Novel microarrays for DNA genotyping in the presence of excess background DNA

用于在过量背景 DNA 存在下进行 DNA 基因分型的新型微阵列

基本信息

批准号：
8714643
负责人：
Alfredo Andres Celedon
金额：
$ 35万
依托单位：
SCANOGEN, INC.
依托单位国家：
美国
项目类别：
财政年份：
2014
资助国家：
美国
起止时间：
2014-05-15 至 2016-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8714643
关键词：
Antineoplastic Agents BRAF gene Biomedical Research Biosensor Clinical Research Codon Nucleotides Colorectal DNA DNA Fingerprinting DNA Microarray Chip Detection Development Devices Diagnostic Procedure Electronics Equipment Evaluation Formalin Genes Genetic Genomic DNA Genotype Goals Hour Hybrids KRAS2 gene Label Lung Malignant Neoplasms Mass Spectrum Analysis Medicine Microbiology Mutate Mutation Nucleotides Oligonucleotides Oncogenes Pancreas Paraffin Embedding Performance Phase Physicians Preparation Property Reproducibility Research Resolution Sampling Sensitivity and Specificity Small Business Innovation Research Grant Somatic Mutation Stress System Techniques Technology Testing Time Tumor Cell Line base cancer type clinical application cost cost effective digital fetal flexibility genetic analysis improved melanoma multiplex detection mutant new technology novel novel strategies prenatal prototype public health relevance sensor single molecule synthetic construct tool tumor

项目摘要

DESCRIPTION (provided by applicant): DNA microarrays are a primary tool for DNA genotyping because of their capacity to detect multiple targets simultaneously. However, DNA microarrays require fluorescent labeling and long incubation steps which limit their applicability. Furthermore, DNA microarrays are not capable of detecting DNA targets in the presence of excess background DNA. This capacity is essential in multiple biomedical applications, such as tumor genotyping, microbiology testing and non-invasive prenatal genetic analysis. Here, we propose development of a novel microarray based on Twist-Biosensor (TBS) technology that will be label-free and more than 20 times faster than standard microarrays. Additionally, the new technique will be capable of detecting DNA in the presence of 20-fold excess background DNA, making it ideal for difficult genotyping situations. In this application, we propose the development of novel microarrays to dramatically improve and simplify tumor genotyping for research and clinical applications. Available techniques used to detect somatic mutations are either limited in their multiplexing capacity, such as real-time PCR and mass spectroscopy, or time consuming and labor intensive such as Next-Gen, Sanger and Pyro-sequencing. Twist-Biosensor is a novel microarray technique in which hybridization is detected with single molecule resolution. In addition, Twist- Biosensor applies disrupting torsional stress to DNA hybrids, a novel strategy that gives the microarrays extremely high sequence selectivity. Using these unique properties, we will develop highly multiplexed microarrays to detect somatic mutations that will be accurate, rapid, and cost- effective. The overall aim of this proposal is to demonstrate the capabilities of Twist-Biosensor microarrays for the detection of multiple mutations in the presence of excess background DNA. We will develop prototype devices to detect mutations of the BRAF and KRAS genes and test them using tumor samples. Aim 1 focuses on demonstrating rapid (3 hours) and accurate detection of a single mutation in samples containing 20-fold excess background DNA. Aim 2 focuses on improving TBS detection by integrating the biosensors in a digital sensor array. The new system will have high multiplexing capability, will use inexpensive equipment and will be easy to automate. Our ultimate goal is to produce a flexible platform for biomedical research and clinical applications.

描述（由申请人提供）：DNA 微阵列是 DNA 基因分型的主要工具，因为它们能够同时检测多个目标。然而，DNA 微阵列需要荧光标记和较长的孵育步骤，这限制了其适用性。此外，DNA 微阵列无法在存在过量背景 DNA 的情况下检测 DNA 靶标。这种能力在多种生物医学应用中至关重要，例如肿瘤基因分型、微生物学检测和非侵入性产前遗传分析。在这里，我们建议开发一种基于 Twist-Biosensor (TBS) 技术的新型微阵列，该技术无需标记，并且比标准微阵列快 20 倍以上。此外，新技术将能够在背景 DNA 过量 20 倍的情况下检测 DNA，使其成为困难的基因分型情况的理想选择。在此应用中，我们建议开发新型微阵列，以显着改善和简化研究和临床应用的肿瘤基因分型。用于检测体细胞突变的现有技术要么受限于多重分析能力（例如实时 PCR 和质谱法），要么耗时且费力密集（例如 Next-Gen、Sanger 和 Pyro 测序）。 Twist-Biosensor 是一种新型微阵列技术，可以以单分子分辨率检测杂交。此外，Twist-Biosensor 对 DNA 杂交体施加破坏性扭转应力，这是一种赋予微阵列极高序列选择性的新颖策略。利用这些独特的特性，我们将开发高度多重的微阵列来准确、快速且经济高效地检测体细胞突变。该提案的总体目标是展示了 Twist-Biosensor 微阵列在存在过量背景 DNA 的情况下检测多种突变的能力。我们将开发原型设备来检测 BRAF 和 KRAS 基因的突变，并使用肿瘤样本进行测试。目标 1 侧重于展示对含有 20 倍过量背景 DNA 的样品中的单个突变的快速（3 小时）和准确检测。目标 2 侧重于通过将生物传感器集成到数字传感器阵列中来改进 TBS 检测。新系统将具有高复用能力，将使用廉价的设备并且易于自动化。我们的最终目标是为生物医学研究和临床应用创建一个灵活的平台。