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Lorenzo Berti with the University of California, Berkeley |
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Date: July 31, 2003
Time: 10:30am
Location: NASA LaRC, Bldg 1192C, Rm 102
Speaker: Lorenzo Berti with the University of California, Berkeley
Subject: "Probe Design for High-throughput DNA Detection"
The completion of the Human Genome was just the starting point for an even more challenging undertaking. Massive amounts of data still need to be collected in order to compile genomics databases, characterize diseases, develop recombinant therapies, characterize and exploit enzymatic processes in many organisms and develop new diagnostic assays to name but a few. To complete this otherwise daunting task in a reasonable time, we are now facing the development of high-throughput, ultra-sensitive cost-effective assays. The remarkable physico-chemical characteristics of DNA fall short of providing the properties needed for achieving ultra-sensitive detection. DNA must be chemically modified, in a way that does not change its properties in terms of base-pairing, but imparts to the molecule the necessary characteristics for obtaining high-sensitivity detection.
DNA modification with fluorescent labels proved to be an extremely valuable tool. Not only is fluorescence an intrinsically sensitive technique, but becomes exquisitely sensitive when a laser is employed as the excitation source. Furthermore, Fluorescence Resonance Energy Transfer (FRET) is a property that can be exploited for achieving multiplexing detection capabilities and overcoming the labeling complexities associated with modern instrumentation. This has been the major focus of our work. We have successfully employed FRET in developing primers used in DNA sequencing. In order to overcome the problems associated with the high synthetic costs and low versatility of such primers, we have also developed a universal FRET label, that can be linked to any primer or other target bearing an appropriate functionality. The design and synthesis of this label have been optimized, employing a combination of solid-phase automated synthesis and bio-conjugation chemistry. Also, a protocol for coupling this label to the target of interest has been developed. Our universal FRET label has been successfully employed for a variety of DNA-based diagnostic assays such as fragment sizing, allele-specific amplification and SNP detection. On another front, electrochemical detection has also been explored. By coupling redox labels to DNA probes it is possible to develop compact and inexpensive systems for DNA detection, although lacking the sensitivity of the fluorescence based approach.
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