章节3-4PPT.ppt

Chapter 3 Cutting and joining DNA molecules;一、Types of restriction and modification system;二、Nomenclature of restriction endonuclease;三、Recognition sequence of restriction endonuclease;Isoschizomer (同裂酶): the restriction endonucleases with the same recognition sequence and cut site.Neoschizomer (新裂酶): the restriction endonucleases with the same recognition sequence but different cut site.Star activity (星号活性): under extreme conditions (high pH or low ionic intensity), restriction endonucleases are capable of cleaving sequences which are similar but not identical to their defined recog-nition sequence, this altered specificity is known as star activity. For example, EcoRI﹡: N/AATTN;五、The importance of eliminating restriction systems in E.coli strains used as hosts for recombinant molecules;六、The importance of enzyme quality;Part 2 Joining DNA molecules;一、DNA ligase;二、Cohesive-end ligation ;To elevate ligation efficiency or recombination efficiency by:1. ligation reaction at a high concentration of DNA fragments. In dilute solutions the circularization of linear fragments is relatively favoured because of the reduced frequency of intermolecular reactions.2.treating linear vector fragments with alkaline phosphatase to remove 5’-terminal phosphate groups, both recircularization and plasmid dimer formation are prevented.;三、 Artificial linker or adapter ligation;Adapter: Artificial DNA with one cohesive end not phosphorylated and one blunt end bearing a 5’ phosphate group.;四、Homopolymeric-tail ligation ;五、Joining PCR products;2. Another solution is incorporation of restriction sites at each end of PCR products;六、Joining DNA molecules without DNA ligase;Chapter 4 Plasmid and phage vectors;;一、Host range of plasmids;Replication regulation of Col E1;The ori region of pSC101;三、Partitioning and segregative stability of plasmids Plasmids are stably maintained at each cell division because they contain a partitioning function region (par region). 四、Inco