Author: James Malcolm Waters
Waters, James Malcolm, 2004 INVESTIGATION OF DNA PROFILING METHODS FOR FORENSIC EXAMINATION OF SOIL EVIDENCE., Flinders University, School of Biological Sciences
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In this thesis, an investigation of the potential of two DNA based profiling techniques for the analysis of forensic soil evidence is presented. These profiling techniques were: - The previously described technique, Terminal Restriction Fragment Length Polymorphism Analysis (TRFLP) of 16S DNA (Chpt 3.1) and - A semi-novel profiling technique, Arbitrarily Amplified DNA (AAD) profiling (Chpts 3.2 and 3.3), analysed by both: o conventional length polymorphism of DNA fingerprints (AADLP) (Chpt 3.2) and o a completely novel method, DNA sequence similarity (AADSS) (Chpts 3.2 and 3.3), which was investigated using -- Southern Hybridisation (Chpt 3.2) and -- Microarray Technology (Chpt 3.3). These methods were successful at distinguishing samples of soil to varying degrees. TRFLP analysis was capable of generating low but significant differences in similarity statistics between replicate and distinct soil profiles, while both AAD analyses (AADLP and AADSS) generated large and significant differences in similarity statistics between replicate and distinct soil profiles. The potential for significant differences between similarity statistics to be generated enables classification of soil samples as either having common origins (matching, if there is no significant difference) or not (excluded from matching, if there is a significant difference). The affect of several technical, environmental and practical variables on the biological profiles generated using these techniques was investigated further. These variables included sampling and processing of soils (assessed with TRFLP, AADLP and AADSS), time in situ (TRFLP and AADSS), time ex situ under a number of storage conditions, as well as spatial variations of microbial communities over small distances (AADSS only). The most capable method for distinguishing soils of different origin was AADSS, closely followed by AADLP, with TRFLP obtaining only marginal success relative to the other two methods. The preferred format of the AADSS technique is the microarray technology as it is capable of generating a good deal more data from soil DNA profiles than Southern hybridisation. However, as both are capable of distinguishing soils, the low cost Southern hybridisation technique may provide a suitable entry point technology for many forensic laboratories. The molecular mechanism of the arbitrary amplification profiling system (AAD) was investigated (Chpt 3.4), allowing insight into the way these profiles are generated and potential ways to control the process in order to optimise profiles for various purposes. Many potential improvements and developments are suggested which may further enhance the utility of the techniques presented in this thesis. The findings presented in this thesis demonstrate the potential for biological profiling of soil communities as a relatively simple, high resolution, objective tool that permits stringent statistical analysis, is not reliant on expert interpretation and is complementary to existing strategies for the forensic examination of soil evidence.
Keywords: Soil,DNA profiling,TRFLP
Subject: Biological sciences thesis
Thesis type: Doctor of Philosophy
School: School of Biological Sciences
Supervisor: Prof. Leigh A. Burgoyne