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 (QuantifilerTM 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 (GlobalFilerTM 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

Alla scoperta dei misteri relativi alla conservazione del DNA estratto. Stato dell'arte e prospettive future

Prendendo spunto da un caso giudiziario italiano, sono stati indagati i misteri della conservazione del DNA estratto a diverse temperature, in diverse condizioni ambientali e a diverse tempistiche

NIPAT as Non-Invasive Prenatal Paternity Testing Using a Panel of 861 SNVs

In 1997, it was discovered that maternal plasma contains Cell-Free Fetal DNA (cffDNA). cffDNA has been investigated as a source of DNA for non-invasive prenatal testing for fetal pathologies, as well as for non-invasive paternity testing. While the advent of Next Generation Sequencing (NGS) led to the routine use of Non-Invasive Prenatal Screening (NIPT or NIPS), few data are available regarding the reliability and reproducibility of Non-Invasive Prenatal Paternity Testing (NIPPT or NIPAT). Here, we present a non-invasive prenatal paternity test (NIPAT) analyzing 861 Single Nucleotide Variants (SNV) from cffDNA through NGS technology. The test, validated on more than 900 meiosis samples, generated log(CPI)(Combined Paternity Index) values for designated fathers ranging from +34 to +85, whereas log(CPI) values calculated for unrelated individuals were below −150. This study suggests that NIPAT can be used with high accuracy in real cases

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® SeqStudioTM 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

"Biological identikit": Development of a SNPs-panel for the analysis of forensic DNA phenotyping and ancestry

Personal identification in mass disasters and in crimes is essential for humanitarian, ethical and legal reasons. In these contexts, when individuals cannot be identified by standard forensic DNA analysis, the Forensic DNA Phenotyping and the analysis of the biogeographical ancestry could help. The aim of this study was to evaluate the potential of a new panel of 891 SNPs in predicting phenotypic traits and biogeographical origin to create a “biological identikit”. In addition to fresh biological material, old evidence found at the crime scene or extracted and long-term stored DNA were tested with 41 SNPs for phenotyping and 850 SNPs for ancestry. All the SNPs were successfully incorporated into a single two-step multiplex PCR reaction using the IonAmpliSeq ™ Library Plus and applied for massive parallel sequencing with the Ion S5 platform using up to 0.05 ng/µL of DNA. The analysis of the results was carried out with an in-house predictive algorithm and consulting 20 population databases. By comparing the results obtained with identikit or video-photographic surveys, it was possible to predict phenotype and ancestry with an accuracy greater than 90%. While these new markers cannot identify a specific individual, they can be a valuable investigative tool