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: 3173387
• 负责人: ROGER Wallace GIESE
• 金额: $ 18.03万
• 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/3173387
• 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式定量人体样本中的DNA加合物 气相色谱-电子捕获检测器（GC-ECD）和 通过负化学电离质谱法（GC-NCI-MS）检测。 通过用双标记物标记加合物获得高灵敏度。 对于嘧啶碱基和核苷的标准品， 目前观察到低FG水平。 总体而言，该方法包括 以下步骤：（1）纯化DNA;（2）水解（酸或 （3）分离损伤（损伤） 通过高效液相色谱（HPLC）从正常DNA单体; 或者（4a）用“直接”标记的损伤单体的化学标记 这些化合物的表征和定量 通过GC-ECD/GC-NICI-MS法测定的三聚体标记单体，或（4 b）化学 用“释放标签标记物”标记病变单体， 通过HPLC表征和GC-ECD定量。 首先 将采用的方法，包括正在进行的工作， 加合物：O 6-烷基-鸟嘌呤，O 4-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 将确定暴露于环氧乙烷引起的风险。 最后 将测定N6-羟甲基-腺嘌呤和蛋白质-（赖氨酰）-核苷 可能是由于接触甲醛而产生的加合物。 本研究是 需要帮助确定人类的致癌和致突变风险， 暴露于化学物质。