In-field triaging of hair samples using fluorescent in-situ detection

Abstract

Shed hairs are the most common type of hair samples found at crime scenes, however, they lack high levels of nuclear DNA and are generally not processed for further nuclear DNA analysis. Multiple studies have reported the benefits of using Diamond™ Nucleic Acid Dye (DD), a nucleic acid binding dye, that can be used to visually identify the presence and amount of DNA in biological samples. Shed hairs pose challenges in the identification of DNA even when stained with DD due to the lack of nuclei present. This project aims to apply existing fluorescent techniques to develop a screening method to allow for easy identification of DNA presence and categorisation of shed hairs based on the fluorescent signal observed from DD staining. This research evaluated factors with potential effects on the fluorescence signal, such as time since staining and the Extended Depth of Field function on the Optico N300F Fluorescent Microscope. A grading scale was developed to help in categorising samples based on the type of hair and the strength of the fluorescent signal. The categories given to each sample were compared against the DNA profile obtained to determine the efficiency of the grading system. Furthermore, multiple studies were conducted to determine the best methods in processing shed hairs. These included evaluation of multiple extraction and amplification kits, assessment of direct amplification, and effects of AmpliTaq Gold™ 360 DNA Polymerase. Finally, the staining and collection of shed hairs was assessed for its application in field with the use of a mobile laboratory and a portable fluorescent microscope. The results of this project demonstrated the efficiency of the grading scale in determining the best shed hair samples for DNA analysis. Observations were made regarding the time since staining of shed hairs as the fluorescent signal rapidly declined after a few minutes. the PrepFiler® BTA extraction kit was best for extracting shed hair samples, whereas the VeriFiler™ Plus Kit was best for amplification. Furthermore, direct amplification was found to increase allelic information from DNA profiles of shed hairs. Although the in-field application was not as successful as the laboratory setting, a new collection method was developed for ease of use and further sampling in a laboratory.

Date of Thesis submission	2024
Original language	English
Awarding Institution	Western Sydney University
Supervisor	Alicia Haines (Supervisor) & Brenden Riley (Supervisor)