High Throughput Technology for Assessing Global DSB Repair Capacity

描述(由申请人提供)： 项目摘要/摘要建议开发和现场测试一种健壮、高通量、廉价的方法来测量人类个体的全球DNA修复动力学。潜在的应用是与癌症治疗策略有关的流行病学研究，以及促进各种疾病的预防策略的制定--无论是在独立的流行病学研究中，还是在补充基于分子的关联研究中。所提出的方法建立在目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，该系统使用基于手指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系统目前只检测一种DNA修复蛋白(3-H2AX)，并且每个样本只检测一次；该系统将被调整为通过以下方式来表征DNA修复动力学：A)增加自动辐照仪，将DNA双链断裂引入手指样本，B)扩展基于机器人的分析系统，以允许自动测量辐射挑战后的时间函数，C)扩展系统以允许同时测量不止一种DNA修复蛋白。该系统将能够以高通量(E1000个样本/天)表征全球DSB修复动力学，如通过全自动免疫组织化学测量3-H2AX、ATM[Ser1981]、53BP1和Mdc1修复蛋白-被选为主要DSB修复途径中的一系列节点的代表。我们的目标如下：1.设计并集成一种创新的紧凑型自动毛细管辐照器，将DNA DSB引入手指血样。由于我们只照射较短的毛细管，并按顺序逐一照射，因此辐照器可以非常紧凑，因此可以使用低活度的90Sr/90Y放射性种子。这些放射性种子的使用将屏蔽需求降至最低，这既是因为这些种子的活性较低，也是因为这些种子释放出短程的贝塔粒子。2.调整RABIT，使其在自动照射后多次(0.5、2、4、8、24小时)自动检测每个手指样本的3-H2AX、ATM[Ser1981]、53BP1和MDc1。3.已经优化了用于我们全自动原位系统的3-H_2AX检测的生物学程序，将对其他三种修复蛋白进行同样的操作。4.建立一种定量方法，用于比较不同生物标志物/不同样本的DNA修复动力学，并能够识别异常值。5.通过首次在健康人群中测量全球DSB修复动力学，证明该系统的实用性和可扩展性。指纹毛细血管血样将从150名健康志愿者身上采集，并使用改进的RABIT系统进行自动分析。这四个修复蛋白的动力学将被分析，它们代表了主要DSB修复途径中的一系列节点。 公共卫生相关性： 提出的项目叙述是开发和现场测试一种强大的、高通量的、廉价的方法来测量人类个体的全球DNA修复动力学。潜在的应用是与癌症治疗策略有关的流行病学研究，也有助于为各种疾病制定预防策略。

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.