Fast Mutation Detection by Tandem SSCP/HA on Microchips

通过微芯片上的串联 SSCP/HA 进行快速突变检测

基本信息

批准号：
7485498
负责人：
Annelise Emily Barron
金额：
$ 17.3万
依托单位：
STANFORD UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2007
资助国家：
美国
起止时间：
2007-12-14 至 2009-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7485498
关键词：
Address Admixture Affect Age Biological Biological Assay Biological Models Blinded Blood capillaries Cancer Detection Cancer Patient Cancer cell line Capillary Electrophoresis Characteristics Chemical Engineering Clinical Collaborations Comprehensive Cancer Center Condition Cost Analysis DNA DNA Fingerprinting DNA Sequence DNA Sequence Alteration DNA analysis Data Detection Devices Disease-Free Survival Drops Drug Formulations Economics Effectiveness Electrophoresis Elements Ensure Evaluation Exons Fluorescence Freezing Funding Gel Generations Genes Genomics Glass Heteroduplex Analysis Homo Hospitals Human Hybridization Array Immunohistochemistry Individual Laboratories Lasers Length Link Malignant Neoplasms Mammary Neoplasms Medical Methodology Methods Microchip Electrophoresis Microfluidic Microchips Microfluidics Migration Assay Molecular Mutate Mutation Mutation Detection Negative Axillary Lymph Node Neoplasm Metastasis Normal Cell Numbers Outcome Patients Pattern Phase Physicians Plastics Polymers Positive Lymph Node Preparation Primary Neoplasm Probability Process Protein p53 Protocols documentation Publishing Range Rate Recurrence Reporting Research Design Research Personnel Running Sampling Screening procedure Sensitivity and Specificity Series Single-Stranded Conformational Polymorphism Single-Stranded DNA Solid Neoplasm Specimen Staining method Stains Standards of Weights and Measures Statistically Significant TP53 gene Techniques Technology Temperature Tissue Sample Translating Tube Tumor Bank Tumor Cell Line Tumor Suppressor Genes Tumor Tissue Tumor-Derived Validation Work anticancer research base blind cancer therapy capillary chemotherapy clinical application clinically relevant conformer cost design ds-DNA electric field genetic analysis genotyping technology innovative technologies interest malignant breast neoplasm medical schools member micro-total analysis system microchip migration mutant novel outcome forecast response success time use tumor

项目摘要

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.

描述（由申请人提供）：建议使用p53基因作为模型系统，通过p53基因作为模型系统来优化，评估和试点快速，可扩展和低成本的微芯片电泳技术（通过串联单链构象多态性（SSCP）/异源性电振贴分析（SSCP）/异源性电蛋白分析（HA），以对癌症进行敏感和特定的分子检测。我们要求1年R21阶段和3年R33阶段。拟议的项目涉及西北Lurie综合癌症中心的成员之间的合作，包括化学工程，医学院和埃文斯顿医院的研究人员。微通道“ Tandem” SSCP/HA是一种新的突变检测方法，该方法最近在我们的实验室中开发，涉及同时生成和分析同型/异源DNA和SSCP构象异构体。对大量样品的研究（32）表明，与单独的SSCP（93％）或单独使用SSCP（75％）相比，串联SSCP/HA可以进行更高的敏感性突变检测（100％）。我们已经开发并发布了优化的样品制备方案，凝胶制剂和毛细管阵列电泳（CAE）的分析条件。在R21阶段，我们将将这些方法转换为微流体电泳芯片，与CAE相比，DNA分析成本的吞吐量和下降大大增加。 p53基因已知在> 50％的人类癌症中被突变，其突变状态可以预测患者对化学疗法的反应，是所选择的重要模型系统。但是，要开发的基于微芯片的基因分析技术应轻松应用于任何与癌症相关的基因。在R21阶段，我们将分析源自肿瘤细胞系的大约60种不同的DNA样品，这些DNA样品代表不同p53外显子中的一系列突变，以确定DNA样品特征和电泳方案对该方法的敏感性和特异性的影响，在由协作生物统治者设计的盲目研究中。当针对微芯片开发了优化的串联SSCP/HA协议时，将通过分析>从Evanston医院的冰冻的实体肿瘤放大的200个选定样品进行分析。通过这项盲目的研究，将首次使用银行肿瘤组织确定和报告敏感性和特异性（预计将达到或接近100％），并为该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可用于医生。