包含的cDNA克隆数目较少课件

生物技术综合实验Comprehensiveexperimentsofbiotechnology生物技术综合实验1主要内容Contents：一、课程简介核酸的分离与纯化IsolationandPurificationofNucleicAcid二、电泳技术AgaroseGelElrctrophoresisandSDS

三、聚合酶链式反应技术PolymeraseChainReaction四、DNA序列测定DNASequencing五、分子杂交技术MolecularHybridization－Southern,NorthernandWesternBlot六、基因文库和cDNA文库的构建ConstructionofcDNALibraryandDNALibrary

七、外源基因的克隆与表达

HeterogenicGeneCloningandExpression八、蛋白质的分离、纯化技术IsolationandPurificationofProtein主要内容Contents：2Part6基因文库和cDNA文库的构建ConstructionofDNA&cDNALibraryPart6基因文库和cDNA文库的构建3基因组文库（genomiclibrary）：包含某种生物体全部基因的随机片段的重组DNA克隆群体称为基因组文库。

cDNA文库（cDNAlibrary）：包含细胞的全部mRNA的信息的重组cDNA克隆群体称为cDNA库。基因组文库（genomiclibrary）：包含某种生物体4一、基因组文库（genomiclibrary）基因工程技术的迅速发展使人们对生物体基因的结构、功能、表达及其调控的研究深入到分子水平，而对特定基因片段的分离和获得是上述研究的基础。完整的基因组文库(genomiclibrary)的构建使任何DNA片段的筛选和获得成为可能。一、基因组文库（genomiclibrary）基因工51.1构建真核细胞基因组文库的载体：λ噬菌体（可插入基因片断约20kb）粘性质粒（可插入基因片断约46kb）酵母人工染色体克隆系统(yeastartificialchromosomecloningsystem),简称YACS。（可插入基因片断约200-500kb）

1.1构建真核细胞基因组文库的载体：6包含的cDNA克隆数目较少课件71.2应用入噬菌体构建基因组文库的基本步骤(1)准备载体DNA。(2)提取高分子量真核细胞DNA：并选择合适的限制性内切酶进行部分降解。(3)分离大小合适的真核DNA片段。1.2应用入噬菌体构建基因组文库的基本步骤(1)8(4)载体DNA与外源DNA连接。(5)连接产物在体外进行包装。(6)检测重组噬菌体的滴度，扩增、分装保存。(4)载体DNA与外源DNA连接。9由于mRNA含有某种细胞的各种RNA分子，因而反转录合成的cDNA将代表各样mRNA拷贝，将其和载体DNA重组，并转化到宿主细菌里或包装成噬菌体颗粒，得到一系列克隆群体。每个克隆只含一种mRNA的信息，足够数目克隆的总和则包含细胞的全部mRNA的信息，这样的克隆群体叫cDNA库。2.1概述二、cDNA文库（cDNAlibrary）由于mRNA含有某种细胞的各种RNA分子，因而10按照筛选方式不同cDNA库可分为：表达型cDNA文库:采用表达型载体。插入的cDNA片段可表达产生融合蛋白，不能采用核苷酸探针筛选的目的基因，可采用能与表达产物发生特异性结合的抗体或化合物进行标记筛选。非表达型cDNA文库：适用于那些采用核苷酸探针进行杂交筛选的基因。按照筛选方式不同cDNA库可分为：11根据载体的不同将cDNA库分为：质粒cDNA库:包含的cDNA克隆数目较少，适于较高丰度的mRNA噬菌体cDNA库:包含的cDNA克隆数目非常多，适用于那些低丰度和极低丰度的mRNA根据载体的不同将cDNA库分为：12在构建一个cDNA库时，首先应考虑的问题是筛选方法。若采用核苷酸探针进行杂交筛选，可构建表达型或非表达型cDNA库；如利用蛋白质的生物活性或免疫原性进行筛选，则只能构建表达型cDNA库。其次应考虑的问题是mRNA的丰度。对于高丰度的mRNA所需构建的cDNA库相对较小；而极低丰度的mRNA，如仅占mRNA总量的1/106的某些mRNA，所需构建的cDNA库则必须很大，尽可能包括其对应的克隆。在构建一个cDNA库时，首先应考虑的问题是筛选方法。若132.2构建cDNA库主要包括以下几个步骤：①mRNA的分离；②cDNA第一链的合成；③cDNA第二条链的合成；④cDNA与载体的连接；⑤噬菌体的包装及转染或质粒的转化。⑥检测重组噬菌体的滴度，扩增、分装保存。

2.2构建cDNA库主要包括以下几个步骤：14cDNA文库构建的具体操作方法见有关实验指南。cDNA文库构建的具体操作方法见有关实验指南。15 UNIT2MANIPULATIONOFDNAANDGENEISOLATIONLECTURES: 9.DNACloningandLibraryConstruction 10.IsolatingGenes UNIT2169.DNACloningandLibraryConstruction

a).DNAcloning i).Restrictionendonucleases ii).Cloningvectors iii).TheprocessofcloningasegmentofDNA b).Libraryconstruction i).Genomiclibraries ii).cDNAlibraries

9.DNACloningandLibraryCon17Howdoesoneisolateageneforaninheriteddisorder? Therearethreeoptions:Startwithacandidateprotein

DNA proteinStartwithacandidatemRNA

DNA mRNADirectpositionalcloning

DNA AllthreeoptionsrequirethecloningofDNA.DNAmRNAproteinHowdoesoneisolateagene18RestrictionendonucleasesRestrictionenzymescutDNAintospecificfragmentsRestrictionenzymesrecognizespecificbasesequencesindouble-stranded DNAandcleavebothstrandsoftheduplexatspecificplacesCharacteristicsofrestrictionenzymes:1.CutDNAsequence-specifically2.Bacterialenzymes;hundredsarepurifiedandavailablecommercially3.Restriction-modificationsystem

BacteriahaveenzymesthatwillcleaveforeignDNA;hence,“restrict”theentryofviral DNA.Topreventthebacteria’sownDNAfrombeingcut,thereisasecondenzymethat

methylatesthesamesitesrecognizedbytherestrictionenzyme(modifiesthatsite).4.Named(e.g.,EcoRI)forbacterialgenus,species,strain,andtype5.Recognizespecific4-8bpsequencessequenceshavesymmetry(theyarepalindromes)aftercuttingtheDNA,thecutendsareeitherbluntstaggered(overhangs)-cohesiveendsfacilitatecloningtheDNA6.Frequencyofcutting4-basecutter 44=256bp5-basecutter 45=1,024bp6-basecutter 46=4,096bp8-basecutter 48=65,536bpRestrictionendonucleases194-basecutter:cutsDNAinto256bpaverage-sizedfragmentsinarandomsequenceevery256bp:NO256bpaverage-sizefragments:YESBar=256bp4-basecutter:every256bp:NO20Productsgeneratedbyrestrictionenzymes COHESIVEENDSEcoRI 5’…GAATTC…3’ 5’…G AATTC…3’ 3’…CTTAAG…5’ 3’…CTTAA G…5’PstI 5’…CTGCAG…3’ 5’…CTGCAG…3’ 3’…GACGTC…5’ 3’…G ACGTC…5’ BLUNTENDSHaeIII 5’…GGCC…3’ 5’…GG CC…3’ 3’…CCGG…5’ 3’…CC GG…5’Productsgeneratedbyrestrict21FormationofrecombinantDNAmoleculescutDNAsmixtogetherfragmentsandannealcohesiveendsseal3’,5’endsbyDNAligaserecombinantDNAsFormationofrecombinantDNAm22VectorsusedinmolecularcloningVector Insert(andhost)

Characteristics sizerangePlasmid SmallcircularDNA <5-10kb(bacteria,yeast)Bacteriophagelambda LinearviralDNA upto~20kborphagelambda(bacteria)Cosmid Hybridofplasmid upto~50kb(bacteria) andphageYeastartificial DNAcontainingyeast ~200to~1000kbchromosomeorYACcentromere,telomeres,(yeast) andoriginsofreplicationVectorsusedinmolecularclon23StructureofpBR322-acommoncloningvectorderivedfromanaturallyoccurringplasmidhasantibioticresistancegenesforselectionof transformantscontainingtheplasmidhasuniquerestrictionenzymecleavagesitesfor insertionofforeignDNAhasoriginofDNAreplication(ori)forpropagationinE.coliEcoRISalIgeneforampicillinresistancegenefortetracyclineresistancePstIoriStructureofpBR322-acommon24CloningasegmentofDNAintoaplasmidvectorbacteriaare“transformed”withtherecombinantplasmidcoloniesthatgrowintetracycline,butnotinampicillinareisolatedPstIHumanDNAcutwithPstIPPpBR322ampR,tetRpBR322(humanclone)tetRPPampRtetRpBR322DNAcutwithPstIinactivatingtheampRgenetetRtetRcombineandligateCloningasegmentofDNAinto25LibraryconstructiontwotypesoflibrariesagenomiclibrarycontainsfragmentsofgenomicDNA(genes)acDNAlibrarycontainsDNAcopiesofcellularmRNAsbothtypesareusuallyclonedinbacteriophagevectorsConstructionofagenomiclibraryvectorDNA(bacteriophagelambda)lambdahasalineardouble- strandedDNAgenometheleftandrightarmsareessential forthephagereplicationcycletheinternalfragmentisdispensable“leftarm”“rightarm”BamHIsitesinternalfragment(dispensableforphagegrowth)LibraryconstructionvectorDNA26NNG

GATCCNN

NNCCTAG

GNNinternalfragmentcutwithBamHI(6-basecutter)removeinternalfragment“leftarm”“rightarm”cutwithSau3A(4-basecutter)whichhasendscompatiblewithBamHI:

NNNGATCNNN

NNNCTAGNNNisolate~20kbfragmentshumangenomicDNA(isolatedfrom manycells)BamHIsites:NNGGATCCNNinternalfragment27combineandtreatwithDNAligase“leftarm”“rightarm”“leftarm”“rightarm”packageintobacteriophageandinfectE.coli123456genomiclibraryofhumanDNAfragmentsinwhicheachphagecontainsadifferenthumanDNAsequence7combineandtreat“leftarm”“ri28isolationof~20kbfragmentsprovidesoptimally sizedDNAsforcloninginbacteriophage

partialdigestionwithafrequent-cutter(4-basecutter)allowsproduction ofoverlappingfragments,sincenoteverysiteiscutoverlappingfragmentsinsuresthatallsequencesinthegenomeareclonedoverlappingfragmentsallowslargerphysicalmapstobeconstructedas contiguouschromosomalregions(contigs)areputtogetherfrom thesequencedatanumberofclonesneededtofullyrepresentthehumangenome(3X109bp) assuming~20kbfragmentstheoreticalminimum=~150,00099%probabilitythateverysequenceisrepresented=~800,000

Partialrestrictionenzymedigestionallowscloningofoverlappingfragmentsa“contig”isolationof~20kbfragments29Allpossiblesites:Resultsofapartialdigestion:=uncut=cutAllpossiblesites:Resultsof30ConstructionofacDNAlibraryreversetranscriptasemakesaDNAcopyofanRNAThelifecycleofaretrovirusdependsonreversetranscriptaseretrovirus1.virusenterscellandloosesenvelope2.thecapsidisuncoated,releasinggenomicRNAandreversetranscriptase3.reversetranscriptasemakesaDNAcopy4.thencopiestheDNAstrandtomakeitdouble-strandedDNA,removingtheRNAwithRNaseH5.theDNAisthenintegratedintothehostcellgenomewhereitistranscribedbyhostRNApolymeraseII6.itistranslatedintoviralproteins,andassembledintonewvirusparticlesnewvirusesConstructionofacDNAlibrary31cDNAlibraryconstructionAAAAA5’3’mRNA(allmRNAsincell)annealoligo(dT)primersof12-18basesinlengthAAAAATTTTT5’3’5’addreversetranscriptaseanddNTPsAAAAATTTTT5’3’3’5’cDNAaddRNaseH(specificfortheRNAstrandofanRNA-DNAhybrid)andcarryoutapartialdigestionAATTTTT5’shortRNAfragmentsserveasprimersforsecondstrandsynthesisusingDNApolymeraseI3’3’cDNAlibraryconstructionAAAA32AAAAATTTTT5’3’shortRNAfragmentsserveasprimersforsecondstrandsynthesisusingDNApolymeraseI

AAATTTTT5’3’DNApolymeraseIremovestheremainingRNAwithits5’to3’exonucleaseactivityandcontinuessynthesisDNAligasesealsthegaps

AAATTTTT5’3’AAAAATTTTT5’3’double-strandedcDNAAAAAA5’shortRNAfragmentsser33AAAAATTTTT5’3’NNNNNNNNGNNNNNNNNCTTAAEcoRIlinkersareligatedtobothends usingDNAligaseAAAAANNNNNNNNGTTTTTNNNNNNNNCTTAAdouble-strandedcDNAcopiesofmRNAwithEcoRIcohesiveendsarenowreadytoligateintoabacteriophagelambdavectorcutwithEcoRI5’3’AATTCNNNNNNNNGNNNNNNNNAAAAA5’NNNNNNNNGEcoRIlinkers34EcoRIsitescombinecDNAswithlambdaarmsandtreatwithDNAligase“leftarm”“rightarm”“leftarm”“rightarm”packageintobacteriophageandinfectE.coli123456cDNAlibraryinwhicheachphagecontainsadifferenthumancDNA7cDNAsEcoRIsitescombinecDNAswith“35cDNALibrarycDNALibrary36TheCentralDogmaDNAmRNAProteinSingle-strandedDouble-strandedPrecursorRNAexonintronAAAAAAAAAAnAAAAAAAAAAnSingle-strandedAAAAAAAAAAndouble-strandedReversetranscriptioncDNATheCentralDogmaDNAmRNAProtei37cDNAAcDNAorcomplementaryDNA,isaDNAcopyofanRNA,usuallymRNAcDNAmRNADoublestrandedSinglestrandedStableunstableEasytomanipulateMoredifficulttomanipulateNeedtobetranscribedintoRNAtomakeaproteinCanbedirectlyusedtomakeaproteincDNAAcDNAorcomplementaryDN38cDNAsynthesisTTTTTTTTTFirststrandNicktranslationcDNAlibrarycDNAsynthesisTTTTTTTTTFirsts39EcoRIGenomicLibraryInfectcellsEcoRIGenomicLibraryInfectce40AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAcDNAsynthesisInfectcellscDNAlibraryAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA41包含的cDNA克隆数目较少课件42GenomiclibrarycDNAlibraryGenomicDNAmRNASourceSpeciesorstrainsSpeciesorstrainsTissuesDevelopmentalstagesVariation12k--20k0.2k--6kInsertsizeEqualCorrelatewithexpressionlevelRepresentationOnlyoneExpressionvs.nonexpressionTypeDNADNAorantibodyorproteinProbeGenestructureInferproteinidentityEncodedproteinInferproteinidentityPurposeGenomiclibrarycDNAlibraryGen43包含的cDNA克隆数目较少课件44EcoRISubcloningofthecDNAinsert+EcoRIEcoRIIsthereabetterwaytosubclonetheinsert?amplifyEcoRISubcloningofthecDNAi45PCR--polymerasechainreactionPCRisoneofthemostpowerfulmolecularbiologytechniques.ItallowsscientiststoamplifyaspecificDNAregioninthetesttubefromextremelytinyamountofDNAsample(evenfromasinglemoleculeofDNA).PCR--polymerasechainreacti46PCR--polymerasechainreactionTaqDNApolymeraseForwardprimerReverseprimerPCR--polymerasechainreacti475’3’5’3’5’3’5’3’5’5’3’3’3’3’5’3’5’5’5’3’3’3’3’5’5’5’5’3’TheidealPCRproductsHeat1stcycleHeat2ndcycle5’3’5’3’5’3’5’3’5’5’3’3’3’3’5’48Heat3’3’5’3’5’5’5’3’3’3’3’5’5’5’5’3’3’5’3’5’3’5’5’3’3’5’5’3’5’3’3’5’5’3’3’5’3’5’3’5’3’5’5’3’5’3’3’5’3rdcycleTheidealPCRproductsHeat3’3’5’3’5’5’5’3’3’3’3’5’5’495’3’3’5’Heat4thcycle5’3’3’5’5’3’3’5’5’3’3’5’5’3’3’5’5’3’3’5’Heat5thcycleHeat6thcycleHeatnthcycle5’3’3’5’2nproducts5’3’3’5’5’3’3’5’5’3’3’5’5’3’3’5’5’3’3’5’5’3’3’5’5’3’3’5’5’3’3’5’5’3’3’5’5’3’3’5’5’3’3’5’5’3’3’5’5’3’3’5’5’3’3’5’Heat4thcycle5’3’3’5’550包含的cDNA克隆数目较少课件51包含的cDNA克隆数目较少课件52CloningoutcDNAinsertbyPCRPCREcoRIEcoRI+EcoRIEcoRIEcoRICloningoutcDNAinsertbyPCR53“ItriedveryhardbutcouldnotisolatethecDNAclonefrom1millioncDNAclones”mRNAlevelisveryverylow“Itriedveryhardbutcouldn54RT-PCRmRNAcDNA(cDNA)nTTTTT--ReverseTranscription+PCRYouneedtoknowatleastpartialsequenceofthecDNAyouwanttoamplify!PCRRT-PCRmRNAcDNA(cDNA)nTTTTT--R55RACEmethodtoisolatefull-lengthcDNARapidamplificationofcDNAendsReversetranscriptaseRNaseHTerminaltransferaseCCCCCOligo(dG)npimerDNApolymeraseCCCCCGGGGGPCRLigatetoTTTTPartialcDNAFull-lengthcDNACCCCCGGGGGCCCCCGGGGGCCCCCGGGGGCCCCCGGGGGCCCCCGGGGGCCCCCGGGGGCCCCCGGGGGCCCCCGGGGGCCCCCGGGGGmRNAistoolongRACEmethodtoisolatefull-le56ExpressionofaclonedgenetostudyitsproteinfunctionsExpressionofaclonedmammaliangeneinmammaliancells Agenomicclone(withintrons)canbeused. AcDNA(withoutintrons)clonecanbeusedExpressionofaclonedgeneinaheterologoussystem a.inbacteria b.inyeast c.ininsectcells ineachcase,cDNAispreferredtobeused,sinceyou don’tknowwhetheramammaliangenecanbespliced correctlyinaheterologoussystem.cDNAmRNAProteinBacterialproteinexpressionvectorsandsystemsExpressionofaclonedgeneto57

-peptideorN-terminusofb-galactosiaseMultiplecloningsitesGGAT

CCCATG