Genotyping Molecular Signatures of CRC using Polymer Microchip-CE and FSCE

使用聚合物微芯片-CE 和 FSCE 对 CRC 进行基因分型分子特征

• 批准号: 7290605
• 负责人: Steven Allan Soper
• 金额: $ 19.03万
• 依托单位: LOUISIANA STATE UNIV A&M COL BATON ROUGE
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-08-06 至 2009-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7290605
• 关键词: Alleles; Architecture; Bedside Testings; Biological Assay; Biological Markers; Blood; Blood capillaries; Cancer Patient; Capillary Electrophoresis; Cells; Characteristics; Charge; Chemotherapy-Oncologic Procedure; Clinical; Colonoscopy; Colorectal Cancer; Cytolysis; DNA; Data; Depth; Detection; Development; Devices; Dinucleotide Repeats; Discrimination; Disease; Disease Management; Effectiveness; Electrophoresis; Enzymes; Exons; Feces; Fluorescence; Friction; Gel; Genome; Genomics; Genotype; Goals; Intervention; K-ras Oncogene; Label; Lasers; Length; Ligase; Ligation; Malignant Neoplasms; Methods; Microchip Electrophoresis; Microfluidic Microchips; Microfluidics; Microsatellite Instability; Molds; Molecular; Molecular Analysis; Molecular Profiling; Monitor; Mutation; Mutation Detection; Noise; Normal tissue morphology; Nuclear; Numbers; Nylons; Operative Surgical Procedures; Performance; Plastics; Point Mutation; Point-of-Care Systems; Polymerase Chain Reaction; Polymers; Polymethyl Methacrylate; Population; Principal Investigator; Process; Production; Reaction; Reading; Reagent; Reporter; Reporting; Research; Resolution; Running; Sampling; Scanning; Score; Screening procedure; Series; Silicon Dioxide; Site; Solutions; Sorting - Cell Movement; Specific qualifier value; Staging; System; TP53 gene; Techniques; Technology; Time; Tissues; aqueous; base; cancer diagnosis; capillary; cost; design; endo V; microchip; mutant; outcome forecast; pressure; programs; repaired; size; tool

DESCRIPTION (provided by applicant): Colorectal cancer (CRC) represents the third-leading killer amongst all cancer-related diseases in the US. While a number of biomarkers and technologies have been evaluated for the management of this disease, few have emerged for use by clinicians in battling CRC, with the predominant screening methods still consisting of monitoring blood in the stool and/or colonoscopy. In this R21 application, research will focus on developing micro-electrophoresis that can monitor the absence/presence of molecular biomarkers originating from nuclear DNA. The molecular assays to be investigated require high-resolution electrophoresis for reading out the results. DNA is typically separated by electrophoresis in a viscous sieving matrix using fused-silica capillaries or microchips (¿-CE). ¿-CE is particularly attractive because it can be integrated to front-end processing steps to provide automated sample processing in a closed architecture free from contamination and envisioned for point-of-care testing. The matrix is loaded into the device using high pressure, which can be time and energy-intensive, must be reloaded between every run, and is expensive. The elimination of sieving matrices and the development of free-solution electrophoresis to sort DNA would drastically decrease the cost associated with molecular assays as well as simplify system set-up and reduce run time. Since both the charge and the friction scale linearly with chain length, the electrophoretic mobility of DNA in free-solution does not change with increased chain length. In order to run free-solution electrophoresis of DNA, the DNA must be conjugated to an uncharged perturbing entity or "drag-tag" producing Free-Solution Conjugate Electrophoresis (FSCE). In this application, FSCE with ¿-CE devices that are fabricated in polymers using replication technology will be used for several different molecular assays, including Ligase Detection Reactions (LDR) for scoring the presence of known point mutations in K-ras oncogenes and an Endo V/LDR assay, a mutation scanning assay to score the presence of sporadic p53 mutations. In addition, microsatellite instability (MSI) will also be evaluated using ¿-CE FSCE. MSI is undertaken using a panel of markers from nuclear DNA that are PCR amplified and analyzed via electrophoresis. Comparisons of electrophoretic mobilities of diseased tissue versus normal tissue provide an indication of MSI status and can be used as a prognosticator for determining effective therapies for treating CRC patients.

描述（由申请人提供）：结直肠癌 (CRC) 是美国所有癌症相关疾病中的第三大杀手，虽然已经评估了许多用于治疗这种疾病的生物标志物和技术，但很少有应用。由与结直肠癌作斗争的战斗人员进行，主要的筛查方法仍然包括监测粪便中的血液和/或结肠镜检查。在这个 R21 应用中，研究将集中于开发可以监测分子生物标志物是否存在的微电泳。待研究的分子测定需要高分辨率电泳来读出结果，通常使用熔融石英毛细管或微芯片 (¿-CE) 在粘性筛分基质中进行电泳来分离 DNA。 -CE 特别有吸引力，因为它可以集成到前端处理步骤中，在无污染的封闭架构中提供自动化样品处理，并可使用高压将基质加载到设备中。 ，这可能是时间和能源密集型的，必须在每次运行之间重新加载，并且价格昂贵。消除筛分基质和开发用于分选 DNA 的自由溶液电泳将大大降低与分子测定相关的成本并简化。系统设置和减少由于电荷和摩擦力均与链长度成线性关系，因此游离溶液中 DNA 的电泳迁移率不会随着链长度的增加而改变。产生自由溶液共轭电泳 (FSCE) 的不带电扰动实体或“拖标签”。在此应用中，FSCE 带有 ¿ -使用复制技术在聚合物中制造的 CE 装置将用于多种不同的分子测定，包括用于对 K-ras 癌基因中已知点突变的存在进行评分的连接酶检测反应 (LDR) 和 Endo V/LDR 测定（一种突变）此外，还将使用 ¿ 评估微卫星不稳定性 (MSI)。 -CE FSCE。MSI 使用一组来自核 DNA 的标记进行，这些标记通过 PCR 扩增并通过电泳进行分析，比较患病组织与正常组织的电泳迁移率，提供 MSI 状态的指示，并可用作确定有效性的预测指标。用于治疗 CRC 患者的疗法。