Increasing the Evidential Value of Biological evidence
With current scientific technologies, a significant amount of genetic information can be obtained from biological evidence found at a crime scene. Not only is it possible to identify the donor of the evidence through routine DNA profiling techniques, but new RNA based methods are being developed to determine the tissue type as well as the physical characteristics of the donor. Despite the information that can be obtained, the ability to determine the age or time the biological material was deposited at the crime scene has eluded the forensic community thus far. Timing is critically important as it could help police determine when the crime was committed. In this body of work an investigation was conducted into whether the degradation rates of nucleic acid macromolecules could serve as molecular clocks for age estimations. An attempt was made to gain a better understanding of the degradation products produced from an internal urban environment and to develop an optimal assay accordingly. A number of different RNA based techniques for ageing both hair and blood samples were also examined. Degradation assays have been traditionally designed around amplicon size however, it was established that testing loci stability is an essential requirement in the optimisation process. The results presented in this thesis suggest the reliability of the data can be increased when the two competing target species are selected from the same loci, which minimised the effect of loci susceptibility to degradation. It was determined that blood stains aged up to 60 days in an internal urban environment were best distinguished (in terms of age estimations) by using targets that differed in size by 170 to 240 base pairs, with one of the targets being between 200 and 300 base pairs in length. Despite using a robust TH01 qPCR assay it was established that an internal “urban” environment was not as stable as predicted and that seasonal temperature variation had a large effect on degradation rates. Interpretation of the results was therefore limited suggesting these optimised target sizes may only be relevant to the winter months. Using a carefully designed hermetically sealed dry swab we were able to remove moisture and inhibit the growth of DNA consuming micro-organisms. It was determined that bacteria alone can cause a 2-fold increase in the degradation rate of a sample aged at room temperature. In terms of integrity, storing samples at room temperature in a moisture free environment was equivalent to storing standard samples (exposed to normal humidity levels) in refrigerated conditions. It was also determined that the effect of bacterial degradation can be halved by lowering the storage temperature from room temperature to 4°C. RNA was examined in an attempt to reduce the large variations that had inhibited previous DNA methodologies. IL-6 and TNF-α were initially selected due to their rapid post-extraction change in expression levels. However, their levels were highly variable, unpredictable and therefore not suitable for this type of analysis even on samples that had been aged for only ten days. It is thought that their dynamic roles in a number of haemopoietic processes could be responsible for the poor results. A new RNA methodology, as described by Nolan et al (2008) was used to analyse samples that had been aged over 80 days. Four targets, AMICA1, MNDA, CASP1 and GAPDH were chosen based on their cell lineage as it was hypothesised that inter-donor variation could be reduced by using targets confined to the granulocytic cell lineage. Using the novel 3’/5’ assay, AMICA1, MNDA and CASP1 all performed poorly and no correlation could be determined between the 3’/5’ ratio and sample age. GAPDH showed some encouraging results with a correlation of 0.912 (age to 3’/5’ ratio) although initial stability over the first 20 days and the inter-donor variation were still limiting factors. It was also thought that the various mRNA degradation processes, in particular the 5’/3’ exonuclease activity, contributed to the poor results generally. A large inter-donor variation was a common aspect to all the blood based methodologies trialled. This meant that none of the methods had any practical value. As a result, an alternative RNA method was used to determine if it was possible to age another forensically important type of biological evidence; hair. Using a Reverse Transcription Quantitative PCR (RT-qPCR) assay, we monitored the Relative Expression Ratio (RER) of two different RNA species (18S rRNA and B-actin mRNA) in hair samples that were aged naturally over a period of three months. Overall the results presented here suggest that the age of hair samples containing follicular tags can be approximated using a second order polynomial (Age = 3.31RER2 - 2.85RER – 0.54), although with limitations.
QH301 Biology, RA1001 Forensic Medicine. Medical jurisprudence. Legal medicine
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