Wood David K, Weingeist David M, Bhatia Sangeeta N, Engelward Bevin P
Harvard-MIT Division of Health Sciences and Technology, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
Proc Natl Acad Sci U S A. 2010 Jun 1;107(22):10008-13. doi: 10.1073/pnas.1004056107. Epub 2010 May 13.
With a direct link to cancer, aging, and heritable diseases as well as a critical role in cancer treatment, the importance of DNA damage is well-established. The intense interest in DNA damage in applications ranging from epidemiology to drug development drives an urgent need for robust, high throughput, and inexpensive tools for objective, quantitative DNA damage analysis. We have developed a simple method for high throughput DNA damage measurements that provides information on multiple lesions and pathways. Our method utilizes single cells captured by gravity into a microwell array with DNA damage revealed morphologically by gel electrophoresis. Spatial encoding enables simultaneous assays of multiple experimental conditions performed in parallel with fully automated analysis. This method also enables novel functionalities, including multiplexed labeling for parallel single cell assays, as well as DNA damage measurement in cell aggregates. We have also developed 24- and 96-well versions, which are applicable to high throughput screening. Using this platform, we have quantified DNA repair capacities of individuals with different genetic backgrounds, and compared the efficacy of potential cancer chemotherapeutics as inhibitors of a critical DNA repair enzyme, human AP endonuclease. This platform enables high throughput assessment of multiple DNA repair pathways and subpathways in parallel, thus enabling new strategies for drug discovery, genotoxicity testing, and environmental health.
由于DNA损伤与癌症、衰老和遗传性疾病直接相关，且在癌症治疗中起关键作用，其重要性已得到充分确立。从流行病学到药物开发等诸多应用领域对DNA损伤的浓厚兴趣，促使人们迫切需要强大、高通量且廉价的工具来进行客观、定量的DNA损伤分析。我们开发了一种用于高通量DNA损伤测量的简单方法，该方法可提供有关多种损伤和途径的信息。我们的方法利用重力将单细胞捕获到微孔阵列中，通过凝胶电泳在形态上揭示DNA损伤。空间编码能够同时对多个并行进行的实验条件进行检测，并实现全自动分析。该方法还具备新功能，包括用于并行单细胞检测的多重标记，以及对细胞聚集体中的DNA损伤进行测量。我们还开发了24孔和96孔版本，适用于高通量筛选。利用这个平台，我们已经对具有不同遗传背景的个体的DNA修复能力进行了量化，并比较了潜在癌症化疗药物作为关键DNA修复酶——人AP核酸内切酶抑制剂的疗效。这个平台能够并行高通量评估多种DNA修复途径和子途径，从而为药物发现、遗传毒性测试和环境卫生开辟新策略。
