生物芯片技术77267课件

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。蛋白芯片(ProteinChips)amicroarray-basedhigh-throughputproteinassaymethodChemiluminescenceorFluorescencebaseddetectionmethodscanbeusedtovisualizeboundantibodies.蛋白芯片应用Diagnosticimmunoassay

thatallowsthesimultaneoushigh-throughputanalysisofknownautoantigens.Inordertoquantifyantibodiesintheseraofpatientswithautoimmunediseases,Recombinantantigensandcontrolproteinswereimmobilizedonslideswithreactivealdehydegroupsasreplicasinserialdilutionsofthevariousantigenstherebyallowingaccuratedeterminationofautoantibodytiterusingminimalamountsofserum.Miniaturizedandhighlyparalleledimmunoassayslikethesewill

reducecost

by

decreasingreagentconsumption

andimproveperformancebygreatlyincreasingthenumberofassaysthatcanbeperformedwithasingleserumsample.Protein-ProteinInteraction.

DNA-ProteinInteraction基因芯片(Genechip)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（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应用之一

基因表达谱（geneexpressionpattern）ResearchUse.ClinicalDiagnosticUse.BiologicalSampleFunctionalInformationOneDisease——OneGeneExpressionPatternResearchUse——

FromSequencetofunction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。ClusteringResearchUse——

FromSequencetofunction庞大的数据库数学模型ChallengesResearchUse——

FromSequencetofunction

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。ClinicalDiagnosticUseAcademicresearchofgeneticdiseasesCancerPrenatalgeneticsGeneralgeneticdiseasesInfectiousdiseasesHAV、HBV、HCV…..HIV

OneDisease=OneExpressionPattern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。ClinicalDiagnosticUsePrototypeAmpliOnc™IBiochip

AmpliOncTMIBiochipafterhybridization;colorcompositeofred,blueandgreenimageThisbiochipcontainsallgenomicregionsthathavebeenreportedtobeamplifiedincancers.RDAProtocolRNAextractionandcDNApreparationfromarchivedtissuespecimens(testeranddriver)GenerationofamplifiedcDNAfragments(‘amplicons’)SubtractivehybridizationofampliconsEnrichmentofcDNAfragmentsfromdifferentiallyexpressedgenes

Microarray用于验证RDAShotgunsubcloningofRDAfragmentsPickingtransformedlibrariesforlong-termpropagationAmplificationofRDAinsertsin96-wellplateformatforarrayingHybridizationofcDNAampliconstomicroarrays

Referencesabout

CouplingofRDA&Microarray

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

SNPs&STRsanalysisSingleNucleotidePolymorphismsShortTandemRepeatsPolymeraseLigaseSNPsTyping——

byLigaseSNPsTyping——

byPolymerase（1）SNPsTyping——

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

LCM俘获细胞的基因表达分析LCM:LaserCaptureMicrodissection原理Geneexpressionprofilesoflaser-capturedadjacentneuronalsubtypesDifferentialGeneExpressionbetweenLarge-andSmall-sizedDorsalRootGanglion(DRG)RatNeurons,NisslstainedLargeDRGNeurons:myelinatedfast-conductingtransmitmechanosensoryinformationSmallDRGNeurons:unmyelinatedslow-conductingtransmitnociceptiveinformationGeneexpressionprofilesoflaser-capturedadjacentneuronalsubtypesDorsalRootGanglion(DRG)RatNeurons,NisslstainedcDNAmicroarrayexpressionpatternsofsmall(S)andlarge(L)neuronsmRNAenrichedinlargeDRGneuronsmRNAenrichedinsmallDRGneurons放射性原位杂交验证结果应用之五——

DevelopmentoftherapeuticDrugsdrugtargetdiscoveryevaluationofanimalmodelsofhumandiseasetestfordrugefficacytestfordrugspecificitytestfordrugtoxicity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方法分类

原位合成

(InSituSynthesis)Lightdirectedoligonucleotidesynthesis.Asolidsupportisderivatizedwithacovalentlinkermoleculeterminatedwithaphotolabileprotectinggroup.Lightisdirectedthroughamasktodeprotectandactivateselectedsites,andprotectednucleotidescoupletotheactivatedsites.Theprocessisrepeated,activatingdifferentsetsofsitesandcouplingdifferentbasesallowingarbitraryDNAprobestobeconstructedateachsite.预先合成后点样

(off-chipsynthesis)接触式点样非接触式点样接触式点样Best!接触式点样：

ChipmakerPin（Telechem专利）ArrayItChipMakerPinsDevelopedinconjunctionwithStanfordUniversitySingledipinsamplemultiplespotsFine(EDM)slotintipofstainlesssteelpinTake-upvolume~250nL(savessample)Spotvolumeof~100to500pL100µmto250µmspotsize(highdensityarrays)Requires10to30pre-printsto“prime”pin非接触式点样：

nQUADTechnologyCharacteristicsofnQUADTechnologyWidedispenserangeLownanolitertohighmicroliterExcellentlinearityPreciseandaccurateCV’stypicallylessthan10%Precisionlessthan5%Non-contactdispensemechanismEasiermechanicalalignment(384,1536,…)“On-the-fly”printingpossibleCapableofdispensingontomembranesorslidesMultipleliquidhandlingmodesAspirate/DispenseContinuousReagentDispensing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-PixSysSeries合成后点样板-CartesianTech.PixSysSeries特点：CombineswithTWOdispensingtechnologiesContactandNon-contactprintingmodeChipMakerquillpinsandnQUADdispensersPinarraySpotSize:75to200umnQUADquantity:10nL10µm