重组DNA与遗传工程ppt课件

1、The Flow of Genetic InformationDNARNAProteinTranslationTranscriptionReverse TranscriptionDNA ReplicationRNA ReplicationDNA DNADNA-dependent DNA Polymerase (DNA Replicase)DNA RNADNA-dependent RNA Polymerase (Transcriptase)RNA DNARNA-dependent DNA Polymerase (Reverse Transcriptase)RNA RNARNA-dependent

2、 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. 根本工具：enzymes2. 基因的來源: purified DNA or cDNA (complementary DNA)3. 傳輸工具：transformation, infection, electroporation, microinjection, gene gun4. 篩選技術: specific antibody screening, hybridization (r

3、adioactive 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, Ham

4、ilton 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穿越載體應用在酵母菌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：繁衍快速、容易操作，缺點：轉錄和轉譯調控機制與

5、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 Tra

6、nsgenic animalsn enlarge valuable partsn resistant to pathagensn improved agricultural products, such as change AAs compositionsTransgenic plantsTi 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

7、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 injection2. 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-2EnzymePolymerase bind single stranded DNA at a point

8、 adjacent to a double-stranded stretch, and binds to dNTPs (deoxynucleotide triphosphates) Components of PCR reaction:Enzyme (Taq, Vent, Pfu)Appropriate buffersAmple dNTPsTemplate DNAPrimersCofactors (eg. Magnesium)The cycle 1Denaturation94 or 92, 30 sec/incubationHalf activity after 30 incubations

9、at 94 for 60 sec each.60-120 sec for the first stepThe cycle 2AnnealingTm = 4 (GC bases) + 2 (AT bases)30-60 sec/incubationThe cycle 3Extension72, 500bp/30 sec; 500-1500/60 secCyclesExample: DenatureAnnealingExtensioncycles944572594557230-35945572 (10 min) 1Condition of temperatureDNA 自動定序儀DNA 自動定序儀

10、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 RFLPDNA fingerprinting DNA電泳DNA指紋辨識Enviro