Intra-Bone Variation of Recoverable Nuclear and Mitochondrial DNA from Human Skeletal Material

As submitted by the proposer: Positive identification of human skeletal remains is not always possible using standard anthropological methods, and thus frequently relies on obtaining DNA from skeletal material. In these cases, cortical osseous tissue from weight bearing long bones (femur, tibia) has been reported to be a better target for obtaining amplifiable DNA than trabecular bone. Since midshaft sections of the femur and tibia contain an abundance of cortical bone, they have become a preferential target for forensic scientists. Research in the Michigan State University Forensic Biology Laboratory has shown statistically significant intra-bone variation in recoverable nuclear and mitochondrial DNA from both cow and pig femora. Overall, significantly more DNA was obtained from the epiphyses and auricular surfaces than diaphysis, contrary to the widely accepted standard. Importantly, these results demonstrate that substantial DNA variation may exist within single bones, however in order to have worthwhile forensic applications, intra-bone variation in DNA quantity and quality must be characterized in human remains. In the proposed research, five pairs of fresh human legs will be obtained from a commercial source. Soft tissue will be excised and skeletal elements macerated. Paired femora and tibias will be divided into longitudinal sections, each drilled to produce bone powder. A homogenous sample from each section will be digested using a standard tissue lysis buffer or a bone specific demineralization buffer. The amount of nuclear and mitochondrial DNA present in each region will be measured using quantitative PCR. Additionally, each extract will undergo DNA quality assessment based on mitochondrial and nuclear amplicon sizes obtained. Following drilling, one half of each paired set of remains will be buried in rich soil and the other half placed on the soil surface. Bones will be retrieved (and reburied) over a period of eight months, and drilled in a systematic fashion to test for both regional DNA loss and micro-variability of DNA loss/retention within bone sections. At period’s end, bones will be sawed transversely and sampled along transverse sections to test for directionality of DNA degradation. The proposed research is designed to assess intra-bone DNA levels and preservation in human skeletal remains, in a manner never before considered, using a systematic, objective approach. The proposed research has the potential to vastly improve our understanding of recoverable DNA in human skeletal material, as well as increase its utility to forensic laboratories by increasing the likelihood that successful typing results are obtained. This project contains a research and/or development component, as defined in applicable law. ca/ncf