Gateway 克隆技术背景原理及其应用

Gateway®克隆技术背景、原理及其应用王宇、余辉首都师范大学TableofContentsOverview1AdvahtagesoftheGatewayTechnology2ApplicationofGatewayTechnology3Examples4Whatis“GatewayTechnology”?‘TheGateway®Technologyisauniversalcloningmethodbasedonthesite-specificrecombinationpropertiesofbacteriophagelambda.’Gateway®TechnologyHandbook.Invtrogen®Keyword：universal、site-specificrecombination、bacteriophagelambdaGeneticRecombinationDefinationofGeneticRecombination:‘Geneticrecombinationisaprocessbywhichamoleculeofnucleicacid(usuallyDNA,butcanalsobeRNA)isbrokenandthenjoinedtoadifferentone.’en.wikipedia.orgTypesofGeneticRecombination：Homologousrecombination（同源重组）Site-specificrecombination（位点专一性重组）HomologousRecombination（同源重组）：‘RecombinationthatrequiresextensivesequencesimilaritybetweentherecombiningDNAs.’RobertF.Weaver.MolecularBiology3th‘同源重组是更为普遍的一种重组机制，它可以发生在任何两种具有相同或相似序列之间，涉及到两个DNA分子在相同区域的断裂和重新连接。’CharacteristicofHomologousRecombinationRandomsite（发生在随机位点）Requireextensivesequencesimilarity（需要一定长度的同源片段，1kb）Site-specificrecombination（位点专一性重组）:‘RecombinationthatalwaysoccursinthesameplaceanddependsonlimitedsequencesimilaritybetweentherecombiningDNAs.’RobertF.Weaver.MolecularBiology3th‘重组反应发生在两个特异序列（不一定同源）之间，如噬菌体整合/外切或转座中共合体结构的解离。’BenjaminLewin.GenesVIIIBacteriophagelambda（λ噬菌体）噬菌体生活史：溶原状态→裂解状态（整合integration）裂解状态→溶原状态（外切excision）溶原状态裂解状态外切（Int、IHF、XIS）整合（Int、IHF）随宿主基因组复制侵染其他宿主（适宜环境）（外界胁迫）att：attachmentsiteO：coresequence，homologous15bpcoreregionsIfthenormalattBsiteisdeletedfromthechromosome,integrasecanbindwithloweraffinitytosecondarysitesonthechromosome,resultinginintegrationoflambdaatadifferentsite.Thefrequencyofintegrationintosecondarysitesis100-1000timesrarerthanintegrationintoattB.Theactualcrossoveroccursbetweenhomologous15bpcoreregionsonthetwosites,butsurroundingsequencesarerequiredastheycontainthebindingsitesfortherecombinationproteins(Landy,1989).ModificationstotheattSitesMutationshavebeenmadetothecoreregionsoftheattsitestoeliminatestopcodonsandtoensurespecificityoftherecombinationreactionstomaintainorientationandreadingframe.Mutationshavebeenintroducedintotheshort(5bp)regionsflankingthe15-bpcoreregionsoftheattBsitestominimizesecondarystructureformationinsingle-strandedformsofattBplasmids(e.g.phagemidssDNAormRNA).A43bpportionoftheattRsitehasbeenremovedtomaketheinvitroattLxattRreactionirreversibleandmoreefficient(Bushmanetal.,1985).MechanismofGatewayBOB’B’OBPOP’BOB’B’OBOB’OP’OPP’P’PPattB1attB2attP1attP2attL1attL2attR1attR2GeneGeneccdBccdBccdBGeneTheccdBproteininterfereswithE.coliDNAgyrase(BernardandCouturier,1992),therebyinhibitinggrowthofmostE.colistrains(e.g.DH5α™,TOP10).CellsthattakeupunreactedvectorscarryingtheccdBgeneorby-productmoleculesretainingtheccdBgenewillfailtogrow.BecauseofthelethaleffectsoftheCcdBprotein,allGateway®vectorscontainingtheccdBgenemustbepropagatedinanE.colistrainthatisresistanttoCcdBeffects.WerecommendusingtheDB3.1™E.colistrainwhichcontainsagyrasemutation(gyrA462)thatrendersitresistanttotheCcdBeffects(BernardandCouturier,1992;Bernardetal.,1993;Mikietal.,1992).MechanismofGatewayWhyweuseEntryClone?UniversalityForsequencingLessredundancyAdvantagesofGatewaytechonology位点特异性重组-一小时自动的亚克隆反应（不需要酶切,连接酶和克隆筛选）保持阅读框和方向克隆重组率高(90%+)一次实验可以转移一个或者多个基因到一个或者多个表达载体任何载体能够改造为Gateway-兼容载体双向亚克隆简单、快速、可自动化-高通量限制位点酶切与Gateway比较在PCR引物上游加入6-10bp碱基，形成限制酶切位点，然后进行PCR使用相应的限制酶消化PCR产物，并纯化(4-6h)使用同样的限制酶消化载体将PCR产物连接到载体上（12h）载体转化与阳性克隆筛选RE酶切位点的选择（目的基因/载体）双酶切体系的选择，酶的星号活性需要考虑的问题假阳性（线性DNA片段的不稳定性）在PCR引物上游加入25bp碱基，形成att位点，然后进行PCRPCR产物与gateway载体反应(1h)载体转化与阳性克隆筛选GatewayMuti-ComponentGatewayDual-directionHigh-throughputApplicationandFuture