一种快速精确测定Tth DNA聚合酶活性的方法  

A Rapid and Accurate Method for Tth DNA Polymerase Activity Assay

作　　者：陈晓雨 张建 张新亚 唐雨婷 邵钰晨 罗志丹 卢辰 CHEN Xiao-yu;ZHANG Jian;ZHANG Xin-ya;TANG Yu-ting;SHAO Yu-chen;LUO Zhi-dan;LU Chen(Jiangsu Key Laboratory of Marine Pharmaceutical Compound Screening,Jiangsu Ocean University,Lianyungang 222005;Jiangsu Institute of Marine Resources Development,Lianyungang 222005)

机构地区：[1]江苏海洋大学江苏省海洋药物活性分子筛选重点实验室,连云港222005 [2]江苏省海洋资源开发研究院(连云港),连云港222005

出　　处：《生物技术通报》2021年第5期281-286,共6页Biotechnology Bulletin

基　　金：江苏高校优势学科建设工程三期项目;连云港市新冠肺炎防治应急专项(SF2006);江苏省研究生科研与实践创新计划项目(KYCX20_2893)。

摘　　要：Tth DNA聚合酶是一种同时具备DNA聚合酶和逆转录酶活性的耐热型工具酶,但在生产和改造过程中,缺乏准确、快速、简便测定酶活的方法。利用高灵敏度双链特异性核酸染料PicoGreen和特殊设计的发夹型探针,建立了一种测定Tth DNA聚合酶活性的新方法。该方法的标准曲线线性关系良好,决定系数R^(2)达到0.99以上,灵敏度和准确度高,操作简单,测定结果与商业化产品标定的活力相符,经荧光定量PCR验证能够反映真实使用环境下的酶活。该方法还可推广到测定其他DNA聚合酶活性,将为DNA聚合酶的生产和改造提供有效的检测手段。Tth DNA polymerase is a thermostable enzyme with both DNA polymerase and reverse transcriptase activities.Traditional methods for activity assay are discontinuous,time consuming,and laborious.We developed a new method for activity assay of Tth DNA polymerase using a highly sensitive double-stranded nucleic acid specific dye PicoGreen,and a specially designed hairpin probe.A good linearity of the standard curve can be established by this method,with the coefficient of determination R^(2)>0.99.This method was of high sensitivity and accuracy,and the results by it were consistent with the activity of commercial products.The verification from fluorescence quantitative PCR confirmed that this method may reveal the enzyme activity in real environment.The method can also be extended to other DNA polymerase activity assay,which will provide an effective tool for the production and transformation of DNA polymerase.

关 键 词：Tth DNA聚合酶 酶活性测定 荧光染料 发夹型探针 

分 类 号：Q55[生物学—生物化学]