Fast Mutation Detection by Tandem SSCP/HA on Microchips

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DESCRIPTION (provided by applicant): It is proposed to optimize, evaluate, and pilot rapid, scalable, and low-cost microchip electrophoresis technologies for sensitive and specific molecular detection of cancer by tandem single-strand conformational polymorphism (SSCP)/heteroduplex analysis (HA), using the p53 gene as a model system. We request a 1-year R21 phase and a 3-year R33 phase. The proposed project involves collaboration between members of Northwestern's Lurie Comprehensive Cancer Center, including researchers in Chemical Engineering, the Medical School, and Evanston Hospital. Microchannel "tandem" SSCP/HA is a novel mutation detection method recently developed in our laboratory, which involves the simultaneous generation and analysis of homo/heteroduplex DNA and SSCP conformers. Studies of a significant number of samples (32) indicate that tandem SSCP/HA allows for much higher-sensitivity mutation detection (100%) than SSCP alone (93%) or HA alone (75%), for p53 samples. We have developed and published optimized sample preparation protocols, gel formulations, and analysis conditions for capillary array electrophoresis (CAE). During the R21 phase, we will translate these methods to microfluidic electrophoresis chips, which offer a large increase in throughput and drop in cost of DNA analysis compared to CAE. The p53 gene, known to be mutated in >50% of human cancers, and whose mutation status can be predictive of patient response to chemotherapy, is the important model system chosen. However, microchip-based genetic analysis technologies to be developed should be easily applied to ANY cancer-related gene. In the R21 phase, we will analyze approximately 60 different DNA samples derived from tumor cell lines, representing a range of mutations in different p53 exons, to determine the impact of DNA sample characteristics and electrophoresis protocols on the sensitivity and specificity of the method, in a blinded study designed by collaborating biostatisticians. When optimized tandem SSCP/HA protocols have been developed for microchips, they will be piloted by the analysis of >200 selected samples amplified from frozen, solid tumors banked at Evanston Hospital. Via this blinded study, sensitivity and specificity (both expected to be at or near 100%) will be determined and reported for the first time using banked tumor tissue, providing necessary validation for clinical application of this technique, and making rapid, low-cost cancer genotyping technology widely available to physicians.