重组DNA与遗传工程

TheFlowofGeneticInformationDNARNAProteinTranslationTranscriptionReverseTranscriptionDNAReplicationRNAReplicationDNADNADNA-dependentDNAPolymerase(DNAReplicase)DNARNADNA-dependentRNAPolymerase(Transcriptase)RNADNARNA-dependentDNAPolymerase(ReverseTranscriptase)RNARNARNA-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,proteinpurificationThetoolkitforDNAtechnology: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：1.cloningvector選殖載體（Bacterialplasmids細菌質體、Bacteriophages噬菌體）2.Shuttlevectors穿梭載體（應用在酵母菌yeast和細菌之上）3.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)Tlymphocyteintravenousinjection2.ADAgenebonemarrowcellDiagnosisofGeneticDiseases遺傳疾病的診斷Cysticfibrosis囊性纖維變性Huntington'sdisease亨氏舞蹈症聚合脢連鎖反應-1聚合脢連鎖反應-2Enzyme•PolymerasebindsinglestrandedDNAatapointadjacenttoadouble-strandedstretch,andbindstodNTPs(deoxynucleotidetriphosphates)ComponentsofPCRreaction:Enzyme(Taq,Vent,Pfu)AppropriatebuffersAmpledNTPsTemplateDNAPrimersCofactors(eg.Magnesium)Thecycle1•Denaturation•94or92℃,30sec/incubation•Halfactivityafter30incubationsat94℃for60seceach.•60-120secforthefirststepThecycle2Annealing•Tm=4(GCbases)+2(ATbases)•30-60sec/incubationThecycle3•Extension•72℃,500bp/30sec;500-1500/60secCyclesExample:DenatureAnnealingExtensioncycles944572594557230-35945572(10min)1ConditionoftemperatureDNA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.Modifypathogen'sgenomePharmaceuticalproducts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RFLPDNAfingerprintingDNA電泳DNA指紋辨識Environment環保Detoxificationmining請舉例說明運用在生物科技中的物理、化學基本技術和原理參考習題：1.怎樣從細胞中分離出我們想要研究的基因片段？2.怎樣鑑定所分離的基因是我們想要找的正確目標？3.如何在試管中複製DNA？4.怎樣證明王小姐未來所生的小孩是鄭先生的？5.請提一個可能解決現代環境問題的遺傳工程技術。