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Molecular genetic investigations of skeleton finds buried in the ground
Forensic Science International: Genetics Supplement Series 3 (2011) e200–e201
Contents lists available at ScienceDirect
Forensic Science International: Genetics Supplement Series journal homepage: www.elsevier.com/locate/FSIGSS
Molecular genetic investigations of skeleton finds buried in the ground K. Thiele a, M. Kohl a,*, H. Bruchhaus b, J. Dreßler a a b
Institute of Forensic Medicine of Leipzig University, Germany Institute of Human Genetics and Anthropology of Friedrich-Schiller-University Jena, Germany
A R T I C L E I N F O
A B S T R A C T
Article history: Received 26 August 2011 Accepted 29 August 2011
In the year 2003, several largely complete skeletons were found on various neighbouring spots during construction works in the vicinity of a Chemnitz detention centre, which had been used for decades. The Authority of the Inquiry did not assign systematic excavation, and so the skeleton material, which was sent in for investigation from the various finding places, also contained skeleton remains of several individuals per finding place. Police investigation, the burial place and some minor clothing remains resulted in the assumption that the graves had been laid out in the first years after World War II. In addition, it was suspected that one of the buried bodies might have been a male person who had been missing at that time. After appropriate mechanical preparation of selected bone materials the DNA-isolation followed, and subsequently quantification with the QuantifierTM Human DNA Quantification kit of the firm Applied Biosystems. The amplification of DNA was carried out with commercially available autosomal and gonosomal amplification kits of various firms. DNA fragment separation was done with ABI PrismTM 3130 Genetic Analyzer of the firm Applied Biosystems.Compared to the pre-investigation, much better and forensically usable results of the analysis were achieved by modified methodology, as will be presented in detail. ß 2011 Elsevier Ireland Ltd. All rights reserved.
Keywords: DNA extraction Bone material
1. Introduction In the year 2003, several largely complete skeletons were found on various neighbouring spots during construction works in the vicinity of a Chemnitz detention centre, which had been used for decades. The Authority of the Inquiry did not assign systematic excavation, and so the skeleton material, which was sent in for investigation from the various finding places, also contained skeleton remains of several individuals per finding place. Police investigation, the burial place and some minor clothing remains resulted in the assumption that the graves had been laid out in the first years after World War II. In addition, it was suspected that one of the buried bodies might have been a male person who had been missing at that time. 2. Materials and methods In 2002/2003, for molecular genetic examinations teeth and bones were cleaned mechanically, then exposed to UV radiation, and crushed; in the subsequent DNA isolation using the InViSorbTM Forensic Kit of the company InViTek, between 0.65 g and 1.65 g were used. Quantification was still performed with the help of the
* Corresponding author. E-mail address: [email protected] (M. Kohl). 1875-1768/$ – see front matter ß 2011 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.fsigss.2011.08.100
Quantiblot1 Human DNA Quantitation Kit of the company Applied Biosystems. DNA amplification was performed using PowerPlex1 16, ES as well as Y System Kits of the company Promega, and DNA fragment separation was carried out in 4.5% denaturalized PAG with the help of ABI PRISM1 377 DNA Sequencer (Applied Biosystems). In the following years the methodology of DNA isolation in the laboratory was modified, and analyses were performed with the help of commercially available DNA test kits. The methodology applied in 2003/2004 did not provide any usable analysis results of the five bone samples (traces 26, 40, 47.1, 60.1, 62) and of one tooth sample (trace 6.1), whereas five tooth samples (traces 4, 16, 19.1, 30.2, 60.2) could be typed, with locus and allelic drop outs to be observed. In 2011, the DNA from the Compacta of the long bones and again of teeth was isolated. At the beginning between 2.35 g and 6.84 g bone powder as well as 1.60 g and 2.41 g tooth powder were produced. The isolated amounts of DNA ranged from 0.002 ng/ml to 0.180 ng/ml for the bone material and from 0.066 ng/ml to 0.629 ng/ml for the tooth material.
3. Results In all the DNA systems, the teeth could be typed using the kits mentioned. Corresponding identification patterns for the teeth of
K. Thiele et al. / Forensic Science International: Genetics Supplement Series 3 (2011) e200–e201
the upper jaw and of the lower jaw were obtained, also considering the respective place, where they were found. The analysis of the bone samples proved to be more complicated. On the right femur from the burial place no. 3 all the analyses had negative results. Two femora did not produce any allelic respectively locus drop outs, eight femora resulted in single allelic or locus drop outs, and with the two tibiae and one femur there was a multiple drop out. The PowerPlex1 S5 Kit provided the best results. The systems using it could be typed in each case. Allelic resp. locus drop outs could be observed occasionally, using the Minifiler Kit, and – as expected – frequently, using PowerPlex1 ESX and ESI Kits as well as the gonosomal kits (Qiagen). For the bone and tooth samples extracted in 2011, the analysis of autosomal INDEL using the DIPplex Kit led to a comparable quality of the results, with STR markers typed by PowerPlex1 S5 and Minifiler Kit. Partly better results were obtained with STR.
e201
4. Conclusion Contemporary investigations resulted in definite identification patterns of eight male individuals. It was discovered that the burial places contained skeleton material of various individuals. YHRD data bank investigations of the individuals’ Haplotypes resulted in their correspondence with two Eurasian-West European metapopulations in Nevada/USA and Austria and one admixed Brazilian metapopulatiom in Brazil. The police suspicion that among the buried persons there was a male individual missing in 1949 (W.T., born in 1892) could not be confirmed, following the comparison with the compared person (G.T., the natural son of the missing W.T.). Conflict of interest statement None.
Contents lists available at ScienceDirect
Forensic Science International: Genetics Supplement Series journal homepage: www.elsevier.com/locate/FSIGSS
Molecular genetic investigations of skeleton finds buried in the ground K. Thiele a, M. Kohl a,*, H. Bruchhaus b, J. Dreßler a a b
Institute of Forensic Medicine of Leipzig University, Germany Institute of Human Genetics and Anthropology of Friedrich-Schiller-University Jena, Germany
A R T I C L E I N F O
A B S T R A C T
Article history: Received 26 August 2011 Accepted 29 August 2011
In the year 2003, several largely complete skeletons were found on various neighbouring spots during construction works in the vicinity of a Chemnitz detention centre, which had been used for decades. The Authority of the Inquiry did not assign systematic excavation, and so the skeleton material, which was sent in for investigation from the various finding places, also contained skeleton remains of several individuals per finding place. Police investigation, the burial place and some minor clothing remains resulted in the assumption that the graves had been laid out in the first years after World War II. In addition, it was suspected that one of the buried bodies might have been a male person who had been missing at that time. After appropriate mechanical preparation of selected bone materials the DNA-isolation followed, and subsequently quantification with the QuantifierTM Human DNA Quantification kit of the firm Applied Biosystems. The amplification of DNA was carried out with commercially available autosomal and gonosomal amplification kits of various firms. DNA fragment separation was done with ABI PrismTM 3130 Genetic Analyzer of the firm Applied Biosystems.Compared to the pre-investigation, much better and forensically usable results of the analysis were achieved by modified methodology, as will be presented in detail. ß 2011 Elsevier Ireland Ltd. All rights reserved.
Keywords: DNA extraction Bone material
1. Introduction In the year 2003, several largely complete skeletons were found on various neighbouring spots during construction works in the vicinity of a Chemnitz detention centre, which had been used for decades. The Authority of the Inquiry did not assign systematic excavation, and so the skeleton material, which was sent in for investigation from the various finding places, also contained skeleton remains of several individuals per finding place. Police investigation, the burial place and some minor clothing remains resulted in the assumption that the graves had been laid out in the first years after World War II. In addition, it was suspected that one of the buried bodies might have been a male person who had been missing at that time. 2. Materials and methods In 2002/2003, for molecular genetic examinations teeth and bones were cleaned mechanically, then exposed to UV radiation, and crushed; in the subsequent DNA isolation using the InViSorbTM Forensic Kit of the company InViTek, between 0.65 g and 1.65 g were used. Quantification was still performed with the help of the
* Corresponding author. E-mail address: [email protected] (M. Kohl). 1875-1768/$ – see front matter ß 2011 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.fsigss.2011.08.100
Quantiblot1 Human DNA Quantitation Kit of the company Applied Biosystems. DNA amplification was performed using PowerPlex1 16, ES as well as Y System Kits of the company Promega, and DNA fragment separation was carried out in 4.5% denaturalized PAG with the help of ABI PRISM1 377 DNA Sequencer (Applied Biosystems). In the following years the methodology of DNA isolation in the laboratory was modified, and analyses were performed with the help of commercially available DNA test kits. The methodology applied in 2003/2004 did not provide any usable analysis results of the five bone samples (traces 26, 40, 47.1, 60.1, 62) and of one tooth sample (trace 6.1), whereas five tooth samples (traces 4, 16, 19.1, 30.2, 60.2) could be typed, with locus and allelic drop outs to be observed. In 2011, the DNA from the Compacta of the long bones and again of teeth was isolated. At the beginning between 2.35 g and 6.84 g bone powder as well as 1.60 g and 2.41 g tooth powder were produced. The isolated amounts of DNA ranged from 0.002 ng/ml to 0.180 ng/ml for the bone material and from 0.066 ng/ml to 0.629 ng/ml for the tooth material.
3. Results In all the DNA systems, the teeth could be typed using the kits mentioned. Corresponding identification patterns for the teeth of
K. Thiele et al. / Forensic Science International: Genetics Supplement Series 3 (2011) e200–e201
the upper jaw and of the lower jaw were obtained, also considering the respective place, where they were found. The analysis of the bone samples proved to be more complicated. On the right femur from the burial place no. 3 all the analyses had negative results. Two femora did not produce any allelic respectively locus drop outs, eight femora resulted in single allelic or locus drop outs, and with the two tibiae and one femur there was a multiple drop out. The PowerPlex1 S5 Kit provided the best results. The systems using it could be typed in each case. Allelic resp. locus drop outs could be observed occasionally, using the Minifiler Kit, and – as expected – frequently, using PowerPlex1 ESX and ESI Kits as well as the gonosomal kits (Qiagen). For the bone and tooth samples extracted in 2011, the analysis of autosomal INDEL using the DIPplex Kit led to a comparable quality of the results, with STR markers typed by PowerPlex1 S5 and Minifiler Kit. Partly better results were obtained with STR.
e201
4. Conclusion Contemporary investigations resulted in definite identification patterns of eight male individuals. It was discovered that the burial places contained skeleton material of various individuals. YHRD data bank investigations of the individuals’ Haplotypes resulted in their correspondence with two Eurasian-West European metapopulations in Nevada/USA and Austria and one admixed Brazilian metapopulatiom in Brazil. The police suspicion that among the buried persons there was a male individual missing in 1949 (W.T., born in 1892) could not be confirmed, following the comparison with the compared person (G.T., the natural son of the missing W.T.). Conflict of interest statement None.