[自然科學]techniqueofmolecularbiology(存儲版)

　　

【正文】 ntent of specific mRNA mRNA in different stages of fungal development Southern and Northern blotting DNA on blot RNA on blot 1. Genomic DNA preparation RNA preparation 2. Restriction digestion 3. Denature with alkali 4. Agarose gel electrophoresis ? 5. DNA blotting/transfer and fixation RNA 6. Probe labeling ? 6. Hybridization (temperature) ? 7. Signal detection (Xray film or antibody) ? 56 Western hybridization ? Proteins from the PAGE gel can be transferred to a membrane, followed by a western hybridization of the target protein by a corresponding antibody. ? Detect the presence of specific protein by antibodyantigen reaction 57 Blot type Target Probe Applications Southern DNA DNA mapping genomic clones estimating gene numbers, etc Northern RNA DNA RNA sizes and abundance (gene expression level) Western Protein Antibodies protein size and abundance (gene expression level) Comparison of Southern, Northern and Western hybridization 58 Are there Eastern hybridization? If you can invent a new technology, you can name it as Eastern hybridization or your own name! 59 4. DNA cloning and gene expression The ability to construct rebinant DNA molecules and maintain them in cells is called DNA cloning. 60 Processes (過程 ) of DNA cloning： 1. Constructing the rebinant DNA molecules (重組DNA) by inserting your interested DNA fragments into a proper vector (載體 ). (Require restriction enzymes and DNA ligase) 2. Transform (轉化 ) the rebinant DNA molecules into petent cells (感受態細胞 ). 3. Propagation of the cells containing the rebinant DNA to form a clone (克隆 ), a set of identical cells containing the same rebinant DNA. 4. Select the desired clones using the selective marker. 1. Restriction digestion of your insert and vector using the same enzyme. 2. Use ligase (連接酶） to join your insert and vector together. 3. Transform the ligation products into E. coli petent cells. 4. Select the desired clones: Grow the cells on a plate containing tetracycline (四環素 ). EcoRI 62 Important ponents of DNA cloning ? Vector (載體） ? Host anisms/cells (宿主細胞） ? Restriction enzymes and DNA ligase(剪刀 +漿糊 ) ? Screening method for selecting the desired clones （篩選陽性克隆） 63 General features of a Vector 1. They contain an origin of replication and can autonomously replicating DNA independent of host’s genome. 2. Easily to be isolated from the host cell. 3. Contains at least one selective marker, which allows host cells containing the vector to be selected amongst those which do not. 4. Contains a multiple cloning site (MCS) to be cut by restriction enzymes for DNA manipulation. 64 Plasmid(質粒 ): most used vector in DNA cloning small, extrachromosomal circular molecules, from 2 to ~200 kb in size, which exist in multiple copies within the host cells Contain an origin of replication, at least one selective marker and multiple cloning site. ? Example of selective marker: ampr gene encoding the enzyme blactamse which degrades penicillin antibiotics such as ampicillin氨芐青霉素抗性基因 65 multiple cloning site(MCS, 多克隆位點 ) MCS selective marker origin of replication 66 Important ponents of DNA cloning ? Vector (載體） ? Host anisms/cells (宿主細胞） ? Restriction enzymes and DNA ligase(剪刀 +漿糊 ) ? Screening method for selecting the desired clones （篩選陽性克隆） 67 ? Host anisms/cells: where the plasmids get multiplied and propagated faithfully, which is crucial for DNA cloning. Prokaryotic host: E. coli ( most cases) Eukaryotic host: Yeast, Saccharomyces cerevisiae Vectors can be introduced into petent cells (感受態細胞 ) by transformation (轉化 ) 68 Important ponents of DNA cloning ? Vector (載體） ? Host anisms/cells (宿主細胞） ? Restriction enzymes and DNA ligase(剪刀 +漿糊 ) ? Screening method for selecting the desired clones （篩選陽性克隆） 69 Screening of positive clones 70 screening (抗生素選擇 )： only the rebinant plasmids grow on the antibioticcontaining plate. screening (藍白斑選擇 ): DNA insertion in the vector shuts down the LacZ gene expression, and turns the colony to white. hybridization screening (菌落雜交篩選 ) Screening methods: 71 (1).Antibiotic screening (抗生素選擇 )： only the rebinant plasmids grow on the antibioticcontaining plate. 72 multiple cloning site(MCS, 多克隆位點 ) MCS selective marker origin of replication 73 antibioticcontaining plate Host cell must be sensitive to this antibiotic! X gene ampR ampR ampR ampR Antibiotic screening can not discriminate the clone containing rebinant pasmid and the clone containing the religated vector 75 (2). Bluewhite screening (藍白斑選擇 ) lacZ encode enzyme bgalactosidase （ b半乳糖苷酶） Xgal (substrate of the enzyme) Blue product Host cell : lacZ△ M15 encoding only the Cterminal of apeptide of bgalactosidase Plasmid: lacZ’, a shortened derivative of lacZ, encoding Nterminal of apeptide of bgalactosidase a plementation (a互補 ) Host cell Plasmid lacZ+ Xgal (substrate of the enzyme) Blue product b半乳糖苷酶 Blue white screening Ampr ori pUC18 (3 kb) MCS (Multiple cloning sites, 多克隆位點） Lac promoter lacZ’ 80 Ampr ori pUC18 (3 kb) MCS (Multiple cloning sites, 多克隆位點） Lac promoter lacZ’ Selfligated vector Ampr ori pUC18 (3 kb) Lac promoter lacZ’ Rebinant plasmid Inserted gene Selfligated vector: blue transformants Rebinant plasmid: containing inserted DNA, whit