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重組 DNA 技術的條件：

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),

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重組 DNA 技術的條件：

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  1. 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

  2. 重組DNA的方法

  3. 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

  4. Restriction enzymes限制脢: 1.Daniel Nathans, Hamilton Smith and Werner Arber (1978 Nobel Prize), DNA ligase 黏合脢

  5. 互補DNA的製造

  6. 製備cDNA

  7. 分離特定基因的方法

  8. 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）

  9. 大腸桿菌的質體

  10. 質體的放大圖

  11. 細菌間質體基因的轉移

  12. 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

  13. 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

  14. GMO：基因重組生物

  15. Diagnosis of Genetic Diseases遺傳疾病的診斷 Cystic fibrosis 囊性纖維變性 Huntington's disease 亨氏舞蹈症

  16. 聚合脢連鎖反應-1

  17. 聚合脢連鎖反應-2

  18. Enzyme • Polymerase bind single stranded DNA at a point adjacent to a double-stranded stretch, and binds to dNTPs (deoxynucleotide triphosphates)

  19. Components of PCR reaction: Enzyme (Taq, Vent, Pfu) Appropriate buffers Ample dNTPs Template DNA Primers Cofactors (eg. Magnesium)

  20. 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

  21. The cycle 2 Annealing • Tm = 4 (GC bases) + 2 (AT bases) • 30-60 sec/incubation

  22. The cycle 3 • Extension • 72℃, 500bp/30 sec; 500-1500/60 sec

  23. Cycles Example: Denature Annealing Extension cycles 94 45 72 5 94 55 72 30-35 94 55 72 (10 min) 1

  24. Condition of temperature

  25. DNA 自動定序儀

  26. DNA 自動定序儀

  27. autosequencer

  28. 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

  29. 轉殖生長基因的老鼠

  30. 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

  31. Vaccines疫苗 Protein coat of virus or bacteria. Modify pathogen's genome

  32. 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）

  33. Forensic 法醫 RFLP DNA fingerprinting

  34. DNA電泳

  35. DNA指紋辨識

  36. Environment 環保 Detoxification mining

  37. 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

  38. 轉殖致瘤基因的植物

  39. 抗除草劑基因的棉花

  40. 利用細菌轉殖植物細胞的方法

  41. 螢光煙草：轉殖作物

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