Long-chain DNA Synthesis Technology

For constructing long-chain DNA, the ordered gene assembly in Bacillus subtilis (OGAB) method, which we developed, was used.^1,2 The OGAB method is a method for assembling multiple DNA fragments using the plasmid transformation system of B. subtilis. Utilizing the specificity of 3 to 4 bases at the end of a DNA fragment, the method enables one-step assembly of a maximum of more than 50 DNA fragments. So far, this synthesis has been outsourced to a DNA synthesis company. When ordering the synthesis of 50 DNA fragments at a time, however, cases such as that where the synthesis of some fragments failed or was rejected occur although many DNA fragments are successfully synthesized. Accordingly, the synthesis sometimes takes as long as 2 months to finally prepare all the necessary DNA fragments.

Therefore, in order to shorten the time taken for DNA fragment preparation, a total system capable of one-stop processing, from the construction of long-chain DNA through chemical synthesis to mass preparation, was established, and technologies were developed from a comprehensive perspective. Individually, through joint research with Nihon Techno Service Co., Ltd., we have developed a high-throughput DNA chemical synthesizer specializing in the synthesis of long-chain DNA, at a low cost. In addition, we have also developed a new PCR method for annealing and elongating chemically synthesized DNA using DNA complementarity. This made it possible to prepare double-stranded DNAs of any sequence in about 3 days. In addition, the cloning process in Escherichia coli, in which this double-stranded DNA fragment is cloned in E. coli and only the selected clones having the correct base sequence are used as a material in the OGAB method, was substantially automated by a liquid dispensing robot. As a result of these research and development activities, it has become possible to produce long-chain DNA of about 30 kb in a short period of about 2 weeks at a cost of several yen per base.

Strengths in the Industrial Arena

The OGAB method allows inexpensive, short-time synthesis of long-chain DNA that could not be synthesized conventionally due to the long period of time required or high GC contents. The achievements from this research and development were commercialized as a long-chain DNA synthesis business by Synplogen Co., Ltd., a business venture spun off from Kobe University.