Comet-Chip: A High-Throughput DNA Damage Sensor for Enviornmental Health Studies

描述(由申请人提供)： DNA损伤是癌症和许多其他疾病的重要风险因素。无论是环境诱导的还是内源性的，对DNA结构的共价修饰都可能是细胞毒性和突变性的。因此，能够测量人类样本中的DNA损伤和修复从根本上来说是有价值的，既可以描绘使细胞容易受到突变影响的环境条件，也可以揭示调节DNA损伤易感性的遗传因素。单细胞凝胶电泳法，或称彗星试验，是测量人类细胞DNA损伤的最灵敏和最通用的方法之一。它的基础是一个简单的原理：当可视化嵌入琼脂糖中的电泳细胞时，未受损的DNA是超级卷曲的且高度致密，而受损的DNA(松弛的环和片段)可以更容易地迁移，从而产生一个明亮的具有彗星状尾巴的类核。尽管彗星试验被证明有效，但在流行病学研究中对环境风险因素的研究没有得到充分利用，主要是因为缺乏标准化，导致研究人员之间的结果不一致，以及执行分析所需的时间/人力。在这里，我们建议应用芯片实验室技术来创造一种“彗星芯片”，它不仅可以克服标准化方面的问题，还可以高通量地并行处理数十个样本。我们预计，拟议的技术将使该检测方法在广泛的临床、流行病学和实验环境中发挥作用。在具体目标一中，将利用液滴阵列方法并行处理数十个样品。具体目标二旨在将细胞构型技术与彗星试验相结合，以评估微环境对细胞间变异性的影响。对于特定的目标III，我们建议创建一个自给自足的彗星设备，以提供急需的一致性。最后，针对特定的目标IV，我们建议将‘彗星芯片’应用于具有不同DNA修复能力的小鼠和人类样本，并严格评估重复性和敏感性。重要的是，虽然我们提议制造的最高端处理器需要复杂的设备，但许多拟议的修改可以以相当低的技术方式应用于彗星测试，因此可以广泛传播。高通量的DNA损伤和反应传感器对于识别危险的环境暴露以及评估旨在减少相关暴露的政策决策的有效性都将是无价的。人们希望，拟议中的技术将产生数据，使政策制定者能够制定明智的干预策略，在疾病发生之前很久就有效地预防癌症和其他疾病。

DESCRIPTION (provided by applicant): DNA damage is an important risk factor for cancer and many other diseases. Whether induced by the environment or created endogenously, covalent modifications to DNA structure can be both cytotoxic and mutagenic. Being able to measure DNA damage and repair in human samples is therefore fundamentally valuable, both for delineating environmental conditions that render cells vulnerable to mutations, and for revealing genetic factors that modulate susceptibility to DNA damage. The single cell gel electrophoresis assay, or 'comet assay' is one of the most sensitive and versatile approaches for measuring DNA damage in human cells. It is grounded on a simple principle: when visualizing electrophoresed cells embedded in agarose, undamaged DNA is supercoiled and highly compact, whereas damaged DNA (relaxed loops and fragments) can more readily migrate, giving rise to the appearance of a bright nucleoid with a comet-like tail. Despite its proven efficacy, the comet assay is underutilized in studies of environmental risk factors in epidemiological studies, mostly because of a lack of standardization that has lead to inconsistent results among researchers and due to the time/labor required to perform the assay. Here, we propose to apply lab- on-a-chip technologies to create a "comet-chip" that will not only overcome problems in standardization, but will also permit high-throughput parallel processing of dozens of samples. We anticipate that the proposed technology will render this assay useful in a broad range of clinical, epidemiological, and experimental settings. In Specific Aim I, droplet-array methodology will be exploited for parallel processing of dozens of samples. Specific Aim II is aimed at combining cell patterning technologies with the comet assay in order to asses the impact of microenvironment on variability among cells. For Specific Aim III, we propose to create a self-contained comet device to provide much needed consistency. Finally, for Specific Aim IV, we propose to apply the 'comet chip' to mouse and human samples of varied DNA repair capacities, and to rigorously evaluate reproducibility and sensitivity. Importantly, while sophisticated equipment will be necessary for the highest-end processor that we propose to create, many of the proposed modifications can be applied to the comet assay in a fairly low-tech fashion, and thus can be widely disseminated. A high- throughput DNA damage and response sensor will be invaluable both for discerning dangerous environmental exposures, and for evaluating the efficacy of policy decisions aimed at reducing relevant exposures. It is hoped that the proposed technology will yield data to empower policy makers in the development of wise intervention strategies that will effectively prevent cancer and other illnesses, long before disease onset.