Forensic trace DNA recovery and amplification from metal and metal-coated surfaces

Abstract

Metals are problematic substrates of interest in frontline forensic practice due to difficulties in obtaining probative DNA evidence from common metal objects and surfaces that are routinely submitted for trace DNA analysis, such as cartridges, bullets, and casings. The low success of trace DNA recovery from metal substrates has been linked to their physicochemical nature, which can degrade DNA following deposition or act as inhibitory contaminants that interfere with PCR amplification. However, the mechanisms behind metal- DNA interactions and how this impacts the efficiency of trace DNA recovery and downstream processes are poorly understood. The research described in this thesis examined trace DNA samples recovered from metal and metal-coated substrates in relation to typical forensic workflows from sample collection through to short tandem repeat profiling. The studies aimed to identify and characterise the negative effect of metal ions on DNA integrity, the collection and/or extraction of trace DNA samples, the co-purification of inhibitory factors with DNA, the interference of metal ions with quantitation, and how these ultimately impact DNA profiling. Seven data chapters illustrate the importance of sampling techniques for the successful recovery of trace DNA from metal substrates. The Isohelix™ swabbing system was shown to be a more effective sampling tool than a Rayon swab. Depending on the chemistry of the qPCR assay, the DNA template input, and the type and quantity of metal ions in the PCR reaction, I observed non-patterned, complex interactions with unexpected DNA quantification results. Additionally, metal ions in qPCR caused direct inhibition or secondary interference of qPCR dye chemistry, leading to under and over-estimation of DNA concentration. I also show that metal-mediated inhibition/degradation of cellular DNA is matrix-dependent, paramagnetic DNA extraction may not be optimum for samples contaminated with ferrous metals, and co-purified metal inhibitors can lead to an imbalance in STR profiles. When exposed to sunlight, self-cleaning metal-coated substrates, such as those coated with titanium dioxide, promote the photocatalytic destruction of trace DNA. Overall, this research highlights the importance of investigating novel trace DNA sampling and quantitation strategies, as well as more sensitive and robust amplification methods, while working with metal substrates.