Wan Wen, Li Lulu, Xu Qianqian, Wang Zhefan, Yao Yuan, Wang Rongliang, Zhang Jia, Liu Haiyan, Gao Xiaolian, Hong Jiong
School of Life Science, University of Science and Technology of China, Hefei, Anhui, People's Republic of China Hefei National Laboratory for Physical Science at the Microscale, Hefei, Anhui, People's Republic of China.
School of Life Science, University of Science and Technology of China, Hefei, Anhui, People's Republic of China Hefei National Laboratory for Physical Science at the Microscale, Hefei, Anhui, People's Republic of China Department of Biology and Biochemistry, University of Houston, Houston, TX, USA
Nucleic Acids Res. 2014 Jul;42(12):e102. doi: 10.1093/nar/gku405. Epub 2014 May 14.
The development of economical de novo gene synthesis methods using microchip-synthesized oligonucleotides has been limited by their high error rates. In this study, a low-cost, effective and improved-throughput (up to 32 oligos per run) error-removal method using an immobilized cellulose column containing the mismatch binding protein MutS was produced to generate high-quality DNA from oligos, particularly microchip-synthesized oligonucleotides. Error-containing DNA in the initial material was specifically retained on the MutS-immobilized cellulose column (MICC), and error-depleted DNA in the eluate was collected for downstream gene assembly. Significantly, this method improved a population of synthetic enhanced green fluorescent protein (720 bp) clones from 0.93% to 83.22%, corresponding to a decrease in the error frequency of synthetic gene from 11.44/kb to 0.46/kb. In addition, a parallel multiplex MICC error-removal strategy was also evaluated in assembling 11 genes encoding ∼21 kb of DNA from 893 oligos. The error frequency was reduced by 21.59-fold (from 14.25/kb to 0.66/kb), resulting in a 24.48-fold increase in the percentage of error-free assembled fragments (from 3.23% to 79.07%). Furthermore, the standard MICC error-removal process could be completed within 1.5 h at a cost as low as $0.374 per MICC.
利用微芯片合成寡核苷酸开发经济的从头基因合成方法一直受到其高错误率的限制。在本研究中，我们开发了一种低成本、高效且通量提高（每次运行多达32条寡核苷酸）的错误去除方法，该方法使用含有错配结合蛋白MutS的固定化纤维素柱，以从寡核苷酸，特别是微芯片合成的寡核苷酸中生成高质量DNA。初始材料中含错误的DNA特异性保留在固定化MutS的纤维素柱（MICC）上，洗脱液中去除错误的DNA被收集用于下游基因组装。值得注意的是，该方法将合成增强型绿色荧光蛋白（720 bp）克隆群体从0.93%提高到83.22%，相应地，合成基因的错误频率从11.44/kb降至0.46/kb。此外，还评估了一种并行多重MICC错误去除策略，用于从893条寡核苷酸组装11个编码约21 kb DNA的基因。错误频率降低了21.59倍（从14.25/kb降至0.66/kb），导致无错误组装片段的百分比增加了24.48倍（从3.23%增至79.07%）。此外，标准的MICC错误去除过程可在1.5小时内完成，每个MICC成本低至0.374美元。
