Methyl-PrepDisc: A Centrifugal Microfluidic Device for Forensic DNA Methylation Sample Preparation

Forensic DNA analysis relies heavily on comparative approaches for human identification, in which unknown evidence samples are compared with known reference materials or database STR profiles from previously identified offenders. For those unknown forensic samples that do not produce a database hit and do not have a reference standard for comparison, forensic DNA phenotyping (FDP) produces an alternative solution to infer externally visible characteristics (EVCs) from unidentified genetic material. The determination of chronological age from human tissue samples is one such FDP technique that has emerged in recent years. This technique relies on the chemical conservation and determination of epigenetic marks, referred to as methylation, that are bound to cytosine residues at specified sites of DNA known to be associated with human aging. Bisulfite conversion (BSC) is currently the gold standard method for methylation sample preparation. This chemical process converts those cytosine bases without methylation to uracil, enabling downstream detection. However, current BSC methods are prone to a high degree of DNA fragmentation and require a centralized laboratory, trained personnel, and several manual tube transfer, pipetting, and centrifugation steps. For this reason, BSC methods in their current form may not be regarded as ideal for forensic applications, in which low levels of input DNA are the norm; time, laboratory space, and resources are limited; and open-tube, manual processes are at a high risk for sample contamination. In response to a solicitation by the National Institute of Justice, this research proposal posits an alternative, rotationally driven microfluidic platform for BSC that would enable rapid, efficient conversion of smaller, forensically relevant DNA input masses in an automated, closed system. The proposed reaction disc will minimize both variability and contamination risk through automation and integration in a closed device. Further, the small size of reaction chambers will enable the use of reduced reagent and DNA input volumes, thus shortening required incubation times known to produce DNA fragmentation and loss with traditional BSC techniques. The Methyl-PrepDisc system will be run on instruments already optimized within the Landers Laboratory and will integrate both sample lysis and magnetic bead-based DNA purification and BSC in one disposable and operational disc capable of completing methylation sample preparation for five samples in tandem. Note: This project contains a research and/or development component, as defined in applicable law, and complies with Part 200 Uniform Requirements - 2 CFR 200.210(a)(14). CA/NCF