Trace DNA evidence dynamics: An investigation into the deposition and persistence of directly- and indirectly-transferred DNA on regularly-used knives

Empirical data on the transfer and persistence of trace DNA are crucial to the evaluation of forensic DNA evidence. This evaluation can be complicated by the occurrence of indirect DNA transfer; the possibility of which is well established, but research into such transfer is often focussed on unrealistic situations, e.g. handling of DNA-free items after participants have shaken hands for 1–2 min. To simulate more realistic scenarios, this study investigated the deposition and persistence of both directly- and indirectly-transferred DNA on knives that had been artificially set up as ‘regularly-used’. Each knife was handled in a prescribed manner by a specific participant over two consecutive days to simulate regular use. Each participant then shook hands for 10 s with a fellow volunteer and immediately stabbed one of their knives into a foam block repeatedly for 60 s. DNA was recovered by mini-taping from triplicate sets of knife handles from four pairings of volunteers after regular use, and at one hour, one day and one week after the handshaking and stabbing events.Total amounts of DNA recovered from the knives, regularly used by a single person, varied among individuals; one volunteer consistently deposited significantly greater amounts than the others, whilst another volunteer did not always leave complete profiles. DNA attributed to the regular user persisted for at least a week, declining with increasing time between DNA deposition and recovery. Non-donor DNA was co-deposited at <5% of the profiles recovered, except for one volunteer, who consistently left DNA from their romantic partner on their knives at ∼25% and ∼11% of the profiles before and after the handshaking and stabbing events, respectively. In three pairings of volunteers, after the handshaking and stabbing events, alleles that could be attributed to the respective handshakers’ profiles were detected as partial minor profiles, equating to ∼10% of the profiles recovered. For the fourth pairing of volunteers, only complete single-source DNA profiles matching the regular user’s profile were recovered. However, it is important to note that, when indirectly-transferred handshaker DNA was detected, it declined with increasing time between DNA deposition and recovery.These data provide an initial insight into the detection and persistence of directly- and indirectly-transferred DNA that extend the data already available on forensic DNA transfer. The results herein suggest that the sooner an item is sampled after an offence has occurred, the greater the chance of recovering indirectly-transferred DNA, which has implications for forensic reconstructions.     

DNA on drugs (part 2): An extended study into the transfer and persistence of DNA onto illicit drug capsules using realistic scenarios

Capsules are now the main form of ecstasy rather than tablets in Australia and therefore their examination is of interest to forensic drug chemists in Australia and possibly elsewhere. Recently, we used controlled experimental conditions to show that capsules may be a source of DNA that can be used to identify those involved in production and distribution of illicit drugs. The question remains: in realistic scenarios where there are more unknowns, can we still detect DNA, and determine whose it is, on the exterior of capsules? The concept of comprehensive forensic intelligence and investigations – utilizing both biological and chemical signatures – relating to illicit drug preparations (i.e., the capsules and their contents) may be of great use to law enforcement. Experiments were conducted with both semi-realistic and realistic scenarios where two volunteers were asked to firstly use an encapsulator and mimic the loading of capsules, then Volunteer 1 would count out the capsules that Volunteer 2 prepared, and vice versa. This was to simulate the scenario where one person was involved in the assembly of the capsules which were then separated into smaller bags of 10 capsules by a second person for distribution. Gelatine and vegetable capsules were tested, with 10 replicates used per capsule type, scenario, and volunteer (total n = 80 capsules). Volunteer 2 was included as a contributor to the DNA profiles generated from 100% of samples handled by them within the semi-realistic scenario, whereas the other volunteer could be included as a contributor in 65% of samples. For the realistic scenario, profiles could be generated with the inclusion of both volunteers as profile contributors in 15% of samples and from just one of the volunteers in a further 50% of samples (therefore in total, either both or one of the volunteers were detected in 65% of realistic samples). Surprisingly, it was not necessarily the case that the last person to handle the capsule was the major or only contributor. The potential variability in the DNA quantities that could be deposited onto the capsules of genuine illicit drugs is high and would vary on a case-by-case basis. Nevertheless, this study has indicated that in realistic scenarios where two people are involved in the later stages of illicit drug capsule preparation, that either one or both individuals may be identified, potentially opening new investigative leads for law enforcement agencies as well as offering new information for intelligence-led policing.     

Secondary and subsequent DNA transfer during criminal investigation

With the introduction of new multiplex PCR kits and instrumentation such as the Applied Biosystems 3500xl, there has recently been a rapid change in technology that has greatly increased sensitivity of detection so that a DNA profile can routinely be obtained from only a few cells. Research to evaluate the risks of passive transfer has not kept pace with this development; hence the risk of innocent DNA transfer at the crime-scene is currently not properly understood. The purpose of this study was to investigate the possibility of investigator-mediated transfer of DNA traces with disposable nitrile-gloves used during crime-scene examinations. We investigated the primary transfer of freshly deposited DNA from touched plastic, wood or metal substrates and secondary and tertiary transfer by a person wearing disposable nitrile-gloves and onto a third object. We show that with use of the new highly sensitive technologies available in forensic DNA analysis there is an enhanced probability to obtain a DNA-profile which has not been directly deposited on the object but is an outcome of one or more transfer events. The nitrile-gloves used by investigators during exhibit examination can act as a vector for DNA transfer from one item to another. We have shown that the amount of DNA deposited on an object affects the probability of transfer. Secondly, the type of substrate material that DNA is deposited onto has an impact on transfer rates.     

Persistence of DNA from laundered semen stains: Implications for child sex trafficking cases

In sexual assault cases, particularly those involving internal child sex trafficking (ICST), victims often hide their semen-stained clothing. This can result in a lag time of several months before the items are laundered and subsequently seized during a criminal investigation. Although it has been demonstrated previously that DNA can be recovered from clothing washed immediately after semen deposition, laundered items of clothing are not routinely examined in ICST cases, due to the assumption that the time delay and washing would result in no detectable DNA. The aim of this study was to examine whether viable DNA profiles could be recovered from laundered semen stains where there has been a significant lag time between semen deposition from one or more individuals and one or more washes of the stained clothing.Items of UK school uniform (T-shirts, trousers, tights) were stained with fresh semen (either from a single donor or a 1:1 mixture from two donors) and stored in a wardrobe for eight months. Stained and unstained items (socks) were then washed at 30 °C or 60 °C and with non-biological or biological detergent. DNA samples extracted from the semen-stained sites and from the unstained socks were quantified and profiled.High quantities of DNA, (6–18 μg) matching the DNA profiles of the semen donors, were recovered from all semen-stained clothing that had been laundered once, irrespective of wash conditions. This quantity,and profile quality,did not decline significantly with multiple washes. The two donor semen samples yielded ∼10-fold more DNA from the T-shirts than from the trousers. This disparity resulted in the T-shirts yielding a ∼1:1 mixture of DNA from the two donors, whereas the trousers yielded a major DNA profile matching only that of the second donor. The quantities of DNA recovered from the unstained socks were an order of magnitude lower, with most of the DNA being attributable to the donor of the semen on the stained clothing within the same wash, demonstrating the transfer of semen-derived DNA among items of clothing in the washing machine.This study demonstrates that complete DNA profiles can be obtained from laundered semen stains on school uniform-type clothing, with an eight-month lag time between semen deposition and laundering, despite multiple washes and stains from two semen donors. These data emphasise the need to recover and examine the clothing of victims for semen and DNA evidence, even if the clothing has been stored for several months or washed multiple times since the sexual offence took place.     

Assessment of the transfer,persistence, prevalence and recovery of DNA traces from clothing: An inter-laboratory study on worn upper garments

Among the various items recovered from crime scenes or persons involved in a crime event, clothing items are commonly encountered and submitted for forensic DNA sampling. Depending on the case circumstances and the activity-of-interest, sampling of the garment may concentrate on collecting DNA from the wearer, or from one or more offenders who have allegedly contacted the item and/or wearer. Relative to the targeted DNA, background DNA already residing on the item from previous contacts, or transferred during or after the crime event, may also be collected during sampling and observed in the resultant DNA profile. Given our limited understanding of how, and from where, background DNA is derived on clothing, research on the transfer, persistence, prevalence, and recovery (TPPR) of DNA traces from upper garments was conducted by four laboratories. Samples were collected from several areas of two garments, each worn on separate working or non-working days and individually owned by four individuals from each of the four laboratories, and processed from DNA extraction through to profiling. Questionnaires documented activities relating to the garment prior to and during wearing, and reference profiles were obtained from the wearer and their close associates identified in the questionnaire. Among the 448 profiles generated, variation in the DNA quantity, composition of the profiles, and inclusion/exclusion of the wearer and their close associates was observed among the collaborating laboratories, participants, garments worn on different occasions, and garment areas sampled.     

DNA transfer in forensic science: A review

Understanding the variables impacting DNA transfer, persistence, prevalence and recovery (DNA-TPPR) has become increasingly relevant in investigations of criminal activities to provide opinion on how the DNA of a person of interest became present within the sample collected. This review considers our current knowledge regarding DNA-TPPR to assist casework investigations of criminal activities. There is a growing amount of information available on DNA-TPPR to inform the relative probabilities of the evidence given alternative scenarios relating to the presence or absence of DNA from a specific person in a collected sample of interest. This information should be used where relevant. However, far more research is still required to better understand the variables impacting DNA-TPPR and to generate more accurate probability estimates of generating particular types of profiles in more casework relevant situations. This review explores means of achieving this. It also notes the need for all those interacting with an item of interest to have an awareness of DNA transfer possibilities post criminal activity, to limit the risk of contamination or loss of DNA.Appropriately trained forensic practitioners are best placed to provide opinion and guidance on the interpretation of profiles at the activity level. However, those requested to provide expert opinion on DNA-related activity level issues are often insufficiently trained to do so. We advocate recognition of DNA activity associated expertise to be distinct from expertise associated with the identification of individuals. This is to be supported by dedicated training, competency testing, authorisation, and regular fit for purpose proficiency testing.The possibilities for experts to report on activity-related issues will increase as our knowledge increases through further research, access to relevant data is enhanced, and tools to assist interpretations are better exploited. Improvement opportunities will be achieved sooner, if more laboratories and agencies accept the need to invest in these aspects as well as the training of practitioners.     

The implications of shedder status and background DNA on direct and secondary transfer in an attack scenario

In court questions are often raised related to how trace DNA was deposited, directly during the crime or innocently for instance by secondary transfer. It is therefore of interest to have knowledge of the probability of transfer or secondary transfer in different situations. Factors that could influence transfer probabilities are background DNA and the shedder status of the involved persons. In this study, we have classified participants as high or low DNA shedders. We observed DNA transfer in a simulated attack scenario, and demonstrated that shedder status has a significant influence of transfer rates. We have examined the background DNA in samples from T-shirts worn in an area with frequent human traffic and detected multiple contributors. We further demonstrated that DNA from co-workers of a T-shirt wearer can be secondarily transferred from the environment and detected in samples, and that the composition of background DNA is correlated with the shedder status of the wearer. Finally, we have illustrated the inference with the results of transfer probabilities and a fictive case with the use of a Bayesian network.     

DNA reviews: the national DNA database of the United Kingdom

The national DNA database in United Kingdom has now been operational for over 10 years. This review looks at the history and development of this investigative resource. From the development of commercial DNA profiling kits to the current statistics for matches obtained in relation to criminal investigation in the United Kingdom, before moving onto discussing potential future direction that national DNA databases might take, including international collaboration on a European and global scale.     

Mitochondrial DNA polymorphisms: a study of 50 French Caucasian individuals and application to forensic casework

The analysis of mitochondrial DNA polymorphisms has proved to be efficient on highly degraded samples or samples having little or no genomic DNA such as hair shafts. In order to use this very sensitive method, the authors first established a database by analysing the mitochondrial DNA polymorphism in 50 French white Caucasian individuals, applied the analysis to different types of samples that can be found in forensic investigations and finally performed this method on two forensic cases involving the discovery of highly putrefied unidentified remains. Received: 10 March 1997 / Received in revised form: 4 February 1998     

The transfer and persistence of DNA under the fingernails following digital penetration of the vagina

Acts of aggression and defence during sexual assaults often lead to transfer of DNA from victim to assailant. Of sexual assault cases recorded by the FSS between 2005 and 2010, approximately 20% and 3% involved digital penetration of the victim's vagina or anus, respectively. Of the 309 cases where digital penetration was alleged, a suspect was arrested within approximately 1 day in 135 cases and fingernail samples were taken in 101 of these cases. In this study the transfer and persistence of DNA under the fingernails after an act of digital penetration were examined, (a) to assist investigators in determining whether to obtain such samples initially and (b) to assist evaluators in assessing the evidential value of female DNA profiles post analysis. Full female profiles were obtained from all swabs collected at 0 and 6 h after digital penetration, indicating that female DNA was always transferred and persisted in the short term. Furthermore, full female profiles were produced from three-quarters of samples collected after 12 h whilst mixed profiles were produced in the majority of samples taken after 18 h. The analysis of several variables indicated that hand washing had a significant effect on the persistence of female DNA profiles.     

Automation of the standard DNA differential extraction on the Hamilton AutoLys STAR system: A proof-of-concept study

For several decades, a common approach for processing sexual assault evidence has been to use the “standard” differential extraction to separate the evidence into a non-sperm-cell fraction and a sperm-cell fraction for further analysis. In this standard approach (P. Gill et al., Nature 318 (1985) 577–579), an initial mild chemical lysis step preferentially digests the mainly epithelial cells, which allows for removing this lysate as a non-sperm-cell fraction. The undigested sperm cells in the remaining fraction may then be purified by a series of wash and centrifugation steps, after which more robust lysis conditions are used to digest this sperm-cell fraction. Although this standard approach has been generally effective, it has been difficult to fully automate, due to the variety of different types of manipulations required for sample processing (e.g., incubation, shaking, substrate separation by centrifugation, and multiple liquid transfers for sperm-pellet centrifugation and washing steps). We describe here a fully automated standard differential extraction procedure that uses the Hamilton AutoLys STAR liquid handling assay-ready workstation, which is configured with on-deck components for sample incubation, shaking and centrifugation steps, and works with unique AutoLys-A^® sample tubes for front-end sample processing. In this proof-of-concept procedure, up to 24 samples may be processed, “hands-free,” in a single automated workflow. The automated procedure was tested by performing differential extractions on mock sexual assault swabs. For comparison, manual differential extractions were performed on identically prepared swabs in a side-by-side manner. DNA quantification and STR typing results showed that similar levels of separation efficiency were achieved for the sperm-cell fractions using both automated and manual procedures, although the results suggest that somewhat higher male DNA yields may be achieved for samples with extremely low semen levels (<∼0.1 μL) using the manual processing procedure. In addition to these mock samples, automated differential extractions were also performed on a set of authentic post-coital swabs (24, 48, 72, and 96 hours, post-coitus); primarily male STR profiles for the sperm-cell fractions were obtained for each sample in this set.     

Combining current knowledge on DNA methylation-based age estimation towards the development of a superior forensic DNA intelligence tool

The estimation of chronological age from biological fluids has been an important quest for forensic scientists worldwide, with recent approaches exploiting the variability of DNA methylation patterns with age in order to develop the next generation of forensic ‘DNA intelligence’ tools for this application. Drawing from the conclusions of previous work utilising massively parallel sequencing (MPS) for this analysis, this work introduces a DNA methylation-based age estimation method for blood that exhibits the best combination of prediction accuracy and sensitivity reported to date. Statistical evaluation of markers from 51 studies using microarray data from over 4000 individuals, followed by validation using in-house generated MPS data, revealed a final set of 11 markers with the greatest potential for accurate age estimation from minimal DNA material. Utilising an algorithm based on support vector machines, the proposed model achieved an average error (MAE) of 3.3 years, with this level of accuracy retained down to 5 ng of starting DNA input (~ 1 ng PCR input). The accuracy of the model was retained (MAE = 3.8 years) in a separate test set of 88 samples of Spanish origin, while predictions for donors of greater forensic interest (< 55 years of age) displayed even higher accuracy (MAE = 2.6 years). Finally, no sex-related bias was observed for this model, while there were also no signs of variation observed between control and disease-associated populations for schizophrenia, rheumatoid arthritis, frontal temporal dementia and progressive supranuclear palsy in microarray data relating to the 11 markers.     

DNA reviews: Ancient DNA

The rapid development, success, and occasional failures of forensic DNA profiling are highly publicised, and as a consequence are well known to the scientific and public communities alike. Over the same period of time that forensic DNA typing has accelerated onto the scene, another related discipline has been born and has made equally, or perhaps, even more groundbreaking achievements in the same arena; that is the science of (bio)molecular archaeology. This review describes the extreme complications of ancient DNA analysis and highlights some of the major achievements that have been accomplished in this field. The nature of DNA degradation and possible solutions to this problem are discussed.     

DNA Profiling and Criminal Justice: A Contribution to a Changing Debate

Forensic DNA profiling is now a routine feature of the criminal justice system in Australia. Its applications in this context continue to increase. Technological advancements and the use of DNA databases have facilitated the uptake of biological evidence into criminal investigations. The development of DNA methodology has progressed through discernable phases that have been paralleled by discussion amongst the legal community. The context of development and the associated debate has changed. It now encompasses broader issues, concerned less specifically with the technology itself and more with the most appropriate means for its use. To contribute more purposefully to this debate and to achieve the most meaningful outcomes from the criminal justice system, we must first understand more holistically the role that DNA evidence plays and the impact that it is capable of. This paper reviews aspects of the forensic and legal contexts of the use of DNA technology in the Justice system. This is a prelude to future research and a justification for the need for such research.     

A forensic case study for body fluid identification using DNA methylation analysis

Recently, a method of identifying body fluids using DNA methylation has been developed (Frumkin et al., 2011). An existing multiplex assay using 9 CpG markers could differentiate 5 body fluids: semen, blood, saliva, menstrual blood, and vaginal fluid. To validate this technique, we evaluated the previously described body fluid identification method by means of single base extension (SBE). DNA methylation was applied to 22 samples in 18 forensic cases; seven of these were semen, three were blood, eight were saliva, three were vaginal fluid, and one was menstrual blood. Total of 18 samples were tested, the DNA methylation profiles were coincident from preliminary tests (acid phosphatase (AP), leucomalachite green (LMG, Sigma Aldrich, St Louis, MO, USA) and SALIgAE®) except one sample which displayed an all-negative result. After applying the DNA methylation method to forensic samples, we determined that it could be very useful for differentiating vaginal secretions from menstrual blood, for which there is no conventional preliminary testing method.     

Crowdsourced and crowdfunded: the future of forensic DNA?

ABSTRACT

Forensic DNA analysis is dependent on comparing the known and the unknown. Expand the number of known profiles, and the likelihood of a successful match increases. Forensic use of DNA is moving towards comparing samples of unknown origin with publicly available genetic data, such as the records held by genetic genealogy providers. Use of forensic genetic genealogy has yielded a number of recent high-profile successes but has raised ethical and privacy concerns. Navigating family trees is complex, even more so when combined with a comparison of genetic relationships. This intelligence-gathering process has led to occasional false leads, and its use also risks a public backlash, similar to concerns over Cambridge Analytica. A cautious approach to use of this technique is therefore warranted.     

DNA reviews: low level DNA profiling

Low copy number (LCN) DNA profiling has recently been scrutinized in the United Kingdom following the comments of Mr Justice Weir made during the trial of suspected terrorist Sean Hoey. Mr Hoey was acquitted of all charges related to the Omagh bombing of 1998, following the inadmissibility of key DNA evidence during this trial. The Association of Chief Police Officers and Crown Prosecution Service, initially suspended the use of this technique, but quickly reinstated its use following an internal enquiry. This review describes the low copy number technique and the sample types that are now routinely collected from suspects, victims, and crime scenes for examination by this method.     

DNA reviews: hair

Human and nonhuman hairs are often recovered during forensic investigation. As with all other biological samples that may be collected, via DNA analysis, hairs have the potential to provide the investigating authority with valuable intelligence pertaining to the identity of offenders, victims and even pets. DNA analysis of hairs is not however a straightforward process. The biochemical make up of hairs provide the DNA analyst with a unique set of challenges that must be overcome before any useful information can be gleaned from the evidence. This short review provides an overview of the location and condition of DNA within hair samples, and discusses the analytical methods that are employed to maximise the information that can be obtained from this sample type.     

DNA transfer: The role of temperature and drying time

It has previously been shown, and reconfirmed here, that biological material on a substrate will transfer readily upon contact with another substrate when wet but hardly when dry. There is however a paucity of data regarding the speed at which body fluids dry and how this may affect its transfer upon contact. Here we conduct transfer experiments at 4 °C, 22 °C and 40 °C at multiple time points during the drying process. The speed at which blood dries is dependent on the temperature, with the drying process complete within 15–60 min. The percentage of deposited DNA transferred upon contact follows an exponential pattern of decline from soon after deposition, decreasing until the sample is dry. There are no differences in transfer rates upon contact among the different temperature conditions within the first 5 min or after 60 min since deposit, but significant variation occurs between these time points. When considering the likelihood of a proposed scenario that incorporates one or more contact situations it is important to consider the timing of the potential transfer event(s) relative to when the biological sample in question was initially deposited. The results of this study will assist the interpretation and evaluation of alternative scenarios involving transfer of biological substances.     

Effect of machine-washing semen-stained fabrics on the persistence of human spermatozoa DNA: A systematic review of five articles

Biological evidence of sexual violence, such as semen, can change due to fabric washing. This can be due to attempts by the perpetrator to eliminate evidence or because the victim feels ashamed of sexual violence. While much research on this topic has been conducted, no systematic review has been attempted. This systematic review explores the effect of fabric machine-washing on the persistence of human spermatozoa deoxyribonucleic acid (DNA). This systematic review seeks in vitro experiments in which semen-stained fabrics were washed by washing machines, published in English, and matched with keywords in PubMed, Europe PMC, ScienceDirect, and Google Scholar. We then assessed the obtained articles with the Joanna Briggs Institute quasi-experimental checklist. This systematic review used the narrative synthesis method. Our search yielded five articles. These articles observe the effect of machine-washing factors on the persistence of human spermatozoa DNA, such as water temperature, washing duration, detergent type, washing repetition, and duration of fabric storage before washing. This systematic review shows that fabric washing insignificantly affects spermatozoa DNA persistence, and DNA might persist after multiple washes. However, variations in the articles indicate that future studies on this topic need to account for more variables and be reported in more detail to reduce bias.