Detection of point mutations in tissues and body fluid DNA have wide-spread implications in studying molecular etiology of cancer as well as in developing new technologies for future clinical applications. In this application we propose to develop a clinically relevant genetic analysis technology that enables multiplex detection of point mutations in unamplified genomic DNA using limited amounts of clinical samples. This amplification-free detection technology will be developed using a combination of two innovative technologies, single-molecule detection (SMD) and quantum dot (QD)-mediated fluorescence resonance energy transfer (qFRET). Preliminary studies have yielded promising results indicating that this integrative SMD-qFRET technology is able to detect DNA targets at extremely low concentrations (~ 5 fM), obviating the need for target amplification. When incorporated with allele-specific oligonucleotide ligation, this technology can enable detection of low- abundance point mutations in unamplified genomic DNA. This project consists of three Specific Aims. First, we will develop an amplification-free point mutation detection method and evaluate it by analyzing four representative point mutations in the KRAS gene (at codon 12 and codon 13) and one commonly occurring mutation in the BRAF gene (at codon 599) in unamplified genomic DNA from ovarian serous tumors. Second, we will enhance the sensitivity and resolution of this new method to 0.5 fM and 0.5 % (mutant/wild-type ratio of 1:200) respectively by optimizing both the design of the QD-mediated fluorescence energy transfer system and the ligation reaction conditions. Third, we will increase the analysis throughput and mass detection efficiency of the assays by implementing this new detection method in a multiplex, microfluidic format. We will design and fabricate a microfluidic array device and use it to dispense and guide micro-volumes of genomic DNA samples for multiplex analysis using SMD spectroscopy for seven mutation assays simultaneously. It is expected that, as compared to conventional PCR-based mutational analysis, this new technology will provide a more rapid and reliable measure in detecting point mutation using a 5 ?l or less assay volume. If successfully established, it could provide a relatively straightforward molecular diagnostic platform for cancer detection and can potentially be performed in many laboratories and clinical settings.
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