Gene synthesis encapsulates two discrete processes: the de novo synthesis of short single-stranded DNA oligonucleotides (oligos) and their subsequent assembly into gene-length dsDNA fragments. The technologies for each process were pioneered concurrently; in fact, the rst synthetic genes were reported before the development of the solid-phase phosphoramidite chemistry that dominates modern oligo synthe­sis platforms. In this section, we review the key advances that inspired the gene synthesis technologies used today.

In the mid-1970s, Letsinger and colleagues debuted a new, highly efcient oligo synthesis approach based on phosphite triester chemistry [7]. This strategy enabled rapid oligo synthesis through the use of highly reactive nucleosides (3′-O-chlorophosphites). Although these intermediates hastened the synthesis reac­tion, they were also difcult to handle [4]. Beaucage and Caruthers [8] addressed this problem by developing the phosphoramidite chemistry that forms the basis of modern chemical oligo synthesis platforms.

Oligos can be synthesized in solution or covalently attached to solid supports (i.e., solid-phase synthesis). These methods differ substantially in the time and resources they require [2]. Whereas solution-based synthesis requires extensive purication at multiple steps in the synthesis cycle, solid-phase synthesis eliminates these time­consuming steps entirely by providing a simple means of separating oligos from reagents, solvents, and by-products. In addition, solid-phase synthesis supports the synthesis of the microgram amounts of DNA—an adequate, yet not excessive, amount for a typical biological experiment.

Interest in silica-based supports—namely, controlled-pore glass (CPG)— renewed in the 1980s alongside the development of phosphite triester and phos­phoramidite chemistries [4]. Unlike earlier supports, CPG was not prone to compaction, making it ideal for use under the continuous ow conditions used in automated synthesizers.

Increasing the efciency of the stepwise, phosphoramidite-based oligo synthesis reaction represents a second area of innovation. Patterned substrates (for light-based chemistries) [24, 25] and highly patterned chips (for inkjet synthesizers) [26] have provided better control over spatially controlled steps in synthesis reaction, reduc­ing the number of errors caused by misalignment. The efciency of the subsequent coupling and capping steps has also improved. A coupling efciency of >99.5% is a requirement for the synthesis of long oligos, and even small (~0.1%) improve­ments to this metric can enable synthesis of longer oligos with fewer errors. (Conversely, even small reductions in coupling efciency can dramatically decrease the length at which quality oligos can be synthesized.) These outsized effects reect the compounding of miniscule inefciencies across the stepwise synthesis of long oligos. In other words, as the length of the synthesized oligo increases, the more apparent small differences in reaction efciencies become. Today’s high- throughput synthesizers are highly efcient, reaching coupling efciencies as high as 99.9% (Twist Bioscience).