FADS-Based Directed Evolution of a Robust Bst DNA Polymerase Adapting High-Temperature Loop-Mediated Isothermal Amplification (HT-LAMP)
Xiao Li, Qiongwei Tang, Jingjie Jiang, Yue‐Peng Shang, Zhaohui Lu, Mingli Chen, J.-J. He, Feng Liu, Sisi Zhu, Zeng-ping Zhang, Hui Han, Xixi Yu, Qiuxian Li, Yuansong Xiu, Yuhong Yang, Ping Gui, Xue‐Feng Wang, Feng Lu, Wei Jing, Long Xu, Yanna Lin, Xinglong Wang, Shu Quan, Xiang Liu, Huancai Yin, Fuqiang Ma
Abstract
Bst DNA polymerase is a key enzyme used in both molecular diagnosis and scientific research. Employing the fluorescence-activated droplet sorting (FADS) technique, we successfully evolved a suboptimal wild-type Bst DNA polymerase into practically valuable mutants through directed evolution. The mutants exhibited significantly improved thermostability and strand displacement capability, enabling much better loop-mediated isothermal amplification (LAMP) performance, with a faster reaction speed (reduced from 40 to 10 min) and a highly stable solid reagent that remained stable for 2 months at 50 °C. Moreover, these robust mutants facilitated high-temperature LAMP assays at 70 °C, thereby eliminating the common issue of false positives in LAMP assays. To better understand the molecular mechanism behind the strand displacement capability, we proposed the strand displacement index (SDI) as a parameter to quantify this property. We also proposed the “hydrophobic blade” hypothesis, providing insights into the mechanism underlying enhanced strand displacement capability. This work serves as a successful example of molecular engineering and LAMP applications of the Bst DNA polymerase.