MALDI TOF TECHNOLOGY FOR MOLECULAR ANALYSIS OF CANCER

描述：(申请人描述) 这项应用是为了开发基于质谱学的技术 用于自动化、多路传输、高吞吐量、灵敏和特定的检测 在大背景下的一小群点突变的肿瘤细胞 野生型细胞。这项工作中开发的技术由三个主要部分组成 台阶。首先，利用多肽核酸扩增临床dna样本。 类似物(PNA)直接的聚合酶链式反应，其中突变的DNA 优先放大。其次，对扩增出的DNA片段进行延伸 通过微型测序产生诊断产品。第三，诊断 使用基质辅助激光识别产品- 解吸-电离飞行时间(MALDI-TOF)质谱仪和 因此，突变的存在和性质是确定的。我们的 初步结果表明，该方法能够鉴定突变株。 存在超过正常等位基因100,000倍的等位基因(在10 Ppm级别)。因此，下一个合乎逻辑的步骤是开发这种方法，并 探索其在癌症研究和检测方面的潜力。两个实验目标 将在这个项目中实现。首先，我们将证明这一点的可行性 使用两种方法识别各种致癌点突变的新技术 单项和多项检测。其次，我们将开发经过验证的检测方法 检测k-ras和p53中的两个热点突变 与癌症相关的最重要的基因。此应用程序由两个 阶段。第1年的研究是R21阶段，第2-3年的工作是R33 相位。这项技术具有巨大的潜力来推进癌症研究和 诊断。早期发现上皮癌是最常见的 这项技术的重要应用领域。上皮源性癌症 构成所有癌症的大多数，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、粪便和 可以采集其他标本，并在正常组织中寻找异常细胞 背景资料。这项技术还可以用来确定 特定的点突变在某些形式的癌症中更准确 举止。这将为癌症与基因的相关性提供洞察力。 细胞功能中的突变和遗传信息的处理。 另一个重要的应用是寻找通常发生在 通过对大量患者进行筛查，发现某些类型的癌症。这些 反过来，突变可以作为早期临床的癌症标记物。 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.