An exploratory study into the effects of DNA and protein degradation in a laboratory based model and naturally aged porcine (S scrofa) teeth

Abstract

In forensic anthropology, laboratory-based (LBM) and field-based (FM) models can be used to develop new methods and to research the stability and rate at which bio-molecules degrade. In this study, both these methods were used to investigate the effects that temperature, time after death (TAD) and other environmental factors had on the concentration of and change in molecular structure (increase in free pyrrole content, ninhydrin reactive nitrogen (NRN) and iron). of collagen, haemoglobin (Hb) and DNA in porcine teeth For the LBM, porcine teeth were heated at 900C for 0-4 hours, 1 hour intervals, (total number of teeth n=35). A porcine FM was established at the Miertjie Le Roux Experimental Farm of the University of Pretoria. From the decomposing pigs, teeth were collected at TAD intervals of 20 days (n=35). The morphology of the teeth were evaluated and recorded. Methods for collagen and DNA isolation, quantification of protein, collagen, haemoglobin (Hb), free pyrrole content (FPC), ninhydrin reactive nitrogen (NRN), total iron, Fe2+ and Fe3+ as well as a real-time PCR method for the detection of mitochondrial cytb gene in porcine teeth were established. These methods were used to determine the concentration and structural integrity of these molecules in the LBM and FM teeth. The morphology of the LBM teeth was regular with only minor changes in colour with time heated. The collagen and Hb concentration did not change with time. A decrease in total iron (not statistically significant) and Fe3+ (p=0.014; R2=0.74) was found and was associated with an increase in Fe2+ (p=0.014; R2=0.965). No change in free pyrolle content was found. The total protein concentration determined using the Biuret method showed a decrease with time (p=0.009; R2=0.99). For DNA, a linear decrease in concentration (p=0.00; R2=0.93) was found. This DNA could still be used for the successful amplification of the cytb gene. As for DNA a similar decrease in NRN (p=0.00; R2=0.99) was also found whether this is related to protein or DNA degradation is unknown. From this data the total protein, DNA and NRN showed a definite time related change in concentration. For the field model the teeth were brown, cracked, weathered and corroded. As for LBM, there were no time related changes in mass and collagen content. A significant decrease in total protein concentration (p=0.00; R2=0.52) and FPC (p=0.01; R2=0.98) was observed. Hb, FPC and iron levels (total iron, Fe3+ and Fe 2) did not change with time but concentrations FPC and iron were higher than those found in the LBM. Also total protein concentration although it decreases with time was also increased when compared to the LBM (sentence is awkward, re-word). This could be due to increase bacteria activity that results in an increase in protein biomass, iron accumulation and pyrolle synthesis. In contrast Hb levels were the same as LBM and are species specific and not related to increased bacterial activity. NRN showed a time related decrease in concentration (p=0.09; R2=0.99) and was also twice that found in the LBM. This is related to decomposition of porcine protein and DNA as well as that derived from bacteria. A decrease in DNA concentration with time was found (p=0.00; R2=0.88). DNA from all samples and could be used for the amplification of cytb. In conclusion the LBM allows for rapid method development and the investigation of the effect of single factors on the integrity of bio-molecules such as protein and DNA. The FM can then be used to further investigate the effect of many additional environmental factors on the concentration and structure of the same bio-molecules. Using both models, it was found that total protein, DNA and NRN showed a time related change in concentration while the concentration of collagen and Hb remained constant. Copyright