Identification of Areas of Oxidative Damage in Human Genomic DNA

描述（由申请人提供）：虽然对许多癌症相关基因中致癌物质诱导突变的定位已经给予了相当多的关注，但对可能导致致突变病变的原始DMA损伤的分布知之甚少。R21项目的目标是开发和验证检测DNA损伤位点的方法，并使我们能够确定这种损伤在全球和基因组DNA中特定位置的分布。我们的假设是DNA损伤的分布不是随机的，而是受基因组DNA的结构和功能特性的影响。为了验证这一假设，我们需要证明我们正在开发的方法将使我们能够可视化DNA损伤位点的分布，并将损伤位点与特定的基因组位置联系起来。一旦我们证明了这种方法的总体可行性，我们将进行实验来评估DNA损伤在特定功能目标中的分布。我们已经证明有能力执行建议分析所需的以下每个研究步骤：(1)我们已经能够使用分子梳理制备用于纤维分析的DNA；(2)我们利用荧光原位杂交（FISH）技术在大量基因组DNA中定位了一个特定的DNA序列；(3)我们使用生物素标记的醛反应探针或荧光探针可视化DNA中apurinic/ ap嘧啶（AP）位点的分布；(4)我们能够在进行复制的区域内直观地识别AP位点；(5)我们确定了正在进行复制的特定区域（所有描述在初步研究部分）。在这项研究中，我们提出以一种新颖的方式将这些技术结合起来是可行的，以确定基因组的某些区域，例如正在复制的位点，是否比其他基因组区域（或者在没有DNA复制的相同位点）对DNA损伤更敏感。为了实现这一目标，我们将使用已知AP位点数量的DNA纤维来确定对精梳DNA中AP位点检测的灵敏度。然后，我们将确定未经处理和处理过的DNA中AP位点的背景分布。最后，我们将确定是否有可能在同一实验样品中检测到所有三种技术（AP位点，FISH， DNA反染）的视觉信号。

DESCRIPTION (provided by applicant): While considerable attention has been given to mapping sites of carcinogen-induced mutations in a number of cancer related genes, less is known about the distribution of the original DMA damage that might have given rise to mutagenic lesions. The goal of this R21 project is to develop and validate methods that would detect sites of DNA damage and allow us to determine the distribution of such lesions both globally and at specific locations in genomic DNA. It is our hypothesis that the distribution of DNA damage is not random, but is influenced by the structure and functional properties of genomic DNA. To test this hypothesis, we need to demonstrate that the methods that we are developing will enable us to visualize the distribution of DNA damage sites and relate the damage sites to specific genomic locations. Once we have demonstrated the general feasibility of this approach, we will perform experiments to evaluate the distribution of DNA damage in specific functional targets. We have demonstrated the ability to perform each of the following research steps necessary for the proposed analysis: (1) We have been able to prepare DNA for fiber analysis using molecular combing; (2) We localized a specific DNA sequence among an excess of genomic DNA using fluorescence in situ hybridization (FISH) on combed DNA fibers; (3) We visualized the distribution of apurinic/apyrimidinic (AP) sites in DNA using either an aldehyde reactive probe tagged with biotin or a fluorescent probe; (4) We were able to visually identify AP sites within regions undergoing replication; and (5) We identified specific regions undergoing replication (all described in the Preliminary Studies section). In this study, we propose to show that it is feasible to combine these techniques in a novel way to establish whether certain areas of the genome, for example sites that are being replicated, are more sensitive to DNA damage than other genomic regions (or, the same sites in the absence of DNA replication). To accomplish this goal, we will determine the sensitivity of detection of AP sites in combed DNA using DNA fibers with a known number of AP sites. We will then determine the background distribution of AP sites in untreated and treated DNA. Finally, we will determine whether it is possible to detect visual signals from all three techniques (AP sites, FISH, DNA counterstain) in the same experimental samples. ASSESSMENT: