Microsphere Array for Lung Cancer Mutation Scanning

描述（申请人的摘要）：用于遗传性疾病的全基因组筛选 基因多态性（SNP）和获得性突变导致癌症或其他疾病。 遗传性疾病有助于了解疾病的原因 以及对治疗的反应然而，检测'en'的能力 这种基因改变目前由于缺乏能够 同时在大的基因库中筛选未知的突变。的目的 这项提议是设计一种新的基于微球的DNA阵列， 结合新的突变扫描技术（ALBUMS）， 在数千个基因中平行筛选未知突变/SNP 同步提出了在肺癌中的试点应用。 将来自癌细胞和正常细胞的cDNA退火并杂交， 在突变/SNP的位置产生错配。ALBUMS附件 分子探针与生成的独特化学基团（醛）共价键连接 通过高度特异性的错配修复糖基化酶来修复错配。突变 然后从正常DNA中分离含有-的DNA，PCR扩增并应用于 一种新的基于微球的DNA阵列，用于单步鉴定 突变的基因区域。ALBUMS与基于微球的阵列的组合 导致了一个快速的预筛选方法的整个cDNA，这可以表明， 多个突变基因区域通常存在于患者群体中， 然后进行排序。这一新方法导致了 需要努力定义对癌症发展至关重要的突变。目标1和2 将设计，工程师和优化一个阵列的100套 适用于微生物技术的、涂敷有多核苷酸的光学编码微球 同时鉴定100个ALBUMS分离的突变DNA片段。 从所有100组微球中复用和提取信息 同时，通过使用流式细胞仪，将被优化用于最小的假 积极/消极。AIM 3将通过工程微球来扩展阵列 适用于同时进行1000-2000个突变/SNP分析 癌症相关基因最后，目标4将把这种方法应用于 来自50名患者的肺腺癌样本的高通量分析。 显示潜在显著趋势的含突变/SNP片段 (e.g.突变通常存在于高比例的患者中），然后将 测序以确定突变的确切位置和性质。如果 该阵列的工程应用证明是成功的，未来的工作 将自动化生产基于微球的DNA阵列， 同时筛选整个人类基因组中的突变/SNP。

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.