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，因为它们能够同时检测多个靶标。然而，DNA微阵列需要荧光标记和长的孵育步骤，这限制了它们的适用性。 此外，DNA微阵列不能在存在过量背景DNA的情况下检测DNA靶。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，我们建议开发一种新的微阵列的基础上扭转生物传感器（TBS）技术，将是无标记和超过20倍的速度比标准的微阵列。此外，新技术将能够在存在20倍过量背景DNA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。在这项应用中，我们提出了新的微阵列的发展，以显着改善和简化肿瘤基因分型的研究和临床应用。用于检测体细胞突变的可用技术要么在其多重能力方面受到限制，例如实时PCR和质谱，要么耗时且劳动密集，例如Next-Gen、桑格和焦磷酸测序。Twist-Biosensor是一种新型的微阵列技术，它可以在单分子水平上检测杂交。此外，Twist-Biosensor将破坏性扭转应力施加到DNA杂交体上，这是一种新的策略，使微阵列具有极高的序列选择性。利用这些独特的性质，我们将开发高度多重化的微阵列来检测体细胞突变，这将是准确的，快速的，和成本效益。本建议的总体目标是 证明了Twist生物传感器微阵列在存在过量背景DNA的情况下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，并使用肿瘤样本进行测试。目标1的重点是证明快速（3小时）和准确的检测含有20倍过量的背景DNA的样品中的单一突变。目标2的重点是通过将生物传感器集成在数字传感器阵列中来改进TBS检测。新系统将具有高度的多路复用能力，将使用廉价的设备，并易于自动化。我们的最终目标是为生物医学研究和临床应用提供一个灵活的平台。