How are custom genes built one nucleotide at a time?

In the field of synthetic biology, the ability to build custom genes from scratch, or de novo, relies on automated chemical synthesis technologies.1

The process of building DNA one nucleotide at a time was pioneered in the 1970s by Har Gobind Khorana, who utilized directed chemical synthesis to generate the first synthetic gene. This approach was significantly refined by subsequent developments, most notably the phosphoramidite method introduced in the early 1980s by Marvin H. Caruthers.2

Modern synthesis is largely automated and involves the following principles:
* Solid-phase synthesis: Building the DNA chain on a solid support, which allows for efficient chemical reactions and the removal of byproducts.2
* Stepwise addition: Nucleotides are added sequentially to the growing chain. The phosphoramidite method provides a stable and efficient way to link these units.2
* Chemical protection: Intermediate steps use specific chemical groups to protect the phosphate and other reactive sites, ensuring that nucleotides are added only at the desired position and preventing undesirable side reactions.2

These advancements allow scientists to manufacture novel DNA sequences that can be used to create complex organic compounds tailored for specific biological functions.1