This research sought to investigate the impact that radioactive materials may have on the analysis and interpretation of forensic DNA evidence. Experiments were designed to examine the effects of ionising radiation, specifically gamma and alpha radiation, on the DNA profiles of forensically-relevant biological matrices. In addition, this work explored issues of both sample contamination and the management of radioactively contaminated evidence in order to provide risk minimisation guidance for the forensic analyst and for the laboratory environment. Initial experimentation investigated the effects of γ-radiation, from a cobalt-60 source, and alpha particles, from a particle accelerator, on DNA from a range of biological matrices. From the experiments, the potential impact of time-to-analysis on the qualitative and quantitative aspects of DNA analysis was examined, in addition to establishing limits of exposure for successful profiling. The purpose of the experiments was to obtain an insight into the stability of the DNA sample post-irradiation, as well as address concerns regarding sample integrity and dose thresholds for DNA degradation. The pattern of DNA profiling results obtained for blood, saliva, bone and standard genomic male DNA following gamma-irradiation demonstrated a progressive loss of the higher molecular weight loci as the radiation dose increased (from 0 to 100,000 Gy). One of the largest target fragments,D18S51 (264-344 bps), was typically absent at both the 50,000 and 100,000 Gy doses. These observations reflect the typical pattern of degraded DNA, where the longer fragments present a greater opportunity for interaction with ionising radiation than the shorter fragments. It is proposed that degradation of the DNA molecule in these cases is likely due to fragmentation of the DNA strand, in addition to inter-strand cross-linking, deamination and dimer formation. This trend was also evident in the findings from the alpha irradiation of blood, saliva, and a human epithelial cell line, HEp-2 (with doses ranging from 0 to 26,400,400 Gy). DNA profile degradation was observed across all matrices at doses at and above 66,000 Gy. Allelic dropout was again observed as first occurring at the higher molecular weight loci. With regards to time-to-analysis, general trends in the data suggest a marginal reduction in DNA profiling response for the gamma-irradiated samples over time, especially between 1-day and 1-week post-irradiation. Therefore, if possible, steps should be taken to process samples within this timeframe. The data was more difficult to interpret for alpha-irradiated samples, although general trends over the three time periods suggest a reduction in response as the time-to-analysis increased. The findings from these experiments demonstrate that gamma-irradiated biological matrices are relatively robust for DNA analysis; little degradation was observed up to an exposure of 1,000 Gy for all samples tested, and it was possible to obtain a full DNA profile at doses at least up to 10,000 Gy. Alpha-irradiated samples proved even more robust at significantly higher doses, an effect likely due to the limited penetrability of the alpha particle. Where observed, the effects of ionising radiation on DNA appear to be consistent with other degradative processes. Therefore, current standard operating procedures used for the interpretation of profiles from degraded DNA can be applied if exposure of the samples to radiation has occurred. This research also critically examined the DNA extraction step to investigate methodologies capable of both effective decontamination of the sample and recovery of purified DNA for downstream profiling. DNA IQ™ and ChargeSwitch® solid-phase extraction systems, as well as conventional Chelex100 resin extraction, were investigated for their effectiveness in the removal of non-radioactive caesium-133 salt. In addition, the contaminant was characterised for its potential interference with DNA extraction efficacy. Confirmatory studies were then conducted using the corresponding radioactive caesium-137 species, with special attention given to establishing guidelines for safe working practices. Both the DNA IQ™ and ChargeSwitch® solid-phase extraction systems proved particularly effective for the purification of DNA samples contaminated with the representative non-radioactive caesium-133 (>99.95% removal for DNA IQ™ and 99.99% for ChargeSwitch®, compared to 98.8% for Chelex100/Microcon® extraction). The findings demonstrate that contamination of the samples with caesium-133 did not result in any significant effects on the quantitation, amplification or profiling of DNA at the concentrations tested. DNA profiling results from all the contaminated samples were consistent with those from the control samples, with no significant effect of the contaminant at the targeted loci/alleles. The amount of remaining caesium-133 in the extraction eluants was extrapolated to reflect dose rates of radioactive caesium-137 (in μSv/h), which demonstrated that both the DNA IQTM and ChargeSwitch® protocols significantly reduced dose rates compared to the Chelex100/Microcon® extraction. Therefore, numerous extracted samples from the DNA IQ™ or ChargeSwitch® systems could be handled before the dose rate limit of 0.5 μSv per work hour for a non-radiation worker is exceeded. The results of the radioactive caesium-137 experimental series confirmed the extraction and decontamination efficacy of these systems, as both the DNA IQ™ and ChargeSwitch® extraction protocols proved capable of removing contamination by caesium-137 at the levels tested. In addition, the presence of caesium-137 did not affect the capability of either protocol to obtain a sample of DNA suitable for profiling. From extraction, the activity was reduced to levels approaching zero, with corresponding exposure dose rates being negligible. The efficiency data generated can be used to provide guidance when planning for the volume of samples processed and the maximum exposure time per laboratory analyst. Workplace risks may be further mitigated through the use of appropriate personal protective equipment, exposure monitoring, contamination monitoring, and waste disposal. This innovative research has contributed to an improved understanding of the effects of ionising radiation on forensic DNA evidence. The findings have revealed the capabilities of select extraction systems in meeting the practical needs of forensic laboratories preparing standard operating procedures for investigations involving radiological incidents. The study has also provided valuable insights into associated operational procedures.
|Date of Award||2009|
|Supervisor||Chris Lennard (Supervisor), Bill Maher (Supervisor) & Jennelle KYD (Supervisor)|