摘要
尿嘧啶DNA糖苷酶(UNG)是防止PCR假阳性反应的工具酶,参照限制性DNA内切酶活力测定法的原理(以完全消化DNA带型的最高稀释度为一个活性单位)和应用凝胶成像分析技术,建立了UNG酶活性的非同位素检测新方法.反应体积为20μL,底物dU ung(dU DNA)为165μg,加UNG后在适当温度下反应10min,使底物去除尿嘧啶,其AP位点经碱降解.通过琼脂糖凝胶电泳和凝胶成像分析,测定底物dU ung的残留量以测定UNG酶活性.酶活力以37℃每分钟降解1ngdU ung的酶量为一个单位,检测灵敏度为1ng,检测线性范围为0～165ng.该法简便易行、客观、精密,重复性好(CV<3%),结果显示直观.
Uracil DNA glycosylase (UNG, EC 3.2.2.3) was effectively used in overcoming carry-over contamination in PCR. UNG acts on double- and single-stranded U-DNA (uracil-containing DNA) by hydrolysis of uracil-glycosidic bonds (base excision), releasing uracil and creating an alkaline-sensitive apurinic site (AP). In order to quickly screen and detect the enzyme activity of UNG, based on the principle of the endonucleasase assay, a nonradioactive enzyme assay using the gel electrophoresis and 1D image analysis system was developed. The dU-ung (UNG gene, 165ng) PCR-amplified, in which dTTP was replaced by dUTP, was used as substract. Total reaction volume was 20 μl. The enzyme reacted for 10 min and an apurinic site (AP) thus produced was hydrolyzed by 0.1 mol/L NaOH solution. The activity of UNG was detected by the gel electrophoresis and 1D image analysis system. One unit of UNG activity is defined as 1 ng dU-ung degraded by the enzyme. The assay range is 0～165 ng and the sensitivity is about 1 ng. This assay is reliable (CV>3%), fast, objective and convenient.
出处
《科技通报》
北大核心
2004年第2期116-120,共5页
Bulletin of Science and Technology
基金
云南省省院合作项目(96S004)
云南省应用基础基金项目(98C095M)
