Microarrays for DNA binding proteins

DNA 结合蛋白微阵列

• 批准号: 7161056
• 负责人: TOMASZ HEYDUK
• 金额: $ 10万
• 依托单位: MEDIOMICS, LLC
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-09-19 至 2009-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7161056
• 关键词: DNA; DNA binding protein; proteins

DESCRIPTION (provided by applicant): Highly multiplexed detection of biological molecules and their activities has been an essential element of modern molecular diagnosis, drug development and research in genomics and proteomics. Well established DNA array methodologies exist which permit highly multiplexed detection of transcripts. Similar methodologies for detecting or measuring activities of proteins are not well developed. We propose in this application development of arrays capable of highly multiplexed detection of sequence-specific DNA binding proteins. DNA binding proteins are an important class of biological molecule which, due to their role in transcription regulation, is of paramount importance for regulation of all cellular processes. The goal of this Phase I proposal will be to demonstrate the feasibility of the approach and to establish the general methodology for preparation of such arrays. This goal will be achieved by accomplishing the following 2 aims: Aim #1. To establish if microarrays for DNA binding proteins can achieve desired sensitivity of detection. We will use purified model DNA binding protein to perform experiments which will establish procedures to maximize detection sensitivity. We will compare several fluorescence labels commonly used in oligonucleotide microarrays for their performance in the microarray for DNA binding proteins. We will test 2 signal amplification schemes for their ability to further enhance detection sensitivity. Finally, we will determine if optimized microarrays will be capable of achieving reproducible detection of the model protein with a desired (pM) sensitivity. Aim #2. To establish if microarrays for DNA binding proteins can analyze DNA binding activities of proteins in complex mixtures. To achieve this goal we will first determine if relative levels of 4 DNA binding proteins could be faithfully reported by the microarray in samples containing varying and known relative amounts of these proteins. Secondly, we will test the detection of SP1 protein (a constitutively expressed transcription factor) at its normal level of expression in its native environment in HeLa cellular extracts. The long term goals of this project will be to develop arrays for highly multiplexed detection of transcription factors involved in all major cellular pathways will be developed and to develop these arrays as diagnostic tools for detecting abnormal cellular states. Microarrays for DNA binding proteins to be developed in this project will be a useful tool for research and diagnosis of human disease. In research, they will facilitate identification of defects in transcription factor activities linked to a disease. In diagnosis, they will provide an opportunity to develop new disease detection tools which will identify disease through a specific pattern of transcription factor activities characteristic for a particular disease.

描述(申请人提供)：生物分子及其活性的高度多元化检测一直是现代分子诊断、药物开发以及基因组学和蛋白质组学研究的基本要素。已有成熟的DNA阵列方法允许对转录本进行高度多重检测。类似的检测或测量蛋白质活性的方法还没有很好地开发出来。在这一应用中，我们建议开发能够对序列特异性DNA结合蛋白进行高度多重检测的阵列。DNA结合蛋白是一类重要的生物分子，由于其在转录调控中的作用，对所有细胞过程的调控都是至关重要的。这项第一阶段提案的目标将是证明这一办法的可行性，并确立编制这类阵列的一般方法。这一目标将通过实现以下两个目标来实现：目标1.确定DNA结合蛋白微阵列是否能达到预期的检测灵敏度。我们将使用纯化的模型DNA结合蛋白进行实验，这些实验将建立程序，以最大限度地提高检测灵敏度。我们将比较在寡核苷酸微阵列中常用的几种荧光标记在DNA结合蛋白微阵列中的性能。我们将测试两种信号放大方案，以进一步提高检测灵敏度。最后，我们将确定优化的微阵列是否能够以所需的(Pm)灵敏度实现对模型蛋白质的可重复检测。目的#2.建立DNA结合蛋白微阵列是否可以分析复杂混合物中蛋白质的DNA结合活性。为了实现这一目标，我们将首先确定在包含不同的和已知的相对数量的这些蛋白质的样本中，微阵列是否能够真实地报告4种DNA结合蛋白的相对水平。其次，我们将在HeLa细胞提取液中检测SP1蛋白(一种结构性表达的转录因子)在其自然环境中的正常表达水平。该项目的长期目标将是开发高度多重检测所有主要细胞途径中涉及的转录因子的阵列，并将这些阵列开发为检测异常细胞状态的诊断工具。该项目将开发的DNA结合蛋白微阵列将成为研究和诊断人类疾病的有用工具。在研究中，它们将有助于识别与疾病有关的转录因子活性缺陷。在诊断方面，他们将提供机会开发新的疾病检测工具，通过特定疾病特有的转录因子活动的特定模式来识别疾病。