Advances in technologies for de novo DNA synthesis， assembly and error correction

doi:10.12211/2096-8280.2020-034

Abstract

Abstract:

DNA is the primary carrier of genetic information in various types of life forms. DNA synthesis is a technology that enables the de novo generation of a blueprint for biological functions. It is one of the key generic technologies in many areas of life sciences and the fundamental tool of biotechnology revolution. Distinct from conventional genetic engineering technologies that can only modify natural DNA, DNA synthesis technologies allow limitless creativity for build of designed nucleotide sequence, rewriting of the organism's genetic information as well as creation of synthetic genomes. Advancements in DNA synthesis have led to remarkable improvements in our ability to understand and engineer biological systems. The process of synthetic creation of DNA involves synthesis of oligonucleotide, assembly of multiple constructs together into longer DNA pieces and the associated error-correction procedures to reduce errors produced during oligo synthesis and subsequent assembly. Here, we review current advancements as well as some of the challenges in the technologies of de novo DNA synthesis, assembly, and error correction. For over six decades, DNA synthesis technologies mainly rely on phosphoramidite chemical synthesis methods which was first invented in the 80s. It has been adopted by both column-based and microarray-based oligo synthesizers. New enzymatic DNA synthesis strategy is poised to revolutionize the field. Despite great potential and recent groundbreaking developments, technical hurdles in enzymatic DNA synthesis methods including blocking technology and protein engineering remain challenging. Due to the limits on length and error rates of the synthesis processes, effective assembly and error correction technologies are required for production of long stretch of DNA. With the rapid development of synthetic biology, there is an urgent and high demand for additional DNA synthesis technologies to produce longer DNA constructs and even complete genomes. Advances in high-throughput, automated, and integrated DNA synthesis technologies will create exponential rates of change in a wide range of fields.

Key words: DNA synthesis, DNA assembly, error correction, synthetic biology, key generic technology

摘要：

DNA设计合成是推动生命科学及其相关领域发展的关键共性底层技术。常规的遗传操作技术仅能对已有的DNA序列进行有限的改造，而DNA合成技术则可从头“书写”生命信息，从另一高度提升我们对生命体理解、预测和操控的能力。DNA合成技术包括寡核苷酸合成技术、DNA组装技术以及DNA纠错技术。本文总结了以上关键技术的特点和发展趋势，经历超过60年的发展后，化学合成法仍然是当前寡核苷酸合成的主流方法，它被广泛应用于柱式及芯片DNA合成仪，酶法DNA合成技术则有望颠覆传统的DNA化学合成方法；现有DNA合成技术在合成能力和准确性上存在局限，难以直接准确合成基因长度的DNA片段，分级的体外与体内组装技术的合理搭配，可将分段合成的寡核苷酸片段装配成长片段DNA，达到基因长度甚至基因组长度DNA序列的合成，它也因此成为长片段DNA合成的关键；寡核苷酸的合成与组装过程都不可避免地引入错误，基于错配结合或错配切除的纠错技术在DNA合成过程不同阶段的应用，不仅能提高DNA合成的准确性，还可有效降低长片段DNA合成的质控成本。近年来合成生物学等相关领域的迅猛发展，对DNA合成相关技术提出了新的要求，正推动DNA合成、组装与纠错相关技术向着高通量、自动化和集成化的方向不断改进和创新。

关键词: DNA合成, DNA组装, 纠错, 合成生物学, 关键共性技术

CLC Number:

Q819

PENG Kai, LU Xiaoyun, CHENG Jian, LIU Ying, JIANG Huifeng, GUO Xiaoxian. Advances in technologies for de novo DNA synthesis， assembly and error correction[J]. Synthetic Biology Journal, 2020, 1(6): 697-708.

彭凯, 逯晓云, 程健, 刘莹, 江会锋, 郭晓贤. DNA合成、组装与纠错技术研究进展[J]. 合成生物学, 2020, 1(6): 697-708.

Figures/Tables 4

Fig. 1 Reaction cycle for solid-phase phosphoramidite synthesis of oligonucleotide[1—Deprotection. The dimethoxytrityl (DMT) ether capping group on the 5’ end of the previous nucleotide is removed to generate the 5’ end hydroxyl group on the growing chain. 2—Coupling. The desired phosphoramidite monomer couples to the chain via the 5’ end hydroxyl group generated in the deprotection step. 3—Capping. Uncoupled 5’ end hydroxyl group is blocked by acylating molecules to prevent further reactions. 4—Oxidation. An oxidizer agent is used to converte phosphite triester linkage to a more stable phosphotriester bond prior to the start of the next cycle. Cleavage: final oligonucleotides are cleaved from their solid support at the end of synthesis]

Fig. 2 The oligo synthesis cycle by TdT-dNTP conjugates mediated reversible termination

Fig. 3 Summary of general schemes of in vitro and in vivo DNA assembly

Fig. 4 Strategies for error-removal based on mismatch cleavage and mismatch binding

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