A Rapid, High-Throughput Method for the Construction of Mutagenesis Libraries
文献类型：期刊论文
| 作者 | Lu, Yuxin1,2; Meng, Shuting3; Guan, Xinyi2,4; He, Pengying2,4; Zhao, Dongxin1,2,3,4 |
| 刊名 | BIOMOLECULES
 |
| 出版日期 | 2025-10-25 |
| 卷号 | 15期号:11页码:18 |
| 关键词 | high-throughput library preparation sequencing normalization oligo-directed mutagenesis |
| DOI | 10.3390/biom15111511 |
| 通讯作者 | Zhao, Dongxin(zhaodongxin@simm.ac.cn) |
| 英文摘要 | As synthetic biology advances toward precise design, the construction of high-quality mutant libraries has become essential for large-scale functional screening. Traditional approaches, such as random and saturation mutagenesis, often suffer from low accuracy, high bias, and limited coverage. An ideal method should offer controlled mutagenesis, comprehensive coverage, high throughput, operational simplicity, and controllable outcomes, enabling effective large-scale screening. Here, we developed a high-throughput, precisely controlled method for constructing a mutagenesis library based on chip-based oligonucleotide synthesis. Using PSMD10 as a model, we constructed a full-length amber codon scanning mutagenesis library with 93.75% mutation coverage. Among the five polymerases evaluated, KAPA HiFi HotStart, Platinum SuperFi II and Hot-Start Pfu DNA Polymerase demonstrated higher amplification efficiency and lower chimera formation rates, making them preferred enzymes for optimized library construction. Analysis of unmapped reads highlighted key technical factors, such as oligonucleotide synthesis errors and chimeric sequence formation caused by incomplete extension of DNA polymerase or synthesis across discontinuous templates during PCR. To improve efficiency and fidelity, we recommend refining PCR conditions and strengthening oligo synthesis quality control. We establish an efficient, scalable, precisely controlled mutagenesis library construction strategy tailored for high-throughput functional research and recommend using a high-fidelity, low-bias polymerase to ensure quality. |
| WOS关键词 | DIRECTED EVOLUTION ; GENE SYNTHESIS |
| 资助项目 | Strategic Priority Research Program of the Chinese Academy of Sciences[XDB0830300] ; National Natural Science Foundation of China[32171447] |
| WOS研究方向 | Biochemistry & Molecular Biology |
| 语种 | 英语 |
| WOS记录号 | WOS:001624039200001 |
| 出版者 | MDPI |
| 源URL | [http://119.78.100.183/handle/2S10ELR8/322085]  |
| 专题 | 中国科学院上海药物研究所 |
| 通讯作者 | Zhao, Dongxin |
| 作者单位 | 1.Nanjing Univ Chinese Med, Sch Chinese Mat Med, Nanjing 210023, Peoples R China 2.Chinese Acad Sci, Shanghai Inst Mat Med, Shanghai 210203, Peoples R China 3.Henan Univ, Sch Pharm, Kaifeng 475004, Peoples R China 4.Univ Chinese Acad Sci, Beijing 100049, Peoples R China |
| 推荐引用方式 GB/T 7714 | Lu, Yuxin,Meng, Shuting,Guan, Xinyi,et al. A Rapid, High-Throughput Method for the Construction of Mutagenesis Libraries[J]. BIOMOLECULES,2025,15(11):18. |
| APA | Lu, Yuxin,Meng, Shuting,Guan, Xinyi,He, Pengying,&Zhao, Dongxin.(2025).A Rapid, High-Throughput Method for the Construction of Mutagenesis Libraries.BIOMOLECULES,15(11),18. |
| MLA | Lu, Yuxin,et al."A Rapid, High-Throughput Method for the Construction of Mutagenesis Libraries".BIOMOLECULES 15.11(2025):18. |
入库方式： OAI收割
来源：上海药物研究所
浏览0
下载0
收藏0
其他版本
除非特别说明，本系统中所有内容都受版权保护，并保留所有权利。