分子生物學(xué)：Section I 基因文庫與篩選

SectionI

Genelibrariesandscreening

基因文庫與篩選

I1Genomiclibraries

基因文庫I2cDNAlibrariescDNA文庫I3Screeningprocedures

篩選流程I1Genomiclibraries

基因組文庫I1-1Representativegenelibraries代表性的基因文庫

I1-2SizeoflibraryI1-3GenomicDNAI1-4Vectors載體Genelibrary:

acollection收集ofdifferentDNAsequencefromanorganism,eachofwhichhasbeenclonedintoavectorforeaseofpurification,storageandanalysis.Genomiclibraries基因組文庫cDNAlibrariescDNA文庫

Genelibrary基因文庫

(madefromgenomicDNA)(madefromcDNA-copyofmRNA)I1-1Representativegenelibraries具代表性的基因文庫

---Containalltheoriginal原初sequencesCertainsequenceshavenotbeencloned. Example:repetitivesequences重復(fù)順序lackingrestrictionsites限制性內(nèi)切位點2.LibrarydoesnotcontainsufficientclonesMissingoriginalsequenceToolongforthevectorusedI1-2Sizeoflibrary

(ensureenoughclones)

文庫大?。ù_保足夠的克隆）mustcontainacertainnumberofrecombinantsfortheretobeahighprobability概率可能性ofitcontaininganyparticularsequence特定序列Theformula公式

tocalculatethenumberofrecombinants重組體數(shù)量:N=ln(1-P)ln(1-f)

P:desiredprobability預(yù)期的可能性

f:thefraction分?jǐn)?shù)ofthegenomeinoneinsertForexample: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.I1-3GenomicDNAlibrariesPurifygenomicDNA

FragmentthisDNA:physicalshearing剪切andrestrictionenzymedigestion

eukaryotes

prokaryotesClonethefragmentsintovectorsTomakearepresentativegenomiclibraries,genomicDNAmustbepurifiedandthenbrokenrandomlyintofragmentsthatarecorrectinsizeforcloningintothechosenvector.

PurificationofgenomicDNA:

Prokaryotes

:extractedDNAdirectlyfromcells

remove

protein,lipidsandotherunwantedmacro-moleculesbyprotease蛋白酶

digestionandphaseextraction

分項提取Eukaryotes

:prepare

cellnuclei細(xì)胞核BreakDNAintofragmentsrandomly:隨機打碎DNA成片斷Physicalshearing:物理剪切

ipeting吸管,mixing混和orsonicaion超聲

Restrictionenzymedigestion:

限制性酶切partialdigestion部分酶切ispreferred首選的togetagreaterlengthsofDNAfragments.常用Sau3A:

5’-/GATC-3’,lessselectivity少用BamH1:5’-G/GATCC-3’,Selectionofrestrictionenzyme限制性內(nèi)切酶的選擇Endsproduced(stickyorblunt)& ThecleavedendsofthevectortobeclonedWhether是否

theenzymeisinhibited抑制byDNAmodifications(CpGmethylationinmammals)Timeofdigestionandratio效率ofrestrictionenzymetoDNAisdependentonthedesiredinsertsizerange想得到的插入片斷大小.I1-4Vectors載體

Accordingtogenome’ssize,wecanselectapropervectortoconstructalibrary.Vectors

PlasmidphageλcosmidYAC

insert(kb)

523451000Themostcommonlychosengenomiccloningvectorsareλrelacementvectors替換載體whichmustbedigestedwithrestrictionenzymestoproducethetwoλendfragmentorλarmsbetweenwhichthegenomicDNAwillbedigestedcoscosLong(left)armshort(right)armExogenousDNA(~20-23kb)λphagevectorincloningcoscosLong(left)armshort(right)armExogenousDNA(~20-23kb)λreplacementvectorcloning3.Packing

withamixtureofthephagecoatproteinsandphageDNA-processingenzymes4.

Infectionandformationofplaques噬菌斑Libraryconstructed2.Ligation1.preparationofarmandgenomicinserts

I2cDNAlibrariescDNA文庫I2-1mRNAisolation,purificationI2-2ChecktheRNAintegrity完整性

I2-3Fractionate分級分離andenrichmRNA

I2-4SynthesisofcDNA

I2-5TreatmentofcDNAends

I2-6LigationtovectorcDNAlibrariescDNA文庫NocDNAlibrarywasmadefromprokaryotic原核

mRNA.

ProkaryoticmRNAisveryunstableGenomiclibrariesofprokaryotesareeasiertomakeandcontainallthegenomesequences.cDNAlibrariesareveryusefulforeukaryotic真核

geneanalysis

Condensed濃縮

proteinencodedgenelibraries,havemuchlessjunksequences.cDNAshavenointrons內(nèi)含子

genescanbeexpressedinE.colidirectlyAreveryusefultoidentify識別newgenesTissueorcelltypespecific特異

(differentialexpressionofgenes)cDNAlibrariesI2-1mRNAisolationmRNA的提取

MosteukaryoticmRNAsarepolyadenylatedattheir3’ends

oligo(dT)canbeboundtothepoly(A)tailandusedtorecoverthemRNA.AAAAAAAAAAn5’cap1.TraditionallymethodwasdonebypassapreparationoftotalRNAdownacolumnofoligo寡聚(dT)-cellulose纖維素2.Morerapidprocedure程序

istoaddoligo(dT)linkedtomagneticbeads磁珠

directlytoacelllysate裂解液and‘pullingout’themRNAusingastrongmagnet磁鐵

3.Alternativeroute路線

ofisolatingmRNAislysing裂解

cellsandthenpreparingmRNA-ribosomecomplexesonsucrose蔗糖

gradientsThreemethodstoisolatemRNA提取mRNA的方法MakesurethatthemRNAisnotdegraded.Methods:Translating翻譯

themRNA:usecell-freetranslationsystem無細(xì)胞翻譯系統(tǒng)

aswheatgermextract麥胚抽提液

orrabbitreticulocytelysate兔網(wǎng)織紅細(xì)胞裂解液toseeifthemRNAscanbetranslatedAnalysisthemRNAsbygelelctrophoresis:useagarose瓊脂糖

orpolyacrylamide聚丙烯酰胺gels

I2-2CheckthemRNAintegrity檢查mRNA的完整性I2-3CloningtheparticularmRNAs克隆特定的mRNAIsusefulespeciallyoneistryingtocloneaparticulargenerathertomakeacompletecDNAlibrary.

Fractionate分餾

onthegel:

performedonthebasisofsize,mRNAsoftheinterestedsizesarerecoveredfromagarosegelsEnrichment富集:

carriedoutbyhybridization雜交

Example:clonethehormone激素

induced誘導(dǎo)的mRNAs(substratedcDNAlibrary,扣除cDNA文庫)I2-4SynthesisofcDNA

cDNA的合成Firststandsynthesis:

materials原料asreversetranscriptase反轉(zhuǎn)錄酶,primer引物(oligo(dT)orhexanucleotides六聚核苷酸)anddNTPs

(Fig1.1)

Secondstrandsynthesis:

bestwayofmakingfull-lengthcDNAisto‘tail’加尾the3’-endofthefirststrandandthenuseacomplementaryprimertomakethesecond.

(Fig2.1)

5’mRNA

AAAAA-3’HO-TTTTTP-5’5’Reversetranscriptase反轉(zhuǎn)錄酶

FourdNTPsAAAAA-3’TTTTTP-5’mRNA

mRNA

cDNAcDNAcDNADuplexcDNA（雙鏈cDNA）AAAAA-3’TTTTTP-5’TTTTTP-5’3’3’-CCCCCCCTerminaltransferase末端轉(zhuǎn)移酶

dCTPAlkali堿

(hydrolyaes水解

RNA)PurifyDNAoligo(dG)KlenowpolymeraseorreverseTranscriptaseFourdNTPs5’-pGGGG-OH5’3’-CCCCCCC5’-pGGGG3’-CCCCCCCTTTTTP-5’-3’Fig1.1第一與第二鏈的合成5’-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-specificnuclease單鏈特異性核酸酶KlenowpolymerasetreatwithE.coRImethylase甲基化酶AddE.coRIlinkers接頭

usingT4DNAligaseE.colRIdigestionLigatetovectorandtransfom轉(zhuǎn)化Fig2.1雙鏈cDNA末端的形成及其接頭添加I2-5TreatmentofcDNAendsBluntandligationoflargefragmentisnotefficient,sowehavetousespecialacidlinkers接頭

tocreatestickyendsforcloning.

Theprocess:Moveprotruding突出

3’-ends(strand-specialnuclease)Fillinmissing3’nucleotide

(klenowfragmentofDNApolyIand4dNTPs)Ligatetheblunt-endandlinkers接頭(T4DNAligase)Restrictionenzymedigestion

(E.coRI)Tailing加尾withterminaltransferase末端轉(zhuǎn)移酶

orusingadaptor銜接

moleculesI2-6Ligationtovector

AnyvectorswithanEcoRIsitewouldsuitableforcloningthecDNA.Theprocess:

Dephosphorylate脫磷酸

thevectorwithalkalinePhosphatase堿性磷酸酶

LigatevectorandcDNAwithT4DNAligase(plasmidorλphagevector)I3Screeningprocedures篩選程序I3-1Screening篩選I3-2Colonyandplaquehybridization

克隆子及噬菌斑雜交

I3-3Expressionscreening

表達篩選I3-4Hybridarrestandrelease

雜交扣留與釋放I3-5Chromosomewalking(repeatscreening)

染色體步移（重復(fù)篩選）I3-1ScreeningTheprocessofidentifying識別oneparticularclonecontainingthegeneofinterestfromamongtheverylargenumberofothersinthegenelibrary.

Usingnucleicacidprobe核酸探針toscreenthelibrarybasedonhybridizationwithnucleicacids.Analyzetheproteinproduct.Screeninglibraries

HybridizationtoidentifytheinterestedDNAoritsRNAproductRadiolabeled放射性同位素probeswhichiscomplementarytoaregionoftheinterestedgeneProbes探針:

AnoligonucleotidederivedfromthesequenceofaproteinproductofthegeneADNAfragment/oligofromarelatedgeneofanotherspeciesBlottingtheDNAorRNAonamembraneHybridizethelabeledprobewithDNA

membrane(Southern)orRNA(Northern)membraneSearchingthegenesofinterestinaDNAlibraryI3Colonyandplaquehybridization噬菌斑雜交TransfertheDNAintheplaqueorcolonytoaNylonornitrocellulose硝化纖維membranePhageDNAbindtothemembranedirectlyBacterialcoloniesmustbelysedtoreleaseDNAonthemembranesurface.Hybridization

(inasolutionContainingNucleicacidprobe)Washtoremove

unhybri-dizationprobeandvisualize

X-rayfilm(radio-activelylabeled)antibodyorenzyme(modifiednucleotidelabeledLineupthehybridizatedregionorrepeatedhybridization(Alkalitreatment)Transfertonitrocellulose硝化纖維ornylonmembraneDenatureDNA(NaOH)Bake烤

ontomembraneProbewith32p-labledDNAcomplementarytogeneofinterestExposetofilmSelectpositive陽性

frommasterplateKeepmasterplate原始平板

Screeningbyplaquehybridization用噬菌斑雜交篩選IdentifytheproteinproductofaninterestedgeneProteinactivity蛋白質(zhì)活性Westernblottingusingaspecificantibody專門的抗體I3-3Expressionscreening表達篩選Expressionscreening(1)Iftheinsertsareclonedintoanexpressionsites,itmaybeexpressed.Therefore,wecanscreenfortheexpressedproteins.However,thisscreeningmaymisstherightcloneExample:theEcoRIsiteoflgt11vector.Theinsertedgeneshaveoneinsixchange(1/6)tobeinboththecorrectorientation(2possibilities;

)andreadingframe(threepossibilities;threenucleotidecodeXXX).Expressionscreening(2)Theprocedure

hassimilaritiestotheplaquehybridizationprotocol.‘Plaquelift’(takenbyplacingamembraneonthedishofplaque)

Immersed浸入

inasolutionoftheantibodyDetected

byotherantibodies

RepeatcyclesofscreeningtoisolatepureplaquesAntibodiescanbeusedtoscreentheexpressionlibrary.I3-4HybridarrestandscreenIndividualcDNAclonesorpoolsofclonescanbeusedtohybridizetomRNApreparationHybridarrest:translatethemRNApo