Abstract
In forensic genetics, the analysis of postmortem bones is one of the most challenging due to the low quantity of degraded endogenous DNA. The most widely used approach for sample preparation, in those cases, is pulverizing the bone. However, processing pulverized bone is extremely delicate, requiring strict laboratory conditions and operating procedures. In fact, several recent publications have focused on non-powder approaches. The objectives of this study were, thus, to validate a non-powder protocol for DNA extraction from forensic bones and an alternative pretreatment, large fragment demineralization (LFD). Thirty human femurs and tibiae received by the Legal Medicine Institute of Brescia, Italy, were included in the study. Bone powder and one transversal section of the diaphysis were sampled from each bone. DNA extraction from the powder was carried out using PrepFiler BTA (BTA), while the transversal section was submitted to the alternative demineralizing pretreatment (LFD) followed by DNA extraction using the QIAamp DNA Investigator. DNA extracts were assessed for human DNA quantity and degradation by means of a validated in-house qPCR assay and amplified with commercial kits. Inhibition assessment was carried out through Quality Sensor analysis using 24plex QS Kit. The differences in quantity, quality of human DNA, and number of alleles detected between both methods were comparable and not statistically significant. We propose the use of the LFD protocol as a complementary approach capable of confirming the genotypes or detect alleles not observed using BTA, without the need for pulverization.
Similar content being viewed by others
References
Hochmeister MN, Budowle B, Borer UV, Eggmann U, Comey CT, Dirnhofer R (1991) Typing of deoxyribonucleic acid (DNA) extracted from compact bone from human remains. J Forensic Sci. 36:1649–1661
Parsons TJ, Huel RML, Bajunović Z, Rizvić A (2019) Large Scale DNA identification: the ICMP experience. Forensic Sci Int Genet. 38:236–244. https://doi.org/10.1016/j.fsigen.2018.11.008
Loreille OM, Diegoli TM, Irwin JA, Coble MD, Parsons TJ (2007) High efficiency DNA extraction from bone by total demineralization. Forensic Sci Int Genet. 1:191–195. https://doi.org/10.1016/j.fsigen.2007.02.006
Zeng X, Elwick K, Mayes C, Takahashi M, King JL, Gangitano D, Budowle B, Hughes-Stamm S (2019) Assessment of impact of DNA extraction methods on analysis of human remain samples on massively parallel sequencing success. Int J Legal Med. 133:51–58. https://doi.org/10.1007/s00414-018-1955-9
Pajnič IZ (2016) Extraction of DNA from human skeletal material. Methods Mol Biol. 1420:89–108. https://doi.org/10.1007/978-1-4939-3597-0_7
Kitayama T, Ogawa Y, Fujii K, Nakahara H, Mizuno N, Sekiguchi K, Kasai K, Yurino N, Yokoi T, Fukuma Y, Yamamoto K, Oki T, Asamura H, Fukushima H (2010) Evaluation of a new experimental kit for the extraction of DNA from bones and teeth using a non-powder method. Leg Med (Tokyo). 12:84–89. https://doi.org/10.1016/j.legalmed.2009.12.004
Caputo M, Irisarri M, Alechine E, Corach D (2013) A DNA extraction method of small quantities of bone for high-quality genotyping. Forensic Sci Int Genet. 7:488–493. https://doi.org/10.1016/j.fsigen.2013.05.002
Harrel M, Mayes C, Gangitano D, Hughes-Stamm S (2018) Evaluation of a powder-free DNA extraction method for skeletal remains. J Forensic Sci. 63:1819–1823. https://doi.org/10.1111/1556-4029.13749
Huynen L, Lambert DM (2015) A Concentrated hydrochloric acid-based method for complete recovery of DNA from bone. J Forensic Sci. 60:1553–1557. https://doi.org/10.1111/1556-4029.12846
PrepFiler® and PrepFiler® BTA Forensic DNA Extraction Kits User Guide (2012). http://www3.appliedbiosystems.com/cms/groups/applied_markets_support/documents/generaldocuments/cms_096102.pdf
QIAamp® DNA Investigator Handbook (2012). https://b2b.qiagen.com/us/resources/resourcedetail?id=dcc5a995-3743-4219-914d-94d6a28e49b3&lang=en
Corrêa HSD, Brescia G, Cortellini V, Cerri N, Verzeletti A (2020) DNA quantitation and degradation assessment: a quantitative PCR protocol designed for small forensic genetics laboratories. Electrophoresis. 41:714–719. https://doi.org/10.1002/elps.201900360
Miloš A, Selmanović A, Smajlović L, Huel RLM, Katzmarzyk C, Rizvić A, Parsons TJ (2007) Success rates of nuclear short tandem repeat typing from different skeletal elements. Croat Med J. 48:486–493
Calacal GC, Apaga DLT, Salvador JM, Jimenez JAD, Lagat LJ, Villacorta RPF, Lim MCF, Fortum RR, Datar FA, De Ungria MCA (2015) Comparing different post-mortem human samples as DNA sources for downstream genotyping and identification. Forensic Sci Int Gen. 19:212–220. https://doi.org/10.1016/j.fsigen.2015.07.017
Johnston E, Stephenson M (2016) DNA profiling success rates from degraded skeletal remains in Guatemala. J Forensic Sci. 61:898–902. https://doi.org/10.1111/1556-4029.13087
Emmons AL, Davoren J, DeBruyn JM, Mundorff AZ (2020) Inter and intra-individual variation in skeletal DNA preservation in buried remains. Forensic Sci Int Genet. 44:102193–102163. https://doi.org/10.1016/j.fsigen.2013.08.001
Mundorff A, Davoren JM (2004) Examination of DNA yield rates for different skeletal elements at increasing post mortem intervals. Forensic Sci Int Genet. 8:55–63
Andronowski JM, Mundorff AZ, Pratt IV, Davoren JM, Cooper DML (2017) Evaluating differential nuclear DNA yield rates and osteocyte numbers among human bone tissue types: a synchrotron radiation micro-CT approach. Forensic Sci Int Genet. 28:211–218. https://doi.org/10.1016/j.fsigen.2017.03.002
Corrêa HSD, Cortellini V, Brescia G, Verzeletti A (2020) Human identification through DNA analysis of restored postmortem teeth. Forensic Sci Int Genet. 47:102302
Corrêa HSD, Pedro FLM, Volpato LER, Pereira TM, Siebert Filho G, Borges ÁH (2017) Forensic DNA typing from teeth using demineralized root tips. Forensic Sci Int. 280:164–168. https://doi.org/10.1016/j.forsciint.2017.10.003
Miquelestorena-Standley E, Jourdan ML, Collin C, Bouvier C, Larousserie F, Aubert S, Gomez-Brouchet A, Guinebretière JM, Tallegas M, Brulin B, le Nail LR, Tallet A, le Loarer F, Massiere J, Galant C, de Pinieux G (2020) Effect of decalcification protocols on immunohistochemistry and molecular analyses of bone samples. Mod Pathol. 33:1505–1517. https://doi.org/10.1038/s41379-020-0503-6
Vanek D, Budowle B, Dubska-Votrubova J, Ambers A, Frolik J, Pospisek M, al Afeefi AA, al Hosani KI, Allen M, al Naimi KS, al Salafi D, al Tayyari WAR, Arguetaa W, Bottinelli M, Bus MM, Cemper-Kiesslich J, Cepil O, de Cock G, Desmyter S, el Amri H, el Ossmani H, Galdies R, Grün S, Guidet F, Hoefges A, Iancu CB, Lotz P, Maresca A, Nagy M, Novotny J, Rachid H, Rothe J, Stenersen M, Stephenson M, Stevanovitch A, Strien J, Sumita DR, Vella J, Zander J (2017) Results of a collaborative study on DNA identification of aged bone samples. Croat Med J. 58:203–213. https://doi.org/10.3325/cmj.2017.58.203
Klempner S, Williams D, Sanchez K, Li R (2016) Processing skeletal samples for forensic DNA analysis. In Forensic Science (eds Katz E, Halámek J). https://doi.org/10.1002/9783527693535.ch9
Harrel M, Hughes-Stamm S (2020) A powder-free DNA extraction workflow for skeletal samples. J Forensic Sci. 65:601–609. https://doi.org/10.1111/1556-4029.14197
Author information
Authors and Affiliations
Corresponding author
Ethics declarations
Conflict of interest
The authors declare that they have no conflict of interest.
Additional information
Publisher’s note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Supplementary Information
ESM 1
(XLSX 28 kb)
Rights and permissions
About this article
Cite this article
Corrêa, H., Cortellini, V., Franceschetti, L. et al. Large fragment demineralization: an alternative pretreatment for forensic DNA typing of bones. Int J Legal Med 135, 1417–1424 (2021). https://doi.org/10.1007/s00414-021-02531-0
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00414-021-02531-0