Accelerated aging of forensically relevant biological materials on swabs.

The preservation of DNA in biological samples is important for forensic testing, as samples can be tested years or even decades after collection. Generally, the DNA within biological evidence is stable over shorter time frames but can degrade over extended periods. In this work, we evaluated accelerated aging as a method to reduce the duration of studies examining the stability of DNA in forensic evidence-type samples. Evaluation of the DNA extracted from cells stored at 37 and 50°C for 194 or 79 days, respectively, showed similar quality metrics to cells stored at 25°C for 548 days.

Evaluating differential nuclear DNA yield rates and osteocyte numbers among human bone tissue types: A synchrotron radiation micro-CT approach.

Molecular human identification has conventionally focused on DNA sampling from dense, weight-bearing cortical bone tissue, typically from femora or tibiae. A comparison of skeletal elements from three contemporary individuals demonstrated that elements with high quantities of cancellous bone yielded nuclear DNA at the highest rates, suggesting that preferentially sampling cortical bone may be suboptimal (Mundorff & Davoren, 2014). Despite these findings, the reason for the differential DNA yields between cortical and cancellous bone tissues remains unknown. The primary goal of this work is to ascertain whether differences in bone microstructure can be used to explain differential nuclear DNA yield among bone tissue types observed by Mundorff and Davoren (2014), with a focus on osteocytes and the three-dimensional (3D) quantification of their associated lacunae. Osteocytes and other bone cells are recognized to house DNA in bone tissue, thus examining the density of their lacunae may explain why nuclear DNA yield rates differ among bone tissue types. Lacunae were visualized and quantified using synchrotron radiation-based micro-Computed Tomographic imaging (SR micro-CT). Volumes of interest (VOIs) from cortical and cancellous bone tissues (n=129) were comparatively analyzed from the three skeletons sampled for Mundorff and Davoren's (2014) study. Analyses tested the primary hypothesis that the abundance and density of osteocytes (inferred from their lacunar spaces) vary between cortical and cancellous bone tissue types. Results demonstrated that osteocyte lacunar abundance and density vary between cortical and cancellous bone tissue types, with cortical bone VOIs containing a higher lacunar abundance and density. We found that the osteocyte lacunar density values are independent of nuclear DNA yield, suggesting an alternative explanation for the higher nuclear DNA yields from bones with greater quantities of cancellous bone tissue. The use of SR micro-CT allowed for a scale of analysis that revealed a high range of variation in lacunar abundance in both tissue types. Moreover, high-resolution SR micro-CT imaging revealed potential soft tissue remnants within marrow spaces not visible macroscopically. It is hypothesized that soft tissue remnants observed among the trabeculae of skeletal elements with high quantities of cancellous bone tissue are responsible for the high nuclear DNA yields. These findings have significant implications for bone-sample selection for nuclear DNA analysis in a forensic context when skeletal remains are recovered from the ground surface.

The effect of common imaging and hot water maceration on DNA recovery from skeletal remains.

Identifying human remains often begins with cleaning and imaging the material. Hot water maceration is used to remove adherent soft tissue from bone and radiographs are taken to better visualize osseous details. Heat and radiation are known to have harmful effects on DNA, but their ability to degrade DNA when used for cleaning and imaging has not been well studied. To better understand their individual and combined effects on the recoverability of DNA from bone, skeletal samples were subjected to (1) hot water maceration (62 °C for 45 min); (2) CT scanning (0.6mm slices, 120 kV, 10.4s); (3) X-ray (50 kVp, 150 mA, 0.03 s, 40 in); and (4) all 3 treatments combined. Forty-eight DNA samples were extracted, quantified and amplified with the AmpFLSTR(®) Identifiler(®) system. Nearly all of the processed samples had reduced RFU values relative to the unprocessed samples, indicating some amount of genetic loss. This loss did not always translate into loss of profile completeness, since only a few samples had a reduction in the number of loci detected after processing. DNA yields were not significantly reduced by any one of the processing methods, however the results indicate that the damaging effects are additive. It is possible that processing may reduce a bone's DNA reservoir and as more procedures are preformed, the pool of available genetic information might be diminished. Many intrinsic and extrinsic factors can affect the recoverability of DNA from bone. Collecting a DNA sample prior to processing avoids the negative effects from hot water maceration and radiological imaging.

Examination of DNA yield rates for different skeletal elements at increasing post mortem intervals.

Identification of contemporary human remains by DNA STR testing is mainly limited by the ability to isolate sufficient amounts of DNA from the skeletal samples. A key part of this work relies on selection of the skeletal element with the best chance of obtaining a DNA STR profile. DNA was extracted from 55 bone samples, from 3 recently skeletonized individuals, representing most element types in the human body. Comparison of DNA yields from samples within an individual showed that the small cancellous bones on average have much higher amounts of DNA per unit mass than dense cortical bones. Complete 16 locus STR profiles were obtained for all 3 individuals from 36 of the element types, 10 had full profiles for 2 of the 3 individuals, 3 had full profiles for 1 of the 3 and 5 did not have any full profiles. The sample types with the least STR loci were from the arms. Ten skeletal elements were tested from 12 additional skeletons ranging from 3-21 years post mortem interval (PMI). At increasing PMI the small cancellous bones continued to yield more DNA and STR loci than the cortical bones. These findings suggest that the current recommendation for selection of long cortical bone samples for DNA testing of skeletal remains should be re-evaluated.