DNA合成、组装与纠错技术研究进展

doi:10.12211/2096-8280.2020-034

合成生物学 ›› 2020, Vol. 1 ›› Issue (6): 697-708.DOI: 10.12211/2096-8280.2020-034

DNA合成、组装与纠错技术研究进展

彭凯¹^,², 逯晓云¹, 程健¹, 刘莹¹, 江会锋¹, 郭晓贤¹

^1.中国科学院天津工业生物技术研究所系统微生物工程重点实验室，天津 300308
^2.天津科技大学生物工程学院，天津 300457

收稿日期:2020-03-23 修回日期:2020-10-22 出版日期:2020-12-31 发布日期:2021-01-15
通讯作者: 江会锋,郭晓贤
作者简介:彭凯（1995—），男，硕士研究生。主要研究方向为DNA纠错。E-mail： pengk@tib.cas.cn
江会锋（1981—），男，博士，研究员。主要研究方向为代谢合成生物学。E-mail： jiang_hf@tib.cas.cn
郭晓贤（1982—），男，博士，副研究员。主要研究方向为酶法DNA合成。E-mail： guoxx@tib.cas.cn
基金资助:
国家重点研发计划(2020YFC1316400);天津市合成生物技术创新能力提升行动(TSBICIP-KJGG-007-01)

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

PENG Kai¹^,², LU Xiaoyun¹, CHENG Jian¹, LIU Ying¹, JIANG Huifeng¹, GUO Xiaoxian¹

^1.Tianjin Institute of Industrial Biotechnology，Chinese Academy of Sciences，Tianjin 300308，China
^2.School of Biological Engineering，Tianjin University of Science and Technology，Tianjin 300457，China

Received:2020-03-23 Revised:2020-10-22 Online:2020-12-31 Published:2021-01-15
Contact: JIANG Huifeng, GUO Xiaoxian

摘要/Abstract

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

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

中图分类号:

Q819

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.

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.

图/表 4

图1 固相亚磷酰胺寡核苷酸合成反应循环[1—脱保护，用三氯乙酸脱去固相载体上核苷酸5' 位的DMT（dimethoxytrityl）保护基团，获得供下一步缩合反应的游离5' 羟基；2—偶联，将亚磷酰胺保护的核苷酸单体与四氮唑活化剂混合，形成亚磷酰胺四唑活性中间体(其3' 端已被活化，但5' 端仍受DMT保护)，与脱保护后生成的5' 羟基发生缩合反应，寡核苷酸链向前延长一个核苷酸；3—加帽，缩合反应后，通过乙酰化来封闭未参与反应的5' 羟基，防止其在随后的循环反应中被延伸，以减少产物中单核苷酸缺失片段的比例；4—氧化，用碘的四氢呋喃溶液将连接3',5' 的不稳定亚磷酰胺三酯氧化为磷酸三酯，得到稳定的寡核苷酸链]

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]

图2 TdT-dNTP 交联体介导的可逆终止用于寡核苷酸合成循环[TdT (terminal deoxynucleotidyl transferase) is specifically conjugated with the desired dNTP to form TdT-dNTP conjugate before the reaction cycle. The conjugate is then incorporated into the 3' end of the primer in the polymerization reaction. The subsequent deprotection reaction removes TdT and unreacted TdT-dNTP conjugate to release the primer for next round of polymerization. The cycle is iterated to extend a primer by a defined sequence]

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

图3 常见体外和体内DNA组装技术及流程（Assembly of synthesized overlapping single-stranded oligos usually employ LCR and PCA based approaches. These assembly process turn oligos into longer double-stranded DNA constructs which can then be further assembled into longer fragments by Gibson assembly and Golden gate. In vivo assembly is typically achieved by feeding yeast cell with large fragments that have overlapping ends through transformation. The efficient DNA recombination machinery eventually string the fragments for assemblies that span hundreds of thousands or even millions of bases）

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

图4 基于错配切割和错配结合的纠错策略（Error rich DNA sequences are denatured and randomly re-hybridized to expose the errors introduced during oligo synthesis and subsequent assembly as bulging mismatches. The mismatches are then removed by error correction strategies using either mismatch-cleaving enzymes or mismatch-binding proteins. Fragments with fewer errors are recovered by overlap assembly extension PCR or filtering)

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

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DNA合成、组装与纠错技术研究进展

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

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