zj基因工程第6章(重组DNA的方法)

第六章重组DNA技术

基因工程的操作过程切接转增检表DNA的酶切与连接策略

重组率

第一节DNA的体外重组

（切与接）1DNA的酶切与连接策略

在选择外源DNA同载体分子连接反应的程序时，需要考虑到下列三个因素：

实验步骤要尽可能简单易行；

连接形成的接点序列，最好能被一定的核酸内切限制酶重新切割，以便回收插入的外源DNA片段；

对转录和转译过程中密码结构的阅读不发生干扰；同种内切酶生产的粘性末端的连接5'

5'GAATTCCTTAAGGAATTCCTTAAGEcoRI5'GCTTAAAATTCG5'

5'

GCTTAA

5'5'

AATTCG5'

退火GCTTAAAATTCG5'

GCTTAA

5'

AATTCGGCTTAAAATTCG5'

GCTTAA

5'

AATTCGT4-DNAligaseGAATTCCTTAAG5'

5'GAATTCCTTAAGGAATTCCTTAAG5'

5'GAATTCCTTAAG同尾酶生产的粘性末端的连接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片段定向插入载体分子BamHI5'

5'GAATTCCTTAAGGGATCCCCTAGG5'GCCTAGAATTCG5'

5'

GCTTAA

5'5'

GATCCG5'

退火GCCTAGAATTCG5'

TCTTAA5'

GATCCGT4-DNAligaseTAATTCCTTAAG5'

5'GGATCACCTAGT5'

GAATTCCTTAAG5'

EcoRIGCTTAAGATCCG5'

TCTTAA5'

GATCATGGATCCCCTAGGBamHIEcoRI×1.2非互补粘性末端DNA分子间的连接BamHI5'5'GGATCCCCTAGGGGATCCCCTAGG5'GCCTAGGATCCG5'5'CTGCAG5'GACGTC3'T4-DNAligaseCGATCCGCTAGG5'5'GGATCGCCTAGC5'CTGCAGGACGTC5'PstI3'T4-DNApol切平5'CG5'GCKlenow补平5'GGATCCCTAGGATCC5'CTAGGCGATCCGCTAGG5'5'GGATCGCCTAGC1.2.1同聚物加尾法连接(5`突出末端)5'GCCTAGGATCCG5'

5'

GCTTAA5'5'5'

AATTCGT4-DNAligase5'

5'Klenow补平Klenow补平5'GGATCCCTAGGATCC5'

CTAGG5'

GAATTCTTAA5'5'5'

AATTCTTAAGTdTTdTdGTPdCTPGAATTGGGGGGCTTAAAATTCGGGGGGTTAAGGGATCCCCCCCCCTAGGATCCCCCCCCCTAGGGAATTGGGGGGGATCCCTTAACCCCCCCTAGGGGATCCCCCCCAATTCCCTAGGGGGGGTTAAG5'

5'GAATTGGGGGGGATCCCTTAACCCCCCCTAGGGGATCCCCCCCAATTCCCTAGGGGGGGTTAAG退火BamHIBamHIEcoRIBamHI1.2.1同聚物加尾法连接(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加热GTCTTTTTTTTTGAAAAAAACCAGAAAAAAAAACTTTTTTTGTGACCAGT1.2.1同聚物加尾法连接(平头末端)1.2.2衔接物连接法BamHI5'

5'GGATCCCCTAGGT4-DNAligaseKlenow补平GATCC5'

G5'GCCTAG5'5'

5'GGATCCCTAG5'

GATCCCTAGG5'5'GGATCGGAATTCCGATCCCCTAGCCTTAAGGCTAGG5'

5'EcoRIGGAATTCCCCTTAAGGEcoRIlinker1.2.3DNA接头连接法2重组率2.1含义：重组率=含有外源DNA的重组分子数/载体分子总数2.2提高重组率的方法提高外源DNA片段与载体的分子比：5:1-10:1；载体DNA分子在连接前先除去磷酸基团；使用同聚物加尾连接技术；应用柯斯质粒载体DNA分子在连接前先除去磷酸基团：

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碱性磷酸单酯酶碱性磷酸单酯酶

基因工程的操作过程切接转增检表EcoRITheBeta-GalactosidaseenzymeiscodedforbytheLacZgeneTheEcoRIsiteinterruptstheLacZgeneTestTransformationInsertDNAPLASMIDVECTORDNAEcoRIStickyEndsonBothMolecules如何赋于体外连接DNA分子以生命力？CompetentcellsandTransformation第二节重组DNA分子的转化

和扩增（转与增）1受体细胞（receptorcell)：1.1定义：又称为宿主细胞或寄主细胞(hostcell)等，从实验技术上讲是能摄取外源DNA并使其稳定维持的细胞；从实验目的上讲是有应用价值和理论研究价值的细胞。1.2选择受体细胞的基本原则①便于重组DNA分子的导入。②便于重组体的筛选。③遗传稳定性高，易于进行扩大培养或易于进行高密度发酵而不影响外源基因的表达效率。④受体细胞内源蛋白水解酶基因缺失或蛋白酶含量低，利于外源蛋白表达产物在细胞内积累，可促进外源基因高效表达。⑤安全性高，无致病性，不会对外界环境造成生物污染。⑥能使重组DNA分子稳定存在于细胞中。⑦受体细胞在遗传密码子的应用上无明显偏倚性。⑧具有较好的转译后加工机制等，便于真核目的基因的高效表达。⑨在理论研究和生产实践上有较高的应用价值。1.3常用的受体细胞原核生物酵母菌动物细胞植物细胞2重组DNA分子导入受体细胞的方法（转）转化:指将质粒DNA或以它为载体构建的重组质粒导入细菌中的过程。转染:指病毒及其重组子导入受体细胞的过程微粒子转化：将DNA包裹上“外衣”，通过“发射”或融合的方式导入受体细胞。电击转化：利用电场使受体细胞膜出现“微孔隙”，从而吸收DNA分子进行转化。UPTAKEOFDNATransformationcellmadecompetenttotakeupDNATransfectionwhenthecloningvectorusedhasaspectsofavirus,thehostcellcanbeinfected(transfected)toinserttherecombinantmoleculeMicroprojectilesparticlescoatedwithDNAare"fired"atacellandpenetratethemembraneElectroporationthecellisplacedinanelectricfieldsuchthatsmallporesaretemporarilyopenedinthemembrane.AddedDNAcanenterthroughtheseporesCompetentCells“Competent”toTakeupDNABacterialCellPlasmidsandotherDNAmolecules+Ice+HeatShockCaCl2ACompetentCellMayTakeUpOneorMorePiecesofDNAPlasmidDNAmolecules+CaCl2+Ice+HeatShockBacterialCell感受态细胞(competentcell):当受体细胞处于容易吸收外源DNA的状态时,就称为感受态细胞。

2.1Ca2+诱导的完整细菌细胞的转化

Ca2+诱导的完整细胞的转化适用于革兰氏阴性细菌（如大肠杆菌等），1970年建立此技术，其原理是Ca2+与细菌外膜磷脂在低温下形成液晶结构，后者经热脉冲发生收缩作用，使细胞膜出现空隙，细菌细胞此时的状态叫做感受态

大肠杆菌感受态细胞的制备：100ml菌体培养至OD600=0.5，离心收集菌体用10ml冰冷的100mMCaCl2溶液悬浮菌体，离心收集菌体用1ml冰冷的100mMCaCl2溶液悬浮菌体冰浴放置12-24小时，备用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.大肠杆菌感受态细胞的质粒转化：取100ml感受态细胞，加入相当于50ng载体的重组DNA连接液，混匀冰浴放置半小时在42℃保温90s（热脉冲）快速将转化细胞转移至冰浴中放置1-2分钟加入1ml新鲜培养基，于37℃培养1小时（扩增）涂在合适的固体培养基平板上进行筛选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?

革兰氏阳性细菌（如枯草杆菌、链霉菌等）接纳外源DNA的主要屏障是细胞壁，因而这类细菌通常采用原生质体（细胞去壁后的形态）转化的方法转移质粒或重组DNA分子2.2细菌原生质体的转化2.3噬菌体DNA的转染感受态细胞的培养：将大肠杆菌在含有麦芽糖（不含葡萄糖）和MgCl2的培养基中培养至OD600=0.5吸附：加入经体外包装好的重组噬菌体悬浮液，30℃保温10分钟转染裂解：加入20倍体积的新鲜培养基，30℃培养2小时，直至培养液中菌体裂解，培养液澄清度增加，然后涂板筛选基因重组转染体外包装噬菌体噬菌体DNA的转染USINGAVIRUSTOINSERTDNAINTOACELLThegeneisinsertedintothegeneticmake-upofharmlessvirusesthattheninvadecells,carryingthegeneintothecells.2.4电穿孔转化将待转化的质粒或DNA重组连接液加在电穿孔转化仪的样品池中，两极施加高压电场。在强大电场的作用下，细菌细胞壁和细胞膜产生缝隙，质粒或DNA重组分子便可进入细胞内；

2.5借助于载体的基因转移2.6磷酸钙沉淀法2.7脂质体介导法2.8血影细胞介导法

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转化率3.1转化率含义：每微克载体DNA在最佳转化条件下进入受体细胞的分子数。3.2转化率的用途:

利用转化率和重组率等参数可以帮助设计DNA重组实验规模。例如，某一DNA重组实验的重组率为20%，转化率为107个/µg载体，经切接处理后的载体转化率比天然的载体低100倍，欲获得104个重组克隆，需投入多少载体DNA进行重组实验？

若重组率为100%，则转化后产生104个重组克隆需要：104／(107

×10-2)=0.1µg载体DNA

考虑到实验系统的重组率为20%，所以实际投入载体应为：0.1÷20%=0.5µg载体DNA载体投入量确定后，外源DNA的投入量即可按10∶1的要求算出（5µg）3.3转化率的影响因素载体及DNA重组分子方面：受体细胞方面：转化方法：4转化细胞的扩增（增）4.1含义：是指转化完成之后细胞的短时间培养；4.2目的：

增殖转化细胞，使得有足够数量的转化细胞用于筛选程序；扩增和表达载体分子上的标记基因，便于筛选。Doesawhitecolony,pickedfromyourTestTransformationplates,andthengrownonampicillin,reallycontaintheForeignGeneDNA?Isthewhitecolonyjustanampicillin-resistantcontaminant?DidtheForeignGeneDNAligateintotheplasmidcorrectly,ordidtheplasmid/insertgetdamagedduringtheligationreaction?第三节重组子的筛选和鉴定（检）由于重组率和转化率不可能达到理想极限，因此必须借助各种筛选和鉴定方法区分转化子与非转化子、重组子与非重组子、目的重组子与非目的重组子转化子重组子目的重组子重组子的筛选直接筛选间接筛选DNA鉴定载体筛选重组子大小鉴定重组子酶切图谱鉴定DNA序列分析同源性分析鉴定质粒载体的抗性标记筛选噬菌体包装容量的正性筛选质粒载体的α-互补筛选标记补救筛选翻译产物（westernblotting)转录产物（Norternblotting)其它方法（报告基因等)1.1抗药性筛选法例：插入失活筛选法（Insertionalinactivation）1载体遗传标记检测ROPROISalIBamHI