重组DNA与遗传工程.pptVIP

The Flow of Genetic Information 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 ? 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） 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  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 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 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 ? 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