基因工程基因文庫的構(gòu)建

Genelibrary(Genomiclibrary)：Acollectionofindependentclonesrepresentingtheentiregenomeofanorganism.Firststep

toisolateatargetDNAsequencefromanorganism’sgenome.

1976L.Clark,J.Carbon

n=ln(1-p)/ln(1-f)n:numbersofrecombinantrequiredinfulllibraryp:probabilityoftargetgeneinlibraryf:rateofaverageinsertionDNAsizestogenomeMammal

3×109bpIfp=99%clonedfragment20kbf=20kb/3×109

n=690773.2E.coli

4639221bpp=99%f=20kb/4600kbn=1056.9p=99%f=10kb/4600kbn=2116

cDNAlibrary:AcollectionofindependentclonesrepresentingtheentirecDNAreverselytranscriptedfrommRNAinaspecifictissueofanorganisminaspecificphage.BacterialcoloniesconsistingofgenomicDNAlibraryChoosingvectorStepsoflibraryconstructionPlasmid<10kbPhage0-23kbCosmid~45kbBAC~100kb（250-300kb?）YAC~300kb-1.2MbSUP4基因：酵母細胞Trp-tRNA基因的赭色突變抑制基因，含有克隆位點。在發(fā)生Ade2-赭色突變的宿主細胞中，如果沒有外源基因的插入，則突變基因表達受抑制受體菌為Ade+，形成白色菌落；當(dāng)有外源基因插入時，SUP4表達將被阻斷，抑制作用被消除，受體菌為Ade-，菌落為紅色。Cosmid

Halfofphage.If700000,cosmid350000(菌落數(shù))CosmidisusuallyappliedinlargesizeDNAcloning,butitismuchmoredifficultthanphage，becausethereisnomuchexperience.（pJB8andc2RBaremoreusedingenelibraryconstruction)phagevector1970’s,likeCharon4Aandgt-WES，15～20kbPresently,

EMBLlines,like2001，DASH，Charon38-40，GEM-11etc.(replacementvectors),20-24kbBasicprinciples

inchoosing

vectorEasilyrecoverforeignDNAfrom;

Easilyprepare2arms;Availabilityofvectorsequence,REmapping;Ambermutation(琥珀突變)invectorarmsasspecialscreeningmodel;Activegamgeneinrecombinants；Chisequenceinvectorarms.Gam蛋白與核酸外切酶(ExonucleaseV)結(jié)合，抑制了宿主的RecBCD的核酸外切酶V活性，使得外來DNA分子不會被切割降解。Enterlyticcyclereplicationgiveriseto50genome,thenrollingcyclereplicatingtoproduceconcatemer,andfinallyitiscutandpackagedintoparticles.recB/recC/recDproducts,controllingrecombination,andhaveactivitiesofExnuleaseV,inhibitingDNAreplicationtransfertorollingcycle.gamgene

products

bindandinactivateexnucleaseV.Ifgam-,recAproductisrequiredtopackage,butformsmallplaques(小噬斑),sothehostmustberecA+.ButrecA+maycauseotherrecombination,thusweusevectorrecA-/gam+,torealizeproliferationinrecA-host.Charon32-35,40chisite：8bp,innaturalE.Coli,1copyofevery5-10Kb,butdon’texistinwildλDNA,notrequiredforrecombination.red-gam-，foreigninsertionDNAwithchisitewouldformclearplaques.Ifnot,formsmallplaques.BecauseofuncertaintyofchisiteinforeignDNA,therecombinantsformlargeandsmallplaques（addingchisiteinvector,suchas2001,DASH,F1X,EMBLlines）野生型以及red-gam-噬菌體之蝕菌斑表型噬菌體寄主菌Rec+recA-recA-recBC-P2溶源細菌red-gam-red-gam-χ+非常小小+--++++微小小-LibraryconstructionbyphageTotalDNAextractionInitiallengthofDNA

≥4timeofclonedDNA;otherwisetoofewefficientfragments.

Preparationofvectorarms

buyingpreparationandpurification(gradientcentrifugationwithsucroseorNaCl)REdigestionBamHI/EMBL

XhoI/GEM-11DigestionofgenomeDNA

generallybySau3AIrecover20-24kb（agaroseorgradientcentrifugation)REdigestionproducestickyends,butwithgreatrandom,sothenumberoflibraryshouldbelargerthanthetheoreticvalue.Physicalmethods,splicingDNAbysonication.LigationandpackagingLongconcatemer

，packagingparticles（storein4℃for6months）.Particles

≥100-1000timesofnakedDNA

intransfectionrate.Dephosphoration

ofvectorincreaseligationandpackagingefficiencyDoubleREdigestionofvectorEMBLlines，2001，XDASH，Charson40,35,34）EMBL3A:SalIBamHIEcoRIE1B1-S1Inligationreaction

phage108pfu/gDNADigestionbyBamHIandEcoRIandremovementofsmallfragment,decreasethenumberofrecombinantstoonehundredth.Combinationwithothermethods(suchasSpiscreening)onethousandthtoonehundredth

Partialfulfillmentof3’recessedendifgenomeDNA-GATC--CTAG--GATC--CTAG-Sau3AI-GAGATC--CTAGAG-KlenowdATP/dGTP-CTCGAG--GAGCTC-XhoIKlenowdCTP/dTTP-CTCTCGAG--GAGCTCTC-互補GEM-11GenomeDNAVector

LysisplaquesoflambdaphageonE.colibacteria

PlaquenumberindifferentplatedishesAmplificationandstore

E.coliFormplaquesIdentificationofrecombinantwithforeignDNACharacterizationofrecombinantswithDNACharacterizationofrecombinants

insitublotofplaques(噬斑原位雜交)BlotscreeningwithprobePurificationAnalyzing

Southernblot,REcut,sequencing,immunologicalmethodConstructionofsubgenomiclibraryAtypeoflibrarycontainingspecialpartofgenomeDNA.e.g.plasmidDNA,mitochondriaDNA,specialREDNAfragment.Example:cutgenomicDNAbydifferentREs,Southernblotfoundtargetgenein8.3kbSpeIfragment.recoverthisfragmentforsubgenomiclibraryconstructionIsolatingmRNAConstructionofcDNAlibraryIntegrityofmRNAlargemolecularweightprotein

cell-freetranslationsystemtargetpeptide

immunoprecipitation(免疫沉淀)andSDSmRNAsizes

（500bp～8kb，mainly1.5～2kb）removingsmallfragmentSynthesizingfirststrandofcDNAtheproducedcDNAshouldcorrespondtotheaboverange.

mRNAabundancehigh-abundancemRNA

珠蛋白，免疫球蛋白，卵清蛋白.50-90%

low(rare)…0.5%Causinglargelibrary,anddifficultiestocharacterizationoftargetgeneEnrichmentofmRNAisolationofmRNAbymolecularsizesfirstlydenature,thengradientcentrifugation(sucrose）

invitrotranslationtodetectitsactivityIsolationofcDNAbymolecularsizesrecentlyapplied,especiallyforlargemRNA

ImmunologicalpurificationofpolyribosomeAntibodyagainsttargetpeptideInimmunoaffinitypurification(proteinA-Sepharose)column,monocloneantibodymakepolyribosomebindproteinA-spharosecolumn,EDTAwash,andoligo(dT)enrichmRNA.Only0.01-0.05