EFFECTS OF SELECTED METAL SALTS ON THE FIDELITY OF DNA SYNTHESIS IN VITRO

人体接触痕量金属的生理效应 工作场所和环境中的环境都有很好的记录。几种金属都有 被鉴定为诱变剂和致癌物。值得注意的是，锰、钴、镉、 已发现银、钡、铬、铅、镍和砷会增加碱基取代 在体外DNA复制试验中，误差至少为30% 检测与A相反的错误配对的核苷酸的错误结合 合成多核苷酸DNA模板。 在这项提案中，全面分析了沙门氏菌的诱变效应 金属盐对DNA合成保真度的影响将使用 体外保真度测定。该检测方法可以检测到各种 在合成250个核苷酸序列过程中出现的不同错误 包含在天然DNA模板中。利用这个系统，我们将 系统地研究了各种金属盐类对水的影响。 体外DNA合成的保真度。具体来说，我们将测量剂量 所选金属对体外DNA合成和保真度的影响 确定特定类型的突变频率和错误率 突变，并构建所选金属的突变谱。这个 预计这项工作的结果将为 所选金属的诱变特异性和剂量水平，并且可以 对金属诱变的机制提供进一步的洞察。 这个项目将包括各个方面的学生。学生们将学到 DNA操作基础、空斑分析、体外DNA 复制、微生物技术、DNA测序、数据管理、 以及危险金属盐和放射性核素的安全使用。

The physiological effects of exposure to trace quantities of metals in the workplace and in the environment are well documented. Several metals have been identified as mutagens as well as carcinogens. Notably, Mn, Co, Cd, Ag, Ba, Cr, Pb, Ni, and As have been found to increase base substitution errors by at least 30% in an in vitro DNA replication assay designed to detect misincorporation of an incorrectly paired nucleotide opposite a synthetic polynucleotide DNA template. In this proposal, a comprehensive analysis of the mutagenetic effects of metal salts on the fidelity of DNA synthesis will be undertaken using an in vitro fidelity assay. The assay allows detection of a variety of different errors occurring during synthesis of a 250 nucleotide sequence contained within a natural DNA template. Utilizing this system, we will systematically study the effects of a variety of metal salts on the fidelity of in vitro DNA synthesis. Specifically, we will measure dose effects of the selected metals on in vitro DNA synthesis and fidelity, determine mutation frequencies and error rates for specific types of mutations, and construct mutational spectra for the selected metals. The results of this work are expected to provide valuable information on mutagenic specificity and dose levels for the selected metals, and may provide further insight into the mechanism of metal-induced mutagenesis. This project will include students in every aspect. Students will learn fundamentals of DNA manipulations, plaque assays, in vitro DNA replication, microbiological techniques, DNA sequencing, data management, and safe use of hazardous metal salts and radionuclides.