DNA degradation poses a significant challenge in forensic analysis when biological evidence is exposed to environmental conditions that compromise DNA integrity. Although prior studies have evaluated individual factors affecting degradation, limited research has compared biological fluid type, environmental exposures, and genotyping methods within a single controlled study. This study aimed to quantitatively and comparatively assess how these variables influence DNA stability and overall downstream profile interpretation.

Blood, saliva, and mixed blood–saliva samples from three donors were deposited onto glass slides and exposed to heat, heat with ultraviolet (UV) radiation, and cold conditions over six timepoints (0, 7, 20, 35, 60, and 91 days). DNA was extracted using an organic phenol–chloroform method and quantified using the Qiagen Quantiplex Pro RGQ Kit to obtain DNA concentration and degradation index (DI) values. A subset of samples meeting concentration thresholds were amplified using the GlobalFiler PCR Amplification Kit for STR analysis and a custom 38-plex InDel panel. Amplified products were separated by capillary electrophoresis and assessed for completeness of the DNA profiles, allelic retention, and mixture interpretation.

Results demonstrated a progressive increase in DNA degradation over time, reflected by decreasing DNA concentrations and increasing DI values. Environmental conditions strongly influenced degradation rates, with UV and heat exposures producing the most rapid degradation, while cold storage effectively preserved DNA integrity. Biological fluid type further impacted degradation patterns, as saliva samples showed faster degradation, higher DI values, and earlier STR allelic dropout compared to blood. In mixed samples, differential degradation led to increasing contributor imbalance, with blood contributors retaining a greater number of alleles over time.

Comparison of genotyping methods revealed distinct trade-offs. STR analysis provided higher discriminatory power and clearer contributor differentiation but was more susceptible to degradation, resulting in partial or incomplete profiles under prolonged exposure. In contrast, InDel markers demonstrated greater stability, maintaining high profile completeness across all conditions due to shorter amplicon sizes; however, their biallelic nature limited resolution in complex mixture interpretation.

Overall, these findings demonstrate that DNA degradation is strongly influenced by the combined effects of environmental exposure and biological fluid type, with direct implications for genotyping success. This study supports the combined use of STR and InDel markers to optimize forensic DNA analysis, particularly for degraded and mixed samples, and provides valuable data to inform evidence interpretation in environmentally compromised casework.

Authors

• Filipa Simao (Towson University, United States of America)
• Lindsey Pirchio (Towson University, United States of America)