Evaluation of a fluorescent dye to visualize touch DNA on various substrates.

A wide variety of items are submitted as evidence in a forensic investigation. Identifying the location of DNA on such items is central to maximizing DNA profiling success and thus the ability to link a person of interest to a particular item or crime. Recent publications describe a fluorescent staining method using Diamond™ Dye (DD) to visualize cellular material on the surface of non-porous items (e.g., glass, plastic). However, substrates of varying porosity and background color have not yet been examined. Varying porous substrates (i.e., paper bank note, stamp, cigarette, wooden matchstick, and fabric) and non-porous substrates (i.e., enamel tooth and plastic bank note) were examined for their suitability with the use of DD. To improve the visualization of cellular material on the porous substrates, we also explored two DD diluents and adjusting image contrast. The results suggest the optimal diluent depends on the absorbent nature of the substrate. For example, ethanol was sufficient for visualization on the non-porous substrates, whereas water was better for the porous substrates. While cellular material was detected on the paper bank note, tooth, and stamp, background fluorescence or autofluorescence and surface type of matchstick prevented clear visualization on this substrate. It was also determined that by adjusting the contrast of images for tooth, paper bank note and matchstick aided in the detection of cellular material. Overall, this study extends the use of DD for latent DNA detection to absorbent substrates, highlights the limitations associated with these substrate types, and suggests modifications to improve visualization on these challenging substrates.

How many cells are required for successful DNA profiling?

Through advances in fluorescent nucleic acid dye staining and visualisation, targeted collection of cellular material deposited, for example by touch or within a saliva deposit, is possible. In regard to the potential evidentiary value of the deposit the questions remain: 'How many cells are required to generate an informative DNA profile?'; 'How many visualised corneocytes within a touch deposit compared to typical nucleated cells are required in order to achieve successful DNA profiling?'. Diamond TM Nucleic Acid Dye (DD) staining of cellular material, and subsequent visualisation utilising portable fluorescence microscopy, was performed for touch and saliva samples to target defined numbers of cells for collection, by swab and tapelift, and subsequent processing via direct PCR and PCR post-extraction. The resulting DNA quantification data and alleles generated within subsequent DNA profiles could be correlated to the number of cells initially collected to determine cellular threshold requirements for DNA profile generation for each workflow. Full profiles were consistently generated using direct PCR when the template was ≥40 buccal cells collected by either a swab or tapelift. By contrast ≥800 corneocytes collected by swabbing or ≥4,000 corneocytes collected by a tapelift were required to generate same number of STR alleles from touch samples. When samples were processed through a DNA extraction workflow, ≥80 buccal cells were required to generate full profiles from both swab and tapelift, while touch samples required ≥4,000 corneocytes collected by a swab and >8,000 corneocytes collected by a tapelift. The data presented within this study allow for informative sample triage and workflow decisions to be made to optimise STR amplification based on the presence and visual quantification of stained cellular material.

An assessment of tape-lifts.

Tape-lifting is a non-destructive alternative to swabbing for collection of biological materials deposited on surfaces, especially on porous substrates. While there have been a number of studies looking at the efficiency of tapes in terms of recovery and their effect on downstream processes, none has been able to visually monitor cellular material collection. We report on a comparative study of a range of tapes regarding their collection efficiency of cellular material visualised using fluorescence microscopy, their background fluorescence after staining with DD diluted with three different solvent types and inhibition of subsequent PCR using direct PCR. Three of the fourteen tapes were selected for further testing. These were brown packing tape (Packmate™), clear tape (Sellotape®), and DNA-free tape (Lovell Surgical Solutions). These did not inhibit direct STR amplification; the other eleven tapes exhibited either high background fluorescence and/or inhibition of subsequent PCR. The effectiveness of the three tapes for the collection of cellular material was tested after tape-lifting a fingermark once, twice and ten times. The amount of cellular material within fingermarks stained with Diamond™ dye (DD) was recorded using fluorescence microscopy before and after tape-liftings. The DNA-free tape (Lovell) used in many forensic laboratories gave poor recovery compared to the other two tapes. After a tape-lift, an average of 30 % of cellular material was recovered by using DNA-free tape (Lovell), contrasting with an average recovery of 59.5 % for the clear tape (Sellotape®) and 88.8 % for the brown packing tape (Packmate™). The results presented show that standard crime scene sampling tape does collect DNA but is less effective than shop-bought tapes. Full DNA profiles can be generated from all of touched fabric samples that were collected DNA using the three tapes, triaged by DD staining and amplified by direct PCR approach.

Successful STR amplification of post-blast IED samples by fluorescent visualisation and direct PCR.

Improvised explosive devices (IEDs) present a number of challenges in terms of the generation of forensically relevant information. Inhibition to PCR from sub-optimal sample types as well as from specific substrates has historically meant that extraction prior to PCR has been required. Improvements to STR kit buffers lead to the successful introduction of direct PCR to the analysis of IED-relevant samples, however none of these samples have been exposed to detonations. This study presents data to support the use of direct PCR in the analysis of IED components post-detonation. VeriFiler™ Plus generated informative profiles, containing ≥ 12 autosomal alleles, from samples touched for a maximum of 15 s that were then exposed to a detonation from plastic explosive placed as close as 100 mm. Of the 37 recovered touched items or fragments, 28 contained autosomal alleles from the donor with 18 (49 %) presenting informative profiles that matched the DNA donor. This compared with results following STR PCR post-extraction with one of 11 amplified post-detonation touch DNA samples being informative. The use of Diamond™ Nucleic Acid Dye (DD) staining and visualisation before and after detonation allowed for analysis as to cell loss or damage as a result of the detonation itself and aided in the triaging of samples to be selected for DNA profiling. This is the first record of cellular visualisation and comparison before and after detonation with accompanying STR results on a range of sample types typical of IED constituents. Following comparison of DD visualised cells and STR amplification success, chemical analysis of plastic and electrical tape samples supported substrate-specific inhibition. These data represent the first instance of informative DNA profiles being produced from post-detonation samples using direct PCR, as close as 100 mm from the charge.

Detection of cellular material in lip-prints.

We report on the visualization of cellular material within lip-prints using Diamond™ dye (DD). The transfer of cellular material via the lips can occur in cases of contact with food or drinking items as well as cases of alleged sexual assault involving oral contact. DD can effectively detect cellular material transferred by touch. Here we investigate if lip-prints can be detected and whether there is consistency within, or variability between, a person's propensity to shed cells within lip-prints. Ten volunteers were asked to press their lips against a glass slide with medium pressure for 15 s after not eating or drinking for at least 30 min. Both upper and lower lips were observed, and all tests were performed in five replicates, giving in total 900 observed areas. Consistency in the amount of cellular material deposited by lip-prints for each of the 10 individuals was observed, with each individual being associated with a 'lip shedder' status between the extremes of heavy and light. The majority of females shed more cells than the majority of males. No correlation was observed between the lip-prints shedder-status compared to deposition of cellular material from a thumb. Further, no correlation was observed between lip morphology and the 'lip shedder' status. Visualization of cellular material was not affected by lip-balm but was adversely affected by cosmetics such as lipstick. This technique demonstrates the visualization of deposited cells from parts of the body other than fingers and how cellular material can be visualized allowing targeted collection of DNA.

Detection of latent DNA.

Touch DNA is one of the most common sample types submitted for DNA profiling. There is currently no process to visualise the presence of such DNA deposited when a person makes direct contact with items of forensic relevance. This report demonstrates the effective use of Diamond Dye to bind to DNA and allow visualisation of deposited cellular material using a mini-fluorescence microscope. Volunteers made contact with a range of items typical of those submitted as part of a forensic investigation. Contact was for less than 5 s and occurred either 15 min after hands were washed to remove any traces of DNA, and therefore under controlled conditions, or at an undefined time post handwashing to mimic real-world scenarios. Diamond Dye bound to cellular material on all the items used and in all cases it was clear where the volunteers had made this brief contact. It was also clear where no contact had been made. DNA profiling was performed on a sub-set of samples to confirm that the cellular material viewed under the microscope was human in origin and deposited by the person contacting the item; this was the result obtained in every sample tested. Diamond dye is relatively inexpensive, simple to apply, binds to the DNA in 3 s or less, has no mutagenic effects at the concentrations used, does not affect subsequent DNA profiling, and does not bind effectively to bacterial DNA. In combination with a mini-fluorescence microscope, this proof-of-concept study shows that otherwise invisible DNA deposited by touch can be visualised. The position and amount of cellular material deposited during even brief contact can be recorded allowing targeted sampling in any further DNA typing of forensically-significant items.

Shedding light on shedders.

All previous examinations of the shedder status of individuals have been based on conclusions inferred from the amount of DNA deposited by donors after they have held an object for a fixed period of time. In all interpretations of shedder status experiments have involved a range uncertainties, especially in regards to results arising from studies carried out in different laboratories. These apply to the efficiency of the swab collecting DNA from the item touched, the amount of DNA left on the swab after attempts to recover it, and the percentage loss of DNA during the lysis and extraction processes. No previous study has attempted to mitigate these uncertainties or verify how much of the DNA deposited was collected through swabbing, how much DNA present on the swab was recovered or how much DNA is lost during the extraction process. We present a study that accurately measures the deposition, collection and amplification of DNA deposited by a range of donors allowing for an accurate determination of the shedder status of individuals. Eleven donors were asked to wash their hands and then deposit a thumbprint onto glass slides by making pressure for 15 seconds 0, 15, 60 and 180 minutes after handwashing. Both left and right thumbs were used and all testing was performed in triplicate. Measurement of the quantity of cellular material deposited on the slides was carried out using DiamondTM Nucleic Acid Dye and fluorescence microscopy on each of 264 thumbprints. Fluorescence microscopy was then used to demonstrate that all the DNA present on the slides was recovered by the swabbing operations and then direct PCR, using the Identifiler™ Plus kit, was used to ensure that none of the DNA present on swabs was lost during DNA profiling. The combination of using a DNA binding dye and direct PCR allowed an accurate means of measuring the extent to which individuals exhibit different extents of shedding. This small study, 11 donors, showed that individuals fell into one of three distinct groups: heavy, intermediate, and light shedders, regardless of the hand used.