Nanobiosensing Method for Point Mutation Detection of Cancer

描述（由申请人提供）：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开发用于未来临床应用的新技术方面具有广泛的意义。在本申请中，我们提出开发一种临床相关的遗传分析技术，该技术能够使用有限量的临床样品对未扩增的基因组DNA中的点突变进行多重检测。这种免扩增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）两种创新技术开发。初步研究已经产生了有希望的结果，表明这种整合的SMD-qFRET技术能够检测极低浓度（~ 5 fM）的DNA靶标，从而避免了对靶标扩增的需要。当与等位基因特异性寡核苷酸连接结合时，该技术能够检测未扩增的基因组DNA中的低丰度点突变。该项目包括三个具体目标。首先，我们将开发一种无扩增点突变检测方法，并通过分析来自卵巢浆液性肿瘤的未扩增基因组DNA中KRAS基因（密码子12和密码子13）中的四个代表性点突变和BRAF基因（密码子599）中的一个常见突变来对其进行评估。其次，我们将通过优化量子点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0.5 fM和0.5%（突变体/野生型比例为1：200）。第三，我们将通过以多重、微流体形式实施这种新的检测方法来提高分析通量和测定的质量检测效率。我们将设计和制造一个微流控阵列装置，并使用它来分配和引导微体积的基因组DNA样品，用于同时使用SMD光谱进行七种突变检测的多重分析。预计，与传统的基于PCR的突变分析相比，这种新技术将提供一种更快速和可靠的检测点突变的措施，使用5？l或更少的测定体积。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.