| Popis: |
Our novel, enzymatic DNA synthesis (EDS) technology was made through engineering of three critical components: a DNA polymerase, a solid support, and reversible terminators. Terminal deoxynucleotidyl-Transferase (TdT), which performs poly-nucleotide additions in a template-free manner, was engineered for improved expression, stability, and the ability to incorporate non-natural deoxynucleotide triphosphate bases. Novel surface chemistries have been developed to allow TdT to incorporate DNA bases on paramagnetic beads, resins, and glass as solid supports for EDS. Synthesis is enabled by the use of 3â€(2)-O-blocked reversible terminators, which suppresses poly-nucleotide addition by TdT until the 3'OH group on the extended polymer is deprotected. Here, we describe the development and key functionality of a benchtop EDS system for lab use. Unlike phosphoramidite synthesis, EDS produces DNA in the 'biological' orientation (i.e. 5'->3'), with an intact 5'-phosphate group. EDS provides a scalable synthesis system that eliminates the need for solvents, like acetonitrile, minimizing organic waste and decreasing research's carbon footprint, allowing users to make oligos without the need for specialized training and delivering oligos for iterative experiments without having to wait for a centralized oligo manufacturer. Current data indicates that purity levels comparable to those of existing technologies can be achieved, with an average per-position error rate |
