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Bone fragment or bone powder? ATR-FTIR spectroscopy–based comparison of chemical composition and DNA preservation of bones after 10 years in a freezer

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Abstract

Freezing bone samples to preserve their biomolecular properties for various analyses at a later time is a common practice. Storage temperature and freeze–thaw cycles are well-known factors affecting degradation of molecules in the bone, whereas less is known about the form in which the tissue is most stable. In general, as little intervention as possible is advised before storage. In the case of DNA analyses, homogenization of the bone shortly before DNA extraction is recommended. Because recent research on the DNA yield from frozen bone fragments and frozen bone powder indicates better DNA preservation in the latter, the aim of the study presented here was to investigate and compare the chemical composition of both types of samples (fragments versus powder) using ATR-FTIR spectroscopy. Pairs of bone fragments and bone powder originating from the same femur of 57 individuals from a Second World War mass grave, stored in a freezer at − 20 °C for 10 years, were analyzed. Prior to analysis, the stored fragments were ground into powder, whereas the stored powder was analyzed without any further preparation. Spectroscopic analysis was performed using ATR-FTIR spectroscopy. The spectra obtained were processed and analyzed to determine and compare the chemical composition of both types of samples. The results show that frozen powdered samples have significantly better-preserved organic matter and lower concentrations of B-type carbonates, but higher concentrations of A-type carbonates and stoichiometric apatite. In addition, there are more differences in the samples with a low DNA degradation index and less in the samples with a high DNA degradation index. Because the results are inconsistent with the current understanding of bone preservation, additional research into optimal preparation and long-term storage of bone samples is necessary.

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Acknowledgements

The authors thank the Slovenian Government Commission on Concealed Mass Graves for the support in the exhumations of Second World War victims.

Funding

This study was financially supported by the Slovenian Research Agency (the project “Determination of the most appropriate skeletal elements for molecular genetic identification of aged human remains,” J3-8214, and Research Group Archaeology, 0581–012).

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Authors and Affiliations

  1. Institute of Forensic Medicine, Faculty of Medicine, University of Ljubljana, Korytkova 2, 1000, Ljubljana, Slovenia

    Irena Zupanič Pajnič

  2. Centre for Interdisciplinary Research in Archaeology, Department of Archaeology, Faculty of Arts, University of Ljubljana, Zavetiška 5, 1000, Ljubljana, Slovenia

    Tamara Leskovar

  3. National Institute of Chemistry, Hajdrihova 19, 1000, Ljubljana, Slovenia

    Ivan Jerman

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  1. Irena Zupanič Pajnič
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Correspondence to Tamara Leskovar.

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Zupanič Pajnič, I., Leskovar, T. & Jerman, I. Bone fragment or bone powder? ATR-FTIR spectroscopy–based comparison of chemical composition and DNA preservation of bones after 10 years in a freezer. Int J Legal Med 135, 1695–1707 (2021). https://doi.org/10.1007/s00414-021-02620-0

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