A SEMI-AUTOMATED METHODOLOGY FOR THE EXTRACTION OF DNA FROM HUMAN SKELETAL REMAINS

Purpose: The conventional method used to extract DNA from skeletal remains is not readily amenable to automation. The numerous steps in the current procedure increase the risk of both sample loss and the potential for contamination with extraneous DNA. An automated DNA extraction procedure will be evaluated and optimized for the quantity and quality of DNA recovered from bone samples on the AutoMate Express™ (Life Technologies). The goal of this project is to determine if this process will yield equivalent amounts of DNA that will produce autosomal STR profiles and mitochondrial DNA sequence data that are comparable to those obtained with the conventional method of organic extraction. Methods: Varying amounts of bone powder, from different age groups of remains, were incubated with BTA Lysis Buffer (Life Technologies) which contains DTT and Proteinase K from two hours to overnight. After the incubation any remaining bone powder was spun down and 230µL of the lysate was loaded onto the AutoMate Express™ columns containing the PrepFiler™ magnetic beads. Total run-time for the instrument is 30 minutes and DNA is eluted in 50µL of buffer. The extracts were quantified and amplifications were performed. Autosomal STR profiles and mitochondrial DNA sequence data generated from samples processed using the AutoMate Express™ were compared to the data generated by the conventional organic extraction method. Results: No significant difference was observed between a 2-hour incubation and overnight incubation when using the AutoMate Express. The overall quantity and quality of the DNA extracted using the AutoMate Express™ was equal to or better than the DNA extracted using the conventional organic method. Conclusions: To this point the DNA obtained using the AutoMate Express, which takes a total of 4 hours, generates genetic data that is comparable or better than the data generated using the conventional methodology which takes 2-3 days.