Evaluation of cell capture and collection from bone

Current processing techniques utilized to harvest DNA from osseous elements are destructive and success rates widely vary. In addition, few studies have explored whether pulverization of bone sample contributes to DNA damage observed in profiles obtained from skeletal remains. This work proposes to implement cell capture methods in demineralized bone slices from degraded cortical skeletal remains for the collection of nucleated cells and subsequent retrieval and recovery of genomic sequences for human identification. By eliminating the homogenization of bone, the likelihood of introducing intrinsic or extrinsic co-purified inhibitory substances to the sample may be minimized due to the direct isolation of cells. The project will be conducted over 12 months and will use demineralization followed by enzymatic digestion of collagen to loosen cells from their matrix connections and may allow for streamlined DNA isolation without the need for post-extraction purification methods for co-isolated impurities, as is commonly performed at present within the forensic and ancient DNA communities. Subsequent evaluation of several cell capture approaches will be conducted to identify the most effective method, including chemical excision through partial and total collagen digestion, optical tweezers, and micromanipulation techniques; and will assess the developed methodologies using aged and degraded human skeletal remains. Transferring captured cells to a microcentrifuge tube allows for direct integration into existing DNA analysis workflows and may be less susceptible to inhibitory and competitive effects from native and foreign components, often found in degraded skeletal remains. Additionally, evaluation of the use of optical tweezers and micromanipulation techniques for subsequent picking of nucleated cells from demineralized bone slices may provide significantly better profiles than traditional pulverization of calcified tissue and may enable more confident and rapid identifications. We will investigate concordance of genetic markers by means of traditional and emerging genotyping applications, including length-base and targeted sequencing technologies with capillary electrophoresis and high-throughput sequencing to maximize the recovery of DNA for human identification. In addition to the annual and final technical reports, expected scholarly products include one doctoral dissertation, two publications in peer-reviewed journals, and at least one presentation at a scientific meeting. CA/NCF