Miniaturized Integrated Genotyping Microsystems

描述（由申请人提供）： 该资助的目的是开发新型微制造遗传分析微系统和相关方法，可用于研究，发现和/或诊断环境中的癌症基因型的高性能分析。最初的工作将集中在完善的仪器，试剂和方法，需要应用多态性比率测序（PRS）的高通量遗传分析的线粒体DNA的变化，在肿瘤组织中使用传统的毛细管阵列电泳。我们将优化标记和合并方法，并开发方便的PRS数据分析软件。然后，约翰霍普金斯大学（JHU）的科学家将接受培训，在UCB执行PRS。最后，我们将过渡到JHU的常规高通量应用的技术。其次，我们将设计，构建和评估一个完全集成的线粒体PRS芯片。该晶片级设备采用RCA（滚环扩增）制备的线粒体DNA，并将模板解析到96个单独的DNA测序模块中，包括延伸反应器和CE分离通道，以在1小时内产生整个线粒体PRS分析。一旦该系统开发完成，将构建第二代版本，然后在JHU用于高通量分析。第三，我们将开发一种完全集成的微型设备，用于从基因组DNA进行SNP和其他基因分型。该装置将接受纯化的基因组DNA作为输入，并将单个样品解析到96个不同的PCR反应器中，用于多重等位基因特异性扩增和多态性或癌症标志物的分析。这种微型设备将允许对少量DNA进行基因分型，并且具有完全集成大部分重要样本制备过程的优势，从而提供低成本、高通量的肿瘤样本基因分型。最后，我们将开发一种便携式基因分型设备，用于实时分析信息线粒体或基因组DNA变异或诊断标记。该系统将是有价值的（i）用于即时遗传分析以鉴定癌症标志物的存在和/或监测可能的复发，和（ii）用于执行组织样品的实时分子病理学以确定癌症侵袭的程度。

DESCRIPTION (provided by applicant): 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.