Bone Marrow as a Source of DNA in Forensic Genetics: An Optimized Nucleic Acids Extraction Protocol

Background: low-quantity or degraded samples are often studied in forensic genetics. Therefore, it is important to efficiently obtain all the available DNA from the biological sample analyzed to provide the most reliable results. This is particularly challenging in bone marrow processing due to its hydrophobic molecular structure, as for other lipid-rich tissues, especially if rancid. In fact, during adipose tissue decomposition, the putrefaction of fatty acids can in some instances give a compact cerous consistency to the lipidic tissue, hardly susceptible to the nucleic acid extraction mechanisms. According to environmental circumstances, this condition is notably observable in submerged bodies or in putrefied bone marrow. Thus, this study is focused on developing an optimized nucleic acids extraction protocol for putrefied bone marrow. Methods: genetic analyses were performed on putrefied yellow bone marrow collected from 20 human femora recovered from bodies in different decomposition stages. The optimized method was developed by integrating additional steps, reagents and time intervals on a silica-based column commercial kit. This strategy was compared in DNA yield to a standard extraction protocol, represented by the same commercial kit, but following the manufacturer’s directions. Both these strategies were tested in nucleic acid isolation efficiency by performing DNA typing, including real-time PCR quantification, Short Tandem Repeats (STR) amplification and fragments analysis steps. The analytical parameters evaluated were allele count, DNA concentration (ng/µL) and Degradation Index (DI). Results: for allele count and DNA concentration parameters, the optimized protocol showed clear and significant qualitative and quantitative improvements compared with the standard protocol, supporting its potential applicability in forensic casework and laying the foundation for future studies. Conclusions: prior to appropriate laboratory internal validation, the optimized protocol can be used for tough lipid-rich tissues processing without the need to purchase a dedicated system and using a same commercial kit routinely adopted for other forensic genetics matrices.