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: 7479576
• 负责人: Steven Allan Soper
• 金额: $ 14.92万
• 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 2010-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7479576
• 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。虽然许多生物标志物和技术已经被评估用于这种疾病的管理，但很少有临床医生用于对抗CRC，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。在这项R21应用中，研究将集中在开发微电泳，可以监测来自核DNA的分子生物标志物的存在/不存在。待研究的分子测定需要高分辨率电泳来阅读结果。DNA通常通过使用熔融二氧化硅毛细管或微芯片（？-CE）在粘性筛分基质中电泳来分离。EC-CE特别有吸引力，因为它可以集成到前端处理步骤中，以在无污染的封闭架构中提供自动化样品处理，并可用于即时检测。使用高压将基质加载到装置中，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，并且是昂贵的。筛选矩阵的消除和自由溶液电泳的发展来分选DNA将大大降低与分子测定相关的成本，以及简化系统设置和减少运行时间。由于电荷和摩擦力都与链长呈线性关系，因此DNA在自由溶液中的电泳迁移率不随链长的增加而改变。为了进行DNA的自由溶液电泳，DNA必须与不带电荷的扰动实体或产生自由溶液缀合电泳（FSCE）的“拖拽标签”缀合。在本申请中，使用复制技术在聚合物中制造的带有？-CE装置的FSCE将用于几种不同的分子测定，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，这是一种突变扫描测定，用于对散发性p53突变的存在进行评分。此外，微卫星不稳定性（MSI）也将使用<$-CE FSCE进行评估。MSI使用来自核DNA的一组标记物进行，所述标记物通过PCR扩增并通过电泳分析。患病组织与正常组织的电泳迁移率的比较提供了MSI状态的指示，并且可以用作确定用于治疗CRC患者的有效疗法的指示器。

项目成果

Ligase detection reaction for the analysis of point mutations using free-solution conjugate electrophoresis in a polymer microfluidic device.

• DOI: 10.1002/elps.200800197
• 发表时间: 2008-12
• 期刊: ELECTROPHORESIS
• 影响因子: 2.9
• 作者: Sinville, Rondedrick;Coyne, Jennifer;Meayher, Robert J.;Cheng, Yu-Wei;Barany, Francis;Barron, Annelise;Soper, Steven A.
• 通讯作者: Soper, Steven A.