Low-template deoxyribonucleic acid (DNA) samples are commonly found within forensic biological evidence. Low amounts of DNA become increasingly difficult to analyze as the allelic peaks become less distinguishable from instrumental noise. Forensic laboratories currently try to increase allele signal intensity through additional polymerase chain reaction (PCR) cycles or enhancing capillary electrophoresis injection times or potentials. Purification of the post-PCR product may also be conducted as PCR reagents can compete with DNA fragments during electrokinetic injection. Though these strategies have proven useful, resulting in a higher signal to noise ratio, low-template samples continue to exhibit allele drop-out due to the stochastic variation induced by the forensic DNA laboratory process. Further complicating analysis is the fact that low-template DNA samples are often exhausted as the full amount is needed for analysis. Thus, PCR can be considered a destructive technique. Since allele drop-out is hypothesized to be the result of 1) insufficient levels of amplicons and 2) sampling effects, it is desirable to obtain the original DNA template after amplification for future analysis. This would minimize the impact of 1) above. Thus, a novel method which isolates genomic DNA after PCR amplification has been developed. Amplification products were produced using biotinylated primers and cleaned from the solution with streptavidin-coated magnetic beads. Filtration was then used to remove remaining PCR reagents and primers. The result is a recovered sample containing the original genomic DNA. Re-amplification was then performed showing the method is successful. Although the method is capable of re-amplifying isolated DNA after PCR, there are points within the procedure that need to be optimized. For example, significant amounts of DNA are lost during the cleaning process and there is a high retention of the original amplified product. This study describes the optimization steps taken to reduce DNA loss, specifically through the filtration step. When method optimization is complete, low-template DNA samples could be analyzed recursively without being destroyed during PCR.

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.

Reliable detection of low template DNA (LTDNA) is dependent on reproducible allelic peaks and use of appropriately set thresholds. Analyzing LTDNA at different thresholds than high template DNA samples is discouraged by some scientists [1]. Thus, enhancing LTDNA samples is preferred if thresholds set for high template DNA are maintained for LTDNA samples. Post-amplification purification with the use of silica columns increases peak heights of single source LTDNA, while maintaining heterozygote balances of the sample before purification. LTDNA samples are difficult to interpret due to stochastic effects: allelic dropout and heterozygote imbalance. Interpretation of LTDNA mixtures is even more complex due to allelic sharing between contributors and allelic masking by artifacts. To determine if post-amplification purification of DNA from two-person LTDNA mixtures can improve profile interpretation, variable concentrations and mixture ratios of saliva extracts were applied to Macherey-Nagel NucleoSpin® and Qiagen MinElute® columns. The peak heights and heterozygote balance of two-person LTDNA mixtures were compared before and after purification with each silica column. Contributor proportion and heterozygote balance were not significantly affected by purification, and the peak heights of samples improved with use of either silica column. However, peak heights were higher in samples purified with Qiagen MinElute® columns. Given the higher peak heights, fewer dropouts occurred in samples purified with Qiagen MinElute® columns, but the occurrence of locus dropout reduced after purification with either column. The performance of each silica column was characterized by comparing fold increase and their individual effect on the primer front. The fold increase of samples purified with Qiagen MinElute® columns was higher than samples purified with Macherey-Nagel NucleoSpin® columns, but replicate purifications of Macherey-Nagel NucleoSpin® columns were more precise than replicate purifications of Qiagen MinElute® columns. Samples purified with Macherey-Nagel NucleoSpin® columns removed more primers than samples purified with Qiagen MinElute® columns. Post-amplification purification with silica columns is a useful investigative tool for LTDNA, especially for LTDNA mixtures. Both silica columns increased peak height, maintained contributor proportion, maintained heterozygote balance, and reduced primer front, but the degree of fold increase and reduction in primer front must be considered to facilitate the decision of which silica column to use for post-amplification purification of LTDNA. Due to its precision, Macherey-Nagel NucleoSpin® columns are ideal for LTDNA mixture samples while Qiagen MinElute® columns are ideal for single source LTDNA due to its ability to elevate peak heights.

In traditional forensic DNA casework, the inclusion or exclusion of individuals who may have contributed to an item of evidence may be dependent upon the assumption on the number of individuals from which the evidence arose. Typically, the determination of the minimum number of contributors (NOC) to a mixture is achieved by counting the number of alleles observed above a given analytical threshold (AT); this technique is known as maximum allele count (MAC). However, advances in polymerase chain reaction (PCR) chemistries and improvements in analytical sensitivities have led to an increase in the detection of complex, low template DNA (LtDNA) mixtures for which MAC is an inadequate means of determining the actual NOC. Despite the addition of highly polymorphic loci to multiplexed PCR kits and the advent of interpretation softwares which deconvolve DNA mixtures, a gap remains in the DNA analysis pipeline, where an effective method of determining the NOC needs to be established. The emergence of NOCIt -- a computational tool which provides the probability distribution on the NOC, may serve as a promising alternative to traditional, threshold- based methods. Utilizing user-provided calibration data consisting of single source samples of known genotype, NOCIt calculates the a posteriori probability (APP) that an evidentiary sample arose from 0 to 5 contributors. The software models baseline noise, reverse and forward stutter proportions, stutter and allele dropout rates, and allele heights. This information is then utilized to determine whether the evidentiary profile originated from one or many contributors. In short, NOCIt provides information not only on the likely NOC, but whether more than one value may be deemed probable. In the latter case, it may be necessary to modify downstream interpretation steps such that multiple values for the NOC are considered or the conclusion that most favors the defense is adopted. Phase I of this study focused on establishing the minimum number of single source samples needed to calibrate NOCIt. Once determined, the performance of NOCIt was evaluated and compared to that of two other methods: the maximum likelihood estimator (MLE) -- accessed via the forensim R package, and MAC. Fifty (50) single source samples proved to be sufficient to calibrate NOCIt, and results indicate NOCIt was the most accurate method of the three. Phase II of this study explored the effects of template mass and sample complexity on the accuracy of NOCIt. Data showed that the accuracy decreased as the NOC increased: for 1- and 5-contributor samples, the accuracy was 100% and 20%, respectively. The minimum template mass from any one contributor required to consistently estimate the true NOC was 0.07 ng -- the equivalent of approximately 10 cells' worth of DNA. Phase III further explored NOCIt and was designed to assess its robustness. Because the efficacy of determining the NOC may be affected by the PCR kit utilized, the results obtained from NOCIt analysis of 1-, 2-, 3-, 4-, and 5-contributor mixtures amplified with AmpFlstr® Identifiler® Plus and PowerPlex® 16 HS were compared. A positive correlation was observed for all NOCIt outputs between kits. Additionally, NOCIt was found to result in increased accuracies when analyzed with 1-, 3-, and 4-contributor samples amplified with Identifiler® Plus and with 5-contributor samples amplified with PowerPlex® 16 HS. The accuracy rates obtained for 2-contributor samples were equivalent between kits; therefore, the effect of amplification kit type on the ability to determine the NOC was not substantive. Cumulatively, the data indicate that NOCIt is an improvement to traditional methods of determining the NOC and results in high accuracy rates with samples containing sufficient quantities of DNA. Further, the results of investigations into the effect of template mass on the ability to determine the NOC may serve as a caution that forensic DNA samples containing low-target quantities may need to be interpreted using multiple or different assumptions on the number of contributors, as the assumption on the number of contributors is known to affect the conclusion in certain casework scenarios. As a significant degree of inaccuracy was observed for all methods of determining the NOC at severe low template amounts, the data presented also challenge the notion that any DNA sample can be utilized for comparison purposes. This suggests that the ability to detect extremely complex, LtDNA mixtures may not be commensurate with the ability to accurately interpret such mixtures, despite critical advances in software-based analysis. In addition to the availability of advanced comparison algorithms, limitations on the interpretability of complex, LtDNA mixtures may also be dependent on the amount of biological material present on an evidentiary substrate.