From synthetic biology and gene therapy to data storage and biofuel production, the demand for synthetic DNA is rising.

Chemical synthesis

Phosphoramidite oligonucleotide synthesis is the gold-standard method for generating synthetic DNA. Scientists use the method to assemble modified nucleotides—phosphoramidite nucleosides—with the help of chemicals.1

          Infographic showing chemical DNA synthesis.

(1) Chemicals remove the 5’-hydroxyl protecting group.

The nucleosides are rich in exposed hydroxyl and amino groups, so scientists use protecting groups to prevent unintended reactions with compounds used in DNA synthesis. Chemicals remove the protecting groups to facilitate nucleoside coupling.

(2) Chemicals displace the diisopropylamino group.

(3) Chemicals cap unreacted 5’-hydroxyl groups.

An oxidation step creates a more stable coupling. The oligonucleotide is ready for another round of synthesis.

(4) Chemicals stabilize coupling.

Once the oligonucleotide reaches the desired length, additional chemicals remove the remaining protecting groups and cleave the chain from its solid support.

(5) Chemicals cleave the oligonucleotide and remove protecting groups.

(6) Continued exposure to harsh chemicals damages the DNA and reduces yields.  Scientists use short oligonucleotides as primers for polymerase chain reaction and next-generation sequencing, in DNA microarrays and fluorescence in situ hybridization, and in antisense therapies.

Enzymatic synthesis

Enzymatic DNA synthesis is an emerging technology that offers several advantages over chemical methods. Scientists use two main approaches to achieve template-independent oligonucleotide assembly.2

          Infographic showing enzymatic DNA synthesis

(1) Protected deoxynucleotide triphosphate (dNTP) and tethered dNTP approaches both use terminal deoxynucleotidyl transferase (TdT), a unique and specialized DNA polymerase that does not require a primer template to construct DNA.

(2) An incoming protected dNTP or tethered TdT-dNTP conjugate couples to the previously added base. 

(3) Mild, aqueous wash reagents.

The deblocking or untethering step is followed by a wash step to remove any lingering reagents, including blocking agents, enzymes, and unbound dNTP. The oligonucleotide is ready for another round of synthesis.

(4) A cleavage reagent separates the oligonucleotide from the base.

Once the desired length is reached, chemicals cleave the oligonucleotide from the solid support.

(5) Reactions occur in mild, aqueous conditions, which limits the use of harsh chemicals that cause DNA damage. Therefore, enzymatic approaches can generate longer strands, such as gene fragments, with a lower error rate.

  1. Hoose A, et al. DNA synthesis technologies to close the gene writing gap. Nat Rev Chem. 2023;7(3):144-161.
  2. Jensen MA, Davis RW. Template-independent enzymatic oligonucleotide synthesis (TiEOS): Its history, prospects, and challenges. Biochemistry. 2018;57(12):1821-1832.

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