A Novel Magnetic Bead-Based Differential DNA Extraction Method with Potential for High-Throughput Automation in...

Background/Objectives: Sexual offences remain a global challenge, disproportionality affecting developing and conflict-stricken countries. Differential DNA extraction (DDE) is standardly applied to intimate swabs collected in these cases to separate and purify sperm and epithelial fractions ahead of DNA profiling. Whilst DNA purification steps using magnetic bead technology are routinely automated in general forensic workflows, the separation step within DDE workflows usually relies on centrifugation, which is challenging to automate in a high-throughput manner. This proof-of-concept study aimed to develop a novel method to separate sperm and epithelial fractions using magnetic bead technology to enable a fully automated and high-throughput DDE workflow. Methods: First, the Macherey-Nagel NucleoMag^® Forensic DNA kit's protocol for forensic samples was modified to be DDE-based, and different magnetic beads for the separation and purification steps were assessed on mock sexual offence samples. Once a working protocol was established, variables within the protocol were systematically adjusted to improve quality metrics and DNA profiling outcomes. The top-performing method was then assessed with decreased input biological material and on a post-coital swab. Results: The DDE protocol developed in this study identified Cytiva SeraSil-Mag^TM magnetic beads to successfully separate epithelial and sperm fractions, which will enable a fully automated and high-throughput DDE workflow for the first time. DNA extracted from the sperm fraction of mock sexual offence samples prepared with 1 μL of semen input yielded a mean Y-target DNA yield of 25.04 ng (SD = 15.11 ng), a median M:F ratio of 1:3.53 (range = 1:1.08-1:22.46) and a mean log(LR) of 12.01 (SD = 5.41) when the female contributor's DNA profile was unknown. Conclusions: The proof-of-concept of a magnetic bead-based DDE method was successfully demonstrated across a range of different semen input volumes, and the benefit of information-preserving genotyping using the TrueAllele^® system was demonstrated.