Rational Design of High-Number DNA Fragments for Gene Construction

Gene synthesis is frequently used in modern molecular biology research either to create novel genes or to obtain natural genes when the synthesis approach is more flexible and reliable than cloning. DNA chemical synthesis has limits on both its length and yield, thus full-length genes have to be hierarchically constructed from synthesized DNA fragments. Gibson Assembly and its derivatives are the simplest methods to assemble multiple DNA fragments. Previously, up to 52 oligonucleotides have been assembled at once with Gibson Assembly, but that success is specific to a viral genome. Generally, the number of single-stranded oligonucleotides or double-stranded DNA fragments that can be assembled in a single reaction are much lower. We have developed a rational design method for gene construction that allows high-number DNA fragments to be assembled into full-length genes in a single reaction. Using this new design method and a modified version of the traditional Gibson Assembly protocol, we have assembled three different genes with up to 45 DNA fragments at once. Our design method uses the thermodynamic analysis software Picky that identifies all unique junctions in a gene where consecutive DNA fragments are specifically made to connect to each other. Our novel method is generally applicable to any gene sequences, and can improve both the efficiency and cost of gene assembly. This technology can be used to construct bacterial, animal and plant genes.