2022年生物技術(shù)專(zhuān)業(yè)英語(yǔ)翻譯

Lesson1DNACLONING:ANOVERVIEW第一課克?。焊乓狣NAcloning：DNAdoningfacilitatestheisolationandmanipulationoffragmentsofanorganism'sgenomebyreplicatingthemindependentlyaspartofanautonomousvector.DNA克?。篋NA克隆通過(guò)獨(dú)立復(fù)制可以用分離和操作措施使生物體基因組片段成為自發(fā)性載體的一部分。Hostsandvectors:MostoftheroutinemanipulationsinvolvedingenecloninguseEscherichiacoliasthehostorganism.PlasmidsandbacteriophagesmaybeusedascloningvectorsinE.coli.Vectorsbasedonplasmids,virusesandwholechromosomeshavebeenusedtocarryforeigngenesintootherprokaryoticandeukaryoticorganisms.宿主和載體：大多數(shù)的基因克隆使用的常規(guī)操作，以大腸桿菌為宿主生物體。質(zhì)粒和噬菌體可作為大腸桿菌的克隆載體。以質(zhì)粒、病毒和整條染色體為載體，將外源基因?qū)肫渌撕驼婧松铩ubcloning:SubcloningisthesimpletransferofaclonedfragmentofDNAfromonevectortoanother;itservestoillustratemanyoftheroutinetechniquesinvolvedingenecloning.亞克?。嚎寺∈强寺〉腄NA片段從一種載體到另一種載體的簡(jiǎn)樸傳遞；它可以用來(lái)闡明基因克隆中的許多常規(guī)技術(shù)。DNAlibraries:DNAlibraries,consistingofsetsofrandomclonedfragmentsofeithergenomicorcDNA,eachinaseparatevectormolecule,areusedintheisolationofunknowngenes.基因庫(kù)：基因庫(kù)，由兩組隨機(jī)克隆片段構(gòu)成，無(wú)論是基因組還是基因，每一種在一種單獨(dú)的載體分子中，都被用來(lái)分離未知的基因。Screeninglibraries:Librariesarescreenedforthepresenceofagenesequencebyhybridizationwithasequencederivedfromitsproteinproductorarelatedgene,orthroughthescreeningoftheproteinproductsoftheclonedfragments.篩選庫(kù)：通過(guò)與來(lái)自其蛋白產(chǎn)物或有關(guān)基因的序列雜交，或者通過(guò)克隆片段的蛋白產(chǎn)物的篩選，篩選出一種基因序列的存在。Analysisofaclone:Onceidentified,aclonedgenemaybeanalyzedbyrestrictionmapping,andultimatelybyDNAsequencing,beforebeingusedinanyofthediverseapplicationsofDNAcloning.克隆分析:在用于基因克隆的多種應(yīng)用中之前，一旦發(fā)現(xiàn)某個(gè)克隆基因可以通過(guò)限制性圖譜進(jìn)行分析，則用這措施進(jìn)行DNA序列測(cè)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?。阂话闵?，由于大多數(shù)生物體的蛋白質(zhì)或其她成分的稀缺性及其在大量的凈化過(guò)程中的困難，導(dǎo)致它們的具體分子分析變得困難甚至不也許。一種措施是分離負(fù)責(zé)蛋白質(zhì)體現(xiàn)的基因或者其產(chǎn)品的構(gòu)成。然而每個(gè)生物體的基因組都是大而復(fù)雜的（見(jiàn)第4節(jié)），每個(gè)細(xì)胞中任何目的序列一般只發(fā)生一次或兩次。因此，原則的化學(xué)或生化措施不能用于分離特定區(qū)域的基因組進(jìn)行研究，特別是所需的DNA序列與所有其她的序列的化學(xué)性質(zhì)相似。這種難點(diǎn)的解決措施是放置一種也許涉及目的基因或序列相對(duì)短的基因組片段在一種自主復(fù)制片段的基因，稱為載體，在另一種宿主物種中它可以復(fù)制獨(dú)立的原始基因組，形成重組DNA。具有重組DNA的宿主生物體的繁殖形成一組基因相似的生物體，或克隆。這個(gè)過(guò)程就是DNA克隆。AmongsttheexplodingnumbersofapplicationsofDNAcloning,oftencollectedtogetherunderthetermgeneticengineering,arethefollowing:在長(zhǎng)期的基因工程中，克隆的應(yīng)用程序的數(shù)量激增，這是如下幾項(xiàng)：DNAsequencing,andhencethederivationofproteinsequence(seeTopicJ2).DNA序列測(cè)定，也即蛋白質(zhì)序列的推導(dǎo)（見(jiàn)主題J2）。Isolationandanalysisofgenepromotersandothercontrolsequences(seeTopicJ4).基因啟動(dòng)子與其他控制序列的分離與分析（見(jiàn)主題J4）。Investigationofprotein/enzyme/RNAfunctionbylarge-scaleproductionofnormalandalteredforms(seeTopicJS).蛋白質(zhì)/酶/RNA功能的正常大規(guī)模生產(chǎn)和變化的研究（見(jiàn)主題J5）。Identificationofmutations,forexamplegenedefectsleadingtodisease(seeTopicJ6).突變的辨認(rèn)，例如基因缺陷導(dǎo)致的疾病（見(jiàn)主題J6）。Biotechnology;thelarge-scalecommercialproductionofproteinsandothermoleculesofbiologicalimportance,forexamplehumaninsulinandgrowthhormone(seeTopicJ6).生物技術(shù)；蛋白質(zhì)和其她分子生物學(xué)意義的大規(guī)模商業(yè)化生產(chǎn)，例如人胰島素和生長(zhǎng)激素（見(jiàn)主題J6）。Engineeringanimalsandplants,andgenetherapy(seeTopicJ6).轉(zhuǎn)基因動(dòng)物和植物，和基因治療（見(jiàn)主題J6）。Engineeringproteinstoaltertheirproperties(seeTopicJ6).通過(guò)蛋白質(zhì)工程來(lái)變化它們的性質(zhì)（見(jiàn)主題J6）Hostsandvectors:TheinitialisolationandanalysisofDNAfragmentsisalmostalwayscarriedoutusingthebacteriumE.coliasthehostorganism,althoughtheyeastSaccharomycescerevisiaeisbeingusedtomanipulateverylargefragmentsofthehumangenome(seeTopicH3).Awidevarietyofnaturalrepliconshavethepropertiesrequiredtoallowthemtoactascloningvectors.Vectorsmustnormallybecapableofbeingreplicatedandisolatedindependentlyofthehost'sgenome,althoughsomearedesignedtoincorporateDNAintothehostgenomeforlongertermexpressionofclonedgenes.Vectorsalsoincorporateaselectablemarker,agenewhichallowshostcellscontainingthevectortobeselectedfromamongstthosewhichdonot,usuallybyconferringresistancetoatoxin(seeTopicG2),orenablingtheirsurvivalundercertaingrowthconditions(seeTopicH3).宿主和載體DNA片段的初步分離和分析幾乎總是使用大腸桿菌作為宿主生物體，雖然釀酒酵母被用來(lái)操縱人類(lèi)基因組大片段（見(jiàn)主題H3）。多種各樣的自然的復(fù)制，規(guī)定她們擁有作為克隆載體的性質(zhì)。載體一般是可以被復(fù)制和獨(dú)立的主機(jī)的基因組中分離，雖然某些被設(shè)計(jì)成將核酸到宿主基因組中的長(zhǎng)期體現(xiàn)的克隆基因。載體也將選擇標(biāo)記容許宿主細(xì)胞中沒(méi)有相似序列載體的基因，一般用抗毒素（見(jiàn)主題G2），或使她們?cè)谝欢ǖ纳L(zhǎng)條件生存（見(jiàn)主題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).第一種大腸桿菌質(zhì)粒染色體外（從染色體分離）的環(huán)狀質(zhì)粒（見(jiàn)主題G2），和某些噬菌體（感染細(xì)菌的病毒；見(jiàn)主題R2）也被用于大腸桿菌。噬菌體K可用于克隆片段不小于質(zhì)粒，噬菌體M13和容許克隆的DNA被孤立單鏈形式（見(jiàn)主題H2）。更專(zhuān)業(yè)的載體已被設(shè)計(jì)為使用質(zhì)粒和噬菌體方面，如質(zhì)粒，噬菌體K雜交稱為粘粒（見(jiàn)主題H3）。非常大的基因組片段從人類(lèi)和其她物種已被克隆的釀酒酵母的酵母人工染色體（YAC克??；seetopicH3）。質(zhì)粒和噬菌體載體已被用于在一種范疇內(nèi)的其她比大腸桿菌，細(xì)菌和噬菌體的基因體現(xiàn)。某些也許被用來(lái)將DNA插入宿主基因組，例如噬菌體K（見(jiàn)主題H2）。質(zhì)粒載體已經(jīng)開(kāi)發(fā)使用的酵母（酵母著絲粒質(zhì)粒），而植物；細(xì)菌質(zhì)粒（根癌農(nóng)桿菌Ti質(zhì)粒）可以用來(lái)整合進(jìn)基因組DNA。在文化的其她真核細(xì)胞中，載體一般是根據(jù)其自然感染病毒所需的物種，或者通過(guò)保持她們的DNA染色體外或整合到宿主基因組（涉及SV40，桿狀病毒，逆轉(zhuǎn)錄病毒；見(jiàn)主題H4）。Subcloning:Thesimplestkindofcloningexperiment,whichexemplifiesmanyofthebasictechniquesofDNAcloning,isthetransferofafragmentofclonedDNAfromonevectortoanother,aprocessknownassubcloning.Thismightbeusedtoinvestigateashortregionofalargeclonedfragmentinmoredetail,ortotransferagenetoavectordesignedtoexpressitinaparticularspecies,forexample.InthecaseofplasmidvectorsinE.coli,themostcommonsituation,theprocessmaybedividedintothefollowingsteps,whichareconsideredingreaterdetailinTopicsG2-G5:亞克隆：最簡(jiǎn)樸的克隆實(shí)驗(yàn)，這體現(xiàn)了許多DNA克隆的基本技術(shù)，是克隆的片段從一種載體到另一種載體的轉(zhuǎn)移，這一過(guò)程稱為亞克隆。這可更具體的用來(lái)研究一種大克隆片段的短區(qū)域，例如說(shuō)將基因放入載體使其轉(zhuǎn)移到一種特定物種中進(jìn)行體現(xiàn)。一般狀況下，在質(zhì)粒載體轉(zhuǎn)移到大腸桿菌的案例中，該措施可分為如下幾種環(huán)節(jié)，其中在主題g2-g5的闡明更具體:IsolationofplasmidDNAcontainingtheclonedsequenceofinterest(seeTopicG2).質(zhì)粒分離涉及目的克隆序列（見(jiàn)主題G2）。Digestion(cutting)oftheplasmidintodiscretefragmentswithrestrictionendonucleases(seeTopicG3).用限制性內(nèi)切酶切（割）離散片段的質(zhì)粒（見(jiàn)主題G3）。Separationofthefragmentsbyagarosegelelectrophoresis(seeTopicG3).通過(guò)瓊脂糖凝膠電泳分離片段（見(jiàn)主題G3）。Purificationofthedesiredtargetfragment(seetopicG3)純化所需的目的片段（見(jiàn)主題G3）。Ligation(joining)ofthefragmentintoanewplasmidvector,toformanewrecombinantmolecule(seeTopicG4).將片段插入新的質(zhì)粒載體中，以形成新的重組分子（見(jiàn)主題G4）。(6)TransferoftheligatedplasmidintoanE.Coilstrain(transformation)(seetopicG4).把連接質(zhì)粒轉(zhuǎn)移到大腸桿菌菌株（變換）（見(jiàn)主題G4）。(7)selectionoftransformedbacteria(seetopicG4).轉(zhuǎn)化菌的篩選（見(jiàn)主題G4）。(8)Analysisofplasmids(seeTopicG4).質(zhì)粒分析（見(jiàn)主題G4）。DNAlibraries:克隆庫(kù):TherearetwomainsourcesfromwhichDNAisderivedforcloningexperimentsdesignedtoidentifyanunknowngene-bulkgenomicDNAfromthespeciesofinterestand,inthecaseofeukaryotes,bulkmRNAfromacellortissuewherethegeneisknowntobeexpressed.TheyareusedintheformationofgenomiclibrariesandcDNAlibrariesrespectively(seeTopicI2).有2個(gè)重要來(lái)源，一是基因的克隆實(shí)驗(yàn)，目的是鑒定未知的基因——來(lái)自目的物種的大部分基因組DNA。二是就真核生物來(lái)說(shuō)，大多數(shù)mRNA來(lái)自已知其基因體現(xiàn)的細(xì)胞或組織。它們分別用于基因組文庫(kù)和基因庫(kù)的形成（見(jiàn)主題I2）。DNAlibrariesaresetsofDNAclones(acloneisageneticallydistinctindividualorsetofidenticalindividuals),eachofwhichhasbeenderivedfromtheinsertionofadifferentfragmentintoavectorfollowedbypropagationinthehost.GenomiclibrariesarepreparedfromrandomfragmentsofgenomicDNA.However,genomiclibrariesmaybeaninefficientmethodoffindingagene,particularlyinlargeeukaryoticgenomes,wheremuchoftheDNAisnoncoding(seeTopicD4).ThealternativeistouseasthesourceofthelibrarythemRNAfromacellortissuewhichisknowntoexpressthegene.DNAcopies(cDNA)aresynthesizedfromthemRNAbyreversetranscriptionandaretheninsertedintoavectortoformacDNAlibrary,cDNAlibrariesareefficientforcloningagenesequence,butyieldonlythecodingregion,andnotthesurroundinggenomicsequences.克隆庫(kù)是一組基因的克?。寺∈且环N基因不同的個(gè)體或相似的個(gè)體），每一種都來(lái)自于一種不同的片段插入到載體中，然后在宿主中傳播?；蚪M文庫(kù)是從基因組的隨機(jī)片段制備的。然而，基因組文庫(kù)中也許是一種低效率的尋找基因的措施，特別是在大型的真核生物的基因組，其中大部分是非編碼DNA（見(jiàn)主題D4）。另一種措施是運(yùn)用細(xì)胞或組織中的細(xì)胞或組織來(lái)體現(xiàn)基因的來(lái)源。通過(guò)反轉(zhuǎn)錄合成基因的復(fù)制品，并將其插入到載體中，形成一種文庫(kù)，文庫(kù)是有效的，用于克隆一種基因序列，但只產(chǎn)生編碼區(qū)，而不是周邊的基因組序列。Screeninglibraries:篩選庫(kù):Sinceitisnotapparentwhichcloneinalibrarycontainsthegeneofinterest,amethodforscreeningforitspresenceisrequired.ThisisoftenbasedontheuseofaradioactivelylabeledDNAprobewhichiscomplementaryorpartiallycomplementarytoaregionofthegenesequence,andwhichcanbeusedtodetectitbyhybridization(seeTopicC3).Theprobesequencemightbeanoligonucleotidederivedfromthesequenceoftheproteinproductofthegene,ifitisavailable,orfromarelatedgenefromanotherspecies(seeTopicI3).Anincreasinglyimportantmethodforthegenerationofprobesisthepolymerasechainreaction(PCR;.seeTopicJ3).Otherscreeningmethodsrelyontheexpressionofthecodingsequencesoftheclonesinthelibrary,andidentificationoftheproteinproductfromitsactivity,orwithaspedficantibody,forexample(seeTopicI3).由于在克隆庫(kù)中目的基因是不明顯的，因此其篩選的措施的存在是必需的。這一般基于應(yīng)用的放射性標(biāo)記DNA探針是互補(bǔ)的或部分互補(bǔ)的一種區(qū)域的基因序列，可以通過(guò)雜交來(lái)檢測(cè)它（見(jiàn)主題C3）。探針序列也許是來(lái)自于基因蛋白質(zhì)產(chǎn)物序列的一種寡核苷酸，也也許來(lái)自另一種有關(guān)基因（見(jiàn)主題I3）。聚合酶鏈反映是一種日益重要的探針生成措施（PCR；。見(jiàn)主題J3）。其她篩選措施依賴于在克隆庫(kù)中的編碼序列的體現(xiàn)，例如說(shuō)用特異抗體進(jìn)行蛋白質(zhì)產(chǎn)物的活性鑒定（見(jiàn)主題I3）。Analysisofaclone:克隆分析:Onceaclonecontainingatargetgeneisidentified,thestructureoftheclonedfragmentmaybeinvestigatedfurtherusingrestrictionmapping,theanalysisofthefragmentationoftheDNAwithrestrictionenzymes(seeTopicJ1),orultimatelybythesequencingoftheentirefragment(seeTopicJ2).Thesequencecanthenbeanalyzedbycomparisonwithotherknownsequencesfromdata-bases,andthecompletesequenceoftheproteinproductdetermined(seeTopicJ2).Thesequenceisthenavailableformanipulationinanyoftheapplicationsofcloningdescribedabove.一旦一種克隆具有目的基因，克隆的片段的構(gòu)造可以用限制性內(nèi)切酶圖譜進(jìn)一步調(diào)查，分析酶切的DNA碎片（見(jiàn)主題J1），或通過(guò)整個(gè)片段的順序進(jìn)行分析（見(jiàn)主題J2）。那么序列可通過(guò)與其她已知序列數(shù)據(jù)庫(kù)進(jìn)行對(duì)比分析，進(jìn)而擬定蛋白質(zhì)產(chǎn)物的完整序列（見(jiàn)主題j2）。因而序列可用于操縱上述的任何克隆應(yīng)用。ManyenzymesareusedinvitroinDNAcloningandanalysis.ThepropertiesofthecommonenzymesaregiveninTable1,alongwiththesectionwheretheiruseisdiscussedinmoredetail.許多酶應(yīng)用于基因克隆與分析中。常用酶的性質(zhì)見(jiàn)表1，根據(jù)她們的使用部分進(jìn)行更具體的討論。

Lesson3RESTRICTIONENZYMESANDELECTROPHORESIS第三課限制酶和電泳Restrictionendonucleases:Restrictionendonucleasesarebacterialenzymeswhichcut(hydrolyze)DNAintodefinedandreproduciblefragments.Inbacteria,theyformpartoftherestriction-modificationdefensemechanismagainstforeignDNA.Theyarethebasictoolsofgenecloning.限制性內(nèi)切酶：限制性內(nèi)切酶是把DNA切割或者水解成明確的且可反復(fù)片段的細(xì)菌酶。在細(xì)菌中，它們構(gòu)成部分限制性修飾對(duì)抗外源基因的防御機(jī)制。它們是基因克隆的基本工具。Recognitionsequence:RestrictionenzymescleaveDNAsymmetricallyinbothstrandsatshortpalindromic(symmetrical)recognitionsequencestoleavea5'-phosphateanda3'-OH.Theyleavebluntends,orprotruding5'-or3'-termini.辨認(rèn)序列：限制內(nèi)切酶在短的對(duì)稱辨認(rèn)序列的位點(diǎn)上對(duì)稱性地切割雙鏈DNA，而留下一種5端的磷酸和一種3端的羥基。它們留下遲鈍的末端或者突出5'末端或3’末端。Cohesiveends:Restrictionenzymeproductswithsingle-strandedterminiaresaidtohavecohesiveor'sticky'ends,sincetheycanannealbybasepairingtoanyotherfragmentwithacomplementaryterminus.粘性末端：由限制酶切割產(chǎn)生的單鏈終點(diǎn)處據(jù)說(shuō)是擁有有粘著力的的末端，后來(lái)它們可以退火，通過(guò)其她片段與末端進(jìn)行堿基互補(bǔ)配對(duì)。Restrictiondigests:CommerciallysuppliedenzymesareusedtodigestplasmidDNAbeforeanalysisorpurificationofthefragmentsbyagarosegelelectrophoresis.限制性消化：在分析或者通過(guò)瓊脂糖凝膠電泳進(jìn)行片段的提純和分析之前，質(zhì)粒DNA要用市場(chǎng)上能買(mǎi)到的酶消化分解。Agarosegelelectrophoresis:AgarosegelsseparatelinearDNAonthebasisofsize,bythemigrationofDNAthroughamatrixundertheinfluenceofanelectricfield.Electrophoresismaybeusedtodeterminethegrossorganizationofplasmidmolecules.瓊脂糖凝膠電泳：瓊脂糖凝膠根據(jù)大小分離線狀DNA，在電場(chǎng)的作用下將DNA遷移到基質(zhì)中。電泳可以用于擬定質(zhì)粒分子總的組織。Isolationoffragment:SpecificDNAfragmentsmaybecutoutofagarosegelsandpurifiedforuseinsubsequentcloningexperiments.分離片段：特殊DNA片段可以被瓊脂糖凝膠切割開(kāi)來(lái)以及純化，用在隨后的克隆實(shí)驗(yàn)。Restrictionendonucleases:限制性內(nèi)切酶：ToincorporatefragmentsofforeignDNAintoaplasmidvector,methodsforthecuttingandrejoiningofdsDNAarerequired.Theidentificationandmanipulationofrestrictionendonucleasesinthe1960sandearly1970swasthekeydiscoverywhichallowedthecloningofDNAtobecomeareality.Restriction-modificationsystemsoccurinmanybacterialspecies,andconstituteadefensemechanismagainsttheintroductionofforeignDNAintothecell.Theyconsistoftwocomponents;thefirstisarestrictionendonuclease,whichrecognizesashort,symmetricalDNAsequence(Fig.1),andcuts(hydrolyzes)theDNAbackboneineachstrandataspecificsitewithinthatsequence.ForeignDNAwillhencebedegradedtorelativelyshortfragments.Thesecondcomponentofthesystemisamethylase,whichaddsamethylgrouptoaCorAbasewithinthesamerecognitionsequencesinthecellularDNA(seeTopicCf).ThismodificationrendersthehostDNAresistanttodegradationbytheendonuclease.將外來(lái)DNA片段整合到質(zhì)粒載體中需要雙鏈DNA切割和再結(jié)合的措施。在上世紀(jì)60年代和上世紀(jì)70年代初期，限制性內(nèi)切酶的辨認(rèn)和操縱是使DNA克隆成為事實(shí)的重要發(fā)現(xiàn)。限制修飾系統(tǒng)在許多細(xì)菌物種中均有，并且構(gòu)成了一種抵制外來(lái)DNA進(jìn)入細(xì)胞中。它們由兩個(gè)要素構(gòu)成；第一種是限制性內(nèi)切酶。限制性內(nèi)切酶辨認(rèn)短的對(duì)稱DNA序列，并在每條鏈的那段序列的特殊位點(diǎn)上切割。因此外來(lái)DNA將被退化到相對(duì)短的片段。系統(tǒng)的第二個(gè)重要要素是甲基化酶。在細(xì)胞DNA中，甲基化酶在相似的辨認(rèn)序列中對(duì)C或者A添加一種甲基基團(tuán)。這種變化使宿主DNA能抵制由于內(nèi)切酶而導(dǎo)致的降解。Recognitionsequences:Theactionofrestrictionendonudeases(restrictionenzymesforshort)isillustratedinFig.laincludingthearchetypalenzymeEcoRIasanexample.Thisenzyme,whichactsasadimer,willonlyrecognizea6bppalindromicsequence(thesequenceisthesame,reading5'→3',oneachstrand).TheproductofthecuttingreactionatthissiteonalinearDNAistwodouble-Strandedfragments(restrictionfragments),eachwithanidenticalprotrudingsingle-stranded5'-endwithaphosphategroupattached.The3'-endshavefreehydroxylgroups.辨認(rèn)序列：在圖1a闡明了限制性內(nèi)切酶的行動(dòng)特性還以原型的EcoRI酶作為例子。這種酶是一種二聚物，只能辨認(rèn)6個(gè)堿基對(duì)的復(fù)發(fā)序列（此類(lèi)序列都是同樣的，在每條單鏈上都是從5'→3'閱讀）。在每個(gè)線狀DNA上的特殊位點(diǎn)切割得到的產(chǎn)物是兩個(gè)雙鏈片段，每個(gè)片段帶有相似的突出的單鏈帶有磷酸基團(tuán)的5’末端。3’端具有自由的羥基基團(tuán)。A6bprecognitionsequencewilloccuronaverageevery46=4096bpinrandomsequenceDNA;hence,averylargeDNAmoleculewillbecutintospecificfragmentsaveraging4kbbysuchanenzyme.Hundredsofrestrictionenzymesarenowknown,andalargenumberarecommerciallyavailable.Theyrecognizesitesranginginsizefrom4to8bpormore,andmaygiveproductswithprotruding5'-or3'-tailsorbluntends.Thenewlyformed5'-endsalwaysretainthephosphategroups.TwofurtherexamplesareillustratedinFig.1.TheextremelyhighspecificityofrestrictionenzymesfortheirsitesofactionallowslargeDNAmoleculesandvectorstobecutreproduciblyintodefinedfragments.在隨機(jī)的DNA序列里平均每46=4096bp就會(huì)浮現(xiàn)一種6bp的辨認(rèn)序列；因此，一種人非常大的DNA分子會(huì)被這樣的酶切割成明確的平均具有4kb的片段。目前許許多多的限制性內(nèi)切酶已經(jīng)被懂得，并且大多數(shù)用在市場(chǎng)上。它們辨認(rèn)在4到8bp的大小范疇或者更多的位點(diǎn)，并且能給出帶有突出5’尾巴或者3’尾巴或者是遲鈍末端的產(chǎn)物。新形成的5’末端總是保存磷酸基團(tuán)。在圖1中闡明了兩個(gè)更深一層的例子。限制性內(nèi)切酶對(duì)于它們位置解決的高度特異性容許DNA分子和載體可再生地被切割成為明確的片段。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的帶有突出末端的產(chǎn)物具有深一步的特性；這些末端是粘性末端，因此它們能和相似的懸垂序列通過(guò)單鏈尾部的堿基配對(duì)與其她末端連接在一起。因此，例如任何由EcoRI切割得到的片段能通過(guò)相似的措施退火成任何其她片段，并且隨后可以被加到共價(jià)連接。事實(shí)上，在某些狀況上由酶形成的DNA末端與不同辨認(rèn)序列可以兼容，使單鏈的尾部能與堿基互補(bǔ)。Restrictiondigests:DigestionofplasmidorgenomicDNA(seeTopicI1)iscarriedoutwithenzymesforanalyticalorpreparativepurposes,usingcommercialenzymesandbuffersolutions.AllrestrictionenzymesrequireMg2+,usuallyataconcentrationofupto10mM,butdifferentenzymesrequiredifferentpHs,NaC1concentrationsorothersolutionconstituentsforoptimumactivity.Thebuffersolutionrequiredforaparticularenzymeissuppliedwithitasaconcentrate.Thedigestionofasampleplasmidwithtwodifferentrestrictionenzymes,BamHIandEcoRI,isillustratedinFig.2.限制性消化：質(zhì)?；蛘呋蚪MDNA的消化是通過(guò)酶進(jìn)行的，為了分析或制備的目的，運(yùn)用市場(chǎng)上的酶和緩沖液。所有限制性內(nèi)切酶都需要Mg2+,時(shí)常達(dá)到10mM的濃度，但為了達(dá)到最適活力，不同的酶需要不同的pHs，NaCl濃度或其她溶液成分。需要特殊酶的緩沖液作為一種濃縮液供應(yīng)給它。在圖2闡明了一種樣本質(zhì)粒通過(guò)兩種限制性內(nèi)切酶BamHI和EcoRI的消化。Thedigestionofafewhundrednanograms(<1μg)ofplasmidDNAissufficientforanalysisbyagarosegelelectrophoresis;preparativepurposesmayrequireafewmicrograms.Theformeramountcorrespondstoafewpercentofaminiprepsample,asdescribedinTopicG2.TheDNAisincubatedwiththeenzymeandtheappropriatebufferattheoptimumtemperature(usually37℃通過(guò)瓊脂糖凝膠電泳，幾百微毫克的質(zhì)粒的消化足以讓分析；制備目的也許需要幾微克。如在TopicG2所描述，前面的數(shù)量相應(yīng)于小量制備樣本的很少的比例。DNA是在最適溫度（一般是37℃）由酶和合適緩沖液培養(yǎng)的。Inavalumeofperhaps20μl.Adyemixtureisthenaddedtothesolution,andthesampleisloadedonanagarosegel.然后將染色的混合物加到溶液中，接著樣本裝到瓊脂糖凝膠上。Agarosegelelectrophoresis:Agaroseisapolysaccharidederivedfromseaweed,whichformsasolidgelwhendissolvedinaqueoussolutionatconcentrationsbetween0.5and2%(w/V).Agaroseusedforelectrophoresisisamorepurifiedformoftheagarusedtomakebacterialcultureplates.瓊脂糖凝膠電泳：瓊脂糖是一種來(lái)自于海草的多聚糖并且當(dāng)以0.5到2%的濃度溶解在水溶液的時(shí)候它能形成固體凝膠。用于電泳的瓊脂比用于制作細(xì)菌培養(yǎng)基的是更加純化的形式。Whenanelectricfieldisappliedtoanagarosegelinthepresenceofabuffersolutionwhichwillconductelectricity,DNAfragmentsmovethroughthegeltowardsthepositiveelectrode(DNAishighlynegativelycharged;seeTopicC1)ataratewhichisdependentonitssizeandshape(Fig.3).Smalllinearfragmentsmovemorequicklythanlargeones,whichareretardedbyentanglementwiththenetworkofagarosefibersformingthegel.Hence,thisprocessofelectrophoresismaybeusedtoseparatemixturesofDNAfragmentsonthebasisofsize.Differentconcentrationsofgel[1%,1.5%(w/v),etc.]willallowtheoptimalresolutionoffragmentsindifferentsizeranges.TheDNAsamplesareplacedinwellsinthegelsurface(Fig.3),thepowersupplyisswitchedonandtheDNAisallowedtomigratethroughthegelinseparatelanesortracks.當(dāng)一種電場(chǎng)是應(yīng)用于有緩沖溶液存在的瓊脂糖凝膠時(shí)其會(huì)導(dǎo)電，DNA片段以根據(jù)自身大小和形狀形成的速率從瓊脂糖向正極移動(dòng)（DNA是高度帶負(fù)電的，看TopicC1）小的線狀片段比大的移動(dòng)得更快，由于的大的會(huì)被瓊脂糖纖維形成的網(wǎng)所阻礙。因此，電泳的過(guò)程可以根據(jù)大小被用于分里DNA片段的混合物。不同濃度的凝膠容許片段在不同大小范疇里有最佳的辨別率。DNA樣本被放在凝膠表面的井里，打開(kāi)電源后DNA被容許在分隔開(kāi)的分道或軌道上遷移通過(guò)凝膠。Theaddeddyealsomigrates,andisusedtofollowtheprogressofelectrophoresis.TheDNAisstainedbytheinclusionofethidiumbromide(seeTopicC4)inthegel,orbysoakingthegelinasolutionofethidiumbromideafterelectrophoresis.TheDNAshowsupasanorangebandonilluminationbyUVlight.加進(jìn)染料也遷移，接著背用于緊隨電泳過(guò)程。在凝膠中，DNA被內(nèi)含物溴化乙錠染色或者在電泳后在溴化乙錠里浸染凝膠。在紫外燈的照射下DNA以橙黃色的帶浮現(xiàn)。Figure4aillustratestheresultofgelelectrophoresisofthefragmentsformedbythedigestionsinFig.2.Theplasmidhasbeenrunonthegelwithoutdigestion(trackU),andafterdigestionwithBamHI(trackB)andEcoRI(trackE).AsetoflinearmarkerDNAfragmentsofknownsizes(tracksM)havebeenrunalongsidethesamplesattwodifferentconcentrations;thesizesaremarkedonthefigure.Anumberofpointsmaybenoted.圖4a闡明片段的凝膠電泳的成果，而通過(guò)消化形成的片段在圖二中。沒(méi)有消化的質(zhì)粒已經(jīng)在凝膠上面跑起來(lái)（軌道U），之后被BamHI（軌道B）和EcoRI（軌道E）消化。一整套已知大小的線狀標(biāo)記的DNA片段和兩個(gè)不同濃度的樣本已經(jīng)跑起來(lái)；在圖中大小有標(biāo)記。點(diǎn)的數(shù)量可以記下來(lái)。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.在瓊脂糖凝膠上運(yùn)營(yíng)的未消化的質(zhì)粒DNA一般由兩條帶構(gòu)成。較低移動(dòng)更快的帶由從細(xì)胞中完好無(wú)缺分離出來(lái)的負(fù)螺旋質(zhì)粒DNA構(gòu)成。由于它自身高湊的體型構(gòu)造，這樣具有高度的移動(dòng)性。（見(jiàn)主題4）上一條帶是開(kāi)環(huán)或者是微缺的DNA，形成于一套破損超螺旋的DNA帶；這種帶有帶開(kāi)了的循環(huán)構(gòu)象和較低的遷移。帶有小胡DNA非得軌道深深地揭發(fā)單一的片段和5個(gè)片段，片段的大小能通過(guò)跟標(biāo)記軌道M比較被估計(jì)得到。在軌道E中，帶的強(qiáng)度跟片段的大小成比例，在給定的摩爾濃度里由于每小片段擁有小量DNA。這對(duì)標(biāo)記是真實(shí)的，由于這樣的狀況她們通過(guò)噬菌體K的48.5kb線狀DNA消化而形成。在軌道U，B和E上DNA的數(shù)量分布是不均勻的；擬定的數(shù)量最佳化清晰地呈現(xiàn)所有片段。AmoreaccuratedeterminationofthesizesofthelinearfragmentscanbemadebyplottingacalibrationcurveofthelogofthesizeoftheknownfragmentsintrackMagainstthedistancemigratedbyeachfragment.Thisplot(Fig.4b)isafairlystraightline,oftenwithadeviationatlargefragmentsizes.Thismaybeusedtoderivethesizeofanunknownlinearfragmentonthesamegelfromitsmobility,byreadingoffthelog(size)asshown.Itisnotpossibletoderivethesizesofundigestedcircularplasmidsbythesamemethod,sincetherelativemobilityofcircularandlinearDNAonageldependsontheconditions(temperature,electricfield,etc.).更多線狀片段的大小的精確測(cè)定能通過(guò)在軌道M上依托每一種片段遷移距離測(cè)繪成已知片段的校準(zhǔn)曲線的記錄制作。這種狀況是一條相稱直的線，常常在龐大的片段大小時(shí)有背離。這可以在相似的凝膠上根據(jù)片段的遷移被用于導(dǎo)出未知大小的線狀片段，根據(jù)出來(lái)的成果讀取數(shù)據(jù)。通過(guò)相似的措施導(dǎo)出未消化的環(huán)狀質(zhì)粒是不也許的，由于在凝膠上的環(huán)狀和線狀DNA的有關(guān)遷移性是需要條件的。Isolationoffragments:Agarosegelsmayalsobeusedpreparativelytoisolatespecificfragmentsforuseinsubsequentligationandothercloningexperiments.Fragmentsareexcisedfromthegel,andtreatedbyorieofanumberofprocedurestopurifytheDNAawayfromthecontaminatingagaroseandethidiumbromidestain.IfweassumethattheEcoRIfragmentcontainingthegeneX(Fig.2)isthetargetDNAforasubcloningexperiment(seeTopicGl),thenthethirdlargestfragmentintrackEofFig.4acouldbepurifiedfromthegelreadyforligationintoanewvector(seeTopicG4).分離片段：瓊脂糖凝膠也能被用于分離特殊片段，從而用在隨后的連接和其她克隆實(shí)驗(yàn)。從凝膠上切除的片段，接著通過(guò)大量環(huán)節(jié)從污染的瓊脂糖中純化出DNA，再用溴化乙錠染色。如果我們假定具有基因X的EcoRI片段是亞克隆實(shí)驗(yàn)的目的DNA，圖4a的在軌道上的第三大的片段能從凝膠中被純化出來(lái)，為連接進(jìn)一種新的載體做準(zhǔn)備（看主題G4）Lesson4LIGATION,TRANSFORMATIONANDANALYSISOFRECOMBINANTS第4課結(jié)扎，重組轉(zhuǎn)型分析DNAligation:T4DNAligaserepairsbreaksinadsDNAbackboneandcancovalentlyrejoinannealedcohesiveendsinthereverseofarestrictionenzymereaction,tocreatenewDNAmolecules.DNA連接：T4DNA連接酶在雙鏈DNA的分水嶺處修復(fù)破損的地方，然后在反向限制酶反映中，能共價(jià)地再加入退火的粘性末端，從而形成新的DNA分子。RecombinantDNAmolecules:Theuseofarestrictionenzyme,followedbyDNAligase,cancreaterecombinantplasmids,withatargetDNAfragmentinsertedintoavectorplasmid.重組DNA分子：限制性內(nèi)切酶的應(yīng)用，緊隨DNA連接酶，能通過(guò)將目的片段插入一種人載體質(zhì)粒形成重組質(zhì)粒。Alkalinephosphatase:Treatmentofthelinearvectormoleculewithalkalinephosphatasewillremovethe5'-phosphatesandrenderthevectorunabletollgateintoacirclewithoutaninsertedtarget,soreducingtheproportionofrecreatedvectorinthemixture.堿性磷酸酶：堿性磷酸酶對(duì)線狀載體分子的解決是移除5’的磷酸鍵和會(huì)使沒(méi)帶出入目的基因的載體連接成一種環(huán)，從而混合物中減少重新形成的載體的比率。Transformation:Transformationistheprocessoftake-upofforeignDNA,normallyplasmids,bybacteria.PlasmidsareclonedbytransferintostrainsofE.coliwithdefinedgeneticproperties.TheE.colicellscanbemadecompetenttotakeupplasmidDNAbytreatmentwithCa2+.Thecellsareplatedoutonagarandgrowntoyieldsinglecolonies,orclones.轉(zhuǎn)換：轉(zhuǎn)換是提取外來(lái)DNA的過(guò)程，一般是在細(xì)菌里的質(zhì)粒。帶有明確遺傳特性的質(zhì)粒是通過(guò)轉(zhuǎn)移到大腸桿菌菌株才被克隆的。大腸桿菌細(xì)胞能通過(guò)Ca2+的解決提取質(zhì)粒DNA。細(xì)胞被平鋪在瓊脂上，然后逐漸產(chǎn)出單一的菌落或是克隆體。Selection:Bacteriawhichhavetakenupaplasmidareselectedbygrowthonaplatecontaininganantibiotictowhichtheplasmidvectorencodesresistance.篩選：已經(jīng)提取了質(zhì)粒的細(xì)菌是通過(guò)具有看抗生素的生長(zhǎng)培養(yǎng)基選出來(lái)的，而這種抗生素是阻礙質(zhì)粒編碼的。Transformationefficiency:Theefficiencyofthetransformationstepisgivenbythenumberofanti-biotic-resistantcoloniespermicrogramofinputplasmidDNA.轉(zhuǎn)換效率：轉(zhuǎn)換環(huán)節(jié)的效率是通過(guò)輸出質(zhì)粒DNA每微克抗生素抗性菌落的數(shù)量所定的。Screeningtransformants:Inmanycases,suchaswhenusingDNAlibraries,plasmidandothervectorshavebeendesignedtofacilitatethescreeningoftransformantsforrecombinantplasmids.Inthecaseofasimplesubcloningexperiment,transformantsarescreenedmosteasilybydigestingtheDNAfromminipreparationsofthetransformants,followedbyanalysisonanagarosegel.篩選轉(zhuǎn)化株：在許多狀況里，如運(yùn)用DNA文庫(kù)、質(zhì)粒和其她載體是已經(jīng)設(shè)計(jì)好增進(jìn)篩選重組質(zhì)粒的轉(zhuǎn)換株的。在亞克隆實(shí)驗(yàn)狀況里，轉(zhuǎn)換株通過(guò)消化來(lái)自轉(zhuǎn)換株的小量準(zhǔn)備的DNA最容易被篩選出來(lái)，它是緊隨瓊脂糖凝膠分析的。Growthandstorageoftransformants:Singlecoloniesfromatransformationplatearegrowninliquidmedium,maintainingtheant