Download - 重組 DNA 技術的條件:
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DNA 1. enzymes2. : purified DNA or cDNA (complementary DNA)3. transformation, infection, electroporation, microinjection, gene gun4. : specific antibody screening, hybridization (radioactive isotope or fluorescent agent label),5. expression vectors, protein purification
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DNA
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The toolkit for DNA technology: 1.Restriction enzymes: Daniel Nathans, Hamilton Smith and Werner Arber (1978 Nobel Prize) 2.DNA vector3. Host organisms
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Restriction enzymes: 1.Daniel Nathans, Hamilton Smith and Werner Arber (1978 Nobel Prize), DNA ligase
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DNA
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cDNA
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DNA vector cloning vector Bacterial plasmidsBacteriophagesShuttle vectorsyeastRetroviruses
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Host organisms: (1) Bacteria: (2) Yeast: (3) Animal or plant cell: DNA
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Diagnosis of Genetic Diseases Human Gene Therapy Vaccines Pharmaceutical products Forensic Environment Agricultural
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GMO
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Diagnosis of Genetic Diseases Cystic fibrosis Huntington's disease
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EnzymePolymerase bind single stranded DNA at a point adjacent to a double-stranded stretch, and binds to dNTPs (deoxynucleotide triphosphates)
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Components of PCR reaction:Enzyme (Taq, Vent, Pfu)Appropriate buffersAmple dNTPsTemplate DNAPrimersCofactors (eg. Magnesium)
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The cycle 1Denaturation94 or 92, 30 sec/incubationHalf activity after 30 incubations at 94 for 60 sec each.60-120 sec for the first step
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The cycle 2AnnealingTm = 4 (GC bases) + 2 (AT bases)30-60 sec/incubation
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The cycle 3Extension72, 500bp/30 sec; 500-1500/60 sec
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CyclesExample: DenatureAnnealingExtensioncycles944572594557230-35945572 (10 min) 1
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Condition of temperature
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DNA
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DNA
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autosequencer
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Human Gene Therapy SCID (severe combined immunodeficiency)1. retroviral vector + ADA()T lymphocyte intravenous injection2. ADA gene bone marrow cell
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HDLs: LDLs:
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Vaccines Protein coat of virus or bacteria.Modify pathogen's genome
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Pharmaceutical products InsulinGrowth hormonesEPO (erythropoietin) Interferon
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Forensic RFLPDNA fingerprinting
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DNA
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DNA
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Environment Detoxification mining
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Agricultural Transgenic animalsn enlarge valuable partsn resistant to pathagensn improved agricultural products, such as change AAs compositionsTransgenic plantsTi plasmid (Tumor-inducing plasmid)Agrobacterium tumefaciensAntisense genes
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DNA