Next Generation DNA Synthesis

Ever large pieces of DNA such as genes and gene clusters are required for Synthetic Biology, and these are normally made by a combination of chemical and biochemical methods. The chemical methodology is required at the start of the process to generate very short pieces of DNA (oligonucleotides) by automated solid-phase methods. These are then used to build bigger pieces of DNA by biochemical methods that are based on the polymerase chain reaction (PCR amplification). The chemical synthesis of DNA can lead to damage which results in mistakes (mutations) in the final DNA product, and to avoid this the DNA has to be repaired by various enzymes. This is tedious and slows down the overall process, increasing costs and limiting the size of DNA that can be made. In this project we will analyse DNA made by modern ultra high throughput chemical methods and optimise the process to minimise mutations. We will also explore a different way to make large pieces of DNA; enzymatic ligation. In this process DNA constructs with modified bases can be made, which are very useful in gene expression and biomedical studies. These cannot be made by PCR amplification which erazes the modifications. Such modified DNA can only be properly made from highly pure oligonucleotides in very large numbers, placing stringent requiremenst on high-throughput oligonucleotide synthesis. Overall this project will greatly increase the capacity, quality and efficiency of DNA synthesis and is highly relevant to Synthetic Biology Centres in the UK and beyond.

Current DNA synthesis strategies start by building a series of 1000-mer DNA duplexes by PCR from synthetic ODNs, then using these to produce increasingly large DNA fragments. This process is made tedious by the limited size of the initial PCR products. Although PCR can synthesise much larger constructs the choice of length is influenced by the number of mutations present in the original ODNs, and the difficulty in editing these out. No matter what detailed PCR strategy is used, every base in the PCR product originates directly from a synthetic ODN, or utilises a nucleobase in a synthetic template in the initial cycles of amplification. This places stringent requirements on synthetic ODNs that are used in DNA synthesis. Using current ODN synthesis methods, around one mutation in 500-1000 bases is generated via chemical damage to the ODN. The mutations have to be corrected, and specific techniques have been developed for this, none of which are highly efficient. Consequently this is a time consuming and expensive bottleneck in DNA synthesis. The problem will become more severe with increasing pressure for cheap high-throughput ODN synthesis. Therefore there is an urgent requirement for a systematic study of the nature and frequency of mutations arising from synthetic ODNs. If the chemical lesions that occur in synthetic ODNs can be characterised in detail it should be possible to modify the methodology to minimise them. There is another pressing need for improved ODN synthesis; DNA synthesis by ligation will become increasingly important in the near future when DNA constructs containing epigenetic markers and other modified nucleobases are required. Clearly such constructs cannot be made entirely by PCR. Ligation-based DNA synthesis is particularly sensitive to ODN purity and integrity of length, so improved synthesis methods need to be developed. We will undertake a systematic study on the mutations occurring during ODN synthesis and then optimise the procedure.

We will play a leading role in the development of improved methods of DNA synthesis for Synthetic Biology applications through evaluation and optimisation of oligonucleotide synthesis chemistry. Our goal is to contribute to the expansion and deployment of Synthetic Biology in healthcare, agriculture and other areas to the advantage of industry, academic researchers and society. Our DNA synthesis capacity and new developments will be relevant to the following sectors:
1. Industry. Advances in DNA synthesis will help enable UK based pharmaceutical and agrochemical industries to obtain maximum benefit from adoption of Synthetic Biology, ensuring that societal benefits are delivered quickly and effectively. Materials, tools and methods created by us (including cheap large DNA constructs and modified aptamers) will allow more robust control of cellular processes such as gene expression, bringing about a step change in the effectiveness of Synthetic Biology in microbial, animal and plant host organisms and impacting applications in the pharmaceutical and agricultural sectors in particular.
2. Academic. We will help to ensure that the UK maintains and enhances its leading international position in this vitally important new field by improving methodologies and increasing DNA synthesis capacity. Synthetic Biology has the potential to provide solutions to key societal challenges including those in healthcare and food science: our work will impact on existing UK and International Synthetic Biology Centres, thereby leading to an increase in our knowledge of fundamental biology and of how the many different aspects of cellular physiology interact and are regulated. Some of this new knowledge will find therapeutic and diagnostic applications.
5. Training. We will train early career researchers from a variety of backgrounds in nucleic acid chemistry and DNA synthesis. They will receive outstanding training in interdisciplinary research that is linked to recent developments in Synthetic Biology. They will be ideally positioned to translate Synthetic Biology research into industrial practice and to continue to enhance the UK's leading position in Synthetic Biology research. We will contribute to a world-leading concentration of research excellence that will underpin recent RCUK investment in doctoral training, in particular through the Oxford/Bristol/Warwick EPSRC & BBSRC CDT in Synthetic Biology.
6. Sustainability. We will expand beyond the boundaries of the initial funding to develop a major Centre of Excellence in DNA chemistry and DNA synthesis for Synthetic Biology, seeking funding from a variety of sources including industry. We will welcome visiting scientists from industry, including pharma, biotech, emerging SMEs and from other academic research centres.