利用DNA芯片技术高效合成Ostreococcus Tauri叶绿体基因组(英文)

Chemical Synthesis of the Ostreococcus Tauri Chloroplast Genome on a Microchip

目的:21世纪以来,随着合成生物学的高速发展及其所遇到的问题,开发下一代DNA合成技术已经成为了必然趋势。基因芯片技术和DNA大片段组装技术是建立下一代DNA合成平台的关键技术力量。方法:为了开发具有工业化标准的DNA芯片-基因组合成平台,我们首次利用电化学DNA芯片和DNA大片段组装技术合成了72kb的Ostreococcus tauri的全叶绿体基因组。结果:首先,我们使用电化学DNA芯片合成仪合成了564条150bp的Oligo Mix,并成功扩增分离了其中96%的Oligo序列,剩下的基因组序列是通过传统的固相亚磷酰胺三脂合成法合成。在此基础上,我们利用DNA重组技术将564条150bp Oligo片段分三步克隆到了一个pGSYN系统。通过高通量测序,我们证实叶绿体基因组被成功地人工合成。整个合成成本大约是目前传统基因合成成本的10%-20%。结论:研究证实基因芯片技术和DNA大片段组装技术的应用是能够明显的降低现阶段基因组合成工艺的成本。新技术的成熟推广和成本的有效控制也会进一步加速科学家对基因组功能的深入研究以及合成生物学的质的飞跃。

Objective： The era of synthetic biology is approaching. A combination of the cutting-edge technologies of the mi- crochip-based oligonucleotide synthesis and the manipulation of genomic sized fragments is the key element to the evolution of the next generation DNA synthesis technology. Methods： To evaluate the efficiency of the DNA chip-based genome synthesis, we established a microchip-based approach to synthesizing the entire chloroplast genome sequences. Results： In this study, the entire chloroplast genome was divided into 150 mer oligonucleotides and the PCR amplification success rate from the chip synthesized oligo pool is 96%. We also established an efficient strategy to assemble these oligonucleotides into an entire chloroplast genome. The overall cost of this approach is 10%-20% of the traditional gene synthesis process. Conclusions： This study demonstrated a practical approach to synthesizing whole genome sequences in a single DNA microchip that could significantly decrease the cost of genome synthesis. This strategy indicated an approach to a cost-and time effective genome synthesis that paves way to the future application for synthesis of a biological pathway and phenotyoe.