ULTRATRACE ANALYSIS OF DNA LESIONS WITH ELECTROPHORES

该项目的目标是开发超灵敏的方法， 测量环境致癌物对DNA造成的损伤。 气相 作为受损DNA的标记剂， 当通过具有电子捕获的气相色谱法测量时的超灵敏度 检测（GC-ECD）或采用负离子化学品的气相色谱法 离子化质谱（GC-NICI-MS）。 初步结果显示，在 至少对于标准品，破坏DNA单体（碱基、核苷或 核苷酸）可以在低的10-16摩尔水平下定量， 这将允许每个细胞只能测量100个DNA修饰， 人类显微活检样本。 总体而言，该方法包括： 步骤如下：（1）纯化DNA;（2）水解（酸或 酶）的DNA下降到DNA单体;（3）分离病变 高性能液体从正常的DNA单体（受损） （4a）病变的化学标记 单体与“直接酯化物”，然后进行表征， 通过GC-ECD/GC-NICI-MS定量这些经双氢呋喃标记的单体， 或（4 b）用“释放标签”化学标记损伤单体 随后使用HPLC表征并通过HPLC定量。 GC-ECD。 具体目标是首先将这种新方法应用于建模 损伤DNA单体涉及甲基/乙基损伤，然后选择 DNA单体的苯并（a）芘和乙酰氨基芴加合物，以及最后 对应受损的DNA聚合物 DNA提取分析 还将进行在组织培养中处理的细胞。 超灵敏 需要进行分析，以确定人类暴露于低浓度 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。

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.