1、IV. DNA recombinant technology1.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 clo
2、nal 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 cells trait, that is called gene engineering. Above technology process is also called a community name- DNA recombinant.2.Technology steps of DN
3、A 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 tr
4、ansformated 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 bionts cells (much from bacteria). These enzymes
5、 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:
6、 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 ) re
7、action activity RE activity unit (u): under comfortable con- dition, the 1 u enzyme volume can digest 1 ug DNA in 50 ul reaction solution in 1 h time. reaction results Blunt end: Hap I digested: 5GTT AAC3 3CAA TTG5 Cohesive end, EcoR I digested: 5GAATTC3 3CTTAAG5 *the different RE can identify the d
8、ifferent seque- nces in DNA, and have the different digested result. (2) DNA polymerase。鄂 p305. DNA polymerase can catalysis the nucleo- tides (dNTP) polymerazatioin obey the DNA template strand to synthesis a new DNA strand. There are different types of DNA polymerase, that have different function.
9、 For example: E.coli DNA polymerase I. Taq DNA polymerase.(3)Ligase 鄂 p306. Ligase can catalysis the ligation （连接反应） of DNAs or RNAs nick.that divided to DNA ligase and RNA ligase. for example, the function of T4 DNA ligase are to ligate:* 1single strand nick of dsDNA. 2cohesive end of two dsDNA. 3blunt end of two dsDNA. 4single strand nick of DNA/RNA hybrid molecular.
