使之快速进入实际应用。 Prototype AmpliOnc™ I Biochip This biochip contains all genomic regions that have been reported to be amplified in cancers. AmpliOncTM I Biochip after hybridization; color composite of red, blue and green image Oncogene Targets On the AmpliOnc™ I Biochip FGR NMYC MYCL1 RAF1 HRAS1 EGFR1 KRAS2 FGFR1 REL PDGFRA WNT1 GLI MDM2 CDK4 CND1 INT2 MYB MET PIK3CA MYC 3 1 4 2 9 8 7 6 ABL FGFR2 10 11 12 5 JUNB HER2 AKT2 YES1 FES AKT1 13 14 15 16 19 17 18 AR PDGFB 20q13 20 21 22 Y X RDA Protocol • RNA extraction and cDNA preparation from archived tissue specimens(tester and driver) • Generation of amplified cDNA fragments (‘amplicons’) • Subtractive hybridization of amplicons • Enrichment of cDNA fragments from differentially expressed genes Microarray 用于验证RDA • Shotgun subcloning of RDA fragments • Picking transformed libraries for long-term propagation • Amplification of RDA inserts in 96-well plate format for arraying • Hybridization of cDNA amplicons to microarrays References about Coupling of RDA & Microarray • Schena, M. et al. (1995)Science, 270, 467 • Lockhart,et al.(1996).Nature Biotechnology, 14, 1675 • DeRisi, et al. (1996). Nature Genet., 14, 457. Microarray 还可用于验证 • SSH • Differential Display PCR 应用之三—— SNPs & STRs analysis • • • • Single Nucleotide Polymorphisms Short Tandem Repeats Polymerase Ligase SNPs Typing —— by Ligase SNPs Typing —— by Polymerase（1） SNPs Typing —— by Polymerase（2） STRs Typing（1） STRs Typing（2） STRs Typing（3） 应用之四 —— LCM俘获细胞的基因表达分析 LCM: Laser Capture Microdissection 原理 Gene expression profiles of lasercaptured adjacent neuronal subtypes Differential Gene Expression between Large- and Small-sized Dorsal Root Ganglion (DRG) Rat Neurons, Nissl stained Large DRG Neurons: •myelinated •fast-conducting •transmit mechanosensory information Small DRG Neurons: •unmyelinated •slow-conducting •transmit nociceptive information Gene expression profiles of lasercaptured adjacent neuronal subtypes Dorsal Root Ganglion (DRG) Rat Neurons, Nissl stained cDNA microarray expression patterns of small (S) and large (L) neurons mRNA enriched in large DRG neurons mRNA enriched in small DRG neurons 放射性原位杂交验证结果 应用之五—— Development of therapeutic Drugs • drug target discovery • evaluation of animal models of human disease • test for drug efficacy • test for drug specificity • test for drug toxicity 其它应用 在预防医学方面，可以使人们尽早地认识自 身潜在的疾病，并实施有效的防治措施 法医学中，进行基因指纹快速识别，以及亲 子鉴定 其它应用 监测流行病和传染病扩散 监测有害微生物的发生和传播 农、林、牧、鱼等产业的品种改良和 病虫防治 生物芯片制作技术 及相关产品介绍 生物芯片制作方法分类 片基 显色及检测方式 钢性片基如玻片、半导 荧光，激光共聚焦扫 原位合成 体硅片等 描、定量分析；生物 (in situ synthesis) 传感器等 玻片、薄膜片基如 NC、 荧光，激光共聚焦扫 预先合成后点样 Nylon 膜等 描、定量分析；生物 (off-chip synthesis) 传感器等 原位合成 (In Situ Synthesis) Light directed oligonucleotide synthesis. A solid support is derivatized with a covalent linker molecule terminated with a photolabile protecting group. Light is directed through a mask to deprotect and activate selected sites, and protected nucleotides couple to the activated sites. The process is repeated, activating different sets of sites and coupling different bases allowing arbitrary DNA probes to be constructed at each site. 预先合成后点样 (off-chip synthesis) • 接触式点样 • 非接触式点样 接触式点样 Best! 接触式点样： Chipmaker Pin （Telechem专利） ArrayIt ChipMaker Pins • Developed in conjunction with Stanford University • Single dip in sample multiple spots • Fine (EDM) slot in tip of stainless steel pin • Take-up volume ~250 nL (saves sample) • Spot volume of ~100 to 500 pL • 100 µm to 250 µm spot size (high density arrays) • Requires 10 to 30 pre-prints to “prime” pin 非接触式点样： nQUAD Technology Switching Valve Connecting Tubing High-Speed MicroSolenoid Valve Syringe Pump Removable Tip Orifice Reservoir Controller Characteristics of nQUAD Technology • Wide dispense range – Low nanoliter to high microliter – Excellent linearity • Precise and accurate – CV’s typically less than 10% – Precision less than 5% • Non-contact dispense mechanism – Easier mechanical alignment (384, 1536,…) – “On-the-fly” printing possible – Capable of dispensing onto membranes or slides • Multiple liquid handling modes – Aspirate/Dispense – Continuous Reagent Dispensing 生物芯片点样系统必备性能 • 多针头自动同时取样，取样板为96孔或384孔板 • 单针取样量为250到500nL，每次点样量为100到 150pL • 点样直径为100到150um，系统点阵分辨率为10um • 可程控进行连续点样操作 • 固相介质如玻璃片须通过适当机制进行位置固定 • 点样针尖必须具备单次取样连续点样的性能 Cartesian - PixSys Series 合成后点样板 - Cartesian Tech. PixSys Series 特点： • Combines with TWO dispensing technologies • Contact and Non-contact printing mode • ChipMaker quill pins and nQUAD dispensers • Pin array Spot Size: 75 to 200um • nQUAD quantity: 10nL • 10 µm positioning resolution • 50 slide capacity Methods in Microarray Detection •CCD •Confocal Scanning Microscopy General Scanning Inc. 权威的Scanner 专业供应商 ScanArray5000/4000/3000 SCANARRAY3000/4000/5000特点： • 全自动检测及分析系统 • 适用于DNA微阵列生物芯片应用 • 共聚焦激光扫描探测系统 • 灵敏度最高：<0.5分子/u2 • 极高分辨率：5um像素点 • 多种激发及检测通道 • 适用于多种生物标记荧光探针 GENERAL SCANNING - ScanArray System Summary(1)：系统工程 Summary(2)：生物芯片应用优势 • Experiments can be performed at a faster rate • Users can simultaneously screen hundreds to thousands of targets(104 - 106) in a single experiment • Analysis of biochips can be automated through hardware/software • Less reagents are needed for experiments • Low cost per target with biochips Summary(3)：生物芯片现有技术及发展趋势 • Photolithography (Affymetrix) • Drop-on-demand (Cartesian) • Surface contact (TeleChem, Cartesian) • Greater density • Accelerated automation • Cost reduction