基因的轉(zhuǎn)移與重組體的檢測課件

基因的轉(zhuǎn)移與重組體的檢測PartI:GeneTransferTransduction,TransfectionandTransformationTransduction-transferofbacterialgenefromonebacteriumtoanotherbyabacteriophage.Transfection-theuptakeofDNAbyaeukaryoticcell,followedbytheincorporationofgeneticmarkerspresentintheDNAintothecell’sgenome.Transformation-geneticalterationofanorganismbroughtaboutbytheincorporationofforeignDNAintocells.基因的轉(zhuǎn)移與重組體的檢測Streptococcuspneumoniaestains基因的轉(zhuǎn)移與重組體的檢測TypeColonyMorphologyCapsuleVirulenceReactionwithAntiserumPresentedAgainstAppearanceSizeTypeIISTypeIIISIIRRoughSmallAbsentAvirulentNoneNoneIISSmoothLargePresentVirulentAgglutinationNoneIIIRRoughSmallAbsentAvirulentNoneNoneIIISSmoothLargePresentVirulentNoneAgglutinationStreptococcus

pneumoniae基因的轉(zhuǎn)移與重組體的檢測TransformationInStreptococcuspneumoniae基因的轉(zhuǎn)移與重組體的檢測轉(zhuǎn)化transformation1.制備感受態(tài)細(xì)胞（competentcells）容易吸收外源DNA的細(xì)菌細(xì)胞

Ca2++4℃

甘油（20%）-70℃保存

2.轉(zhuǎn)化感受態(tài)細(xì)胞連接的DNA

4℃30mim42℃90sec熱激（heat-shock）

復(fù)蘇（37℃

培養(yǎng)）

涂平板（Ampr）基因的轉(zhuǎn)移與重組體的檢測CaCl2inducedtransformation基因的轉(zhuǎn)移與重組體的檢測PhageDNAandPackaging基因的轉(zhuǎn)移與重組體的檢測RecombinantCosmidDNAandPackaging基因的轉(zhuǎn)移與重組體的檢測PartII:Selection,screeningand

analysisofrecombinantsSelection:somesortofpressure(e.g.thepressureofanantibiotic)isappliedduringthegrowthofhostcellscontainingrecombinantDNA.Screeningisaprocedurebywhichapopulationofviablecellsissubjectedtosomesortofanalysisthatenablethedesiredsequencestobeidentified.基因的轉(zhuǎn)移與重組體的檢測a.Antibioticresistanceselectioninplasmid基因的轉(zhuǎn)移與重組體的檢測Insertioninactivationamprtet

sinsertDNAamprtetrpBR322AmpicillinTetracycline影?。≧eplicaplate）基因的轉(zhuǎn)移與重組體的檢測AntibioticresistanceandinsertioninactivationinplasmidpBR322基因的轉(zhuǎn)移與重組體的檢測AntibioticresistanceandinsertioninactivationinlacZgene基因的轉(zhuǎn)移與重組體的檢測b.Complementation:CloneandselectionoflacYgene基因的轉(zhuǎn)移與重組體的檢測c.Antibioticresistanceselectionandgeneticcomplementation基因的轉(zhuǎn)移與重組體的檢測d.αcomplementationsystem

InsertioninactivationselectioninplasmidpUC18基因的轉(zhuǎn)移與重組體的檢測Insertioninactivationinthe

αcomplementationsystempUC119ampr-lacZ/insertDNAAmp+IPTG+X-galampr-lacZ/基因的轉(zhuǎn)移與重組體的檢測StructureofX-gal基因的轉(zhuǎn)移與重組體的檢測Lactose5-bromo-4-chloro-3-indolyl-β-D-galactopyranosideβ-galactosidase5,5’-dibromo-4,4’-dichloroindigoDifferentcoloniesintheuseofX-gal基因的轉(zhuǎn)移與重組體的檢測e.Insertioninactivationin

vectorλgt10andCharon16A

基因的轉(zhuǎn)移與重組體的檢測Insertioninactivationinvectorλgt10

基因的轉(zhuǎn)移與重組體的檢測CharonRecombinantPackagingandSelection基因的轉(zhuǎn)移與重組體的檢測f.SpiSelectionsystemforBacteriophagevectorEMBL4

基因的轉(zhuǎn)移與重組體的檢測ColoniesofE.coli基因的轉(zhuǎn)移與重組體的檢測Coloniesandplaques基因的轉(zhuǎn)移與重組體的檢測g.SelectionprinciplefortheTACloning基因的轉(zhuǎn)移與重組體的檢測h.Selectionprinciplefortheyeastartificialchromosome

YAC載體的選擇標(biāo)記主要采用營養(yǎng)缺陷型基因，如色氨酸、亮氨酸和組氨酸合成缺陷型基因trp1、leu2和his3、尿嘧啶合成缺陷型基因ura3等，以及赭石突變抑制基因sup4。與YAC載體配套工作的宿主酵母菌（如AB1380）的胸腺嘧啶合成基因帶有一個(gè)赭石突變ade2-1。帶有這個(gè)突變的酵母菌在基本培養(yǎng)基上形成紅色菌落，當(dāng)帶有赭石突變抑制基因sup4的載體存在于細(xì)胞中時(shí)，可抑制ade2-1基因的突變效應(yīng)，形成正常的白色菌落。利用這一菌落顏色轉(zhuǎn)變的現(xiàn)象，可用于篩選載體中含有外源DNA片段插入的重組子?；虻霓D(zhuǎn)移與重組體的檢測i.Selectionprincipleforthebacterial

artificialchromosome

裝載外源DNA后的重組質(zhì)粒可通過：（1）氯霉素抗性和（2）LacZ基因的α–互補(bǔ)篩選。基因的轉(zhuǎn)移與重組體的檢測j.酶切鑒定：插入片段的大小和方向基因的轉(zhuǎn)移與重組體的檢測j.酶切鑒定：插入片段的大小和方向EXSHEHEEXABCEHSEXDSHE400020007006000AHES/HBS/HHHE/HCDM基因的轉(zhuǎn)移與重組體的檢測k.PurifyingclonedDNA基因的轉(zhuǎn)移與重組體的檢測k.

基因的轉(zhuǎn)移與重組體的檢測l.Identifyingandisolationclonesofinterestsl.

基因的轉(zhuǎn)移與重組體的檢測m.ScreeningalibraryofclonesbycolonyinsituhybridizationtoalabeledprobeCPSF基因在真核表達(dá)載體上的克隆

研究表明,至少6種蛋白復(fù)合體參與前體mRNA3’端加工即切割與多聚腺苷酸化特異性因子(cleavageandpolyadenylationspecificityfactor,CPSF)poly(A)多聚酶(poly(A)polymerase,PAP)切割刺激因子（cleavagestimulationfactor,CstF）切割因子I和II(cleavagefactorIandII,CFImandCFIIm)核內(nèi)多聚腺苷酸結(jié)合蛋白1（polyadenylatebinding-proteinnuclear1,PABPN1）。

其中，CPSF被認(rèn)為在前體mRNA3’端加工過程中起著核心作用，直接與切割/多聚腺苷酸化信號(hào)AAUAAA結(jié)合，在兩個(gè)反應(yīng)中都是必需的?；虻霓D(zhuǎn)移與重組體的檢測切割和多聚腺苷酸化信號(hào)示意圖

USE:upstreamelementsPAS:poly-AsignalsequenceDSE:downstreamelements基因的轉(zhuǎn)移與重組體的檢測C末端與N末端TAP-tagTandemAffinityPurification，TAP基因的轉(zhuǎn)移與重組體的檢測TAP技術(shù)純化蛋白質(zhì)復(fù)合體流程示意圖基因的轉(zhuǎn)移與重組體的檢測切割復(fù)合體模型

基因的轉(zhuǎn)移與重組體的檢測多聚腺嘌呤化復(fù)合體模型

基因的轉(zhuǎn)移與重組體的檢測載體pTRE2hyg的形體結(jié)構(gòu)和多克隆位點(diǎn)基因的轉(zhuǎn)移與重組體的檢測載體pTRE2hyg的形體結(jié)構(gòu)和多克隆位點(diǎn)圖

基因的轉(zhuǎn)移與重組體的檢測載體pTRE2hyg-TAP-tag的形體結(jié)構(gòu)和

多克隆位點(diǎn)圖

基因的轉(zhuǎn)移與重組體的檢測載體pTRE2hyg-TAP-tag-CPSF30K的形體結(jié)構(gòu)和多克隆位點(diǎn)基因的轉(zhuǎn)移與重組體的檢測載體pTRE2hyg-TAP-tag-CPSF73K的形體結(jié)構(gòu)和多克隆位點(diǎn)基因的轉(zhuǎn)移與重組體的檢測載體pTRE2hyg-TAP-tag-CPSF100K的形體結(jié)構(gòu)和多克隆位點(diǎn)基因的轉(zhuǎn)移與重組體的檢測載體pTRE2hyg-TAP-tag-CPSF160K的形體結(jié)構(gòu)和多克隆位點(diǎn)圖

基因的轉(zhuǎn)移與重組體的檢測質(zhì)粒pBS1761的形體結(jié)構(gòu)圖

基因的轉(zhuǎn)移與重組體的檢測質(zhì)粒pUK的形體結(jié)構(gòu)圖

基因的轉(zhuǎn)移與重組體的檢測TAP-tag片段的PCR擴(kuò)增

Primer1：5’CGGGATCCATGGBamHI切點(diǎn)

CAGGCCTTGCGCAAC3’

Primer2：5’GTCGACGGCTAGNheI切點(diǎn)CTTATCGTCATCATCAAGTGC3’

基因的轉(zhuǎn)移與重組體的檢測CPSF基因的PCR擴(kuò)增

CPSF30kPrimer1：

5’GCTAGCCATGCAGGAAATCATCGCC3’NheI切點(diǎn)CPSF73kPrimer1：

5’GCTAGCCATGTCCGCGATTCCCGC3’NheI切點(diǎn)CPSF100kPrimer1：5’GCTAGCCATGACATCTATTATCAAATTAACTA3’

NheI切點(diǎn)CPSF160kPrimer1：5’GCTAGCCATGTA