PROJECT SUMMARY Genomic instability, induced by DNA damage, and partly mitigated by DNA repair and antioxidants, plays a critical role in the pathogenesis of many major human diseases, such as neurodegeneration, cancer and cardiovascular disease, together with aging. There is a critical need for automated assays that can perform these assessments, and bring them into greater routine use, which will help advance our understanding of disease, and its environmental causes. We propose to address this void by combining our expertise in automated liquid handling and medical device engineering, with our innovations in single cell gel electrophoresis (the comet assay). Due to its relative simplicity, the comet assay continues to gain increasing popularity as a means of quantifying DNA damage/repair/antioxidant capacity. In addition to regulatory applications, the comet assay is used widely for human biomonitoring, assessing environmental and occupational exposures, and understanding basic biological mechanisms. Although we, and others, have described innovations that have increased sample throughput for the comet assay, it remains time-consuming (up to three days) and, for the most part, labor intensive (multiple steps, and multiple sample slides processed individually), which can also introduce errors and variation. We have developed a method for high throughput processing of comet assay slides (HTP comet; patent pending). However, this method, as with all comet assay methods, requires operator involvement at numerous key steps, which increases the total cost of the assay, and limits its more widespread use. We have further developed a patented, proof-of-principle device that fully automates the majority of the steps in the comet assay, making it a fully walkaway system, that decreases assay run time; removes all slide manipulations, and the need for a dedicated dark room, increases safety, control and standardization of conditions, and minimizes operator involvement. This STTR application represents a strategic effort between Engineering Resources Group (an NSF I-corps experienced, SBC), and Florida International University, to determine the technical merit, feasibility, and commercial potential of an automated HTP (AHTP) comet device through refinement and miniaturization. The availability of such a self-contained, benchtop device, as proposed, will accelerate existing applications of the comet assay, as well as increase the number of sites and contexts in which DNA damage/repair/antioxidants status can be assessed. In turn, this will advance translational discovery, stimulate biomonitoring, and promote new applications for the assessment of DNA damage, repair, and cellular antioxidant capacity.

PROJECT NARRATIVE DNA damage, caused by environmental agents, or the body’s normal processes, plays a critical role in the development of many major human diseases, with profound public health implications. We propose the development of an automated device, which is simple, rapid, with increased throughput, and affords increased precision, to support the assessment of DNA damage and repair. Facilitating such measures will help advance our knowledge of the role of DNA damage in health and disease.