TaqDNA聚合酶的制备及其活性、纯度的检测.doc

Taq DNA 聚合酶的制备及活性检测 肖碧山 （泉州师院化学与生命科学学院 生物科学专业2002级 学号020304005） 指导老师：孙亮先 （副教授） 摘要：用含有Taq DNA 聚合酶基因的pTaq 表达质粒转化E.coli DH5α菌株, IPTG诱导表达Taq DNA 聚合酶。利用该酶的热稳定性,反复两次- 70 ℃、75 ℃交替冻融以裂解细胞释放胞浆;以高速离心除去冻融变性的细胞碎片及核酸蛋白的复合物以达到快速纯化Taq DNA 聚合酶的目的。以商品酶作对照，用PCR 扩增反应和SDS - PAGE 检测所制备的Taq DNA 聚合酶的活性及纯度。最后用GIS凝胶图像处理系统测定所纯化Taq DNA 聚合酶的分子量。 关键词：交替冻融；Taq DNA 聚合酶；PCR反应；SDS电泳；活性；纯度；GIS凝胶图像处理系统 The purification and activity determination of Taq DNA Polymerase Xiao Bi-Shan (Department of Biology,school of chemical＆biology science,Quanzhou Normal university. Quanzhou,Fujian province, 362000,China) Abstract : The strain of E.coli DH5α was transformed with pTaq expressing plasmid within which the Taq DNA polymerase gene was inserted. The expression of the gene could be induced by IPTG. Exploiting the thermal stability property of this enzyme , the we developed a rapid method to isolated this enzyme by two cycles of -70℃ freezing and 75℃ thawing , which ruptured the cell membrane and released the cell content . After a high speed centrifugation , the denatured macromolecules precipitated and the Taq DNA polymerase could be collected in the supernatant. Take commercially available enzyme as and contrast, PCR amplify reacting and SDS - PAGE measures activity and purity of Taq DNA polymerase prepared. Use GIS gel picture processing system measures the molecular weight of Taq DNA polymerase finally. Key words : freezing and thawing；Taq DNA polymerase；polymerase chain reaction；SDS；activity； purity；GIS gel picture processing system. 1 引言 1958年科学家分离出DNA聚合酶后,就曾设想利用DNA聚合酶扩增大量DNA。1983年kary Mullis发明了聚合酶链式反应(PCR) [1]。1985年RandallK.Saiki等利用Klenow DNA聚合酶相应建立了聚合酶链式反应(Polymerase Chain Reaction),简称PCR技术[3]，由于klenowDNA聚合酶在高温下很快就失活，故每一轮PCR都要追加Klenow DNA聚合酶，这限制了PCR技术的推广与应用。1986年Erlich分离纯化出热稳定的适用于PCR技术的Taq DNA聚合酶，Taq DNA聚合酶最初是从耐热细菌Thermus aquaticus 中纯化而得[2]，现已有其基因工程酶[3]。1988 年RandallK.Saiki等也从水生栖热菌(Thermus aquatics)中分离纯化到了Taq DNA聚合酶并将它应用于PCR技术[3]。自此以后PCR技术才真正地得到了推广，被广泛地应用于基因诊断、定点诱变、DNA序列测定以及DNA多态性研究等各个方面。随着PCR技术的推