A novel ChIP-on-Chip technology for ENCODE

描述：(申请人提供)为响应ENCODE倡议，我们建议开发一种新的芯片上技术来绘制人类基因组中功能DNA元件的图谱。这项拟议的技术是将标准染色质免疫沉淀与一种新的方法相结合，以扩增和排列丰富的DNA。这项拟议的技术旨在提高现有芯片上技术的灵敏度，并采用新的实验设计，以允许未来的大规模应用。 我们计划分四步系统地开发这项技术。(1)我们将从已有的启动子阵列平台入手，确定是否可以使用签名寡核苷酸来取代阵列中PCR扩增的启动子片段。这可能会提高现有系统的杂交效率和特异性。基于寡核苷酸的阵列将更容易用于未来的大规模测绘研究。更重要的是，第一个目标中的实验将为下一阶段的技术开发奠定基础。(2)我们将实施一项新的策略来扩增和标记免疫沉淀DNA。我们的程序将结合在其他基因组学方法中成功使用的一些策略，包括寡聚连接、固相选择和以相对公正的方式进行信号放大。根据设计，该技术有可能成为各种功能基因组应用的高特异性和敏感性的通用平台。(3)在第三个目标中，我们将通过进行现有技术无法解决的三个实验，来记录正在开发的技术的力量。由于每组实验都将由单独的团队实验室并行进行，因此我们最终将对该技术进行广泛的功能验证。实验设计还旨在创造参与实验室之间的协同效应，以便我们的阵列系统将逐步扩大，这将为我们未来更大的项目做好准备。(4)最后，我们将测试一种通用策略，以无偏见地在ENCODE项目定义的染色体区域中发现参与转录调控和转录-剪接耦合的功能DNA元件。拟议的研究将是一种力量 以一种方式说明这项技术在解决广泛的重要生物学问题方面的效用。

DESCRIPTION: (provided by applicant) In response to the ENCODE initiative, we propose to develop a novel ChiP-on-Chip technology to map functional DNA elements in the human genome. The proposed technology is to couple standard chromatin immunoprecipition with a new way to amplify and array the enriched DNA. The proposed technology aims to improve the sensitivity of an existing ChiP-on-Chip technology with new experimental design to permit future large-scale applications. We plan to systematically develop the technology in four steps. (1) We will start with the existing promoter array platform already in hand and determine whether we can use signature oligos to replace PCR-amplified promoter fragments in the array. This may improve hybridization efficiency and specificity of the existing system. The oligo-based array would be easier for future large scale mapping studies. More importantly, the experiments in the first aim will set the stage for the next phase of technology development. (2) We will implement a new strategy to amplify and label immunoprecipitated DNA. Our procedure will incorporate a number of strategies successfully used in other genomics approaches, including oligo ligation, solid phase selection, and signal amplification in a relatively unbiased way. According to the design, the technology has the potential to become a general platform with high specificity and sensitivity for a variety of functional genomics applications. (3) We will in the third aim to document the power of the technology under development by conducting three lines of experiments that can not be addressed with any existing technologies. Because each set of the experiments will be carried out in parallel by individual team labs, we will have extensive functional validation of the technology in the end. The experimental design also aims to create synergies among participating labs such that our array system will be progressively enlarged, which will prepare us for larger projects in the future. (4) Finally, we will test a general strategy for unbiased discovery of functional DNA elements involved in transcriptional regulation and transcription-splicing coupling in a chromosomal region defined by the ENCODE project. The proposed studies would be a power way to illustrate the utility of the technology in addressing a wide range of important biological questions.