? ? 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. ? ? ? ?
