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

Abstract

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.

Keywords

Phi29 DNA polymerase / Rolling-circle amplification efficiency / High-salt conditions / Droplet sorting / Fluorescence intensity

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Funding

National Key Research and Development Program of China(2022YFC3401303)

Shandong Province Key R & D Program(2023CXGC010714)

Natural Science Foundation of Jiangsu Province(BK20240202)

Key Technological Project of Jiangxi Province(20244AFH82001)

RIGHTS & PERMISSIONS

Jiangnan University