Hierarchical gene synthesis using DNA microchip oligonucleotides

‘Shotgun DNA synthesis’ for the high-throughput construction of large DNA molecules

“…EarI and BtsI) were used in common flanking sequences to ensure that at least one set of microchip oligonucleotides remained intact after restriction enzyme digestion. We carried out selective amplification of sub-pool oligonucleotides cleaved from a 55 K Agilent DNA microchip using two sets of flanking primer pairs (Supplementary Sequence 4: primer set 1), and removed common sequences using the Type IIS restriction enzymes as previously illustrated (Figure 2a and b) (8,9). We then carried out shotgun DNA synthesis by utilizing microchip oligonucleotides with cleaved flanking sequences.…”

“…Oligonucleotides cleaved from DNA microchips have previously been utilized to synthesize low-cost DNA (5–9). However, the low assembly efficiency of chip-derived oligonucleotides hinders target gene construction, and a laborious DNA assembly optimization process is consequently required (Supplementary Note 1) (8). To overcome the challenges associated with microchip oligonucleotides, only a small sub-pool of oligonucleotides (i.e.…”

“…To overcome the challenges associated with microchip oligonucleotides, only a small sub-pool of oligonucleotides (i.e. <20) are often amplified to ensure high assembly efficiency (7,8). Recently, a highly efficient isothermal DNA assembly method based on in vitro recombination and amplification was successfully used to synthesize the mouse mitochondrial genome (10).…”

Next generation gene synthesis: From microarrays to genomes

“…Their approach, which was also used by Kim et al. , involved synthesizing additional primer binding sites barcoding different fragments, which could be used for amplification with the corresponding primer set. Both groups showed that the incomplete removal of primer sites by enzymatic cleavage leads to failures in assembly.…”

“…Kosuri et al showed that the selective amplification of barcoded oligos belonging to one fragment (subpool) can decrease the complexity of the pool and leads to more successful assemblies [25]. Their approach, which was also used by Kim et al [16], involved synthesizing additional primer binding sites barcoding different fragments, which could be used for amplification with the corresponding primer set. Both groups showed that the incomplete removal of primer sites by enzymatic cleavage leads to failures in assembly.…”