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

描述(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治疗这种疾病，但很少出现临床医生用于对抗CRC的情况，主要的筛查方法仍然是监测粪便中的血液和/或结肠镜检查。在R21的这一应用中，研究将专注于开发能够监测源于核DNA的分子生物标记物的缺失/存在的微电泳法。要研究的分子分析需要高分辨率的电泳法来读出结果。DNA通常通过使用熔融硅毛细管或微芯片(？-CE)在粘性筛分基质中进行电泳法分离。CE特别有吸引力，因为它可以集成到前端处理步骤中，在封闭的架构中提供自动化的样品处理，没有污染，并可用于护理点测试。矩阵使用高压加载到设备中，这可能是时间和能量密集型的，必须在每次运行之间重新加载，而且成本很高。消除筛选基质和发展自由溶液电泳法来分选DNA，将大大降低与分子分析相关的成本，并简化系统设置和减少运行时间。由于电荷和摩擦标度都与链长呈线性关系，因此DNA在自由溶液中的电泳率不随链长的增加而变化。为了进行DNA的自由溶液电泳法，DNA必须连接到一个不带电荷的扰动实体上，或产生自由溶液共轭电泳法(FSCE)的“拖拽标签”。在这项应用中，使用复制技术在聚合物中制造的带有-CE装置的FSCE将被用于几种不同的分子分析，包括用于评分K-ras癌基因点突变存在的连接酶检测反应(LDR)和用于评分零星p53突变存在的突变扫描分析Endo V/LDR分析。此外，微卫星不稳定性(MSI)也将使用CE FSCE进行评估。MSI是使用一组来自核DNA的标记进行的，这些标记被聚合酶链式反应扩增，并通过电泳法进行分析。比较病变组织和正常组织的电泳迁移率可以作为MSI状态的一个指标，并可作为确定治疗结直肠癌患者的有效治疗方法的预测指标。

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.