Table 1.

Examples of companies applying enzymatic and hybrid approaches to DNA synthesis

Company	Business type	Synthesis platform	Lengtha (kb)b	Prosc	Consc
ANSA Biotechnologies132	Service provider	TdT reversibly tethered to NTPs	0.3–3	Tethering prevents formation of homopolymeric tracts Low reliance on chemistry	Tethers can leave ‘chemical scars’ upon removal Incomplete tether removal affects downstream applications
Camena Bioscience101,128,133,134	Service provider	TiEOS with combinations of enzymes	0.3–3	High sequence accuracy (full-length sequences at 85% vs 23% by POS) Combination of different enzymes avoids dependence on TdT	Accuracy limited to <3 kb sequences Secondary structure formation of long G-rich sequences
DNA Script23,94,95	DNA printer	TdT with reversibly terminated 3ʹ-protected NTPs	0.01–3	Customized reagent kits, off the shelf solution for newcomers High-throughput benchtop synthesizers (96 oligos a run)	Printers print sequences of up to 80 nucleotides in length Secondary structure formation of long G-rich sequences
Evonetix51,52,149,150	DNA printer	Thermally controlled parallel synthesis	0.01–10	Site-selective, highly parallelized, high-density on-chip production Effective for the synthesis of GC-rich and repeat sequences	Insufficient thermolysis can reduce purity Challenging to differentiate product from mismatched sequences for long DNA
Molecular Assemblies129–131	Service provider	TdT with reversibly terminated 3ʹ-protected NTPs	1–10	Steric control over elongation and random polymerization Option for protected NTPs with free 3ʹ-OH	Subject to compatibility between re-engineered TdT and terminators Limited choice of cleavable terminators compatible with TdT
Ribbon Biolabs153	Service provider	Convergent Gibson assembly	1–10	High accuracy assembly under robotic control Synthesis of large DNA: first >10 kb DNA made	Reliance on large libraries of highly pure oligonucleotides High impact of mutations and frameshifts on synthesis
Touchlight Genetics173–176	Service provider	Templated rolling circle amplification	1–10	Rapid manufacturing of multigram DNA quantities DNA of practically unlimited lengths	Dependence on template production ‘Cleavage scars’ in resulting dbDNA
Twist Bioscience47–50	Service provider	Hybrid approach of POS, PCR and Gibson assembly	0.3–5	Low error rates in high-density microarray format Efficient solution to reduce depurination in each cycle	Low quantities of produced oligonucleotides Reliance on PCR to amplify DNA for gene assembly

3ʹ-OH, 3ʹ-hydroxyl group of the deoxyribose ring; C, cytosine; dbDNA, doggybone DNA; G, guanosine; NTP, nucleoside 5ʹ-triphosphate; POS, phosphoramidite oligonucleotide synthesis; TdT, terminal deoxynucleotidyl transferase; TiEOS, template-independent enzymatic oligonucleotide synthesis. ^aLengths depend on sequence composition and complexity. ^bConservative estimates of conceivable lengths by the given technology, and POS and TiEOS remain limited to <0.3 and <3 kb, respectively. ^cFew exemplars given in each case for simplicity, the exemplars may be common for different vendors using similar underpinning technologies.