Characterising the fluctuation of microRNA expression throughout a full menstrual cycle

When processing a crime scene, obtaining a DNA profile that can identify an individual is extremely important. However, the identification of the body fluid that the sample was obtained from could provide key information of the events that occurred. microRNA (miRNA) expression analysis is a technique that has the potential to differentiate body fluids. The presence and expression of body fluid specific miRNA would provide a fast and effective tool for progressing crime scene investigation, especially alleged sexual assault cases. Forensic case work lacks methods for identifying vaginal material, venous blood, menstrual blood and aspermatozoic seminal fluid within samples. A large screening study followed by a 31 day study on five female volunteers was performed utilising RT-qPCR on a large panel of body fluid markers. Screening showed a selection of markers were suitable to differentiate each body fluid, in some cases however, expression fluctuated when analysed over a 31 day period. The data shows that hsa-miR-412 may be suitable for identifying menstrual blood, expression from markers hsa-miR-124 and hsa-miR-205 varied significantly over the 31 day period and between individuals and therefore were less suitable for body fluid identification. The data supports the use of miRNA markers for the identification of certain body fluids such as menstrual and venous blood; however markers for the identification of body fluids such as vaginal material and saliva require further investigation

Evaluating the effect of multiple displacement amplification upon low-level multiple source DNA profiles

The interpretation of low-level DNA mixtures, where there is DNA present from more than one individual in a sample, is of great difficulty. This can be due to the low-level nature of the result and the complexity inherent in DNA results with multiple sources. These two elements in one sample only further compound the difficulty. Multiple displacement amplification (MDA) techniques, with or without a macromolecular crowding (MDA-MC) agent, have been suggested as potential tools for improving these results. The aim of this study was to investigate whether the proposed techniques could be of use to forensic genetics casework. Buccal swabs underwent extraction, and then mixtures of samples were prepared with a range of known mixing ratios, from 1:1 through to 50:1. These samples were split into three prior to undergoing standard DNA analysis, MDA or MDA-MC. DNA quantification was carried out using real-time PCR and the Human DNA Quantifiler kit, and STR analysis was carried out using the AMPfSTR NGMSElect kit. For each set of results, the mean mixing ratio was calculated and compared with the actual mixing ratio. The incorporation of MDA and MDA-MC did not sufficiently improve the mixed DNA results for all mixing ratios. Although there were significant improvements at mixing ratios of 10:1 and 50:1, this is insufficient, as knowledge of the actual mixing ratio prior to analysis is required. Consequently, as it stands, MDA and MDA-MC is not suitable for forensic casework. However, despite a range of actual mixing ratios from 1:1 through to 50:1, no mixing ratios above ~10:1 could be obtained, suggesting that this is the threshold at which the major contributor masks the minor contributor. This is thought to be due to drop-outs from the minor contributor, which increases as the mixing ratio increases

Differentiating between monozygotic twins through DNA methylation specific high resolution melt curve analysis

Although STR profiling is extremely powerful in identifying individuals from crime scene stains, it is unable to differentiate between monozygotic (MZ) twins. Efforts to address this include mutation analysis through whole genome sequencing and through DNA methylation studies. Methylation of DNA is affected by environmental factors; thus, as MZ twins age, their DNA methylation patterns change. This can be characterised by bi-sulfite treatment followed by pyrosequencing. However, this can be time consuming and expensive, thus unlikely to be widely used by investigators. If the sequences are different, then, in theory, the melting temperature should be different. Thus the aim of this study is to assess whether high resolution melt curve analysis can be used to differentiate between MZ twins. Five sets of MZ twins provided buccal swabs which underwent extraction, quantification, bi-sulfite treatment, PCR amplification and high resolution melting curve analysis targeting two markers, Alu-E2F3 and Alu-SP. Significant differences were observed between all MZ twins, targeting Alu-E2F3, and in four out of five MZ twins, targeting Alu-SP (p<0.05). Thus it has been demonstrated that bi-sulfite treatment followed by high resolution melting curve analysis could be used to differentiate between MZ twins