Miniaturized Integrated Genotyping Microsystems

描述(由申请人提供)： 这笔赠款的目标是开发可用于研究、发现和/或诊断环境中的癌症基因类型的高性能分析的新型微制遗传分析微系统和相关方法。最初的工作将集中于改进所需的仪器、试剂和方法，以便将多态比率测序(PRS)应用于使用常规毛细管阵列电泳对肿瘤组织中的线粒体DNA变异进行高通量遗传分析。我们将优化标签和合并方法，开发便捷的无线数据传输数据分析软件。然后，约翰霍普金斯大学(JHU)的科学家将接受在UCB进行PR的培训。最后，我们将把这项技术转移到JHU，用于其常规的高通量应用。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。这种晶片规模的设备采用RCA(滚圈扩增)准备的线粒体DNA，并将模板解析为96个单独的DNA测序模块，包括延伸反应器和CE分离通道，在不到1小时的时间内完成完整的线粒体PRS分析。一旦该系统被开发，第二代版本将被构建，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设备，从基因组DNA中进行SNP和其他基因分型。该设备将接受纯化的基因组DNA作为输入，并将单个样本解析到96个不同的PCR反应中，以进行多重等位基因特异性扩增和多态或癌症标记的分析。这种微型设备将允许从少量DNA中进行基因分型，并具有充分整合重要样本准备过程的大部分的优势，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，用于实时分析信息丰富的线粒体或基因组DNA变异或诊断标记。该系统将是有价值的(I)的护理点遗传分析，以确定癌症标记物的存在和/或监测可能的复发和(Ii)进行实时分子病理学组织样本，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.