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Evaluation of postmortem DNA degradation using the comet assay Johnson, Laura Alexandra


One of the most important longstanding problems in the field of forensic medicine is the determination of time of death upon the discovery of a possible homicide victim. With a majority of homicide victims discovered within the first 48 hours, it is critically important to be able to determine time of death quickly and with accuracy and precision. Current methods of determining postmortem interval vary, but none can provide better than an 8-hour window time estimate. In this paper, the potential application of single-cell gel electrophoresis (also known as the comet assay) to evaluate postmortem cell death processes, specifically nuclear DNA fragmentation, is assessed. Upon the death of an organism, internal nucleases contained within the cells should cause chromosomal DNA to degrade into increasingly smaller fragments over time, and if these fragments can be isolated and visualized, the fragmentation should prove to be measurable and quantifiable. An original study providing proof of the concept of postmortem DNA fragmentation between early and late time periods was conducted using human leukocytes. With an established trend of DNA degradation seen in the leukocyte results, this study was then expanded using a porcine animal model, over a longer time period, with more frequent timepoints evaluated. DNA degradation in all samples was revealed by single-cell gel electrophoresis (also known as the comet assay) and quantified by the use of the DNA-specific quantitative stains propidium iodide and silver nitrate, as measured by digital camera affixed to a microscope. The comet 'tail moment' gave a measure of the proportion of fragmented to non-fragmented DNA, while the 'tail-length' provided the relative size of degraded DNA fragments. In both models, an increase in DNA fragmentation was found to correlate with increased postmortem interval from 0-56 hours postmortem as evaluated by comet tail-moment and by comet tail-length, with tail-length providing the strongest statistical correlation, based on regression analysis. The postmortem DNA fragmentation observed in this study reveals a sequential, time-dependant process with the potential for use as a predictor of postmortem interval in homicide cases.

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