一种基于定量荧光的 DNA 聚合酶稳态分析检测方法。
A quantitative fluorescence-based steady-state assay of DNA polymerase.
出版信息
FEBS J. 2014 Apr;281(8):2042-50. doi: 10.1111/febs.12760.
Abstract
Fluorescent dyes that bind DNA have been demonstrated as a useful alternative to radionucleotides for the quantification of DNA and the in vitro measurement of the activity of DNA polymerases and nucleases. However, this approach is generally used in a semi-quantitative way to determine relative rates of reaction. In this report, we demonstrate a method for the simultaneous quantification of DNA in both its single-strand and double-strand forms using the dye PicoGreen. This approach is used in a steady-state assay of DNA polymerase Klenow fragment exo(−), where we determine kcat and Km values for the DNA polymerase that are in excellent agreement with literature values.
摘要
荧光染料与 DNA 结合已被证明是放射性核素的一种有用替代品，可用于定量 DNA 以及体外测量 DNA 聚合酶和核酸酶的活性。然而，这种方法通常用于半定量地确定相对反应速率。在本报告中，我们展示了一种使用染料 PicoGreen 同时定量单链和双链 DNA 的方法。该方法用于 Klenow 片段外切酶 DNA 聚合酶 exo(-)的稳态测定，其中我们确定了 DNA 聚合酶的 kcat 和 Km 值，与文献值非常吻合。
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