DNA Repair-on-a-Chip: Spatially Encoded Microwell Arrays

DESCRIPTION provided by applicant Preserving genomic integrity is essential in order to suppress cancer neurodegeneration aging and other diseases At odds with genomic preservation is DNA damage which can drive mutations sequence rearrangements and cellular toxicity DNA damage is unavoidable as DNA damaging agents are present in our environment and in our cells To counteract the deleterious effects of DNA damage we have evolved sophisticated DNA repair systems It is now known that every major DNA repair pathway suppresses cancer Furthermore since cancer is often treated using DNA damaging agents it is not surprising that the DNA repair capacity of tumors modulates sensitivity to chemotherapy Despite its importance measurements of DNA damage and repair are far from routine primarily due to the lack of reliable and rapid DNA damage assays Here by bringing together convergent expertise among engineers biologists and computer programmers we propose to meet this need by developing a platform for rapid semi automated single cell DNA damage quantification that can be broadly distributed and readily applied by researchers in public health academia industry and medicine As defined in the Phase I submission we created and tested a prototype for a well CometChip platform and have optimized the engineering design and a production apparatus to produce spatially encoded and well demonstrated that supplementation of the Microwell Comet gels with extracellular matrix proteins EMPs supports the growth of human cells for up to two weeks and the EMPs do not impact the formation of comets To enable characterization of the genotoxicity of chemicals used commercially those found in the environment or newly developed pharmaceuticals and to quantify DNA repair capacity without the need to identify specific DNA Repair technology This proposal to develop the andapos DNA Repair on a Chipandapos technology combines the use of agarose based Microwell arrays spatially encoded cellular recognition automated data processing and extra cellular matrix proteins to optimize validate and commercialize a series of Spatially Encoded Microwell Arrays We will demonstrate that we have significantly advanced the manufacturing process Aim have developed a macrowell former to produce well and welll CometChips Aim and propose the implementation of a graphical user interface for data analysis Aim Finally we will rigorously validate this new technology by analyzing the genotoxic effects of a range of compounds from the NTP library for their impact on DNA damage and repair responses and to reveal inter individual and inter cell type variation in DNA damage responses Aim Through the integration of traditional methods in biology and engineering the DNA Repair on a Chip platform described here represents a significant technological advance providing high throughput objective and quantitative measurements that have the potential to become a new standard in DNA damage analysis PUBLIC HEALTH RELEVANCE We describe a new methodology that provides for robust high throughput DNA damage and repair analysis by exploiting gravity capture of single cells into a Microwell array DNA damage levels are revealed morphologically by single cell gel electrophoresis The Microwell array enables fully automated DNA damage and DNA repair measurement of multiple experimental conditions simultaneously This technological advance opens the door to new strategies for drug discovery genotoxicity testing and environmental health research through objective quantitative analyses