Partenaires et organisations internationales
(Anglais)
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INSERM U468 (F), CNR-IBRM (I), MPIGM-Berlin (D), InnoSense (I), Imstar (F)
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Résumé des résultats (Abstract)
(Anglais)
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The objectives of this proposal are to develop new and powerful parallel genetic analysis procedures based on the use of the DNA mimic, peptide nucleic acid (PNA) as probes for mutations in high throughput capillary electrophoresis (CE) and microarray biochip systems. They can be summarised as follows:
1. To produce a library of PNA probes designed to detect specific gene mutations in microarray- and capillary electrophoresis-based diagnostic systems. To this end, new methods to attach PNAs on glass slides are under development and capillary electrophoresis procedures suited to distinguish normal and mutant sequences in the Cystic fibrosis gene will be implemented.
2. To improve the performance of capillary electrophoresis procedures by developing new coatings and matrices, and by adapting to a fully automated 96-capillary device the mutation screening methods developed for a single capillary unit.
3. To design and build robotic systems for PNA array manufacturing.
4. To develop new fluorophores, and build innovative, robust and fully automated detection systems.
The deliverable produced will be evaluated in the clinical laboratory setting at various steps of the project.
Results and Milestones:
During the first year of work, the proposed workpackages have been completed. This project exploits the inherent advantages of a new kind of probe, Peptide Nucleic Acids (PNA). Because of the nature of its neutrally charged chemical backbone, the PNA-DNA bond is stronger than the DNA-DNA bond with normal probes.
Several PNA probes, corresponding to various CFTR (cystic fibrosis transmembrane conductance regulator) gene mutations have been synthesised. They were tested in hybridisation with DNA samples bearing or not specific Cystic Fibrosis mutations, and the hybrid molecules were separated using capillary electrophoresis. Our results show that a good discrimination of the various molecular species can be achieved, paving the way for further developments based on this approach.
In parallel, with the aim to improve capillary electrophoresis, new polymers with a low viscosity and high sieving capacity have been produced along with new coatings molecules. These reagents were shown to be suitable replaceable matrices, enlarging the panel of sieving polymers available for separation of DNA.
Another challenge of this project is to build new robotic systems for microarray fabrication and analysis. A first prototype of a microarrayer was designed, built, and evaluated in comparison to several instruments that are presently commercially available. A 96 channels capillary electrophoresis device, suited to high throughput mutation detection, is being improved. A confocal laser scanner is under construction and a prototype of a DNA chip reader, based on the use of a cooled CCD camera, was built and is currently evaluated. The design and development of a set of appropriate imaging software are in progress.
Finally, two new dyes, suited to the analysis of DNA hybrid molecules, were developed (called FAR-Fuchsia and FAR-Blue). Their evaluation against commercially available comparable reagents demonstrated their usefulness and advantages.
Benefits and Beneficiaries:
The instruments and reagents under development are expected to improve the efficiency, reliability and throughput of molecular diagnostic procedures. The beneficiaries are molecular diagnostics laboratories, research centres, and all organisations involved in genotyping.
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