Development of a standard high throughput comet assay

描述(申请人提供)：单细胞凝胶电泳法(SCGE)或彗星试验现在是全世界公认的一种检测不同类型DNA损伤的简单且廉价的方法。迄今为止，由于缺乏标准化的分析平台和程序，以及多样本分析的劳动密集型性质，彗星试验在遗传毒性研究中的采用一直受到阻碍。与检测相关的另一个问题是，为了避免引入人造DNA对细胞的损害，需要对新分离的细胞或组织进行检测。这使得很难直接比较在不同时间收集的细胞，或者在无法立即进行分析的现场收集样本。Trevigen在第一阶段的应用中开发了一种高通量彗星载玻片，用于标准化彗星载玻片的分析准备。这些载玻片目前有单孔分析格式(2孔格式)和高通量分析格式(20孔格式和96孔格式)。特雷维根还在初步研究中证明，有可能在不造成DNA损伤的情况下保存细胞，供以后分析或用作后续分析的标准。第二阶段应用的目标是：1.通过为细胞中受损的DNA开发一个受控的电泳环境，使彗星分析的电泳平台和程序标准化，这将消除与彗星分析在日常操作中的重复性有关的问题。2.建立以不同浓度DNA损伤剂处理的细胞为基础的彗星试验标准品，并将这些标准品或其他生物材料长期保存，作为彗星试验的参照物。这些目标的实现将导致标准化的彗星分析程序，该程序可以在任何实验室使用，并可以开发为高通量的自动化系统。在不破坏DNA的情况下保存细胞将使彗星测试的标准得以制定，并将使生物材料能够被存档以供未来研究或在实验室之间共享。它还将允许现场收集的样本被安全运输，供以后进行分析。单细胞凝胶电泳法(SCGE)或彗星试验是一种简单、廉价的检测不同类型DNA损伤的方法。该项目的长期目标是开发标准化的平台和程序，包括用于该检测的DNA标准。

DESCRIPTION (provided by applicant): The single cell gel electrophoresis (SCGE) or comet assay is now recognized worldwide as a simple and inexpensive method for detection of different types of DNA damage. Adoption of the comet assay for genotoxic studies has been hampered to date by the lack of a standardized platform and procedures for the assay, as well as the labor-intensive nature of the assay for multiple-sample analysis. Another problem associated with the assay is that to avoid introduction of artificial DNA damage to cells, the assay needs to be run of freshly isolated cells or tissues. This make it difficult to direct compare cells collected at various times, or to collect samples in the field where the assay cannot immediately performed. Trevigen in the Phase I application developed a high throughput comet slides for standardization of the comet slide preparation for the assay. These slides are available at present in formats for single assay analysis (2 well format) and also for high throughput analysis (20 and 96 well format). Trevigen also demonstrated in preliminary studies that it is possible to preserve cells without DNA damage for analysis later or to use as standards in subsequent assays. The goals of the Phase II application are: 1. To standardize the electrophoretic platform and procedures for the comet assay by developing a controlled environment for electrophoresis of the damaged DNA in the cells, which will eliminate the problems, associated with reproducibility of the comet assay in the day to day operation. 2. To develop comet assay standards based on cells, which were treated with various concentration of DNA damaging agents, and long term preservation of these standards or other biological materials, to be used as reference material in the comet assay. The accomplishment of these aims will results in a standardized comet assay procedure, which can be used in any laboratory and can be developed as a high throughput automatic system. The preservation of cells without DNA damage will allow development of standards for the comet assay and will allow that biological material could be archived for future studies or to be shared between laboratories. It would also allow field collected samples to be safely transported for analysis later. The single cell gel electrophoresis (SCGE) or comet assay is a simple and inexpensive method for detection of different types of DNA damage. The long-term objective of this project is to develop standardized platform and procedures, including DNA standards for this assay.