AHTP-comet: development of an automated, high throughput comet assay system

PROJECT SUMMARY Genomic instabilityinduced by DNA damageand partly mitigated by DNA repair and antioxidantsplays a critical role in the pathogenesis of many major human diseasessuch as neurodegenerationcancer and cardiovascular diseasetogether with agingThere is a critical need for automated assays that can perform these assessmentsand bring them into greater routine usewhich will help advance our understanding of diseaseand its environmental causesWe propose to address this void by combining our expertise in automated liquid handling and medical device engineeringwith our innovations in single cell gel electrophoresisthe comet assayDue to its relative simplicitythe comet assay continues to gain increasing popularity as a means of quantifying DNA damage repair antioxidant capacityIn addition to regulatory applicationsthe comet assay is used widely for human biomonitoringassessing environmental and occupational exposuresand understanding basic biological mechanismsAlthough weand othershave described innovations that have increased sample throughput for the comet assayit remains time consumingup to three daysandfor the most partlabor intensivemultiple stepsand multiple sample slides processed individuallywhich can also introduce errors and variationWe have developed a method for high throughput processing of comet assay slidesHTP cometpatent pendingHoweverthis methodas with all comet assay methodsrequires operator involvement at numerous key stepswhich increases the total cost of the assayand limits its more widespread useWe have further developed a patentedproof of principle device that fully automates the majority of the steps in the comet assaymaking it a fully walkaway systemthat decreases assay run timeremoves all slide manipulationsand the need for a dedicated dark roomincreases safetycontrol and standardization of conditionsand minimizes operator involvementThis STTR application represents a strategic effort between Engineering Resources Groupan NSF I corps experiencedSBCand Florida International Universityto determine the technical meritfeasibilityand commercial potential of an automated HTPAHTPcomet device through refinement and miniaturizationThe availability of such a self containedbenchtop deviceas proposedwill accelerate existing applications of the comet assayas well as increase the number of sites and contexts in which DNA damage repair antioxidants status can be assessedIn turnthis will advance translational discoverystimulate biomonitoringand promote new applications for the assessment of DNA damagerepairand cellular antioxidant capacity PROJECT NARRATIVE DNA damagecaused by environmental agentsor the body s normal processesplays a critical role in the development of many major human diseaseswith profound public health implicationsWe propose the development of an automated devicewhich is simplerapidwith increased throughputand affords increased precisionto support the assessment of DNA damage and repairFacilitating such measures will help advance our knowledge of the role of DNA damage in health and disease