Advances in trace DNA analysis, in combination with increased ability to detect minute amounts of DNA, necessitated research investment into the understanding of the factors relevant to DNA-TPPR, including individuals’ ability to deposit their trace DNA, termed their shedder status. Variability in shedder tests and assessment methods, in combination with limited data routinely published, currently limits a scientist’s ability to utilize such data in casework circumstances while also raising questions on which of the myriads of methods is the most appropriate going forward. In the present study, we investigated trace DNA deposition by 5 individuals gripping a plastic tube using several of the most common conditions related to duration since handwashing (unwashed, 15 min post-wash, and 1-hour post-wash). Five replicates were undertaken per method, with the aim of developing a method for determining the best shedder test when limited data is available. Hierarchical Bayesian modelling (HBM) was used to compare five different modelling structures for the data. The most supported model was chosen and used to provide insights about the performance of each shedder test method and ultimately choose the best method for determining shedder status. The Bayesian modelling approach offers advantages in certain situations by providing posterior parameter distributions that avoid binary significance interpretations. The results from the models described in this study showed that Method 3 (testing 1-hour after a hand wash) was best for shedder classification of those tested. This method most closely resembles personal natural behaviour while still allowing for some standardisation of the protocol. We use the data from this study to generate a shedder distribution and show how the knowledge of where an individual falls on the shedder distribution can be incorporated into an evaluation framework such as a Bayesian network that might be used in an evaluation of observations given activity level propositions.

Swabs are commonly used in forensic contexts to recover biological material from surfaces. Previous investigations have shown that there is no single best swab and wetting agent combination for DNA recovery. Touch DNA is often present in low quality and quantity, and the recovery and processing methods used during casework can result in DNA loss, therefore even small improvements in recovery could influence successful detection. Therefore, a DNA recovery method tailored for a touch DNA workflow is fundamental to effective forensic casework. In this study, combinations of five swabs (viscose, cotton, nylon, foam and polyester) and three wetting agents (water, 100

This study details the detection of blood remote from a laboratory using a direct recombinase polymerase amplification reaction integrated with a lateral flow assay. The assay targets the ALAS 2 gene; a marker used in mRNA assays for the identification of blood in body fluid assignments. The process can be used at scenes as no specialized equipment is needed; the assay is efficient between 17°C and at least at 45°C. A result can be obtained in 10 min if the ambient temperature is more than 25°C but this time increases to 40 min if cooler than 25°C. Like any test for a body fluid, the assay needs to be highly sensitive, positive results were obtained with this assay from blood dilute to 0.0005 μL. The test was found to be specific for blood as positive results were only recorded in the presence of blood and no other body fluid. The reagents as a solution were stable for 3 days at 4°C adding to their potential use outside of laboratory conditions. The assay is straightforward to perform yielding results that can be interpreted easily. While not replacing presumptive tests, this can be used to further give confidence in assigning a sample as blood.

Trace DNA can be deposited onto a wide range of surface types which can include substrates with a coating of oil: examples being firearm components, power tools, and kitchen utensils. We report on the impacts of oil on DNA recovery and downstream DNA processing. Thumbprints were made on 60 separate glass slides, then stained with Diamond Dye and fluorescent cellular material counted as an estimation of the cells deposited. Aliquots of five different mineral oils, chosen due to their common uses and variation in viscosity, were spread across the entire deposited thumbprint. The thumbprints coated with one of the five oils were left at room temperature for either 1 day or 7 days. A swab was used to collect cellular material which was then processed manually through a DNA extraction process, quantified and STR alleles amplified using the VeriFiler™ Plus STR kit. It was observed that all five oils impacted the initial DNA recovery process by saturating the swab. The presence of oils also impacted the DNA extraction process by interacting with the magnetic resin. Oils with higher viscosity accentuated these observed effects during the DNA extraction process, as the presence of a white precipitate was seen to be carried over into the final eluate. A total of 56% of the 1-day samples and 72% of the 7-day samples resulted in DNA profiles comprised of 12 or more alleles. The results of this study highlighted the possible impacts of collecting samples with oils present on the surface.

This review focusses on the use of DNA binding dyes to detect and record the presence of latent DNA on items of forensic relevance. Latent DNA can be crucial in forensic investigations and remains invisible unless an enhancement method is applied. Latent DNA is deposited on items of forensic relevance through various modes of transfer, with direct contact between skin and the item being the most common. Skin cells, otherwise called dead keratinocytes or corneocytes, have been shown to contain highly variable amounts of DNA. There is no standardised presumptive test for skin cells, but the advent of DNA-binding dyes allowed for the first time, the presence and number of stained corneocytes to be recorded. A commonly used DNA binding dye is Diamond™ Nucleic Acid Dye (DD). The dye has been used to detect the presence of latent DNA within biological deposits on a range of substrates and has been used to assess shedder status. This review discusses the many potential benefits of staining a substrate with a dye to detect latent DNA and then being able to target collection of a sample only where there is cellular material present. Despite advantages, the use of dyes to detect cellular material has not transitioned into forensic science practice; the reasons for this are discussed including some of the problems of dye staining of substrates. The review concludes by highlighting opportunities for conducting research to monitor cell deposition, persistence and transfer.

In instances of direct physical contact between individuals involved in criminal activity, body samples can provide significant and relevant information to aid in criminal investigations and court proceedings. Fingernails are one such forensically relevant body area that is capable of providing evidence of direct contact and potentially revealing whether the interaction involved was of a forceful kind. Several studies have investigated the prevalence of non-self DNA under fingernails after different crime-related scenarios; however, few have assessed the types of DNA profiles found after everyday activities. Furthermore, the comparability of the fingernail samples to those deposited on contacted surfaces remains unknown. In this study, we examined the composition of self- and non-self-DNA in samples collected from under the fingernails and held tubes from the same set of individuals. Additionally, the potential use of fingernails samples for shedder assessment was evaluated through comparison with two common shedder categorisation tests. For these purposes, samples were collected from both hands of 25 individuals of different demographics, without any prior restrictions on activities. Direct deposits were made by holding a 50 mL tube (for DNA shedder testing) and placing index fingers onto a slide (for Diamond™ dye cell counting shedder testing). Fingernail samples from both hands were taken immediately after tube-holding deposits. Reference DNA samples were collected from the participants as well as their cohabitating partners and other adults. Qualitative and quantitative data on DNA and cell deposits were collected to support activity-level evaluations. In our study, mixture inversions were rare, with non-self DNA, when detected, usually present as a minor component. More non-self DNA was detected after participants' contact with the tube compared to fingernail samples. Partners' DNA was frequently detected in both sample types, but more so in fingernail samples. Comparisons of the three shedder testing methods (fingernails, tube holding and cell count) showed that the categorisation results of these methods are not interchangeable and that DNA methods (tube vs. fingernails) were more consistent (64 % of deposited classified into the same shedder category) with each other than with cell counts (tube vs. cell count: 52 % classified into the same shedder category) (fingernails vs. cell count: 40 % remained in the same category). We anticipate that these datasets will serve as a valuable resource for activity-level evaluations and encourage other investigators to contribute to the growing data collection.

The analysis of illicit drugs for the presence of human DNA provides an opportunity to gain greater information on ‘who’ is involved in the illicit drug supply chain, complementing the existing chemical profiling techniques to identify ‘what’ and ‘where’. Given the known inhibitory effect of numerous illicit drugs and related compounds on PCR, it is crucial to understand the effectiveness of currently used DNA extraction kits to successfully remove these compounds while extracting sufficient DNA for downstream DNA profiling. To do this, four common illicit drugs (methamphetamine HCl, heroin HCl, MDMA HCl and GHB Na) in addition to two precursors of methamphetamine (pseudoephedrine HCl and phenyl-2-propanone) were spiked with saliva and DNA extracted. Two DNA extraction kits were selected to evaluate columns compared to beads, the QIAGEN QIAamp® DNA Investigator kit and the Promega DNA IQ™ system. DNA quantification of these samples yielded significantly more DNA recovery for each drug, except P2P, when extracted using DNA Investigator. Similarly, peak heights and heterozygous peak height balance were, on average, improved when samples were extracted with DNA Investigator compared to DNA IQ. Regardless of drug or extraction kit, no inhibition was detected during DNA quantification or profiling, indicating reduced DNA yields were caused by inefficient DNA extraction rather than inhibition of the PCR processes. All samples extracted using DNA Investigator yielded complete DNA profiles while DNA IQ yielded near-complete profiles, ultimately resulting in extremely strong likelihood ratio values from all samples. The generation of informative DNA profiles, despite the presence of illicit drugs, using two commercially available DNA extraction kits highlights the potential to implement these sample types into operational laboratories.

Illicit drugs are often made in less-than-sterile environments and can be stored in ways which can be detrimental to any DNA present, such as whether they are exposed to UV radiation. Previously, analysis of how exposure to UV impacted DNA for forensic applications has been in controlled laboratory conditions isolating a single component of UV radiation and often on DNA-rich samples such as bloodstains or saliva. To evaluate DNA persistence in more realistic conditions, capsules, such as those used to distribute controlled substances, were manually made and then packed into ziplock bags. The persistence of DNA deposited on capsules was examined when left indoors in either, complete darkness, direct sunlight in high UV conditions (summer) or in low UV conditions (winter) for three weeks in ambient room temperature. The DNA yield, STR DNA profile quality and degradation index were all analysed to determine the impact of varied UV exposure on DNA in a semi-temperature-controlled environment. Capsule samples exposed to high UV conditions had significantly reduced DNA yields, a lower number of alleles from the capsule handler and, thus, reduced likelihood ratios compared to capsules exposed to darkness and low UV conditions. Samples exposed to either darkness or low UV had little-to-no differences in all DNA quality measures tested. Despite a decreased DNA yield and poorer quality DNA profiles, capsules left in high UV conditions for three weeks have sufficient DNA for DNA profiles with over half the genetic information present. The storage conditions of drug capsules, either before or after seizure by law enforcement, can impact the DNA persistence in as little as three weeks, which is problematic for often already low concentrations of DNA in trace samples.

Saliva is a frequently encountered body fluid at crime scenes, however currently there are no definite means to rapidly identify a body fluid as being saliva. In this study, a novel detection method for saliva using a modified Loop-mediated Isothermal Amplification (LAMP) integrated with CRISPR-Cas (Clustered Regularly Interspaced Short Palindromic Repeat-CRISPR associated protein) and LFA (Lateral Flow Assay) was developed to detect the expression of a saliva-specific gene: follicular dendric cell secreted protein (FDCSP). To determine the specificity of the assay, RNA from saliva plus other commonly encountered body fluids was tested (peripheral blood, semen, vaginal fluid, and menstrual blood): positive results were only observed from RNA extracted from known saliva samples and RNA from all the other body fluids exhibited a negative result. To assess the reproducibility, triplicates were used from one saliva sample, and the assay was performed on three different days: positive results were observed from all triplicates. The limit of detection was 2-6 (0.3906 ng RNA) or 2-7 (0.1953 ng RNA). This preliminary study for the identification of saliva requires no complex equipment and is easy to perform, offering an alternative means for body fluid identification.

The successful analyses of DNA obtained from cellular deposits on metal substrates is an on-going issue with many metallic substrates inhibiting downstream enzymatic reactions. To examine this problem further, we report on the monitoring of persistence of cells deposited by touch on a range of metal surfaces using the DNA binding dye, Diamond Dye. Fingerprints were deposited in defined areas on metal substrates and stained with Diamond Dye. The cells were recorded at time points from initial deposition through to four weeks. Cells deposited on a glass microscope acted as a control. Little cell loss was recorded over the 4-week period on cells deposited on glass, nickel, stainless steel, and zinc. Unusual patterns of cell loss were recorded for cells deposited on copper and brass. Cells deposited on aluminium showed the greatest cell loss, nearly 22 %, contrasting with a loss of 4.5 % for cells deposited on glass (control). Whole thumbprints were deposited on the same substrates and stored for four weeks after which cellular material was removed using a swab and the DNA analysed using quantification and STR amplification. While cells deposited on copper did not record the greatest cell loss over the four weeks compared to the other metal substrates, when quantified and profiled, the whole thumbprints produced the least informative DNA profiles. No notable inhibition was recorded by qPCR for any sample, but degradation was indicated for both the brass and copper deposits. The data confirms that there are interactions between metallic surfaces and DNA and the substrate and DNA binding dye, which made cell visualisation difficult on brass and copper substrates. However, it also highlights that these metal-DNA interactions are causing DNA degradation on the copper and brass substrates that affect subsequent profile quality.

Hairs are encountered commonly in a forensic investigation such as at crime scenes, in automobiles or on clothing, which can originate from humans, domestic species or wildlife. Hairs can be a source of DNA although the amount of DNA associated with a single hair is limited and often less than the optimal amount required for DNA profiling. The root sheath contains most of the nuclear DNA with trace amounts of mitochondrial DNA in the shaft of the hair. Nuclear DNA testing targets highly repetitive DNA regions called microsatellites or short tandem repeats. The required amount of DNA for profiling may be present in the root sheath but not often within the hair shaft. Mitochondrial DNA loci are targeted on hair shafts due to the greater copy number of this cytoplasmic organelle compared to nuclear DNA. New technologies are increasing the sensitivity of DNA typing from a range of sample types including hairs.

Menstrual blood and vaginal fluid are frequently encountered in conjunction with semen in alleged sexual assault cases. There are few tests with high specificity for these two body fluids and here we report on a 4-plex MSRE-PCR system for the detection of menstrual blood and vaginal fluid. The assay uses four markers in a multiplex PCR: a positive control (PC), digestive control (DC), menstrual blood-specific (MB) marker and vaginal fluid-specific (VF) marker. Menstrual blood and vaginal fluid samples were identified by the MB and VF markers as expected. Additionally, the results using samples from saliva, semen and blood were also correctly identified, with an absence of both the MB and VF markers as expected, even though MB and VF signals can occur within a few non-target body fluids (urine, nasal fluid and skin). It was found that the methylation levels at the markers for both menstrual blood and vaginal fluid have differences between individuals. The methylation level of the marker for menstrual blood was affected by the day during the menstruation cycle, which also affected the sensitivity of the test. DNA required for correct identification of menstrual blood increased from 1 ng at day 1 to 1.5 ng for day 5. The sensitivity of the assay using samples from vaginal fluid was at least 0.5 ng of DNA for confident identification. Menstrual blood samples deposited on seven types of materials frequently encountered in forensic investigations were all correctly assigned. Also, samples of simulated mixed menstrual blood and vaginal fluid were tested and the results were all concordant as expected. These data help to verify the applicability of the 4-plex MSRE-PCR system in forensic casework.

Background/Objectives: DNA profiling can fail, or produce poor results, when naturally occurring materials are present during the amplification step. This study demonstrates that simple modifications to the reaction setup can overcome this obstacle. PCR inhibition is caused by a range of compounds including haem, humic acid and dyes. Various strategies to overcome this inhibitory effect have been explored, such as improving extraction methods to remove these compounds, diluting samples to reduce inhibitor concentration, or using inhibitor-tolerant DNA polymerases. In this study, we evaluate whether modified setups can help mitigate the effects of humic acid, a common inhibitor that induces various inhibition mechanisms. Methods: We combined the GlobalFiler STR kit with Investigator Quantiplex Pro into a single reaction. Supplementing the amplification GlobalFiler with additional reagents creates altered amplification environments that use additional DNA polymerase and reaction buffer. Results: The modified setups outperformed the standard GlobalFiler protocol, even at the highest concentration of humic acid tested. The STR reactions supplemented with qPCR reagents produced higher-quality profiles with improved allele amplification and an even peak balance, indicating that a dual-DNA polymerase system offers a more robust and inhibitor-tolerant environment for STR amplification. In addition to demonstrating the value of this combined approach, these data provide a comprehensive dataset characterising the impact of increasing humic acid concentrations on profile quality from an ideal DNA input. Conclusions: For PCR inhibitors with similar mechanisms this approach offers broader applicability in forensic casework and a promising step toward more reliable and robust profiling of inhibited samples.

An initial step in the development of a smart PCR machine, capable of amending the cycling parameters when amplifying STR alleles, is to monitor PCR progression in real-time. Performing qPCR allows for the real-time monitoring and recording of amplification of control loci: comprised of a small and large amplicon, a positive control, and a section of the Y chromosome. This qPCR data enables the recording of degradation and inhibition, as the fluorescence during qPCR theoretically should follow an exponential increase. Hypothetically, combining qPCR with STR amplification would allow real-time quantification of fluorescence such that the parameters of the PCR could be modified to optimise STR amplification: fluorescence below expectation would indicate a need to amend the PCR parameters to improve the DNA amplification. In this study, two different commercially available qPCR kits were combined separately with one of four different STR kits, and the resulting STR profile quality was recorded. Controls were performed by amplifying the same quantity of DNA template for each of the four STR kits, with all standard single and combined amplifications performed five times, resulting in 60 amplifications in total. No significant decrease in profile quality or likelihood ratios were recorded for any of the combinations. There were no adverse effects on the STR amplification when performed on a real-time PCR machine, despite two different enzymes and the presence of additional primers requiring differing temperatures to bind. These data are needed as the first step towards a smart PCR machine that can adjust cycling parameters in real-time.

“Shedder status” describes the inherent variation between individuals to leave touch DNA on a surface through direct contact. Depending on the amount and quality of DNA or cellular deposition, individuals are typically deemed high, intermediate, or low shedders. Although many shedder tests have been described, variability in study design and categorisation criteria has limited the ability of researchers to accurately compare results, as well as accrue the necessary population data. As activity level reporting becomes more common, the need for reliable and standardised testing increases. To assess reproducibility, this study compared shedder status data generated by six participants using three different shedder tests, as modified from the literature. This involved DNA quantification and profiling of a handprint made on a glass plate, DNA quantification and profiling of a grip mark made on a plastic conical tube, and cell scoring of a Diamond™ Dye-stained fingermark. All participants washed and dried their hands fifteen minutes before each deposit. To assess the impact of behaviour on shedder designation, participants either refrained from activity or went about their daily tasks during this wait. The shedder status of participants changed between tests, as DNA-based testing often generated lower shedder statuses than cell scores. Further, when different categorisation methods were applied to a single test, intra-person variability increased as the number of shedder designations increased from two (low/high) to five (low/low-intermediate/intermediate/intermediate-high/high). Moving forward, the utilisation of a single shedder test and standardised categorisation criteria is needed to employ shedder testing in forensic casework.

Our ultimate aim is to develop a smart PCR system that can monitor amplification in real-time and amend PCR conditions on-the-fly to obtain better quality DNA profiles from challenging samples. For such a system to exist, a method is needed to monitor the progress of an amplification reaction in real-time without compromising the quality of the DNA profile. This study used a combination of GlobalFilerTM and the Investigator Quantiplex Pro RGQ Kit for PCR and found the combination-multiplex amplified the highly polymorphic regions (STRs) used for DNA profiling and quantification regions used for DNA quantification in a single reaction. By using this combination, it is now possible to amplify STRs for DNA profiling whilst also monitoring the kinetics of the amplification reaction in real-time. The combination of these reactions was performed on an open-source qPCR machine to allow the real-time fluorescence data to be collected before DNA profiles were generated using capillary electrophoresis.

PCR has transformed DNA profiling, providing the opportunity to analyse trace DNA. Trace DNA often contains degraded DNA, such that many samples generate no meaningful STR data. The PCR process has changed little since first use despite the drive for greater sensitivity: the same steps of denaturation, annealing and extension used in the first STR amplifications are recognizable in current processes. Our aim is to move towards a smart PCR that can monitor amplification and amend cycling conditions. To achieve this, DNA amplification needs to be performed on a real-time PCR machine. Initially, this was the adjustment of the time at the denaturation and annealing steps. Standard STR profiling was performed using the GlobalFilerTM kit on an open-source qPCR machine and reduced in step changes the denaturation time to 2 s from 10 and time of annealing from 90 s to 30. The comparison of the STR profiles resulting from the modified amplification parameter to those of the control set indicated a loss of allelic amplification although the resulting likelihood ratios were extremely high. We identify the groundwork required to attain the ambitious goal of creating a smart PCR system that can respond in real time.

The use of PCR is widespread in biological fields. Some fields, such as forensic biology, push PCR to its limits as DNA profiling may be required in short timeframes, may be produced from minute amounts of starting material, and may be required to perform in the presence of inhibitory compounds. Due to the extreme high-throughput of samples using PCR in forensic science, any small improvement in the ability of PCR to address these challenges can have dramatic effects for the community. At least part of the improvement in PCR performance could potentially come by altering PCR cycling conditions. These alterations could be general, in that they are applied to all samples, or they could be tailored to individual samples for maximum targeted effect. Further to this, there may be the ability to respond in real time to the conditions of PCR for a sample and make cycling parameters change on the fly. Such a goal would require both a means to track the conditions of the PCR in real time, and the knowledge of how cycling parameters should be altered, given the current conditions. In Part 1 of our work, we carry out the theoretical groundwork for the ambitious goal of creating a smart PCR system that can respond appropriately to features within individual samples in real time. We approach this task using an open qPCR instrument to provide real-time feedback and machine learning to identify what a successful PCR ‘looks like’ at different stages of the process. We describe the fundamental steps to set up a real-time feedback system, devise a method of controlling PCR cycling conditions from cycle to cycle, and to develop a system of defining PCR goals, scoring the performance of the system towards achieving those goals. We then present three proof-of-concept studies that prove the feasibility of this overall method. In a later Part 2 of our work, we demonstrate the performance of the theory outlined in this paper on a large-scale PCR cycling condition alteration experiment. The aim is to utilise machine learning so that throughout the process of PCR automatic adjustments can be made to best alter cycling conditions towards a user-defined goal. The realisation of smart PCR systems will have large-scale ramifications for biological fields that utilise PCR.