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Lesson1DNACLONING:ANOVERVIEW第一课克隆：概要DNAcloning：DNAdoningfacilitatestheisolationandmanipulationoffragmentsofanorganism'sgenomebyreplicatingthemindependentlyaspartofanautonomousvector.DNA克隆：DNA克隆通过独立复制可以用分离和操作方法使生物体基因组片段成为自发性载体的一部分。Hostsandvectors:MostoftheroutinemanipulationsinvolvedingenecloninguseEscherichiacoliasthehostorganism.PlasmidsandbacteriophagesmaybeusedascloningvectorsinE.coli.Vectorsbasedonplasmids,virusesandwholechromosomeshavebeenusedtocarryforeigngenesintootherprokaryoticandeukaryoticorganisms.宿主和载体：大多数的基因克隆使用的常规操作，以大肠杆菌为宿主生物体。质粒和噬菌体可作为大肠杆菌的克隆载体。以质粒、病毒和整条染色体为载体，将外源基因导入其他原核和真核生物。Subcloning:SubcloningisthesimpletransferofaclonedfragmentofDNAfromonevectortoanother;itservestoillustratemanyoftheroutinetechniquesinvolvedingenecloning.亚克隆：克隆是克隆的DNA片段从一个载体到另一个载体的简单传递；它可以用来说明基因克隆中的许多常规技术。DNAlibraries:DNAlibraries,consistingofsetsofrandomclonedfragmentsofeithergenomicorcDNA,eachinaseparatevectormolecule,areusedintheisolationofunknowngenes.基因库：基因库，由两组随机克隆片段组成，无论是基因组还是基因，每一个在一个单独的载体分子中，都被用来分离未知的基因。Screeninglibraries:Librariesarescreenedforthepresenceofagenesequencebyhybridizationwithasequencederivedfromitsproteinproductorarelatedgene,orthroughthescreeningoftheproteinproductsoftheclonedfragments.筛选库：通过与来自其蛋白产物或相关基因的序列杂交，或者通过克隆片段的蛋白产物的筛选，筛选出一个基因序列的存在。Analysisofaclone:Onceidentified,aclonedgenemaybeanalyzedbyrestrictionmapping,andultimatelybyDNAsequencing,beforebeingusedinanyofthediverseapplicationsofDNAcloning.克隆分析:在用于基因克隆的各种应用中之前，一旦发现某个克隆基因可以通过限制性图谱进行分析，则用这方法进行DNA序列测定。DNAcloning:detailedmolecularanalysisofproteinsorotherconstituentsofmostorganismswasrendereddifficultorimpossiblebytheirscarcityandtheconsequentdifficultyoftheirpurificationinlargequantities.Oneapproachistoisolatethegene(s)responsiblefortheexpressionofaproteinortheformationofaproduct.However,everyorganism'sgenomeislargeandcomplex(seeSectionD),andanysequenceofinterestusuallyoccursonlyonceortwicepercell.Hence,standardchemicalorbiochemicalmethodscannotbeusedtoisolateaspecificregionofthegenomeforstudy,particularlyastherequiredsequenceofDNAischemicallyidenticaltoalltheothers.Thesolutiontothisdilemmasistoplacearelativelyshortfragmentofagenome,whichmightcontainthegeneorothersequenceofinterest,inanautonomouslyreplicatingpieceofDNA,knownasavector,formingrecombinantDNA,whichcanbereplicatedindependentlyoftheoriginalgenome,andnormallyinanotherhostspeciesaltogether.PropagationofthehostorganismcontainingtherecombinantDNAformsasetofgeneticallyidenticalorganisms,oraclone.ThisprocessishenceknownasDNAcloning.DNA克隆：一般上，因为大多数生物体的蛋白质或其他成分的稀缺性及其在大量的净化过程中的困难，导致它们的详细分子分析变得困难甚至不可能。一种方法是分离负责蛋白质表达的基因或者其产品的构成。然而每个生物体的基因组都是大而复杂的（见第4节），每个细胞中任何目标序列通常只发生一次或两次。因此，标准的化学或生化方法不能用于分离特定区域的基因组进行研究，特别是所需的DNA序列与所有其他的序列的化学性质相同。这种难点的解决方法是放置一个可能包含目标基因或序列相对短的基因组片段在一个自主复制片段的基因，称为载体，在另一个宿主物种中它可以复制独立的原始基因组，形成重组DNA。含有重组DNA的宿主生物体的繁殖形成一组基因相同的生物体，或克隆。这个过程就是DNA克隆。AmongsttheexplodingnumbersofapplicationsofDNAcloning,oftencollectedtogetherunderthetermgeneticengineering,arethefollowing:在长期的基因工程中，克隆的应用程序的数量激增，这是以下几项：(1)DNAsequencing,andhencethederivationofproteinsequence(seeTopicJ2).DNA序列测定，也即蛋白质序列的推导（见主题J2）。(2)Isolationandanalysisofgenepromotersandothercontrolsequences(seeTopicJ4).基因启动子与其它控制序列的分离与分析（见主题J4）。(3)Investigationofprotein/enzyme/RNAfunctionbylarge-scaleproductionofnormalandalteredforms(seeTopicJS).蛋白质/酶/RNA功能的正常大规模生产和改变的研究（见主题J5）。(4)Identificationofmutations,forexamplegenedefectsleadingtodisease(seeTopicJ6).突变的识别，例如基因缺陷导致的疾病（见主题J6）。(5)Biotechnology;thelarge-scalecommercialproductionofproteinsandothermoleculesofbiologicalimportance,forexamplehumaninsulinandgrowthhormone(seeTopicJ6).生物技术；蛋白质和其他分子生物学意义的大规模商业化生产，例如人胰岛素和生长激素（见主题J6）。(6)Engineeringanimalsandplants,andgenetherapy(seeTopicJ6).转基因动物和植物，和基因治疗（见主题J6）。(7)Engineeringproteinstoaltertheirproperties(seeTopicJ6).通过蛋白质工程来改变它们的性质（见主题J6）Hostsandvectors:TheinitialisolationandanalysisofDNAfragmentsisalmostalwayscarriedoutusingthebacteriumE.coliasthehostorganism,althoughtheyeastSaccharomycescerevisiaeisbeingusedtomanipulateverylargefragmentsofthehumangenome(seeTopicH3).Awidevarietyofnaturalrepliconshavethepropertiesrequiredtoallowthemtoactascloningvectors.Vectorsmustnormallybecapableofbeingreplicatedandisolatedindependentlyofthehost'sgenome,althoughsomearedesignedtoincorporateDNAintothehostgenomeforlongertermexpressionofclonedgenes.Vectorsalsoincorporateaselectablemarker,agenewhichallowshostcellscontainingthevectortobeselectedfromamongstthosewhichdonot,usuallybyconferringresistancetoatoxin(seeTopicG2),orenablingtheirsurvivalundercertaingrowthconditions(seeTopicH3).宿主和载体DNA片段的初步分离和分析几乎总是使用大肠杆菌作为宿主生物体，虽然酿酒酵母被用来操纵人类基因组大片段（见主题H3）。各种各样的自然的复制，要求他们拥有作为克隆载体的性质。载体通常是能够被复制和独立的主机的基因组中分离，虽然一些被设计成将核酸到宿主基因组中的长期表达的克隆基因。载体也将选择标记允许宿主细胞中没有相同序列载体的基因，通常用抗毒素（见主题G2），或使他们在一定的生长条件生存（见主题H3）。ThefirstE.colivectorswereextrachromosomal(separatefromthechromosome)circularplasmids(seeTopicG2),andanumberofbacteriophages(virusesinfectingbacteria;seeTopicR2)havealsobeenusedinE.coli.Phagekcanbeusedtoclonefragmentslargerthanplasmidvectors,andphageM13allowsclonedDNAtobeisolatedinsi