We attempted DNA extraction from 14 bone and dental specimens found at Wadi Maqoch site, which is located in the Jordan Valley and dated to the Chalcolithic period. DNA was isolated from tiny amounts of bone and tooth powder following a slightly modified silica extraction protocol. Stringent precautions were undertaken to prevent possible contamination of the samples. Each DNA sample was subjected to a number of PCR reactions. Sex identification was performed using primers and conditions described by Sullivan and colleagues, but failed to provide positive results. Hypervariable segment I (HS-I) of the mitochondrial DNA control region was amplified in three overlapping PCR reactions. One of the DNA samples yielded amplification products of 153 bp, 232 bp and 271 bp that together comprised almost the complete HS-I. Automated sequencing revealed the following changes as compared to the Cambridge Reference Sequence: G to C at position 16,129, C to T at position 16,223. In addition, equal peaks of C and T were observed at position 16,311, suggesting possible heteroplasmy. Notably, the presence of heteroplasmy in position 16,311 has been already revealed in a number of studies. Cloning of PCR products will allow us to verify this finding. It is important to mention that two PCR reactions overlap at 16,223. We repeatedly observed a T at this position. Furthermore, none of the investigators in the laboratory carries this mtDNA haplotype or any of these polymorphisms. An extensive search in different mtDNA databases has revealed no exact matches to the sequence obtained in the Chalcolithic specimen. Three individuals (Moroccan Jew, Cantonese and Swiss) were found to differ at position 16,129 by possessing A and not C.
We are currently investigating the phenomenon of DNA decay in human bone with the aim of understanding the precise role played by environmental factors such as the action of soil bacteria, fungi, and other microorganisms. With this in view, we sampled a group of 32 human femurs dating back to 1800 ad and submitted them to a series of analyses to check the following parameters: state of preservation of collagen; racemization of aspartic acid; state of preservation of mitochondrial DNA; presence of bacterial DNA; presence of the DNA of lower eukaryotes. The results are consistent with the hypothesis that most of the endogenous human DNA vanishes long before the bone structure undergoes extensive diagenesis and soil microorganisms penetrate in a massive way.
In the last few years, identifications of drugs in archaeological human remains were reported several times but comments on the reliability of the data were often missing. To obtain valid data, in particular on nicotine residues in ancient bones, two skeletal series were analyzed and environmental influences on the results estimated in an exposure experiment. Bone samples from the early modern age (18th century, Goslar, Germany) and from the Bronze Age (Lichtenstein Cave, Germany) were analyzed for the tobacco alkaloid and its major metabolite, cotinine. In 22 out of 34 femur samples of the Goslar series, trace amounts of nicotine were found, but no cotinine, the major metabolite. Its finding would have proved the use of tobacco in the historic population, whereas the mere discovery of nicotine cannot discriminate between endogenous material and that resulting from a contamination during the sample treatment or museum storage. This points to the necessity of further pilot studies for long-term persistence, since it was not clarified whether insufficient accumulation or poor stability may have prevented the detection of cotinine. The magnitude of a possible nicotine deposition was estimated in a control exposure experiment. A Bronze Age bone sample that definitely contained no nicotine, as proven by pretests, was exposed to environmental tobacco smoke for six weeks and then analyzed. In one part, 11.6 ng g m 1 nicotine were estimated and, in another, washed before the analysis, 35.9 ng g m 1 were estimated. The higher amount of nicotine can be explained by the fact that tobacco smoke deposits were rinsed from the surface into the inner parts during the washing step. The results highlight the importance of additional analyses for metabolites; new patterns must be created that incorporate all now widespread drugs.
Here, we report new radiocarbon dates and palaeodietary data ( i 13 C and i 15 N measurements) from human bone collagen of two Early Neolithic populations in Primorye (Maritime) Province, in the Russian Far East. We found that the coastal people of the Boisman 2 site ( n =10) had isotope values consistent with a diet of (likely hunted) sea mammals, while the inland population of the Chertovy Vorota site ( n =2) had isotope values consistent with a diet that was a mixture of both terrestrial and marine (mostly fish) food resources. Our results suggest that advanced sea mammal hunting already existed in Primorye at ca. 5800 BP (ca. 6600 cal years BP). Marine reservoir corrections were required for the radiocarbon dates on the human remains, due to the consumption of "old" marine-based carbon.
When ancient DNA is obtained from dental remains by bone milling, crushing and sectioning, the morphologically informative crown and roots sustain damage which renders them of little or no further use to other investigators. The newly patented Reverse-Root-Canal technique makes it possible to obtain dentin rich in nucleic acids without damaging the teeth. This new non-destructive method to obtain dentin containing ancient DNA could, in effect, multiply the meager supply of ancient dental remains that are available. Keywords: Ancient DnaDentinReverse-root-canalShangTeeth
Cyprinid remains were collected from an archaeological site at Wolin, Poland. DNA has been extracted from a 1000 years old bone of common bream, and a 172 bp fragment of the cytochrome b gene of the mitochondrial DNA (mtDNA) successfully amplified by the polymerase chain reaction. The obtained fragment has been sequenced, and showed one point mutation (G M A) at the position 123 of the gene.
In order to determine whether the morphometric criteria used for separating wild and domestic caprines are expressed at the genetic level, Capra bones from the Neolithic site of Abu Gosh, located 12 km west of Jerusalem, Israel were examined. Archaeological excavations at the site revealed the presence of two occupations; the Pre-Pottery Neolithic B (PPNB) dated to circa 9500-8000 bp , and the Pottery Neolithic (PN) dated to circa 7500-5500 bp . The quantity of goat remains in the PPNB assemblage and their morphometric characteristics suggest that they represent wild animals undergoing an early phase of domestication. In contrast, the faunal remains from the PN levels are predominantly those of domestic goats ( Capra hircus ). For the purpose of examining the relationship between morphometry and genetics, mitochondrial DNA (mtDNA) was extracted and cytochrome b and D-loop sequences run on a small sample of caprine bones from both the PPNB and PN levels at the site. Overall, the ancient DNA findings agreed with those obtained from the morphometric studies and successfully distinguished wild from domestic goats. However, two species of wild goat--the bezoar goat ( Capra aegagrus ) and the Nubian ibex ( Capra ibex nubiana )--that were not differentiated on the basis of the morphometric analysis, were identified on the basis of their mtDNA sequences. This suggests that post-cranial morphometric criteria for distinguishing between these two species still need to be refined. The findings also show that at Abu Gosh the domestic goat, C. hircus , was established by the PN period, and that the genetic changes that accompanied the shift in status from wild to domestic goats occurred within a relatively short period of time; circa 2000 years.
We investigated 31 samples of charred archaeological cereal grains from the Northern Alpine region, dating from Neolithic to medieval times. One hundred and twenty five DNA extracts from 23 extraction series were screened for the presence of authentic DNA by PCR amplification of a 240 bp fragment from the high molecular weight glutenin subunit gene promoter (HGP) region. Criteria of authenticity adjusted to the features of charred cereal remains were applied. No PCR products were amplified in most extracts. Extraction series with positive results were further analysed with additional primer sets directed at the single-copy HGP region and with primers for the ribosomal (ITS2) or the chloroplast ( rbcL ) DNA. Most positive results of the HGP regions were subsequently considered not authentic, mainly because of molecular inconsistency. In four extracts the successful amplification of the rbcL region, the ITS2 region, or the HGP and the ITS2 region suggested the presence of authentic DNA but further and independent data would be needed to verify the results. In this study the unambiguous extraction of authentic DNA from charred cereal remains could not be shown.
One of the crucial steps during ancient DNA purification is the removal of inhibitory substances from hard tissue extracts. We present a cheap and easy-to-perform method for the purification of ancient DNA using nitrocellulose membranes in order to remove inhibitory inorganic (EDTA, calcium, etc.) and organic (humic acid, fulvic acids, etc.) compounds from tissue extracts in a single step procedure. Subsequent ethanol precipitation provides purified and concentrated ancient DNA, suitable for multiplex PCR amplification.
We prepared extracts from a sample of charred wheat grains, 2000 years in age, using a standard DNA preparation method in combination with various post-purification procedures designed to remove inhibitors of the polymerase chain reaction (PCR). The resulting extracts gave amplification products when PCRs were directed at the Glu genes, which are located on the wheat nuclear genome. However, repeated attempts to amplify the chloroplast rbcL locus were unsuccessful with these extracts, even though the rbcL PCR system gave positive results with as little as 8.3 pg of modern DNA, compared with a detection limit of 53 pg for the Glu PCRs. We conclude that there is a differential preservation of nuclear and chloroplast DNA in charred wheat grains.
The study is carried out on a randomly drawn sample of 200 individuals from the early medieval graveyard at Weingarten, Germany, dating from the 5th-8th century A.D. This skeletal population comprises 800 individuals, who show variation of their social standing through characteristic sets of grave goods that display wealth and rank differentials. Several DNA typing approaches are applied to reconstruct residence patterns in this socially stratified population. For one, the simultaneous amplification of nine autosomal short tandem repeats (STRs) generates the genetic fingerprint that is unique to an individual. Furthermore, through the analysis of Y-chromosomal STRs individuals of the same paternal lineage can be identified, whereas sequencing analysis of the hypervariable region of the mitochondrial DNA can determine which members of a population belong to the same matrilineage. Comparisons between the social groups will show if differing variabilities can be detected for the analysed DNA sequences. In the case of patrilocal residence, a high variability of mitochondrial DNA sequences should be detectable, whereas a population practising matrilocal residence should be discernible by a high number of deviating Y haplotypes. With the results of the ancient DNA typing, it should be determinable if different patterns of residence were applied in the social groups of this historical population.
Pompeii is a unique archaeological site because of the natural disaster occurred there makes it possible to perform many kinds of studies, including the biological and naturalistic. Morphological and morphometric analyses were conducted on five equine skeletons dated to 79 BC , found in a stable near the "Casti Amanti" house, to assess their taxonomic assignment. These studies classified four of the skeletons as donkeys and one as a mule. We report the molecular characterisation of the five equine skeletons based on their ancient DNA extraction and sequencing. Attention was focused on the D-loop region of mitochondrial DNA (mtDNA), compared to DNA from the living equine species Equus asinus and Equus caballus . The results of molecular analyses do not fit exactly with previous morphological and morphometric studies. Our data indicates that two of the skeletons could be horses or mules and three could be donkeys or hinnies. This result demonstrates the relevant contribution of modern molecular approaches to solve interesting taxonomic problems.
macromolecular material in the Murchison meteorite J.S. WATSON, M.A. SEPHTON, G.D. LOVE, A.B. VERCHOVSKY, C.E. SNAPE AND I. GILMOUR 1 Planetary and Space Sciences Research Institute, Open University, Milton Keynes, Buckinghamshire, MK7 6AA, United Kingdom (j.watson@open.ac.uk) 2 Fossil Fuels and Environmental Geochemistry, NRG, Drummond Building, University of Newcastle, Newcastle upon Tyne, NE1 7RU, United Kingdom 3 School of Chemical, Environmental and Mining Engineering, University of Nottingham, University Park, Nottingham, NG7 2RD, United Kingdom
Interspecific sequence polymorphisms in the mitochondrial cytochrome b gene were analyzed by PCR-RFLP to determine the species origin of Bronze Age animal skeletal remains. Existing techniques were refined by targeted primer design focusing on a DNA fragment shorter than 200 bp, an approach allowing us to identify up to six animal species at the same time. Possible contaminants, such as human DNA, were reliably ruled out. For routine applications in archaeometry, food or material analyses, PCR-RFLP may thus provide a simple alternative to sequencing of PCR products, allowing discrimination between species, even if the template DNA is degraded or contains traces of DNA from various species.
The study of ancient human remains can be approached today through polymerase chain reaction (PCR) analysis, but one of the main difficulties involves overcoming strong PCR inhibitions. We studied skeletal remains from six archaeological sites. The remains studied include a total of 27 individuals and span between 2500 and 400 years before the present. DNA was extracted from 35 samples and sex-specific amelogenin sequences were amplified to identify the sex of the samples. Only consistent results for which molecular and anthropometrical data agreed were considered. Notwithstanding the success of the sex identification, the samples were analysed using ultraviolet (UV) spectrophotometry and high performance liquid chromatography (HPLC) in order to establish a rapid protocol to predict the possible presence and state of ancient DNA. A combination of UV and HPLC peaks seems to indicate a strong degradation of endogenous DNA. A PCR inhibitor was also isolated through HPLC in a set of six samples.
We compared five silica-based methods for their efficiency at extracting DNA from human bones from 18th to mid-19th century London. Polymerase chain reactions (PCRs) were directed at two mitochondrial and one nuclear loci. Methods that used commercially available silica columns were more efficient than methods that used a silica slurry. Extraction with Qiagen QIAquick columns and the Qiagen QIAamp DNA Mini Kit were equally efficient, though we recommend the former as this method involves fewer steps.
