生物技术专业英语翻译

Lesson1DNACLONING:ANOVERVIEW第一课克隆：概要DNAcloning：DNAdoningfacilitatestheisolationandmanipulationoffragmentsofanorganism'sgenomebyreplicatingthemindependentlyaspartofanautonomousvector.DNA克隆：DNA克隆经过独立复制能够用分离和操作方法使生物体基因组片段成为自发性载体一部分。Hostsandvectors:MostoftheroutinemanipulationsinvolvedingenecloninguseEscherichiacoliasthehostorganism.PlasmidsandbacteriophagesmaybeusedascloningvectorsinE.coli.Vectorsbasedonplasmids,virusesandwholechromosomeshavebeenusedtocarryforeigngenesintootherprokaryoticandeukaryoticorganisms.宿主和载体：大多数基因克隆使用常规操作，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，将外源基因导入其余原核和真核生物。Subcloning:SubcloningisthesimpletransferofaclonedfragmentofDNAfromonevectortoanother;itservestoillustratemanyoftheroutinetechniquesinvolvedingenecloning.亚克隆：克隆是克隆DNA片段从一个载体到另一个载体简单传递；它能够用来说明基因克隆中许多常规技术。DNAlibraries:DNAlibraries,consistingofsetsofrandomclonedfragmentsofeithergenomicorcDNA,eachinaseparatevectormolecule,areusedintheisolationofunknowngenes.基因库：基因库，由两组随机克隆片段组成，不论是基因组还是基因，每一个在一个单独载体分子中，都被用来分离未知基因。Screeninglibraries:Librariesarescreenedforthepresenceofagenesequencebyhybridizationwithasequencederivedfromitsproteinproductorarelatedgene,orthroughthescreeningoftheproteinproductsoftheclonedfragments.筛选库：经过与来自其蛋白产物或相关基因序列杂交，或者经过克隆片段蛋白产物筛选，筛选出一个基因序列存在。Analysisofaclone:Onceidentified,aclonedgenemaybeanalyzedbyrestrictionmapping,andultimatelybyDNAsequencing,beforebeingusedinanyofthediverseapplicationsofDNAcloning.克隆分析:在用于基因克隆各种应用中之前，一旦发觉某个克隆基因能够经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：通常上，因为大多数生物体蛋白质或其余成份稀缺性及其在大量净化过程中困难，造成它们详细分子分析变得困难甚至不可能。一个方法是分离负责蛋白质表示基因或者其产品组成。然而每个生物体基因组都是大而复杂（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。所以，标准化学或生化方法不能用于分离特定区域基因组进行研究，尤其是所需DNA序列与全部其余序列化学性质相同。这种难点处理方法是放置一个可能包含目标基因或序列相对短基因组片段在一个自主复制片段基因，称为载体，在另一个宿主物种中它能够复制独立原始基因组，形成重组DNA。含有重组DNA宿主生物体繁殖形成一组基因相同生物体，或克隆。这个过程就是DNA克隆。AmongsttheexplodingnumbersofapplicationsofDNAcloning,oftencollectedtogetherunderthetermgeneticengineering,arethefollowing:在长久基因工程中，克隆应用程序数量激增，这是以下几项：DNAsequencing,andhencethederivationofproteinsequence(seeTopicJ2).DNA序列测定，也即蛋白质序列推导（见主题J2）。Isolationandanalysisofgenepromotersandothercontrolsequences(seeTopicJ4).基因开启子与其它控制序列分离与分析（见主题J4）。Investigationofprotein/enzyme/RNAfunctionbylarge-scaleproductionofnormalandalteredforms(seeTopicJS).蛋白质/酶/RNA功效正常大规模生产和改变研究（见主题J5）。Identificationofmutations,forexamplegenedefectsleadingtodisease(seeTopicJ6).突变识别，比如基因缺点造成疾病（见主题J6）。Biotechnology;thelarge-scalecommercialproductionofproteinsandothermoleculesofbiologicalimportance,forexamplehumaninsulinandgrowthhormone(seeTopicJ6).生物技术；蛋白质和其余分子生物学意义大规模商业化生产，比如人胰岛素和生长激素（见主题J6）。Engineeringanimalsandplants,andgenetherapy(seeTopicJ6).转基因动物和植物，和基因治疗（见主题J6）。Engineeringproteinstoaltertheirproperties(seeTopicJ6).经过蛋白质工程来改变它们性质（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片段初步分离和分析几乎总是使用大肠杆菌作为宿主生物体，即使酿酒酵母被用来操纵人类基因组大片段（见主题H3）。各种各样自然复制，要求他们拥有作为克隆载体性质。载体通常是能够被复制和独立主机基因组中分离，即使一些被设计成将核酸到宿主基因组中长久表示克隆基因。载体也将选择标识允许宿主细胞中没有相同序列载体基因，通惯用抗毒素（见主题G2），或使他们在一定生长条件生存（见主题H3）。ThefirstE.colivectorswereextrachromosomal(separatefromthechromosome)circularplasmids(seeTopicG2),andanumberofbacteriophages(virusesinfectingbacteria;seeTopicR2)havealsobeenusedinE.coli.Phagekcanbeusedtoclonefragmentslargerthanplasmidvectors,andphageM13allowsclonedDNAtobeisolatedinsingle-strandedform(seeTopicH2).Morespecialistvectorshavebeenengineeredtouseaspectsofplasmidsandbacteriophages,suchastheplasmid-bacteriophagekhybridsknownascosmids(seeTopicH3).VerylargegenomicfragmentsfromhumansandotherspecieshavebeenclonedinS.cerevisiaeasyeastartificialchromosomes(YACs;seeTopicH3).PlasmidandphagevectorshavebeenusedtoexpressgenesinarangeofbacteriaotherthanE.coli,and.somephagesmaybeusedtoincorporateDNAintothehostgenome,forexamplephagek(seeTopicH2).Plasmidvectorshavebeendevelopedforuseinyeast(yeastepisomalplasmids),whileinplants;abacterialplasmid(AgrobacteriumtumefaciensTiplasmid)canbeusedtointegrateDNAintothegenome.Inothereukaryoticcellsinculture,vectorshaveoftenbeenbasedonviruseswhichnaturallyinfecttherequiredspecies,eitherbymaintainingtheirDNAextrachromosomallyorbyintegrationintothehostgenome(examplesincludeSV40,baculovirus,retroviruses;seeTopicH4).第一个大肠杆菌质粒染色体外（从染色体分离）环状质粒（见主题G2），和一些噬菌体（感染细菌病毒；见主题R2）也被用于大肠杆菌。噬菌体K可用于克隆片段大于质粒，噬菌体M13和允许克隆DNA被孤立单链形式（见主题H2）。更专业载体已被设计为使用质粒和噬菌体方面，如质粒，噬菌体K杂交称为粘粒（见主题H3）。非常大基因组片段从人类和其余物种已被克隆酿酒酵母酵母人工染色体（YAC克隆；seetopicH3）。质粒和噬菌体载体已被用于在一个范围内其余比大肠杆菌，细菌和噬菌体基因表示。一些可能被用来将DNA插入宿主基因组，比如噬菌体K（见主题H2）。质粒载体已经开发使用酵母（酵母着丝粒质粒），而植物；细菌质粒（根癌农杆菌Ti质粒）能够用来整合进基因组DNA。在文化其余真核细胞中，载体通常是依照其自然感染病毒所需物种，或者经过保持他们DNA染色体外或整合到宿主基因组（包含SV40，杆状病毒，逆转录病毒；见主题H4）。Subcloning:Thesimplestkindofcloningexperiment,whichexemplifiesmanyofthebasictechniquesofDNAcloning,isthetransferofafragmentofclonedDNAfromonevectortoanother,aprocessknownassubcloning.Thismightbeusedtoinvestigateashortregionofalargeclonedfragmentinmoredetail,ortotransferagenetoavectordesignedtoexpressitinaparticularspecies,forexample.InthecaseofplasmidvectorsinE.coli,themostcommonsituation,theprocessmaybedividedintothefollowingsteps,whichareconsideredingreaterdetailinTopicsG2-G5:亚克隆：最简单克隆试验，这表现了许多DNA克隆基本技术，是克隆片段从一个载体到另一个载体转移，这一过程称为亚克隆。这可更详细用来研究一个大克隆片段短区域，比如说将基因放入载体使其转移到一个特定物种中进行表示。通常情况下，在质粒载体转移到大肠杆菌案例中，该方法可分为以下几个步骤，其中在主题g2-g5说明更详细:IsolationofplasmidDNAcontainingtheclonedsequenceofinterest(seeTopicG2).质粒分离包含目标克隆序列（见主题G2）。Digestion(cutting)oftheplasmidintodiscretefragmentswithrestrictionendonucleases(seeTopicG3).用限制性内切酶切（割）离散片段质粒（见主题G3）。Separationofthefragmentsbyagarosegelelectrophoresis(seeTopicG3).经过琼脂糖凝胶电泳分离片段（见主题G3）。Purificationofthedesiredtargetfragment(seetopicG3)纯化所需目标片段（见主题G3）。Ligation(joining)ofthefragmentintoanewplasmidvector,toformanewrecombinantmolecule(seeTopicG4).将片段插入新质粒载体中，以形成新重组分子（见主题G4）。(6)TransferoftheligatedplasmidintoanE.Coilstrain(transformation)(seetopicG4).把连接质粒转移到大肠杆菌菌株（变换）（见主题G4）。(7)selectionoftransformedbacteria(seetopicG4).转化菌筛选（见主题G4）。(8)Analysisofplasmids(seeTopicG4).质粒分析（见主题G4）。DNAlibraries:克隆库:TherearetwomainsourcesfromwhichDNAisderivedforcloningexperimentsdesignedtoidentifyanunknowngene-bulkgenomicDNAfromthespeciesofinterestand,inthecaseofeukaryotes,bulkmRNAfromacellortissuewherethegeneisknowntobeexpressed.TheyareusedintheformationofgenomiclibrariesandcDNAlibrariesrespectively(seeTopicI2).有2个主要起源，一是基因克隆试验，目标是判定未知基因——来自目标物种大部分基因组DNA。二是就真核生物来说，大多数mRNA来自已知其基因表示细胞或组织。它们分别用于基因组文库和基因库形成（见主题I2）。DNAlibrariesaresetsofDNAclones(acloneisageneticallydistinctindividualorsetofidenticalindividuals),eachofwhichhasbeenderivedfromtheinsertionofadifferentfragmentintoavectorfollowedbypropagationinthehost.GenomiclibrariesarepreparedfromrandomfragmentsofgenomicDNA.However,genomiclibrariesmaybeaninefficientmethodoffindingagene,particularlyinlargeeukaryoticgenomes,wheremuchoftheDNAisnoncoding(seeTopicD4).ThealternativeistouseasthesourceofthelibrarythemRNAfromacellortissuewhichisknowntoexpressthegene.DNAcopies(cDNA)aresynthesizedfromthemRNAbyreversetranscriptionandaretheninsertedintoavectortoformacDNAlibrary,cDNAlibrariesareefficientforcloningagenesequence,butyieldonlythecodingregion,andnotthesurroundinggenomicsequences.克隆库是一组基因克隆（克隆是一个基因不一样个体或相同个体），每一个都来自于一个不一样片段插入到载体中，然后在宿主中传输。基因组文库是从基因组随机片段制备。然而，基因组文库中可能是一个低效率寻找基因方法，尤其是在大型真核生物基因组，其中大部分是非编码DNA（见主题D4）。另一个方法是利用细胞或组织中细胞或组织来表示基因起源。经过反转录合成基因复制品，并将其插入到载体中，形成一个文库，文库是有效，用于克隆一个基因序列，但只产生编码区，而不是周围基因组序列。Screeninglibraries:筛选库:Sinceitisnotapparentwhichcloneinalibrarycontainsthegeneofinterest,amethodforscreeningforitspresenceisrequired.ThisisoftenbasedontheuseofaradioactivelylabeledDNAprobewhichiscomplementaryorpartiallycomplementarytoaregionofthegenesequence,andwhichcanbeusedtodetectitbyhybridization(seeTopicC3).Theprobesequencemightbeanoligonucleotidederivedfromthesequenceoftheproteinproductofthegene,ifitisavailable,orfromarelatedgenefromanotherspecies(seeTopicI3).Anincreasinglyimportantmethodforthegenerationofprobesisthepolymerasechainreaction(PCR;.seeTopicJ3).Otherscreeningmethodsrelyontheexpressionofthecodingsequencesoftheclonesinthelibrary,andidentificationoftheproteinproductfromitsactivity,orwithaspedficantibody,forexample(seeTopicI3).因为在克隆库中目标基因是不显著，所以其筛选方法存在是必需。这通常基于应用放射性标识DNA探针是互补或部分互补一个区域基因序列，能够经过杂交来检测它（见主题C3）。探针序列可能是来自于基因蛋白质产物序列一个寡核苷酸，也可能来自另一个相关基因（见主题I3）。聚合酶链反应是一个日益主要探针生成方法（PCR；。见主题J3）。其余筛选方法依赖于在克隆库中编码序列表示，比如说用特异抗体进行蛋白质产物活性判定（见主题I3）。Analysisofaclone:克隆分析:Onceaclonecontainingatargetgeneisidentified,thestructureoftheclonedfragmentmaybeinvestigatedfurtherusingrestrictionmapping,theanalysisofthefragmentationoftheDNAwithrestrictionenzymes(seeTopicJ1),orultimatelybythesequencingoftheentirefragment(seeTopicJ2).Thesequencecanthenbeanalyzedbycomparisonwithotherknownsequencesfromdata-bases,andthecompletesequenceoftheproteinproductdetermined(seeTopicJ2).Thesequenceisthenavailableformanipulationinanyoftheapplicationsofcloningdescribedabove.一旦一个克隆含有目标基因，克隆片段结构能够用限制性内切酶图谱深入调查，分析酶切DNA碎片（见主题J1），或经过整个片段次序进行分析（见主题J2）。那么序列可经过与其余已知序列数据库进行对比分析，进而确定蛋白质产物完整序列（见主题j2）。因而序列可用于操纵上述任何克隆应用。ManyenzymesareusedinvitroinDNAcloningandanalysis.ThepropertiesofthecommonenzymesaregiveninTable1,alongwiththesectionwheretheiruseisdiscussedinmoredetail.许多酶应用于基因克隆与分析中。常见酶性质见表1，依照他们使用部分进行更详细讨论。

Lesson3RESTRICTIONENZYMESANDELECTROPHORESIS第三课限制酶和电泳Restrictionendonucleases:Restrictionendonucleasesarebacterialenzymeswhichcut(hydrolyze)DNAintodefinedandreproduciblefragments.Inbacteria,theyformpartoftherestriction-modificationdefensemechanismagainstforeignDNA.Theyarethebasictoolsofgenecloning.限制性内切酶：限制性内切酶是把DNA切割或者水解成明确且可重复片段细菌酶。在细菌中，它们组成部分限制性修饰反抗外源基因防御机制。它们是基因克隆基本工具。Recognitionsequence:RestrictionenzymescleaveDNAsymmetricallyinbothstrandsatshortpalindromic(symmetrical)recognitionsequencestoleavea5'-phosphateanda3'-OH.Theyleavebluntends,orprotruding5'-or3'-termini.识别序列：限制内切酶在短对称识别序列位点上对称性地切割双链DNA，而留下一个5端磷酸和一个3端羟基。它们留下迟钝末端或者突出5'末端或3’末端。Cohesiveends:Restrictionenzymeproductswithsingle-strandedterminiaresaidtohavecohesiveor'sticky'ends,sincetheycanannealbybasepairingtoanyotherfragmentwithacomplementaryterminus.粘性末端：由限制酶切割产生单链终点处听说是拥有有粘着力末端，日后它们能够退火，经过其余片段与末端进行碱基互补配对。Restrictiondigests:CommerciallysuppliedenzymesareusedtodigestplasmidDNAbeforeanalysisorpurificationofthefragmentsbyagarosegelelectrophoresis.限制性消化：在分析或者经过琼脂糖凝胶电泳进行片段提纯和分析之前，质粒DNA要用市场上能买到酶消化分解。Agarosegelelectrophoresis:AgarosegelsseparatelinearDNAonthebasisofsize,bythemigrationofDNAthroughamatrixundertheinfluenceofanelectricfield.Electrophoresismaybeusedtodeterminethegrossorganizationofplasmidmolecules.琼脂糖凝胶电泳：琼脂糖凝胶依照大小分离线状DNA，在电场作用下将DNA迁移到基质中。电泳能够用于确定质粒分子总组织。Isolationoffragment:SpecificDNAfragmentsmaybecutoutofagarosegelsandpurifiedforuseinsubsequentcloningexperiments.分离片段：特殊DNA片段能够被琼脂糖凝胶切割开来以及纯化，用在随即克隆试验。Restrictionendonucleases:限制性内切酶：ToincorporatefragmentsofforeignDNAintoaplasmidvector,methodsforthecuttingandrejoiningofdsDNAarerequired.Theidentificationandmanipulationofrestrictionendonucleasesinthe1960sandearly1970swasthekeydiscoverywhichallowedthecloningofDNAtobecomeareality.Restriction-modificationsystemsoccurinmanybacterialspecies,andconstituteadefensemechanismagainsttheintroductionofforeignDNAintothecell.Theyconsistoftwocomponents;thefirstisarestrictionendonuclease,whichrecognizesashort,symmetricalDNAsequence(Fig.1),andcuts(hydrolyzes)theDNAbackboneineachstrandataspecificsitewithinthatsequence.ForeignDNAwillhencebedegradedtorelativelyshortfragments.Thesecondcomponentofthesystemisamethylase,whichaddsamethylgrouptoaCorAbasewithinthesamerecognitionsequencesinthecellularDNA(seeTopicCf).ThismodificationrendersthehostDNAresistanttodegradationbytheendonuclease.将外来DNA片段整合到质粒载体中需要双链DNA切割和再结合方法。在上世纪60年代和上世纪70年代早期，限制性内切酶识别和操纵是使DNA克隆成为事实主要发觉。限制修饰系统在许多细菌物种中都有，而且组成了一个抵制外来DNA进入细胞中。它们由两个要素组成；第一个是限制性内切酶。限制性内切酶识别短对称DNA序列，并在每条链那段序列特殊位点上切割。因另外来DNA将被退化到相对短片段。系统第二个主要要素是甲基化酶。在细胞DNA中，甲基化酶在相同识别序列中对C或者A添加一个甲基基团。这种改变使宿主DNA能抵制因为内切酶而造成降解。Recognitionsequences:Theactionofrestrictionendonudeases(restrictionenzymesforshort)isillustratedinFig.laincludingthearchetypalenzymeEcoRIasanexample.Thisenzyme,whichactsasadimer,willonlyrecognizea6bppalindromicsequence(thesequenceisthesame,reading5'→3',oneachstrand).TheproductofthecuttingreactionatthissiteonalinearDNAistwodouble-Strandedfragments(restrictionfragments),eachwithanidenticalprotrudingsingle-stranded5'-endwithaphosphategroupattached.The3'-endshavefreehydroxylgroups.识别序列：在图1a说明了限制性内切酶行动特征还以原型EcoRI酶作为例子。这种酶是一个二聚物，只能识别6个碱基正确复发序列（这类序列都是一样，在每条单链上都是从5'→3'阅读）。在每个线状DNA上特殊位点切割得到产物是两个双链片段，每个片段带有相同突出单链带有磷酸基团5’末端。3’端具备自由羟基基团。A6bprecognitionsequencewilloccuronaverageevery46=4096bpinrandomsequenceDNA;hence,averylargeDNAmoleculewillbecutintospecificfragmentsaveraging4kbbysuchanenzyme.Hundredsofrestrictionenzymesarenowknown,andalargenumberarecommerciallyavailable.Theyrecognizesitesranginginsizefrom4to8bpormore,andmaygiveproductswithprotruding5'-or3'-tailsorbluntends.Thenewlyformed5'-endsalwaysretainthephosphategroups.TwofurtherexamplesareillustratedinFig.1.TheextremelyhighspecificityofrestrictionenzymesfortheirsitesofactionallowslargeDNAmoleculesandvectorstobecutreproduciblyintodefinedfragments.在随机DNA序列里平均每46=4096bp就会出现一个6bp识别序列；所以，一个人非常大DNA分子会被这么酶切割成明确平均具备4kb片段。现在许许多多限制性内切酶已经被知道，而且大多数用在市场上。它们识别在4到8bp大小范围或者更多位点，而且能给出带有突出5’尾巴或者3’尾巴或者是迟钝末端产物。新形成5’末端总是保留磷酸基团。在图1中说明了两个更深一层例子。限制性内切酶对于它们位置处理高度特异性允许DNA分子和载体可再生地被切割成为明确片段。Cohesiveends:Thoseproductsofrestrictionenzymedigestionwithprotrudingendshaveafurtherproperty;theseendsareknownascohesive,or'sticky'ends,sincetheycanbindtoanyotherendwiththesameoverhangingsequence,bybasepairing(annealing)ofthesingle-strandedtails.Hence,forexample,anyfragmentformedbyanEcoRIcutcanannealtoanyotherfragmentformedinthesameway(Fig.lb),andmaysubsequentlybejoinedcovalentlybyligation(seeTopicE4).Infact,insomecases,DNAendsformedbyenzymeswithdifferentrecognitionsequencesmaybecompatible,providedthesingle-strandedtailscanbase-pairtogether.粘性末端：那些由限制性酶消化得到带有突出末端产物具备深一步特征；这些末端是粘性末端，所以它们能和相同悬垂序列经过单链尾部碱基配对与其余末端连接在一起。所以，比如任何由EcoRI切割得到片段能经过相同方法退火成任何其余片段，而且随即能够被加到共价连接。实际上，在一些情况上由酶形成DNA末端与不一样识别序列能够兼容，使单链尾部能与碱基互补。Restrictiondigests:DigestionofplasmidorgenomicDNA(seeTopicI1)iscarriedoutwithenzymesforanalyticalorpreparativepurposes,usingcommercialenzymesandbuffersolutions.AllrestrictionenzymesrequireMg2+,usuallyataconcentrationofupto10mM,butdifferentenzymesrequiredifferentpHs,NaC1concentrationsorothersolutionconstituentsforoptimumactivity.Thebuffersolutionrequiredforaparticularenzymeissuppliedwithitasaconcentrate.Thedigestionofasampleplasmidwithtwodifferentrestrictionenzymes,BamHIandEcoRI,isillustratedinFig.2.限制性消化：质粒或者基因组DNA消化是经过酶进行，为了分析或制备目标，利用市场上酶和缓冲液。全部限制性内切酶都需要Mg2+,时常达成10mM浓度，但为了达成最适活力，不一样酶需要不一样pHs，NaCl浓度或其余溶液成份。需要特殊酶缓冲液作为一个浓缩液供给给它。在图2说明了一个样本质粒经过两种限制性内切酶BamHI和EcoRI消化。Thedigestionofafewhundrednanograms(<1μg)ofplasmidDNAissufficientforanalysisbyagarosegelelectrophoresis;preparativepurposesmayrequireafewmicrograms.Theformeramountcorrespondstoafewpercentofaminiprepsample,asdescribedinTopicG2.TheDNAisincubatedwiththeenzymeandtheappropriatebufferattheoptimumtemperature(usually37℃经过琼脂糖凝胶电泳，几百微毫克质粒消化足以让分析；制备目标可能需要几微克。如在TopicG2所描述，前面数量对应于小量制备样本极少百分比。DNA是在最适温度（通常是37℃）由酶和适当缓冲液培养。Inavalumeofperhaps20μl.Adyemixtureisthenaddedtothesolution,andthesampleisloadedonanagarosegel.然后将染色混合物加到溶液中，接着样本装到琼脂糖凝胶上。Agarosegelelectrophoresis:Agaroseisapolysaccharidederivedfromseaweed,whichformsasolidgelwhendissolvedinaqueoussolutionatconcentrationsbetween0.5and2%(w/V).Agaroseusedforelectrophoresisisamorepurifiedformoftheagarusedtomakebacterialcultureplates.琼脂糖凝胶电泳：琼脂糖是一个来自于海草多聚糖而且当以0.5到2%浓度溶解在水溶液时候它能形成固体凝胶。用于电泳琼脂比用于制作细菌培养基是愈加纯化形式。Whenanelectricfieldisappliedtoanagarosegelinthepresenceofabuffersolutionwhichwillconductelectricity,DNAfragmentsmovethroughthegeltowardsthepositiveelectrode(DNAishighlynegativelycharged;seeTopicC1)ataratewhichisdependentonitssizeandshape(Fig.3).Smalllinearfragmentsmovemorequicklythanlargeones,whichareretardedbyentanglementwiththenetworkofagarosefibersformingthegel.Hence,thisprocessofelectrophoresismaybeusedtoseparatemixturesofDNAfragmentsonthebasisofsize.Differentconcentrationsofgel[1%,1.5%(w/v),etc.]willallowtheoptimalresolutionoffragmentsindifferentsizeranges.TheDNAsamplesareplacedinwellsinthegelsurface(Fig.3),thepowersupplyisswitchedonandtheDNAisallowedtomigratethroughthegelinseparatelanesortracks.当一个电场是应用于有缓冲溶液存在琼脂糖凝胶时其会导电，DNA片段以依照本身大小和形状形成速率从琼脂糖向正极移动（DNA是高度带负电，看TopicC1）小线状片段比大移动得更加快，因为大会被琼脂糖纤维形成网所妨碍。所以，电泳过程能够依照大小被用于分里DNA片段混合物。不一样浓度凝胶允许片段在不一样大小范围里有最好分辨率。DNA样本被放在凝胶表面井里，打开电源后DNA被允许在分隔开分道或轨道上迁移经过凝胶。Theaddeddyealsomigrates,andisusedtofollowtheprogressofelectrophoresis.TheDNAisstainedbytheinclusionofethidiumbromide(seeTopicC4)inthegel,orbysoakingthegelinasolutionofethidiumbromideafterelectrophoresis.TheDNAshowsupasanorangebandonilluminationbyUVlight.加进染料也迁移，接着背用于紧随电泳过程。在凝胶中，DNA被内含物溴化乙锭染色或者在电泳后在溴化乙锭里浸染凝胶。在紫外灯照射下DNA以橙黄色带出现。Figure4aillustratestheresultofgelelectrophoresisofthefragmentsformedbythedigestionsinFig.2.Theplasmidhasbeenrunonthegelwithoutdigestion(trackU),andafterdigestionwithBamHI(trackB)andEcoRI(trackE).AsetoflinearmarkerDNAfragmentsofknownsizes(tracksM)havebeenrunalongsidethesamplesattwodifferentconcentrations;thesizesaremarkedonthefigure.Anumberofpointsmaybenoted.图4a说明片段凝胶电泳结果，而经过消化形成片段在图二中。没有消化质粒已经在凝胶上面跑起来（轨道U），之后被BamHI（轨道B）和EcoRI（轨道E）消化。一整套已知大小线状标识DNA片段和两个不一样浓度样本已经跑起来；在图中大小有标识。点数量能够记下来。UndigestedplasmidDNA(trackU)runonanagarosegelcommonlyconsistsoftwobands.Thelower,moremobile,bandconsistsofnegativelysupercoiledplasmidDNAisolatedintactfromthecell.Thishasahighmobility,becauseofitscompactconformation(seeTopicC4).Theupperbandisopen-circular,ornickedDNA,formedfromsupercoiledDNAbybreakageofonestrand;thishasanOpened-outcircularconformationandlowermobility.ThelanescontainingthedigestedDNAdearlyrevealasinglefragment(trackB)andfivefragments(trackE),whosesizescanbeestimatedbycomparisonwiththemarkertracks(M).TheintensitiesofthebandsintrackEareproportionaltothesizesofthefragments,sinceasmallfragmenthaslessmassofDNAatagivenmolarconcentration.Thisisalsotrueofthemarkers,sinceinthiscasetheyareformedbydigestionofthe48.5kblinearDNAofbacteriophagek(seeTopicH2).TheamountofDNApresentintracksU,BandEisnotequal;thequantifieshavebeenoptimizedtoshowallthefragmentsclearly.在琼脂糖凝胶上运行未消化质粒DNA通常由两条带组成。较低移动更加快带由从细胞中完好无缺分离出来负螺旋质粒DNA组成。因为它本身高凑体型结构，这么具备高度移动性。（见主题4）上一条带是开环或者是微缺DNA，形成于一套破损超螺旋DNA带；这种带有带开了循环构象和较低迁移。带有小胡DNA非得轨道深深地揭露单一片段和5个片段，片段大小能经过跟标识轨道M比较被估量得到。在轨道E中，带强度跟片段大小成百分比，在给定摩尔浓度里因为每小片段拥有小量DNA。这对标识是真实，因为这么情况他们经过噬菌体K48.5kb线状DNA消化而形成。在轨道U，B和E上DNA数量分布是不均匀；确定数量最好化清楚地展现全部片段。AmoreaccuratedeterminationofthesizesofthelinearfragmentscanbemadebyplottingacalibrationcurveofthelogofthesizeoftheknownfragmentsintrackMagainstthedistancemigratedbyeachfragment.Thisplot(Fig.4b)isafairlystraightline,oftenwithadeviationatlargefragmentsizes.Thismaybeusedtoderivethesizeofanunknownlinearfragmentonthesamegelfromitsmobility,byreadingoffthelog(size)asshown.Itisnotpossibletoderivethesizesofundigestedcircularplasmidsbythesamemethod,sincetherelativemobilityofcircularandlinearDNAonageldependsontheconditions(temperature,electricfield,etc.).更多线状片段大小精准测定能经过在轨道M上依靠每一个片段迁移距离测绘成已知片段校准曲线统计制作。这种情况是一条相当直线，经常在庞大片段大小时有背离。这能够在相同凝胶上依照片段迁移被用于导出未知大小线状片段，依照出来结果读取数据。经过相同方法导出未消化环状质粒是不可能，因为在凝胶上环状和线状DNA相关迁移性是需要条件。Isolationoffragments:Agarosegelsmayalsobeusedpreparativelytoisolatespecificfragmentsforuseinsubsequentligationandothercloningexperiments.Fragmentsareexcisedfromthegel,andtreatedbyorieofanumberofprocedurestopurifytheDNAawayfromthecontaminatingagaroseandethidiumbromidestain.IfweassumethattheEcoRIfragmentcontainingthegeneX(Fig.2)isthetargetDNAforasubcloningexperiment(seeTopicGl),thenthethirdlargestfragmentintrackEofFig.4acouldbepurifiedfromthegelreadyforligationintoanewvector(seeTopicG4).分离片段：琼脂糖凝胶也能被用于分离特殊片段，从而用在随即连接和其余克隆试验。从凝胶上切除片段，接着经过大量步骤从污染琼脂糖中纯化出DNA，再用溴化乙锭染色。假如我们假定含有基因XEcoRI片段是亚克隆试验目标DNA，图4a在轨道上第三大片段能从凝胶中被纯化出来，为连接进一个新载体做准备（看主题G4）Lesson4LIGATION,TRANSFORMATIONANDANALYSISOFRECOMBINANTS第4课结扎，重组转型分析DNAligation:T4DNAligaserepairsbreaksinadsDNAbackboneandcancovalentlyrejoinannealedcohesiveendsinthereverseofarestrictionenzymereaction,tocreatenewDNAmolecules.DNA连接：T4DNA连接酶在双链DNA分水岭处修复破损地方，然后在反向限制酶反应中，能共价地再加入退火粘性末端，从而形成新DNA分子。RecombinantDNAmolecules:Theuseofarestrictionenzyme,followedbyDNAligase,cancreaterecombinantplasmids,withatargetDNAfragmentinsertedintoavectorplasmid.重组DNA分子：限制性内切酶应用，紧随DNA连接酶，能经过将目标片段插入一个人载体质粒形成重组质粒。Alkalinephosphatase:Treatmentofthelinearvectormoleculewithalkalinephosphatasewillremovethe5'-phosphatesandrenderthevectorunabletollgateintoacirclewithoutaninsertedtarget,soreducingtheproportionofrecreatedvectorinthemixture.碱性磷酸酶：碱性磷酸酶对线状载体分子处理是移除5’磷酸键和会使没带出入目标基因载体连接成一个环，从而混合物中降低重新形成载体比率。Transformation:Transformationistheprocessoftake-upofforeignDNA,normallyplasmids,bybacteria.PlasmidsareclonedbytransferintostrainsofE.coliwithdefinedgeneticproperties.TheE.colicellscanbemadecompetenttotakeupplasmidDNAbytreatmentwithCa2+.Thecellsareplatedoutonagarandgrowntoyieldsinglecolonies,orclones.转换：转换是提取外来DNA过程，通常是在细菌里质粒。带有明确遗传特征质粒是经过转移到大肠杆菌菌株才被克隆。大肠杆菌细胞能经过Ca2+处理提取质粒DNA。细胞被平铺在琼脂上，然后逐步产出单一菌落或是克隆体。Selection:Bacteriawhichhavetakenupaplasmidareselectedbygrowthonaplatecontaininganantibiotictowhichtheplasmidvectorencodesresistance.筛选：已经提取了质粒细菌是经过含有看抗生素生长培养基选出来，而这种抗生素是妨碍质粒编码。Transformationefficiency:Theefficiencyofthetransformationstepisgivenbythenumberofanti-biotic-resistantcoloniespermicrogramofinputplasmidDNA.转换效率：转换步骤效率是经过输出质粒DNA每微克抗生素抗性菌落数量所定。Screeningtransformants:Inmanycases,suchaswhenusingDNAlibraries,plasmidandothervectorshavebeendesignedtofacilitatethescreeningoftransformantsforrecombinantplasmids.Inthecaseofasimplesubcloningexperiment,transformantsarescreenedmosteasilybydigestingtheDNAfromminipreparationsofthetransformants,followedbyanalysisonanagarosegel.筛选转化株：在许多情况里，如利用DNA文库、质粒和其余载体是已经设计好促进筛选重组质粒转换株。在亚克隆试验情况里，转换株经过消化来自转换株小量准备DNA最轻易被筛选出来，它是紧随琼脂糖凝胶分析。Growthandstorageoftransformants:Singlecoloniesfromatransformationplatearegrowninliquidmedium,maintainingtheantibioticselectionfortheplasmid,andaportionofthecultureisstoredforlateruseasafrozenglycerolstock.转换株生长和储存：来自转换平板单一菌落是在一体培养基中生长，继续抗生素对质粒选择，也是被储存到冰冻丙三醇库存放存起来为以后使用培养菌一部分。Gelanalysis:Recombinantplasmidscanbedistinguishedfromvectorsbysizeonanagarosegelandbyexcisi