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.

When offenders commit crime there is the potential that they may leave behind trace amounts of their DNA, even when there has been no apparent body fluid spill. During the examination of crime scenes, scene investigators try to identify areas that may be sampled to locate these traces. Specialist techniques are then required within the laboratory to enable such small amounts to be analysed to obtain a profile. These techniques are referred to as Low Template DNA analysis (LTDNA), of which Low Copy Number DNA (LCN DNA) is one instance. In 2008, following the Omagh Bombing trial, and comments made by Judge Weir, the UK Forensic Regulator commissioned a review of the science of LTDNA analysis. The subsequent report made specific mention of the fact that there was no available information on the success rate of the use of such DNA techniques and that there seemed to be confusion over what constituted a success. The report went on to state that there was no information on where such trace amounts of DNA were likely to be found, or what factors could influence the likelihood of obtaining a trace DNA profile (Caddy, 2008). This research considered the outcomes of LCN DNA analysis from 3,552 samples to try to establish where trace amounts of DNA could be found, whether some areas sampled were more successful in generating profiles than others, and the likelihood of the profiles obtained being of use to a criminal investigation. Analysis of results identified areas that were more successful in generating profiles of use to an investigation and highlighted significant differences in results across a variety of items from which samples were taken. DNA samples taken from items associated with communication such as mobile phones were much more likely to produce a profile useful to a criminal investigation than those taken from fixed surfaces within premises. The results obtained showed that obtaining a DNA profile did not necessarily correlate with the profile being of use to a criminal investigation. This was due to the fact that a large number of these profiles were anticipated eliminations from legitimate sources. Items that produced high numbers of profiles but were anticipated eliminations, and therefore of no value to an investigation, came from items associated with skin samples and clothing. The research went further to identify key factors that affected the profiling rates. Factors that had a positive influence on the ability to obtain a profile included: any area that had been in close proximity to saliva (direct contact was not required); samples that had been recovered from the inside of premises or vehicles and therefore protected from the elements; those that were dry; items that were of a porous nature; and those that had a rough texture. No differences were found between the actual surface materials (plastic, glass, wood, metal), as all showed a propensity to generate profiles. Other factors that were considered but proved to have no effect on the profiling rates included seasonal differences and whether the area targeted for sampling was clearly defined. Items that had had high contact with a victim, were recovered from outside or had been wet, all proved to be less useful to an nvestigation. A further finding of the research was that swabs that had been recovered and stored frozen appeared to deteriorate in their ability to profile. This was particularly notable if they were submitted later than 5 months after recovery. Items stored in dry conditions did not deteriorate in this way. Overall the research can be used to provide investigators with the knowledge of what areas of crime scenes are most likely to yield trace DNA material, the key factors that can affect the likelihood of obtaining a profile, and those areas that are more likely to produce profiles useful to criminal investigations.

It is common practice for DNA STR profiles to be analyzed using an analytical threshold (AT), but as more low template DNA (LT-DNA) samples are tested it has become evident that these thresholds do not adequately separate signal from noise. In order to confidently examine LT-DNA samples, the behavior and characteristics of the background noise of STR profiles must be better understood. Thus, the background noise of single source LT-DNA STR profiles were examined to characterize the noise distribution and determine how it changes with DNA template mass and injection time. Current noise models typically assume the noise is independent of fragment size but, given the tendency of the baseline noise to increase with template amount, it is important to establish whether the baseline noise is randomly found throughout the capillary electrophoresis (CE) run or whether it is situated in specific regions of the electropherogram. While it has been shown that the baseline noise of negative samples does not behave similarly to the baseline noise of profiles generated using optimal levels of DNA, the ATs determined using negative samples have shown to be similar to those developed with near-zero, low template mass samples. The distinction between low-template samples, where the noise is consistent regardless of target mass, and standard samples could be made at approximately 0.063 ng for samples amplified using the Identifiler^TM Plus amplification kit (29 cycle protocol), and injected for 5 and 10 seconds. At amplification target masses greater than 0.063 ng, the average noise peak height increased and began to plateau between 0.5 and 1.0 ng for samples injected for 5 and 10 seconds. To examine the time dependent nature of the baseline noise, the baselines of over 400 profiles were combined onto one axis for each target mass and each injection time. Areas of reproducibly higher noise peak heights were identified as areas of potential non-specific amplified product. When the samples were injected for five seconds, the baseline noise did not appear to be time dependent. However, when the samples were injected for either 10 or 20 seconds, there were three areas that exhibited an increase in noise; these areas were identified at 118 bases in green, 231 bases in yellow, and 106 bases in red. If a probabilistic analysis or AT is to be employed for DNA interpretation, consideration must be given as to how the validation or calibration samples are prepared. Ideally the validation data should include all the variation seen within typical samples. To this end, a study was performed to examine possible sources of variation in the baseline noise within the electropherogram. Specifically, three samples were prepared at seven target masses using four different kit lots, four capillary lots, in four amplification batches or four injection batches. The distribution of the noise peak heights in the blue and green channels for samples with variable capillary lots, amplifications, and injections were similar, but the distribution of the noise heights for samples with variable kit lots was shifted. This shift in the distribution of the samples with variable kit lots was due to the average peak height of the individual kit lots varying by approximately two. The yellow and red channels showed a general agreement between the distributions of the samples run with variable kit lots, amplifications, and injections, but the samples run with various capillary lots had a distribution shifted to the left. When the distribution of the noise height for each capillary was examined, the average peak height variation was less than two RFU between capillary lots. Use of a probabilistic method requires an accurate description of the distribution of the baseline noise. Three distributions were tested: Gaussian, log-normal, and Poisson. The Poisson distribution did not approximate the noise distributions well. The log-normal distribution was a better approximation than the Gaussian resulting in a smaller sum of the residuals squared. It was also shown that the distributions impacted the probability that a peak was noise; though how significant of an impact this difference makes on the final probability of an entire STR profile was not determined and may be of interest for future studies.