克隆技术及基因表达专题剖析.ppt

克隆技术及基因表达专题 6. DNA cloning, analysis and gene expression * Nucleic acids- sequencing The ability to construct recombinant DNA molecules and maintain them in cells is called DNA cloning. Processes (过程) of DNA cloning： Forming the recombinant DNA molecules (重组DNA) by inserting your interested DNA fragments into a proper vector (载体). (Require restriction enzymes and ligase) Transform (转化) the recombinant DNA molecules into competent cells (感受态细胞). Propagation of the cells containing the recombinant DNA to form a clone (克隆), a set of identical cells containing the same recombinant DNA. Select the desired clones using the selective marker. Restriction digestion of your insert and vector using the same enzyme. Use ligase to join your insert and vector together. Transform the ligation products into E. coli. competent cells. Grow the cells on a plate containing tetracycline (四环素). Host organisms/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 (large fragments of human genome) General features of a Vector They contain an origin of replication and can autonomously replicating DNA independent of host’s genome. Easily to be isolated from the host cell. Most are circular, some are linear (e.g. YAC vector). Contains at least one selective marker, which allows host cells containing the vector to be selected amongst those which do not. Contains a multiple cloning site (MCS) to be cut by restriction enzymes for DNA manipulation. Cloning vectors (克隆载体): allowing the exogenous DNA to be inserted, stored, and manipulated at DNA level. E. coli cloning vector (circular): plasmids (质粒) bacteriophages (l and M13) (噬菌体) plasmid-bacteriophage l hybrids (cosmids) (考斯质粒,质粒和噬菌体杂和体). Yeast cloning vector: yeast artificial chromosomes (YACs，酵母人工染色体) (Linear) Plasmids: small, extrachromosomal circular molecules, from 2 to ~200 kb in s