Title

Exploring Soil Bacterial Communities for Forensic Applications: A Genomics Approach

Date of Completion

January 2010

Keywords

Biology, Molecular|Biology, Genetics|Biology, Bioinformatics

Degree

Ph.D.

Abstract

It is accepted that soil evidence can be used in forensic investigations, where bacteria in soil are used to generate DNA profiles. The research presented in this thesis investigates how soil can be best used for forensic applications. Although bacterial profiles can be generated using several molecular methods, terminal restriction fragment length polymorphism (T-RFLP) analysis has been used most frequently to produce forensically relevant profiles. The second chapter proposes an alternative to T-RFLP analysis: comprehensive restriction fragment length polymorphism analysis (C-RFLP). This alternate typing method utilizes high performance liquid chromatography (HPLC) to separate and visualize unlabeled DNA fragments. However, neither method readily allows forensic scientists to extrapolate which types of bacteria are present in the soil sample in question. Knowing the molecular identity of a peak in a profile (i.e. which bacterial group is responsible for the presence of observed peaks) provides an additional layer of potentially informative information. In chapter three, 454 high throughput sequencing was used to survey fourteen soil samples, cataloging the major and minor components to soil bacterial communities. From these extensive DNA libraries, five bacterial groups were selected as candidates for group-specific bacterial typing. The main goal of chapter four was to determine the forensic potential of using such targeted analysis. DNA from soils was amplified using group-specific primers, digested with a restriction enzyme, and resolved using HPLC. HPLC was used because of its potential shown in chapter two and also to demonstrate that fragments could be collected and identified by sequence. The data show that group-specific profiles can be generated and used for forensic comparison due to the sufficient genetic variability within groups tested. This suggests that targeted molecular analysis of bacteria has great potential as a forensic soil typing tool and should be explored further. Ultimately, research on group-specific typing will aid in the development of a multiplex kit to be used in crime labs nationwide. ^