04-博士研究生课-高级生物化学与分子生物学-专题-肿瘤功能基因组学-许丽艳

ProgressofFunctionalGenomicsLi-YanXuDNARecombinationTechnologyGeneChipStrategyofFunctionalGenomicsRNAinterferenceDNArecombinationtechnology1DNAcloning2toolenzyme3targetgene4genevector1DNAcloningItisaprocessofDNAmolecularamplification.Usually,thefirstatargetDNAfragmentisinsertedtoavectorandarecombinant(replicon)isconstructed.Thesecondtherecombinantistransformedintohostcellandscreenoutthecellcontainingtherecombinant.Thelastthatcellisamplified,namelyamassoftargetDNAmoleculeisgained.2toolenzymerestrictionendonucleaseDNAligaseDNApolymeraseIreversetranscriptasepolynucleotidekinaseend-transferasealkalinephosphatasestructuralcharacterofcuttingsiterecognizedbyrestrictionenzymerestrictionendonucleaserecognizedsequenceandcutBamHIEcoRIGAATTCCTTAAGGGATCCCCTAGG5’PvuISstIGAGCTCCTCGAGCGATCGGCTAGC5’AluISmaIAGCTTCGACCCGGGGGGCCC5’5’5’5’5’5’5’5’5’5’3targetgeneTheinterestedgeneisthetargetgenesourceofthetargetgene*ItisfromgenomicDNAdirectly,thisisprokaryoticgeneonlygenerally.*Itisfromartificialsynthesis,thisissimplepolypeptidegenegenerally.*ItisfrommRNA.*ItisfromgenomiclibraryorcDNAlibrary.*PolymeraseChainReaction(PCR).synthesizecDNAfrommRNAAAA…AAA5’3’mRNAAAA…AAA3’5’AAA…AAATTT...TTT5’3’5’3’primer:oligodTreversetranscriptaseTTT...TTT5’3’basichydrolysisTTT...TTT5’3’?TTT...TTT5’3’AAA…AAADNApolymeraseITTT...TTT5’3’AAA…AAA3’5’S1nucleasegenomiclibrarygenomicDNAfragment50-200kbextractionrestrictivelycutgenefragmentsrecombinationrecombinanttransformationgenomiclibrarytargetgenecDNAlibraryrecombinationrecombinanttransformationcDNAlibraryextractionmRNAcDNAdoublestrands5'3'extension3'5'PCRProcess5'3'5'3'3'5'denaturationannealing3'5'5'3'5'3'3'5'5'3'5'5'5'3'3'5'denaturationNextcycle5'3'5'3'4genevectorThegenevectorsareDNAmolecules,whichstructureisreconstructed.TheycancarrytargetDNAfragmentThetargetgeneorDNAfragmentisamplifiedandexpressed.vector*plasmid*cosmid*phage*M13phage*insectvirusDNA(autographcalifornicavirus,ACNPV)*yeastartificialchromosomeDNA*vacciniavirusDNA*simianvirus40DNA3-10kb40kb29-48.5kb5.243kb180kb6.407kb20kb4-8kb15kb0.3-1.0kb2.5kb128kb100kb25kb*bovinepapillomavirusDNA8.0kb10kb0.2-2.2Mb0.3-1.2Mb*retrovirusDNA*fowlpoxvirusDNA*adenovirusDNA*herpessimplexvirusDNA*cytomegalovirusDNA*Epstein-BarrvirusDNA240kb170kb6.407kb233-238kb8-10kb24-36kbXmnI39662034XmnIPstI36122067PvuII1424AvaI650SalI375BamHIplasmidpBR3224.36kb29HindIIIEcoRI0AoriginAscreeninggeneAsinglerestrictionsiteconditionoriplasmidpUC192.69kbEcoRISacIKpnISmaIBamHIXbaIHincIIPstISphIHindIIIpolylinker52bpPlaclacIlacZ’theprocedureofgenecloningseparatetargetgeneaswellasvector1cuttargetgeneandvectorrestrictedly2jointargetgeneandvector3recombinanttransformation4separatetargetgeneaswellasvector1cuttargetgeneandvectorrestrictedly2ligatetargetgeneandvector3recombinantscreening5recombinantscreening5recombinanttransformation4recombinantscreening5goastepfurther...targetgeneamplify6incomplete1231+3incompletedigestion123SmaIcomplete1+2+31------2,22------5,33------9,44------14,556nn+n(n+1)/2n+1separatetargetgenecutandligatetargetgeneandvectorCGGCCGGCHpaIICCGGGGCCCCGGGGCCHpaIIgenomeDNACGGCCGGCHpaIILigaserecombinantCCGGGGCCplasmidHpaIIrecombinanttransformationvectorsandrecombinantscompetentcellsrecombinantscreeningamportetetc+plasmidextractiondigistwithrestrictionenzeme1212marker-+12marker-+targetgeneamplification食管癌细胞NGAL基因5’端转录调控区不同长度片段PCR扩增结果200bpM1431113794565741615211242000bp1000bp重组子pGEM-1431~152XhoI和BglII双酶切后，琼脂糖凝胶电泳鉴定结果200bpM1143111379456574161521124M21000bp←947bp←5.0kb←2.0kb重组子pGLP-1431~152XhoI和BglII双酶切后，琼脂糖凝胶电泳鉴定结果M143111379456574161525000bp1375bp564bptargetgeneexpressionprokaryoticexpressionsystemDExpressionanalysisoffourexpressionvectorsinE.colibySDS-PAGEeukaryoticexpressionsystem21kDa→←25kDa12345678910DNARecombinationTechnologyGeneChipStrategyofFunctionalGenomicsRNAinterferenceDNAAnalysisSouthernBlot:telomerelength;amplification;deletionPCR:mutation;DNAmethylationInsituhybridization:genelocation;translocationDNAChip:SNPBioinformaticsDNAAnalysisSouthernBlot:telomerelength;amplification;deletionPCR:mutation;DNAmethylationInsituhybridization:genelocation;translocationDNAChip:SNPBioinformaticsRNAAnalysisNorthernBlot:expressedgenesRT-PCR:expressedgenesInsituhybridization:genelocationcDNAmicroarray:differentiallyexpressedgenesBioinformaticsProteinAnalysisWesternBlot:proteinconcentrationImmunohistochemicalTechnique:proteinlocation2-DElectrophoresis:differerentiallyexpressedproteinsBioinformaticsCoverofScience1992199319941995199619971998199920002001FirstMicroarrayPatentIssued1stCatalogGeneChip®ProductRocheEasyAccessCommerciallaunchWestSacramento2002RocheAmpliChipTMlaunched2003U133Set10K19912004100KWorld'sFirstDiagnosticMicroarraySystemLaunchedbyAffyinEurope11µmMillionsofcopiesofaspecificoligonucleotideprobeImageofHybridizedGeneChipArray500,000differentcomplementaryprobesSinglestranded,labeled‘target’Oligonucleotide‘probe’GeneChip®ArrayHybridizedProbeCell1.28cm1.28cmPerfectMatchPMMMMismatch独具创新的PM-MM探针设计Oligo探针ATCGGTAGCCATGCATGAGTTACTAATCGGTAGCCATCCATGAGTTACTA13M