重组DNA与遗传工程.ppt

TheFlowofGeneticInformation,DNADNADNA-dependentDNAPolymerase(DNAReplicase)DNARNADNA-dependentRNAPolymerase(Transcriptase)RNADNARNA-dependentDNAPolymerase(ReverseTranscriptase)RNARNARNA-dependentRNAPolymerase(RNAReplicase)DNA合成酶、轉錄酶、反轉錄酶、RNA合成酶,TheCentralDogma分子生物學的中心教條:,遺傳工程（重組DNA),重組DNA技術的條件：,1.基本工具：enzymes2.基因的來源:purifiedDNAorcDNA(complementaryDNA)3.傳輸工具：transformation,infection,electroporation,microinjection,genegun4.篩選技術:specificantibodyscreening,hybridization(radioactiveisotopeorfluorescentagentlabel),5.基因表現：expressionvectors,proteinpurification,ThetoolkitforDNAtechnology:,1.Restrictionenzymes限制脢:DanielNathans,HamiltonSmithandWernerArber(1978NobelPrize)2.DNAvector載體3.Hostorganisms宿主,Restrictionenzymes限制脢:,1.DanielNathans,HamiltonSmithandWernerArber(1978NobelPrize),DNAligase黏合脢,互補DNA的製造,製備cDNA,分離特定基因的方法,DNAvector載體：,cloningvector選殖載體（Bacterialplasmids細菌質體、Bacteriophages噬菌體）Shuttlevectors穿梭載體（應用在酵母菌yeast和細菌之上）Retroviruses反轉錄病毒（藉由感染的方式）,大腸桿菌的質體,質體的放大圖,細菌間質體基因的轉移,Hostorganisms宿主:,(1)Bacteria:優點：繁殖快速、容易操作，缺點：轉錄和轉譯調控機制與真核細胞不同(2)Yeast:優點：真核細胞的調控，缺點：不易操作(3)Animalorplantcell:優點：高等生物細胞的表現，缺點：不易接納外來DNA,重組DNA的方法,遺傳工程的應用,一、DiagnosisofGeneticDiseases遺傳疾病的診斷二、HumanGeneTherapy人類基因療法三、Vaccines疫苗四、Pharmaceuticalproducts藥品製造五、Forensic法醫六、Environment環保七、Agricultural農業,GMO：基因重組生物,轉殖生長基因的老鼠,Agricultural農業,Transgenicanimalsnenlargevaluablepartsnresistanttopathagensnimprovedagriculturalproducts,suchaschangeAAscompositionsTransgenicplantsTiplasmid(Tumor-inducingplasmid)Agrobacteriumtumefaciens根瘤土壤桿菌Antisensegenes反義基因抑制番茄成熟,利用細菌轉殖植物細胞的方法,轉殖致瘤基因的植物,抗除草劑基因的棉花,螢光煙草：轉殖作物,HumanGeneTherapy人類基因療法,SCID(severecombinedimmunodeficiency)重度混合免疫不全症1.retroviralvector+ADA(腺甘脫胺脢)Tlymphocyteintravenousinjection2.ADAgenebonemarrowcell,DiagnosisofGeneticDiseases遺傳疾病的診斷,Cysticfibrosis囊性纖維變性Huntingtonsdisease亨氏舞蹈症,聚合脢連鎖反應-1,聚合脢連鎖反應-2,Enzyme,PolymerasebindsinglestrandedDNAatapointadjacenttoadouble-strandedstretch,andbindstodNTPs(deoxynucleotidetriphosphates),ComponentsofPCRreaction:,Enzyme(Taq,Vent,Pfu)AppropriatebuffersAmpledNTPsTemplateDNAPrimersCofactors(eg.Magnesium),Thecycle1,Denaturation94or92,30sec/incubationHalfactivityafter30incubationsat94for60seceach.60-120secforthefirststep,Thecycle2,AnnealingTm=4(GCbases)+2(ATbases)30-60sec/incubation,Thecycle3,Extension72,500bp/30sec;500-1500/60sec,Cycles,Example:DenatureAnnealingExtensioncycles944572594557230-35945572(10min)1,Conditionoftemperature,DNA自動定序儀,DNA自動定序儀,autosequencer,脂蛋白與膽固醇,HDLs:高密度脂蛋白將膽固醇運送到肝臟加以代謝LDLs:低密度脂蛋白將膽固醇運送到利用或儲存細胞,膽固醇所引起的冠狀動脈阻塞,Vaccines疫苗,Proteincoatofvirusorbacteria.Modifypathogensgenome,Pharmaceuticalproducts藥品製造,Insulin胰島素Growthhormones生長激素EPO(erythropoietin)紅血球生長素Interferon（干擾素）,Forensic法醫,RFLPDNAfingerprinting,DNA電泳,DNA指紋辨識,Environment環保,Detoxific