Sample storage is of paramount importance in forensic genetics laboratories since only optimal storage enables successful recovery of DNA from old bones that contain very low amount of severely degraded DNA. When identification of missing persons from skeletal remains is completed, bone sample is routinely stored at -20°C for long-term storage for retesting in future, if necessary. After molecular genetic analyses of Slovenian Second World War (WWII) victims, small fragments of femurs were stored at -20°C. Reduction in DNA recovery has been observed in frozen liquid DNA extracts by some authors and the goal of our study was to explore how freezing of bone samples affects the preservation of DNA. To achieve this goal, the difference in DNA yield in extracts obtained from WWII bones analyzed in 2009 (data from published paper) and DNA yield in extracts obtained from the same bones (piece sampled next to the one used in 2009) taken out of the freezer after long-term storage on -20°C for 10 years was examined, using the same extraction method and the same quantification kit. Up to 100 ng DNA/g of bone powder was obtained from 57 WWII femurs and up to 31 ng DNA/g of bone powder from the same femurs investigated after long-term storage in this study. 0,5 g of bone powder was decalcified using full demineralization extraction method. The DNA was purified in a Biorobot EZ1 device (Qiagen) and DNA quantity determined with the Human Quantifiler kit (TFS). Statistical analysis showed significant difference in DNA yield in extracts obtained from WWII bones in 2009 and extracts obtained from the same bones stored at -20°C after 10 years. As reported for frozen liquid DNA extracts, reduction in recovery of DNA was confirmed for frozen bone samples as well.

Bone samples may yield low-quality and low-quantity DNA and duplicated analyses of different genetic markers have to be performed for identification of missing persons. Mostly no DNA extract is left after analyses and efficient storage of bones is needed to ensure the stability of the sample over time for retesting using new markers and new technologies. Usually not all of the bone powder prepared in grinder is used for extraction and rest can be stored for future analyses. After molecular genetic analyses of 88 victims of Second World War (WWII) Konfin I mass grave in Slovenia (performed in 2009), fragments of femurs and bone powder that were left were stored at -20°C. Some authors reported that long-term storage of powder results in the reduction of DNA preservation and its degradation (even at low temperature), explained by an increase in oxidative damage as a result of the enormous increase in exposed surface area. Consequently, grinding of bones as shortly prior to DNA extraction was recommended. The goal of our study was to explore the difference in DNA yield between bone fragment and bone powder frozen for 10 years. 57 WWII femurs were examined and DNA extracted from each of them using bone fragment (piece sampled next to the one used in 2009) and bone powder obtained in 2009, both taken out of freezer after 10 years of storage. Half gram of bone powder was decalcified using full demineralization extraction method. The DNA was purified in a Biorobot EZ1 (Qiagen) and quantified with PowerQuant kit (Promega). Statistical analysis showed significant difference at the 0.05 level in DNA yield comparing fragments of bones and bone powder stored at -20°C for 10 years. The results show there is more DNA stored in the bone powder than in the bone fragments. Because of time -consuming powdering procedure we recommend to store not only the fragment of the bone, but obtained bone powder as well.