The objective of this portion of the PPG 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 and/or diagnostic settings. Initial work will focus on the refinement of the apparatus, reagents and methods used to apply Polymorphism Ratio Sequencing (PRS) to the high-throughput genetic analysis of mitochondrial DNA variations in tumor tissue. We will optimize the labeling and pooling methods and develop convenient PRS data analysis software. Then JHU scientists will be trained at UCB on the performance of PRS, and we will transition the technique to JHU for its routine high throughput application. Second, we will work on the design, construction and evaluation of a fully integrated mitochondrial sequencing chip. This wafer scale device starts with RCA (rolling circle amplification) or long-range PCR amplified mitochondrial DNA and parses the template into 96 individual DNA sequencing modules, consisting of extension reactors and CE separation channels, to produce an entire mitochondiral sequence in under 1 hr. Once this system is developed, it will be transitioned to JHU for routine application. Third, we will develop a fully integrated microdevice for the performing SNP or other genetic typing from genomic DNA. This device will start with purified genomic DNA as the input and will parse the sample to 96 different PCR reactors for multiplex amplification and analysis of polymorphisms or markers. This microdevice will permit genetic typing from very small nL 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 mitochondiral DNA or genomic DNA. This system will be useful for point-of-care genetic analysis and for performing real-time molecular pathology of tissue samples.
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