High Throughput Technology for Assessing Global DSB Repair Capacity

描述（由申请人提供）： 项目摘要/摘要提议开发和现场测试一种强大的高通量廉价方法，用于测量人类个体整体 DNA 修复动力学。潜在的应用包括与癌症治疗策略相关的流行病学研究，以及促进各种疾病预防策略的制定——无论是在独立的流行病学研究中，还是补充基于分子的关联研究。所提出的方法建立在当前的全自动高通量生物剂量测定系统 RABIT（快速自动化生物剂量测定工具）的基础上，该系统使用基于指尖采血的血液样本和原位多孔板平台来测量淋巴细胞中 3-H2AX 焦点的产量。 RABIT 系统目前仅检测一种 DNA 修复蛋白 (3-H2AX)，并且每个样本仅检测一次；该系统将通过以下方式进行调整，以表征 DNA 修复动力学：A) 添加自动辐照器，将 DNA 双链断裂引入指尖样本中，B) 扩展基于机器人的分析系统，以允许在辐照挑战后自动测量时间函数，C) 扩展系统以允许同时测量多个 DNA 修复蛋白。该系统将能够以高通量（e1,000 个样本/天）表征全局 DSB 修复动力学，通过对 3-H2AX、ATM[ser1981]、53BP1 和 Mdc1 修复蛋白（选择作为主要 DSB 修复途径中一系列节点的代表）的全自动免疫组织化学测量进行分析。我们的目标如下： 1. 设计一种创新型紧凑型自动毛细管辐照器并将其集成到 RABIT 中，以将 DNA DSB 引入指尖采血样本中。由于我们仅照射短毛细管，并且是逐一连续照射的，因此照射器可以非常紧凑，因此可以使用低活度 90Sr/90Y 放射性种子。使用这些放射性种子可以最大限度地减少屏蔽要求，这既是因为放射性种子的活性低，又是因为这些种子发射短程β粒子。 2. 调整 RABIT，使其在自动照射后多次（0.5、2、4、8、24 小时）自动分析每个指尖样本的 3-H2AX、ATM[ser1981]、53BP1 和 Mdc1。 3. 已经优化了 3-H2AX 测定的生物方案以用于我们的全自动原位系统，对其他三种修复蛋白也将进行同样的操作。 4. 建立定量方法来比较不同生物标志物/不同样品的测量 DNA 修复动力学，并能够识别异常值。 5. 通过首次测量健康人群中的全局 DSB 修复动力学，展示系统的实用性和可扩展性。将从 150 名健康志愿者身上采集指尖毛细血管血样，并使用改进的 RABIT 系统进行自动分析。将分析其动力学的四种修复蛋白代表主要 DSB 修复途径中的一系列节点。 公共卫生相关性： 拟议的项目叙述是开发和现场测试一种强大的高通量廉价方法，用于测量人类个体整体 DNA 修复动力学。潜在的应用包括与癌症治疗策略相关的流行病学研究，以及促进各种疾病预防策略的制定。

DESCRIPTION (provided by applicant): Project Summary/Abstract Proposed is development and field testing of a robust high-throughput inexpensive approach for measuring individual global DNA repair kinetics in humans. Potential applications are for epidemiological studies relating to cancer therapy strategies, and also to facilitate development of preventive strategies for a variety of diseases - either in standalone epidemiological studies, or to complement molecularly-based association studies. The proposed approach builds on a current fully-automated high-throughput biodosimetry system, the RABIT (Rapid Automated BIodosimetry Tool), which measures yields of 3-H2AX foci in lymphocytes, using fingerstick-based blood samples and an in-situ multi-well plate platform. The RABIT system currently assays only one DNA repair protein (3-H2AX), and at only one time for each sample; the system will be adapted to characterize DNA repair kinetics by A) adding an automated irradiator, to introduce DNA double strand breaks into the fingerstick sample, B) extending the robotically-based analysis system to allow automated measurements of a function of time after the irradiation challenge, C) extending the system to allow simultaneous measurements of more than one DNA repair protein. The system will be able to characterize, with high throughput (e1,000 samples / day) global DSB repair kinetics, as assayed by fully automated immunohistochemical measurements of 3-H2AX, ATM[ser1981], 53BP1, and Mdc1 repair proteins - chosen as representative of a range of nodal points in the main DSB repair pathways. Our goals are as follows: 1. Design and incorporate into the RABIT an innovative compact automated capillary irradiator, to introduce DNA DSBs into the fingerstick blood sample. Because we irradiate only short capillary tubes, and on a one- by-one sequential basis, the irradiator can be very compact, so low-activity 90Sr/90Y radioactive seeds can be used. Use of these radioactive seeds minimizes the shielding requirements, both because of the low activity and because these seeds emit short-ranged beta particles. 2. Adapt the RABIT so that it automatically assays each fingerstick sample for 3-H2AX, ATM[ser1981], 53BP1, and Mdc1, and at multiple times (0.5, 2, 4, 8, 24 hrs) after the automated irradiation. 3. Having already optimized the biological protocol for the 3-H2AX assay for use in our fully automated in-situ system, the same will be done for the other three repair proteins. 4. Establish a quantitative approach for comparing the measured DNA repair kinetics of different biomarkers / different samples, and to be able to identify outliers. 5. Demonstrate the practicality and scalability of the system by measuring, for the first time, global DSB repair kinetics in a healthy human population. Fingerstick capillary blood samples will be collected from 150 healthy volunteers, and automatically analyzed using the modified RABIT system. The four repair proteins whose kinetics will be analyzed represent a range of nodal points in the main DSB repair pathways. PUBLIC HEALTH RELEVANCE: Project Narrative Proposed is development and field testing of a robust high-throughput inexpensive approach for measuring individual global DNA repair kinetics in humans. Potential applications are for epidemiological studies relating to cancer therapy strategies, and also to facilitate the development of preventive strategies for a variety of diseases.