一种基于T7核酸内切酶I纠错的病毒基因组高效组装方法

An efficient assembly method for a viral genome based on T7 endonuclease I-mediated error correction

摘要:

基因合成技术是支撑合成生物学发展的使能技术。现有的基因从头合成技术存在操作步骤多、效率低、错误率高且长度有限等问题，难以支撑合成生物学日益拓展的庞大需求。其中DNA片段的组装和纠错是基因合成的关键环节。本研究首先通过平衡序列设计软件能力、PCR扩增能力以及组装酶组装能力等参数，将约10 kb病毒基因组序列进行合理拆分后设计寡核苷酸序列；然后使用高保真聚合酶进行两步PCR反应，完成3.0 kb DNA片段的从头合成，并使用T7核酸内切酶I分别对不同阶段的PCR产物进行纠错反应；最后将从头合成并经过纠错的3.0 kb DNA片段进行约10 kb片段的组装并测序验证。结果表明，本方法可成功获得约10 kb DNA片段，有效降低组装过程中的大片段突变概率，组装错误率最低可至0.36 errors/kb。综上，本研究开发了一种高效从头合成约10 kb病毒基因组方法，辅以T7核酸内切酶I纠错，可1 d内获得约10 kb病毒基因组片段，5 d内获得病毒基因组的正确质粒。该方法优化了基因从头合成流程，降低了错误率，简化了合成与组装步骤，并进一步降低了病毒基因组组装成本。

Abstract:

Gene synthesis is an enabling technology that supports the development of synthetic biology. The existing approaches for de novo gene synthesis generally have tedious operation, low efficiency, high error rates, and limited product lengths, being difficult to support the huge demand of synthetic biology. The assembly and error correction are the keys in gene synthesis. This study first designed the oligonucleotide sequences by reasonably splitting the virus genome of approximately 10 kb by balancing the parameters of sequence design software ability, PCR amplification ability, and assembly enzyme assembly ability. Then, two-step PCR was performed with high-fidelity polymerase to complete the de novo synthesis of 3.0 kb DNA fragments, and error correction reactions were performed with T7 endonuclease I for the products from different stages of PCR. Finally, the virus genome was assembled by 3.0 kb DNA fragments from de novo synthesis and error correction and then sequenced. The experimental results showed that the proposed method successfully produced the DNA fragment of about 10 kb and reduced the probability of large fragment mutations during the assembly process, with the lowest error rate reaching 0.36 errors/kb. In summary, this study developed an efficient de novo method for synthesizing a viral genome of about 10 kb with T7 endonuclease I-mediated error correction. This method enabled the synthesis of a 10 kb viral genome in one day and the correct plasmid of the viral genome in five days. This study optimized the de novo gene synthesis process, reduced the error rate, simplified the synthesis and assembly steps, and reduced the cost of viral genome assembly.