Abstract:

DNA-based data storage technology has many considerable advantages, and been suggested as one of the most promising technologies to cope up with future crisis in information storage. It involves the conversion of real information into A/T/C/G sequences, synthesis of preservable DNA polymers by DNA synthesis technology, and data deciphering by DNA sequencing technology. Nevertheless, the current cost of DNA synthesis is still high, which greatly limits the rapid development and industrial application of DNA data storage. As the key technology of DNA data storage, DNA synthesis lays the foundation for the practical application of DNA data storage. Since the first oligonucleotides were made in the 1950 s, DNA synthesis technology has been rapidly developed and commercialized, spawning the emergence of DNA synthesizers with different throughput, which achieved oligonucleotides synthesis with dozens of nucleotides to MB-level microbial genomes. In this review, we systematically summarized the key research progress of DNA synthesis technology in terms of its historical development, which includes column-based chemical oligonucleotide synthesis, chip-based chemical oligonucleotide synthesis, oligonucleotide purification, oligonucleotide assembly, error correction and gene cloning, large fragment gene synthesis, genome synthesis and next generation enzymatic DNA synthesis. Currently, the widely used DNA synthesis technology starts from chemical synthesis of oligonucleotide. Although a number of chemical technologies have been proposed, the one typically used is the "phosphoramide" method, which includes the steps of "deprotection","coupling","capping" and "oxidation". The chemical synthesis generally produces single-stranded oligonucleotide with less than 200 nt. For double-stranded DNA synthesis, the single-stranded oligonucleotides need to be assembled. The oligonucleotide assembly technologies including ligase chain assembly (LCA) and polymerase chain assembly (PCA) were thus developed, and have been well applied in the commercialized gene synthesis. Following the development of chemical oligonucleotide synthesis technology and gene synthesis technology, several bacterial genomes and yeast chromosomes have been successfully synthesized, by employing the strategies of "one time de novo synthesis" or "gradual replacement synthesis". Meanwhile, new enzymatic DNA synthesis technology has also made considerable progress in the recent years, opening up a new path for synthetic biologists. In addition to these key research developments, we further summarized and analyzed the impact of key parameters of DNA synthesis technology, such as length, cost and speed, on DNA data storage, in order to provide some references and ideas for the development and the practical application of the entire DNA data storage process. Finally, we envisioned the future trend of DNA synthesis technology, including cost reduction, further development of genome synthesis technology and enzymatic DNA synthesis technology, as well as the establishment of a faster DNA synthesis technology with a longer fragment and lower-cost for DNA data storage.

Key words: oligonucleotide synthesis, gene synthesis, genome synthesis, enzymatic DNA synthesis, DNA data storage

摘要：

DNA数据存储由于在存储应用上的诸多优点而日渐受到广泛关注。DNA数据存储流程包括将0/1二进制信息转换为A/T/C/G碱基序列，利用人工DNA合成技术将碱基序列合成为DNA多聚物分子，以及通过测序技术进行数据读出等环节。然而，目前的人工DNA合成成本依然高昂，严重制约了以DNA为介质的数据存储技术的快速发展及其产业化应用。人工DNA合成作为DNA数据存储的基础技术和成本关键，是决定DNA数据存储从理论走向应用的主要因素。本文以DNA合成的发展历程出发，系统地总结了其关键技术的研究进展，包括柱式化学寡核苷酸合成、芯片化学寡核苷酸合成、寡核苷酸纯化、寡核苷酸拼装、基因合成纠错与克隆筛选、大片段基因合成组装及基因组合成，以及新一代酶法合成等。同时，进一步总结和分析了DNA合成技术关键参数长度、成本及速度对DNA数据存储商业化发展的影响，以期为DNA数据存储的全流程技术开发和应用研究提供一定的参考和思路。降低DNA合成成本，开发更加高效的基因组合成策略，进一步发展新一代酶法DNA合成技术，以及建立面向DNA数据存储的长片段、低成本、快写入等功能应用的DNA合成技术等是未来DNA合成技术的重要发展趋势。

关键词: 寡核苷酸合成, 基因合成, 基因组合成, 酶法DNA合成, DNA存储