Abstract
Methods for quantifying DNA damage, as well as repair of that damage, in a high-throughput format are lacking. Single cell gel electrophoresis (SCGE; comet assay) is a widely-used method due to its technical simplicity and sensitivity, but the standard comet assay has limitations in reproducibility and throughput. We have advanced the SCGE assay by creating a 96-well hardware platform coupled with dedicated data processing software (CometChip Platform). Based on the original cometchip approach, the CometChip Platform increases capacity ~200 times over the traditional slide-based SCGE protocol, with excellent reproducibility. We tested this platform in several applications, demonstrating a broad range of potential uses including the routine identification of DNA damaging agents, using a 74-compound library provided by the National Toxicology Program. Additionally, we demonstrated how this tool can be used to evaluate human populations by analysis of peripheral blood mononuclear cells to characterize susceptibility to genotoxic exposures, with implications for epidemiological studies. In summary, we demonstrated a high level of reproducibility and quantitative capacity for the CometChip Platform, making it suitable for high-throughput screening to identify and characterize genotoxic agents in large compound libraries, as well as for human epidemiological studies of genetic diversity relating to DNA damage and repair.
Highlights
There is compelling evidence that genomic instability plays a prominent role in the initiation of carcinogenesis and it has been linked to aging as well as to a variety of adverse health conditions such as neurodegenerative syndromes and birth defects
The CometChip Platform utilizes the basic principles of single cell gel electrophoresis (SCGE)
A priori designation Known genotoxicant Known genotoxicant Known genotoxicant Known genotoxicant Known genotoxicant Known genotoxicant Known genotoxicant Known genotoxicant Known genotoxicant Known genotoxicant Known genotoxicant Known genotoxicant Known genotoxicant Known genotoxicant Possible genotoxicant Possible genotoxicant Possible genotoxicant Possible genotoxicant Possible genotoxicant Possible genotoxicant Possible genotoxicant Possible genotoxicant Possible genotoxicant Possible genotoxicant Possible genotoxicant Possible genotoxicant Possible genotoxicant Non-genotoxicant Non-genotoxicant Non-genotoxicant Non-genotoxicant Non-genotoxicant Non-genotoxicant Non-genotoxicant Non-genotoxicant Non-genotoxicant Non-genotoxicant newly developed CometChip hardware and software platform presented here represents a significant leap from the traditional comet assay used to measure DNA damage, with far greater sensitivity and throughput
Summary
There is compelling evidence that genomic instability plays a prominent role in the initiation of carcinogenesis and it has been linked to aging as well as to a variety of adverse health conditions such as neurodegenerative syndromes and birth defects (for reviews[1,2]). To combat the effect of DNA damage, cells have evolved multiple, often overlapping DNA repair pathways to ensure that damage is efficiently and accurately repaired. The ability to measure both endogenous levels of DNA damage and genotoxicant-induced DNA damage is important. Diverse methods for measuring genomic damage have been developed including alkaline unwinding[3], DNA fiber analysis[4], direct-damage microscopy[5] and long amplification PCR6. All the methods developed far have shortcomings, including challenges to be scaled up to a high-throughput format, and a laborious work-flow that makes DNA damage quantification challenging and often difficult to accurately reproduce
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