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Tilingarrayandexonarray第十一讲Tiling芯片•全基因组测序的完成，使新的全基因组实验手段成为可能•TilingArrays提供了一个解读基因组序列的方法（aphysicalreadoutofagenome）•Tiling指贴，盖，搭的意思，Tiling芯片指叠瓦式阵列芯片，也指嵌合芯片，是非常适合全基因组分析的针对所有转录本的DNA微阵列•密集的探针数才能保证对整个基因组全转录本的扫描，保证一个都不会少genometiling探针来源•Oligos：synthesizeddirectlyontheslidesorpreparedinsolutionandthendeposited•PCRproductstilingtypicallyof~1-kbinlength•芯片上的双链形态对杂交的影响•难以规模化，制备~1-kbPCR产物的tiling芯片仅以chromosome22(roughly1%ofthehumangenome)为例就需要20000次PCR反应。APCRtilingoftheentirehumangenomewouldrequireapproximatelytwomillionPCRreactionsatthesameresolution。不仅PCR反应量大，而且相应的生物信息学处理要求也很大。•分辨率低PCR产物用于tiling的缺点GeneChipTilingArraysfeature•25-meroligonucleotidesforoptimalhybridizationspecificity•High-resolutionarraysforthemostaccuratedetection•usedtobuildannotations,particularlyingenomicregionspreviouslythoughttobe“genomedeserts.”•一般采用等长移位法，按照靶序列从头到尾选取一定长度（16－25nt－36nt）的互补核苷酸序列形成一组探针组合，其中相邻探针序列相差一个或多个碱基，覆盖的碱基数目是恒定的。TilingArrayDesignTilingArrayDesign高分辨率分辨率：一条探针的正中间的碱基与相邻探针正中间的碱基之间的距离，取决于两探针相隔的距离（gap）的大小TilingArrayDesign•Probes：non-repetitive（RepetitiveelementswereremovedbyRepeatMasker），throughoutcodingandnon-codingsequences，•Tilingarrayslabeledwith“R”arecomplementarytothereverse(-)strandofagenomeandFarraysarecomplementarytotheforward(+)direction.PromotertilingarrayWhole-GenomeTilingArraySets•TheGeneChipHumanTiling1.0RArraySetisa14-arraysetdesignedfortranscriptmappingorotheranalysesthatbenefitfrombothperfectmatchandmismatchprobes.•TheGeneChipHumanTiling2.0RArraySetisdesignedforChIPexperimentsandfeaturesalloftheperfectmatchprobesfromtheHumanTiling1.0RArraySet.•Eacharraywithinthesetscontainsover6.5millionprobestospecificallyinterrogategenomicregions2004scienceResults•oligonucleotidemicroarraysrepresenting1.5GbofnonrepetitivegenomicDNAfromeachstrandofthehumangenome•Atotalof51,874,38836-nucleotide(nt)probes,positionedevery46ntonaverage,wereselectedtointerrogatesenseandantisensestrandsofthegenomeandsynthesizedatafeaturedensityofabout390,000probesperarray•发现了上千条新的转录区，验证了全基因组水平经序列预测得到的基因转录产物的表达。•得到比以前更为详尽的关于基因表达的注释（extensiveanddiverse）•很多新的转录产物与在小鼠中已经充分研究的蛋白序列上保守，证明这些新发现的转录产物中的绝大部分都是功能性的转录产物。•很多保守的转录序列都位于已知基因的远端，而且相当一部分的蛋白编码产物在300个氨基酸以上。ChromatinImmunoprecipitation•multipleregulationpathwaysinteracting,regulatorynetworks.•Chromatinimmunoprecipitation(ChIP)assayshavebeenusedtostudyprotein/DNAinteractions.•ChIPprotocolsinconjunctionwithwhole-genometiledmicroarraystounderstandDNAregulationbymappingsitesoftranscriptionfactorsbindingtoDNA.Generegulationbackground•Whatischromatin?–ChromatinistheportionofthecellnucleusthatcontainsalloftheDNAofthenucleus–DNAisalwaysfoundinassociationwithabundanthistoneproteins,whichareusedbythecelltoregulategeneexpressionthroughaseriesofmodificationevents(acetylationandde-acetylation)thataltertheDNA’sconformation(compression)–Ingeneral,acetylationofhistonetailsseemstocause“relaxation”oftheDNA,whichenablesproteins(transcriptionfactors)toaccesstheDNAandinitiatetranscriptionChIPbackground•ChIPisamethodusedtopurifyandanalyzesmallgenomicfragmentsusingaprocessoffixation,DNAshearingandantibody-basedprecipitation•Classically–ChIPhasbeenperformedagainstmodificationsofhistonetails•i.e.DNAfragmentsareenrichedusingantibodiesspecificforacetylatedhistones–However,whilethisisanindicatorofpromoteractivityitdoesnotidentifytheregulatingfactor(TF)involved•Alternatively–ChIPcanbeperformedusingantibodiesspecificforregulatingproteinsi.e.transcriptionfactors–However,thismethodistechnicallydemandinganddifficulttovalidatewithoutprovenreagentsbecauseTFsarefarlessabundantthanhistoneproteinsChIP:Whatisitusedfor?ChIPisusedtostudyProtein/DNAInteractions,mainlyinthefieldoftranscriptionalactivity®ulation:•TranscriptionFactors(TF)•RNApolymerase,Histones,…•Otherprotein/DNAinteractions:DNAreplication,DNARepair,methylation,telomerelength,etc.LocationAnalysis(ChIP-on-chip)Majorapplications•Determinationofmechanismofactionfortranscriptionfactorsandtargetgenesinnormalanddiseasestates•Identificationofnoveltargets•Validationofbioassaysandtoxicantsignatures•Mappingregulatoryswitchesincellcircuitryandsignalingnetworks•Understandingprocessesoftranscription,histonemodification,andDNAreplication,modification,andrepairLocationAnalysis(ChIP-on-chip)ChIP-on-chipWorkflow•染色质免疫共沉淀芯片将染色质沉淀和芯片技术结合在一起，简单的说，在体内DNA和蛋白质在甲醛作用下交联，并且通过与目标蛋白对应的抗体实现免疫沉淀。通过超声波作用将DNA降解为0.2-2kb的片段。需要对超声波参数进行设定，以获得长度在一定范围内的DNA片段。Pull-downDNA和合适的对照用荧光标记，加在载玻片上，用于芯片分析，可以找到特异性蛋白在基因组中的结合位点。StemCellTranscriptionalRegulationNetworksAdditionalStudyApplication:ChIP-on-chip•Comprehensiveandgenome-widelocalizationof18,000humangenes•co-occupancy•biologicallyrelevantgenomictargets•OCT4,SOX2,NANOGpathways•DeconstructionofEScellphenotypetoidentifycircuitscontrollingstemcellself-renewalandpluripotency•IdentificationofpotentialtherapeutictargetsanddrugscreeningcandidatesTILING芯片的应用•最精确的定位转录产物mappingnoveltranscripts的方法•蛋白/DNA相互作用：转录因子与DNA上的哪个区域结合（Chromosome-IPcDNAbindingregion）•表观遗传学的甲基化分析，组蛋白乙酰化•SNP分析•DNAcopy-numberalterations(arrayCGH)•protein-bindingmotifs(PBMs)小结•Tilingarray的基本概念•Tilingarray的应用•Chip-on