多重位移放大
重组酶聚合酶扩增
DNA聚合酶
生物
聚合酶
基因组
PCR的应用
DNA纳米球测序
计算生物学
分子生物学
单细胞测序
DNA
聚合酶链反应
基因组文库
遗传学
基因
DNA提取
数字聚合酶链反应
外显子组测序
突变
基序列
作者
Jia Zhang,Xiaolu Su,Yefei Wang,Xiaohang Wang,Shiqi Zhou,Hui Jia,Xiaoyan Jing,Yanhai Gong,Jichao Wang,Jian Xu
标识
DOI:10.3389/fbioe.2023.1233856
摘要
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.
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