NEW METHODS FOR CANCER DETECTION

（申请人描述） 这项申请的目标是开发两种创新的突变 检测系统，可能成为强大的临床工具， 人类癌症的诊断和分期。 癌症是由 多个基因突变的积累，但是没有一个基因是 总是在成人癌症中发生突变因此，为了鉴定索引突变， 因此，迫切需要开发一种方法， 快速调查许多基因的突变。一种特殊的突变被发现 在原发肿瘤中可作为肿瘤细胞的克隆性标志物; 然而，微转移的筛查带来了额外的问题， 在大量正常细胞的背景中检测突变。 我们已经召集了一个调查小组， 这两个问题的综合解决方案。我们专注于 两个基因，K-ras和p53，这是最常见的突变， 结肠癌和乳腺癌。在结肠癌中，这两个基因的突变 高度聚集到特定的密码子，因此它们是理想的模型 证明我们癌症的“原理证明”的系统 相关突变检测方法。 我们的战略方针是：（一）开发一种多重聚合酶链 反应/连接酶检测反应（PCR/LDR）系统，用于检测 肿瘤中的K-ras和p-53突变。 (ii)发展能力， 以100，000个癌细胞中1个的灵敏度鉴定突变- 使用我们的PCR/限制性内切酶/LDR（PCR/RE/LDR）检测100万个正常细胞 法(iii)设计和合成核苷酸类似物， 将特异性DNA序列插入限制性内切酶位点，以及 增加Tth DNA连接酶的保真度。(iv)发展能力， 以100 - 1，000个癌细胞中1个的灵敏度检测多种突变 使用突变Tth DNA连接酶的正常细胞，含有核苷酸的引物 类似物或其他栖热菌DNA连接酶。 (v)制定方法， 同时检测大量的LDR，产品使用可寻址 寡核苷酸或肽核酸阵列。 (vi)探索能力 为了多重PCR/LDR以筛选原发性结肠肿瘤中的突变， 粪便和结肠灌洗样本。(vii)探索PCR/RE/LDR的能力 鉴定已知突变作为隐匿性肿瘤的克隆标记物 结肠癌扩散（淋巴结、粪便和盆腔冲洗液） 以及乳腺癌（淋巴结、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).