Reconstitution of the DNA amplification system in microcompartments is the primary step toward artificial cell construction through a bottom-up approach. However, amplification of >100 kbp DNA in micrometer-sized reactors has not yet been achieved. Here, implementing a fully reconstituted replisome of Escherichia coli in micrometer-sized water-in-oil droplets, we developed the in-droplet replication cycle reaction (RCR) system. For a 16 kbp template DNA, the in-droplet RCR system yielded positive RCR signals with a high success rate (82%) for the amplification from single molecule template DNA. The success rate for a 208 kbp template DNA was evidently lower (23%). This study establishes a platform for genome-sized DNA amplification from a single copy of template DNA with the potential to build more complex artificial cell systems comprising a large number of genes.

中文翻译：

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从 Femtoliter 液滴中的单个模板分子扩增超过 100 kbp DNA

在微区室中重建 DNA 扩增系统是通过自下而上方法构建人工细胞的首要步骤。然而，在微米大小的反应器中扩增 >100 kbp DNA 尚未实现。在这里，在微米大小的油包水液滴中实施完全重组的大肠杆菌复制体，我们开发了液滴内复制循环反应 (RCR) 系统。对于 16 kbp 模板 DNA，in-dropletRCR 系统产生阳性 RCR 信号，从单分子模板 DNA 扩增成功率高 (82%)。208 kbp 模板 DNA 的成功率明显较低 (23%)。这项研究建立了一个从模板 DNA 的单拷贝中扩增基因组大小的 DNA 的平台，有可能构建包含大量基因的更复杂的人工细胞系统。