Post-mortem environment and DNA quality: studies addressing the forensic utility of routine molecular analyses

Forensic anthropology involves the identification of human skeletal remains in the context of a medico-legal setting, achieved through the application of physical anthropological skills and methods (Byers 2011). Routine anthropological metric and non-metric assessments are performed and factors such as age, sex, ethnicity and stature can be relatively accurately estimated, which assists towards the positive identification of a victim (Franklin 2010). By performing such investigations, the pool of potential victims is reduced, allowing for more rapid identification through established biological processes (e.g. DNA sequencing). Many taphonomic processes (such as animal scavenging, climate variations and burial environment) have the potential to alter skeletal morphology and DNA and therefore the feasibility of subsequent molecular analysis. Disaster victim identification relies on both molecular analysis as well as anthropological assessment to achieve positive identification following skeletal exposure to extreme environmental conditions.

The present study examines the quality and quantity of skeletal DNA extracted from porcine long bones following prolonged exposure to varied taphonomic environments. Bones were subject to one of three test conditions; fortnightly cycles of wet/dry conditions, fortnightly cycles of freeze/thawing or submersion within 1% sulphuric acid. Medullary DNA extraction were subsequently performed on both intact and fragmented remains within each group at one, three and five month intervals. This was assessed against test control bones as well as fresh control bones at each time point. Preliminary quantification was achieved through nanodrop spectrophotometry; this was followed by gel electrophoresis. These results indicated that organic DNA extraction provided higher quantities of DNA compared to commercial extraction kits. DNA qualifications were measured following sequencing using three primers; two mitochondrial DNA primers and one nucleic (Pig growth hormone) primer. Trace scores (phred scores), QV20+ values and continuous read lengths were used to gauge DNA quality. Each measure of DNA quality was assessed against five factors; treatment group, length of exposure to environment, method of extraction, bone fragmentation and primer selection, using one-way ANOVA.

Bones within acidic environments consistently elicited the lowest quality DNA, similarly the quality of DNA obtained through sequencing using Nucleic DNA was significantly lower than that obtained through MtDNA sequencing. While unsurprisingly the test control bones provided the highest quantities and quality of skeletal DNA. The result of the present study indicate that taphonomic processes not only negatively influence the morphology of skeletal remains, but also have the potential to promote DNA degradation following prolonged exposure. Primer selection proved to be the single greatest factor influencing the success of DNA amplification with mitochondrial primers providing higher quality DNA for every treatment group at all three extraction points. The conclusions of the present study have the potential to influence processes utilised for victim identification following natural or man made disasters. Despite bone diagenesis, skeletal molecular analysis has the potential to yield high quality sequences (in the case of freeze/thaw and wet/dry bones). With primer selection crucial to this success, the combined sequencing of nucleic and mitochondrial DNA would be recommended in order to achieve positive identification regardless of the taphonomy surrounding the remains.