Journal of Forensic Pathology

In forensic science, touch DNA is used to measure DNA loss through the creation of eccrine fingerprints in a Lab

Naveen Negi^{* *}Correspondence: Naveen Negi, Department of Forensics, India, Email:

Author info »

Introduction

The Locard Exchange Principle, a fundamental idea in the study of fingerprints, states that whenever two objects come into touch, there will be a material exchange. The traditional information-bearing component of the exchange, sweat and oil acting as ridge detail, is transmitted via the fingers as transmission vectors. The science of touch DNA, however, has allowed forensic scientists to recognise the additional capability of extracting "DNA fingerprints from fingerprints," i.e., that the sweat and oil exchange contains a second information-bearing component in the DNA-containing cells that support genetic profiling. Touch samples are made when an object is handled or touched, and they include DNA that cannot be linked to a specific body fluid. Although the origins of the human DNA in a touch sample have not been clearly established, numerous lines of evidence suggest that they are probably made up of transplanted or indigenous nucleated epithelial cells as well as shed corneocytes.cell-free DNA, fragmented cells, and nuclei. The quantity and quality of DNA in a touch sample can vary widely between and within individuals, depending on numerous variables like the donor's activities, sex, age, substrate, temperature, and humidity. As a result, it is challenging to identify a real fingerprint as the positive control for gathering and examining touch data. The objectives of the work described here were to begin bridging this gap by establishing a method to create control fingerprints that were standardised, created in a lab, and contained a known amount of DNA; quantifying loss at critical failure points during collection and analysis, providing an empirical basis for the improvement of touch sample methodology; and changing one of the variables (surface) to show how the mock fingerprint method was used. Previous touch DNA experiments have attempted to standardise the deposition of the biological components, but none have successfully created a clear positive control. In previous research, participants deposited touch samples by contacting sterile tubes, glass plates, or the hand of another volunteer for a predetermined amount of time, such as 3, 10, or 60 s. Other experimental techniques required volunteers to wear a piece of clothing or rub their hands over a substrate for a predetermined amount of time and instances. The DNA deposited is not constant though because there is so much inter- and intra-person variability and even a single

person can be either an excellent or a lousy shedder depending on his or her personal circumstances at the time of sampling. There have also been experiments with methods that use non-epidermal cell types as a DNA source. Researchers claimed it was useless to standardise sampling with several collections from the same donor since people don't carry a constant amount of cellular material in their fingerprints across time. Other methods have used measured, naked DNA or known amounts of other bodily fluids like blood or saliva. Such methods have been used by a number of labs, but one drawback is that these controls have quite different biology from touch samples. The relevance and extension of results employing these controls from study to crime lab casework may be constrained by variations in cell wall, size, and type of cells from those typically deposited in real touch DNA cases. A different approach includes creating the biological material through cell culture for the standardisation of touch DNA controls. Human dermal fibroblasts obtained from skin were grown by Feine et al. Through the use of successive dilutions of cell suspensions, DNA was indirectly measured. There have also been experiments with methods that use non-epidermal cell types as a DNA source. Researchers claimed it was useless to standardise sampling with several collections from the same donor since people don't carry a constant amount of cellular material in their fingerprints across time. Other methods have used measured, naked DNA or known amounts of other bodily fluids like blood or saliva. Such methods have been used by a number of labs, but one drawback is that these controls have quite different biology from touch samples. The relevance and extension of results employing these controls from study to crime lab casework may be constrained by variations in cell wall, size, and type of cells from those typically deposited in real touch DNA cases. A different approach includes creating the biological material through cell culture for the standardisation of touch DNA controls. Human dermal fibroblasts obtained from skin were grown by Feine et al. Through the use of successive dilutions of cell suspensions, DNA was indirectly measured. The required volumes were deposited on glass slides for collection by tape-lifting or swabbing after the quantification results were utilised to determine how much DNA was present in the cell suspension. However, using actively dividing connective tissue cells as a comparison for a touch sample may not be the best option given that their DNA content has not been properly examined. Because the sample is a complex mixture of elements that can change over time, it presents a challenge to provide a positive control for the collection and analysis of touch DNA. By defining the fundamental elements of an eccrine touch sample to include human diploid cells in an inorganic solution, we were able to reduce this complexity in the work reported here. This simplification created an initial set of circumstances that allowed us to assess the impact of a basic eccrine background on DNA collection and loss. In order to gain a better knowledge of the ideal data collecting and processing methods, we can independently assess the effects of specific variables in future studies, such as the addition of fatty acids or various DNA sources, such as microbial or cell-free. The laboratory-produced, or fictitious, eccrine fingerprint used as the touch sample positive control mentioned in this article. The basic planning process is as follows: Create a suspension of human diploid cells; count the number of cells per microliter; and then use the number of cells per litre to calculate how much DNA, such as 2.85 ng, should be present in the cell suspension; combine the target suspension volume with an inorganic fingerprint solution; place the dummy fingerprint on a surface, collect, and analyse. If the count was 110 cells/l, then 4.3 l of suspension had 475 cells, or 2.85 ng DNA. The laboratory-produced, or fictitious, eccrine fingerprint used as the touch sample positive control mentioned in this article. The basic planning process is as follows: Create a suspension of human diploid cells; count the number of cells per microliter; and then use the number of cells per litre to calculate how much DNA, such as 2.85 ng, should be present in the cell suspension; combine the target suspension volume with an inorganic fingerprint solution; place the dummy fingerprint on a surface, collect, and analyse. If the count was 110 cells/l, then 4.3 l of suspension had 475 cells, or 2.85 ng DNA. Each method of counting was taken into account separately. Separate averages were calculated for the nine (LUNATM) and the eighteen cell counts. The data displayed here was created using the LUNATM cell counts. The volume of suspension necessary to deliver a particular amount of DNA was calculated using the mean number of cells per microliter of suspension. In 1X PBS, the volume was increased to 18 l, and 2 l of 10X Fingerprint Solution was added. A surface measuring roughly 3.63 cm2 was pipetted with the entire 20 l volume, which was then left to air dry at ambient temperature. A sterile cotton-tipped swab was used to completely swab the region after being moistened with 20 l 2% SDS. 300 mock fingerprints were created and analysed for each of the four surfaces, for a total of 1200 pretend fingerprints. A common phenol-chloroform technique was used to extract DNA from the samples. A cotton tip was taken from a swab and incubated in 400 l of DNA extraction buffer for an overnight period at 56°C. The tube was centrifuged after the swab was transferred to a Spin-X filter (Corning, Tewksbury, MA). Following the manufacturer's instructions, the phases were separated in a Phase Lock Gel Tube (2 ml, heavy, Eppendorf, Boulder, CO) using 400 microliters of 25:24:1 phenol/chloroform/isoamyl alcohol (Fisher, Scientific, Norcross, GA). DNA was centrifuged after being precipitated for at least 1 hour in 1 ml (2.5 vol) of 100% ethanol at 20°C. The pellet was twice rinsed in 1 ml (2.5 vol) 70% ethanol before being dried in an incubator at 56°C. After being incubated in a 56°C water bath for an entire night, the DNA was re-solubilized in 30 l of sterile water. Attempt Blue. In either 1X PBS (Fisher Scientific) or 1X AccumaxTM (Innovative Cell Technologies, Inc., San Diego CA), buccal epithelial cells were suspended. A volume of 0.4% dye and ten microliters of the cell solution were combined. The entire volume was pipetted onto a clean glass slide and allowed to dry for 1 hour under a biosafety hood after being incubated for 3 minutes at room temperature. The slides were heated to 105°C for 1.5 minutes to fix them. The slides were mounted with PermountTM after cooling, and they were left to dry overnight before being examined under a bright field microscope. Authentic fingerprints were left on spotless glass slides. Volunteers continued with their regular daily activities up until the donation, although they skipped washing their hands for at least an hour. The thumb, index, and middle fingers each left a print. The glass slides were touched by the donors' fingertips, who moved them back and forth for 10 seconds. After applying 10 l of 0.4% trypan blue to the slide, it was instantly visible under a bright field microscope at 40X or 100X magnification. H&E (Hematoxylin and Eosin). Both fake and real fingerprints were heat-fixed for 1.5 minutes at 105°C after drying for an hour in a biosafety hood. The slides were placed in slide holders and allowed to cool before being stained. Gill 2 Hematoxylin (3 minutes in the staining chamber); 1 minute of running tap water; a quick rinse with 1% acid alcohol (3% HCl, 95% ethanol) (Fisher Scientific); 1 minute of running tap water; 45 seconds of Scott's Tap Water Substitute; and 1 minute of running tap water are the steps in the staining process.; (g) Eosin Y, an alcoholic stain, was used for 10 seconds in the staining chamber; (h) 95% ethanol was used for 1 minute; (i) 100% ethanol was used for 1 minute; and (j) Xylene was used for 1 minute. Before being seen under a bright field microscope, the slides were mounted in PermountTM and allowed to dry at room temperature for an entire night.