This PhD project describes the development of two novel forensic approaches the use of trace DNA isolation and profiling from fired gun cartridges, and the development and application of epigenetic analyses of the autosomal and mitochondrial genomes in order to estimate age.In the first part of the work two novel swabs (forensiX) and a standard cotton swab (EUROTUBE/Deltalab) were compared for the collection of forensic samples. Additionally, the effect of long-term storage of these samples was assessed. The forensiX swabs generally produced a higher (two- to four-fold) DNA yield compared to standard swab (around 750 pg and 250 pg, respectively, from 0.5 μL of saliva). These findings demonstrate the importance of ‘active drying’ performance in the preservation of DNA and swab selection. Using the forensiX swabs, we subsequently produced data that indicated that DNA samples deposited on cartridges during loading can survive firing from eight different types of gun.It has been shown that the methylation status of certain human DNA loci correlates with ageing. Blood sample DNA from a cohort of 82 women aged 18 to 91 years was obtained. We used Illumina MiSeq next generation sequencing platform to investigate the promoter regions of 23 genes suggested to have age-correlated methylation levels. Methylation levels at three CpGs located in the ASPA, ITGA2B and PDE4C genes showed an epigenetic signature of ageing with only a 6 year error range from chronological age.The methylation of mitochondrial DNA (mtDNA) is a new and incompletely described phenomenon with unknown biological control and significance. We describe the bisulphite sequencing of mtDNA from the same cohort of individuals. We detected low and variable levels of mtDNA methylation at 54 of 133 CpG sites interrogated. Regression analysis of methylation levels at two CpG sites (M1215 and M1313) located within the 12S ribosomal RNA gene showed an inverse relationship with age.We wished to create a fast and simple forensic tool (compared to next generation sequencing) for practical age estimation. The EpiTect Methyl II PCR system (QIAGEN) was used to compare methylation levels of CpG islands of the promoter regions of 4 age related genes (NPTX2, KCNQ1DN, GRIA2 and TRIM58). The data obtained from DNA methylation quantification showed successful estimation of subject age (11 year accuracy).This thesis describes practical steps to obtain forensic DNA samples while also applying novel techniques to explore the use of epigenetic profiling as a means to age prediction. The data generated suggest that the analysis of methylation patterns of both specific autosomal and mitochondrial gene sequences from biological evidence left at crime scene may help build up an accurate picture of an offender’s age and may help investigators obtain better descriptive information about a contributor from DNA, regardless of database inclusion.
|Date of Award||1 Oct 2015|
- University Of Strathclyde
|Supervisor||Benjamin Pickard (Supervisor) & Lynn Dennany (Supervisor)|