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

• 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
• 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

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，因为它们可以同时检测多个靶标。但是，DNA微阵列需要荧光标记和较长的孵化步骤，以限制其适用性。 此外，在存在多余的背景DNA的情况下，DNA微阵列无法检测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，微生物学测试和非侵入性产前遗传分析。在这里，我们提出了基于扭曲生物传感器（TBS）技术的新型微阵列的开发，该微阵列将是无标签的，并且比标准微阵列快20倍以上。此外，新技术将能够在存在20倍多余的背景DNA的情况下检测DNA，这是难以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，以极大地改善并简化研究和临床应用的肿瘤基因分型。用于检测体突变的可用技术要么受到其多重能力的限制，例如实时PCR和质谱法，要么是耗时和劳动量的，例如下一代，Sanger和Pyro-seversing。扭曲生物传感器是一种新型的微阵列技术，其中通过单分子分辨率检测到杂交。此外，扭曲生物传感器将破坏扭转应力应用于DNA杂种，这是一种新型策略，使微阵列的序列选择性极高。使用这些独特的属性，我们将开发高度多重的微阵列来检测将是准确，快速和成本效益的体突变。该提议的总体目的是 证明在存在过量背景DNA的情况下，扭曲生物传感器微阵列检测多个突变的能力。我们将开发原型设备来检测BRAF和KRAS基因的突变，并使用肿瘤样品进行测试。 AIM 1专注于证明快速（3小时），并准确检测包含20倍过量背景DNA的样品中的单个突变。 AIM 2专注于通过将生物传感器集成到数字传感器阵列中来改善TBS检测。新系统将具有较高的多路复用功能，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提供灵活的平台。