Advancing DNA Diagnostics: Breakthrough in Bst Polymerase Evolution Enhances LAMP Efficiency

Loop-mediated isothermal amplification (LAMP), a widely used DNA amplification method, is prized for its speed, accuracy, and ease of use. It plays a crucial role in detecting diseases and identifying genetically modified organisms. Central to its performance is the Bst DNA polymerase enzyme, which enables the isothermal amplification process typically carried out at 60–65°C. Key properties like strand displacement activity, thermal stability, and catalytic efficiency determine the enzyme's effectiveness.

A team led by Ma Fuqiang at the Suzhou Institute of Biomedical Engineering and Technology (SIBET), part of the Chinese Academy of Sciences, has made significant progress in enhancing Bst polymerase through directed evolution. Their efforts were supported by a fluorescence-activated droplet sorting (FADS) platform, a cutting-edge tool built on droplet microfluidics. This system enables ultra-high-throughput screening of enzyme variants at the single-cell level, capable of evaluating over 10 million mutants each day.

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Using this platform, the researchers developed a novel high-throughput screening protocol for Bst polymerase, integrating fluorescence-based detection with microfluidic technology. For the first time, this method was used to guide the enzyme’s directed evolution.

The result was the discovery of Bst polymerase mutants with significantly improved enzymatic activity. When applied in LAMP reactions, these enhanced enzymes not only reduced the time required for the reaction to reach its peak but also exhibited greater thermal stability, allowing for longer shelf life during storage.
This breakthrough marks a significant leap from the performance of the wild-type enzyme to enzymes with commercial application potential. It highlights the transformative capability of FADS technology in accelerating the evolution of molecular enzymes, paving the way for more efficient and reliable diagnostic tools.