ULTRATRACE ANALYSIS OF DNA LESIONS WITH ELECTROPHORES

使用电泳仪对 DNA 损伤进行超微量分析

• 批准号: 3173390
• 负责人: ROGER Wallace GIESE
• 金额: $ 11.39万
• 依托单位: NORTHEASTERN UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1983
• 资助国家: 美国
• 起止时间: 1983-09-01 至 1986-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3173390
• 关键词: DNA; carcinogenesis; drug metabolism; environment related neoplasm /cancer; ions; mass spectrometry; monomer; polymers; trace elements

The objective of this project is to develop ultrasensitive methodology for measuring damage to DNA caused by environmental carcinogens. Gas phase electrophores as labeling agents for the damaged DNA will provide the ultrasensitivity when measured by gas chromatography with electron capture detection (GC-ECD) or gas chromatography with negative ion chemical ionization mass spectrometry (GC-NICI-MS). Preliminary results suggest, at least for standards, that damaged DNA monomers (bases, nucleosides, or nucleotides) potentially can be quantitated at the low 10-16 mole level, which would allow only 100 modifications of DNA per cell to be measured in human microbiopsy samples. Overall the methodology consists of the following steps: (1) purification of the DNA; (2) hydrolysis (acid or enzymes) of the DNA down to DNA monomers; (3) separation of lesion (damaged) from normal DNA monomers by high performance liquid chromatography (HPLC); and either (4a) chemical labeling of the lesion monomers with "direct electrophores", followed by characterization and quantitation of these electrophore-labeled monomers by GC-ECD/GC-NICI-MS, or (4b) chemical labeling of the lesion monomers with "release tag electrophores", followed by characterization using HPLC and quantitation by GC-ECD. The specific aims are to apply this new methodology first to model lesion DNA monomers involving methyl/ethyl damage, then to selected benzo(a)pyrene and acetylaminofluorene adducts of DNA monomers, and finally to corresponding damaged DNA polymers. Analysis of DNA extracted from cells treated in tissue culture also will be undertaken. Ultrasensitive analysis is needed to determine the importance of human exposure to low levels of carcinogens. The project involves the scientific disciplines of analytical chemistry and biochemical pharmacology.

这个项目的目标是开发超灵敏的方法学 测量环境致癌物质对DNA的损伤。气相 作为受损DNA的标记剂的电泳仪将提供 电子捕获气相色谱测定的超高灵敏度 负离子化学检测(GC-ECD)或气相色谱 电离质谱仪(GC-NICI-MS)。初步结果表明，在 至少就标准而言，破坏DNA单体(碱基、核苷或 核苷酸)潜在地可以在低10-16摩尔水平上定量， 这将只允许测量每个细胞的100个DNA修饰 人体显微活组织检查样本。总体而言，该方法由 以下步骤：(1)DNA的纯化；(2)水解(酸或 酶)向下延伸到DNA单体；(3)病变的分离 用高效液体对正常DNA单体进行破坏 或(4a)病变的化学标记 具有“直接电泳法”的单体，然后进行表征和 用GC-ECD/GC-NICI-MS对这些凝胶标记单体进行定量， 或(4b)用“释放标签”对病变单体进行化学标记 电泳法“，然后使用高效液相色谱进行表征，并通过 GC-ECD联用。具体的目标是首先将这种新的方法应用于建模 涉及甲基/乙基损伤的损伤DNA单体，然后选择 DNA单体的苯并(A)芘和乙酰氨基荧加合物，最后 与相应的受损DNA聚合物有关。从沙棘中提取的DNA的分析 在组织培养中处理的细胞也将被采用。超灵敏 需要进行分析以确定人类暴露于低氧环境的重要性 致癌物质的水平。该项目涉及以下科学学科： 分析化学和生化药理学。