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TECHNOLOGY FOR MUTATION ANALYSIS OF CANCER

癌症突变分析技术

基本信息

批准号：
6174335
负责人：
G. Mike Makrigiorgos
金额：
$ 16.79万
依托单位：
DANA-FARBER CANCER INST
依托单位国家：
美国
项目类别：
财政年份：
1999
资助国家：
美国
起止时间：
1999-08-24 至 2001-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/6174335
关键词：
biotechnology carcinogenesis gene mutation genetic polymorphism genetic screening genotype human genetic material tag human tissue neoplasm /cancer diagnosis neoplasm /cancer genetics neoplastic cell nucleic acid hybridization nucleic acid probes nucleic acid quantitation /detection nucleic acid sequence p53 gene /protein polymerase chain reaction technology /technique development

项目摘要

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.

将癌症诱导与特定突变关联起来关键取决于能否同时有效筛选多个基因中的未知突变的技术。该提案将优化和简化一项新开发的技术（ALBUMS），用于敏感和大规模的碱基替换筛选，碱基替换是主要由多种诱变剂产生的突变，在几种人类癌症中发现。来自癌细胞和正常细胞的 DNA 退火并杂交，在碱基替换位置产生错配。 ALBUMS 利用高度特异性错配识别酶在错配位置产生的独特化学基团（醛）上新分子的共价和特异性结合，以分离含有突变的 DNA。基于微孔板的检测设计可以同时筛选数百或数千个不同的基因，分离出具有突变的基因，并将其应用到现有的大规模杂交DNA阵列上，以一步鉴定突变基因。 R21 阶段（可行性）将 (a) 定义检测和分离含有突变的基因（基因型选择）的最佳操作条件。 ALBUMS 将用于在正常等位基因数量不断增加的情况下从单个突变基因 (p53) 中分离 cDNA，并用于检测已知含有 p53 突变的细胞系中的 p53 突变体。 (b) 将建立通过结合 ALBUMS 和商业 DNA 杂交阵列（芯片）对数百或数千个人类基因进行单步筛选的可行性。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数百或数千个基因的突变。将通过常规测序来绘制和验证突变组，以便确定新技术在定义癌症分子谱方面的效用。在最后一步中，这种高通量技术将得到简化，以便为研究人员和临床医生提供一种方便的程序，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 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。