Analysis of postmortem DNA degradation by single-cell gel electrophoresis.

摘要

One of the most important longstanding problems in the field of forensic medicine is the determination of the time of death upon the discovery of a possible homicide victim. With a majority of homicide victims discovered within the first 48h, it is critically important to be able to determine time of death quickly, and with accuracy and precision. Current methods of determining postmortem interval (PMI) vary, but none can provide better than an 8-h window time estimate. In this paper, the potential application of single-cell gel electrophoresis (SCGE), 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 seen in the leukocyte results, this study was then expanded using a porcine animal model, over a longer time period, with more frequent time-points evaluated. DNA degradation in all samples was revealed by SCGE and quantified by the use of DNA-specific quantitative stains, 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 an increased PMI from 0 to 56h postmortem, as evaluated by comet-tail-moment and by comet-tail-length, with tail-length providing the strongest statistical correlation, based upon regression analysis. The postmortem DNA fragmentation observed in this study, reveals a sequential, time-dependent process with the potential for use as a predictor of PMI in homicide cases.