2014.05.14,Professor Gao Xiaolian/Associate Professor Hong Jiong Published a paper entitled:“Error removal in microchip-synthesized DNA using immobilized MutS” in Nucleic Acids Research,online

  • Author:Wan Wen、Li Lulu、Xu Qianqian、Wang Zhefan、Yao Yuan、Wang Rongliang、Zhang Jia、Liu Haiyan、Gao Xiaolian、Hong Jiong
    Abstract:
    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.
    DOI: 10.1093/nar/gku405

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