DNA recovered from skeletal material is often used to establish positive identification of severely decomposed or fragmented remains. DNA is preferentially sought from long bones like the femur, usually from cortical bone of the midshaft diaphysis. Although this strategy is often successful, the femur has extensive differences in morphology, tissue composition, and points of articulation that may differ in DNA content. In the research presented, intra-femoral heterogeneity was assessed in a proximal/distal manner to determine variation in DNA quantity and quality. Mitochondrial and nuclear DNA yields were compared across nine regions of the diaphysis, and the distal and proximal epiphyses, using fresh bovine and porcine femora and two tissue digestion protocols (non-demineralization and demineralization). In addition, bovine femora were subjected to burial and surface exposure over a six month interval to assess how DNA heterogeneity was affected by environments where forensically relevant remains are often discovered. The epiphyses had significantly more DNA than the metaphyses, which had more than the diaphysis, DNA quality was consistent among all regions tested, and mitochondrial and nuclear DNA had similar regional variation. Bone demineralization resulted in more DNA recovered at the mid-diaphysis, while the non-demineralization protocol did the same for nuclear DNA at the epiphyses. Environmental exposure affected DNA quantity and quality, and burial influenced inter-regional DNA yields over time. These findings indicate that intra-bone DNA heterogeneity can be as important as inter-bone DNA heterogeneity, and should be considered when choosing a sampling location for DNA isolation.
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