Browsing by Author "Varljen, Tatjana (24734171400)"

Previous studies have revealed that the European Roma share close genetic, linguistic and cultural similarities with Indian populations despite their disparate geographical locations and divergent demographic histories. In this study, we report for the first time Y-chromosome distributions in three Roma collections residing in Belgrade, Vojvodina and Kosovo. Eighty-eight Y-chromosomes were typed for 14 SNPs and 17 STRs. The data were subsequently utilized for phylogenetic comparisons to pertinent reference collections available from the literature. Our results illustrate that the most notable difference among the three Roma populations is in their opposing distributions of haplogroups H and E. Although the Kosovo and Belgrade samples exhibit elevated levels of the Indian-specific haplogroup H-M69, the Vojvodina collection is characterized almost exclusively by haplogroup E-M35 derivatives, most likely the result of subsequent admixture events with surrounding European populations. Overall, the available data from Romani groups points to different levels of gene flow from local populations. © 2010 Wiley-Liss, Inc.

Previous studies have revealed that the European Roma share close genetic, linguistic and cultural similarities with Indian populations despite their disparate geographical locations and divergent demographic histories. In this study, we report for the first time Y-chromosome distributions in three Roma collections residing in Belgrade, Vojvodina and Kosovo. Eighty-eight Y-chromosomes were typed for 14 SNPs and 17 STRs. The data were subsequently utilized for phylogenetic comparisons to pertinent reference collections available from the literature. Our results illustrate that the most notable difference among the three Roma populations is in their opposing distributions of haplogroups H and E. Although the Kosovo and Belgrade samples exhibit elevated levels of the Indian-specific haplogroup H-M69, the Vojvodina collection is characterized almost exclusively by haplogroup E-M35 derivatives, most likely the result of subsequent admixture events with surrounding European populations. Overall, the available data from Romani groups points to different levels of gene flow from local populations. © 2010 Wiley-Liss, Inc.

Tissue formalin fixation and paraffin embedding (FFPE) is a standard method for long-term preservation and morphological and molecular analysis. The aim of this study was to analyze the effect of storage time on the integrity of DNA isolated from three different healthy FFPE tissues. DNA was isolated from FFPE heart, liver and brain tissues obtained from autopsy and archived from 1988 to 2017 using two different methods of DNA isolation: phenol–chloroform-isoamyl alcohol (PCI) and PureLink Genomic DNA Kit. The quantification and purity of DNA was measured spectrophotometrically at 260 nm and 280 nm. The quality of isolated DNA was evaluated by PCR amplification of GPD1 (150 bp), ACTB (262 bp) and RPL4 (407 bp) genes. The histomorphological characteristics of FFPE tissues were not significantly changed during 30 years of storage. Higher yield (272.9 ± 10.3 µg) and purity (A260/280 = 2.05) of DNA was obtained using the PCI method for DNA isolation from FFPE liver tissue. The PCI extraction method showed reproducible and consistent results in PCR amplification of all of three examined genes. The GPD1 gene can be amplified up to 30 years, the ACTB gene in the same samples up to 26 years and the RPL4 gene up to 6 years of storage in FFPE blocks. Although the best yield and purity of isolated DNA (using both isolation methods) was obtained from FFPE liver tissue, the DNA with the most preserved integrity was obtained from brain tissue archived up to 30 years. This is the first report using long-term archived healthy FFPE tissues (up to 30 years) that shows that the DNA isolated from these tissues is of preserved integrity and can be used in molecular autopsy. © 2020, Springer Science+Business Media, LLC, part of Springer Nature.

To develop in house protocol for DNA analyses of contact traces, we conducted a series of experiments using low copy number (LCN-PCR) [P. Gill, Application of low copy number DNA profiling, Croatian Med. J. 42(3)(2001) 229-232] amplification of DNA isolated from touched objects. In each experiment, touched objects were swabbed using double swab technique, DNA was extracted by organic extraction protocol, and pooled DNA extracts from both wet and dry swabs were amplified using AmpFlSTR Identifiler kit in 34-cycle PCR. In the first part of this study, seven volunteers held sterile plastic tubes for 10 s, 15 min after washing their hands. From seven tested subjects, we recovered three full and four partial profiles. One of the partial profiles differed from volunteer's reference profile. In the second experiment, volunteers held each other's ankles in order to investigate success of DNA analyses from material transferred by interpersonal skin contact. Mixtures of both persons were obtained from all swabs, and the ratio of each person in the isolate, depended both on shedding status, as well as on hand dominance. The third part of this research tested the effect of the period after the deposition on both quantity and quality of DNA extract. Full DNA profiles were obtained even after 24 h since deposition for two good shedders. In conclusion, LCN-PCR technology provides a valuable approach in DNA typing of trace amounts of biological material, left on even shortly touched objects. © 2008.

To develop in house protocol for DNA analyses of contact traces, we conducted a series of experiments using low copy number (LCN-PCR) [P. Gill, Application of low copy number DNA profiling, Croatian Med. J. 42(3)(2001) 229-232] amplification of DNA isolated from touched objects. In each experiment, touched objects were swabbed using double swab technique, DNA was extracted by organic extraction protocol, and pooled DNA extracts from both wet and dry swabs were amplified using AmpFlSTR Identifiler kit in 34-cycle PCR. In the first part of this study, seven volunteers held sterile plastic tubes for 10 s, 15 min after washing their hands. From seven tested subjects, we recovered three full and four partial profiles. One of the partial profiles differed from volunteer's reference profile. In the second experiment, volunteers held each other's ankles in order to investigate success of DNA analyses from material transferred by interpersonal skin contact. Mixtures of both persons were obtained from all swabs, and the ratio of each person in the isolate, depended both on shedding status, as well as on hand dominance. The third part of this research tested the effect of the period after the deposition on both quantity and quality of DNA extract. Full DNA profiles were obtained even after 24 h since deposition for two good shedders. In conclusion, LCN-PCR technology provides a valuable approach in DNA typing of trace amounts of biological material, left on even shortly touched objects. © 2008.

The aim of this study is to investigate the effects of formalin fixation on the degradation of DNA molecules in five different healthy tissues exempted during the autopsy, as well as the selection of the method that is most suitable for the DNA isolation. Heart muscle, liver, brain, lung and kidney tissue obtained from the healthy people who suddenly died from a violent death were used. The parts of tissue were fixed in 10% phosphate-buffered formalin as well as in 4% unbuffered formalin at room temperature. Morphology of tissue was studied using H&E staining. The DNA was isolated 6 h, 1–7 days (every 24 h), 10, 14, 28 days and 2 months after fixation using two different methods: extraction with phenol-chloroform-isoamyl alcohol as well as with PureLink Genomic DNA Kit. Yield and purity of the DNA samples were measured spectrophotometrically at 260 nm and 280 nm. The PCR amplifications of the glycerol-3-phosphate dehydrogenase 1 (GPD1, 150 bp), ß actin (ACTB, 262 bp) and ribosomal protein L4 (RPL4, 407 bp) genes were performed to evaluate the degree of DNA fragmentation. The RPL4 gene was amplified up to 72 h, ACTB gene up to 14 days and GPD1 gene up to 28 days from tissue fixed in phosphate-buffered formalin using phenol-chloroform-isoamylalcohol protocol for DNA isolation. Liver and kidney gave better results of PCR amplification, but statistical significance between tissues was not found. Preserving period, fixative and DNA extracting method are important factors for successful PCR amplification. The healthy tissue, fixed in phosphate-formalin up to 28 days, can be useful source in molecular studies. Tissues fixed in unbuffered formalin are suitable for molecular analysis up to 7 days. © 2018 Elsevier GmbH

The aim of this study is to investigate the effects of formalin fixation on the degradation of DNA molecules in five different healthy tissues exempted during the autopsy, as well as the selection of the method that is most suitable for the DNA isolation. Heart muscle, liver, brain, lung and kidney tissue obtained from the healthy people who suddenly died from a violent death were used. The parts of tissue were fixed in 10% phosphate-buffered formalin as well as in 4% unbuffered formalin at room temperature. Morphology of tissue was studied using H&E staining. The DNA was isolated 6 h, 1–7 days (every 24 h), 10, 14, 28 days and 2 months after fixation using two different methods: extraction with phenol-chloroform-isoamyl alcohol as well as with PureLink Genomic DNA Kit. Yield and purity of the DNA samples were measured spectrophotometrically at 260 nm and 280 nm. The PCR amplifications of the glycerol-3-phosphate dehydrogenase 1 (GPD1, 150 bp), ß actin (ACTB, 262 bp) and ribosomal protein L4 (RPL4, 407 bp) genes were performed to evaluate the degree of DNA fragmentation. The RPL4 gene was amplified up to 72 h, ACTB gene up to 14 days and GPD1 gene up to 28 days from tissue fixed in phosphate-buffered formalin using phenol-chloroform-isoamylalcohol protocol for DNA isolation. Liver and kidney gave better results of PCR amplification, but statistical significance between tissues was not found. Preserving period, fixative and DNA extracting method are important factors for successful PCR amplification. The healthy tissue, fixed in phosphate-formalin up to 28 days, can be useful source in molecular studies. Tissues fixed in unbuffered formalin are suitable for molecular analysis up to 7 days. © 2018 Elsevier GmbH

Southeastern Europe and, particularly, the Balkan Peninsula are especially useful when studying the mechanisms responsible for generating the current distribution of Paleolithic and Neolithic genetic signals observed throughout Europe. In this study, 404 individuals from Montenegro and 179 individuals from Serbia were typed for 17 Y-STR loci and compared across 9 Y-STR loci to geographically targeted previously published collections to ascertain the phylogenetic relationships of populations within the Balkan Peninsula and beyond. We aim to provide information on whether groups in the region represent an amalgamation of Paleolithic and Neolithic genetic substrata, or whether acculturation has played a critical role in the spread of agriculture. We have found genetic markers of Middle Eastern, south Asian and European descent in the area, however, admixture analyses indicate that over 80% of the Balkan gene pool is of European descent. Altogether, our data support the view that the diffusion of agriculture into the Balkan region was mostly a cultural phenomenon although some genetic infiltration from Africa, the Levant, the Caucasus, and the Near East has occurred. © 2010 Wiley-Liss, Inc.

Southeastern Europe and, particularly, the Balkan Peninsula are especially useful when studying the mechanisms responsible for generating the current distribution of Paleolithic and Neolithic genetic signals observed throughout Europe. In this study, 404 individuals from Montenegro and 179 individuals from Serbia were typed for 17 Y-STR loci and compared across 9 Y-STR loci to geographically targeted previously published collections to ascertain the phylogenetic relationships of populations within the Balkan Peninsula and beyond. We aim to provide information on whether groups in the region represent an amalgamation of Paleolithic and Neolithic genetic substrata, or whether acculturation has played a critical role in the spread of agriculture. We have found genetic markers of Middle Eastern, south Asian and European descent in the area, however, admixture analyses indicate that over 80% of the Balkan gene pool is of European descent. Altogether, our data support the view that the diffusion of agriculture into the Balkan region was mostly a cultural phenomenon although some genetic infiltration from Africa, the Levant, the Caucasus, and the Near East has occurred. © 2010 Wiley-Liss, Inc.