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)用何為生物芯片?生物芯片主要指通過(guò)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(duì)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(cè)。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。生物芯片分類蛋白芯片(ProteinChips)amicroarray-basedhigh-throughputproteinassaymethodChemiluminescenceorFluorescencebaseddetectionmethodscanbeusedtovisualizeboundantibodies.蛋白芯片應(yīng)用Diagnosticimmunoassay

thatallowsthesimultaneoushigh-throughputanalysisofknownautoantigens.Inordertoquantifyantibodiesintheseraofpatientswithautoimmunediseases,Recombinantantigensandcontrolproteinswereimmobilizedonslideswithreactivealdehydegroupsasreplicasinserialdilutionsofthevariousantigenstherebyallowingaccuratedeterminationofautoantibodytiterusingminimalamountsofserum.Miniaturizedandhighlyparalleledimmunoassayslikethesewill

reducecost

by

decreasingreagentconsumption

andimproveperformancebygreatlyincreasingthenumberofassaysthatcanbeperformedwithasingleserumsample.Protein-ProteinInteraction.

DNA-ProteinInteraction基因芯片(Genechip)技術(shù)是指通過(guò)微陣列(Microarray)技術(shù)將高密度DNA片段陣列通過(guò)高速機(jī)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(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(cè)等方面研究的最新革命性技術(shù)基因芯片（Genechip）基因芯片發(fā)展歷史Southern&NorthernBlotDotBlotMacroarrayMicroarrayTypesofDNAChipsComparisonofDNAChipTechnologies

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,givesfullunderstandingofdiseaseprocessDNAChipTechnologySolidsupport(glass,plastic,metal,silicon)MiniaturizedarrayofDNA(geneticmaterial)WorkonthebiochemicalprincipleofDNA/DNAhybridizationHybridizedprobes(DNAmolecules)arefluorescentlylabeledAssayFormats

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

基因表達(dá)譜（geneexpressionpattern）ResearchUse.ClinicalDiagnosticUse.BiologicalSampleFunctionalInformationOneDisease——OneGeneExpressionPattern

ResearchUse——

FromSequencetofunction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。ClusteringResearchUse——

FromSequencetofunction龐大的數(shù)據(jù)庫(kù)數(shù)學(xué)模型ChallengesResearchUse——

FromSequencetofunction

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(guò)比較不同個(gè)體或物種之間以及同一個(gè)體在不同生長(zhǎng)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(guò)程中的規(guī)律?；蛐酒蔀檠芯坎煌瑢哟味嗷騾f(xié)同作用的生命過(guò)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。ResearchUse——

FromSequencetofunctionClinicalDiagnosticUseAcademicresearchofgeneticdiseasesCancerPrenatalgeneticsGeneralgeneticdiseasesInfectiousdiseasesHAV、HBV、HCV…..HIV

OneDisease=OneExpressionPattern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(guò)渡，使之快速進(jìn)入實(shí)際應(yīng)用。ClinicalDiagnosticUsePrototypeAmpliOnc?IBiochip

AmpliOncTM

IBiochipafterhybridization;colorcompositeofred,blueandgreenimageThisbiochipcontainsallgenomicregionsthathavebeenreportedtobeamplifiedincancers.12345678910111213141516171819202122XYOncogeneTargetsOntheAmpliOnc?IBiochipPDGFBEGFR1PDGFRAMETFGFR2WNT1MYBHER2YES1HRAS1CND1RAF1GLIMYCMDM220q13RELMYCL1FGRFESABLINT2PIK3CANMYCAKT2FGFR1JUNBAKT1KRAS2CDK4ARRDAProtocolRNAextractionandcDNApreparationfromarchivedtissuespecimens(testeranddriver)GenerationofamplifiedcDNAfragments(‘a(chǎn)mplicons’)SubtractivehybridizationofampliconsEnrichmentofcDNAfragmentsfromdifferentiallyexpressedgenes

Microarray

用于驗(yàn)證RDAShotgunsubcloningofRDAfragmentsPickingtransformedlibrariesforlong-termpropagationAmplificationofRDAinsertsin96-wellplateformatforarrayingHybridizationofcDNAampliconstomicroarrays

Referencesabout

CouplingofRDA&Microarray

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

Microarray

還可用于驗(yàn)證SSHDifferentialDisplayPCR應(yīng)用之三——

SNPs&STRsanalysisSingleNucleotidePolymorphismsShortTandemRepeatsPolymeraseLigaseSNPsTyping——

byLigaseSNPsTyping——

byPolymerase（1）SNPsTyping——

byPolymerase（2）STRsTyping（1）STRsTyping（2）STRsTyping（3）應(yīng)用之四——

LCM俘獲細(xì)胞的基因表達(dá)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(yàn)證結(jié)果應(yīng)用之五——

DevelopmentoftherapeuticDrugsdrugtargetdiscoveryevaluationofanimalmodelsofhumandiseasetestfordrugefficacytestfordrugspecificitytestfordrugtoxicity在預(yù)防醫(yī)學(xué)方面，可以使人們盡早地認(rèn)識(shí)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(shí)別，以及親子鑒定其它應(yīng)用監(jiān)測(cè)流行病和傳染病擴(kuò)散監(jiān)測(cè)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)用生物芯片制作技術(shù)及相關(guān)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(yù)先合成后點(diǎn)樣

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

ChipmakerPin（Telechem專利）ArrayItChipMakerPinsDevelopedinconjunctionwithStanfordUniversitySingledipinsample

multiplespotsFine(EDM)slotintipofstainlesssteelpinTake-upvolume~250nL(savessample)Spotvolumeof~100to500pL100μmto250μmspotsize(highdensityarrays)Requires10to30pre-printsto“prime”pin非接觸式點(diǎn)樣：

nQUADTechnologyCharacteristicsofnQUADTechnologyWidedispenserangeLownanolitertohighmicroliterExcellentlinearityPreciseandaccurateCV’stypicallylessthan10%Precisionlessthan5%Non-contactdispensemechanismEasiermechanicalalignment(384,1536,…)“On-the-fly”printingpossibleCapableofdispensingontomembranesorslidesMultipleliquidhandlingmodesAspirate/DispenseContinuousReagentDispensing生物芯片點(diǎn)樣系統(tǒng)必備性能多針頭自動(dòng)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(guò)適當(dāng)機(jī)制進(jìn)行位置固定點(diǎn)樣針尖必須具備單次取樣連續(xù)點(diǎn)樣的性能Cartesian-PixSysSeries合成后點(diǎn)樣板-CartesianTech.PixSysSeries特點(diǎn)：CombineswithTWOdispensingtechnologiesContactandNon-contactprintingmodeChipMakerquillpinsandnQUAD