A safety and absolute activity measurement method for Phi29 DNA polymerase based on chemiluminescent detection of dATP consumption

Abstract

Background

Phi29 DNA polymerase serves as a cornerstone enzyme in molecular biology, enabling critical applications such as rolling-circle amplification, multiple strand-displacement amplification, and single-molecule real-time sequencing. Despite its widespread use, traditional methods for assessing its activity—including radioactive labeling and fluorescence-based quantification—suffer from limitations such as operational complexity, low precision, and safety risks. These challenges have hindered standardized quality control in both academic and industrial settings.

Results

To address these limitations, we developed a chemiluminescence-based absolute quantitation method that directly measures dATP consumption during polymerization. This method streamlines operational workflows by eliminating the need for multi-step purification procedures or specialized equipment, enabling the quantification of Phi29 DNA polymerase activity within 2 h. It demonstrates robust linearity and sensitivity across a broad dynamic range (25–200 μg/mL), while employing chemiluminescence-based detection of dATP to replace ³H-labeled dTTP, thereby eliminating biohazard risks associated with radioactive materials and enhancing feasibility for routine laboratory implementation.

Significance

This method introduces a novel approach for determining DNA polymerase activity by pioneering the correlation between dATP stoichiometry and enzymatic activity. It expands the applicability of activity assays to routine molecular biology laboratories, enabling rapid inter-batch consistency testing in commercial enzyme production. This advancement establishes a new benchmark for polymerase quality control.