Recent advances in DNA assembly technologies

Abstract

DNA assembly is one of the most important foundational technologies for synthetic biology and metabolic engineering. Since the development of the restriction digestion and ligation method in the early 1970s, a significant amount of effort has been devoted to developing better DNA assembly methods with higher efficiency, fidelity, and modularity, as well as simpler and faster protocols. This review will not only summarize the key DNA assembly methods and their recent applications, but also highlight the innovations in assembly schemes and the challenges in automating the DNA assembly methods.

Bricks and blueprints: methods and standards for DNA assembly

“…This acceleration in the scale of DNA assembly enables construction projects too complex to be drawn out on the back of an envelope, which instead require an engineering approach. In the past decade important 1 assembly methods such as Gibson assembly and Golden Gate have been developed 7,8 , which define new protocols for joining together DNA parts. Alongside these methods, researchers have also developed various physical standards such as MODAL (Modular Overlap-Directed Assembly with Linkers) 9 and MoClo (Modular Cloning system) 12 that define rules for the format of DNA parts that can be used with them.…”

“…1 assembly methods such as Gibson assembly and Golden Gate have been developed 7,8 , which define new protocols for joining together DNA parts. Alongside these methods, researchers have also developed various physical standards such as MODAL (Modular Overlap-Directed Assembly with Linkers) 9 and MoClo (Modular Cloning system) 12 that define rules for the format of DNA parts that can be used with them.…”

Directed evolution combined with synthetic biology strategies expedite semi-rational engineering of genes and genomes

“…24 Especially today, the rapid development of synthetic biology 25 and metabolic engineering 26 require efficient, flexible, and faithful DNA assembly approaches because of the low capacity of traditional restriction, digestion, and ligation methods. In fact, modular and combinatorial assemblies of various genetic segments, particularly the assembly of large DNA fragments without scars by restriction, digestion, and ligation methods are extremely difficult.…”

“…In fact, modular and combinatorial assemblies of various genetic segments, particularly the assembly of large DNA fragments without scars by restriction, digestion, and ligation methods are extremely difficult. 24,27 As a result, several in vitro methods, such as circular polymerase extension cloning, 28 sequence and ligation-independent cloning, 29 Gibson assembly method, 30 Ligase Cycling Reaction (LCR), 31 and in vivo methods including DNA assembly with homologous recombination in Saccharomyces cerevisiae (DNA assembler-yeast) 27,32 have been developed recently. The Gibson assembly method is the most well-known method, and involves the generation of singlestranded complementary overhangs by T5 exonuclease and covalent joining by fusion DNA polymerase and Taq DNA ligase.…”

High molecular weight DNA assembly in vivo for synthetic biology applications

“…[1,2,[4][5][6]12] In addition to Gibson isothermal assembly, SLIC, CPEC, SLiCE, OGAB, and LCR, recently developed methods for short DNA fragments assembly include Golden Gate, [35,36] MASTER: methylation-assisted tailorable ends rational method, [37] Golden Braid, [38] MODAL: modular overlap directed assembly with linkers, [39] and MoClo, [40] which have been reviewed elsewhere. [41] The Golden Gate and MASTER methods rely on type II restriction enzymes and MspJI endonuclease, respectively. In addition, MASTER requires PCR amplification of the DNA fragments.…”