PhD in Molecular Biosciences, School of Biosciences (PhD Studentship)
Reference Number: R1052
Key Studentship Information
Project title: Digital BART: simple ultra-quantification of DNA targets
Project description: This project aims to develop a novel technique called Digital BART (Bioluminescent Assay in Real-Time) for detecting and quantifying specific DNA sequences at very low levels. Digital BART will enable real-time detection of DNA amplification reactions to give highly accurate quantitation of target DNA or RNA. BART uses light emission as the detection system, allowing far simpler instrumentation and thus greatly lower cost. Applications are in all areas of diagnosis and detection requiring accurate and sensitive determination of copy number, including copy number variation in disease diagnosis, viral load determination in animal, plant and human disease, and detection of food pathogens and contaminants.
Molecular diagnostics detects unique DNA or RNA sequences, normally using PCR (polymerase chain reaction) in a real-time variant known as qPCR (quantitative PCR). This requires sophisticated and expensive equipment, and has a limit of quantitation of around 50 copies, below which it cannot provide a reproducible measure of copy number present. A new technology known as digital PCR uses statistical presence and absence in thousands of simultaneous reactions at limiting dilution to calculate copy number with high accuracy, but is limited by high cost and sophistication of the instrumentation required.
BART is a well-established technology developed by the CASE partner Lumora Ltd based on light emission by firefly luciferase when a specific target DNA sequence is detected (further information can be seen here). An isothermal DNA amplification is used in which DNA is rapidly amplified in an exponential reaction using primer sequences specific for the target. Pyrophosphate is released during DNA synthesis which is converted to ATP and simultaneously detected by a temperature-stable firefly luciferase. A peak of light is emitted at a time dependent on the initial DNA target amount. This provides a limit of quantitation similar to qPCR.
BART is a simple yet well proven and commercialised technology that can be readily developed for digital approaches, since it is based on continuous isothermal amplification, not requiring thermal cycling. Secondly, the DNA amplification is reported by light emission which is readily detected by CCD chip or photodiodes. The project will develop the digital BART system and other appproaches to single copy DNA molecule detection. Parameters underlying the reactions will be investigated to provide the basis for establishing robust quantitative assays. The final output will be a new robust platform for measuring DNA target amounts to single copy at extremely high levels of accuracy, and at much lower cost than current technologies.
The two co-supervisors (Prof Jim Murray at Cardiff University and Dr Laurence Tisi at Lumora) were winners of the BBSRC Commercial Innovator of the Year 2012 for BART and this project represents a new direction for the technology. Murray’s lab occupies newly constructed and purpose-designed space with a very high standard of equipment and support. PhD students are supported by the presence of 3 Senior Research Associates and four experienced postdoctoral researchers. The group has wide collaborations with leading groups, and provides an exciting and stimulating environment for research training.
Eligibility and funding
This is a four year BBSRC Industrial CASE Award with Lumora Ltd starting on 1st October 2013. The annual stipend wil be at least £13,726.
Applicants will need a 2:1 (or equivalent) in their undergraduate degree in a relevant subject.
In general, UK and EU nationals resident in the UK for three years prior to the programme start date are eligible to apply (full details are available here).
How to Apply
Applications for this PhD studentship can be made through our Online Application Service. Please ensure you state in the 'Funding' section of the application that you intend to apply for this studentship.
Further application guidance from the School of Biosciences is available on this page.
For further information, please contact Professor Jim Murray via email (MurrayJA1@cardiff.ac.uk).