The Optimisation and Validation of Fluorescence in situ Hybridisation and Laser Microdissection for Forensic Casework Analysis

Abstract

Forensic evidence recovered from sexual assault crime often comprises mixtures of body fluids from the complainant and assailant. These samples can be deposited on surfaces or taken directly from an individual via a medical examination. The Forensic Biology Group at the Institute of Environmental Science and Research (ESR) often receives such evidence for DNA profiling in order to determine the profiles of the contributors of the biological mixture. Spermatozoa (sperm) are often the main component of interest if they can be successfully separated from epithelial cells (cells that line body cavities and surfaces) for DNA analysis. Where a biological sample does not contain sperm, for example in a mixture of blood, epithelial cells or when a donor's semen does not contain sperm, the separation of male and female cells prior to DNA analysis can be unsuccessful as these cells are morphologically identical in both males and females. It is a goal of forensic biologists to produce single source DNA profiles from biological mixtures as the analysis of mixed profiles is difficult and extremely time consuming. A technique which allows the separation of morphologically identical cells from males and females is required for forensic biology analysis. Fluorescent in situ hybridisation (FISH) is a technique that can be applied to epithelial cells in order differentiate based on the gender origin of the cells by applying different coloured fluorescent probes to the X and Y sex determining chromosomes (X/Y FISH). Coupling X/Y FISH with laser microdissection (LMD) can allow the direct visualisation, targeting, and collection of pure populations of male and female cells from biological mixtures. The aim of this research was to optimise and validate a robust protocol combining X/Y FISH and LMD for the gender identification, collection and DNA analysis of mixed cell populations for forensic casework at ESR. This research combined the use of the Vysis CEP® X SpectrumOrange/Y SpectrumGreen DNA Probe Kit and the Leica 6000 Laser Microdissector to optimise a robust protocol for the separation of male and female epithelial cells. A large data set is obtained and analysed to provide recommendations of the optimal cell number to be collected, and the limits of detection for these techniques with the different multiplex amplification kits available for forensic casework DNA analysis at ESR. The compatibility of the techniques with specialist multiplex amplification kits, amplification enhancing agents and with mock forensic samples is also assessed. The research conducted as part of this thesis will ultimately provide the experiments necessary for the casework implementation of a robust and reliable technique for the separation of male and female epithelial cells typically encountered in forensic evidence.