The broad, long-term objectives of this proposal are to develop a new highly sensitive micro array technology to enable the accurate detection of weakly expressed genes from very limited amounts of biological material. This will enlarge the field of application of the DMA chip technology significantly.
The specific aims are 1) to develop a robust protocol to increase micro array sensitivity by seven orders of magnitude compared to current technology, 2) to design and validate a library of tag sequences used to generate a combinatorial library to be used in the amplification process and 3) to conduct three series of experiments to validate our approach and demonstrate its broad range of application. The health relatedness of this project is to provide molecular biologists with a unique resource to detect weakly expressed genes with an unmatched sensitivity from a very low amount of starting material. Numerous studies on the initiation and progression of diseases where key genes are expressed at levels below current detection limit will greatly benefit from this new technology. A better understanding of molecular mechanisms involved in these diseases will undoubtedly speed up the discovery of new drugs. Our technology will also make possible the development of new molecular diagnosis tests compatible with the low amount of material available from the lesser invasive biopsy techniques. The research plan consists of three stages of amplification. The first amplification is during the reverse transcription by using gene specific primers combined to a reporter sequence, the second amplification is by doing an RNA amplification of this reporter and the final amplification is by using a high-sensitivity signal detection based on the resonance light scattering properties of metal nanoparticles. The experimental approach will involve the development of techniques to label mRNA with specific tags and amplify these tags with the T7 RNA polymerase. This will be followed by an indirect labeling with gold and silver particles, which generate a higher signal than fluorescent dyes. A collection of tag sequences will be validated by hybridization after T7 amplification. Repeating a published experiment using a much lower amount of material will validate our technology. Further validation will be by conducting new experiments to detect weakly expressed genes involved in adipogenesis, and to monitor bacterial gene expression in infected host cells. ? ? ?
