Degradation of DNA can lead to either poor quality, imbalanced, or even no profiles. Therefore, appropriate collection and storage methods are critical to minimize its impact. If the DNA is degraded prior to sample collection, then the degradation process can only be arrested and other methods have to be employed to try to improve the quality of the DNA profile. The major aims of this thesis were to assess alternative methods for molecular preservation of muscle tissue samples and to obtain better DNA profiles from degraded samples. Assessment of DNA degradation was undertaken using an in-house PCR assay which amplifies four amplicons from 70 bp to 384 bp. DNA degradation was evaluated in whole pig carcasses exposed to hot and humid environmental conditions. A full DNA profile could be generated for 24 hours, but some full profiles were obtained from samples taken as late as 72 hours. It was determined that when collecting tissue samples from partially decomposed bodies, those should be preferentially from the surface of the body in touch with the ground, as the results show that DNA persistence is improved. In order to compare field and laboratory degradation patterns, muscle tissue samples were incubated in the laboratory at 25 °C and 37 °C. The persistence of DNA was increased when compared to field, most likely due to the lack of insect activity and of variations in temperature and humidity. Partially degraded muscle samples were preserved with 96% ethanol, cell lysis solution, or cell lysis solution with 1% sodium azide, which had been stored at room temperature for seven years. Samples were re-extracted to assess the long-term efficacy of these storage solutions. The results show that ethanol and cell lysis solution with 1% sodium azide were successful in preserving DNA for this period. Fresh muscle tissue samples were stored at 25 °C and 37 °C for up to 42 days using vodka and 37.5% ethanol as preservatives. Complete amplification profiles were obtained up to the last time point from samples that had any preservative solution, while samples left untreated had dropouts after 14 days. It is recommended that the use of drinking ethanol should be considered in situations where the stock of absolute ethanol is limited. The possibility of using vacuum for preservation was tested on fresh muscle tissue samples incubated at 25 °C and 37 °C. The results show that even if there was a limited amount of air inside the storage bag, and not complete vacuum, DNA persistence was enhanced when compared to samples incubated at the same conditions in plastic tubes. Some approaches were attempted to improve degraded DNA profiles. First, degraded DNA was selectively extracted from agarose gels to manipulate the proportion of longer and smaller DNA fragments present. Despite promising preliminary results, this technique showed no usefulness in improving DNA profiles. Purification columns were used with the same aim, but when comparing the original sample with the processed samples, the best results obtained were of equivalence. As an alternative approach, a protocol of DNA Capture was developed in an attempt to preferentially extract the fragments to be analysed in a degraded DNA sample in equal amounts. Whilst the DNA capture method worked in preliminary experiments, it was not applied to degraded profiles. The results obtained have allowed recommendations around collection (i.e. how long samples could be viable for DNA analysis) and storage to be refined. Attempts to rebalance already degraded profiles were not successful. Future field experiments planned as a follow up to the work presented involve testing collection methods and the effectiveness of vacuum body bags.

Ground penetrating radar (GPR) is a non‐invasive, geophysical tool used for the detection of clandestine graves. GPR operates by detecting density differences in soil by the transmission of high frequency electromagnetic (EM) waves from an antenna. A 500 Megahertz (MHz) frequency antenna is typically used for forensic investigations, as it provides a suitable compromise between depth of penetration and sub‐surface resolution. Domestic pig (Sus scrofa) carcasses were clothed in 100% cotton t‐shirts and 50% cotton/50% polyester briefs, and buried at a consistent depth at three field sites of contrasting soil texture (silty clay loam, fine sand and fine sandy loam) in southern Ontario. GPR was used to detect and monitor the graves for a period of 14 months post burial. Analysis of collected data revealed that GPR had applicability in the detection of clandestine graves containing remains in silty clay loam and fine sandy loam soils, but was not suitable for detection in fine sandy soil. Specifically, within a fine sandy loam soil, there is the potential to estimate the post burial interval (PBI), as hyperbolic grave response was well defined at the beginning of the 14 month burial duration, but became less distinctive near the completion of the study. Following the detection of a clandestine grave containing a carcass, collection of gravesoil, tissue and textile samples is important for the estimation of the stage of decomposition and the post burial interval (PBI) of the remains. Throughout the decomposition process of a carcass, adipose tissue is subjected to hydrolytic enzymes that convert triglycerides to their corresponding unsaturated, saturated and salts of fatty acids. The composition of fatty acids in the decomposed tissue will vary with the post mortem period, but it is unknown what affect the soil texture has on lipid degradation. As decomposition proceeds, fatty acids can leach from the tissues into the surrounding burial environment. Fatty acid analysis of gravesoil, tissue and textile samples, exhumed at two, eleven and fourteen month post burial intervals, was conducted using diffuse reflectance infrared Fourier transform spectroscopy (DRIFTS), attenuated total reflectance – Fourier transform infrared (ATR‐FTIR) spectroscopy and gas chromatography – mass spectrometry (GC‐MS). Infrared (IR) spectroscopy analysis of the samples provided a qualitative profile of lipid degradation. Analysis of gravesoil samples did not reveal IR spectroscopy bands attributable to fatty acid degradation or adipocere formation. IR spectroscopy analysis of tissue samples is applicable for the estimation of carcass decomposition in all of the soil textures tested. Results of textile IR spectroscopy analysis revealed limited potential to estimate the stage of carcass decomposition in silty clay loam soil. GC‐MS was used to quantify the peak area ratio (area/int std area) (PAR) of myristic (C14:0), palmitic (C16:0), palmitoleic (C16:1), stearic (C18:0) and oleic (C18:1) acids. GC‐MS results revealed that analysis of both tissue and textile samples can be useful in the estimation of the stage of decomposition and the PBI of carcasses in all three of the soil textures tested. The results of this research may have applicability within forensic investigations involving decomposing bodies by aiding in the location of clandestine graves in silty clay loam and fine sandy loam soil through the use of GPR. Infrared spectroscopy and GC‐MS analysis of the fatty acid composition of tissue and textile samples may also be incorporated into investigational protocols to aid in the estimation of the stage of decomposition and the PBI of a body. / UOIT

Macro and micro nutrients

This project was designed and developed in response to the need to improve the methodology employed in the DNA profiling of the Kuwaiti victims of the First Gulf War (1990-1991). The main challenges have involved developing the methodology in an attempt to increase the DNA recovery from the skeletal remains and also assess the preservation of DNA in the remains. In addition, work was undertaken to assess two commercial STR amplification kits, the Identifiler® and MiniFilerTM, to establish allele frequency databases for use in Kuwait and to assess the concordance of the two kits. In order to assess the methodology for DNA extraction and prediction of DNA preservation two sources of materials were used: simulated casework samples and actual casework samples. To obtain simulated casework samples, bones from sheep and teeth from human were buried at three different sites within Kuwait. These were sampled over a period of 60 weeks. In addition, samples from the femur and humerus of 25 individuals who were killed during the Gulf War, but had not yet been identified, were taken for analysis. These were exhumed from five gravesites, three in Iraq and two in Kuwait. Previous attempts to generate DNA profiles from the samples had failed. Different extractions protocols and purification methods were assessed including: a phenol:chloroform-based extraction; the GENECLEAN® Kit (Obiogene); QlAquick Gel Extraction kit (Qiagen); the QIAamp DNA Blood Maxi kit (Qiagen), using a protocol based on Davoren et al (2007); and a modified silica-based extraction using the DNeasy® Blood and Tissue Kit (Qiagen). PCR amplification of the extracts and the real-time quantification results showed that the modification of a silica-based method, using the Qiagen DNeasy® kit, was successful in removing inhibitors that were present in the extracts and obtained with all the other extraction methods. This allowed the successful profiling of 19 out of the 25 samples that had previously failed. In an attempt to further improve the DNA extraction efficiency, the effect of Nphenacylthiazolium bromide (PTB) was assessed. PTB has been reported previously to improve DNA extraction from ancient DNA samples (Paabo, 1989; Poinar et al., 1998) by releasing DNA that has become cross-linked with proteins. In this study, the effect of PTB, while statistically significant when used with samples from some sites, was minimal. The power of different methods to allow an effective system of triage (sorting of samples based on the likelihood of successful analysis) was examined. Three parameters were assessed: gross morphology, histology, and chemical status of the bones were compared with the amount and quality of DNA that was recovered from different samples. The simulated casework samples displayed only minor changes in gross morphology and histology over the period of the study, whereas the casework samples displayed varying degrees of change. The samples from Iraqi sites generally displayed good morphological and histological preservation. In contrast, the samples from the two sites within Kuwait displayed an almost complete lack of histological features and changes (pitting/cracks) to the surface. The morphological and histological preservation correlated closely with the success rate when extracting DNA from casework samples that were buried in Iraq and Kuwait. Nitrogen content in all samples was very similar and the results showed that it was not a useful indicator of preservation. The MiniFilertM (Applied Biosystems) is designed for the analysis of degraded DNA. Before applying this to casework, it is important to carry out a concordance study in order to ensure the results with the MiniFilerTM are comparable to the Identifiler® (Applied Biosystems) DNA profiles. The reference database with relatives' DNA profiles are all generated using Identifiler®. To assess the concordance, the MiniFilerTM profiles from 200 unrelated Kuwaiti samples were compared to Identifiler® profiles. Concordance was observed for 99.875% of the compared loci (1598 of 1600). The two discordant profiles displayed allelic dropout: one at the Dl 35317 locus due to non-amplification of allele 10 in the MiniFilerTM profile, and one at the D18S51 locus due to nonamplification of allele 18 in the Identifiler® profile. Finally, since the population of Kuwait is heterogeneous, with a strong tribal system, the possibility of subpopulation effect within the Kuwaiti population was examined. Allele frequencies for the 15 STR loci included in Identifiler® kit were ascertained in a sample population of 502 unrelated Kuwaiti individuals. The results were compared with 6 different populations. The Kuwaiti population was very similar to neighboring Iraqi and Saudi populations. These data are now used in casework undertaken in Kuwait, to calculate the statistical significance of matching DNA profiles (the results of the reference database work are included in Appendix 1 rather than in the main body of the thesis).

Proper interpretation of DNA profiles depends on the quality of the DNA samples, the amplification efficiency and the success of post-PCR processing. Chemicals associated with forensic samples can affect the amplification process while random errors occurring during pipetting and electrokinetic injection can cause variability. This can lead to either a reduced signal or lack of DNA profiles. As recommended by the SWGDAM, laboratories carrying out DNA profiling have to adopt standardise and validate procedures, which lead to high levels of quality assurance and control. During amplification, monitoring is restricted to the use of exogenous controls, which are unable to identify issues associated with individual samples. To address this limitation, four PCR Internal Controls (PJC5) i.e. two Internal Amplification Controls (IACs) and two Internal Non-Amplifiable Control (INAC5), to be used with the AmpFtSTR® SGM Plus® kit were developed. The IACs (90 bp and 410 bp) and INACs (80 bp and 380 bp) fragments were generated from the plasmid pBR322 and added along with human DNA in a 12.5 gl PCR volume. During the reaction the IAC% and 1AC 41 0 are amplified non-competitively with ROX labelled primers, while the pre-labelled INAC80 and 1NAC 380 were not involved with the amplification process. Both sets of fragments were detected as red peaks on the electropherogram flanking the human DNA profile. To study the behaviour of the markers within the system and their effect on the performance and sensitivity of the assay, the PICs were used during the amplification of human DNA of different quantity and quality and with the addition of three common inhibitors. Initial experiments involving the individual development of the fragments showed that both the INACs and IACs can be successfully applied to the amplification of human DNA with SGM Plus® reaction under optimised conditions, without significantly impacting the quality of human DNA profile. As the INACs fragments are designed not to amplify during the reaction, they gave a stable signal that can be used to monitor the post-PCR sample processing. The peak height ratios (PHRs) of human DNA with that of the LNAC8 0 or 1NAC380 can also be used to normalise the signal and assess the amplification efficiency of human DNA samples within replicates of the same sample run under the same conditions. The JACs on the other hand gave more information on the process of the PCR. They were able to monitor changes in the amplification efficiency and detect presence of inhibitors with a minimum inhibitory concentration closer to the SGM Plus® as compared to the Quantifiler® WC system. The IAC90 and 1AC410 ratios were also used to distinguish between partial profiles obtained from degraded DNA and those resulting from partial inhibition. Combining the two sets of fragments together with the amplification of human DNA needed further reaction optimisation, involving the addition of dNTPs and MgCl2. The presence of PICs provided information on the amplification performance and post-PCR processing and can assist with the interpretation of human DNA profiles. The position of the fragments also allowed PICs to be used as sizing standard. Compared to the GSTh 500 ROXTM size standard, PICs showed a slightly lower sizing precision, with standard deviations lower than 0.3 bp. Even though this resulted in allelic bins higher than 1 bp limit for 99.7% confidence, all samples sized with PICs were correctly genotyped. The addition of PICs would be a valuable tool, in particular, for the analysis of compromised DNA. In particular it would be useful when analysing DNA recovered from skeletal remains, which are prone to accumulation of PCR inhibitors and DNA degradation.

In Malaysia, as well as other forensic laboratories in tropical climates many of the crime scene samples received at the forensic laboratory are less than ideal. They are often present low amounts and/or degraded due to environmental exposure to high temperatures, sun and humidity for days or even months. STR analysis is widely accepted by forensic community, but sometimes this technique gives unreliable results when profiling degraded samples as the amplicons size are relatively larger (100 bp to 450 bp). While, miniSTR is a reduced size of STR amplicons which enables higher recovery of information from degraded samples, but only a few loci are amplified and allele drop out still may occur, as the amplicons are up to 200 bp. The percentage of recovery DNA profile from degraded DNA using mtDNA is much higher due to its present in cells at a much higher copy number than the nuclear DNA. However, the major drawback for mtDNA is labour intensive and has a low information value (i.e. it is not highly discriminating). Insertion/deletion polymorphisms (INDELs) are relatively new class of a DNA marker used in forensic casework; used most commonly as a supplementary method to STR (Short Tandem Repeat) based typing. INDELs, like SNPs (Single Nucleotide Polymorphisms), are particularly useful for the analysis of highly degraded DNA as the amplicon sizes are typically below 160 bp; they can also be valuable as an additional tool to help resolve kinship cases, with the advantage over STRs that do not have high mutation rates. INDELs have an advantage over SNPs in that they are length polymorphisms and so can be analysed by simply measuring the length of the allele(s). The Qiagen Investigator DIPplex® kit is currently one of two commercially available kits for the amplification of INDEL polymorphisms; it amplifies 30 biallellic INDEL loci and the amelogenin locus. The primers used are fluorescence labelled with 6-FAM, BTG, BTY and BTR. This technique is robust, relatively simple, and the results are analysed using the same capillary electrophoresis equipment and software as used for STR typing. The INDEL markers have simple biallelic structure and combine the advantages of STR and SNP assays. This study has established that the INDEL technique, using the Investigator® DIPplex PCR kit, is a simple, informative and sensitive approach for the typing of degraded DNA, as compared to STRs and SNPs. In this research, allele frequencies for 30 autosomal INDEL loci were studied in 500 unrelated individuals (100 each) from Malay, Malay-Chinese (M-Chinese), Malay-Indian (M-Indians), Iban and Bidayuh. The PCR amplification used the Qiagen Investigator® DIPplex kit. These population groups represent the majority of the population in Malaysia. No significant departure from Hardy Weinberg Equilibrium (HWE) expectations were observed for most of the INDEL loci analyzed (p-value >0.05) on the Malaysian population samples. The exceptions were HLD101 for Malay (p = 0.0009), HLD133 for M-Indian (p=0.005), HLD125 for Iban (p=0.028) and HLD93 for Bidayuh (p = 0.014). However, when the Bonferroni correction for multiple testing performed on the population samples, none of the previous p-values was significant. There were no Malaysian population studies was carried out using the Qiagen Investigator® Investigator DIPplex kit at the time of the research. This INDEL assay have undergo an extensive valdidation process and novelty report of the allele frequencies of INDELs would serve as reference database for individual identification in the Malaysian population in the future. Even the match probability of the STR is higher, INDEL still gives an acceptable value for forensic identification; e.g. linking different pieces of evidence or re-association of body parts in the case of human identification. Biological samples received in Malaysia forensic laboratory have often been exposed to unfavourable environmental conditions. This can lead to DNA degradation and end up in incomplete DNA profiles. It is difficult to distinguish between low template DNA that are producing no or partial profiles because of DNA degradation and those that produce no or incomplete profile because of PCR inhibition. Even though real-time PCR methods are available for quantification and detection of PCR inhibitors, the information received is limited as real-time PCR targets amplicons that are much smaller than those typically targeted in forensic analysis. To gain more information on the quality of extracted DNA, a new multiplex PCR assay comprising a Mini 4-plex targeting amplicons of 50 base pairs (bp), 70 bp, 112 bp and 154 bp along with two Internal Amplification Controls (IACs) of 90 bp and 170 bp was developed. The primers were redesigned from a 4 plex & IACs system developed by previous PhD UCLan students. This multiplex was optimised so that it worked efficiently on DNA template as low as 0.009 ng, which highlighted the strength of the Mini 4-plex system. The IACs were effective in detecting PCR inhibitors. The Mini 4-plex system (Mini 4-plex & IACs) was demonstrated to be an effective tool for identifying degraded and inhibited samples, which could be used to triage forensic samples in a casework laboratory. Therefore, this study has led to the improvement of new and novel markers assessing DNA degradation and PCR inhibition on forensic samples. This will demonstrate the compatibility with forensic laboratory workflows. The need of this Mini 4-plex assay in forensic laboratory can reduce time and cost of DNA analysis. Besides it will contribute to a good management samples, where after being assessed the samples can be decided to analyse using appropriate kit (e.g. miniFiler or INDEL). Indirectly, this will increase the quality of the sample itself. In order to increase the power of a 15 Mini-INDEL multiplex, which was developed earlier by UCLan PhD student, a total of 9 autosomal INDEL markers that are not part of the Qiagen Investigator DIPplex® kit were selected and redesigned from (Pereira et al. 2009). In this study a simple and sensitive INDEL multiplex was successfully developed for human identification. However, the discrimination power is still low when compared to STR systems, but has potential value when analysing highly degraded material. By combining the 15 Mini-INDELs and 9 Mini-INDELs allele frequency data, it will give beneficial by increasing the match probability values in future analysis.

Body fluids are commonly recovered from crime scenes by forensic investigators and their identification are necessary part of forensic casework study. Current body fluid identification techniques rely on enzymatic tests, which have limited sensitivity and specificity, they require large amount of template, use separate assays for various body fluids, and are prone to contamination. Various genes are expressed in different body fluids that could be used as genetic markers for body fluid identification, and are used in forensic investigations. The aim of this study was to use mRNA markers to identify human body fluids, which included blood, semen, saliva, vaginal secretion and menstrual blood. Initially, ten reference genes (UCE, TEF, GAPDH, 18S rRNA, ACTB, B2M, B-Actin, OAZ1, RPS 29 and S15) were studied to establish an appropriate reference gene in body fluid identification. These are constitutive genes used for normalisation of gene expression data and control of variations in experiments. qRT-PCR efficiency, sensitivity and limit of detection (LOD) were investigated using SYBR Green and Taqman probes. The results of the SYBR Green efficiency test experiment displayed five markers, UCE, TEF, ACTB, B2M, and RPS29 with 90-110% efficiency with a slope -3.33 ± 10%. Subsequently, Taqman probes were designed for the five markers and then used for the Taqman probe experiment. Reference gene stability test was carried out on body fluid samples stored up to 6 months at room temperature using the designed Taqman probe assays. The results established ACTB and UCE as best candidate reference gene markers in this study as both were most stable in samples stored for 6 months. Furthermore, to identify each of the five body fluids, thirty-two (32) body fluid specific mRNA markers were evaluated, optimised and validated. The experiment was initially carried out with non-fluorescent makers to determine the specificity of the markers. These were analysed using agarose gel electrophoresis. Further optimization was then carried out using fluorescently labelled markers. This was done in five separate multiplexes for each body fluid; –semen-plex, saliva-plex, vaginal secretion-plex, menstrual blood-plex and blood-plex. An attempt was made to combine all the five-separate multiplex into a single multiplex. All body fluids were identified unambiguously with no cross-reactions of non-target body fluids using the combined multiplex assays. Following further evaluation and validation tests, a total of 14 markers were selected and a capillary electrophoresis (CE) based, multiplex assay was developed to identify blood, saliva, semen and vaginal secretion samples simultaneously. The markers in the developed multiplex assay included ALAS2 and PF4 (blood), STATH and HTN3 (saliva), PRM1, TGM4, MSMB, NKX3-1 (semen), ACTB and UCE (reference genes), CRYP2B7P1, SFTA2, MUC4 and L. crispatus (vaginal secretion). Extensive validation, which include sensitivity, specificity, reduced volume reactions, degradation, reproducibility, mixtures, cycle number and mastermix, was carried out in accordance with the guidelines detailed in Scientific Working Group in DNA Analysis (SWGDAM). The 14-marker CE-based assay displayed high specificity and sensitivity. Each body fluid was detected down to 1:3000 dilution of mRNA except vaginal secretion that was detected down to 1:1500 dilutions of sensitivity. Specificity experiments showed no cross reactions of the assay with non-target body fluids. Reproducibility study displayed similar results reported from an independent laboratory. All body fluids exposed to environmental insult were identified up to at least day 30 of 51, with blood being identified up to day 51. In the mixture study, all body fluids were identified unambiguously using the developed multiplex assay. In conclusion, the results of this study have led to the development of a new and novel capillary electrophoresis-based mRNA marker assay for forensic body fluid identification, demonstrating its compatibility with forensic laboratory workflows. The use of this assay to profile forensic casework samples for body fluid identification would be a future application of this work.

Sudden unexpected death in infancy (SUDI) remains a global concern and is a particular burden in South Africa. Infections have been previously linked to SUDI deaths, but empirical data in a South African context is lacking. This study aimed to explore the burden and risk factors of infection-related infant death at Salt River Mortuary (SRM). To identify the types of infections associated with SUDI in a local setting, medico-legal files from SRM between 1 January 2017 and 31 December 2018 were reviewed. Included cases involved infants between 1 day and 365 days old where an infectious cause of death was either suspected or confirmed (n=288). Variables pertaining to cause of death, scope of post-mortem investigation, clinical history and risk factors were collected from case files and assessed. Most infants (73.6%) demised within four months of age. The major modifiable risk factors were cosleeping (95.0%, n=264/278), side or prone sleeping position of the infant (73.3%, n=195/266), as well as tobacco smoke exposure (46.9%, n=122/260). Respiratory infection was the leading cause of death in this population, followed by gastroenteritis. Philippi area recorded the most gastroenteritis and respiratory infection-related deaths at 25.0% (n= 8/32) and 23.4% (n= 45/192), respectively. Milnerton and Gugulethu recorded 18.8% (n=6/32) and 15.6% (n=5/32) of gastroenteritis-related deaths, respectively. Nyanga and Mitchells Plain recorded 11.5% (n= 22/192) and 9.9% (n=19/192) of respiratory infection-related deaths, respectively. Despite infections being diagnosed as cause of death, microbial analysis was only requested in 22.9% (n= 66/288) and histology was only performed in 14.9% (n= 43/288) of the cases. Where microbial analyses were requested, Staphylococcus aureus bacteria was the most common organism found, followed by Cytomegalovirus. However, due to the small numbers of microbial analyses, geographical hotspots could not be identified at the pathogen level. There is therefore a need to adopt a standard protocol for the investigation of SUDI to optimise the translation of mortality data into targeted public health interventions. The promotion of awareness in at-risk areas should be harnessed in a local context to develop preventive strategies and ultimately reduce infant death.

No description available.

No description available.