NEW METHODS FOR CANCER DETECTION

(申请人描述) 该应用程序目标是开发两个创新的突变 检测系统可能成为治疗艾滋病的强大临床工具 人类癌症的诊断和分期。癌症是由 多个基因的突变累积，但没有单一基因 在成人癌症中总是发生突变。因此，要识别索引突变， 对于原发肿瘤，迫切需要开发一种方法来 快速检测许多基因的FIR突变。发现了一种特殊的突变 在原发肿瘤中可作为肿瘤细胞的克隆性标志物； 然而，对微转移的筛查带来了额外的问题 在正常细胞的大量背景中检测突变。 我们已经组建了一支调查小组，他们的专业知识将被 朝着这两个问题的综合解决方案迈进。我们一直专注于 两个基因，K-ras和p53，是最常见的突变之一 结肠癌和乳腺癌。在结肠癌中，这两个基因的突变 高度聚集在特定的密码子上，因此它们是一个理想的模型 为我们的癌症证明“原则性证据”的系统 相关的突变检测方法。 我们的战略方针是：(I)开发多聚合酶链条 反应/连接酶检测反应(PCR/LDR)体系的建立 K-ras和p-53基因在肿瘤中的突变。(2)发展以下能力 识别敏感性为10万个癌细胞中有1个的突变- 1,000,000个正常细胞使用我们的PCR/限制性内切酶/LDR(PCR/RE/LDR) 方法。(Iii)设计和合成核苷酸类似物以转化为 将特定的DNA序列插入限制性内切酶位点，并 提高TTH DNA连接酶的保真度。(四)发展以下能力 以100-1000个癌细胞中1个的灵敏度检测多个突变 正常细胞使用突变的TTH DNA连接酶，含核苷酸的引物 类似物，或其他Thermus DNA连接酶。(V)制定方法，以便 使用可寻址技术同时检测大量LDR、产品 寡核苷酸或多肽核酸阵列。(六)探索能力 为了多重PCR/LDR以筛查原发结肠肿瘤中的突变， 粪便和结肠灌洗样本。(七)探索PCR/RE/LDR的能力 确定已知突变为隐匿性肿瘤的克隆标记物 转移到结肠癌(淋巴、大便和盆腔冲洗) 乳腺癌(淋巴、骨髓)。

(Applicant's description) The goal of this application is to develop two innovative mutation detection systems that may become powerful clinical tools for the diagnosis and staging of human cancers. Cancer results from the accumulation of mutations in multiple genes, however no single gene is always mutated in adult cancers. Thus, to identify an index mutation in primary tumors, there is an urgent need to develop a method that can rapidly survey fir mutations in many genes. A specific mutation identified in the primary tumor can be used as a clonal marker for tumor cells; however, screening for micrometastases poses the additional problem of detecting the mutation in a vast background of normal cells. We have assembled a team of investigators whose expertise will be directed toward an integrated solution to these two problems. We have focused on two genes, K-ras and p53, which are among the most commonly mutated in colon and breast cancers. In colon cancers, mutations in these two genes are highly clustered to specific codons, and thus they are an ideal model system in which to demonstrate "proof of principle" for our cancer associated mutation detection methods. Our strategic approach is to: (i) Develop a multiplex polymerase chain reaction/ligase detection reaction (PCR/LDR) system for the detection of K-ras and p-53 mutations in tumors. (ii) Develop the capability to identify mutations at a sensitivity of 1 cancer cell in 100,000 - 1,000,000 normal cells using our PCR/restriction enzyme/LDR (PCR/RE/LDR) method. (iii) Design and synthesize nucleotide analogues to convert specific DNA sequences into restriction endonuclease sites, and to increase the fidelity of Tth DNA ligase. (iv) Develop the capability to detect multiple mutations at a sensitivity of 1 cancer cell in 100 - 1,000 normal cells using mutant Tth DNA ligases, primers containing nucleotide analogues, or other Thermus DNA ligases. (v) Develop methods for the simultaneous detection of large amounts of LDR, products using addressable oligonucleotide or peptide nucleic acid arrays. (vi) Explore the ability to multiplex PCR/LDR to screen for mutations in primary colon tumors, stool and colonic lavage samples. (vii) Explore the ability of PCR/RE/LDR to identify known mutations as clonal markers of occult tumor dissemination to colon cancer (lymph nodes, stool, and pelvic washings) and in breast cancer (lymph nodes, bone marrow).