摘要
利用分子信标被切割后荧光信号的变化,发展了一种简便的检测限制性内切酶活性的新型荧光分析方法.以限制性内切酶XspⅠ为例,将其识别位点嵌入具有茎环结构的分子信标探针(Molecular Beacon,MB)的环状部分,利用这种分子信标在溶液中形成的瞬时二聚体结构,实现了限制性内切酶对分子信标的切割.在优化的条件下,反应初速度与酶的浓度成正比,线性范围为0.05～50U/mL,检测限为0.05U/mL.通过改变分子信标环部的识别位点的序列,还可以检测其他限制性内切酶如AluⅠ的活性.
A novel and simple fluorescence assay for the activity of Xsp I restriction endonuclease was proposed based on molecular beacons cutting by restriction endonuclease. The temporary and dynamic double DNA structure was suggested as the possible cleavage mechanism. Under the optimal condition, the initial velocity was proportional to the concentration of endonuclease in the range of 0.05 to 50 U/mL and the detection limit was 0.05 U/mL. This method can also be extended to other restriction endonueleases, such as A lu I , by changing the reorganization sequence in the loop part of MB （Molecular Beacon, MB）.
出处
《生命科学研究》
CAS
CSCD
2009年第1期6-10,共5页
Life Science Research
基金
国家自然科学基金资助项目(90606003)
湖南省科技计划项目(2008FJ3205)
湖南省科研计划重点项目(03SSY1006)
湖南省杰出青年科学基金资助项目(08JJ1002)
关键词
分子信标
荧光分析
限制性内切酶
molecular beacon
fluorescence assay
restriction endonucleases
