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<p>Phi29 DNA polymerase (Phi29 Pol) has emerged as a powerful tool in the third-generation sequencing technology such as DNA nanoball-based sequencing. However, natural Phi29 Pol with low amplification activity under high-salt conditions needs to be engineered to meet specific sequencing demands, which are usually achieved through a time-consuming and iterative trial-and-error process. Herein, we develop a high-throughput screening methodology for efficiently detecting Phi29 Pol mutants with high rolling-circle amplification (RCA) efficiency under high-salt conditions. The method uses a nucleic acid gel stain sensitive to oligonucleotides to achieve the input conversion from enzymatic amplification efficiency to fluorescence intensity in micron-sized droplets. We further demonstrate the potential of this methodology in the first high-throughput droplet sorting of Phi29 Pol. The RCA efficiency of sorted mutant S6 is 1.39-fold that of initial enzyme M2 in 300 mM KCl. Overall, this study provides a cost-effective and rapid solution for improving the performance of Phi29 Pol under high-salt conditions.</p>

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A high-throughput screening platform for improving the rolling-circle amplification efficiency of Phi29 DNA polymerase under high-salt conditions

  • 期刊论文

  • 发表日期：

    2025年6月4日

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  • Xirui Hua,
  • Wenwen Yu,
  • Yangyang Li,
  • Xianhao Xu,
  • Yaokang Wu,
  • Yanfeng Liu,
  • Jianghua Li,
  • Guocheng Du,
  • Long Liu,
  • Xueqin Lv

摘要

Phi29 DNA polymerase (Phi29 Pol) has emerged as a powerful tool in the third-generation sequencing technology such as DNA nanoball-based sequencing. However, natural Phi29 Pol with low amplification activity under high-salt conditions needs to be engineered to meet specific sequencing demands, which are usually achieved through a time-consuming and iterative trial-and-error process. Herein, we develop a high-throughput screening methodology for efficiently detecting Phi29 Pol mutants with high rolling-circle amplification (RCA) efficiency under high-salt conditions. The method uses a nucleic acid gel stain sensitive to oligonucleotides to achieve the input conversion from enzymatic amplification efficiency to fluorescence intensity in micron-sized droplets. We further demonstrate the potential of this methodology in the first high-throughput droplet sorting of Phi29 Pol. The RCA efficiency of sorted mutant S6 is 1.39-fold that of initial enzyme M2 in 300 mM KCl. Overall, this study provides a cost-effective and rapid solution for improving the performance of Phi29 Pol under high-salt conditions.