Trace DNA: a review,discussion of theory,and application of the transfer of trace quantities of DNA through skin contact

Advances in STR PCR DNA profiling technology allow for the analysis of minute quantities of DNA. It is frequently possible to obtain successful DNA results from cellular material transferred from the skin of an individual who has simply touched an object. Handling objects, such as weapons or other items associated with a crime, touching surfaces, or wearing clothing, may represent sufficient contact to transfer small numbers of DNA bearing cells, or trace DNA, which can be successfully analyzed. With this minimal amount of contact required to yield a suspect profile comes tremendous crime solving potential, and a number of considerations for prudent application, and the maximization of evidentiary value. Evidentiary materials not previously considered must be recognized and preserved, and the resulting DNA type profiles interpreted in their proper forensic context.     

Investigation into the usefulness of DNA profiling of earprints

DNA profiling of biological trace evidence has been used for many years. The application of this technique specifically to the DNA profiling of earprints has not to date been thoroughly investigated. This report presents the results of 60 earprints collected from three healthy adult volunteers under controlled laboratory conditions. DNA profile analysis revealed that high levels of non-donor alleles are observed when earprints are collected for DNA profiling. The source of these non-donor alleles is investigated and the impact that their presence within the profile may have on the use of this technique is discussed.     

Comparison of Commercial Kits for Recovery and Analysis of Bacterial DNA From Fingerprints

In forensic science, fingerprints are a common source of evidentiary information. However, latent examination is not always successful and trace human DNA cannot always be obtained. Thus, examining the fingerprint microbiome may offer a suitable alternative to more traditional methods of forensic identification. The Zymo Research ZR Bacterial/Fungal DNA MicroPrep™ Kit, Qiagen QIAmp^® DNA Mini Kit, Promega Wizard^® Genomic DNA Purification Kit, and the MPBio FastDNA^® Spin Kit were compared for their ability to yield a sufficient amount of bacterial DNA for next-generation sequencing in order to obtain a microbiome profile. Prints were deposited onto slides, allowed to sit for up to 1 month, and total DNA isolated and quantified using each kit. The kit from Zymo Research yielded the most concentrated DNA sample (0.0084 ng/µL) in the least amount of time as compared to other kits examined. Although this amount of DNA was far below the recommended DNA concentration threshold recommended for next-generation sequencing, a microbiome profile was successfully obtained. As interest in using the microbiome of an individual as a forensic tool continues to increase, there is the possibility that the microbiome of a fingerprint could complement traditional human DNA profiling in the future. This study provides evidence that trace amounts of bacterial DNA from fingerprints is quantifiable and sufficient for microbiome analysis.     

DNA Profiles from Fingerprint Lifts—Enhancing the Evidential Value of Fingermarks Through Successful DNA Typing

This study evaluated the compatibility of the most common enhancement methods and lifting techniques with DNA profiling. Emphasis is placed on modern lifting techniques (i.e., gelatin lifters and Isomark?) and historical fingerprint lifts for which limited research has been previously conducted. A total of 180 fingerprints were deposited on a glass surface, enhanced, lifted, and processed for DNA typing. DNA could be extracted and profiled for all the powders and lifts tested and from both groomed fingerprints and natural prints with no significant difference in the percentage of profile recovered. DNA profiles could also be obtained from historical fingerprint lifts (79.2% of 72 lifts) with one or more alleles detected. These results demonstrate the compatibility between different powder/lift combinations and DNA profiling therefore augmenting the evidential value of fingerprints in forensic casework.     

Investigation into "normal" background DNA on adult necks: implications for DNA profiling of manual strangulation victims

Others have investigated the role that DNA profiling could play as a method for identifying the perpetrator of manual strangulation. These studies have demonstrated that it is possible to collect offender DNA from the skin surface of a victim following physical contact. It is not known whether nonself biological material is normally present on the skin surface due to adventitious transfer occurring during innocent everyday interactions. To test the hypothesis that detectable amounts of nonself DNA are normally present on the skin surface of healthy adult individuals due to the adventitious transfer of DNA occurring during normal day-to-day social interactions, we designed an experiment in three phases. Phase 1 was used to deduce which DNA collection, extraction, and amplification methods were suited to investigating this question. During phase 2, the neck surface of 24 healthy adult volunteers was swabbed. DNA was extracted using the QIAamp DNA mini kit and amplified using the SGM Plus PCR amplification kit, using 28 PCR cycles. The work carried out during phase 3 involved a simulated assault to investigate primary and secondary transfer of DNA during physical contact. It was found that 23% of neck areas swabbed during phase 2 of this investigation showed nondonor alleles in the resulting DNA profile, with 5% of areas showing six or more nondonor alleles. The results of phase 3 showed that primary, secondary, and zero transfer of victim and/or offender DNA could be observed after physical contact and that alleles from an unknown source could still be detected in this more controlled experiment. The data presented in this paper demonstrate that DNA profiles generated after swabbing the skin surface of healthy adults can include components of an unknown source, present due to adventitious transfer. These components, if present in large quantities, have the potential to interfere with DNA profile interpretation of swabs taken for the investigation of physical assault by DNA profiling.     

Visualising DNA transfer: Latent DNA detection using Diamond Dye

A central question is ‘how did DNA get there’? To help answer this, we visually monitored and recorded DNA transfer from one substrate to another. When an individual touches a substrate, traces of their DNA are transferred (primary/direct) which can then subsequently be transferred to a second substrate (secondary/indirect). Currently DNA transfer and how much remains can only be determined by collecting the biological material from the substrate, isolating the DNA and quantifying the amount recovered. However, Diamond™ Dye (DD) enables such DNA transfer events to be visualised by monitoring the movement of cellular material.We examined primary and secondary DNA transfer using aluminium as a primary substrate with cotton, polyester, aluminium and plastic as secondary substrates and four contact types between two substrates (passive, pressure, friction and friction with pressure). Participants pressed their index finger against the aluminium for 15 s and then DD was applied to the area of contact; cellular material was detected via a fluorescence microscope. Contact between that substrate and a second substrate was performed, using one of the four contact types. After this contact between substrates each was viewed microscopically and transfer of cellular material was recorded.Cellular material could be recorded as having transferred from one substrate to another. Substrate and contact type had an effect on the extent DNA transfers. DNA transferred at a high rate with aluminium as a primary substrate and cotton, polyester and aluminium as secondary substrates when pressure with friction was applied. This information expands our understanding of how DNA transfers and which factors affect it, thus assisting greatly with activity level reporting as to how DNA came to be where it was found.     

Experiences with single locus DNA probes in casework.

DNA profiling in this laboratory has been employed primarily in cases of sexual assault and the largest category of items examined has been internal vaginal swabs. 79% of these gave a profile which was different from that of the victim. Results have been obtained from swabs taken up to 70 h after intercourse. In cases where DNA results were obtained, one or more suspects were excluded in 29% of the cases.     

The prevalence of mixed DNA profiles on fingernail swabs

There is a general acceptance that cellular material will transfer from one person to another person's fingernails through everyday contact. However, the level or degree of contact required to transfer sufficient cellular material in order to obtain a DNA profile is not known. This study examined swabs from the fingernails of 40 volunteers and compared the DNA profiles obtained to the daily activities of that individual. The majority (78%) of high level profiles obtained were associated with recent intimate contact. However, high level profiles were also obtained from the fingernails of individuals who shared accommodation with their partner, flatmates and/or children. Low level profiles and single profiles were associated with all levels of contact.     

An innovative transfer DNA experimental design and qPCR assay to identify primary,secondary, and tertiary DNA transfer

“Touch DNA” is a form of trace DNA that is presumed to be deposited when an individual touches something and leaves behind DNA-containing skin cells, sweat, or other fluids. While touch DNA is often the result of direct contact (i.e., primary transfer), it can also be indirectly transferred between surfaces or individuals (e.g., secondary or tertiary transfer). Even experts cannot distinguish between different types of transfer and do not fully understand which variables affect direct versus indirect transfer or how often each type of transfer occurs. In this study, we utilize an innovative protocol that combines a paired male and female transfer DNA experimental design with an Amelogenin qPCR assay to generate data on primary, secondary, and tertiary DNA transfer. We report frequencies of indirect DNA transfer and also investigate the potential effects of participant age, self-identified ethnicity, and skin conditions on DNA transfer. Out of 22 experimental trials, we detected primary transfer (male + female) in 71% of trials, secondary DNA transfer in 50% of trials, and tertiary DNA transfer in 27% of trials. No significant associations were found between primary DNA transfer and age, self-identified ancestry, or skin conditions, however, all individuals with sloughing skin conditions demonstrated primary DNA transfer and we suggest this variable be explored in larger samples. These results contribute to a better understanding of the conditions under which secondary and tertiary DNA transfer occurs and can be used to propose realistic DNA transfer scenarios in court cases.     

Prevalence of mixed DNA profiles in fingernail swabs from autoptic cases

Physical contact between two or more persons can give rise to the transfer of DNA from one person to another and biological material can accumulate under the fingernail hyponichium. The purpose of this study is to value the normal levels of foreign DNA profiles under the fingernail of individuals deceased of a non-violent death, in order to define the usefulness of this approach in the recovery of a suspect's DNA profile. No foreign DNA was found in the majority of the cases.     

Comparison of the variables affecting the recovery of DNA from common drinking containers

As the boundaries of forensic DNA profiling continue to expand, less obvious sources of biological evidence are being collected at crime scenes for DNA profiling. One example is the recovery of biological evidence from common drink containers, such as bottles and cans, which have been found at crime scenes. There are many variables that may have an impact on recovering a DNA profile from such exhibits. In this research, the effects of person to person variation, time, type of drink (including alcoholic and non-alcoholic beverages), and type of drink container, were assessed for their impact on the major analytical outcomes of the DNA process. The results show that the alpha-amylase activity varies from individual to individual and is reduced in the presence of some alcoholic drinks. A reasonable DNA yield was obtained from all samples, however, the concentrations exhibited significant person to person variation. The type of drink container influenced the DNA yield with cans giving a higher yield than bottles of the same drink type. To a reduced extent the presence or absence of alcohol affected the overall DNA yield and when partial or failed DNA profiles were produced they were more likely to be associated with alcoholic drinks than non-alcoholic drinks.     

Single source DNA profile recovery from single cells isolated from skin and fabric from touch DNA mixtures in mock physical assaults

The ability to obtain DNA profiles from trace biological evidence is routinely demonstrated with so-called ‘touch DNA evidence’, which is generally perceived to be the result of DNA obtained from shed skin cells transferred from a donor's hands to an object or person during direct physical contact. Current methods for the recovery of trace DNA employ swabs or adhesive tape to sample an area of interest. While of practical utility, such ‘blind-swabbing’ approaches will necessarily co-sample cellular material from the different individuals whose cells are present on the item, even though the individuals' cells are principally located in topographically dispersed, but distinct, locations on the item. Thus the act of swabbing itself artifactually creates some of the DNA mixtures encountered in touch DNA samples. In some instances involving transient contact between an assailant and victim, the victim's DNA may be found in such significant excess as to preclude the detection and typing of the perpetrator's DNA. In order to circumvent the challenges with standard recovery and analysis methods for touch DNA evidence, we reported previously the development of a ‘smart analysis’ single cell recovery and DNA analysis method that results in enhanced genetic analysis of touch DNA evidence. Here we use the smart single cell analysis method to recover probative single source profiles from individual and agglomerated cells from various touched objects and clothing items belonging to known donors. We then use the same approach for the detection of single source male donor DNA in simulated physical contact/assault mixture samples (i.e. male ‘assailant’ grabbing the wrist, neck or clothing from the female ‘victim’, or being in transient contact with bedding from the ‘victim’). DNA profiles attributable to the male or female known donors were obtained from 31% and 35% of the single and agglomerated bio-particles (putative cells) tested. The known male donor ‘assailant’ DNA profile was identified in the cell sampling from every mixture type tested. The results of this work demonstrate the efficacy of an alternative strategy to recover single source perpetrator DNA profiles in physical contact/assault cases involving trace perpetrator/victim cellular admixtures.     

The propensity of individuals to deposit DNA and secondary transfer of low level DNA from individuals to inert surfaces

We have shown that there is a difference between individuals in their tendency to deposit DNA on an item when it is touched. While a good DNA shedder may leave behind a full DNA profile immediately after hand washing, poor DNA shedders may only do so when their hands have not been washed for a period of 6h. We have also demonstrated that transfer of DNA from one individual (A) to another (B) and subsequently to an object is possible under specific laboratory conditions using the AMPFISTR SGM Plus multiplex at both 28 and 34 PCR cycles. This is a form of secondary transfer. If a 30 min or 1h delay was introduced before contact of individual B with the object then at 34 cycles a mixture of profiles from both individuals was recovered. We have also determined that the quantity and quality of DNA profiles recovered is dependent upon the particular individuals involved in the transfer process. The findings reported here are preliminary and further investigations are underway in order to further add to understanding of the issues of DNA transfer and persistence.     

Novel cellular signatures for determining time since deposition for trace DNA evidence

With the increase in sensitivity of DNA profiling, questions about how and when the DNA was deposited have become a driving issue in forensic cases. To address this, we propose a novel method to determine time since deposition of trace DNA samples based on morphological and autofluorescence properties of individual epithelial cells which can change as the sample ages. To develop this signature, a series of trace DNA samples were generated by contact/handling a substrate and then allowed to age anywhere between one day and more than one year prior to collection. Imaging flow cytometry (IFC) was then used to characterize the morphology and autofluorescence profiles of individual cells within each sample followed by multivariate modelling and predictive classification.Resultsshowed that epithelial cell populations could be classified with high accuracy (∼90%) into one of three time-since-deposition groups: < 1 week, between 1 week and 2 months, and > 2months. Differences across age groups were largely driven by decreases in brightfield contrast and increases in the intensity of autofluorescence. To further test this approach for forensic casework, 47 individual donor cell populations spanning each time deposition group were classified blindly against the remaining data set. Samples containing at least 75 cells and a posterior probability greater than 0.90 showed classification accuracies ∼95%. Accuracies for individual time groups were 97% (<1 week), 92% (1week-2months), 98% (>2 months) with an average posterior probability for all time groups ∼0.96. This indicates that autofluorescence and morphological analyses may provide probative information regarding time since deposition for many types of trace DNA samples in forensic casework.     

The tendency of individuals to transfer DNA to handled items

This research investigates factors influencing the transfer of DNA to handled objects and the process known as 'shedding'. Volunteers were recruited to hold sterile plastic tubes using experiments originally designed by Lowe et al. [A. Lowe, C. Murray, J. Whitaker, G. Tully, P. Gill, The propensity of individuals to deposit DNA and secondary transfer of low level DNA from individuals to inert surfaces, Forensic Sci. Int. 129 (2002) 25-34]. Transferred cellular material was collected from the tubes and STR profiles generated using the AmpFlSTR SGM Plus multiplex with 28 and 34 PCR cycles. Volunteers were asked to hold the tubes with each hand, and to participate in a series of handwashing experiments. The DNA profiling results obtained from the transferred skin cells were compared. An attempt was made to characterize the volunteers as 'good' or 'bad' shedders and to establish which, if any, of the experimental variables were associated with 'good' shedding. Our results suggest that many factors significantly influence shedding, including which hand an individual touches an item with and the time that has elapsed since they last washed their hands. We have found that it may be more complicated than previously reported to categorise a person as being either a 'good' or a 'bad' shedder and that if truly 'good' shedders exist they may be significantly rarer than some have estimated. In the current research no 'good' shedders were observed in a group of 60 volunteers. Given these results, it seems that rather than being applied to individual forensic cases, knowledge of shedding characteristics will be most useful in providing general background data for the interpretation of trace DNA evidence.     

Recovery of Trace DNA on Clothing: A Comparison of Mini‐tape Lifting and Three Other Forensic Evidence Collection Techniques

Trace DNA is often found in forensic science investigations. Experience has shown that it is difficult to retrieve a DNA profile when trace DNA is collected from clothing. The aim of this study was to compare four different DNA collection techniques on six different types of clothing in order to determine the best trace DNA recovery method. The classical stain recovery technique using a wet cotton swab was tested against dry swabbing, scraping and a new method, referred to as the mini‐tape lifting technique. Physical contact was simulated with three different “perpetrators” on 18 machine‐washed garments. DNA was collected with the four different DNA recovery methods and subjected to standard PCR‐based DNA profiling. The comparison of STR results showed best results for the mini‐tape lifting and scraping methods independent of the type of clothing. The new mini‐tape lifting technique proved to be an easy and reliable DNA collection method for textiles.     

Forensic comparison of soils by bacterial community DNA profiling

This preliminary investigation has shown that a soil microbial community DNA profile can be obtained from the small sample of soil recovered from the sole of a shoe, and from soil stains on clothing. We have also shown that these profiles are representative of the site of collection and therefore could potentially be used as associative evidence to prove a link between suspects and crime scenes. Soil community profiles were obtained using the T-RFLP fingerprinting method that uses fluorescent primer technology and semi-automated analysis techniques similar to those used in human DNA profiling in forensic laboratories.     

An investigation of two methods of DNA recovery from fired and unfired 9 mm ammunition

Cartridge cases are often recovered from crime scenes involving firearms and, in the United Kingdom (where gun possession is strictly controlled), these are commonly from 9 mm calibre ammunition. The ability to obtain informative DNA profiles from touch DNA on recovered cartridges could have a significant impact on the investigation of that type of offence. However, this avenue may not be routinely considered as investigators in the UK have historically had a low expectation of obtaining useful DNA profiles. This stance may not be unreasonable given that (a) only trace amounts of DNA are likely to have been transferred onto the cartridge cases through handling; and (b) when the cartridge is spent, the potential deterioration of that DNA caused by the act of discharging the weapon.We introduce a novel semi-automatable method using direct lysis for the recovery of DNA from ammunition and compare it with a traditional double-swabbing method (using wet and dry swabs). DNA profiling of the DNA recovered using both methods was carried out using the ESI17 FAST STR system (Promega). This demonstrated a significant increase in DNA recovery using the direct lysis approach, and correspondingly improved STR results.We also investigated the effect on the recovery and profiling of DNA from fired, and unfired, 9 mm cartridges using the direct lysis technique. These results demonstrate that DNA suitable for STR analysis can still be recovered from fired ammunition with only slightly reduced yields compared to unfired ammunition. In these experiments, the handler of the ammunition was most commonly either the sole contributor or the major contributor to the recovered DNA profile.     

DNA profiling of disaster victim identification in Trenggalek shipwreck case

DNA analysis is one of the primary methods of identification in DVI practices. The external environment of a mass disaster often results in severe fragmentation, decomposition and intermixing of the remains. However, DNA profiling still can be achieved even on cases involving partial, severely decomposed remains. This report shows the DNA profile of shipwreck victims using identifiler plus marker from tissue sample exposed to environmental conditions.     

Natural DNA mixtures generated in fraternal twins in utero

Analysis of multiple genetic loci using short tandem repeats (STR) is widely used in human identity testing because the extensive polymorphism at these loci allows for a high degree of discrimination among individuals. We recently received a forensic case that included several pieces of evidence and reference blood samples. Upon initial testing, one of the suspects had a DNA profile that included three alleles at four of the nine loci tested (vWA, FGA, TH01, and D5S818). At each locus, two of the alleles appeared to be "major" alleles with a third "minor" allele present. The profile appeared to be a mixture of two people. Contamination of this first reference sample was suspected and a second, unopened blood specimen was requested from this individual. The DNA profile from this second reference specimen was identical to that of the original specimen at each locus. One of the evidence samples also displayed an identical mixed DNA profile matching that of the reference specimens mentioned above. The relative peak heights of the two "major" and one "minor" allele remained constant in all three samples. Additional background information revealed that the suspect had not received a bone marrow transplant or blood transfusion. However, it was disclosed that this individual is a fraternal (dizygotic) twin. We hypothesize that an exchange of blood cells between the fetuses occurred in utero and that the additional alleles present in these reference samples are derived from cells contributed by his twin sibling. No additional specimens from the suspect or his twin could be obtained for confirmation, and our hypothesis remains untested. Forensic scientists should be aware of this possibility when faced with a DNA profile in which extra alleles at multiple loci are detected.