2种制备Taq DNA聚合酶方法的比较  

Comparasion two methods of preparing Taq DNA polymerase

作　　者：秦川[1] 杨献军[1] 杨瑞[1] 王天云[2] 

机构地区：[1]新乡医学院分子生物学研究室,河南新乡453003 [2]新乡医学院生物化学与分子生物学教研室,河南新乡453003

出　　处：《新乡医学院学报》2008年第5期469-471,共3页Journal of Xinxiang Medical University

基　　金：河南省科技厅自然科学基金(编号:511042300);河南省科技攻关资助项目(编号:624410041)

摘　　要：目的比较2种制备纯化Taq DNA聚合酶的方法,为PCR提供实验用酶。方法Taq DNA聚合酶基因的pTaq表达质粒转化E.coli菌株,IPTG诱导12 h后收集菌体,分别用硫酸铵沉淀法和冻融法进行纯化,SDS-PAGE电泳和PCR扩增分析比较其纯度和活性。结果硫酸铵沉淀法制备的Taq DNA聚合酶活性能可有效扩增DNA片段;冻融法制备的Taq DNA聚合酶活性较低。结论硫酸铵沉淀法制备的Taq DNA聚合酶优于冻融法,能够达到分子生物学实验中基因扩增的要求。Objective To compare two methods of preparing purified Taq DNA polymerase in order to provide experimental enzyme for polymerase chain reaction （PCR）. Methods Using Taq DNA polymerase expression plasmid to transfer E. coli strain, the cells were collected after inducing with Isopropyl β-D-1-Thiogalactopyranoside for 12 hours. The enzyme was purified by ammonium sulfate precipitation and freeze-thaw method ,respectively, and then the activity and purity were analysed by PCR and SDS-PAGE. Results The activity of Taq DNA polymerase that prepared by ammonium sulfate precipitation was higher than that prepared by freeze-thaw method. Conclusion The Taq DNA polymerase purified by ammonium sulfate precipitation is better than the freeze-thaw method, which can meet the requirement of molecular biology experiment of gene amplification.

关 键 词：TAQ DNA聚合酶 表达 纯化 

分 类 号：Q786[生物学—分子生物学]