第六章重組DNA技術(shù)

基因工程的操作過(guò)程切接轉(zhuǎn)增檢表DNA的酶切與連接策略

重組率

第一節(jié)DNA的體外重組

（切與接）1DNA的酶切與連接策略

在選擇外源DNA同載體分子連接反應(yīng)的程序時(shí)，需要考慮到下列三個(gè)因素：

實(shí)驗(yàn)步驟要盡可能簡(jiǎn)單易行；

連接形成的接點(diǎn)序列，最好能被一定的核酸內(nèi)切限制酶重新切割，以便回收插入的外源DNA片段；

對(duì)轉(zhuǎn)錄和轉(zhuǎn)譯過(guò)程中密碼結(jié)構(gòu)的閱讀不發(fā)生干擾；同種內(nèi)切酶生產(chǎn)的粘性末端的連接5'

5'GAATTCCTTAAGGAATTCCTTAAGEcoRI5'GCTTAAAATTCG5'

5'

GCTTAA

5'5'

AATTCG5'

退火GCTTAAAATTCG5'

GCTTAA

5'

AATTCGGCTTAAAATTCG5'

GCTTAA

5'

AATTCGT4-DNAligaseGAATTCCTTAAG5'

5'GAATTCCTTAAGGAATTCCTTAAG5'

5'GAATTCCTTAAG同尾酶生產(chǎn)的粘性末端的連接BamHI5'

5'GGATCCCCTAGGGGATCCCCTAGG5'GCCTAGGATCCG5'

5'

TACTAG

5'5'

GATCAT5'

退火GCCTAGGATCCG5'

TACTAG

5'

GATCATT4-DNAligaseTGATCCACTAGG5'

5'GGATCACCTAGT5'

TGATCAACTAGT5'

BclIGCCTAGGATCCG5'

TACTAG

5'

GATCATTGATCCACTAGG5'

5'GGATCACCTAGT1.1外源DNA片段定向插入載體分子BamHI5'

5'GAATTCCTTAAGGGATCCCCTAGG5'GCCTAGAATTCG5'

5'

GCTTAA

5'5'

GATCCG5'

退火GCCTAGAATTCG5'

TCTTAA5'

GATCCGT4-DNAligaseTAATTCCTTAAG5'

5'GGATCACCTAGT5'

GAATTCCTTAAG5'

EcoRIGCTTAAGATCCG5'

TCTTAA5'

GATCATGGATCCCCTAGGBamHIEcoRI×1.2非互補(bǔ)粘性末端DNA分子間的連接BamHI5'5'GGATCCCCTAGGGGATCCCCTAGG5'GCCTAGGATCCG5'5'CTGCAG5'GACGTC3'T4-DNAligaseCGATCCGCTAGG5'5'GGATCGCCTAGC5'CTGCAGGACGTC5'PstI3'T4-DNApol切平5'CG5'GCKlenow補(bǔ)平5'GGATCCCTAGGATCC5'CTAGGCGATCCGCTAGG5'5'GGATCGCCTAGC1.2.1同聚物加尾法連接(5`突出末端)5'GCCTAGGATCCG5'

5'

GCTTAA5'5'5'

AATTCGT4-DNAligase5'

5'Klenow補(bǔ)平Klenow補(bǔ)平5'GGATCCCTAGGATCC5'

CTAGG5'

GAATTCTTAA5'5'5'

AATTCTTAAGTdTTdTdGTPdCTPGAATTGGGGGGCTTAAAATTCGGGGGGTTAAGGGATCCCCCCCCCTAGGATCCCCCCCCCTAGGGAATTGGGGGGGATCCCTTAACCCCCCCTAGGGGATCCCCCCCAATTCCCTAGGGGGGGTTAAG5'

5'GAATTGGGGGGGATCCCTTAACCCCCCCTAGGGGATCCCCCCCAATTCCCTAGGGGGGGTTAAG退火B(yǎng)amHIBamHIEcoRIBamHI1.2.1同聚物加尾法連接(3`突出末端)CTGCA3'

GG3'

ACGTC5'

GCATG3'C5'C3'GTACGT4-DNAligaseTdTTdTdCTPdGTPGCATGCCCCCC3'C退火PstIPstIC3'

CCCCCCGTACGCTGCAGGGGGG

3'

GG3'

GGGGGGACGTC5'

5'GCATGCCCCCC

GCGGGGGGACGTCCTGCAGGGGGGCGCCCCCCGTACG5'

5'GCATGCCCCCC

GCGGGGGGACGTCCTGCAGGGGGGCGCCCCCCGTACG5'

5'GCATGCCCCCCTGCAGCGTACGGGGGGACGTCCTGCAGGGGGGCATGCGACGTCCCCCCGTACG5'

5'GCATGCCCCCCTGCAGCGTACGGGGGGACGTCCTGCAGGGGGGCATGCGACGTCCCCCCGTACGKlenowPstISphIC3'

GG5'

G3'C5'C3'GT4-DNAligaseTdTTdTdTTPdATPGTTTTTTTTTT3'C退火C3'

TTTTTTTTTTGCAAAAAAAAAA

3'

GG3'

AAAAAAAAAAC5'

5'GTTTTTTTTTTGCAAAAAAAAAACCAAAAAAAAACGTTTTTTTTTTGS1C3'

5'

5'GTTTTTTTTTTGCAAAAAAAAAACCAAAAAAAAACGTTTTTTTTTTG加熱GTCTTTTTTTTTGAAAAAAACCAGAAAAAAAAACTTTTTTTGTGACCAGT1.2.1同聚物加尾法連接(平頭末端)1.2.2銜接物連接法BamHI5'

5'GGATCCCCTAGGT4-DNAligaseKlenow補(bǔ)平GATCC5'

G5'GCCTAG5'5'

5'GGATCCCTAG5'

GATCCCTAGG5'5'GGATCGGAATTCCGATCCCCTAGCCTTAAGGCTAGG5'

5'EcoRIGGAATTCCCCTTAAGGEcoRIlinker1.2.3DNA接頭連接法2重組率2.1含義：重組率=含有外源DNA的重組分子數(shù)/載體分子總數(shù)2.2提高重組率的方法提高外源DNA片段與載體的分子比：5:1-10:1；載體DNA分子在連接前先除去磷酸基團(tuán)；使用同聚物加尾連接技術(shù)；應(yīng)用柯斯質(zhì)粒載體DNA分子在連接前先除去磷酸基團(tuán)：

5'

5'GAATTCCTTAAGGAATTCCTTAAGEcoRI5'GCTTAApAATTCG5'

p5'

GCTTAAOH

5'5'

AATTCG5'

HO退火5'

G-A-A-T-T-CC-T-T-A-AG5'GA-A-T-T-CC-T-T-A-A-GOHHOOHHO堿性磷酸單酯酶堿性磷酸單酯酶

基因工程的操作過(guò)程切接轉(zhuǎn)增檢表EcoRITheBeta-GalactosidaseenzymeiscodedforbytheLacZgeneTheEcoRIsiteinterruptstheLacZgeneTestTransformationInsertDNAPLASMIDVECTORDNAEcoRIStickyEndsonBothMolecules如何賦于體外連接DNA分子以生命力？CompetentcellsandTransformation第二節(jié)重組DNA分子的轉(zhuǎn)化

和擴(kuò)增（轉(zhuǎn)與增）1受體細(xì)胞（receptorcell)：1.1定義：又稱為宿主細(xì)胞或寄主細(xì)胞(hostcell)等，從實(shí)驗(yàn)技術(shù)上講是能攝取外源DNA并使其穩(wěn)定維持的細(xì)胞；從實(shí)驗(yàn)?zāi)康纳现v是有應(yīng)用價(jià)值和理論研究?jī)r(jià)值的細(xì)胞。1.2選擇受體細(xì)胞的基本原則①便于重組DNA分子的導(dǎo)入。②便于重組體的篩選。③遺傳穩(wěn)定性高，易于進(jìn)行擴(kuò)大培養(yǎng)或易于進(jìn)行高密度發(fā)酵而不影響外源基因的表達(dá)效率。④受體細(xì)胞內(nèi)源蛋白水解酶基因缺失或蛋白酶含量低，利于外源蛋白表達(dá)產(chǎn)物在細(xì)胞內(nèi)積累，可促進(jìn)外源基因高效表達(dá)。⑤安全性高，無(wú)致病性，不會(huì)對(duì)外界環(huán)境造成生物污染。⑥能使重組DNA分子穩(wěn)定存在于細(xì)胞中。⑦受體細(xì)胞在遺傳密碼子的應(yīng)用上無(wú)明顯偏倚性。⑧具有較好的轉(zhuǎn)譯后加工機(jī)制等，便于真核目的基因的高效表達(dá)。⑨在理論研究和生產(chǎn)實(shí)踐上有較高的應(yīng)用價(jià)值。1.3常用的受體細(xì)胞原核生物酵母菌動(dòng)物細(xì)胞植物細(xì)胞2重組DNA分子導(dǎo)入受體細(xì)胞的方法（轉(zhuǎn)）轉(zhuǎn)化:指將質(zhì)粒DNA或以它為載體構(gòu)建的重組質(zhì)粒導(dǎo)入細(xì)菌中的過(guò)程。轉(zhuǎn)染:指病毒及其重組子導(dǎo)入受體細(xì)胞的過(guò)程微粒子轉(zhuǎn)化：將DNA包裹上“外衣”，通過(guò)“發(fā)射”或融合的方式導(dǎo)入受體細(xì)胞。電擊轉(zhuǎn)化：利用電場(chǎng)使受體細(xì)胞膜出現(xiàn)“微孔隙”，從而吸收DNA分子進(jìn)行轉(zhuǎn)化。UPTAKEOFDNATransformationcellmadecompetenttotakeupDNATransfectionwhenthecloningvectorusedhasaspectsofavirus,thehostcellcanbeinfected(transfected)toinserttherecombinantmoleculeMicroprojectilesparticlescoatedwithDNAare"fired"atacellandpenetratethemembraneElectroporationthecellisplacedinanelectricfieldsuchthatsmallporesaretemporarilyopenedinthemembrane.AddedDNAcanenterthroughtheseporesCompetentCells“Competent”toTakeupDNABacterialCellPlasmidsandotherDNAmolecules+Ice+HeatShockCaCl2ACompetentCellMayTakeUpOneorMorePiecesofDNAPlasmidDNAmolecules+CaCl2+Ice+HeatShockBacterialCell感受態(tài)細(xì)胞(competentcell):當(dāng)受體細(xì)胞處于容易吸收外源DNA的狀態(tài)時(shí),就稱為感受態(tài)細(xì)胞。

2.1Ca2+誘導(dǎo)的完整細(xì)菌細(xì)胞的轉(zhuǎn)化

Ca2+誘導(dǎo)的完整細(xì)胞的轉(zhuǎn)化適用于革蘭氏陰性細(xì)菌（如大腸桿菌等），1970年建立此技術(shù)，其原理是Ca2+與細(xì)菌外膜磷脂在低溫下形成液晶結(jié)構(gòu)，后者經(jīng)熱脈沖發(fā)生收縮作用，使細(xì)胞膜出現(xiàn)空隙，細(xì)菌細(xì)胞此時(shí)的狀態(tài)叫做感受態(tài)

大腸桿菌感受態(tài)細(xì)胞的制備：100ml菌體培養(yǎng)至OD600=0.5，離心收集菌體用10ml冰冷的100mMCaCl2溶液懸浮菌體，離心收集菌體用1ml冰冷的100mMCaCl2溶液懸浮菌體冰浴放置12-24小時(shí)，備用E.ColibacteriumE.coliisthemostcommonbacteriuminthehumangutE.colihasbeenextensivelystudiedReproduceveryrapidlyAsinglecellcandivideandgiveriseto106cellsovernightThegrowthrateofabacterialcultureisnotconstant.Intheearlyhours(lagphase),growthisveryslowbecausethestartingnumberofdividingcellsissmall.Thisisfollowedbyatimeofrapidcelldivisionknownasthelogphase.Theactuallengthofeachphasedependsonthetemperatureatwhichthecellsareincubated.RecombinantTransformationCompetentCellProcedureLabelasterile1.5mltubewith“CC”andyourgroup’sname.FromtheMid-Logsuspension,transfer1mlofculturetoyoursterile1.5mltube.RecombinantTransformationCompetentCellProcedurePlacethetubeinthemicrocentrifuge(MicroVmicrocentrifuge,shown)andspinfor2minutesat10,000RPM.RecombinantTransformationCompetentCellProcedureSterilelydrainofftheLBbroth,leavingavisiblepelletofbacteriainthetube.RecombinantTransformationCompetentCellProcedureUseasterileplasticpipettetotransfer0.5ml(10drops)ofcalciumchloridetothetubecontainingthecells.RecombinantTransformationCompetentCellProcedureResuspendthepelletedcellbyvigorouslyfinger-flicking(vortexing)thetube.Placeonicefor20minutes.RecombinantTransformationCompetentCellProcedureAfter20minutes,placethetubeinamicrocentrifugeandspinfor2minutesat10,000RPM.RecombinantTransformationCompetentCellProcedureSterilelydrainoffthecalciumchloride,beingcarefulnottodisturbthecellpellet.RecombinantTransformationCompetentCellProcedureSterilelyadd2dropsoffreshcalciumchlorideandfingervortextoresuspend.RecombinantTransformationCompetentCellProcedurePlacecellssuspendedincalciumchlorideoniceintherefrigerator(approx.0oC)andstoreuntilyouarereadytousethem.DONOTFREEZE.RecombinantTransformationCompetentCellProcedureHoldingthecellsat0oCfor24hoursincreasesthecellcompetency.Cellscanbeheldon“wetice”(combinationofmeltedandsolidice)forseveraldayswhileyoumaketherecombinantconstructs.大腸桿菌感受態(tài)細(xì)胞的質(zhì)粒轉(zhuǎn)化：取100ml感受態(tài)細(xì)胞，加入相當(dāng)于50ng載體的重組DNA連接液，混勻冰浴放置半小時(shí)在42℃保溫90s（熱脈沖）快速將轉(zhuǎn)化細(xì)胞轉(zhuǎn)移至冰浴中放置1-2分鐘加入1ml新鮮培養(yǎng)基，于37℃培養(yǎng)1小時(shí)（擴(kuò)增）涂在合適的固體培養(yǎng)基平板上進(jìn)行篩選TransformationprocedureSCREENINGPlasmidvectorcontainsanampicillinresistancegenemakingthecellresistant.Growthoftransformedcells(cellsreceivingtheplasmid)canbeidentifiedonagarmediumcontaining(e.g.)ampicillin.LB-AmpPlate100ul1:5V=1000ul200ul1:100LB-AmpPlate100ul1000ulControlplateTransformationsLB-AmpPlate100ul1:10010ul1000ulLBPlate100ul1:10010ul1000ulTestTransformation1000ul10ulUncutplasmidaddedNoplasmidaddedNoplasmidaddedForeignGeneDNAligatedplasmidaddedControlplate:

UncutpBluescriptplasmid,incubated,andaddedtocompetentcells.

MediaonlyMedia+AmpicillinTestTransformation:

EcoRI-cutForeignGeneDNAligatedtoEcoRI-linearizedpBluescript,thenaddedtocompetentcells.Media+AmpicillinDoesawhitecolony,pickedfromyourTestTransformationplates,andthengrownonampicillin,reallycontaintheForeignGeneDNA?Isthewhitecolonyjustanampicillin-resistantcontaminant?DidtheForeignGeneDNAligateintotheplasmidcorrectly,ordidtheplasmid/insertgetdamagedduringtheligationreaction?

革蘭氏陽(yáng)性細(xì)菌（如枯草桿菌、鏈霉菌等）接納外源DNA的主要屏障是細(xì)胞壁，因而這類細(xì)菌通常采用原生質(zhì)體（細(xì)胞去壁后的形態(tài)）轉(zhuǎn)化的方法轉(zhuǎn)移質(zhì)粒或重組DNA分子2.2細(xì)菌原生質(zhì)體的轉(zhuǎn)化2.3噬菌體DNA的轉(zhuǎn)染感受態(tài)細(xì)胞的培養(yǎng)：將大腸桿菌在含有麥芽糖（不含葡萄糖）和MgCl2的培養(yǎng)基中培養(yǎng)至OD600=0.5吸附：加入經(jīng)體外包裝好的重組噬菌體懸浮液，30℃保溫10分鐘轉(zhuǎn)染裂解：加入20倍體積的新鮮培養(yǎng)基，30℃培養(yǎng)2小時(shí)，直至培養(yǎng)液中菌體裂解，培養(yǎng)液澄清度增加，然后涂板篩選基因重組轉(zhuǎn)染體外包裝噬菌體噬菌體DNA的轉(zhuǎn)染USINGAVIRUSTOINSERTDNAINTOACELLThegeneisinsertedintothegeneticmake-upofharmlessvirusesthattheninvadecells,carryingthegeneintothecells.2.4電穿孔轉(zhuǎn)化將待轉(zhuǎn)化的質(zhì)?；駾NA重組連接液加在電穿孔轉(zhuǎn)化儀的樣品池中，兩極施加高壓電場(chǎng)。在強(qiáng)大電場(chǎng)的作用下，細(xì)菌細(xì)胞壁和細(xì)胞膜產(chǎn)生縫隙，質(zhì)?；駾NA重組分子便可進(jìn)入細(xì)胞內(nèi)；

2.5借助于載體的基因轉(zhuǎn)移2.6磷酸鈣沉淀法2.7脂質(zhì)體介導(dǎo)法2.8血影細(xì)胞介導(dǎo)法

OTHERTRANSFOMATIONMETHODSCalciumphosphateprecipitationandphagocytosis:usedtotransformcellsofmammals.Lipofection:usedtotransformcellsofanimals,yeast,plantsandbacteria.TheDNAtobetransferredisplacedintoliposomes.Sincetheliposomesaremadeupoflipids,theybecomepartofthecellmembraneofthecellsandthecontents-thenewDNA-entersthecells.BIOLISTICSAspeciallydesignedgenegunusingcompressedheliumgasfiresdozensofmetalpiecesattargetcells.Thetinypellets,usuallyoftungstenorgold,aremuchsmallerthenthetargetcell,andcoatedwithDNA.Inthe"biolistic"(acrossbetweenbiologyandballistics)or"genegun"method,microscopicgoldbeadsarecoatedwiththegeneofinterestandshotintotheplantcellwithapulseofhelium.

Onceinsidethecell,thegenecomesoffthebeadandintegratesintothecell'sgenome.ModelfromBioRad:

Biorad'sHeliosGeneGun3轉(zhuǎn)化率3.1轉(zhuǎn)化率含義：每微克載體DNA在最佳轉(zhuǎn)化條件下進(jìn)入受體細(xì)胞的分子數(shù)。3.2轉(zhuǎn)化率的用途:

利用轉(zhuǎn)化率和重組率等參數(shù)可以幫助設(shè)計(jì)DNA重組實(shí)驗(yàn)規(guī)模。例如，某一DNA重組實(shí)驗(yàn)的重組率為20%，轉(zhuǎn)化率為107個(gè)/μg載體，經(jīng)切接處理后的載體轉(zhuǎn)化率比天然的載體低100倍，欲獲得104個(gè)重組克隆，需投入多少載體DNA進(jìn)行重組實(shí)驗(yàn)？

若重組率為100%，則轉(zhuǎn)化后產(chǎn)生104個(gè)重組克隆需要：104／(107

×10-2)=0.1μg載體DNA

考慮到實(shí)驗(yàn)系統(tǒng)的重組率為20%，所以實(shí)際投入載體應(yīng)為：0.1÷20%=0.5μg載體DNA載體投入量確定后，外源DNA的投入量即可按10∶1的要求算出（5μg）3.3轉(zhuǎn)化率的影響因素載體及DNA重組分子方面：受體細(xì)胞方面：轉(zhuǎn)化方法：4轉(zhuǎn)化細(xì)胞的擴(kuò)增（增）4.1含義：是指轉(zhuǎn)化完成之后細(xì)胞的短時(shí)間培養(yǎng)；4.2目的：

增殖轉(zhuǎn)化細(xì)胞，使得有足夠數(shù)量的轉(zhuǎn)化細(xì)胞用于篩選程序；擴(kuò)增和表達(dá)載體分子上的標(biāo)記基因，便于篩選。Doesawhitecolony,pickedfromyourTestTransformationplates,andthengrownonampicillin,reallycontaintheForeignGeneDNA?Isthewhitecolonyjustanampicillin-resistantcontaminant?DidtheForeignGeneDNAligateintotheplasmidcorrectly,ordidtheplasmid/insertgetdamagedduringtheligationreaction?第三節(jié)重組子的篩選和鑒定（檢）由于重組率和轉(zhuǎn)化率不可能達(dá)到理想極限，因此必須借助各種篩選和鑒定方法區(qū)分轉(zhuǎn)化子與非轉(zhuǎn)化子、重組子與非重組子、目的重組子與非目的重組子轉(zhuǎn)化子重組子目的重組子重組子的篩選直接篩選間接篩選DNA鑒定載體篩選重組子大小鑒定重組子酶切圖譜鑒定DNA序列分析同源性分析鑒定質(zhì)粒載體的抗性標(biāo)記篩選噬菌體包裝容量的正性篩選質(zhì)粒載體的α-互補(bǔ)篩選標(biāo)記補(bǔ)救篩選翻譯產(chǎn)物（westernblotting)轉(zhuǎn)錄產(chǎn)物（Norternblotting)其它方法（報(bào)告基因等)1.1抗藥性篩選法例：插入失活篩選法（Insertionalinactivation）1載體遺傳標(biāo)記檢測(cè)ROPROISalIBamHI