Touch DNA Research Articles

The Effect of Surface Type, Collection and Extraction Methods On Touch DNA

The Effect of Surface Type, Collection and Extraction Methods On Touch DNA

Study on reproducibility evaluation of STR typing of Touch DNA

Short Tandem Repeat (STR) typing plays an important role in forensic science. Currently, many laboratories return the residual unextracted DNA evidence sample to police stations according to regulations in Japan. However, it has not been clarified how well the results of the original DNA analysis can be reproduced in the case of retest of trace or mixed DNA from remaining unextracted DNA evidence samples. In this study, simulated touch DNA samples were prepared, and STR typing was performed to verify the reproducibility of the results. Touch DNA samples were produced by having 25 individuals hold a doorknob, a 50 mL tube, and another personʼs clothing tightly for 1 minute each. These touch DNA samples were divided into two parts and STR typing was performed on each and the results were compared. The results indicated that the number of alleles and peak heights may vary between the original DNA test and retest, and the results are not necessarily identical. The discordance in the results may be due to changes in the amount of DNA extracted from the material, changes in the mixture ratio, or the appearance of alleles of unknown origin. This is due to the heterogeneity of touch DNA samples collected by swabbing, etc., and is not considered to negate the reliability of the respective result of DNA tests. The reproducibility of results when re-extracting from residual touch DNA evidence sample was expected to be difficult due to trace or mixed DNA, but to our knowledge, there are no previous reports verifying this fact. This study reveals for the first time the reality of this situation.

The Effect of Time and Environmental Conditions on Touch DNA

The Effect of Time and Environmental Conditions on Touch DNA

Analysis of Touch DNA

Analysis of Touch DNA

The Impact of Deposition Area and Time on Touch DNA Collected from Fabric

The Impact of Deposition Area and Time on Touch DNA Collected from Fabric

Touch DNA Sampling Methods: Efficacy Evaluation and Systematic Review.

Collection and interpretation of "touch DNA" from crime scenes represent crucial steps during criminal investigations, with clear consequences in courtrooms. Although the main aspects of this type of evidence have been extensively studied, some controversial issues remain. For instance, there is no conclusive evidence indicating which sampling method results in the highest rate of biological material recovery. Thus, this study aimed to describe the actual considerations on touch DNA and to compare three different sampling procedures, which were "single-swab", "double-swab", and "other methods" (i.e., cutting out, adhesive tape, FTA® paper scraping), based on the experimental results published in the recent literature. The data analysis performed shows the higher efficiency of the single-swab method in DNA recovery in a wide variety of experimental settings. On the contrary, the double-swab technique and other methods do not seem to improve recovery rates. Despite the apparent discrepancy with previous research, these results underline certain limitations inherent to the sampling procedures investigated. The application of this information to forensic investigations and laboratories could improve operative standard procedures and enhance this almost fundamental investigative tool's probative value.

Evaluating the sensitivity of presumptive and confirmatory tests for body fluids

Most exhibits received in the forensic genetics laboratory of Ras Al Khaimah (United Arab Emirates) are swabs or clothing with stains suspected of being either blood, semen or saliva; swabs from touch DNA are also reasonably common. Routine practice has been to use presumptive tests to characterize the materials before DNA extraction. In this study we evaluated the presumptive methods currently used for blood (Hemastix® and Kastle-Meyer), semen (Phosphatesmo KM) and saliva (Phadebas®) in comparison to the confirmatory tests OBTI Hexagon and RSID™ Blood, RSID™ Semen and RSID Saliva™. Results from this study showed that, as expected, presumptive tests were generally more sensitive in detecting body-fluids than confirmatory tests. The presumptive tests were at least two-fold more sensitive than the RSID™ Blood, Semen and Saliva confirmatory tests. However, the OBTI Hexagon test showed comparable sensitivity to Kastle-Meyer and Hemastix® for the detection of blood; this test utilizes an anti-hemoglobin antibody, which enables the high level of sensitivity. DNA was extracted from different dilutions and quantified using real-time PCR with the Quantifiler™ Human kit. Except for the RSID™-Blood and Saliva, the limit of detection for the tests was at dilutions where recovery of sufficient DNA for STR analysis was not likely.

A customized protocol to generate STR profiles from latent fingerprints

For decades, dactyloscopical and DNA analyses have both played a key role in forensic investigations involving friction skin patterns and/or human biological material. In many occasions, friction ridge impressions may hold little discriminatory power due to low quality of ridge patterns and/or insufficient area extension of such patterns. In these cases, an appropriate human DNA collection and a high-quality DNA extraction become crucial steps to yield a genetic identity from an unsuitable latent fingerprint pattern. Indeed, over the past few years, it has been proven that complete Short Tandem Repeat (STR) profiles can be obtained from a touch DNA sample. In this study, a protocol has been customized to maximize the performance of genetic profiling from latent fingerprints. Six participants provided two sets of finger impressions on pre-cleaned glass surfaces. These impressions were generated by the participant’s dominant (DH) and non-dominant hand (NDH). Genetic material from fingerprints was pooled using a cotton swab for each donor and hand, combining 1–5 depositions consecutively. This was followed by DNA extraction, Real-Time PCR for DNA quantification, capillary electrophoresis (CE) for sequencing, and genotyping software for STR profiling. DNA yield was measured by ng/cm2 (DNA/fingerprint area). Statistical tests detected DNA yield differences by donor’s sex, age distribution, handedness and fingerprint pooling. Results revealed that DNA quantities from DH was dependent on the number of pooled fingerprints. However, NDH yielded similar DNA quantities across all fingerprint pooled combinations. With the aid of a customized protocol, DNA titers was improved and meaningful STR profiles were produced for donors’ DH and NDH.

Y-STR mixture deconvolution by single-cell analysis.

Since Y-STR typing only amplifies male Y chromosomal DNA, it can simplify the interpretation of some DNA mixtures that contain female DNA. However, if there are multiple male contributors, mixed Y-STR DNA profiles will often be obtained. Y-STR mixture analysis cases are particularly challenging though as, currently, there are no validated probabilistic genotyping (PG) software solutions commercially available to aid in their interpretation. One approach to fully deconvoluting these challenging mixtures into their individual donors is to conduct single-cell genotyping by isolating individual cells from a mixture prior to conducting DNA typing. In this work, a physical micromanipulation technique involving a tungsten needle and direct PCR with decreased reaction volume and increased cycle number was applied to equimolar 2- and 3-person buccal cell male DNA mixtures and a mock touch DNA case scenario involving the consecutive firing of a handgun by two males. A consensus DNA profiling approach was then utilized to obtain YFiler™ Plus Y-STR haplotypes. Buccal cells were used to optimize and test the direct single-cell subsampling approach, and 2-3 person male buccal cell mixtures were fully deconvoluted into their individual donor Y-STR haplotypes. Single-cell (or agglomerated cell clump) subsampling from the gun's trigger recovered single-source Y-STR profiles from both individuals who fired the gun, the owner, and the other unrelated male. Only the non-owner's DNA was found in the cells recovered from the handle. In summary, direct single-cell subsampling as described represents a potential simple way to analyze and interpret Y-STR mixtures.

Detection and analyses of latent DNA

Latent DNA detection has the potential to transform aspects of DNA collection at scenes and from items. In the absence of being able to visualise the location of cellular material, all collection of samples at crime scenes is currently performed blind. With the advent of the application of a nucleic acid staining dye, the DNA within skin cells (commonly called keratinocytes and corneocytes) can be visualised. Diamond Dye fluoresces when it binds to the backbone of DNA. This fluorescence can be recorded using a simple mini-microscope allowing the location and number of cells to be recorded. The potential to visualise cells on a wide range of substrates opens the possibility to target sample collection and to triage samples for further analyses to only those containing DNA. Diamond Dye has been found to be safe at the concentration used, inexpensive, available commercially, easy to apply, is highly sensitive, and does not inhibit further analyses such as PCR. This work presented at the ISFG congress gives an overview of the current developments on using DNA staining dyes to record the number of cells present on a wide range of substrates. It is essential to firstly understand the composition of cellular material deposited by touch, where it originates and the relative composition of corneocytes and cell-free DNA. Insight into the origins of touch DNA will be presented along with the staining of nuclei using a range of dyes to show corneocyte degradation. The presentation will cover how DNA binding dyes can be used to effectively triage sample collection, monitor cell collection using different swabs and tapes.

Detection of touch DNA evidence on swab by SYBR®Green I Nucleic Acid Gel Stain

Invisible touch DNA sample is one of the most common sample types received in the DNA laboratory. However, the appearance of DNA from touch sample collection cannot be observed until the DNA detection process is completed. The objective of this study focused on effectiveness of SYBR®Green I Nucleic Acid Gel Stain (SYBR Green I, Invitrogen, USA) using four types of swabs; including cotton, rayon, nylon, and foam swab, for collecting touch DNA from objects. Touch DNA samples were collected from 1 male and 1 female volunteers and stained with 5x SYBR Green I. This study demonstrated 3 experimental designs; including effectiveness, stability and visualizing of touch DNA mockup study. The results showed that the foam swab was the only swab type that could distinguish between touch DNA with and without SYBR Green I fluorescence; therefore, the foam swab was studied in the mockup experiment step and was appropriate for the study of the stability of SYBR Green I stained swab. Furthermore, our results on stability study showed that touch DNA on foam swab stained with 5x SYBR Green I fluorescence immediately faded on Day 1 , but remained until Day 28 in the dark at room temperature. The mockup study using a foam swab that collected touch DNA on objects and stained it with 5x SYBR Green I revealed that SYBR Green I fluorescence was visualized differently from that without touch DNA. These outcomes indicated that SYBR Green I can be used as a stain in the touch DNA screening method on foam swabs for forensic laboratory, which would increase productivity by reducing time and resources cost.

The impact of deposition area and time on Touch DNA collected from fabric

Touched items at crime scenes are frequently analysed to help link suspects to crimes, for example, Touch DNA is collected from victims’ clothes in cases such as sexual assault, homicide, theft etc. Tape lifting is the preferred collection method of choice for trace DNA from clothes, fabric items and porous surfaces such as paper, therefore this study investigated the impact of deposition area and time on Touch DNA collected from fabric using minitapes. The amount of Touch DNA collected from the fabric was significantly affected by deposition area (p < 0.05), time (p < 0.05) and the interaction between the deposition area and time (p < 0.05), with the quantity of DNA collected decreasing over time. Also, the buttocks area of the trouser compared to the chest area is more prone to friction from an activity like repeatedly sitting on different surfaces which reduces the amount of Touch DNA available. In conclusion, it is more effective to collect trace DNA from victim clothes as soon as possible after the crime is committed.

Development and Validation of MPS-Based System for Human Appearance Prediction in Challenging Forensic Samples.

Forensic DNA phenotyping (FDP) provides the ability to predict the human external traits from unknown sample donors, directly from minute amounts of DNA found at the crime scene. We developed a MPS multiplex assay, with the aim of genotyping all 41 DNA markers included in the HIrisPlex-S system for simultaneous prediction of eye, hair and skin colours. Forensic samples such as blood, skeletal remains, touch DNA, saliva swab, artificially degraded samples together with individuals with known phenotypes and a set of 2800 M control DNA were sequenced on the Ion Torrent platform in order to evaluate the concordance testing results and the forensic suitability of the 41-plex MPS assay. The panel was evaluated by testing a different number of PCR cycles and the volume of reagents for library preparation. The study demonstrated that full and reliable profiles were obtained with 0.1–5 ng, even with high degraded DNA. The increment of the number of PCR cycles results in an improvement of correctly genotyping and phenotyping for samples with low amounts of degraded DNA but higher frequencies of artefacts were found. The high DNA degradation level did not influence the correct genotyping and phenotyping and the critical parameter affecting the result is the quantity of input DNA. Eye and hair colour was predicted in 92.60% of individuals and skin colour in 85.15% of individuals. The results suggest that this MPS assay is robust, highly sensitive and useful for human pigmentation prediction in the forensic genetic field.

Monitoring cell loss through repetitive deposition.

Touch DNA deposited on items can be visualised by using fluorescent nucleic acid staining dyes. It might be expected that if a person contacts items multiple times, then at each contact fewer cells should be transferred and deposited. Here we report on the use of Diamond Dye (DD) to monitor any reduction in cellular deposition during multiple contacts. A volunteer, who was assigned as a heavy shedder, was asked to deposit a thumbprint using both left and right thumbs for 15 s onto separate clean glass slides. Thumbprints were collected in triplicate with each mark made for 15 s. Immediately after deposition, a second and then third mark was made in the same way. The three consecutive depositions were repeated 30 times to create 90 tested thumbprints. The number of cells within each entire thumbprint was scored using a cell-counting program, developed in-house. The number of cells deposited by the second and third depositions showed a decrease in the cell number compared to the first deposition, by ~70% and ~85% respectively. There was no difference between the percentage of cell persistence from whole thumbprints (16 frames) compared to only scoring part of thumbprints (4 frames). The data obtained provide insight into how cells are deposited by touch and how many cells remain on the thumb for subsequent contact events. Such information may account for cell deposition when performing actions (e.g. loading a firearm or repetitively opening a closing zip-lock bag).

Development of a multiplex assay for detection of autosomal and Y-chromosomal STRs, assessment of the degradation state of mitochondrial DNA and presence of mitochondrial length heteroplasmies

The current focus in most routine forensic casework is detection of autosomal or gonosomal Short Tandem Repeats (STRs). With increasing degradation, STR analysis tends to be less successful up to complete failure. For challenging samples such as telogen hair roots and shafts, touch DNA samples or skeletal remains, mitochondrial DNA (mtDNA) analysis provides a powerful tool. Determination of DNA quantity is an important part in the casework workflow. Several ready-to-use kits are commercially available for nuclear DNA targets. However, quantification of mtDNA targets requires the establishment of an in-house method. Some assays even contain assessment of degradation, which alleviates the choice of target enrichment for sequencing through medium or small amplicons. As Sanger-type Sequencing (STS) still remains the golden standard in many laboratories, identification of heteroplasmies in C-tract regions prior to the sequencing reaction is advantageous. Firstly, primer selection can be expanded with primers binding near the C-tract and secondly, determination of the dominant variant is straightforward. All those quantity (nuclear and mtDNA) and quality (degradation and length heteroplasmies) evaluations usually require at least two separate reactions. Therefore, the aim of this project was the combination of all these targets in one multiplex assay using capillary electrophoresis to spare valuable sample extract. Amplification of representative autosomal and Y-chromosomal STRs allows estimate of success of (Y-)STR analysis. Simultaneously, five length heteroplasmies in the mitochondrial control region are targeted as well as three conservative regions of differing fragment lengths for assessment of the mitochondrial degradation state. Based on the outcome of this assay, forensic examiners can decide if STR analysis may be suitable. In case of absent STR peaks, appropriate proceeding of mtDNA sequencing can be determined.

Collaborative swab performance comparison and the impact of sampling solution volumes on DNA recovery

The collection of DNA traces marks the first step determining the success of genetic analysis. This study aimed to identify and validate a suitable alternative to the currently used ForensiX Evidence Collection Kit containing a cardboard box for swab storage. This box has to be folded at the crime scene, which is time-consuming and carries the risk of potential contamination and handling difficulties.A collaboration study involving three police departments and one laboratory for forensic genetics was performed to compare the currently used swab against three challenger swabs: ForensiX SafeDry, Copan 4N6FLOQSwab™ Genetics and Copan 4N6FLOQSwab™ Crime Scene. Mock samples consisted mainly of touch DNA, but also blood, saliva and semen were applied to twelve items with different surfaces. Every organisation contributed with three DNA collectors, whose individual collection efficiencies were investigated. The challenge of preparing homogenous traces, especially touch DNA, was addressed by enhancing hand contact frequency and sampling area. As a further part of the swab comparison study, we describe for the first time the influence of different swabbing solution volumes on the sampling efficiency of the different swabs.The application of touch DNA was also tested for a further swab type, the Sarstedt Forensic Swab, which yielded such low DNA concentrations that it was excluded from the collaboration study. The Copan Genetics and Copan Crime Scene swabs yielded significantly lower DNA concentrations than the currently used ForensiX Evidence Collection Kit and ForensiX SafeDry swab. The inter-individual performance results of the operators revealed significant differences in sampling skills. Comparing different swabbing solution volumes showed higher DNA yields or no significant difference for the ForensiX Evidence Collection Kit and ForensiX SafeDry than the Copan Genetics, depending on the item or trace type swabbed.Our results highlight the importance of validating first-step components that are decisive to the success of DNA typing in the context of specific sampling procedures and laboratory methods. Also, the significance of individuals' securing variations, principally unknown for crime scene investigation and laboratory teams, is emphasised for the first time, offering a practical approach for improving and training DNA collecting activities and ensuring the optimal securing evidence process. These findings increase the knowledge of impacts on DNA collection and, thus, benefit other laboratories and forensic services, particularly when using the same extraction methods.

The Impact of Collection Method on Touch DNA Collected from Fabric

The Impact of Collection Method on Touch DNA Collected from Fabric

DNA deposited in whole thumbprints: A reproducibility study

Touch DNA is pivotal in forensic science therefore understanding the mechanisms and variations of deposition and composition of genetic material in touched deposits is essential. Shedder status is still poorly understood, and the consistency and cohesiveness of research is less developed compared to other transfer and persistence considerations. In this study, the inter- and intra-variations between shedder categories and individuals were investigated by use of a nucleic acid binding dye. Ten volunteers deposited 30 thumbprints under two different time points post handwashing: 15 after a period of 15 min post handwashing (defined) and 15 after a period of at least 60 min post handwashing (undefined). Thumbprints were made on glass slides, marked with a grid of 55 squares, then the marks were stained with Diamond Dye and the cells that fluoresced in each square (7500 total) counted to determine the total number of cells. Shedders were less consistent in cellular deposition when thumbprints were made in the defined condition compared to waiting at least 60 min. Heavy shedders consistently generated informative profiles (defined here as 12 or more alleles); this occurred 73% of the time in the defined condition and 87% in the undefined. Intermediate shedders produced informative profiles, which occurred 50% of the time in the defined and 80% in the undefined condition. Light shedders consistently produced uninformative profiles with only 33% being considered uploadable to a DNA database for the defined condition and 27% informative for the undefined condition.

Analysis of mixed DNA profiles from the RapidHIT™ ID platform using probabilistic genotyping software STRmix™

Rapid DNA instruments are gaining interest in the forensic community as a means to generate DNA profile information more quickly than standard laboratory workflows, and with the potential to be carried out at the scene where samples are taken. Due to the many years that DNA profiles have been generated in a standard laboratory workflow, there have been numerous studies into profiling performance. These have flowed into probabilistic means of evaluating DNA profiles produced in those laboratories. In this study we examined the DNA profiling performance of the RapidHIT™ ID system on artificial mixtures constructed from raw DNA, mixtures constructed from body fluids, and touch DNA samples. We calibrated the probabilistic genotyping system STRmix™ for use on results produced by the RapidHIT ID system. Identical DNA samples were split, with half analysed on the RapidHIT ID system and the other half analysed in the standard laboratory workflow. Profiles produced from these paired samples were compared with regards to their composition and discrimination power. In general, profiles produced using the RapidHIT ID system showed good discrimination power, but less than those produced via the standard laboratory workflow. This is the expected trade-off for the advantages of speed and portability.

Analysis of rapid HIT application to touch DNA samples.

Rapid DNA technology is being utilized for reference profiles worldwide. There is also strong data in the literature to support its use for high-template DNA sources, the same is not true for low-template sources, such as touch DNA; this is a requirement before wider implementation to forensic casework is considered. We report on the Rapid HIT Intel cartridge's ability to facilitate successful amplification of touch DNA to obtain profiles from template deposited on items commonly encountered in forensic casework. Eight items were touched in ten replicates- two were tapelifted, three swabbed, and three directly inserted. Significance was observed in the alleles amplified and RFU with respect to sample type. Three samples performed well: cable tie, fabric, and matchstick. As two of these were directly inserted, this should be considered for any sample small enough. Placement of highly absorbent substrates into the cartridge is not advised as it can cause a lysate-pull error. Heterozygote loci often presented as homozygous (32%-78% loci per profile); this was influenced by substrate type and profile RFU. Loci with larger masses exhibited higher false homozygosity also. Comparison of the donor's profile analyzed was performed against previous datasets analyzing touch DNA through standard workflow, including manual DNA extraction, PCR, and CE separation. These data show that for all substrates, except for a fabric swatch, standard processing is preferential to Rapid HIT analysis. In its current form, rapid DNA technology is not fit for the routine analysis of touch DNA samples in forensic casework.