DNA ADDUCT SPECTROMETRY

我们通常认为化学突变是一组DNA加合物， 致突变前病变、细胞反应系统诱导物，或 两者 我们建议开发一种通用技术来测试这一命题。 我们打算绘制位置图，清点人数，并最终确定 序列特异性DNA加合物的化学结构形成于 也研究突变作为DNA功能的实验 顺序 我们的策略是首先通过变性分离加合的DNA序列 梯度凝胶电泳 第二，我们建议找出 通过DNA扩增在凝胶上定位加合的DNA序列， 单个凝胶切片。 最后，我们建议绘制 通过使用DNA聚合酶在鉴定的DNA序列中的加合物，或 外切核酸酶阻断，或者在某些情况下，通过定位在 DNA扩增 我们建议重点关注诱变剂MNNG和H2O2，以支持 我们的项目合作者在烷基化和氧化剂的研究 诱发突变，因为这些化学物质的预期加合物将 挑战我们的分析能力。 特别是，我们建议进行投入产出分析，加合 二倍体人MNNG和H_2O_2谱图和突变谱图 细胞 如果我们能够实现这些目标，我们进一步建议发展 提供足够位点特异性DNA加合物的适当技术 我们估计可以通过质谱法直接研究的材料 1012到1013个分子。

We commonly think of chemical mutation as a set of DNA adducts which are either premutagenic lesions, inducers of cellular response systems, or both. We propose to develop a general technology to test this proposition. We intend to map the position, count the number and, ultimately, identify the chemical structure of sequence specific DNA adducts formed in experiments in which mutation is also studied as a function of DNA sequence. Our strategy is to first separate adducted DNA sequences by denaturing gradient gel electrophoresis. Secondly, we propose to identify the position of adducted DNA sequences on the gel by DNA amplification of individual gel slices. Finally, we propose to map the positions of the adducts in the identified DNA sequences by use of DNA polymerase or exonuclease blocking or, in some cases, by mapping mutations arising during DNA amplification. We propose to focus on the mutagens MNNG and H2O2 to support the efforts of our Program collaborators in the study of alkylating and oxidizing agent induced mutation and because the expected adducts from these chemicals will challenge our analytical capability. In particular, we propose to perform an input-output analysis, adduct spectra map and mutation spectra map, for MNNG and H2O2 in diploid human cells. If we can reach these goals, we further propose to develop appropriate technology to provide sufficient site specific DNA adduct material for direct study by mass spectrometry which we estimate to be 1012to1013 molecules.