Nanobiosensing Method for Point Mutation Detection of Cancer

描述(申请人提供)：检测组织和体液DNA中的点突变在研究癌症的分子病因学以及为未来的临床应用开发新技术方面具有广泛的意义。在这项应用中，我们建议开发一种与临床相关的基因分析技术，该技术能够使用有限数量的临床样本对未扩增基因组DNA中的点突变进行多重检测。这种无需放大的检测技术将结合单分子检测(SMD)和量子点(QD)介导的荧光共振能量转移(QFRET)这两项创新技术来开发。初步研究结果表明，这种集成的SMD-qFRET技术能够在极低的浓度(~5fM)下检测DNA靶，而不需要靶扩增。当与等位基因特异的寡核苷酸连接结合时，这项技术可以检测到未扩增基因组DNA中的低丰度点突变。该项目由三个具体目标组成。首先，我们将建立一种无扩增点突变检测方法，并通过分析卵巢浆液性肿瘤未扩增基因组DNA中KRAS基因的四个有代表性的点突变(密码子12和密码子13)和常见的BRAF基因突变(密码子599)对其进行评估。其次，我们将通过优化QD介导的荧光能量转移系统的设计和连接反应条件，将这种新方法的灵敏度和分辨率分别提高到0.5fM和0.5%(突变体/野生型比例为1：200)。第三，通过以多路微流控格式实现这种新的检测方法，我们将提高分析吞吐量和分析的质量检测效率。我们将设计和制造一种微流控阵列设备，并使用它来分配和引导微量基因组DNA样本，用于同时进行七种突变检测的SMD光谱多重分析。与传统的基于聚合酶链式反应的突变分析方法相比，这一新技术将提供一种更快速、更可靠的方法，使用5？L或更少的试剂盒进行点突变检测。如果成功建立，它可以为癌症检测提供一个相对简单的分子诊断平台，并有可能在许多实验室和临床环境中进行。

DESCRIPTION (provided by applicant): Detection of point mutations in tissues and body fluid DNA have wide-spread implications in studying molecular etiology of cancer as well as in developing new technologies for future clinical applications. In this application we propose to develop a clinically relevant genetic analysis technology that enables multiplex detection of point mutations in unamplified genomic DNA using limited amounts of clinical samples. This amplification-free detection technology will be developed using a combination of two innovative technologies, single-molecule detection (SMD) and quantum dot (QD)-mediated fluorescence resonance energy transfer (qFRET). Preliminary studies have yielded promising results indicating that this integrative SMD-qFRET technology is able to detect DNA targets at extremely low concentrations (~ 5 fM), obviating the need for target amplification. When incorporated with allele-specific oligonucleotide ligation, this technology can enable detection of low- abundance point mutations in unamplified genomic DNA. This project consists of three Specific Aims. First, we will develop an amplification-free point mutation detection method and evaluate it by analyzing four representative point mutations in the KRAS gene (at codon 12 and codon 13) and one commonly occurring mutation in the BRAF gene (at codon 599) in unamplified genomic DNA from ovarian serous tumors. Second, we will enhance the sensitivity and resolution of this new method to 0.5 fM and 0.5 % (mutant/wild-type ratio of 1:200) respectively by optimizing both the design of the QD-mediated fluorescence energy transfer system and the ligation reaction conditions. Third, we will increase the analysis throughput and mass detection efficiency of the assays by implementing this new detection method in a multiplex, microfluidic format. We will design and fabricate a microfluidic array device and use it to dispense and guide micro-volumes of genomic DNA samples for multiplex analysis using SMD spectroscopy for seven mutation assays simultaneously. It is expected that, as compared to conventional PCR-based mutational analysis, this new technology will provide a more rapid and reliable measure in detecting point mutation using a 5 ?l or less assay volume. If successfully established, it could provide a relatively straightforward molecular diagnostic platform for cancer detection and can potentially be performed in many laboratories and clinical settings.