Protein-mediated error correction for de novo DNA synthesis -: art. no. e162
Protein-mediated error correction for de novo DNA synthesis -: art. no. e162
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DOI：
10.1093/nar/gnh160
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发表时间：
2004-01-01
影响因子：
14.9
通讯作者：
Jacobson, JM
中科院分区：
文献类型：
--
作者：
Carr, PA;Park, JS;Jacobson, JM
廉价的、按需合成的DNA的可用性使得生物技术中的许多强大应用成为可能，从而推动了目前对越来越长的DNA构建体的从头合成的相当大的兴趣。从寡核苷酸到产物的DNA合成，甚至与小病毒基因组一样大的产物已经完成。尽管取得了这样的成就，但迄今为止，产生这样长的构建体所需的成本和时间使得基因长度（和更长）的DNA合成不能以与PCR和DNA测序相同的方式成为日常研究工具。低成本、高通量从头DNA合成的一个关键障碍是最终产物中错误的频率。在这里，我们采用DNA错配结合蛋白MutS（来自水生栖热菌）来去除合成基因中的失败产物。相对于传统的基因合成技术，这种方法将错误减少了>15倍，产生的DNA每10000个碱基对有一个错误。该方法是通用的，可扩展的，并且可以迭代多次以获得更高的保真度。证明了2.5 kb基因的合成所需的成本和时间的减少。
The availability of inexpensive, on demand synthetic DNA has enabled numerous powerful applications in biotechnology, in turn driving considerable present interest in the de novo synthesis of increasingly longer DNA constructs. The synthesis of DNA from oligonucleotides into products even as large as small viral genomes has been accomplished. Despite such achievements, the costs and time required to generate such long constructs has, to date, precluded gene-length (and longer) DNA synthesis from being an everyday research tool in the same manner as PCR and DNA sequencing. A critical barrier to low-cost, high-throughput de novo DNA synthesis is the frequency at which errors pervade the final product. Here, we employ a DNA mismatch-binding protein, MutS (from Thermus aquaticus) to remove failure products from synthetic genes. This method reduced errors by >15-fold relative to conventional gene synthesis techniques, yielding DNA with one error per 10 000 base pairs. The approach is general, scalable and can be iterated multiple times for greater fidelity. Reductions in both costs and time required are demonstrated for the synthesis of a 2.5 kb gene.