Development of a chemiluminescent detection method for absolute activity measurement of Taq DNA polymerase through dATP consumption

Abstract

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.