基因克隆-表达及功能研究(Gene-cloning--expression-and-function

Genecloning,expressionandfunctionalstudy基因克隆，表达及功能研究vectorsCloningvectors:克隆载体tocloneageneinavectorExpressionvectors:表达载体toexpressagenefromavectorIntegrationvectors:整合载体tointegrateageneinagenomethroughavectorCloningvectors1Plasmidvecters2Bacteriophagevectors3Cosmids&BACs4EukaryoticvectorsCloningvectors:allowingtheexogenousDNAtobeinserted,stored,andmanipulatedmainlyatDNAlevel.expressionvectors:allowingtheexogenousDNAtobeinserted,stored,andexpressed.1.Containsanoriginofreplication,allowingforreplicationindependentofhost’sgenome.2.ContainsSelectivemarkers:Selectionofcellscontainingaplasmidtwinantibioticresistanceblue-whitescreening3.Containsamultiplecloningsite(MCS)4.Easytobeisolatedfromthehostcell.AplasmidvectorforcloningAmpicillinresistant?yesyesTetracyclineresistant?NoyesBXBBBXAmproriAmprTcroripBR322AmprTcrori-ScreeningbyinsertionalinactivationofaresistancegeneTwinantibioticresistancescreeningReplicaplating:transferofthecoloniesfromoneplatetoanotherusingabsorbentpadorVelvet(绒布).transferofcolonies+ampicillin+ampicillin+tetracyclinethesecolonieshavebacteriawithrecombinantplasmidBluewhitescreeningAmproripUC18(3kb)MCS(Multiplecloningsites,多克隆位点）LacpromoterlacZ’ScreeningbyinsertionalinactivationofthelacZgeneTheinsertionofaDNAfragmentinterruptstheORFoflacZ’gene,resultinginnon-functionalgeneproductthatcannotdigestitssubstratex-gal.Recreatedvector:bluetransformantsRecombinantplasmidcontaininginsertedDNA:whitetransformantsRecreatedvector(noinsert)Recombinantplasmid(containinsert)backMultiplecloningsitesMultiplerestrictionsitesenabletheconvenientinsertionoftargetDNAintoavectorAmproripUC18(3kb)MCS(Multiplecloningsites,多克隆位点）LacpromoterlacZ’…ACGAATTCGAGCTCGGTACCCGGGGATCCTCTAGAGTCGACCTGCAGGCATGCA….ThrAsnSerSerValProGlyAspProLeuGluSerThrCysArgHisAlaSer…EcoRISacIKpnISmaIXmaIBamHIXbaISalIHincIIAccIPstISphILacZAplasmidvectorforgeneexpressionExpressionvectors:allowingtheexogenousDNAtobeinserted,storedandexpressed.1.PromoterandterminatorforRNAtranscriptionarerequired.2.IntactORFandribosomalbindingsites(RBS)arerequiredfortranslation.3.Include：(1)bacterialexpressionvectors,(2)yeastexpressionvectors,(3)mammalianexpressionvectorT7promoterRBSStartcodonMCSTranscriptionterminatorAmproriT7expressionvectorAnbacterialexpressionvectorMCSAyeastexpressionvectorBacteriophagevectorTwoexamples:1.λphage•bacteriophageλ•λreplacementvector2.M13phage•M13phagevector•CloninginM13•Hybridplasmid-M13vectors•virusesthatcaninfectbacteria.•48.5kbinlength•Linearorcirculargenome(cosends)•Lyticphase(Replicateandrelease)•Lysogenicphase(integrateintohostgenome)λphageAnalysisofeukaryoticgenesandthegenomeorganizationofeukaryotesrequiresvectorswithalargercapacityforclonedDNAthanplasmidsorphage.Humangenome(3x109bp):largegenomeandlargegenedemandvectorswithalargesizecapacity.CloninglargeDNAfragments(EukaryoticGenomeproject)GenomiclibraryVScDNAlibraryCosmidvectors1.Utilizingthepropertiesofthephagecossitesinaplasmidvector.2.AcombinationoftheplasmidvectorandtheCOSsitewhichallowsthetargetDNAtobeinsertedintothehead.3.Theinsertcanbe37-52kbDigestionLigationC)PackagingandinfectFormationofacosmidcloneYACvectorsAccommodatesgenomicDNAfragmentsofmorethan1Mb,andcanbeusedtoclonetheentirehumangenome,butnotgoodinmappingandanalysis.(yeastartificialchromosome)EssentialcomponentsofYACvectors:•Centromers(CEN),telomeres(TEL)andautonomousreplicatingsequence(ARS)forproliferationinthehostcell.•amprforselectiveamplificationandmarkerssuchasTRP1andURA3foridentifyingcellscontainingtheYACvectorinyeastcells.•Recognitionsitesofrestrictionenzymes(e.g.,EcoRIandBamHI)YACCloningBACvectors细菌人工染色体1.MorestablethanYAC2.Capacityis300-350kb3.Onetotwocopiesineachcell4.Easytohandle5.MorepopularingenomicmappingI1GenomiclibrariesI1-1RepresentativegenelibrariesI1-2SizeoflibraryI1-3GenomicDNAI1-4VectorsGenelibrariesandscreeningGenelibrary:acollectionofdifferentDNAsequencefromanorganism,eachofwhichhasbeenclonedintoavectorforeaseofpurification,storageandanalysis.GenomiclibrariescDNAlibrariesGenelibrary(madefromgenomicDNA)(madefromcDNA-copyofmRNA)I1GenomiclibrariesI1-1Representativegenelibraries---Containalltheoriginalsequences1.Certainsequenceshavenotbeencloned.Example:repetitivesequenceslackingrestrictionsites2.LibrarydoesnotcontainsufficientclonesMissingoriginalsequenceToolongforthevectorusedI1GenomiclibrariesI1-2Sizeoflibrary(ensureenoughclones)mustcontainacertainnumberofrecombinantsfortheretobeahighprobabilityofitcontaininganyparticularsequenceTheformulatocalculatethenumberofrecombinants:N=ln(1-P)ln(1-f)P:desiredprobabilityf:thefractionofthegenomeinoneinsertI1GenomiclibrariesForexample:foraprobabilityof0.99withinsertsizesof20kbthesevaluesfortheE.coli(4.6×106bp)andhuman(3×109bp)genomesare:NE.coli==1.1×103ln(1-0.99)ln[1-(2×104/4.6×106)]Nhuman==6.9×105ln(1-0.99)ln[1-(2×104/3×109)]Thesevaluesexplainwhyitispossibletomakegoodgenomiclibrariesfromprokaryotesinplasmidswheretheinsertsizeis5-10kb,asonlyafewthousandrecombinantswillbeneeded.I1GenomiclibrariesI1-3GenomicDNAlibrariesPurifygenomicDNAFragmentthisDNA:physic