Being one of the most extensively researched and widely used thermostable DNA polymerases, Taq DNA polymerase derived from Thermus aquaticus has played pivotal roles in numerous molecular biology applications. The accurate and convenient determination of enzymatic activity is a fundamental prerequisite for both academic research and the industrial application of Taq DNA polymerase. To address the limitations of traditional methods for polymerase activity determination, such as low accuracy, complicated operation, and radioactive hazards, we have devised an absolute-quantitation method for Taq DNA polymerase based on chemiluminescence detection of the consumption of dATP (CDC-dATP). The CDC-dATP method offers several advantages, including simplicity suitable for routine laboratory use, high sensitivity (detecting as low as 0.0025 U/μL), excellent reproducibility, a broad linear range for Taq DNA polymerase (0.0025–0.1 U/μL) with a strong linear relationship (R² > 0.99), and, significantly, absolute activity quantitation without the associated radioactive hazards. We delved into the molecular behavior of Taq DNA polymerase in the CDC-dATP method. Furthermore, we explored the extensive application potential of the CDC-dATP methodology for measuring the activity of various other enzymes involved in dATP/ATP generation or consumption, encompassing other DNA polymerases, reverse transcriptases, RNA polymerases, hydrolases, and protein kinases, covering all seven major categories of enzymes.

中文翻译：

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开发一种通过 dATP 消耗测量 Taq DNA 聚合酶绝对活性的化学发光检测方法


作为研究最广泛、使用最广泛的热稳定 DNA 聚合酶之一，源自水生热藻的 Taq DNA 聚合酶在众多分子生物学应用中发挥了关键作用。准确、方便地测定酶活性是 Taq DNA 聚合酶学术研究和工业应用的基本前提。为了解决传统聚合酶活性测定方法的局限性，例如准确度低、操作复杂和放射性危害，我们设计了一种基于 dATP 消耗化学发光检测的 Taq DNA 聚合酶绝对定量方法 （CDC-dATP）。CDC-dATP 方法具有多种优点，包括适合常规实验室使用的简单性、高灵敏度（检测低至 0.0025 U/μL）、出色的重现性、Taq DNA 聚合酶的宽线性范围 （0.0025–0.1 U/μL） 具有很强的线性关系 （R² > 0.99），以及显著的绝对活度定量，无相关的放射性危害。我们深入研究了 CDC-dATP 方法中 Taq DNA 聚合酶的分子行为。此外，我们探索了 CDC-dATP 方法的广泛应用潜力，用于测量参与 dATP/ATP 生成或消耗的各种其他酶的活性，包括其他 DNA 聚合酶、逆转录酶、RNA 聚合酶、水解酶和蛋白激酶，涵盖所有七大类酶。