Team:Rutgers

De novo DNA Synthesis is extremely important to the world of Synthetic Biology, but the low efficiency of today's DNA Synthesis technology limits us.

We can only synthesize about 150 bases at a time, so new genes have to be stitched together from smaller strands, which adds a lot to the time and cost required. This needs to change.

We currently use organic (anhydrous) solvents and reactive amidites to synthesize all custom DNA sequences, whether for antibody/enzyme engineering, multi-gene pathway building, or even genome construction. The process looks something like this:

This is one cycle in the process of DNA Synthesis. A solution of blocked nucleotides (whichever A, C, G, or T comes next in the sequence) is added to a group of immobilized DNA strands, and a single nucleotide is incorporated at the end of each strand. The trouble with this highly unnatural method is that each of the chemicals depicted (trichloroacetic acid, 2-benzylthiotetrazole, acetonitrile) causes some harm to the chemical structure of DNA, and this will limit the yield in a way that gets exponentially worse when building longer and longer pieces of DNA.

The process pictured above typically causes enough side-reactions to chemically "break" 1.5% of the existing DNA strands, in each and every cycle (leaving 98.5% of them intact and with correct sequence). This concept is called coupling efficiency, and it's the reason we can only synthesize up to 130-150 bases at a time (or even 200 bases if you're willing to pay a whole lot extra).

We believe that both cost and efficiency could be vastly improved by using enzymes for de novo DNA Synthesis. Enzymes that have evolved alongside nucleic acids for billions of years (polymerases) could be applied to eliminate side-reactions and significantly simplify the process overall. This process simplification could be beneficial on two counts: less complexity in required machinery would reduce capital costs, and less material requirements would reduce operational costs. One way to get huge gains in efficiency for any DNA-related process (see Sequencing, for example) ... With DNA Synthesis, this would mean switching from ... it may be prudent to begin investigating more natural, aqueous environments (that are chemically less harmful to the DNA during synthesis).

This is the enzymatic process that our team has envisioned. Note the

Over the summer we ran a number of assays to investigate the functionality of TdT in the context of our envisioned DNA Synthesis strategy. We used short, single-stranded primers (composed of 15 thymidine residues each) with 5' fluorescent tags (5'-FAM), and visualized the results on 22% polyacrylamide undenaturing gels. The images are oriented with the loading wells above, and the shortest oligos (which travel the fastest) end up furthest down in each lane. Each assay has a control lane with only the 15-length primers for reference.