Date of this Version
For decades, the use of DNA as a biological tool has revolutionized forensic investigations. The primary use of this genetic evidence is for identification of a victim or suspect through short tandem repeat (STR) profiling. However, the usefulness of this evidence can be compromised through inhibition of PCR, damage to the DNA, or low copy number. Here, we investigate damage induced to DNA by environmental factors. UV light is known to damage DNA by the formation of cyclobutane pyrimidine dimers, 6-4 photoproducts, and strand breaks. These lesions can stall polymerase action or misincorporate bases during extension. Oxidative damage is also common to environmentally exposed samples and can occur by microbial digestion or radiation. The primary lesion associated with oxidative damage is the formation of 8-oxoguanine, which can result in base modification. A novel assay involving a glycosylase and S1 enzymatic digestion to convert damage lesions to double strand breaks was developed to investigate damage associated with environmental exposure. Both reactions have enzyme activity in the same buffer, thus samples can be processed in the same tube to minimize the loss of DNA by transfer. Because double strand instead of single strand breaks are evaluated, samples can be evaluated on a native agarose gel which is more sensitive than damage detection techniques such as an alkaline agarose gel. Following optimization of the glycosylase plus S1 reaction, this assay was used as a tool to asses UV and oxidative damage in bloodstains exposed to the environment. The physiological stains were left uncovered to the environment for time points ranging from 1 day to 6 months. A sharp decrease in yield was observed for DNA exposed to the environment for more than 5 days. Samples exposed to environmental insults for 3 and 5 days exhibited both UV and oxidative damage as well as strand breaks. Oxidative damage was determined to constitute a higher number of damage lesions than UV damage. STR profiling revealed this damage did not result in a loss of genetic profile through 5 days of exposure.
Advisor: Ashley Hall