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（由应用程序提供）：结直肠癌（CRC）代表了美国所有与癌症有关的疾病中第三个领先的杀手。尽管已经评估了许多生物标志物和技术来治疗这种疾病，但很少有人出现在与CRC作斗争中的临床医生，而主要的筛查方法仍包括监测凳子和/或结肠镜检查中的血液。在此R21应用中，研究将集中于开发微观流体，以监测源自核DNA的分子生物标志物的吸收/存在。要研究的分子测定需要高分辨率电泳才能读取结果。 DNA通常使用融合的硅毛细血管或微芯片（�-CE）在粘性筛分基质中通过电泳分离。 -CE特别有吸引力，因为它可以集成到前端处理步骤中，以在没有污染的封闭体系结构中提供自动样本处理，并设想进行护理点测试。使用高压将矩阵加载到设备中，这可能是时间和能量密集的，必须在每次运行之间重新加载，并且价格昂贵。消除筛分物质和开发自由溶液电泳来对DNA进行排序，将大大降低与分子组件相关的成本，并简化系统设置并减少运行时间。由于电荷和摩擦尺度均与链长线性线性，因此自由溶液中DNA的电泳迁移率不会随链长的增加而变化。为了运行DNA的自由溶液电泳，必须将DNA结合到无负荷的扰动实体或“阻力标签”，从而产生自由溶液的共轭电泳（FSCE）。在此应用中，使用复制技术在聚合物中制造的FSCE将用于几种不同的分子测定，包括在K -Ras Oncogenes中得分已知点突变的连接酶检测反应（LDR），以及一种eNDO V/LDR ASSAY，一种突变测定，一种突变的扫描测定，以使得Sporad p53突变。另外，还将使用�-CE FSCE评估微卫星不稳定性（MSI）。使用来自PCR扩增和通过电泳分析的核DNA的标记面板进行MSI。相比，解剖组织与正常组织的电泳迁移率提供了MSI状态的指示，可以用作确定有效治疗CRC患者的有效疗法的预后剂。