High-throughput, Label-free Promoter Sequence Discovery

高通量、无标记启动子序列发现

• 批准号: 7149076
• 负责人: M SELIM UNLU
• 金额: $ 23.13万
• 依托单位: BOSTON UNIVERSITY (CHARLES RIVER CAMPUS)
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-07-01 至 2008-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7149076
• 关键词: bioengineering /biomedical engineering; biotechnology; biotinylation; genetic promoter element; high throughput technology; intermolecular interaction; method development; microarray technology; oligonucleotides; optical tomography; protein binding; protein quantitation /detection; silicon compounds; surface coating; surface plasmon resonance; transcription factor

DESCRIPTION (provided by applicant): The proposed work is aimed at demonstrating a high throughput, cost effective, and label-free microarray- based method for detecting macromolecular interactions. In particular, we propose a novel technology that involves the use of microarrays and a new label-free optical detection method based on the Resonant Cavity Imaging Biosensor (RCIB). While the use of optical resonators for sensing applications has been studied for many years, the unique capabilities of RCIB described in this proposal have been enabled by recent innovations developed at Boston University as well as advancements in optical instrumentation. One of the primary enabling factors is the invention of high-quality multi-layer dielectric reflectors developed at Boston University for the study of optoelectronic devices on silicon. This is accompanied by new techniques to sense small changes in material on layered structures. Resonant Cavity Imaging Biosensor (RCIB) promises to open the way to high throughput array based methods for profiling protein expression and for discovering intergenic regulatory regions. This proposal will focus on demonstrating and adapting RCIB detection techniques for protein oligonucleotides arrays, and assessing the sensitivity and specificity of the technology. Promoter sequences and potential promoter sequence regions will be photolithographically synthesized on a DNA microarray. Unlabeled TFs will be introduced to the microarray surface and allowed to bind to their corresponding promoter sequences. The presence of TFs will be detected and quantified using the label-free RCIB method. This work will achieve the following specific aims: 1. Demonstrate RCIB sensing and imaging of 0.1 nm high artificial structures patterned on SiO2; 2. Detect monolayer of streptavidin bound to biotinylated DNA patterned in an array format with 64 features each 50 mu m x 50 mu m in size; 3. Demonstrate specificity and sensitivity of different transcription factors (TFs) binding to their corresponding promoter sequences on a 100-feature microarray.

描述（由申请人提供）：所提议的工作旨在展示一种高通量、成本有效且无标记的基于微阵列的方法来检测大分子相互作用。特别是，我们提出了一种新技术，涉及使用微阵列和基于共振腔成像生物传感器（RCIB）的新型无标记光学检测方法。虽然光学谐振器在传感应用中的使用已被研究多年，但本提案中描述的 RCIB 的独特功能是通过波士顿大学最近开发的创新以及光学仪器的进步而实现的。主要推动因素之一是波士顿大学为研究硅光电器件而开发的高质量多层电介质反射器的发明。伴随着新技术来感知层状结构上材料的微小变化。共振腔成像生物传感器 (RCIB) 有望为基于高通量阵列的蛋白质表达分析和发现基因间调控区域的方法开辟道路。该提案将重点展示和调整蛋白质寡核苷酸阵列的 RCIB 检测技术，并评估该技术的灵敏度和特异性。启动子序列和潜在的启动子序列区域将在DNA微阵列上通过光刻合成。未标记的 TF 将被引入微阵列表面并允许与其相应的启动子序列结合。将使用无标记 RCIB 方法检测和量化 TF 的存在。这项工作将实现以下具体目标： 1. 展示 SiO2 上 0.1 nm 高人工结构的 RCIB 传感和成像； 2. 检测与生物素化 DNA 结合的单层链霉亲和素，其图案化为阵列格式，具有 64 个特征，每个特征尺寸为 50 μ m x 50 μm； 3. 在 100 个特征微阵列上展示不同转录因子 (TF) 与其相应启动子序列结合的特异性和敏感性。