DETECTION OF MUTATIONS IN COLON AND BREAST CANCER

（申请人描述）本项目旨在开发两种新的 与结直肠癌相关的点突变检测技术 癌症和乳腺癌。我们将联合收割机 (PCR)、限制性内切酶消化反应（RE）和连接酶 检测反应（LDR），以检测临床中的K-ras和p53突变 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， 同时快速筛选多个突变等位基因， 100 - 1，000个正常细胞中1个癌细胞的敏感性，以及（ii）对 建立了一种高灵敏度的PCR/RE/LDR方法，以鉴定特异性突变体 等位基因的敏感性为100，000 - 1，000，000正常人中有1个癌细胞 细胞将设计并测试连接引物以优化连接 用于多路复用的保真度和兼容性。突变Tth DNA连接酶和 将测试含有引物的核苷酸类似物以改进PCR/LDR 灵敏度和保真度。对于PCR/RE/LDR， 将限制性位点引入来自野生型的PCR产物中， 型等位基因将被应用并使用K-ras和p53基因进行测试。 多配对和非配对核苷酸类似物以及校对 将测试聚合酶对效率的影响， 限制性位点转换的特异性。 我们打算使用临床样本来证明可行性， K-ras和p53突变检测的潜在临床相关性 PCR/LDR和PCR/RE/LDR在结直肠癌和乳腺癌中的应用 对于结肠癌， 我们将评估多重PCR/LDR作为定量突变的方法， 检测K-ras和p53突变作为播散性骨髓瘤的分子标志物 具有隐匿性微转移风险的组织中的癌细胞（淋巴结， 盆腔冲洗液和骨髓抽吸物）。此外，PCR/LDR和 PCR/RE/LDR将作为结肠癌的诊断检测进行评价， 筛查粪便和结肠灌洗样本中的K-ras突变。

(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.