TECHNOLOGY FOR MUTATION ANALYSIS OF CANCER

将癌症诱导与特定突变相关联，关键取决于能够同时有效筛查几个基因中未知突变的技术的可用性。 该提案将优化和简化一项新开发的技术（ALBUMS），用于灵敏和大规模筛选碱基取代，这些碱基取代是主要由各种诱变剂产生的突变，并在几种人类癌症中发现。 将来自癌细胞和正常细胞的DNA退火并杂交以在碱基取代的位置处产生错配。 ALBUMS利用新型分子在由高度特异性错配识别酶在错配位置处产生的独特化学基团（醛）处的共价和特异性结合，以分离含突变的DNA。 基于微孔板的检测设计允许同时筛选数百或数千个不同的基因，分离具有突变的那些基因，并将它们应用于现有的大规模杂交DNA阵列，用于突变基因的单步鉴定。R21阶段（可行性）将（a）确定检测和分离含突变基因（基因型选择）的最佳操作条件。 ALBUMS将用于在存在增加量的正常等位基因的情况下从单个突变基因（p53）中分离cDNA，并检测已知含有p53突变的细胞系中的p53突变体。 和（B）将建立通过组合ALBUMS和商业DNA杂交阵列（芯片）一步筛选数百或数千个人类基因的可行性。 第二阶段（R33）将开发技术，在DNA芯片上一步筛选人类肿瘤样本中的数百或数千个基因突变。 将通过常规测序绘制和验证突变集，以确定新技术用于定义癌症分子谱的实用性。 在最后一步，这种高通量技术将被简化，以提供一个程序，使研究人员和临床医生能够轻松获得，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ALBUMS应用包括基因分型和多态性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.