The objective of this grant is to develop novel microfabricated genetic analysis microsystems and associated methods that can be used for high-performance analysis of cancer genotypes in the research, discovery and/or diagnostic settings. Initial work will focus on the refinement of the apparatus, reagents and methods needed to apply Polymorphism Ratio Sequencing (PRS) to the high-throughput genetic analysis of mitochondrial DNA variations in tumor tissue using conventional capillary array electrophoresis. We will optimize the labeling and pooling methods and develop convenient PRS data analysis software. Then, Johns Hopkins University (JHU) scientists will be trained to perform PRS at UCB. Finally, we will transition the technique to JHU for its routine high-throughput application. Second, we will design, construct and evaluate a fully integrated mitochondrial PRS chip. This wafer scale device takes RCA (rolling circle amplification) prepared mitochondrial DNA and parses the template into 96 individual DNA sequencing modules, including extension reactors and CE separation channels, to produce an entire mitochondrial PRS analysis in under 1 hr. Once this system is developed, a second-generation version will be constructed and then used at JHU for high-throughput analyses. Third, we will develop a fully integrated microdevice to perform SNP and other genetic typing from genomic DNA. This device will accept purified genomic DNA as the input and will parse the individual sample to 96 different PCR reactors for multiplex allele specific amplification and analysis of polymorphisms or cancer markers. This microdevice will permit genetic typing from small quantities of DNA and has the advantage of fully integrating a large portion of the important sample preparation process thereby providing low-cost, high-throughput genotyping of tumor samples. Finally, we will develop a portable genotyping device for real-time analysis of informative mitochondiral or genomic DNA variations or diagnostic markers. This system will be valuable (i) for point-of-care genetic analysis to identify the presence of cancer markers and/or to monitor possible recurrence and (ii) for performing real-time molecular pathology of tissue samples to determine the extent of cancer invasion.
