Evaluation of the stability of DNA methylation markers in biological stains and its impact on forensic investigations

Background: Study of epigenetic modifications such as DNA methylation has become an important tool in forensic investigations due to its reliability and specificity. DNA methylation is highly dynamic and sensitive to several environmental and lifestyle factors. DNA samples collected from crime scenes can be tested according to their methylation patterns to help identify different types of biological pieces of evidence, including hair, blood, semen, and saliva found at the crime scene. Furthermore, it can help in the identification of sex, age and shed light on the overall identity of the suspect or victim. Objectives: This study aims to validate the use of DNA methylation-specific markers in identifying peripheral blood, menstrual blood, and saliva and investigate the stability of these markers. Additionally, this research assesses the effect of exposure of blood and saliva to different environmental conditions on detecting DNA methylation-specific markers. Methodology: The samples used in this study are peripheral blood, saliva, and menstrual blood. DNA has been extracted from all samples, and its quality has been detected on gel electrophoresis. Then bisulfite conversion and real-time PCR were applied using BLM1 primer to detect peripheral blood samples, MENS1 primer to detect menstrual blood samples, and SPEI1 to detect saliva samples. Dried Stains from the saliva, menstrual blood, and peripheral blood samples have been collected and exposed to different environmental conditions. Results: The results of real-time PCR and statistical analysis of BLM1 and MENS1 primers showed better results than SPEI1 primers in identifying fresh body fluids and those exposed to different environmental conditions of degradation. Conclusion: DNA methylation is highly specific to the person's tissue type, age, and sex. This unique characteristic of DNA methylation is exploited in the identification of victims or culprits during a forensic investigation. The amount and the integrity of DNA used for analysis are often the determining factors in the success of methylation studies. Various factors such as exposure to UV radiation, high temperature, PH, and salt concentration can affect DNA stability.