TECHNOLOGY FOR MUTATION ANALYSIS OF CANCER

将癌症诱导与特定突变联系起来，关键取决于是否有技术可以有效地同时筛查几个基因中的未知突变。这项建议将优化和简化一项新开发的技术(相册)，用于敏感和大规模筛选碱基替换，碱基替换是主要由广泛的诱变剂产生的突变，在几种人类癌症中发现。来自癌细胞和正常细胞的DNA被退火和杂交，以在碱基替换的位置产生错配。Album利用高度特异的错配识别酶在错配位置产生的独特化学基团(醛)上的新分子的共价和特异性结合，以分离包含突变的DNA。该检测的微孔板设计允许同时筛选数百或数千个不同的基因，分离出那些发生突变的基因，并将它们应用于现有的大规模杂交DNA阵列，以一步识别突变基因。R21阶段(可行性)将(A)确定检测和分离含突变基因的最佳操作条件(基因选择)。相册将被用来在存在越来越多的正常等位基因的情况下从单个突变基因(P53)中分离出cDNAs，并在已知含有p53突变的细胞系中检测p53突变。以及(B)将相册和商业DNA杂交阵列(芯片)相结合，建立一步筛选数百或数千个人类基因的可行性。第二阶段(R33)将开发一项技术，在DNA芯片上一步筛查人类肿瘤样本中数百或数千个基因的突变。将通过常规测序绘制和验证突变集，以建立新技术的实用性，以确定癌症的分子图谱。在最后一步中，这项高通量技术将得到简化，为研究人员和临床医生提供一种易于获得的程序，以便对癌症样本进行具有成本效益的大规模突变筛查。进一步设想的相册应用包括基因分型和多态研究，以及突变在癌症以外的疾病中的作用。

Correlating cancer induction with specific mutations is critically dependent on the availability of technologies that can effectively screen for unknown mutations in several genes simultaneously. This proposal will optimize and streamline a newly developed technology (ALBUMS) for the sensitive and large scale screening of base substitutions, which are the mutations predominantly generated by a wide range of mutagens and are found in several human cancers. DNA from cancerous and normal cells are annealed and hybridized to generate mismatches at the positions of base substitutions. ALBUMS utilizes the covalent and specific binding of novel molecules at unique chemical groups (aldehydes) generated at the position of mismatches by highly specific mismatch - recognition enzymes, in order to isolate mutation - containing DNA. The microplate-based design of the assay allows to screen hundreds or thousands of diverse genes simultaneously, isolate those with mutations and apply them on existing large - scale hybridization DNA arrays for a single - step identification of the mutated genes. The R21 phase (feasibility) will (a) define the optimal operating conditions for detecting and isolating mutation-containing genes (genotypic selection). ALBUMS will be used to isolate cDNA from a single mutated gene (p53) in the presence of increasing amounts of normal alleles, and also to detect p53 mutants in cell lines known to contain p53 mutations. And (b) will establish feasibility for the single-step screening of several hundred or thousands of human genes by combining ALBUMS and commercial DNA hybridization arrays (chips). The second phase (R33) will develop technology to screen in a single step hundreds or thousands of genes in human tumor samples for mutations, on DNA chips. Sets of mutations will be mapped and verified by conventional sequencing in order to establish the utility of the new technology to define the molecular profile of cancer. In the last step, this high throughput technology will be streamlined to provide a procedure with easy access to researchers and clinicians for cost - effective, large - scale mutation screening of cancer samples. Further envisioned ALBUMS applications include genotyping and polymorphism studies and role of mutations in diseases other than cancer.