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A Solid-Phase Platform for Combinatorial and Scarless Multipart Gene Assembly

“…The assembly of dsModules using a solid-phase platform was previously described by Dietrich et al, and we optimized the ligation efficiency and assembly cycle time of this method. 17 Using our method, it took only 1 h to ligate two dsModules, whereas the previous method required 5 h. The ligation efficiency of the solid-phase assembly of dsModules (50−60%) is comparable to the method of Dietrich et al (35−70%). We also demonstrate the use of PCR generated modules, which makes it possible to assemble large (>500 bp) modules.…”

“…The use of linkers greatly improved ligation efficiency, up to an average of 93%, and therefore the assembly cycle time could be decreased to 1 h. Compared to Dietrich et al, the ligation efficiency almost doubled, from 35−70% to 80−90%. 17 As mentioned earlier, the ligation efficiency of the 45 nt ssModule could not be determined (Supplementary Figure 1). However, by using linkers, the assembly method either becomes sequencedependent or introduces scars.…”

“…Dietrich et al only demonstrated the ligation of 4 dsModules onto a Head module. 17 For ssODN assembly, the low ligation efficiency hinders the formation of constructs with more than 3 ssModules (Figure 3a; Supplementary Figures 1, 3, 5, 7; Supplementary Tables 1, 3, 5).…”

“…6 Here we present a modular assembly strategy for seamless, sequence-independent and fast combinatorial gene construction at the gene/pathway level. The assembly strategy is based on the ligation of single-or double-stranded oligonucleotides (ssODN or dsODN) using a solid-phase platform, as initially described by Dietrich et al 17 Their method is further optimized by a decreased ligation time and increased amount of ligated dsODNs and expanded by including the assembly of ssODN as well as using PCR-generated dsODN. The use of a solid-phase system facilitates purification of the obtained products and enables automation for high-throughput gene construction.…”

Modern approaches to artificial gene synthesis: aspects of oligonucleotide synthesis, enzymatic assembly, sequence verification and error correction

“…One of the alternative implementation of LCA is blunt-end ligation of double stranded DNA blocks using streptavidincoated magnetic beads and biotin bonded oligonucleotides joined with subsequently added 5′-phosphorylated DNA duplexes (Dietrich et al, 1998). The latest modifications of this approach utilize 5′-phosphorilated single-stranded oligonucleotides (Pengpumkiat et al, 2016).…”