基因芯片的生物信息学

MicroarrayandBioinformatics基因芯片的生物信息学DrJingfuQiu邱景富SchoolofPublicHealth公共卫生学院AimsfortheMicroarrayBioinformaticsUnderstandbasicmicroarraytechnologyanditsuseingeneexpressionanalysis.基因芯片技术与表达谱分析中的应用Learnbasicdataanalysismethodsandhowtoapplythemintheanalysisofgeneexpressiondata基因芯片的数据分析–Dataacquisition数据获得–Datanormalization数据归一化–Dataanalysis数据分析–DataClustering数据聚类Vocabulary-Review回顾Gene基因:hereditaryDNAsequenceataspecificlocationonchromosome.Genetics遗传学:studyofheredity&variationinorganisms.Genome基因组:anorgan'stotalcontent(fullDNAsequence)Genomics基因组学:studyoforganismsintermsoftheirgenome.2002年2月12日,历时10载耗资20亿美元的人类基因组计划最终完成,并报道了99%的人类基因组序列.Vocabulary-Review回顾Protein蛋白质:sequenceofaminoacidsthat“doessomething”Proteomics蛋白质组学:studyofalloftheproteinsthatcancomefromanorganismsgenomeBioinformatics生物信息学:thecollection,organization&analysisoflarge-scale,complexbiologicaldata.FunctionalGenomics功能基因组学:studyofobtaininganoverallpictureofgenomefunctions,includingtheexpressionprofilesatthemRNAlevelandtheproteinlevelMicroarray基因芯片–AhighthroughputtechnologythatallowsdetectionofthousandsofgenesSimultaneously–genechip,biochip,array–Muchrelyoncomputeraids–CentralplatformforfunctionalgenomicsTypesofMicroarrays芯片的种类DNAmicroarrays,suchascDNAmicroarraysandoligonucleotidemicroarraysMMChips,forsurveillanceofmicroRNApopulationsProteinmicroarraysTissuemicroarraysCellularmicroarrays(alsocalledtransfectionmicroarraysChemicalcompoundmicroarraysAntibodymicroarraysTypesofDNAMicroarrays1.cDNAchip(DNAmicroarray,two-channelarray)cDNA芯片:–ProbecDNA(500~5,000baseslong)isimmobilizedtoasolidsurfacesuchasglass–Usingrobotspotting–TraditionallycalledDNAmicroarray–FirstlydevelopedatStanfordUniversity2.Genechip(DNAchip,Affymetrixchip)基因芯片:–Oligonucleotide(20~80-meroligos)issynthesizedeitherinsitu(on-chip)orbyconventionalsynthesisfollowedbyon-chipimmobilization–HistoricallycalledDNAchips–DevelopedatAffymetrix,Inc.,undertheGeneChip®trademark–ManycompaniesaremanufacturingoligonucleotidebasedchipsusingalternativetechnologiesHistory历史HGP(humangenomeproject):suggestedbyDelbeccoonMar.7,1986，startedinOct.1990,rapidandsensitivetechniquesforhumangenomeinformationanalysis80S:suggestionbasedoncomputerchip,WBrainstrieditfirstly.90S:StephenFodor(PresentofAffymetrixnow)madeitsuccessfully.1995:QuantitativemonitoringofgeneexpressionpatternswithacomplementaryDNAmicroarrayEndof1996:thefirstDNAchipMicroarraysarePopular芯片技术的普及AtNYUMedCenternowcollectingabout3GBofmicroarraydataperweek(60chips,6-10differentexperiments)PubMedsearchmicroarray=24,431papers352218529683815872441357845375341569701000200030004000500060001995199619971998199920002001200220032004200520062007PapersofMicroarrayindifferentyearWhatproblemscanitsolve?基因芯片的应用–Differingexpressionofgenesovertime,betweentissues,anddiseasestates基因表达差异–Identificationofcomplexgeneticdiseases复杂性基因疾病的诊断–Drugdiscoveryandtoxicologystudies药理与毒理学研究–Mutation/polymorphismdetection(SNP’s)SNP检测–Pathogenanalysis诊断病原Features特点–Parallelism高平行•Thousandsofgenessimultaneously–Miniaturization小型化•Smallchipsize–Multiplexing高通量•Multiplesamplesatthesametime–Automation自动化•Chipmanufacturing•ReagentsDifferentialGeneExpression基因表达差异AFewExamples:Celltypespecific-e.g.skincellvs.braincellDevelopmentalstage-e.g.embryonicskincellvs.adultskincellDiseasestate-e.g.normalskincellvs.skintumorcellEnvironment-specific-e.g.skincelluntreatedvs.treateddrugs,toxinsWhatisitspitfall缺陷与不足?–DetecttranscriptionmRNAlevel,nottranslationproteinlevel–Manyfactors(variations)canaffecttheresult：影响因素众多•Chipandprobedesign•Experimentdesign•Samplepreparation•Imageacquisition•Datanormalization•Dataanalysis•….–Successcrucial成功关键:•Youknowboththebiologyproblemandthecomputeraids(software,statistics).RequrimentsArrayspotter点样仪Arrayscanner扫描仪Chemistrysystems杂交体系Softwares软件Marketpredict市场预期At1999：1billionUSDLessthan5yrs:20billions2005：5billions(USA)2010：40billions(USA)Don’tincludediseasediagnosticThelargestindustryinsteadofmicroelectricsPrinciple原理•SimilartoNorthern–Base-Pairing,hybridizationbetweennucleiccids•MajordifferencesfromNorthern–Detectsthousandsofgenessimultaneously/individual–Probesfixationonglassslide/nylonmembrane–Targetsampleslabelingwithfluorescent/radioactivedNTPDesigningtheProbes探针的设计Theprobesneedtobeofhighspecificitytoavoidhybridizationwithwrongtargetmolecules.特异性Theprobesneedtogenerateanoutputthatiseasytoread(spotslieindefinedpositionsandbeofregularsizeandshapeandevenspacing).杂交结果容易判读TheprobeshavetohavehighsensitivitytodetectthemRNAandtheintensityofthespotlightmustbedifferentiablefrombackgroundnoise.敏感性Resultsmustbereproducibleacrossmultipleexperiments.重复性SpottingProcess点样过程点样针Spotrobot点样仪Cheungetal.1999Affymetrix基因芯片表达差异检测ComparisonofProbeTypes两种探针比较Advantages•NoneedtoisolateandpurifycDNAsbecauseoligonucleotidescanbesynthesized.•Shortoligonucleotidesarelesslikelytohavecross-reactivitywithothersequencesinthetargetDNA.•DensityofchipsishigherthanwithcDNAs.Limitations•Thesequencehastobeknown.•Synthesiscanbeexpensiveandtime-consuming.•Theshortsequen