Enzymatic de novo DNA Synthesis Methods

Synthetic DNA is required for nearly all aspects of biological research and bioengineering. Essentially all contemporary de novo DNA synthesis begins with oligonucleotides produced using the nucleoside phosphoramidite method. Despite its remarkable performance for a stepwise synthesis chemistry — often achieving >99% stepwise yields — the phosphoramidite method has certain limitations, including that it is in practice limited to the direct synthesis of ~200mers by side reactions and produces hazardous waste. Synthetic DNA molecules longer than 200 bp must be assembled from oligonucleotides, but the assembly process can introduce errors and is not amenable to all sequences, so new de novo DNA synthesis methods are needed.Here, I present two novel de novo DNA synthesis methods based on enzymes. Unlike the phosphoramidite method, these enzymatic methods operate in aqueous buffers and need not create hazardous waste. They also both take advantage of the exquisite specificity of enzymes to eliminate side reactions. The first method is based on deoxyribonucleoside triphosphate (dNTP) conjugates of the template-independent polymerase TdT, which extend a single-stranded DNA molecule by one nucleotide. The second method is based on ligation of small duplex DNA “extension units” that extend a growing DNA molecule by a defined sequence. Both methods can be iterated to synthesize a defined sequence.A single DNA molecule can be amplified more than a million-fold without many errors, so in principle only one “perfect” molecule must be synthesized. Exploiting the unique possibilities offered by this ability, I present a new principle for “real-time error correction” of single-molecule stepwise DNA synthesis that aims to enable direct synthesis of longer DNA molecules than is currently possible with any method