Development of a standard high throughput comet assay

标准高通量彗星测定的开发

• 批准号: 7287399
• 负责人: Jay George
• 金额: $ 44.86万
• 依托单位: TREVIGEN, INC.
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-01-01 至 2009-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7287399
• 关键词: Achievement; Acute; Address; Adoption; Affect; Age; Alkaline Single-Cell Gel Electrophoresis Assay; Anodes; Antigens; Antineoplastic Agents; Appendix; Archives; Area; Attention; Automation; Benzopyrenes; Biological; Biological Assay; Biological Monitoring; Biological Preservation; Buffers; Carcinogens; Cell Count; Cell Cycle; Cell Cycle Arrest; Cell Death; Cell Line; Cells; Chemicals; Chronic; Cleaved cell; Collecting Cell; Comet Assay; Communities; Computer software; Condition; Consensus; Contracts; Controlled Environment; Critiques; Cytolysis; DNA; DNA Adducts; DNA Damage; DNA Repair; DNA analysis; DNA glycosylase; Data; Data Analyses; Deposition; Detection; Detergents; Development; Development Plans; Device or Instrument Development; Educational workshop; Electrophoresis; Engineering; Ensure; Environmental Monitoring; Epidemiology; Epoxy Compounds; Equipment; Exposure to; Feedback; Fluorescence; Fluorescent Dyes; Fluorescent in Situ Hybridization; Funding; Future; Gel; Genes; Genetic; Germany; Glass; Glycol; Goals; Grant; Guidelines; Hand; Height; Imagery; Immobilized Cells; Immunohistochemistry; In Situ; In Situ Hybridization; Individual; International; Laboratories; Lead; Learning; Letters; Manuals; Manufacturer Name; Market Research; Marketing; Metabolic; Methods; Microscope; Modification; Monitor; Movement; Mutagens; Mutation; Mycoplasma; Nature; Normal Cell; Numbers; Nutritional; Olives - dietary; Operative Surgical Procedures; Patients; Pattern; Performance; Persons; Pharmaceutical Preparations; Pharmacologic Substance; Phase; Phase I Clinical Trials; Positioning Attribute; Preclinical Drug Evaluation; Preparation; Price; Procedures; Process; Progress Reports; Protocols documentation; Publications; Published Comment; Publishing; Pump; Purpose; Radiation; Range; Rate; Reagent; Recommendation; Records; Reporting; Reproducibility; Research; Research Personnel; Resistance; Retirement; Risk Assessment; Running; Sales; Sampling; Scientist; Screening procedure; Sepharose; Signal Transduction; Site; Slide; Small Business Funding Mechanisms; Small Business Innovation Research Grant; Sodium Chloride; Source; Specific qualifier value; Staining method; Stains; Standardization; Standards of Weights and Measures; Structure; Suggestion; System; Tail; Techniques; Technology; Temperature; Testing; Text; Time; Tissues; Toxicology; Treatment Effectiveness; Treatment outcome; United States Food and Drug Administration; Validation; Variant; Virus; Work; adduct; anticancer research; base; cancer cell; cancer risk; cancer therapy; carcinogenesis; cell preparation; cell type; chemotherapeutic agent; commercialization; cost; cost effective; crosslink; day; design; drug development; drug discovery; electric field; endonuclease; gel electrophoresis; genotoxicity; high throughput analysis; improved; instrument; instrumentation; interest; melting; migration; neoplastic cell; pre-clinical; programs; prototype; reagent standardization; repaired; research study; response; sensor; therapy resistant; tumorigenesis; voltage

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.

描述（由申请人提供）：单细胞凝胶电泳（SCGE）或彗星测定现在被全世界公认为是检测不同类型DNA损伤的简单且廉价的方法。迄今为止，由于缺乏标准化平台和检测程序，以及多样本分析检测的劳动密集型特性，彗星检测在基因毒性研究中的采用受到了阻碍。与该测定相关的另一个问题是，为了避免对细胞引入人工 DNA 损伤，该测定需要对新鲜分离的细胞或组织进行。这使得直接比较在不同时间收集的细胞或在无法立即进行测定的现场收集样本变得困难。 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制备的测定。这些载玻片目前有用于单次测定分析（2 孔格式）和高通量分析（20 和 96 孔格式）的格式。 Trevigen 还在初步研究中证明，可以保存没有 DNA 损伤的细胞，以便以后进行分析或在后续测定中用作标准品。 II 期应用的目标是： 1. 通过开发细胞中受损 DNA 电泳的受控环境，标准化彗星测定的电泳平台和程序，这将消除与日常操作中彗星测定重现性相关的问题。 2. 以不同浓度DNA损伤剂处理的细胞为基础，制定彗星实验标准品，并长期保存这些标准品或其他生物材料，作为彗星实验的参考材料。这些目标的实现将产生标准化的彗星测定程序，该程序可用于任何实验室，并可开发为高通量自动系统。保存没有 DNA 损伤的细胞将有助于制定彗星测定标准，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。它还允许安全运输现场收集的样本以供稍后分析。单细胞凝胶电泳 (SCGE) 或彗星测定是检测不同类型 DNA 损伤的简单且廉价的方法。该项目的长期目标是开发标准化平台和程序，包括该检测的 DNA 标准。