Fluorometric methods employing low concentrations of ethidium bromide for DNA topoisomerase and endonuclease assays.
Fluorometric methods employing low concentrations of ethidium bromide for DNA topoisomerase and endonuclease assays.
复制标题
使用低浓度溴化乙锭进行 DNA 拓扑异构酶和核酸内切酶测定的荧光方法。
DOI：
10.1016/0003-2697(89)90595-2
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发表时间：
1989
影响因子：
2.9
通讯作者：
Foglesong,PD
中科院分区：
文献类型：
--
作者：
Foglesong,PD
通过测量酶处理封闭双链DNA后溴化乙锭荧光强度的变化，可以快速检测DNA拓扑异构酶活性。当溴化乙锭浓度降低10倍至0.1 μg/ml时，荧光测定的灵敏度提高了3倍。荧光测定的结果与反应DNA的琼脂糖凝胶电泳分析密切一致。在pH7.0和pH12.0条件下，用0.1 μg/ml溴化乙锭测定DNA复合物的荧光强度，建立了一种灵敏的荧光分析方法。这些方法使得DNA拓扑异构酶和核酸内切酶活性的非常快速和灵敏的测量成为可能。
DNA topoisomerase activity can be rapidly assayed by measuring the change in ethidium bromide fluorescence intensity after treatment of closed duplex DNA with enzyme. The sensitivity of the fluorometric assay has been enhanced 3-fold by a 10-fold reduction in ethidium bromide concentration to 0.1 μg/ml. The results of the fluorometric assays are in close agreement with agarose gel electrophoretic analyses of reacted DNA. A sensitive fluorometric method using 0.1 μg/ml ethidium bromide has also been developed to determine the fraction of nicked and linear DNAs in a mixture containing closed duplex DNA by measuring the fluorescence intensities of ethidium-DNA complexes at pH 7.0 and pH 12.0. These methods make possible very rapid and sensitive measurements of DNA topoisomerase and endonuclease activities.