Ligase-Free Synthetic Gene Assembly

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CONTEXTSynthetic biology is a pioneering avenue of research that traverses all of the disciplines of science to deliver new materials, pharmaceuticals, food and consumer products. An essential component to fabricating and applying biological systems in both research and commercial sectors is the ability to generate and manipulate DNA as a repository for genetic information. In order to make and modify proteins for biotechnological applications, the appropriate genetic code can be presented to an organism, however, chemists are still unable to synthesise sufficiently long DNA sequences to make entire genes and hence their useful protein products. In the past, biologists harnessed multiple enzymes, and a process called ligation, to fuse DNA segments into usable formats. Whilst this approach proved effective, there are still limitations in terms of time, efficiency and cost when applied to the large-scale synthesis of multiple genes. Hence there is now a clear opportunity to develop new, faster, accurate and cost-effective strategies for the complete synthesis of large DNA molecules using synthetic chemistry alone.AIMS AND OBJECTIVESOur project aims to employ readily available and inexpensive chemicals to connect small, overlapping strands of DNA in tandem to assemble synthetic genes. We will exploit our knowledge of how these chemicals behave and react in water to generate simple, efficient protocols for the gene assembly process. A range of chemical and biochemical techniques will be used to confirm that our ligation approach to strand joining has been successful. Ultimately we will prepare a synthetic gene and use it to make the protein that it encodes. The success of our research plan is based upon clear experimental evidence that demonstrates the validity of our strand ligation strategy. Our goal is to further enhance ligation efficiency by generating new and improved chemical building blocks to make the short DNA strands (known as oligonucleotides). These component oligonucleotides will be tested, the ligation conditions optimised, and then we will target our ultimate goal of a synthetic gene, to demonstrate the complete concept.POTENTIAL APPLICATIONS AND BENEFITSOur oligonucleotide ligation technology can be readily applied to DNA assembly to speed up and reduce the costs of gene and genome manufacture. Our approach will initially be targeted at improving gene synthesis, however, it could also benefit a wide range of nucleic acid-based approaches from molecular diagnostics to medical therapies and even in crop protection. Because our approach offers rapid chemistry, it will be ideal for monitoring large numbers of samples in parallel (high-throughput). It will also offer advantages to large-scale applications, such as the generation of larger quantities of ligated nucleic acids for gene therapies, and even for RNA-based crop protection agents.These applications offer economic, environmental, societal and medical benefits across a wide range of users.