武大分子生物学课件application1(精)

1武大分子生物学课件application1(精)2vectorsCloningvectors:

克隆载体

tocloneageneinavectorExpressionvectors:

表达载体

toexpressagenefromavectorIntegrationvectors:

整合载体

tointegrateageneinagenomethroughavector3

Cloningvectors1Plasmidvecters2Bacteriophagevectors3Cosmids&BACs4Eukaryoticvectors4Cloningvectors:

allowingtheexogenousDNAtobeinserted,stored,andmanipulatedmainlyatDNAlevel.expressionvectors:

allowingtheexogenousDNAtobeinserted,stored,andexpressed.5Containsanoriginofreplication,allowingforreplicationindependentofhost’sgenome.ContainsSelectivemarkers:Selectionofcellscontainingaplasmid

twinantibioticresistance

blue-whitescreeningContainsamultiplecloningsite(MCS)Easytobeisolatedfromthehostcell.Aplasmidvectorforcloning6Ampicillinresistant?yes

yesTetracyclineresistant?NoyesBXBBBXAmproriAmprTcroripBR322AmprTcrori-ScreeningbyinsertionalinactivationofaresistancegeneTwinantibioticresistancescreening7Replicaplating:transferofthecoloniesfromoneplatetoanotherusingabsorbentpadorVelvet(绒布).transferofcolonies+ampicillin+ampicillin+tetracyclinethesecolonieshavebacteriawithrecombinantplasmid8BluewhitescreeningAmproripUC18(3kb)MCS

(Multiplecloningsites,

多克隆位点）LacpromoterlacZ’ScreeningbyinsertionalinactivationofthelacZgeneTheinsertionofaDNAfragmentinterruptstheORFoflacZ’gene,resultinginnon-functionalgeneproductthatcannotdigestitssubstratex-gal.9Recreatedvector:bluetransformantsRecombinantplasmidcontaininginsertedDNA:whitetransformantsRecreatedvector(noinsert)Recombinantplasmid(containinsert)back10MultiplecloningsitesMultiplerestrictionsitesenabletheconvenientinsertionoftargetDNAintoavectorAmproripUC18(3kb)MCS

(Multiplecloningsites,

多克隆位点）LacpromoterlacZ’…ACGAATTCGAGCTCGGTACCCGGGGATCCTCTAGAGTCGACCTGCAGGCATGCA….ThrAsnSerSerValProGlyAspProLeuGluSerThrCysArgHisAlaSer…EcoRISacIKpnISmaIXmaIBamHIXbaISalIHincIIAccIPstISphILacZ11AplasmidvectorforgeneexpressionExpressionvectors:

allowingtheexogenousDNAtobeinserted,storedandexpressed.PromoterandterminatorforRNAtranscriptionarerequired.IntactORFandribosomalbindingsites(RBS)arerequiredfortranslation.Include：(1)bacterialexpressionvectors,(2)yeastexpressionvectors,(3)mammalianexpressionvector12T7promoterRBSStartcodonMCSTranscriptionterminatorAmproriT7expressionvectorAnbacterialexpressionvector13MCSAyeastexpressionvector14BacteriophagevectorTwoexamples:λphage

bacteriophageλλreplacementvectorM13phageM13phagevectorCloninginM13Hybridplasmid-M13vectors15virusesthatcaninfectbacteria.48.5kbinlengthLinearorcirculargenome(cosends)Lyticphase(Replicateandrelease)Lysogenicphase(integrateintohostgenome)λphage16Analysisofeukaryoticgenesandthegenomeorganizationofeukaryotesrequiresvectorswitha

largercapacityforclonedDNAthanplasmidsorphage.Humangenome(3x109bp):largegenomeandlargegenedemandvectorswithalargesizecapacity.CloninglargeDNAfragments(EukaryoticGenomeproject)GenomiclibraryVScDNAlibrary17CosmidvectorsUtilizingthepropertiesofthephagelcossitesinaplasmidvector.AcombinationoftheplasmidvectorandtheCOSsitewhichallowsthetargetDNAtobeinsertedintothelhead.Theinsertcanbe37-52kb18DigestionLigationC)PackagingandinfectFormationofacosmidclone19YACvectorsAccommodatesgenomicDNAfragmentsofmorethan1Mb,andcanbeusedtoclonetheentirehumangenome,butnotgoodinmappingandanalysis.(yeastartificialchromosome)20EssentialcomponentsofYACvectors:Centromers(CEN),telomeres(TEL)andautonomousreplicatingsequence(ARS)forproliferationinthehostcell.amprforselectiveamplificationandmarkerssuchasTRP1andURA3foridentifyingcellscontainingtheYACvectorinyeastcells.Recognitionsitesofrestrictionenzymes(e.g.,EcoRIandBamHI)21YACCloning22BACvectors细菌人工染色体1.MorestablethanYAC2.Capacityis300-350kb3.Onetotwocopiesineachcell4.Easytohandle5.Morepopularingenomicmapping23I1Genomiclibraries

I1-1RepresentativegenelibrariesI1-2SizeoflibraryI1-3GenomicDNAI1-4VectorsGenelibrariesandscreening24Genelibrary:

acollectionofdifferentDNAsequencefromanorganism,eachofwhichhasbeenclonedintoavectorforeaseofpurification,storageandanalysis.GenomiclibrariescDNAlibraries

Genelibrary

(madefromgenomicDNA)(madefromcDNA-copyofmRNA)I1Genomiclibraries

25I1-1Representativegenelibraries---ContainalltheoriginalsequencesCertainsequenceshavenotbeencloned. Example:repetitivesequenceslackingrestrictionsites

2.LibrarydoesnotcontainsufficientclonesMissingoriginalsequenceToolongforthevectorusedI1Genomiclibraries

26I1-2Sizeoflibrary

(ensureenoughclones)

mustcontainacertainnumberofrecombinantsfortheretobeahighprobabilityofitcontaininganyparticularsequenceTheformulatocalculatethenumberofrecombinants:N=ln(1-P)ln(1-f)

P:desiredprobability

f:thefractionofthegenomeinoneinsertI1Genomiclibraries

27Forexample: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==6.9×105

ln(1-0.99)ln[1-(2×104/3×109)]Thesevaluesexplainwhyitispossibletomakegoodgenomiclibrariesfromprokaryotesinplasmidswheretheinsertsizeis5-10kb,asonlyafewthousandrecombinantswillbeneeded.

I1Genomiclibraries

28I1-3GenomicDNAlibrariesPurifygenomicDNA

FragmentthisDNA:physicalshearingandrestrictionenzymedigestion

eukaryotes

prokaryotesClonethefragmentsintovectorsI1Genomiclibraries

29Tomakearepresentativegenomiclibraries,genomicDNAmustbepurifiedandthenbrokenrandomlyintofragmentsthatarecorrectinsizeforcloningintothechosenvector.PurificationofgenomicDNA:

Prokaryotes

:extractedDNAdirectlyfromcells

remove

protein,lipidsandotherunwantedmacro-moleculesbyproteasedigestionandphaseextraction.Eukaryotes

:prepare

cellnucleiI1Genomiclibraries

30BreakDNAintofragmentsrandomly:Physicalshearing:

pipeting,mixingorsonicaion

Restrictionenzymedigestion:

partialdigestionispreferredtogetagreaterlengthsofDNAfragments.I1Genomiclibraries

31Sau3A:5’-/GATC-3’,lessselectivity BamH1:5’-G/GATCCSelectionofrestrictionenzymeEndsproduced(stickyorblunt)& ThecleavedendsofthevectortobeclonedWhethertheenzymeisinhibitedbyDNAmodifications(CpGmethylationinmammalsTimeofdigestionandratioofrestrictionenzymetoDNAisdependentonthedesiredinsertsizerange.I1Genomiclibraries

32I1-4VectorsAccordingtogenome’ssize,wecanselectapropervectortoconstructalibrary.Vectors

PlasmidphageλcosmidYAC

insert(kb)

523451000ThemostcommonlychosengenomiccloningvectorsareλrelacementvectorswhichmustbedigestedwithrestrictionenzymestoproducethetwoλendfragmentorλarmsbetweenwhichthegenomicDNAwillbedigestedI1Genomiclibraries

33coscosLong(left)armshort(right)armExogenousDNA(~20-23kb)λphagevectorincloningcoscosLong(left)armshort(right)armExogenousDNA(~20-23kb)34λreplacementvectorcloning2.Packing

withamixtureofthephagecoatproteinsandphageDNA-processingenzymes3.

InfectionandformationofplaquesLibraryconstructedLigation0.preparationofarmandgenomicinserts35

I2cDNAlibrariesI2-1mRNAisolation,purificationI2-2ChecktheRNAintegrity

I2-3FractionateandenrichmRNA

I2-4SynthesisofcDNA

I2-5TreatmentofcDNAends

I2-6LigationtovectorGenelibrariesandscreening36cDNAlibrariesNocDNAlibrarywasmadefromprokaryoticmRNA.

ProkaryoticmRNAisveryunstableGenomiclibrariesofprokaryotesareeasiertomakeandcontainallthegenomesequences.I2cDNAlibraries37cDNAlibrariesareveryusefulforeukaryoticgeneanalysis

Condensedproteinencodedgenelibraries,havemuchlessjunksequences.cDNAshavenointronsgenescanbeexpressedinE.colidirectlyAreveryusefultoidentifynewgenesTissueorcelltypespecific(differentialexpressionofgenes)cDNAlibrariesI2cDNAlibraries38I2-1mRNAisolationMosteukaryoticmRNAsarepolyadenylatedattheir3’endsoligo(dT)canbeboundtothepoly(A)tailandusedtorecoverthemRNA.AAAAAAAAAAn5’capI2cDNAlibraries39I2cDNAlibraries401.TraditionallymethodwasdonebypassapreparationoftotalRNAdownacolumnofoligo(dT)-cellulose2.Morerapidprocedureistoaddoligo(dT)linkedtomagneticbeadsdirectlytoacelllysateand‘pullingout’themRNAusingastrongmagnet3.AlternativerouteofisolatingmRNAislysingcellsandthenpreparingmRNA-ribosomecomplexesonsucrosegradientsThreemethodstoisolatemRNA.I2cDNAlibraries41MakesurethatthemRNAisnotdegraded.Methods:TranslatingthemRNA:usecell-freetranslationsystemaswheatgermextractorrabbitreticulocytelysatetoseeifthemRNAscanbetranslatedAnalysisthemRNAsbygelelctrophoresis:useagaroseorpolyacrylamidegels

I2-2CheckthemRNAintegrityI2cDNAlibraries42I2-3CloningtheparticularmRNAsIsusefulespeciallyoneistryingtocloneaparticulargenerathertomakeacompletecDNAlibrary.Fractionateonthegel:

performedonthebasisofsize,mRNAsoftheinterestedsizesarerecoveredfromagarosegelsEnrichment:

carriedoutbyhybridizationExample:clonethehormoneinducedmRNAs(substratedcDNAlibrary)I2cDNAlibraries43I2-4SynthesisofcDNA:Firststandsynthesis:

materialsasreversetranscriptase,primer(oligo(dT)orhexanucleotides)anddNTPs(Fig1.1)

Secondstrandsynthesis:

bestwayofmakingfull-lengthcDNAisto‘tail’the3’-endofthefirststrandandthenuseacomplementaryprimertomakethesecond.(Fig2.1)

I2cDNAlibraries445’mRNA

AAAAA-3’HO-TTTTTP-5’5’ReversetranscriptaseFourdNTPsAAAAA-3’TTTTTP-5’mRNA

mRNA

cDNAcDNAcDNADuplexcDNAAAAAA-3’TTTTTP-5’TTTTTP-5’3’3’-CCCCCCCTerminaltransferasedCTPAlkali(hydrolyaesRNA)PurifyDNAoligo(dG)KlenowpolymeraseorreverseTranscriotaseFourdNTPs5’-pGGGG-OH5’3’-CCCCCCC5’-pGGGG3’-CCCCCCCTTTTTP-5’-3’Fig1.1ThefirststrandsynthesisI2cDNAlibraries455’-pGGGG3’-CCCCCCCHO-CCGAATTCGGGGGG3’-GGCTTAAGCCCCCC5’-pAATTCGGGGGGTTTTTGGCTTAAGCC-OHCCGAATTCGG-3’3’-CCCC3’-CCCCCCC3’-CCC5’-pGGGG5’-pGGGGTTTTTp-5’-3’TTTTTp-5’TTTTTp-5’-3’-3’TTTTTGGCTTAAp-5’HO-CCG/AATTCGG-3’3’-GGCTTAA/GCC-OHCCG-3’DuplexcDNASinglestrand-specificnucleaseKlenowpolymerasetreatwithE.coRImethylaseAddE.colRIlinkersusingT4DNAligaseE.colRIdigestionLigatetovectorandtransfomFig2.1Secondstrandsynthesis46I2-5TreatmentofcDNAendsBluntandligationoflargefragmentisnotefficient,sowehavetousespecialacidlinkerstocreatestickyendsforcloning.Theprocess:Moveprotruding3’-ends(strand-specialnuclease)Fillinmissing3’nucleotide

(klenowfragmentofDNApolyIand4dNTPs)Ligatetheblunt-endandlinkers(T4DNAligase)Restrictionenzymedigestion

(E.coRI)TailingwithterminaltransferaseorusingadaptormoleculesI2cDNAlibraries47I2-6Ligationtovector

AnyvectorswithanE.coRIsitewouldsuitableforcloningthecDNA.Theprocess:

DephosphorylatethevectorwithalkalinephosphataseLigatevectorandcDNAwithT4DNAligase(plasmidorλphagevector)I2cDNAlibraries48I3ScreeningproceduresI3-1ScreeningI3-2ColonyandplaquehybridizationI3-3ExpressionscreeningI3-4HybridarrestandreleaseI3-5Chromosomewalking(repeatscreening)Genelibrariesandscreening49I3-1ScreeningTheprocessofidentifyingoneparticularclonecontainingthegeneofinterestfromamongtheverylargenumberofothersinthegenelibrary.Usingnucleicacidprobetoscreenthelibrarybasedonhybridizationwithnucleicacids.Analyzetheproteinproduct.I3Screeningprocedures50Screeninglibraries

HybridizationtoidentifytheinterestedDNAoritsRNAproductRadiolabeledprobeswhichiscomplementarytoaregionoftheinterestedgeneProbes:

AnoligonucleotidederivedfromthesequenceofaproteinproductofthegeneADNAfragment/oligofromarelatedgeneofanotherspeciesBlottingtheDNAorRNAonamembraneHybridizethelabeledprobewithDNA

membrane(Southern)orRNA(Northern)membraneSearchingthegenesofinterestinaDNAlibraryI3Screeningprocedures51I3-2ColonyandplaquehybridizationTransfertheDNAintheplaqueorcolonytoaNylonornitrocellulosemembranePhageDNAbindtothemembranedirectlyBacterialcoloniesmustbelysedtoreleaseDNAonthemembranesurface.Hybridization(inasolutionContainingNucleicacidprobe)Washtoremoveunhybri-dizationprobeandvisualize

X-rayfilm(radio-activelylabeled)antibodyorenzyme(modifiednucleotidelabeledLineupthehybridizatedregionorrepeatedhybridization(Alkalitreatment)I3Screeningprocedures52TransfertonitrocelluloseornylonmembraneDenatureDNA(NaOH)BakeontomembraneProbewith32p-labledDNAcomplementarytogeneofinterestExposetofilmSelectpositivefromma