Abstract Mitochondrial DNA sequences from a Croatian gravesite, Šopot- Benkovac, will grant anthropological information about the individuals buried at this site, and also serve as test materials to work with for a new extraction method for bone and tooth (aged and/or degraded) samples. This new extraction method has begun to be examined through the comparison of traditional organic demineralization techniques, such as from the AFDIL protocol (Loreille, 2007) versus the PrepFiler® BTA Forensic DNA Extraction Kit from Applied Biosystems™. The PrepFiler® BTA kit has also shown promising results for successful amplification of the bone extracts. Ultimately, examining the mitochondrial DNA sequences could help in determining relationships between individuals in this gravesite, as well as relationships to individuals at surrounding gravesites and people living in present-day Croatia. Assessing the Ancestral Origin of Ancient Human Remains from Croatian Burial Sites using Mitochondrial DNA Analysis Ayano Fox, Liam Phillips, Mitchell Holland Penn State University, University Park, PA Results First set of successful extractions using AFDIL protocol: 5/11, 9/11, 22/11, 40/11, 57/11 Successful amplification/sequencing (prior runs had inhibition) Low Quality data Preliminary PrepFiler® BTA results: 33/11, 47/11, 38/11, 36/11, 20/11 Successful amplification (no inhibition) Weak bands following amplification Subsequent PrepFiler® BTA results: 22/11, 57/11 – 200 mg, 100 mg, 50 mg Successful amplification (no inhibition) 4 50 mg samples combined using one elution buffer aliquot – best results Sample NameHypervariable Region 1 Hypervariable Region 2 5/11 Haplogroup H1a A>G T>C 73 A>G 263 A>G 315.1C 345 C>CA 9/11 Haplogroup U or H No Mutations73 A>AG 263 A>G 315 dupC 22/11 Haplogroup Unknown A>G G>C delA T>C 73 A>G 152 T>C 217 T>C 40/11 Haplogroup H T>TC73 A>G 152 T>C 57/11 Haplogroup H C>T T>C 263 A>G 263 A>G (AG) 315.1C Background This site is dated in the late Middle Age, around the 13th – 16th century. The first excavation occurred in 1929, when the campaign led to the discovery of the stone fragment of Duke Branimir, which was the only fragment on which he was referred to as the duke and lord of Croats. Skeletal remains were excavated in 1985 and This particular site is of additional interest because pagan burial rituals were discovered in an area where most people were buried according to Christian traditions, suggesting the individuals were not natives of this geographic area. In addition, the fact that some individuals were buried within the same grave indicates a possibility of kinship between individuals in this site. Results from first set of extractions: Source: Google Maps Materials and Method Alterations Materials 46 bone samples 21 tooth, 25 femur Several sets of remains discovered within same grave Some remains discovered individually Methods The AFDIL organic procedure was performed according to their protocol (2011). Life Technologies recommended quantities of 50 mg of bone powder for PrepFiler® BTA. We tested 4 different input amounts to verify the most successful quantity with the greatest efficiency of reagent usage. Adjusted sample input amounts 1 tube, 200 mg – with quadruple reagents also Extract volume = 50 uL 2 tubes, 100 mg each Combine after elution step, extract volume = 100 uL 4 tubes, 50 mg each Combine using one magnetic bead aliquot Extract volume = 50 uL Combine using one elution buffer aliquot Extract volume = uL Tooth, Femur, and Skull from the Croatian Lab Source: Applied Biosystems™ Future Work Extract and sequence remaining samples Phylogenetic analysis Kinship within and between graves Ancestral origins Acknowledgements Zeljana Basic, Ivana Anteric, and Dr. Dragan Primorac Method Comparison Loreille OM, Diegoli TM, Irwin JA, Coble MD, Parsons TJ: High efficiency DNA extraction from bone by total demineralization; Forensic Sci Int Genet 1:191; 2007.