AbstractMitochondrial 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 HollandPenn 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 Name Hypervariable Region 1

Hypervariable Region 2

5/11Haplogroup H1a1

16162 A>G16209 T>C

73 A>G263 A>G315.1C

345 C>CA9/11

Haplogroup U or HNo Mutations 73 A>AG

263 A>G315 dupC

22/11Haplogroup Unknown

16051 A>G16129 G>C16180 delA16189 T>C

73 A>G152 T>C217 T>C

40/11Haplogroup H

16189 T>TC 73 A>G152 T>C

57/11Haplogroup H

16176 C>T16189 T>C

263 A>G263 A>G (AG)

315.1C

BackgroundThis 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 1988. 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 AlterationsMaterials•46 bone samples

• 21 tooth, 25 femur•Several sets of remains discovered within same grave•Some remains discovered individually

MethodsThe 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 amounts1 tube, 200 mg – with quadruple reagents also

Extract volume = 50 uL2 tubes, 100 mg each

Combine after elution step, extract volume = 100 uL4 tubes, 50 mg each

Combine using one magnetic bead aliquotExtract volume = 50 uL

Combine using one elution buffer aliquotExtract volume = 50-60 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.