生物芯片技术77267课件

1、生物芯片技术生物芯片技术生物芯片技术生物芯片技术生物芯片原理和应用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。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生物芯片分类生物芯片分类蛋白芯片(Protein Chips)a microarray-based high-throughput protein assay methodChemiluminescence or Fluorescenc

2、e based detection methods can be used to visualize bound antibodies.蛋白芯片(Protein Chips)a microarra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 an

3、tigens 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 r

4、educe 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蛋白芯片应用Diagnostic immunoassay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

5、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（Gene chip）基因芯片(Gene chip)技术是指通过微阵列(Micro基因芯片发展历史Southern & Northern BlotDot BlotMacroarrayMicroarray基因芯片发展历史Southern & Northern DoTypes of DNA ChipsTypes of DNA ChipsComparison of DNA Chip Technologies Sensitivity of

6、 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) Oligo-Chip cDNA-Ch

7、ip Genomic Chip 8 n or 20 n 50,000 nsequencingexpressionexpressiongenomic analysisComparison of DNA Chip TechnolWhy Genomic Biochips?Screen specimens to determine gene copy number changesEstablish correlations between gene copy number changes and disease biologyDetermine the interaction of multiple

8、genes on the initiation and progression of diseaseAccelerate development of products for genomic disease management to guide therapeutic interventionCombined with expression chips, gives full understanding of disease processWhy Genomic Biochips?Screen spDNA Chip TechnologySolid support (glass, plast

9、ic, metal, silicon)Miniaturized array of DNA (genetic material)Work on the biochemical principle of DNA/DNA hybridizationHybridized probes (DNA molecules) are fluorescently labeledDNA Chip TechnologySolid suppoAssay Formats 1. Extract genomic DNA from tissue4. Hybridize to Chip5. Wash and Image2. La

10、belNormalSampleGenomicExpression1. Extract mRNA from tissue2. Produce cDNA by RT & Label4. Hybridize to Chip5. Wash and Image3. Mix with labeled reference DNA3. Mix with labeled reference cDNATumorSampleAssay Formats 4. Hybridize Areas of Biochip ApplicationsAcademic research of genetic diseasesCanc

11、erPrenatal geneticsGeneral genetic diseasesInfectious diseasesDrug discoveryAnimal farming/veterinary Industrial (fermentation, corrosion)EnvironmentalAreas of Biochip ApplicationsA应用之一 基因表达谱（gene expression pattern）Research Use.Clinical Diagnostic Use.BiologicalSampleFunctional InformationOne Disea

12、seOne Gene Expression Pattern应用之一 基因表达谱（gene expression Research UseFrom 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。ClusteringResearch UseFrom Sequence tResearch UseFrom Sequence to function庞大的数据库数学模型ChallengesResearch UseFrom Sequence tResearch Use From Sequen

13、ce 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 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。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Clinical

14、 Diagnostic UseAcademic research of genetic diseasesCancerPrenatal geneticsGeneral genetic diseasesInfectious diseases HAV、HBV、HCV. HIV One Disease=One Expression PatternClinical Diagnostic UseOne Dis 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

15、之快速进入实际应用。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Prototype AmpliOnc I Biochip AmpliOncTM I Biochip after hybridization; color composite of red, blue and green imageThis biochip contains all genomic regions that have been reported to be amplified in cancers.Prototype AmpliOnc I Biochip 12

16、345678910111213141516171819202122XYOncogene Targets On the AmpliOnc I BiochipPDGFBEGFR1PDGFRAMETFGFR2WNT1MYBHER2YES1HRAS1CND1RAF1GLIMYCMDM220q13RELMYCL1FGRFESABLINT2PIK3CANMYCAKT2FGFR1JUNBAKT1KRAS2CDK4AR123456789101112131415161718192RDA ProtocolRNA extraction and cDNA preparation from archived tissu

17、e specimens(tester and driver)Generation of amplified cDNA fragments (amplicons)Subtractive hybridization of ampliconsEnrichment of cDNA fragments from differentially expressed genesRDA ProtocolRNA extraction and Microarray 用于验证RDAShotgun subcloning of RDA fragmentsPicking transformed libraries for

18、long-term propagationAmplification of RDA inserts in 96-well plate format for arrayingHybridization of cDNA amplicons to microarrays Microarray 用于验证RDAShotgun References aboutCoupling of RDA & Microarray Schena, M. et al. (2019)Science, 270, 467Lockhart,et al.(2019).Nature Biotechnology, 14, 1675DeR

19、isi, et al. (2019). Nature Genet., 14, 457. References abou Microarray 还可用于验证SSHDifferential Display PCR Microarray 还可用于验证应用之三SNPs & STRs analysisSingle Nucleotide PolymorphismsShort Tandem RepeatsPolymeraseLigase应用之三SNPs & STRs analysis生物芯片技术77267课件SNPs Typing by LigaseSNPs Typing by LigaseSNPs Typ

20、ing by Polymerase（1）SNPs Typing by Polymerase（1SNPs Typing by Polymerase（2）SNPs Typing by Polymerase（2STRs Typing（1）STRs Typing（1）STRs Typing（2）STRs Typing（2）STRs Typing（3）STRs Typing（3）应用之四 LCM俘获细胞的基因表达分析LCM: Laser Capture Microdissection应用之四 LCM俘获细胞的基因表达分析LCM: La生物芯片技术77267课件原理原理Gene expression pr

21、ofiles of laser-captured adjacent neuronal subtypesDifferential Gene Expression between Large- and Small-sized Dorsal Root Ganglion (DRG) Rat Neurons, Nissl stainedLarge DRG Neurons: myelinated fast-conductingtransmit mechanosensory informationSmall DRG Neurons: unmyelinatedslow-conductingtransmit n

22、ociceptive informationGene expression profiles of laGene expression profiles of laser-captured adjacent neuronal subtypesDorsal Root Ganglion (DRG) Rat Neurons, Nissl stainedGene expression profiles of lacDNA microarray expression patterns of small (S) and large (L) neuronscDNA microarray expression

23、 patmRNA enriched in large DRG neuronsmRNA enriched in large DRG neumRNA enriched in small DRG neuronsmRNA enriched in small DRG neu放射性原位杂交验证结果放射性原位杂交验证结果应用之五Development of therapeutic Drugsdrug target discoveryevaluation of animal models of human diseasetest for drug efficacytest for drug specifici

24、tytest for drug toxicity应用之五Development of therapeu 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其它应用 监测流行病和传染病扩散其它应用生物芯片制作技术及相关产品介绍生物芯片制作技术生物芯片制作方法分类生物芯片制作方法分类 原位合成(In Situ Synthesis)Light directed oligonucleotide synthesis.A soli

25、d 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

26、 coupling different bases allowing arbitrary DNA probes to be constructed at each site. 原位合成(In Situ Synthesis)Ligh预先合成后点样(off-chip synthesis)接触式点样非接触式点样预先合成后点样(off-chip synthesis)接触接触式点样Best!接触式点样Best!接触式点样：Chipmaker Pin （Telechem专利）接触式点样：Chipmaker Pin （TelechemArrayIt ChipMaker PinsDeveloped in co

27、njunction with Stanford UniversitySingle dip in sample multiple spotsFine (EDM) slot in tip of stainless steel pinTake-up volume 250 nL (saves sample)Spot volume of 100 to 500 pL100 m to 250 m spot size (high density arrays)Requires 10 to 30 pre-prints to “prime” pinArrayIt ChipMaker PinsDevelope非接触

28、式点样：nQUAD Technology非接触式点样：nQUAD TechnologyCharacteristics of nQUAD TechnologyWide dispense rangeLow nanoliter to high microliterExcellent linearityPrecise and accurateCVs typically less than 10%Precision less than 5%Non-contact dispense mechanismEasier mechanical alignment (384, 1536,)“On-the-fly”

29、printing possibleCapable of dispensing onto membranes or slidesMultiple liquid handling modesAspirate/DispenseContinuous Reagent DispensingCharacteristics of nQUAD Techn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

30、具备单次取样连续点样的性能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Cartesian - PixSys SeriesCartesian - PixSys Series合成后点样板 - Cartesian Tech.合成后点样板 - Cartesian Tech.PixSys Series 特点：Combines with TWO dispensing technologiesContact and Non-contact printing modeChipMaker quill pins and nQUAD dispensersPin array Spot Size: 75 to 200umnQUAD quantity: 10nL10 m positioning