Influences of oils on the persistence and recovery of DNA.

Trace evidence characteristics of DNA: A preliminary investigation of the persistence of DNA at crime scenes

The effects of different adhesive tapes and of Hemastix test strips on DNA recovery using magnetic silica beads

In a judiciary setting, questions regarding the mechanisms of transfer, persistence, and recovery of DNA are increasingly more common. The forensic expert is now asked to evaluate the strength of DNA trace evidence at activity level, thus assessing if a trace, given its qualitative and quantitative features, could be the result of an alleged activity. The present study is the reproduction of a real-life casework scenario of illicit credit card use by a co-worker (POI) of its owner (O). After assessing the shedding propensity of the participants, differences in DNA traces' qualitative and quantitative characteristics, given scenarios of primary and secondary transfer of touch DNA on a credit card, a non-porous plastic support, were investigated. A case-specific Bayesian Network to aid statistical evaluation was created and discrete observations, meaning the presence/absence of POI as a major contributor in both traces from direct and secondary transfer, were used to inform the probabilities of disputed activity events. Likelihood Ratios at activity level (LRα) were calculated for each possible outcome resulting from the DNA analysis. In instances where only POI and POI plus an unknown individual are retrieved, the values obtained show moderate to low support in favour of the prosecution proposition.

Sequence-specific eDNA extraction using hydrophobic magnetic ionic liquids attached with oligonucleotide strand

Chapter 5 - DNA Extraction

A comparison of six adhesive tapes as tape lifts for efficient trace DNA recovery without the transfer of PCR inhibitors

The effect of NucleoSpin® Forensic Filters on DNA recovery from trace DNA swabs

DNA transfer to firearms in alternative realistic handling scenarios

Trace DNA detection methods are increasingly applied to biomonitoring, in conservation, biosecurity, and wildlife forensic sciences. Reptiles are high risk invasive alien species that can cause damaging environmental impacts, yet the dynamics around terrestrial reptile DNA deposition and subsequent detection are poorly understood. Concerns around the role of the pet trade in introducing key reptile pests have resulted in calls for greater biosecurity surveillance and biomonitoring tools. Emerging studies have explored the applications of trace DNA and environmental DNA (eDNA) to reptile detectability, but questions remain around DNA persistence, sample storage, and DNA degradation through time influencing biosecurity compliance uptake. Here we consider Pantherophis guttatus (American corn snake), a key vertebrate pest species in Australia, as a model organism for the study of trace DNA detection over time. Our study focussed on the application of a recently developed corn snake LAMP assay for DNA detection from biosecurity relevant samples, with a particular focus on wildlife compliance for captive reptiles and shed skin samples subject to varied storage and environmental conditions. Experiments were conducted with (i) shed skin subject to a range of storage and treatment conditions over a 10-week period, (ii) trace DNA samples deposited on newspaper, plastic, glazed terracotta, and glass for 0 to 168 h post live snake presence, and (iii) shed skin samples subject to varying environmental conditions over an 8-week period. DNA remained detectable for up to 10 weeks under different storage conditions when extracted from shed skin and for up to 168 h after live reptile contact. DNA extracted from shed skin samples subject to varied environmental conditions was detectable up to 8-weeks post deposition with increasing times to detection. Our findings strengthen the case for trace DNA detection methods being applied to monitoring of terrestrial snakes, particularly for rapid compliance, monitoring, and environmental biosecurity.

Chapter 2 - DNA Extraction Methods

A variety of DNA extraction methods have been employed successfully to extract DNA from museum specimens. Toe pads are a common source of ancient DNA in birds, as they are generally not an informative character and can be removed without significant destruction of precious specimens. However, the DNA in these tissues is often highly degraded, both by natural postmortem decay and due to treatment by preservatives. In this case study chapter, we describe the use of both a commercial DNA extraction method and a silica-based method to extract ancient DNA from desiccated toe pads from the extinct passenger pigeon, Ectopistes migratorius. Successful amplification of nuclear DNA was achieved from both methods, representing the first nuclear DNA sequence recovered from this extinct species. We describe simple modifications to both protocols that we employed during the DNA extraction process.

We report on the effects of six dyes used in the detection of DNA on the process of DNA extraction, amplification, and detection of STR loci. While dyes can be used to detect the presence of DNA, their use is restricted if they adversely affect subsequent DNA typing processes. Diamond™ Nucleic Acid Dye, GelGreen™, GelRed™, RedSafe™, SYBR(®) Green I, and EvaGreen™ were evaluated in this study. The percentage of dye removed during the extraction process was determined to be: 70.3% for SYBR(®) Green I; 99.6% for RedSafe™; 99.4% for EvaGreen™; 52.7% for Diamond™ Dye; 50.6% for GelRed™, and; could not be determined for GelGreen™. It was then assumed that the amount of dye in the fluorescent quantification assay had no effect on the DNA signal. The presence of all six dyes was then reviewed for their effect on DNA extraction. The t-test showed no significant difference between the dyes and the control. These extracts were then STR profiled and all dyes and control produced full DNA profiles. STR loci in the presence of GelGreen(TM) at 1X concentration showed increased amplification products in comparison to the control samples. Full STR profiles were detected in the presence of EvaGreen™ (1X), although with reduced amplification products. RedSafe™ (1X), Diamond™ Dye (1X), and SYBR(®) Green I (1X) all exhibited varying degrees of locus drop-out with GelRed™ generating no loci at all. We provide recommendations for the best dye to visualize the presence of DNA profile as a biological stain and its subsequent amplification and detection.

There is a desire in the forensic science community for a robust tool for detecting trace DNA at the crime scene or on collected evidence. Recent research supports the fluorescent DNA-binding dye Diamond Dye (DD) as potentially effective for this purpose. However, additional research is required into the use of DD on trace DNA samples collected from mock evidence (e.g. clothing) to establish its practicality in an operational setting. Most importantly, any tool for detecting trace DNA must have no negative impact on downstream processes and must produce a reliable signal of presence/absence of DNA. Therefore, we aimed to determine whether DD staining of tapelifts taken from mock clothing exhibits impacts DNA yield and STR profiling success, and whether observed fluorescence on stained tapelifts corresponded with DNA yield. Using a paired sample approach of stained/unstained tapelifts from the same item, our results suggest that DD-stained tapelift samples generally recovered less DNA than unstained tapelifts, which resulted in poorer STR profiling success. Further, there was no association between the degree of fluorescence observed for stained tapelifts and DNA yield or STR profiling success. Taken together, these results suggest that DD staining may not be suitable for detecting trace DNA on tapelifts.

Persistence of dead-cell bacterial DNA in ex vivo root canals and influence of nucleases on DNA decay in vitro

Preliminary study on mitochondrial DNA analysis from different sports items