Development of a standard high throughput comet assay

描述（由申请人提供）：单细胞凝胶电泳（SCGE）或彗星分析是目前世界范围内公认的一种简单且廉价的检测不同类型DNA损伤的方法。迄今为止，由于缺乏标准化的分析平台和程序，以及用于多样本分析的分析的劳动密集型性质，在基因毒性研究中采用彗星分析一直受到阻碍。与该试验相关的另一个问题是，为了避免对细胞引入人工DNA损伤，该试验需要在新分离的细胞或组织中进行。这使得很难直接比较在不同时间收集的细胞，或者在不能立即进行分析的现场收集样品。Trevigen在I期申请中开发了一种高通量的彗星载玻片，用于标准化该试验的彗星载玻片制备。目前，这些载玻片可用于单分析（2孔格式）和高通量分析（20孔和96孔格式）。Trevigen还在初步研究中证明，保存没有DNA损伤的细胞以供以后分析或在随后的分析中用作标准是可能的。第二阶段申请的目标是：1。通过开发细胞中受损DNA电泳的受控环境，使彗星测定的电泳平台和程序标准化，这将消除彗星测定在日常操作中重复性相关的问题。2. 以不同浓度的DNA损伤剂处理过的细胞为基础，制定彗星测定标准品，并将这些标准品或其他生物材料长期保存，作为彗星测定的参考材料。这些目标的实现将导致一个标准化的彗星分析程序，它可以在任何实验室使用，并可以开发成一个高通量的自动系统。保存没有DNA损伤的细胞将有助于制定彗星试验的标准，并允许将生物材料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安全运输，以供以后分析。单细胞凝胶电泳（SCGE）或彗星测定是一种简单而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，包括该分析的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.