Degradation of DNA Extracts Stored Under Different Conditions: What We Know and What Is New

Preserving the integrity of forensic evidence through the stabilisation of the biological signature contained in DNA extracts is particularly important, specifically when re-analysis samples after a certain period of time. This translates into the needs for appropriate storage of DNA extracts to ensure the successful outcome of forensic genetic analyses, including human identification through STR typing and the application of phenotype and ancestry panels to infer the morphological characteristics and geographical origin of the contributor. Although guidelines shared by the international scientific community recommend freezing of the DNA extracts for long-term storage, it is possible that samples are preserved in sub-optimal conditions for a variety of reasons (e.g., faulty freezers, moving samples to court office at the case closure). In these circumstances, little is known about the survival of DNA and its suitability for future STR and SNP analyses. In this study, we evaluated the preservation of DNA extracts obtained from buccal swabs collected from two subjects of different sexes using the QIAamp DNA Investigator Kit (Qiagen). The extracts were quantified (Quantifiler™ Trio DNA Quantification Kit – Thermo Fisher Scientific) and aliquots containing 1 ng/µl of DNA were created. In addition, mixtures were set up from the female and male DNA extracts, in a ratio of 1:15 (male/female. All the aliquots were stored at three temperatures (+20°C, +4°C and -20°C) for a period of 90 days. As the results showed a surprising DNA survival rate also at room temperature, revealing similar completeness of the STR profile (GlobalFiler™ PCR Amplification Kit – Thermo Fisher Scientific) at both -20°C and +20°C conditions, the experiment was further extended to 400 days. In addition, after the initial 90 days experiment, a subset of samples was moved to uncontrolled temperature conditions. Surprisingly, DNA extracts did not degrade up to 400 days under any condition, and the ratio in the mixed samples remains unchanged. It was also noted that storing the extracts at +4°C and +20°C causes their evaporation and consequent concentration up to 20-fold. Overall, these results could open new avenues for the analysis of samples from cold cases not always preserved frozen.