DNA Assay-on-a-String: Rapid Detection of Marker Panels Against Sepsis
Lead Research Organisation:
University of Birmingham
Department Name: School of Chemistry
Abstract
Initially the further development of the DNA-assay-on-a-string technique will be performed to contain single stranded "struts" suitable for binding corresponding sequences, unique to the organism's genes of interest. Adaptation of these structures, such as using antibodies, will also be explored so that larger molecules can be analysed. These structures will be analysed through current differentials. As the molecule passes through a nanopore the difference between a bound and unbound "strut" will generate a difference in the current observed, thus illustrating the presence or absence of the marker. To achieve this structure circular plasmid DNA will be used, extracted from bacteria using antibiotic resistance genes for selection, and linearised using restriction enzymes. Using complementary primers with long overhangs, the plasmid DNA can be separated and the primers annealed to complete the probe. The different probes and attachment chemistries, a high level of precision and low tolerance for design errors are particular challenges and highly novel. Initially data will be analysed manually to identify significant features useful for biomarker detection. However, due to the complexity of the data acquired from each experiment, it will be necessary to develop a convolutional neural network to obtain the full content and reduce analysis time. The initial understanding of the data will assist in the development of a neural network. Many repetitions of the acquisition will be performed (unbound DNA strands) to create a set of training data for MATLAB code to learn from. Testing data (bound or unbound DNA strands) will then be applied to the code to assess the ability of the code to identify differences between generated plots. The frequency of occurrence in an unknown sample in comparison to a known concentration of bound analyte could present a method to allow quantification of the analyte. The determination of upregulation and downregulation of specific genes or proteins will be possible. There are many biomarkers for sepsis for which this technique can be adapted to, this research will continue to develop this technique to trial and encompass as many of the most accurate biomarkers as possible. As many biological samples used for diagnostic purposes, such as blood, plasma, saliva and urine, contain a complex mixture of compounds, and present difficulty in direct sampling from the initial site, it is necessary to consider extraction and purification techniques. Initially it is possible to separate large compounds (proteins, metabolites and lipids), DNA and RNA using TRIzol reagent. Large compounds can be further separated using techniques such as ultra-centrifugation, HPLC size exclusion or free-flow electrophoresis. Once purified, it is necessary to consider effects of the solvent used for extraction on the results of the technique. There are several techniques by which to concentrate the analyte for later resuspension which include freeze drying and vacuum centrifugation. Both techniques preserve the analyte whilst removing solvent, and therefore will allow for resuspension in a suitable solvent system. DNA-assay-on-a-string could allow for both diagnostic and prognostic deductions and assist in treatment decisions.
Planned Impact
Discussion and consultation with our industrial partners, including major UK employers and SMEs, reveals a strong need for physical scientists trained at the interface with biology and medicine in order to maintain and increase UK competitiveness in biomedical technologies: an area of rapid growth with a global market already of more than $1 trillion. This same need is apparent in life science and healthcare industries with diverse business focuses, and these partner companies will be direct beneficiaries of our output of graduates trained in our CDT.
Our CDT will address this need by training across the disciplines and equipping the students with the research, career and communication skills needed to be the outstanding, flexible, innovative multidisciplinary researchers our partners require. Our graduates will be seeking to drive forward physical and computer science developments that forge healthcare advances and benefit society through enhanced health treatment and quality of life. They will be generating IP that benefits their employers and the UK economy. They will be excellent communicators able to inform and engage the public on the ethical issues of their work. And to support all these, they will have extensive networks of inter-personal contacts that will enhance and sustain their careers and impact. Thus the impact of our CDT training programme on UK industry and competitiveness will not just be during the CDT lifetime but far into the future.
Beyond our industrial partners and the UK economy, our students themselves will be beneficiaries because of the impact on their future careers and employment prospects. The interdisciplinary taught and research training and the career and communication skills trainings will give our students a unique and sought-after background and professional preparation. They will also benefit from the industrial input embedded in the training, the contacts with industrialists and public sector health researchers, and opportunities for site visits and placements in industry, public sector partners and overseas universities: All of these will provide contacts/platforms for the students to obtain positions (industrial or academic or public sector) on completion of their studies.
We will work in conjunction with our partner Thinktank, to communicate our results to the wider public. Our students will receive explicit training in public communication and put this into practice through a variety of Thinktank events. The ethos of wider communication we promote in our CDT will be embedded in those individual graduates throughout their subsequent careers, giving a sustained longer-term impact. Thinktank will benefit from the events that we hold in the museum and the displays we create with them, and the wider public will benefit not only through the events, but also in the longer term through the training of a generation of researchers who can discuss and engage the public on both the importance and ethical aspects that surround biomedical research and technology.
The research the students will undertake focuses on 3 key UK healthcare challenges: ageing, cardiovascular disease and emergency medicine. The state-of-the-art (2010) Queen Elizabeth acute hospital in Birmingham, one of Europe's largest hospitals and the regional centre for major trauma, is a partner in our programme and our supervisory team includes key on-site clinicians working in nationally important units on these topics. Their involvement, and those of our industry partners, provides a direct pathway to ensure developments can impact immediately on patients or be moved rapidly into clinical trials. Our training programme incorporates examples and requirements to move research from bench to bedside. Patentable outcomes will be exploited with UoB's Alta Innovations KT unit and brought to the attention of EPSRC and our partners. This impact will start during the CDT lifetime with potential to stretch far beyond.
Our CDT will address this need by training across the disciplines and equipping the students with the research, career and communication skills needed to be the outstanding, flexible, innovative multidisciplinary researchers our partners require. Our graduates will be seeking to drive forward physical and computer science developments that forge healthcare advances and benefit society through enhanced health treatment and quality of life. They will be generating IP that benefits their employers and the UK economy. They will be excellent communicators able to inform and engage the public on the ethical issues of their work. And to support all these, they will have extensive networks of inter-personal contacts that will enhance and sustain their careers and impact. Thus the impact of our CDT training programme on UK industry and competitiveness will not just be during the CDT lifetime but far into the future.
Beyond our industrial partners and the UK economy, our students themselves will be beneficiaries because of the impact on their future careers and employment prospects. The interdisciplinary taught and research training and the career and communication skills trainings will give our students a unique and sought-after background and professional preparation. They will also benefit from the industrial input embedded in the training, the contacts with industrialists and public sector health researchers, and opportunities for site visits and placements in industry, public sector partners and overseas universities: All of these will provide contacts/platforms for the students to obtain positions (industrial or academic or public sector) on completion of their studies.
We will work in conjunction with our partner Thinktank, to communicate our results to the wider public. Our students will receive explicit training in public communication and put this into practice through a variety of Thinktank events. The ethos of wider communication we promote in our CDT will be embedded in those individual graduates throughout their subsequent careers, giving a sustained longer-term impact. Thinktank will benefit from the events that we hold in the museum and the displays we create with them, and the wider public will benefit not only through the events, but also in the longer term through the training of a generation of researchers who can discuss and engage the public on both the importance and ethical aspects that surround biomedical research and technology.
The research the students will undertake focuses on 3 key UK healthcare challenges: ageing, cardiovascular disease and emergency medicine. The state-of-the-art (2010) Queen Elizabeth acute hospital in Birmingham, one of Europe's largest hospitals and the regional centre for major trauma, is a partner in our programme and our supervisory team includes key on-site clinicians working in nationally important units on these topics. Their involvement, and those of our industry partners, provides a direct pathway to ensure developments can impact immediately on patients or be moved rapidly into clinical trials. Our training programme incorporates examples and requirements to move research from bench to bedside. Patentable outcomes will be exploited with UoB's Alta Innovations KT unit and brought to the attention of EPSRC and our partners. This impact will start during the CDT lifetime with potential to stretch far beyond.
