生物芯片技术简介

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。生物芯片分类DNAChipsProteinChipsLabChipsBiochips蛋白芯片(ProteinChips)amicroarray-basedhigh-throughputproteinassaymethodChemiluminescenceorFluorescencebaseddetectionmethodscanbeusedtovisualizeboundantibodies.蛋白芯片应用•Diagnosticimmunoassaythatallowsthesimultaneoushigh-throughputanalysisofknownautoantigens.Inordertoquantifyantibodiesintheseraofpatientswithautoimmunediseases,Recombinantantigensandcontrolproteinswereimmobilizedonslideswithreactivealdehydegroupsasreplicasinserialdilutionsofthevariousantigenstherebyallowingaccuratedeterminationofautoantibodytiterusingminimalamountsofserum.Miniaturizedandhighlyparalleledimmunoassayslikethesewillreducecostbydecreasingreagentconsumptionandimproveperformancebygreatlyincreasingthenumberofassaysthatcanbeperformedwithasingleserumsample.•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）基因芯片发展历史Southern&NorthernBlotDotBlotMacroarrayMicroarrayTypesofDNAChipsExpressionChipsGenomicChipsSequencingChipsDNAChipsComparisonofDNAChipTechnologiesSensitivityofDNAchipbasedassaysisafunctionof:–ProbeandtargetDNA/RNA(Complexity)–Chipsurface(autofluorescence&non-spec.bkg)–Attachmentchemistry/methodology(hyb.efficiency&crosshyb.)–Hybridizationefficiency(lotsoffactors)–Detectiontechnology(signaltype,efficiency,noise)Oligo-ChipcDNA-ChipGenomicChip8nor20n2,000n50,000nsequencingexpressionexpressiongenomicanalysisWhyGenomicBiochips?•Screenspecimenstodeterminegenecopynumberchanges•Establishcorrelationsbetweengenecopynumberchangesanddiseasebiology•Determinetheinteractionofmultiplegenesontheinitiationandprogressionofdisease•Acceleratedevelopmentofproductsforgenomicdiseasemanagementtoguidetherapeuticintervention•Combinedwithexpressionchips,givesfullunderstandingofdiseaseprocessDNAChipTechnology•Solidsupport(glass,plastic,metal,silicon)•MiniaturizedarrayofDNA(geneticmaterial)•WorkonthebiochemicalprincipleofDNA/DNAhybridization•Hybridizedprobes(DNAmolecules)arefluorescentlylabeledAssayFormats1.ExtractgenomicDNAfromtissue4.HybridizetoChip5.WashandImage2.LabelNormalSampleGenomicExpression1.ExtractmRNAfromtissue2.ProducecDNAbyRT&Label4.HybridizetoChip5.WashandImage3.MixwithlabeledreferenceDNA3.MixwithlabeledreferencecDNATumorSampleAreasofBiochipApplications•Academicresearchofgeneticdiseases–Cancer–Prenatalgenetics–Generalgeneticdiseases•Infectiousdiseases•Drugdiscovery•Animalfarming/veterinary•Industrial(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。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。ResearchUse——FromSequencetofunctionClinicalDiagnosticUse•Academicresearchofgeneticdiseases–Cancer–Prenatalgenetics–Generalgeneticdiseases•Infectiousdiseases–HAV、HBV、HCV…..–HIVOneDisease=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™IBiochipAmpliOncTMIBiochipafterhybridization;colorcompositeofred,blueandgreenimageThisbiochipcontainsallgenomicregionsthathavebeenreportedtobeamplifiedincancers.12345678910111213141516171819202122XYOncogeneTargetsOntheAmpliOnc™IBiochipPDGFBEGFR1PDGFRAMETFGFR2WNT1MYBHER2YES1HRAS1CND1RAF1GLIMYCMDM220q13RELMYCL1FGRFESABLINT2PIK3CANMYCAKT2FGFR1JUNBAKT1KRAS2CDK4ARRDAProtocol•RNAextractionandcDNApreparationfromarchivedtissuespecimens(testeranddriver)•GenerationofamplifiedcDNAfragments(‘amplicons’)•Subtractivehybridizationofamplicons•EnrichmentofcDNAfragmentsfromdifferentiallyexpressedgenesMicroarray用于验证RDA•ShotgunsubcloningofRDAfragments•Pickingtransformedlibrariesforlong-termpropagation•AmplificationofRDAinsertsin96-wellplateformatforarraying•HybridizationofcDNAampliconstomicroarraysReferencesaboutCouplingofRDA&Microarray•Schena,M.etal.(1995)Science,270,467•Lockhart,etal.(1996).NatureBiotechnology,14,1675•DeRisi,etal.(1996).NatureGenet.,14,457.Microarray还可用于验证•SSH•DifferentialDisplayPCR应用之三——SNPs&STRsanalysis•SingleNucleotidePolymorphisms•ShortTandemRepeats•Polymerase•LigaseSNPsTyping——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:•myelinated•fast-conducting•transmitmecha