生物芯片技術(shù)77267課件

1、生物芯片技術(shù)生物芯片技術(shù)生物芯片技術(shù)生物芯片技術(shù)生物芯片原理和應(yīng)用生物芯片原理和應(yīng)用何為生物芯片?生物芯片主要指通過平面微細(xì)加工技術(shù)在固體芯片表面構(gòu)建的微流體分析單元和系統(tǒng)，以實(shí)現(xiàn)對細(xì)胞、蛋白質(zhì)、核酸以及其他生物組分的準(zhǔn)確、快速、大信息量的檢測。他是繼大規(guī)模集成電路之后的又一次具有深遠(yuǎn)意義的科學(xué)技術(shù)革命。何為生物芯片?生物芯片主要指通過平面微細(xì)加工技術(shù)在固體芯片生物芯片分類生物芯片分類蛋白芯片(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蛋白芯片應(yīng)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蛋白芯片應(yīng)用Diagnostic immunoassay基因芯片(Gene chip)技術(shù)是指通過微陣列(Microarray)技術(shù)將高密度DNA片段陣列通過高速機(jī)器人或原

5、位合成方式以一定的順序或排列方式使其附著在如玻璃片等固相表面，以熒光標(biāo)記的DNA探針，借助堿基互補(bǔ)雜交原理，進(jìn)行大量的基因表達(dá)及監(jiān)測等方面研究的最新革命性技術(shù)基因芯片（Gene chip）基因芯片(Gene chip)技術(shù)是指通過微陣列(Micro基因芯片發(fā)展歷史Southern & Northern BlotDot BlotMacroarrayMicroarray基因芯片發(fā)展歷史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應(yīng)用之一 基因表達(dá)譜（gene expression pattern）Research Use.Clinical Diagnostic Use.BiologicalSampleFunctional InformationOne Disea

12、seOne Gene Expression Pattern應(yīng)用之一 基因表達(dá)譜（gene expression Research UseFrom Sequence to function計(jì)算Ratio 值 (= Cy3/Cy5) 在 0.5-2.0 之外的定義為在兩樣本中有明顯差異表達(dá)。進(jìn)而獲取初步功能信息。ClusteringResearch UseFrom Sequence tResearch UseFrom Sequence to function龐大的數(shù)據(jù)庫數(shù)學(xué)模型ChallengesResearch UseFrom Sequence tResearch Use From Sequen

13、ce to function 可以大大推進(jìn)包括人類（后）基因組計(jì)劃在內(nèi)的各類基因組研究，通過比較不同個(gè)體或物種之間以及同一個(gè)體在不同生長發(fā)育階段、正常和疾病狀態(tài)下基因及其表達(dá)的差異，尋找和發(fā)現(xiàn)新的基因，研究基因的功能以及生物體在進(jìn)化、發(fā)育、遺傳等過程中的規(guī)律。Research Use From Sequence 基因芯片可為研究不同層次多基因協(xié)同作用的生命過程提供手段。 將在研究人類重大疾病如癌癥、心血管疾病等相關(guān)基因及作用機(jī)理方面發(fā)揮巨大的作用。Research Use From Sequence to function 基因芯片可為研究不同層次多基因協(xié)同作用的生命過程提供手Clinical

14、 Diagnostic UseAcademic research of genetic diseasesCancerPrenatal geneticsGeneral genetic diseasesInfectious diseases HAV、HBV、HCV. HIV One Disease=One Expression PatternClinical Diagnostic UseOne Dis 生物芯片能為現(xiàn)代醫(yī)學(xué)科學(xué)及醫(yī)學(xué)診斷學(xué)的發(fā)展提供強(qiáng)有力的手段，促進(jìn)醫(yī)學(xué)從“系統(tǒng)、血管、組織和細(xì)胞層次”(通常稱之為“第二階段醫(yī)學(xué)”)向“DNA、RNA、蛋白質(zhì)及其相互作用層次”(第三階段醫(yī)學(xué))過渡，使

15、之快速進(jìn)入實(shí)際應(yīng)用。Clinical Diagnostic Use 生物芯片能為現(xiàn)代醫(yī)學(xué)科學(xué)及醫(yī)學(xué)診斷學(xué)的發(fā)展提供強(qiáng)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 用于驗(yàn)證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 用于驗(yàn)證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 還可用于驗(yàn)證SSHDifferential Display PCR Microarray 還可用于驗(yàn)證應(yīng)用之三SNPs & STRs analysisSingle Nucleotide PolymorphismsShort Tandem RepeatsPolymeraseLigase應(yīng)用之三SNPs & STRs analysis生物芯片技術(shù)77267課件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）應(yīng)用之四 LCM俘獲細(xì)胞的基因表達(dá)分析LCM: Laser Capture Microdissection應(yīng)用之四 LCM俘獲細(xì)胞的基因表達(dá)分析LCM: La生物芯片技術(shù)77267課件原理原理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放射性原位雜交驗(yàn)證結(jié)果放射性原位雜交驗(yàn)證結(jié)果應(yīng)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應(yīng)用之五Development of therapeu 在預(yù)防醫(yī)學(xué)方面，可以使人們盡早地認(rèn)識自身潛在的疾病，并實(shí)施有效的防治措施 法醫(yī)學(xué)中，進(jìn)行基因指紋快速識別，以及親子鑒定其它應(yīng)用其它應(yīng)用 監(jiān)測流行病和傳染病擴(kuò)散 監(jiān)測有害微生物的發(fā)生和傳播 農(nóng)、林、牧、魚等產(chǎn)業(yè)的品種改良和病蟲防治其它應(yīng)用 監(jiān)測流行病和傳染病擴(kuò)散其它應(yīng)用生物芯片制作技術(shù)及相關(guān)產(chǎn)品介紹生物芯片制作技術(shù)生物芯片制作方法分類生物芯片制作方法分類 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預(yù)先合成后點(diǎn)樣(off-chip synthesis)接觸式點(diǎn)樣非接觸式點(diǎn)樣預(yù)先合成后點(diǎn)樣(off-chip synthesis)接觸接觸式點(diǎn)樣Best!接觸式點(diǎn)樣Best!接觸式點(diǎn)樣：Chipmaker Pin （Telechem專利）接觸式點(diǎn)樣：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非接觸

28、式點(diǎn)樣：nQUAD Technology非接觸式點(diǎn)樣：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生物芯片點(diǎn)樣系統(tǒng)必備性能多針頭自動同時(shí)取樣，取樣板為96孔或384孔板單針取樣量為250到500nL，每次點(diǎn)樣量為100到150pL點(diǎn)樣直徑為100到150um，系統(tǒng)點(diǎn)陣分辨率為10um可程控進(jìn)行連續(xù)點(diǎn)樣操作固相介質(zhì)如玻璃片須通過適當(dāng)機(jī)制進(jìn)行位置固定點(diǎn)樣針尖必須

30、具備單次取樣連續(xù)點(diǎn)樣的性能生物芯片點(diǎn)樣系統(tǒng)必備性能多針頭自動同時(shí)取樣，取樣板為96孔或Cartesian - PixSys SeriesCartesian - PixSys Series合成后點(diǎn)樣板 - Cartesian Tech.合成后點(diǎn)樣板 - Cartesian Tech.PixSys Series 特點(diǎn)：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