分子遗传学理论与技术基础 Chapter IV.DNA recombinant technology.pptVIP

IV. DNA recombinant technology 1.concept DNA recombinant. Gene clone or molecular clone. Gene engineering. *That means to cut, modify and rejoin the different DNA molecules with tool enzyme, to reform a new recon molecular, the recon transfer target gene fragment into the recipient (host) cell to clonal amplify, so, we can get much Of target gene copies,that is called gene cloning. if the target gene can express in host cell in order to reform the host cell’s trait, that is called gene engineering. Above technology process is also called a community name--- DNA recombinant. 2.Technology steps of DNA recombinant.鄂p341,p348图15-5. (1)to isolate the target gene DNA fragment and the vector DNA,then to modify with same tool enzyme. (2) ligase reaction ligate the target gene DNA and vector DNA into recon. (3) the recon transformate the host cell. there is only one recon in one host cell. (4)the transformated host cells are cultured in plate medium, then to screen the target gene cell clone for clone amplify. 3.The key knowledge points: 1) Tool Enzyme. 鄂P299. [concept]: there are many types enzymes, Their function are different, that are got from biont’s cells (much from bacteria). These enzymes work as useful tools in gene operation experiment, so, be called tool enzyme. *types and function of tool enzyme. (1)Restriction Endonuclease (RE). *RE can identify a specific base sequence of dsDNA molecular, then to hydrolysis the phosphodiester bond of dsDNA in endodigetion way. *the types of RE: Type I specificity unstrength. Type III Type II digetion point is specific,very useful. there are near 100 kinds.鄂p304表。 *the bioactivity of RE type II鄂P301. [reaction environment] ~temperature: 37℃ generally, stock at -20℃ and -70 ℃ for long time. ~reaction pH 7.2-7.6 ~helper factors: [Mg]++, BSA(牛血清白蛋白) [reaction activity] RE activity unit (u): under comfortable con- ditio