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: 3173391
• 负责人: ROGER Wallace GIESE
• 金额: $ 14.5万
• 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 1989-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3173391
• 关键词: DNA; carcinogenesis; drug metabolism; electrophoresis; environment related neoplasm /cancer; ions; mass spectrometry; monomer; polymers; trace elements

The goal of this project is to quantitate DNA adducts in human samples by gas chromatography with electron capture detection (GC-ECD) and with detection by negative chemical ionization mass spectrometry (GC-NCI-MS). High sensitivity is obtained by labeling the adducts with electrophores. For standards of pyrimidine bases and nucleosides, detection limits at the low fg level are currently observed. 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 (damage) 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. First the methodology will be applied, including ongoing work, to several alkyl adducts: O6-alkyl-guanine, O4-alkyl-thymine, O2-alkyl-thymine, and O2-alkyl-cytosine. Next the corresponding hydroxyethyl adducts anticipated to arise from exposure to ethylene oxide will be determined. Finally N6-hydroxymethyl-adenine and protein-(lysyl)-nucleoside will be determined as potential adducts caused by exposure to formaldehyde. This research is needed to help determine the carcinogenic and mutagenic risks of human exposure to chemicals.

该项目的目标是通过以下方法对人体样本中的DNA加合物进行定量 气相色谱-电子捕获检测(GC-ECD) 负化学电离质谱仪(GC-NCI-MS)检测。 用电泳法标记加合物可获得较高的灵敏度。 关于嘧啶碱基和核苷的标准，检出限在 目前观察到低Fg水平。总体而言，该方法包括 以下步骤：(1)DNA的纯化；(2)水解(酸或 酶)向下延伸到DNA单体；(3)分离病变(损伤) 用高效液相色谱法从正常DNA单体中提取DNA； 或(4a)用“DIRECT”标记病变单体 《电光》，然后对其进行表征和量化 GC-ECD/GC-NiCI-MS或(4b)化学电泳法标记单体 用“释放标签电泳法”标记病变单体，随后 用高效液相色谱法进行表征，用气相色谱-电子捕获检测器定量。首先是 将对几个烷基化合物采用方法，包括正在进行的工作 加合物：O6-烷基-鸟氨酸、O4-烷基-胸腺嘧啶、O2-烷基-胸腺嘧啶和 O2-烷基胞嘧啶。接下来，预期相应的羟乙基加合物 将确定因接触环氧乙烷而产生的危险。终于 将测定N6-羟甲基腺嘌呤和蛋白质-(赖氨基)-核苷 作为暴露在甲醛中的潜在加合物。这项研究是 需要帮助确定人类致癌和突变的风险 暴露在化学品中。