The differences in the amount of DNA obtained from six different skeletal elements of eight archaeological skeletons were investigated in our laboratory and presented on the world conference in Prague. Current sampling strategy for laboratories typing bones for human identification include samples obtained from femur, tooth and temporal bone. Latest studies suggest that the small bones of the hands and feet were very similar or even better in DNA yield. These bones can be easily sampled with a disposable scalpel and thus reduce potential DNA contamination. The aim of our study was to determine the suitability of metatarsals, metacarpals and phalanges for genetic identification. 48 bone samples from 8 different skeletons (six from 18th century and two from 3rd century) were obtained from 5 archaeological sites in Slovenia. In each skeleton, 6 different skeletal elements were sampled (temporal bone, molar, femur, metacarpal bone, metatarsal bone and proximal phalanx of the hand), and strict precautions followed to prevent contamination. Half of gram of bone powder was decalcified using full demineralization extraction method. The DNA was purified in a Biorobot EZ1 (Qiagen), DNA content determined with the PowerQuant kit (Promega), and autosomal STR typing performed with the Investigator ESSplex Plus kit (Qiagen). Up to 8.75 ng DNA/g of powder was obtained from samples analyzed. The highest yields were detected in temporal bone and the lowest in femur. The success rate of STR typing was evaluated according to the number of successfully typed loci and a strong correlation between the success rate of STR typing and the amount of extracted DNA was confirmed. For all eight skeletons full consensus genetic profiles were determined from skeletal elements analyzed. Our findings suggest it would be suitable to include metatarsal and metacarpal bones in sampling strategy for human identification although further research is needed to substantiate the findings of this study.
B.06 Other
COBISS.SI-ID: 34532825The Prešeren's research, which was awarded by the Faculty of Medicine with the Prešeren’s prize for 2017 was performed in the Laboratory of Molecular Genetics at the Institute of Forensic Medicine, Faculty of Medicine, University in Ljubljana, under mentorship of project leader. The aim of this study was to determine the suitability of small bones of the hands and feet (metacarpals, metatarsals and phalanges) for genetic typing alongside the currently recommended femur, teeth, and the temporal bone. Three hypotheses were set (1. the amount of preserved ancient DNA extracted from the temporal bones, teeth and femurs can be compared to the amount extracted from small bones of the hands and feet (metacarpal bones, metatarsal bones, phalanges), 2. the success rate of autosomal STR typing from ancient DNA extracted from the temporal bones, teeth and femurs can be compared to the rate from small bones of the hands and feet, and 3. the success rate of autosomal STR typing has a strong positive relationship with the amount of extracted ancient DNA). We included 93 bone samples from 13 different skeletons obtained from archaeological sites in Slovenia. Six different skeletal elements were selected from each of the skeletons (temporal bone, teeth, femur, metacarpal bones, metatarsal bones, phalanges). We successfully obtained full or partial genetic profiles from at least one sample per skeleton while half of the samples weren’t eligible for genetic typing. The results confirmed all 3 of our hypothesis. The amount of preserved ancient DNA extracted from the temporal bones, teeth and femurs was comparable to the amount extracted from small bones of the hands and feet. The success rate of autosomal STR typing was comparable between the selected skeletal elements, and had a strong positive relationship with the amount of extracted ancient DNA. These findings suggest it would be appropriate to expand the sampling of skeletal elements for genetic typing of old skeletal remains and include the small bones of the hands and feet. To substantiate the findings of this study further research is needed using a larger amount of skeletons and small bones of the hands and feet.
E.01 National awards
COBISS.SI-ID: 3898388For extracting genomic DNA from Second World War (WWII) victim’s remains, a highly efficient extraction protocol was developed in our laboratory and presented on the conference in Portugal. We analysed more than 150 bones and teeth from WWII mass graves to evaluate this method. Procedures for processing the bone and tooth samples including mechanical and chemical cleaning, cutting and powdering in the presence of liquid nitrogen, complete demineralization of bone and tooth powder, DNA extraction, DNA purification using magnetic beads, DNA quantification and typing, were shown. The most appropriate types of bones and teeth for genetic analyses were described and the measures for preventing contamination in the DNA laboratory were listed. Some examples of identification of the Second World War mass grave victims were presented.
B.03 Paper at an international scientific conference
COBISS.SI-ID: 33673177The 3-day international training course “Ancient DNA extraction course” organised for the forensic researchers from Italy was performed in the Laboratory of Molecular Genetics at the Institute of Forensic Medicine, Faculty of Medicine, University in Ljubljana in October 2017, under organization and leadership of project leader. The training course included experimental individual work with ancient bones and provided the participants first-hand knowledge of how to perform bone DNA typing. Procedures for processing the bone sample (mechanical and chemical cleaning, cutting, and grinding into the powder in presence of liquid nitrogen), decalcification of bone powder, DNA extraction, DNA purification, DNA quantification with real-time PCR, DNA typing of nuclear STRs, electrophoretic separation of amplified fragments and evaluation of DNA typing results were shown on concrete ancient bone samples and the most of the steps were experimentally performed by participants. The course was designed to deliver advanced level training to experienced laboratory based scientists that are familiar to DNA typing technologies. The unique training course was performed in the forensic molecular genetic laboratory equipped specially for processing old bones and teeth.
F.18 Transfer of new know-how to direct users (seminars, fora, conferences)
COBISS.SI-ID: 33490649The master thesis was performed in the Laboratory of Molecular Genetics at the Institute of Forensic Medicine, Faculty of Medicine, University in Ljubljana, under mentorship of project leader. In forensic genetics investigations identification of missing persons is still a challenge, because skeletonized human remains, which we focused on in this thesis, are often in bad condition when discovered, which makes it harder to perform a sampling that would optimize analysis and give the most quality results. Preservance of deoxyribonucleic acid (DNA) in skeletal remains depends on individually specific factors, such as biological sex and age and even more on environmental factors and post mortem interval. In this thesis we analyzed three approximately 70 year-old skeletons, total of 168 skeletal elements from hidden Second World War mass grave Huda jama. With the help of parameters, such as quantity of DNA, degradation rate and typing success, we tried to find the best types of elements to identify the victims. Analysis was composed of cleaning and grinding bones and teeth, extraction and purification of DNA, its quantification with real time polymerase chain reaction (RT-PCR), analysis of short tandem repeats (STR) and creation and interpretation of autosomal genetic profiles. The best parameters were quantity (in nanograms of DNA per gram of bone or tooth powder) and typing success which we defined as a number of successfully amplified loci for specific skeletal elements in three of the examined skeletons. Best 15 skeletal elements to use as a source of DNA are metacarpal bones (I-V), metatarsal bones (I-V), intermediate cuneiform, distal phalanx (foot), talus, petrous bone and tibia from all of which we were able to create complete genetic profile in all three examined skeletons. If we expand this list to skeletal elements that generated partial profiles with drop out of only one STR locus, we can add capitate, first proximal phalanx of both foot and hand, medial cuneiform, patella, navicular, 1st distal hand phalang, calcaneus, lateral cuneiform and cuboid. We also created genetic profiles of everyone involved in any step of the analysis to compare them to sample profiles and exclude potenatial contamination.
D.10 Educational activities
COBISS.SI-ID: 4611185