基因工程-第5章-基因文库的构建-1.ppt

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1、Chapter5ConstructionofDNAlibrariesGenelibrary(Genomiclibrary)：Acollectionofindependentclonesrepresentingtheentiregenomeofanorganism.FirststeptoisolateatargetDNAsequencefromanorganism’sgenome.1976L.Clark,J.Carbonn=ln(1-p)/ln(1-f)n:numbersofrecombinantrequiredinfu

2、lllibraryp:probabilityoftargetgeneinlibraryf:rateofaverageinsertionDNAsizestogenomeMammal3×109bpIfp=99%clonedfragment20kbf=20kb/3×109n=690773.2E.coli4639221bpp=99%f=20kb/4600kbn=1056.9p=99%f=10kb/4600kbn=2116cDNAlibrary:Acollectionofindependentclonesrepresenting

3、theentirecDNAreverselytranscriptedfrommRNAinaspecifictissueofanorganisminaspecificphage.BacterialcoloniesconsistingofgenomicDNAlibraryChoosingvectorStepsoflibraryconstructionPlasmid<10kbPhage0-23kbCosmid~45kbBAC~100kb（250-300kb?）YAC~300kb-1.2MbSUP4基因：酵母细胞Trp-tR

4、NA基因的赭色突变抑制基因，含有克隆位点。在发生Ade2-赭色突变的宿主细胞中，如果没有外源基因的插入，则突变基因表达受抑制受体菌为Ade+，形成白色菌落；当有外源基因插入时，SUP4表达将被阻断，抑制作用被消除，受体菌为Ade-，菌落为红色。CosmidHalfofphage.If700000,cosmid350000(菌落数)CosmidisusuallyappliedinlargesizeDNAcloning,butitismuchmoredifficultthanphage，becausethereisn

5、omuchexperience.（pJB8andc2RBaremoreusedingenelibraryconstruction)phagevector1970’s,likeCharon4Aandgt-WES，15～20kbPresently,EMBLlines,like2001，DASH，Charon38-40，GEM-11etc.(replacementvectors),20-24kbBasicprinciplesinchoosingvectorEasilyrecoverforeignDNAfrom;

6、Easilyprepare2arms;Availabilityofvectorsequence,REmapping;Ambermutation(琥珀突变)invectorarmsasspecialscreeningmodel;Activegamgeneinrecombinants；Chisequenceinvectorarms.Gam蛋白与核酸外切酶(ExonucleaseV)结合，抑制了宿主的RecBCD的核酸外切酶V活性，使得外来DNA分子不会被切割降解。Enterlyticcyclereplicationgiv

7、eriseto50genome,thenrollingcyclereplicatingtoproduceconcatemer,andfinallyitiscutandpackagedintoparticles.recB/recC/recDproducts,controllingrecombination,andhaveactivitiesofExnuleaseV,inhibitingDNAreplicationtransfertorollingcycle.gamgeneproductsbindandinactiva

8、teexnucleaseV.Ifgam-,recAproductisrequiredtopackage,butformsmallplaques(小噬斑),sothehostmustberecA+.ButrecA+maycauseotherrecombination,thusweusevectorrecA-/gam+,toreal