In forensic casework it is often necessary to obtain genetic profiles from crime scene samples that contain increasingly smaller amounts of genetic material, called Low Template DNA (LTDNA). Two examples of LTDNA sources are touch DNA evidence and dust bunnies. Touch DNA refers to DNA that is left behind through casual contact of a donor with an object or another person. Touch DNA can be used to prove a suspect was present at a crime scene. Dust bunnies, or dust conglomerates, typically contain trapped shed skin cells of anyone in the vicinity along with fibers, dirt, hair, and other trace materials. Dust specimens are a potential source of forensic evidence that has been widely underutilized in the forensic community. This is unfortunate because a dust bunny could not only be used to associate a person or crime scene – through trace materials such as fibers – but also to positively identify – through a DNA profile. For example, if a dust specimen is found on a piece of evidence suspected of being moved from its original location, for instance as a body that is too heavy to carry and therefore collects dust while being dragged, then it could be used to link a suspect to a crime scene. Standard methods for obtaining and analyzing touch DNA have been established, but the techniques are not ideal. First, by nature, the 'blind-swabbing' technique, which involves cotton swabs or adhesive tape being applied to an area of interest, can artificially create mixtures of biological material that was originally spatially separated. Second, because the amount of DNA present is typically very low, standard analysis methods may not be sensitive enough to produce probative profiles. In the case of mixtures, the minor component's DNA may go undetected. Dust specimens contain degraded genetic material that has been accumulating for an unknown amount of time. Additionally, dust is usually a conglomeration of genetic material from multiple donors so a mixed profile, if any, is likely to be recovered if standard analysis methods are used. In order to overcome these obstacles presented by LTDNA, a micro-manipulation and combined cell lysis/direct PCR amplification technique has been developed that is sensitive enough to obtain full or probative STR profiles from single or clumped bio-particles collected from touch DNA and dust evidence. Sources of touch DNA evidence such as worn clothing items, touched objects, and skin/skin mixtures are easily sampled using an adhesive material on a microscope slide. Dust specimens can be dispersed onto an adhesive material as well. Targeted bio-particles are then "picked" with a water-soluble adhesive on a tungsten needle and deposited into a micro-volume STR amplification mix. Individual selection and analysis of isolated bio-particles reduces the chance of mixed profile recovery. To aid in the release of genetic material present in the bio-particles, a lysis mix containing a thermostable proteinase is then added to the sample. Samples are then analyzed using standard capillary electrophoresis (CE) methods. In addition to identifying the donor source of these LTDNA sources, it would be beneficial to a criminal investigation to identify the tissue source of the biological material as well. While it is widely speculated that the material originates from shed skin cells, there is little confirmatory evidence proving this assertion. Knowledge of the nature of the evidence could be vital to prevent its misinterpretation during the investigation and prosecution of a crime. Here tissue specific mRNA biomarkers have been evaluated for their use in tissue source determination using a highly sensitive High Resolution Melt (HRM) temperature assay that detects the selectively amplified targets based on their melt temperatures. Using the enhanced genetic analysis technique described above, DNA profile recovery has been markedly enhanced in sources of Touch DNA evidence and dust specimens compared to standard methods. Additionally, the molecular-based characterization method could potentially provide a better understanding of the meaningfulness of the recovered DNA profiles. This optimized strategy provides a method for recovering highly probative data from biological material in low template samples in an efficient and cost effect manner.
| Identifer | oai:union.ndltd.org:ucf.edu/oai:stars.library.ucf.edu:etd-2483 |
| Date | 01 January 2015 |
| Creators | Farash, Katherine |
| Publisher | STARS |
| Source Sets | University of Central Florida |
| Language | English |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | Electronic Theses and Dissertations |
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