Discovering the Mysteries About Degradation of Extracted DNA From Trace Evidence When Stored in Different Conditions: State-of-the-Art and Future Perspectives

Learning Objective: After attending this presentation, attendees will understand the consequences that inappropriate or inaccurate sample preservation can have on judicial caseworks and will gain knowledge on the best isolation and preservation strategies for DNA extracts for forensic genetics. Impact Statement: This presentation will impact the forensic science community by presenting experimental studies on DNA degradation of extracts preserved in non-ideal conditions and by providing guidelines on the best approaches to conserve them without losing any genetic information. The storage of DNA extracts is a debated topic in the forensic arena; despite the existing guidelines on the preservation of biological evidence, there are no clear agreements on the preservation of the extracts originated from the evidence in terms of duration nor temperature. According to NIST guidelines, extracted DNA to be preserved for the long term should be kept frozen.1,2 However, it is not infrequent to encounter situations in which extracts are kept at fridge or room temperature, with unknown implications for its degradation and, consequently, for the final analytical results. In order to evaluate DNA survival and degradation rates, different samples, including blood stains, buccal swabs and blood/saliva mixtures in increasing ratios, were extracted with several methods commonly used in forensic genetics and stored at different temperatures (+20°C, +4°C, and -20°C) for up to 150 days. Aliquots were quantified with qPCR to obtain a quantitative and qualitative assessment at time “0,” then every four days for the first 16 days and every 30 days after one month (from 30 to 150 days). In addition to qPCR, samples were amplified with commercial kits to evaluate their degree of efficiency and typing, on at least two instruments (Applied Biosystems® SeqStudio™ Genetic Analyzer and 3500 Genetic Analyzer) for performance comparisons. The NGS VISAGE panel for phenotype, as well as a new in-house-developed ancestry and phenotype panel, will be additionally applied to selected samples to evaluate the reliability of the NGS data on these samples. Preliminary results indicated that the frozen samples as well as room temperature vacuum-dried samples maintain a complete genetic profile useful for identification purposes and for NGS analyses; the complete results presented at the conference will clarify to what extent mixture samples can be informative on the identity of both contributors to the trace and for how long preservation at different temperatures can provide useful information, and will propose “best practices” that should be applied by forensic genetics laboratories and police forces in order to maximize the preservation of the genetic information in DNA extracts.