Gene synthesis by assembly of short oligonucleotides

In principle, a pre-constructed library of all possible short oligonucleotides could be used to construct many distinct gene sequences. This approach requires computational methods to accurately determine the assembly procedure, but relieves the current technological constraints of custom oligonucleotide synthesis. In order to assess the feasibility of such an approach, I examined T4 DNA Ligase activity on short oligonucleotides and found that ligation is dependent on the formation of a double-stranded DNA duplex of at least five base pairs flanking the site of ligation. However, ligations could be performed with overhangs smaller than five nucleotides and oligonucleotides as small as octamers, in the presence of a second, complementary oligonucleotide. As a proof of principle for DNA synthesis through the assembly of short oligonucleotides, I performed a hierarchical ligation procedure whereby octamers were combined to construct a target 128 bp segment of the human beta–actin gene coding sequence. Thus, the construction of synthetic genes, without the need for custom oligonucleotide synthesis, is feasible. Algorithmic methods were then developed to extend this approach to DNA on the order of thousands of base pairs.Science, Faculty ofGraduat