MALDI TOF TECHNOLOGY FOR MOLECULAR ANALYSIS OF CANCER

描述：（申请人的描述） 本申请旨在开发基于质谱的技术 用于自动化、多路复用、高通量、灵敏和特异性检测 一小群点突变肿瘤细胞在一个大的背景下， 野生型细胞。这项工作中开发的技术包括三个主要方面 步首先，使用肽核酸扩增临床DNA样品， 类似物（PNA）指导的PCR夹持反应，其中突变DNA被 优先放大。第二，PCR扩增的DNA片段被延伸， 通过微型测序来产生诊断产品。三、诊断 使用基质辅助激光识别产品， 解吸电离飞行时间（MALDI-TOF）质谱法和 因此确定了突变的存在和性质。我们 初步结果表明，该方法可以鉴定突变体 等位基因在存在100，000倍过量的正常等位基因（在10 ppm水平）。因此，下一个合乎逻辑的步骤是开发这种方法， 探索其在癌症研究和检测中的潜力。两个实验目标 将在该项目中实现。首先，我们将证明这一点的可行性 一种新技术，利用两种方法来识别各种致癌点突变， 单一和多重测定。其次，我们将开发经过验证的检测方法， 检测k-ras和p53中的“热点”点突变， 与癌症相关的最重要的基因。此应用程序包括两个 阶段。第一年的研究是R21阶段，第二年至第三年的工作是R33阶段 相位这项技术在推进癌症研究和 诊断.早期发现上皮癌是最重要的 这项技术的重要应用领域。 上皮源性癌 占所有癌症的大多数，包括结肠癌和肺癌，其中 支气管镜活检、支气管肺泡灌洗、刷检细胞学、粪便和 可以采取其他标本，并且可以在正常细胞中寻找异常细胞。 背景该技术还可以用于确定 特定癌症中的特定点突变， 方式这将为癌症与基因的相关性提供见解 突变和细胞功能中遗传信息的处理。 另一个重要的应用是寻找突变，通常发生在 通过筛查大量患者来治疗某些类型的癌症。这些 反过来，突变可用作早期临床的癌症标志物 诊断.

DESCRIPTION: (Applicant's Description) This application is proposed to develop a mass spectrometric based technology for automated, multiplexed, high-throughput, sensitive, and specific detection of a small population of point mutation tumor cells in a large background of wild-type cells. The technology developed in this work consists of three major steps. First, the clinical DNA samples are amplified using the peptide nucleic analogues (PNA) directed PCR clamping reactions in which mutant DNA are preferentially amplified. Second, the PCR amplified DNA fragments are extended through mini-sequencing to generate diagnostic products. Third, diagnostic products are identified using matrix-assisted-laser- desorption-ionization time-of-flight (MALDI-TOF) mass spectrometry and therefore, the presence and nature of mutations are determined. Our preliminary results demonstrated that this approach could identify mutant alleles in the presence of 100,000-fold excess of normal alleles (at the 10 ppm level). Thus, the next logical step is to develop this method and to explore its potential in cancer research and detection. Two experimental goals will be achieved in this project. First, we will prove the feasibility of this new technology to identify various cancer-causing point mutations using both single and multiplex assays. Second, we will develop the proven assays for detection of the "hotspot" point mutations in both k-ras and p53, two of the most important genes related to cancers. This application consists of two phases. Research in Year 1 is the R21 phase and work in Year 2-3 is the R33 phase. This technology has great potential to advance cancer research and diagnosis. Earlier detection of epithelial cancers is one of the most important application areas of this technology. Epithelium-derived cancers constitute a majority of all cancers including colon and lung cancers, where bronchoscopic biopsies, bronchoalveolar lavage, brush cytology, stool, and other specimens can be taken and one can look for abnormal cells in the normal background. This technology can also be used to determine the frequency of a particular point mutation in certain forms of cancer in a more accurate manner. This will provide insights into the correlation of cancer with gene mutations and the processing of genetic information in cellular function. Another important application is to seek the mutations that commonly occur in certain forms of cancer by screening a large number of patients. These mutations, in turn, can be used as cancer markers for earlier clinical diagnosis.