The Development of Synthetic Gene Biomarkers for the Field-Based Forensic Detection of Body Fluids and Sex Determination

Abstract

Body fluid and sex determination at the crime scene are important forensic questions where information can be obtained using various approaches. The standard techniques involved are well established and often utilise cheap, rapid tests to presumptively detect the presence of characteristic proteins and chemicals. However, the relatively poor accuracies of these tests compared to laboratory-based techniques that analyse nucleic acids limits their effectiveness and can lead to inefficient sample triage. This presents a need for novel field-based biomarker detection techniques that are both sensitive and specific. Toehold switches are de novo designed RNA/DNA sequences that contain the genetic motifs necessary for coupled transcription-translation of a reporter gene. Gene expression is repressed in the absence of a specific complementary target “trigger” sequence due to switch hairpin formation. Hybridisation of the trigger to the switch initiates hairpin unwinding and enables downstream gene expression. This specific and sensitive approach has led to toehold switches becoming an emerging platform for bio-detection, primarily in the field of viral RNA detection. This thesis identifies a lack of such applications to forensic science alongside a need for novel field-based DNA detection tools amongst forensic end-users with a market research study. To address these issues, a set of toehold switches were designed in-house for the detection of mRNA sequences specific to blood, saliva, semen, and the sex marker amelogenin. A contemporary qPCR assay was internally validated to act as a performance benchmark. To facilitate in vitro expression of toehold switches without expensive commercial cell-free protein synthesis systems, an Escherichia coli cell lysate was developed and optimised in-house. Gene expression from control plasmids was comparable to a commercial equivalent at 37°C and exceeded it at 29°C, with a shelf-life of approximately 6 – 8 months at -80°C. Toehold switches circuit function was unsuccessful with either system, which suggested an issue with toehold switch design. A screening framework utilising a combined melting curve and in silico thermodynamic analysis approach was devised to highlight high-performance toehold switches. This framework was able to predict the performances of six novel toehold switch designs and recommended designs to be discarded or studied further, but further characterisation is required to assess prediction accuracy. Results throughout are discussed in relation to the needs of forensic scientists and the capabilities of toehold switches as bio-detection tools compared to existing techniques.