生物芯片技術77267課件

生物芯片技術生物芯片技術生物芯片原理和應用何為生物芯片?生物芯片主要指通過平面微細加工技術在固體芯片表面構(gòu)建的微流體分析單元和系統(tǒng)，以實現(xiàn)對細胞、蛋白質(zhì)、核酸以及其他生物組分的準確、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意義的科學技術革命。蛋白芯片(ProteinChips)amicroarray-basedhigh-throughputproteinassaymethodChemiluminescenceorFluorescencebaseddetectionmethodscanbeusedtovisualizeboundantibodies.蛋白芯片應用Diagnosticimmunoassay

thatallowsthesimultaneoushigh-throughputanalysisofknownautoantigens.Inordertoquantifyantibodiesintheseraofpatientswithautoimmunediseases,Recombinantantigensandcontrolproteinswereimmobilizedonslideswithreactivealdehydegroupsasreplicasinserialdilutionsofthevariousantigenstherebyallowingaccuratedeterminationofautoantibodytiterusingminimalamountsofserum.Miniaturizedandhighlyparalleledimmunoassayslikethesewill

reducecost

by

decreasingreagentconsumption

andimproveperformancebygreatlyincreasingthenumberofassaysthatcanbeperformedwithasingleserumsample.Protein-ProteinInteraction.

DNA-ProteinInteraction基因芯片(Genechip)技術是指通過微陣列(Microarray)技術將高密度DNA片段陣列通過高速機器人或原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，以熒光標記的DNA探針，借助堿基互補雜交原理，進行大量的基因表達及監(jiān)測等方面研究的最新革命性技術基因芯片（Genechip）TypesofDNAChipsComparisonofDNAChipTechnologies

SensitivityofDNAchipbasedassaysisafunctionof:ProbeandtargetDNA/RNA(Complexity)Chipsurface(autofluorescence&non-spec.bkg)Attachmentchemistry/methodology(hyb.efficiency&crosshyb.)Hybridizationefficiency(lotsoffactors)Detectiontechnology(signaltype,efficiency,noise)

Oligo-Chip cDNA-Chip GenomicChip8nor20n<2,000n>50,000n

sequencingexpressionexpressiongenomicanalysisWhyGenomicBiochips?ScreenspecimenstodeterminegenecopynumberchangesEstablishcorrelationsbetweengenecopynumberchangesanddiseasebiologyDeterminetheinteractionofmultiplegenesontheinitiationandprogressionofdiseaseAcceleratedevelopmentofproductsforgenomicdiseasemanagementtoguidetherapeuticinterventionCombinedwithexpressionchips,givesfullunderstandingofdiseaseprocessAssayFormats

1.ExtractgenomicDNAfromtissue4.HybridizetoChip5.WashandImage2.LabelNormalSampleGenomicExpression1.ExtractmRNAfromtissue2.ProducecDNAbyRT&Label4.HybridizetoChip5.WashandImage3.MixwithlabeledreferenceDNA3.MixwithlabeledreferencecDNATumorSampleAreasofBiochipApplicationsAcademicresearchofgeneticdiseasesCancerPrenatalgeneticsGeneralgeneticdiseasesInfectiousdiseasesDrugdiscoveryAnimalfarming/veterinaryIndustrial(fermentation,corrosion)Environmental應用之一

基因表達譜（geneexpressionpattern）ResearchUse.ClinicalDiagnosticUse.BiologicalSampleFunctionalInformationOneDisease——OneGeneExpressionPatternResearchUse——

FromSequencetofunction計算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。進而獲取初步功能信息。ClusteringResearchUse——

FromSequencetofunction龐大的數(shù)據(jù)庫數(shù)學模型ChallengesResearchUse——

FromSequencetofunction

可以大大推進包括人類（后）基因組計劃在內(nèi)的各類基因組研究，通過比較不同個體或物種之間以及同一個體在不同生長發(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化、發(fā)育、遺傳等過程中的規(guī)律。ClinicalDiagnosticUseAcademicresearchofgeneticdiseasesCancerPrenatalgeneticsGeneralgeneticdiseasesInfectiousdiseasesHAV、HBV、HCV…..HIV

OneDisease=OneExpressionPattern生物芯片能為現(xiàn)代醫(yī)學科學及醫(yī)學診斷學的發(fā)展提供強有力的手段，促進醫(yī)學從“系統(tǒng)、血管、組織和細胞層次”(通常稱之為“第二階段醫(yī)學”)向“DNA、RNA、蛋白質(zhì)及其相互作用層次”(第三階段醫(yī)學)過渡，使之快速進入實際應用。ClinicalDiagnosticUsePrototypeAmpliOnc?IBiochip

AmpliOncTMIBiochipafterhybridization;colorcompositeofred,blueandgreenimageThisbiochipcontainsallgenomicregionsthathavebeenreportedtobeamplifiedincancers.RDAProtocolRNAextractionandcDNApreparationfromarchivedtissuespecimens(testeranddriver)GenerationofamplifiedcDNAfragments(‘a(chǎn)mplicons’)SubtractivehybridizationofampliconsEnrichmentofcDNAfragmentsfromdifferentiallyexpressedgenes

Microarray用于驗證RDAShotgunsubcloningofRDAfragmentsPickingtransformedlibrariesforlong-termpropagationAmplificationofRDAinsertsin96-wellplateformatforarrayingHybridizationofcDNAampliconstomicroarrays

Referencesabout

CouplingofRDA&Microarray

Schena,M.etal.(1995)Science,270,467Lockhart,etal.(1996).NatureBiotechnology,14,1675DeRisi,etal.(1996).NatureGenet.,14,457.應用之三——

SNPs&STRsanalysisSingleNucleotidePolymorphismsShortTandemRepeatsPolymeraseLigaseSNPsTyping——

byLigaseSNPsTyping——

byPolymerase（1）SNPsTyping——

byPolymerase（2）STRsTyping（1）STRsTyping（2）STRsTyping（3）應用之四——

LCM俘獲細胞的基因表達分析LCM:LaserCaptureMicrodissection原理Geneexpressionprofilesoflaser-capturedadjacentneuronalsubtypesDifferentialGeneExpressionbetweenLarge-andSmall-sizedDorsalRootGanglion(DRG)RatNeurons,NisslstainedLargeDRGNeurons:myelinatedfast-conductingtransmitmechanosensoryinformationSmallDRGNeurons:unmyelinatedslow-conductingtransmitnociceptiveinformationGeneexpressionprofilesoflaser-capturedadjacentneuronalsubtypesDorsalRootGanglion(DRG)RatNeurons,NisslstainedcDNAmicroarrayexpressionpatternsofsmall(S)andlarge(L)neuronsmRNAenrichedinlargeDRGneuronsmRNAenrichedinsmallDRGneurons放射性原位雜交驗證結(jié)果應用之五——

DevelopmentoftherapeuticDrugsdrugtargetdiscoveryevaluationofanimalmodelsofhumandiseasetestfordrugefficacytestfordrugspecificitytestfordrugtoxicity在預防醫(yī)學方面，可以使人們盡早地認識自身潛在的疾病，并實施有效的防治措施法醫(yī)學中，進行基因指紋快速識別，以及親子鑒定其它應用監(jiān)測流行病和傳染病擴散監(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用生物芯片制作技術及相關產(chǎn)品介紹生物芯片制作方法分類

原位合成

(InSituSynthesis)Lightdirectedoligonucleotidesynthesis.Asolidsupportisderivatizedwithacovalentlinkermoleculeterminatedwithaphotolabileprotectinggroup.Lightisdirectedthroughamasktodeprotectandactivateselectedsites,andprotectednucleotidescoupletotheactivatedsites.Theprocessisrepeated,activatingdifferentsetsofsitesandcouplingdifferentbasesallowingarbitraryDNAprobestobeconstructedateachsite.預先合成后點樣

(off-chipsynthesis)接觸式點樣非接觸式點樣接觸式點樣Best!接觸式點樣：

ChipmakerPin（Telechem專利）ArrayItChipMakerPinsDevelopedinconjunctionwithStanfordUniversitySingledipinsamplemultiplespotsFine(EDM)slotintipofstainlesssteelpinTake-upvolume~250nL(savessample)Spotvolumeof~100to500pL100μmto250μmspotsize(highdensityarrays)Requires10to30pre-printsto“prime”pin非接觸式點樣：

nQUADTechnologyCharacteristicsofnQUADTechnologyWidedispenserangeLownanolitertohighmicroliterExcellentlinearityPreciseandaccurateCV’stypicallylessthan10%Precisionlessthan5%Non-contactdispensemechanismEasiermechanicalalignment(384,1536,…)“On-the-fly”printingpossibleCapableofdispensingontomembranesorslidesMultipleliquidhandlingmodesAspirate/DispenseContinuousReagentDispensing生物芯片點樣系統(tǒng)必備性能多針頭自動同時取樣，取樣板為96孔或384孔板單針取樣量為250到500nL，每次點樣量為100到150pL點樣直徑為100到150um，系統(tǒng)點陣分辨率為10um可程控進行連續(xù)點樣操作固相介質(zhì)如玻璃片須通過適當機制進行位置固定點樣針尖必須具備單次取樣連續(xù)點樣的性能Cartesian-PixSysSeries合成后點樣板-CartesianTech.PixSysSeries特點：CombineswithTWOdispensingtechnologiesContactandNon-contactprintingmodeChipMakerquillpinsandnQUADdispensersPinarraySpotSize:75to200umnQUADquantity:10nL10μm