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Detection and analyses of latent DNA

Published:September 29, 2022DOI:https://doi.org/10.1016/j.fsigss.2022.09.025

      Abstract

      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.

      Keywords

      1. Introduction

      Cellular material on the skin’s outermost surface is composed of: dead cells in the form of keratinocytes and corneocytes; cells from other people; and cell-free DNA [
      • Burrill J.
      • Daniel J., B.
      • Frascione N.
      A review of trace “Touch DNA” deposits: variability factors and an exploration of cellular composition.
      ]. These biological materials can be deposited to surfaces by touch. This can be on items of crucial forensic relevance, such as cartridge cases, zip-locked bags, knife handles and other weapons. Until very recently, this cellular material was latent, i.e., not visible. The operator, either at a crime scene or in evidence recovery, will collect samples blind. The advent of staining cellular material with a dye has changed this. The application of Diamond™ Nucleic Acid dye (DD) has been shown to detect latent DNA [
      • Kanokwongnuwut P.
      • Kirkbride P.
      • Linacre A.
      Latent DNA detection.
      ,
      • Champion J.
      • Kanokwongnuwut P.
      • van Oorschot R.
      Evaluation of a fluorescent dye to visualise touch DNA on various substrates.
      ], determine shedder status [
      • Kanokwongnuwut P.
      • Martin B.
      • Kirkbride P.
      • et al.
      Shedding light on shedders.
      ,
      • Kaesler T.
      • Kirkbride P.
      • Linacre A.
      DNA deposited in whole thumbprints: a reproducibility study.
      ], and answer the question of how many cells are needed to generate a DNA profile [
      • Kanokwongnuwut P.
      • Martin B.
      • Taylor D.
      • et al.
      How many cells are required for successful DNA profiling?.
      ]. The use of DD as a triage tool has great relevance for forensic practice aiding in the targeting of sample collection. This extended abstract illustrates the use of DD in forensic science to detect latent DNA.

      2. Material and methods

      2.1 Ethics approval

      Approval from the Social and Behavioural Research Committee (reference 8169) was obtained prior to initiating this project.

      2.2 Dye solution and application

      A 20-fold dilution of the stock (10,000×) solution of DD (Promega, Madison, WI, USA) was made in 75 % ethanol (v/v).
      The dye can either be applied using a pipette or a compressed airgun. When using a pipette, aliquot 5 μL of 20× dye dilution onto each substrate and leave to incubate for 5 s. Alternatively, 5 mL of 20× DD can be added to the reservoir of a compressed airgun and apply in one single spray.

      2.3 Control cell deposition

      Donors washed hands under running water and dried with a paper-towel. After a defined time, such as 15 min, contact was made with a pre-cleaned substrate (e.g. a microscope slide) for 15 s and by applying medium pressure.

      2.4 Cell image collection

      Images were collected using a Dino-Lite fluorescence digital microscope (AnMo Electronics.
      Corporation, New Taipei City, TWN) equipped with an emission filter of 510 nm and a blue LED excitation light source (480 nm). Cells were recorded using the Dino-Lite image capture software and scored using an in-house software programme. Scoring of cellular material was performed by counting the number of stained cells in a frame (each 1 mm2) under the microscope at 220× magnification. An example of the microscope and image capture is shown in Fig. 1a with an example of a stained thumbprint in Fig. 1b. Negative controls can be created and recorded by staining and imaging an area of the substrate on which no cells were knowingly deposited.
      Fig. 1
      Fig. 1(a-b) showing the Dino-lite microscope attached to a computer (1a) and a thumbprint showing the corneocytes as green, fluorescent dots on the ridge patterns (1b). (c-g) showing how cell collection can be recorded and monitored. 1c shows stained corneocytes on a section of rope; 1d the cells are removed using tape and the underside of the tape placed under the microscope to visualise cell removal; 1e the area of rope can be placed under the microscope to confirm that many cells were removed and are now on the tape; 1 f the tape can be rolled and placed directly into a PCR tube (1 g) or subjected to an extraction process.

      3. Results

      DD has been found to stain cells such as those deposited by touch: an example of a thumbprint is shown in Fig. 1b. Here a thumbprint was made 15 min after the donor washed their hands. The stained corneocytes can be visualised on the ridge patterns of the thumbprint. This simple method gives an insight into shedder status [
      • Kanokwongnuwut P.
      • Martin B.
      • Kirkbride P.
      • et al.
      Shedding light on shedders.
      ,
      • Kaesler T.
      • Kirkbride P.
      • Linacre A.
      DNA deposited in whole thumbprints: a reproducibility study.
      ] and also the location of corneocytes, which appear to be on the ridge patterns close to pores.
      DD can be applied to evidential items as a screening tool to detect the location and presence of cellular material. An example is shown in Figs. 1c–g. Fig. 1c shows a DD stained section of rope after a donor held the rope – the cells can be seen clearly adhering to the rope fibres. Tape was applied to remove the cells and in Fig. 1d cells can be seen on the side of the tape that was in contact with the rope. The section of rope where the tape removed cells is shown in Fig. 1e – almost all the cells were removed. Fig. 1f shows a small section of the tape, on which there are many corneocytes, that is then placed into a PCR tube (Fig. 1g) for direct PCR (a full single source DNA profile using Identifiler Plus was obtained).

      4. Discussion and conclusion

      The visualisation of cells such as corneocytes deposited by holding items is possible. DD staining can act as a triage tool to determine the location and presence of cells to target the effective collection of cells deposited by touch. The method stains keratinocytes and corneocytes and the varying intensity of fluorescence may be an indication of the chromosomal content, but at magnifications used, cell-free DNA will not be detected.
      The potential problems are if the substrate is highly absorbent, or rough in texture, or has a similar absorbance wavelength of light resulting in auto-fluorescence. The application of DD is quick with stained cells appearing in 10 s or less and as this is non-destructive and not an inhibitor to PCR, there is only a potential benefit.

      Conflict of interest statement

      None Declared.

      Acknowledgements

      Funding was provided by the Attorney General’s Department of South Australia via Forensic Science South Australia.

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