Journal Article

Improved single-cell genome amplification by a high-efficiency phi29 DNA polymerase

Jia, Hui, Gong, Yanhai, Zhou, Shiqi, Xu, Jian, Wang, Yefei, Jing, Xiaoyan, Wang, Xiaohang, Zhang, Jia, Su, Xiaolu, Wang, Jichao

2023

Overview

Single-cell genomic whole genome amplification (WGA) is a crucial step in single-cell sequencing, yet its low amplification efficiency, incomplete and uneven genome amplification still hinder the throughput and efficiency of single-cell sequencing workflows. Here we introduce a process called Improved Single-cell Genome Amplification (iSGA), in which the whole single-cell sequencing cycle is completed in a high-efficient and high-coverage manner, through phi29 DNA polymerase engineering and process engineering. By establishing a disulfide bond of F137C-A377C, the amplification ability of the enzyme was improved to that of single-cell. By further protein engineering and process engineering, a supreme enzyme named HotJa Phi29 DNA Polymerase was developed and showed significantly better coverage (99.75%) at a higher temperature (40°C). High single-cell genome amplification ability and high coverage (93.59%) were also achieved for commercial probiotic samples. iSGA is more efficient and robust than the wild-type phi29 DNA polymerase, and it is 2.03-fold more efficient and 10.89-fold cheaper than the commercial Thermo Scientific EquiPhi29 DNA Polymerase. These advantages promise its broad applications in large-scale single-cell sequencing.

• Publisher: Frontiers Media SA,Frontiers Media S.A
• Subject: Bioengineering and Biotechnology / Chemical bonds / Deoxyribonucleic acid / disulfide bond / DNA / DNA polymerase / DNA-directed DNA polymerase / E coli / Efficiency / Enzymes / GB1 fusion protein / Genomes / Genomics / Microorganisms / Mutation / phi29 DNA polymerase / Plasmids / Potassium / Probiotics / Process engineering / process engineering high-efficiency phi29 DNA polymerase / Protein engineering / Protein expression / Proteins / Reagents / single-cell genome amplification