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.

The criminal use of explosives has increased in recent years. Political instability and the wide spread access to the internet, filled with "homemade recipes," are two conjectures for the increase. C-4 is a plastic bonded explosive (PBX) comprised of 91% of the high explosive RDX, 1.6% processing oils, 5.3% plasticizer, and 2.1% polyisobutylene (PIB). C-4 is most commonly used for military purposes, but also has found use in commercial industry as well. Current methods for the forensic analysis of C-4 are limited to identification of the explosive; however, recent publications have suggested the plausibility of discrimination between C-4 samples based upon the processing oils and stable isotope ratios. This research focuses on the discrimination of C-4 samples based on ratios of RDX to HMX, a common impurity resulting from RDX synthesis. The relative amounts of HMX are a function of the RDX synthetic route and conditions. RDX was extracted from different C-4 samples and was analyzed by ESI-MS-SIM as the chloride adduct, EI-GC-MS-SIM, and NICI-GC-MS. Ratios (RDX/HMX) were calculated for each method. An analysis of variance (ANOVA) followed by a Tukey HSD allowed for an overall discriminating power to be assessed for each analytical method. The C-4 processing oils were also extracted, and analyzed by direct exposure probe mass spectrometry (DEP-MS) with electron ionization, a technique that requires less than two minutes for analysis. The overall discriminating power of the processing oils was calculated by conducting a series of t tests. Lastly, a set of heterogeneous commercial blasting agents were analyzed by laser induced breakdown spectroscopy (LIBS). The data was analyzed by principal components analysis (PCA), and the possibility of creating a searchable library was explored.

Fire incidents are a major contributor to the number of deaths and property losses within the United States each year. Fire investigations determine the cause of the fire resulting in an assignment of responsibility. Current methods of fire debris analysis are reviewed including the preservation, extraction, detection and characterization of ignitable liquids from fire debris. Leak rates were calculated for the three most common types of fire debris evidence containers. The consequences of leaking containers on the recovery and characterization of ignitable liquids were demonstrated. The interactions of hydrocarbons with activated carbon during the extraction of ignitable liquids from the fire debris were studied. An estimation of available adsorption sites on the activated carbon surface area was calculated based on the number of moles of each hydrocarbon onto the activated carbon. Upon saturation of the surface area, hydrocarbons with weaker interactions with the activated carbon were displaced by more strongly interacting hydrocarbons thus resulting in distortion of the chromatographic profiles used in the interpretation of the GC/MS data. The incorporation of an additional sub-sampling step in the separation of ignitable liquids by passive headspace sampling reduces the concentration of ignitable liquid accessible for adsorption on the activated carbon thus avoiding saturation of the activated carbon. A statistical method of covariance mapping with a coincident measurement to compare GC/MS data sets of two ignitable liquids was able to distinguish ignitable liquids of different classes, sub-classes and states of evaporation. In addition, the method was able to distinguish 10 gasoline samples as having originated from different sources with a known statistical certainty. In a blind test, an unknown gasoline sample was correctly identified from the set of 10 gasoline samples without making a Type II error.

The fire debris analyst is often faced with the complex problem of identifying ignitable liquid residues in the presence of products produced from pyrolysis and incomplete combustion of common building and furnishing materials. The purpose of this research is to investigate a modified destructive distillation methodology provided by the Florida Bureau of Forensic Fire and Explosive Analysis to produce interfering product chromatographic patterns similar to those observed in fire debris case work. The volatile products generated during heating of substrate materials are extracted from the fire debris by passive headspace adsorption and subsequently analyzed by GC-MS. Low density polyethylene (LDPE) is utilized to optimize the modified destructive distillation method to produce the interfering products commonly seen in fire debris. The substrates examined in this research include flooring and construction materials along with a variety of materials commonly analyzed by fire debris analysts. These substrates are also burned in the presence of a variety of ignitable liquids. Comparisons of ignitable liquids, pyrolysis products, and products from pyrolysis in the presence of an ignitable liquid are performed by comparing the summed ion spectra from the GC-MS data. Pearson correlation was used to determine if substrates could be discriminated from one another. A pyrolysis products database and GC-MS database software based on comparison of summed ion spectra are shown to be useful tools for the evaluation of fire debris.

Discovering, screening, and associating changes in DNA sequence are important to a broad range of disciplines and play a central role in Forensic Science. The typical types of changes include sequence variations [single nucleotide polymorphisms (SNP)] and length variations [short tandem repeats (STR)]. The steps for forensic DNA sample processing are similar for both types of changes but diverge at the point of detection. A number of approaches are being explored for SNP genotyping while STR analysis primarily consists of size-based analysis by capillary electrophoresis. Limitations exist for all current detection methods that pose significant impacts to forensic analysis. Bi-allelic SNPs result in three possible genotypes with a minimal amount of information generated per marker. Limitations for SNP analysis are due to the inability to amplify a suitable number of SNP markers from low DNA content samples to provide an appropriate level of discrimination. Multi-allelic STR markers are currently the marker of choice for forensic typing but a variety of experimental artifacts are possible that consist of either biology or technology related causes. Molecular genotyping methods developed across other disciplines have potential to alleviate some of these shortcomings but no current approach is capable of genotyping both SNP and STR loci with a single chemistry. The need for a more effective, efficient, and generalized approach led to development of a unique method called Dye Probe Fluorescence Resonance Energy Transfer (dpFRET) and determination of its suitability for forensic analysis. The development phase of the research consisted of synthetic testing to establish proof of concept for the chemistry followed by polymerase chain reaction (PCR) based assays to demonstrate real world applications. Following successful development, the boundaries and limitations for the technology were established (sensitivity, allelic dropout, mixed samples) and efforts were made to improve the approach. In the process, parallel testing for other fields including molecular pathology and conservation biology were incorporated to explore potential widespread application of this new approach. The overall goal of this project was to develop and explore the limitations for a unique approach to genotyping both SNPs and STRs. A majority of the work involved development of the method itself with the ultimate objective of application for forensic science. The focus of this project was to address and alleviate some of the shortcomings of current approaches that result in potential limitations for forensic analysis. It is expected that future applications of this technology might impact a wide range of disciplines to aid in discovery, screening and association of changes in DNA sequence.

Trace textile fiber evidence is found at numerous crime scenes and plays an important role in linking a suspect to the respective scene. Several methods currently exist for the analysis of trace fiber evidence. Microscopy provides information regarding the fibers material, color and weave. For more detailed chemical analysis chromatographic methods are employed and for discrimination between dyes, liquid chromatography coupled with mass spectrometry (LC-MS) is currently the method providing the most discrimination. These methods have primarily focused on the dyes used to color the fibers and have not investigated other components that can potentially discriminate among fibers. This dissertation deals with investigations into the fluorescence of the fiber dyes, (contaminants?) and the fibers themselves, as well as methodology for discriminating between fibers using fluorescence. Initial systematic analysis was conducted on dye standards and extracts taken from fibers colored with the respective dyes of interest. Absorbance, excitation and fluorescence spectra were compared between standards and extracts to determine the optimal area of the fiber to investigate: dyes, fluorescent impurities or the whole fiber. High performance liquid chromatography investigations were performed to give detailed information on the number of dye and fluorescent components present in extracts. Our investigations then focused on the best room-temperature fluorescence (RTF) data format for analysis and discrimination of fiber samples. An excitation emission matrix (EEM) was found to give the greatest amount of spectral information and provide the highest level of discrimination. Successful discrimination between non similar and similar fibers was achieved with the aid of Chemometric analysis. The level of discrimination obtained via RTF-EEM spectroscopy was sufficient to differentiate among fibers obtained from two separate cloths of the same material and colored with the same dye reagent. Final studies deal with examining exposure of the fiber to various environmental contaminants. Clothing fibers are typically exposed to myriad numbers of contaminants, from food stains to cigarette smoke. The challenge then becomes detecting fluorescence signals from trace amounts of these environmental contaminants. We demonstrate the detection and classification of polycyclic aromatic hyrdrocarbons (PAH) present on fibers after exposure to cigarette smoke. This dissertation also investigates the change in fluorescence emission after laundering fibers numerous times. The main drawback of chemical analysis of fibers is the destructive nature of the methods. To extract a dye or contaminant from a fiber essentially destroys the evidence. This leaves the investigator without their original sample in the courtroom. This also provides a finite amount of sample for testing and analysis. This is true of chromatographic methods and for the method detailed in this dissertation which makes use of extracts taken from fiber samples. Lastly, we propose an instrumental setup coupling a microscope to a spectrofluorimeter for the purpose of taking EEM directly from a fiber sample. This setup makes use of the superior optics of the microscope for focusing excitation light onto the fiber sample. Initial studies have been performed on extracts from a single textile fiber and EEM collected from said fiber.

Triacetone Triperoxide (TATP) is a primary high explosive that can be synthesized using commercially available starting materials and has grown in use among terrorists over the past several years. Additives present in the precursors were investigated to see if they carry through the TATP synthesis and can be detected in the final product potentially aiding in the identification of the source. Additives identified in the acetones were also identified in pre-blast and in some post-blast samples. However, these additives are present in trace quantities relative to the TATP, which coupled with the volatility and short lifetimes of some of the additives in TATP samples limit their detection in pre-blast and post-blast material. TATP prepared with different acids in the laboratory could generally be discriminated by observing the change in composition of the headspace of the samples upon heating and by IMS analysis of the crystals. The analysis of TATP synthesized on a larger scale was compared to the laboratory results of pre-blast material and post-blast debris. As in the laboratory samples, organic additives were also detected in the large-scale pre-blast samples and the identification of the additives in post-blast debris was consistent with the results obtained in the laboratory detonations.

Biological evidence may contain any number of cells in any proportion. Extreme low-template DNA samples are often very difficult to interpret due to complex signal or peaks which may be indistinguishable from baseline noise. Current solutions focus on increasing the amount of amplicon detected by adjusting PCR cycle number or capillary electrophoresis injection parameters. Consensus profiling is an additional option. However, the aforementioned solutions are often not helpful for extreme low-template samples due to the high occurrence of allelic drop-out. Additionally, PCR is a destructive technique that causes one amplification to completely exhaust this type of sample, making further typing and analysis impossible. Therefore, a technique that allows for the re-generation of a DNA template in order to amplify it multiple times would be an extremely useful tool. This study outlines the development of a method that allows for the recursive amplification of a DNA sample. Amplification was performed using biotinylated primers for an STR locus and the resulting product was cleaned using streptavidin-coated magnetic beads to sequester the amplicons. Subsequent centrifugal filtration was used to remove the remaining PCR components, thus isolating the original genomic DNA. Re-amplification was then successfully performed at a different STR locus. Though successful, multiple run-throughs of the method indicated retention of signal from the original amplification as well as significant genomic DNA loss during the process. This study outlines experiments seeking to characterize the cause(s) of these imperfections in order to effectively direct method optimization. A computer generated dynamic model was also created and used to simulate the recursive amplification process to assist in development. When optimized, it is expected that recursive amplification can significantly reduce the difficulties associated with low-template DNA analysis and eradicate the concept of an ‘exhaustive’ DNA sample.

The morphoscopic traits used to assign the term Hispanic to a skeleton constitute mainly a mixture of characteristics that have been assigned by anthropologists to Asian and Caucasian ancestry groups. Therefore, the morphological characteristics for the population termed Hispanic are not well defined. The aim of this study is to conduct a biodistance analysis of skeletons of Hispanic ancestry from Puerto Rico. The purpose of this is to assess how similar their morphoscopic characteristics are to other populations termed Hispanic as well as populations termed Asian. The analysis will be conducted by taking craniometric measurements. Pre-Colombian as well as modern skeletons from Puerto Rico will be examined and compared to other Hispanic as well as Asian populations that form part of the Forensic Anthropology Data Bank (FDB). Results from this study will help characterize Hispanic skeletal variation. In addition, this study will discuss the complexities of Hispanic classification in forensic anthropological contexts.

Molecular genetic protocols have been developed to provide evidence in infractions of wildlife statutes in Canada. We have utilized DNA marker systems to address specific questions in wildlife investigations based on their different levels of genetic variability. Multilocus DNA fingerprinting has been applied to poaching infractions to determine if tissue samples associated with a suspected poacher originated from the remains of an animal at a known illegal kill site. The hypervariability of the variable number of tandem repeat (VNTR) loci detected by multilocus DNA fingerprinting allows the individual identification of samples. Highly repetitive satellite DNA markers have been applied to determining the species of origin of unknown tissue samples based on their species-specificity. Satellite DNA profiling have provided evidence in illegal commercialization investigations involving species such as moose (Alcesalces) and white-tailed deer (Odocoileus virginianus), including the illegal addition of game meat in processed meat products. A sex-specific DNA locus, the sex-determining region on the Y-chromosome (Sry), has been utilized to determine the sex of cervid samples that have had gender-specific physical characteristics, antlers and genitalia removed in violation of the validation tag system. Finally, a polymerase chain reaction (PCR) based protocol has been established for the species identification of samples that produce minute amounts of DNA or degraded DNA. Cytochrome b sequences demonstrate low intra-specific levels of sequence divergence and higher inter-specific levels of sequence divergence. Cytochrome b sequence analysis has been applied to fish, game and domestic species commonly involved in wildlife investigations and to the identification o fa number of species, mostly seal species, involved in the trade of animal parts. / Thesis / Master of Science (MSc)