Microsphere Array for Lung Cancer Mutation Scanning

描述(申请人摘要)：全基因组遗传筛查 导致癌症或其他疾病的多态(SNPs)和获得性突变 遗传性疾病有助于了解疾病的原因。 以及对治疗的可能反应。然而，能够检测“集体”的能力 这种基因改变目前由于缺乏技术而受到阻碍 在大型基因库中同时筛查未知突变。的目的是 这项提议是设计一种新型的基于微球的DNA阵列，在 与新的突变扫描技术(专辑)相结合，可以实现高度 数千个基因中未知突变/SNPs的平行筛查 同时。建议将其用于肺癌的中试应用。 来自癌细胞和正常细胞的cDNA经过退火并杂交到 在突变/SNP的位置产生错配。相册附件 分子探针在独特的化学基团(醛)上共价生成 高度特异的错配修复糖基酶造成的错配。突变 然后从正常DNA中提取含有-DNA的DNA，进行PCR扩增并应用于 基于微球的新型DNA阵列一步法鉴定 突变的基因区域。相册与微球阵列的结合 导致了一种对整个cdna的快速预筛选方法，这可以表明 患者群体中普遍存在多个突变基因区域，这 然后进行测序。这一新方法大大减少了 定义对癌症发展至关重要的突变所需的努力。目标1和目标2 将设计、设计和优化100套 寡核苷酸包裹的光学编码微球，适用于 同时鉴定100张专辑中分离的突变DNA片段。 从全部100组微球中多路传输和提取信息 同时，通过使用流式细胞仪，将进行优化，将错误降至最低 积极/消极。Aim 3将通过设计微球来扩展阵列 适用于1000-2000年的同时突变/单核苷酸多态性分析 癌症相关基因。最后，目标4将把这种方法应用于 50例肺腺癌标本的高通量分析。 显示出潜在显著趋势的突变/SNP片段 (例如，高比例患者中常见的突变) 测序以确定突变的确切位置和性质。如果 目前阵列的工程和应用证明是成功的，未来的工作 将自动生产基于微球的DNA阵列，以便 同时筛查整个人类基因组中的突变/SNPs。

DESCRIPTION (Applicant's Abstract): Genome-wide screening for inherited polymorphisms (SNPs) and acquired mutations leading to cancer or to other genetic diseases is instrumental for understanding the causes of the disease and the likely response to treatment. However, the ability to detect 'en masse' such genetic alterations is currently hampered by the lack of technologies able to screen for unknown mutations in large gene pools simultaneously. The aim of this proposal is to engineer a novel microsphere-based DNA array which, in conjunction with a new mutation-scanning technology (ALBUMS), allows highly parallel screening of unknown mutations/SNPs in thousands of genes simultaneously. A pilot application in lung cancer is proposed. cDNAs derived from cancerous and normal cells are annealed and hybridized to generate mismatches at the positions of mutations/SNPs. ALBUMS attaches molecular probes covalently at unique chemical groups (aldehydes) generated at the mismatches by highly specific mismatch-repair glycosylases. Mutation -containing DNA is then isolated from normal DNA, PCR-amplified and applied on novel microsphere-based DNA arrays for single-step identification of the mutated gene regions. The combination of ALBUMS with microsphere-based arrays leads to a rapid pre-screening method for the entire cDNA, which can indicate multiple mutated gene regions commonly present in a patient population, which are then sequenced. This new approach leads to a dramatic reduction in the effort required to define mutations crucial to cancer development. Aims 1 and 2 will design, engineer and optimize an array of 100 sets of oligonucleotide-coated, optically encoded microspheres appropriate for the simultaneous identification of 100 ALBUMS-isolated, mutated DNA fragments. Multiplexing and extracting information from all 100 sets of microspheres simultaneously, by using a flow cytometer, will be optimized for minimal false positives/negatives. Aim 3 will expand the array by engineering microspheres appropriate for the simultaneous mutation/SNP analysis of 1000-2000 cancer-related genes. Finally Aim 4 will apply this approach to the high-throughput analysis of lung adenocarcinoma samples from 50 patients. Mutation/SNP-containing fragments that show potentially significant trends (e.g. mutations commonly present in high percentage of patients) will then be sequenced to define the exact position and nature of the mutation. If the engineering and application of the present array proves successful, future work will automate the production of microsphere-based DNA arrays in order to simultaneously screen for mutations/SNPs in the entire human genome.