DETECTION OF MUTATIONS IN COLON AND BREAST CANCER

(申请人描述)这个项目旨在开发两个新的 与结直肠癌相关的点突变检测技术 癌症和乳腺癌。我们将结合聚合酶链式反应 (PCR)、限制性内切酶消化反应(RE)和连接酶 检测K-ras和P53基因突变的临床应用 癌症患者的样本。 我们的具体目标是：(1)建立一种多重PCR/LDR方法 同时快速筛查多个突变等位基因 100-1000个正常细胞中1个癌细胞的敏感性，以及(Ii)对 建立高灵敏的PCR/RE/LDR方法鉴定特定突变体 等位基因在100,000-1,000,000正常人中的敏感性为1个癌细胞 细胞。将设计和测试连接引物以优化连接 多路传输的保真度和兼容性。突变型TTH DNA连接酶和 将测试含有引物的核苷酸类似物以提高PCR/LDR 敏感性和忠诚度。对于PCR/RE/LDR，一般方法是选择性地 将限制性内切酶切入来自野生动物的- 类型等位基因将使用K-ras和p53基因进行应用和测试。 多配对和非配对核苷酸类似物及其校对 将测试聚合酶对效率和 限制位点转换的特异性。 我们打算用临床样本来证明这种方法的可行性和 联合检测K-ras和P53基因突变的临床意义 大肠癌和乳腺癌的PCR/LDR和PCR/RE/LDR检测对于结肠癌， 我们将评估多重PCR/LDR作为一种定量突变的方法 K-ras和P53基因突变作为播散性肿瘤分子标志物的检测 有隐匿性微转移风险的组织中的癌细胞(淋巴结， 盆腔冲洗和骨髓抽吸)。此外，PCR/LDR和 PCR/RE/LDR将被评估为结肠癌的诊断测试，方法是 对粪便和结肠灌洗液样本进行K-ras突变筛查。

(Applicant's Description) This project aims to develop two new technologies for detection of point mutations associated with colorectal cancer and breast cancer. We will combine the polymerase chain reaction (PCR), restriction endonuclease digestion reaction (RE), and the ligase detection reaction (LDR) to detect K-ras and p53 mutations in clinical samples from cancer patients. Our specific aims are: (1) to develop a multiplex PCR/LDR method to screen rapidly and simultaneously for multiple mutant alleles at a sensitivity of 1 cancer cell in 100 - 1,000 normal cells, and (ii) to develop a highly sensitive PCR/RE/LDR method to identify specific mutant alleles at a sensitivity of 1 cancer cell in 100,000 - 1,000,000 normal cells. Ligation primers will be designed and tested to optimize ligation fidelity and compatibility for multiplexing. Mutant Tth DNA ligases and nucleotide analogue containing primers will be tested to improve PCR/LDR sensitivity and fidelity. For PCR/RE/LDR, a general method to selectively introduce restriction sites into the PCR product derived from the wild- type allele will be applied and tested using the K-ras and p53 genes. Multi-pairing and non-pairing nucleotide analogues as well as proofreading polymerases will be tested for their effect on the efficiency and specificity of the restriction site conversions. We intend to use clinical samples to demonstrate the feasibility and potential clinical relevance of mutation detection in K-ras and p53 using PCR/LDR and PCR/RE/LDR in colorectal and breast cancer. For colon cancer, we will evaluate multiplex PCR/LDR as a method for quantitative mutation detection in K-ras and p53 mutations as molecular markers of disseminated cancer cells in tissues at risk for occult micrometastases (lymph nodes, pelvic washings, and bone marrow aspirates). In addition, PCR/LDR and PCR/RE/LDR will be evaluated as diagnostic tests for colorecal cancer by screening stool and colonic lavage samples for K-ras mutations.