重组DNA与遗传工程

Click to edit Master title style,Click to edit Master text styles,Second level,Third level,Fourth level,Fifth level,*,The Flow of Genetic Information,DNA,RNA,Protein,Translation,Transcription,Reverse Transcription,DNA Replication,RNA Replication,DNA, DNADNA-dependent DNA Polymerase (DNA Replicase),DNA, RNADNA-dependent RNA Polymerase (Transcriptase),RNA DNARNA-dependent DNA Polymerase (Reverse Transcriptase),RNA, RNARNA-dependent RNA Polymerase (RNA Replicase),DNA,合成酶、轉錄酶、反轉錄酶、,RNA,合成酶,The Central Dogma ,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：,enzymes,2.,基因的來源:,purified DNA or cDNA (complementary DNA),3.,傳輸工具：,transformation, infection, electroporation, microinjection, gene gun,4.,篩選技術:,specific antibody screening, hybridization (radioactive isotope or fluorescent agent label),5.,基因表現：,expression vectors, protein purification,The toolkit for DNA technology:,1.,Restriction enzymes,限制脢:,Daniel Nathans, Hamilton Smith and Werner Arber (1978 Nobel Prize),2.DNA vector,載體,3. Host organisms,宿主,Restriction enzymes,限制脢:,1.,Daniel Nathans, Hamilton Smith and Werner Arber (1978 Nobel Prize), DNA ligase,黏合脢,互補,DNA,的製造,製備,cDNA,分離特定基因的方法,DNA vector,載體：,cloning vector,選殖載體（,Bacterial plasmids,細菌質體、,Bacteriophages,噬菌體）,Shuttle vectors,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,Host organisms,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),Animal or plant cell:,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、,Diagnosis of Genetic Diseases,遺傳疾病的診斷,二、,Human Gene Therapy,人類基因療法,三、,Vaccines,疫苗,四、,Pharmaceutical products,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,Transgenic animals,n enlarge valuable parts,n resistant to pathagens,n improved agricultural products, such as change AAs compositions,Transgenic plants,Ti plasmid (Tumor-inducing plasmid),Agrobacterium tumefaciens,根瘤土壤桿菌,Antisense genes,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,Human Gene Therapy,人類基因療法,SCID (severe combined immunodeficiency),重度混合免疫不全症,1.,retroviral vector + ADA(,腺甘脫胺脢),T lymphocyte,intravenous injection,2. ADA gene,bone marrow cell,Diagnosis of Genetic Diseases,遺傳疾病的診斷,Cystic fibrosis,囊性纖維變性,Huntingtons disease,亨氏舞蹈症,聚合脢連鎖反應-1,聚合脢連鎖反應-2,Enzyme,Polymerase bind single stranded DNA at a point adjacent to a double-stranded stretch, and binds to dNTPs (deoxynucleotide triphosphates),Components of PCR reaction:,Enzyme (Taq, Vent, Pfu),Appropriate buffers,Ample dNTPs,Template DNA,Primers,Cofactors (eg. Magnesium),The cycle 1,Denaturation,94 or 92, 30 sec/incubation,Half activity after 30 incubations at 94 for 60 sec each.,60-120 sec for the first step,The cycle 2,Annealing,Tm = 4 (GC bases) + 2 (AT bases),30-60 sec/incubation,The cycle 3,Extension,72, 500bp/30 sec; 500-1500/60 sec,Cycles,Example:,DenatureAnnealingExtensioncycles,9445725,94557230-35,945572 (10 min) 1,Condition of temperature,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,Protein coat of virus or bacteria.,Modify pathogens genome,Pharmaceutical products,藥品製造,Insulin,胰島素,Growth hormones,生長激素,EPO (erythropoietin),紅血球生長素,Interferon （,干擾素）,Forensic,法醫,RFLP,DNA fingerprinting,DNA,電泳,DNA,指紋辨識,Environment,環保,Detoxification,mining,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：,怎樣從細胞中分離出我們想要研究的基因片段？,怎樣鑑定所分離的基因是我們想要找的正確目標？,如何在試管中複製,DNA？,怎樣證明王小姐未來所生的小孩是鄭先生的？,請提一個可能解決現代環境問題的遺傳工程技術。,