Fast Mutation Detection by Tandem SSCP/HA on Microchips

描述(由申请人提供)： 以p53基因为模型系统，优化、评估和试点快速、可扩展、低成本的单链构象多态(SSCP)/异源双链分析(HA)快速、可扩展、低成本的微芯片电泳技术，用于灵敏和特异的癌症分子检测。我们要求1年的R21阶段和3年的R33阶段。拟议的项目涉及西北大学卢里综合癌症中心成员之间的合作，包括化学工程、医学院和埃文斯顿医院的研究人员。微通道串联SSCP/HA是本实验室最近发展起来的一种新的突变检测方法，它涉及到同源/异源双链DNA和SSCP构象的同时产生和分析。对大量样本(32个)的研究表明，串联SSCP/HA对p53样本的突变检测(100%)比单独使用SSCP(93%)或单独使用HA(75%)高得多。我们开发并发布了用于毛细管阵列电泳(CAE)的优化的样品制备方案、凝胶配方和分析条件。在R21阶段，我们将把这些方法转化为微流控电泳芯片，与CAE相比，微流控芯片可大幅提高DNA分析的产量并降低成本。已知在50%的人类癌症中发生突变的p53基因，其突变状态可以预测患者对化疗的反应，是所选择的重要模型系统。然而，将要开发的基于微芯片的基因分析技术应该很容易应用于任何与癌症相关的基因。在R21阶段，我们将分析来自肿瘤细胞系的大约60个不同的DNA样本，代表不同P53外显子的一系列突变，以确定DNA样本特征和电泳方案对该方法的敏感性和特异性的影响，这是由合作的生物统计学家设计的一项盲法研究。当针对微芯片开发了优化的串联SSCP/HA协议后，它们将通过对从埃文斯顿医院储存的冷冻实体肿瘤中扩增的200个精选样本进行分析来试行。通过这项盲法研究，将首次使用储存的肿瘤组织来确定和报告灵敏度和特异度(预计两者都达到或接近100%)，为这项技术的临床应用提供必要的验证，并使快速、低成本的癌症基因分型技术广泛适用于医生。

DESCRIPTION (provided by applicant): It is proposed to optimize, evaluate, and pilot rapid, scalable, and low-cost microchip electrophoresis technologies for sensitive and specific molecular detection of cancer by tandem single-strand conformational polymorphism (SSCP)/heteroduplex analysis (HA), using the p53 gene as a model system. We request a 1-year R21 phase and a 3-year R33 phase. The proposed project involves collaboration between members of Northwestern's Lurie Comprehensive Cancer Center, including researchers in Chemical Engineering, the Medical School, and Evanston Hospital. Microchannel "tandem" SSCP/HA is a novel mutation detection method recently developed in our laboratory, which involves the simultaneous generation and analysis of homo/heteroduplex DNA and SSCP conformers. Studies of a significant number of samples (32) indicate that tandem SSCP/HA allows for much higher-sensitivity mutation detection (100%) than SSCP alone (93%) or HA alone (75%), for p53 samples. We have developed and published optimized sample preparation protocols, gel formulations, and analysis conditions for capillary array electrophoresis (CAE). During the R21 phase, we will translate these methods to microfluidic electrophoresis chips, which offer a large increase in throughput and drop in cost of DNA analysis compared to CAE. The p53 gene, known to be mutated in >50% of human cancers, and whose mutation status can be predictive of patient response to chemotherapy, is the important model system chosen. However, microchip-based genetic analysis technologies to be developed should be easily applied to ANY cancer-related gene. In the R21 phase, we will analyze approximately 60 different DNA samples derived from tumor cell lines, representing a range of mutations in different p53 exons, to determine the impact of DNA sample characteristics and electrophoresis protocols on the sensitivity and specificity of the method, in a blinded study designed by collaborating biostatisticians. When optimized tandem SSCP/HA protocols have been developed for microchips, they will be piloted by the analysis of >200 selected samples amplified from frozen, solid tumors banked at Evanston Hospital. Via this blinded study, sensitivity and specificity (both expected to be at or near 100%) will be determined and reported for the first time using banked tumor tissue, providing necessary validation for clinical application of this technique, and making rapid, low-cost cancer genotyping technology widely available to physicians.