The proposed project aims are two-fold:
the development of alternative DNA labelling methodologies that will reduce the complexity and cost of current conventional DNA sequencing systems;
the parallel development of a new non-gel principle by which fluorescently labelled DNA (generated by polymerase chain reaction (PCR) techniques) can be detected allowing the direct sequencing and mapping of DNA fragments in solution.
A comprehensive feasibility study on the use of the Centre for Applied Microbiology and Research (CAMR) optical aperture detection system for a method to enable rapid, 1-step mapping of deoxyribonucleic acid (DNA) both physical and genetic mapping of large (10sKb) DNA fragments is underway. Initial objectives are:
use of high energy electron beam lithography for fabrication of test submircon aperture in opaque screens; design and construction of optical launch and detection system; immobilization of DNA binding proteins onto model substrate matrices (simulating their future use in apertures on solid substrates to permit monitoring of sequential addition of fluorescently labelled bases to single stranded DNA and hence determine sequence); selection, design and construction of model DNA fragments for calibration purpose for use in the CAMR aperture detection system; and selection of applicable fluorophores for coupling to different components of sequencing systems.
Installation and commissioning of a complete Electron Beam Lithography facility for use in this project is being completed. Facilities include thin film (thermal and spin) coating plants, electron beam pattern generation, plasma and chemical etching systems, SEM, clean room, etc. This facility will enable the rapid and dedicated fabrication of sub-micron structures for use throughout this single DNA molecule characterisation project.
The project will run on three fronts.
The Biotechnology Institute (Copenhagen) and subcontractors will investigate methods of DNA labelling using non-laser excited DNA. Base specific photochemical cleavage of DNA will be used as the basis of alternative methods to classical DNA sequencing.
In parallel, a new method for the direct sequencing of solution phase DNA fragments will be developed by CAMR. The technique will utilise recent advances made in the production of optical apertures of similar size to DNA fragments. Thereby, the optical detection of individual, solution phase DNA molecule fragments generated and labelled by the novel chemistries developed at GEG and using PCR methods, will permit non-gel mediated sequencing of DNA.
This work will rely on the second phase (year two) development of comprehensive data analysis systems (developed by the software house, Optimum Ltd, Greece) employing sophisticated detection (opto)electronics and computer software packages capable of the rapid assimilation of real time generated from solution phase sequencing.
Funding SchemeCSC - Cost-sharing contracts
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