Gateway 克隆技术背景原理及其应用.pdf

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1、Gateway®克隆技术背景、原理及其应用王宇、余辉首都师范大学TableofContents1Overview2AdvahtagesoftheGatewayTechnology3ApplicationofGatewayTechnology4ExamplesWhatis“GatewayTechnology”?‘TheGateway®Technologyisauniversalcloningmethodbasedonthesite-specificrecombinationpropertiesofbacteriophagelambda.’Gate

2、way®TechnologyHandbook.Invtrogen®Keyword：universal、site-specificrecombination、bacteriophagelambdaGeneticRecombinationDefinationofGeneticRecombination:‘Geneticrecombinationisaprocessbywhichamoleculeofnucleicacid(usuallyDNA,butcanalsobeRNA)isbrokenandthenjoinedtoadifferentone.

3、’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

4、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）:‘Recombinationthatalwaysoccursinthesameplaceanddependsonlimitedsequencesimilaritybetwee

5、ntherecombiningDNAs.’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）整合（Int、IHF）溶原状态裂解状态外切（Int、IHF、XIS）侵染其他宿主（外界胁迫）随宿主基因组复制（适宜环境）att：attachmen

6、tsiteO：coresequence，homologous15bpcoreregionsIfthenormalattBsiteisdeletedfromthechromosome,integrasecanbindwithloweraffinitytosecondarysitesonthechromosome,resultinginintegrationoflambdaatadifferentsite.Thefrequencyofintegrationintosecondarysitesis100-1000timesrarerthaninteg

7、rationintoattB.Theactualcrossoveroccursbetweenhomologous15bpcoreregionsonthetwosites,butsurroundingsequencesarerequiredastheycontainthebindingsitesfortherecombinationproteins(Landy,1989).ModificationstotheattSitesMutationshavebeenmadetothecoreregionsoftheattsitestoeliminates

8、topcodonsandtoensurespecificityoftherecombinationreactionstomaintainorienta