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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), - PowerPoint PPT Presentation
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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
DNA
The toolkit for DNA technology: 1.Restriction enzymes: Daniel Nathans, Hamilton Smith and Werner Arber (1978 Nobel Prize) 2.DNA vector3. Host organisms
Restriction enzymes: 1.Daniel Nathans, Hamilton Smith and Werner Arber (1978 Nobel Prize), DNA ligase
DNA
cDNA
DNA vector cloning vector Bacterial plasmidsBacteriophagesShuttle vectorsyeastRetroviruses
Host organisms: (1) Bacteria: (2) Yeast: (3) Animal or plant cell: DNA
Diagnosis of Genetic Diseases Human Gene Therapy Vaccines Pharmaceutical products Forensic Environment Agricultural
GMO
Diagnosis of Genetic Diseases Cystic fibrosis Huntington's disease
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-2
EnzymePolymerase 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 buffersAmple dNTPsTemplate DNAPrimersCofactors (eg. Magnesium)
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
The cycle 2AnnealingTm = 4 (GC bases) + 2 (AT bases)30-60 sec/incubation
The cycle 3Extension72, 500bp/30 sec; 500-1500/60 sec
CyclesExample: DenatureAnnealingExtensioncycles944572594557230-35945572 (10 min) 1
Condition of temperature
DNA
DNA
autosequencer
Human Gene Therapy SCID (severe combined immunodeficiency)1. retroviral vector + ADA()T lymphocyte intravenous injection2. ADA gene bone marrow cell
HDLs: LDLs:
Vaccines Protein coat of virus or bacteria.Modify pathogen's genome
Pharmaceutical products InsulinGrowth hormonesEPO (erythropoietin) Interferon
Forensic RFLPDNA fingerprinting
DNA
DNA
Environment Detoxification mining
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
DNA