Sex identification of ancient DNA samples using a microfluidic device

A microfluidic device has been developed for the sex identification of ancient DNA samples and works by manipulating liquids within an environment of micrometer dimensions. In this work a range of microfluidic DNA extraction methods were evaluated for their compatibility with ancient DNA samples, and the use of streptavidin-coated super paramagnetic particles to isolate biotin-labeled abasic sites within damaged DNA was shown to be the most reproducible. Polymerase chain reaction-based DNA amplification was possible on the microfluidic device when less than 50 pg of template DNA was added. As a proof-of-principle, powdered bone samples were analysed using the integrated methodology developed. Following conventional capillary gel electrophoresis, two out of the three samples produced positive amplification results and were successfully identified as female. These sex identifications were corroborated by independent Amelogenin, anthropological and Y chromosome analysis. The work reported here is the first step in the development of a complete miniaturized microfluidic system that would enable on-site ancient DNA analysis.     

Analysis of ancient mycolic acids by using MALDI TOF MS: response to “Essentials in the use of mycolic acid biomarkers for tuberculosis detection” by Minnikin et al., 2010

The mycobacterial cell wall is consists of several long chain lipid and glycolipid components. Mycolic acids are major and unique fraction of the cell envelope of mycobacteria that include the ancient causative agents of tuberculosis and leprosy. The analytical investigation of these lipid biomarker residues is one of the most promising perspectives in the field of molecular paleopathology. Recently published in this journal, Minnikin et al. present a systematic critique of the MALDI TOF MS analysis for determination of ancient mycolic acids, focusing on our previous paper. In this study, our mass spectrometric investigations by commonly used 2,5 DHB matrix were presented with our comments to the critique authors. On the base of our previous and recent mass spectrometric results we have to realize that the clinical protocols and standards cannot directly be used for the biomolecular paleopathological investigations. The applicability of the recent mycobacterial and clinical results is very limited in the biomolecular archaeology, thus the recent scientific results and protocols should be adapted carefully to bioarchaeological sciences.     

Kinship between burials from Grave Circle B at Mycenae revealed by ancient DNA typing

The richness of the burials in Grave Circle B at Mycenae, Greece indicates that the 35 people interred there held elite status during their lifetimes 3500 years ago. It has been speculated that the groups of burials represent different dynasties or branches of the same family. To test this hypothesis, we carried out an exhaustive ancient DNA (aDNA) study of 22 of the skeletons. We were unable to identify nuclear aDNA in any specimen, but we obtained authentic mitochondrial aDNA sequences for four individuals. The results were compared with facial reconstructions and interpreted within the archaeological context represented by the organisation of the graves and the positions of the burials within the graves. We conclude that the contemporaneous male Γ55 and female Γ58 skeletons, which both possess the UK mitochondrial haplogroup, were brother and sister. The implication is that Γ58 was buried in Grave Circle B not because of a marital connection but because she held a position of authority by right of birth. The results illustrate the difficulty in using aDNA to study kinship relationships between archaeological specimens, but also show that aDNA can advance understanding of kinship when used to test hypotheses constructed from other evidence.     

Digging deeper into the limits of ancient DNA research on syphilis

The search for the origins of syphilis has a long history in the medical and anthropological literatures. If we know more about the emergence of the pathogen that causes the disease in humans we will understand its evolution through time and space as well as shed light on its current state in living populations. Ancient DNA techniques used to isolate Treponema pallidum subsp. pallidum DNA from archaeological human specimens provide direct evidence of its existence in the past. However to date, only Kolman et al. (1999) have been successful in this endeavour, while other attempts have failed (e.g., Barnes and Thomas, 2006; Bouwman and Brown, 2005). Why has there been little success? This paper serves to compliment and add relevant information to Bouwman and Brown's and Barnes and Thomas' discussion concerning our inability to apply ancient DNA techniques to study venereal syphilis in past human populations.Our approach utilized 15 different human specimens from different geographies and different temporal periods: eight samples come from medically diagnosed individuals archived during the American Civil War period; six originate from the United Kingdom and predate 1492 with four of these samples having been previously analyzed by Bouwman and Brown and one sample comes from historic Canada. Human mitochondrial and amelogenin DNA, as well as several genes from the Treponema organism were analyzed revealing the relatively good preservation of human multi-copy and single copy DNA but not treponemal DNA. This study also incorporates a unique molecular experiment using rabbits infected with venereal syphilis to help illustrate that treponemal DNA disseminates to bone early during the first stages of infection but is not present in later stages of the disease using the techniques presented in this study.     

Biomolecular archaeology of ancient tuberculosis: response to “Deficiencies and challenges in the study of ancient tuberculosis DNA” by Wilbur et al. (2009)

It is sixteen years since the first detection of Mycobacterium tuberculosis DNA in archaeological specimens, yet the validity of findings continues to be questioned. Rigorous scientific scrutiny and debate is valuable and has led to a coalescence of procedures and precautions amongst those actively engaged in this work. It is disappointing that these good practices are not recognised by certain scientists whose primary expertise is in the related fields of archaeology, palaeopathology, and eukaryote ancient DNA. There is a danger that by constant repetition, disputable and inadequately justified concerns will assume the status of self-perpetuating myths and misunderstandings. We discuss these issues with reference to a recent article in this journal, in which clear peer-reviewed scientific data were specifically targeted as part of a general critique of the field of the palaeomicrobiology of tuberculosis. We believe we have given sufficient evidence and cogent argument to persuade the unbiased reader that the views in the critique by Wilbur et al. are unjustified.     

Improved methodology for extraction and amplification of DNA from single grains of charred wheat

Comparisons were made between two commonly used methods for the extraction of ancient DNA from charred plant remains. Using artificially charred wheat seeds, we show that silica-binding is the most efficient method for extraction of DNA. We describe a improved silica-binding procedure, including pre-incubation with N-phenacylthiazolium bromide and increased washing of bound DNA, which yields amplifiable DNA from seeds heated at 200 °C for up to 8 h, conditions which promote the formation of Maillard products which often copurify with aDNA and inhibit subsequent PCRs. We believe that this method will be effective in ancient DNA extraction with most types of charred archaeobotanical material. Both cold- and hot-start PCR procedures gave good amplicon yields with extracts prepared in this way, but cold-start PCRs also resulted in synthesis of short artefact products. Addition of bovine serum albumin to PCRs, an inert carrier substance thought to enhance amplification efficiency by binding contaminants, had no advantageous effect and in fact reduced amplicon synthesis.     

The use of the polymerase chain reaction (PCR) to detect Mycobacterium tuberculosis in ancient skeletons

The polymerase chain reaction has been used to extract ancient DNA from a wide range of different types of material. We have considered the possibility of finding residual bacterial DNA in bone that may have been infected with Mycobacterium tuberculosis using this technique. We propose a method of collecting samples and testing for the presence of degraded genetic material in ancient bone. The steps of the polymerase chain reaction are detailed and discussed, as are those for the preparation of the bone sample. Results obtained would suggest that this technique could have wide application in osteoarchaeological research by giving us new information on diseases of antiquity. Future avenues for the investigation of bacterial DNA in ancient bone are suggested.     

Ancient DNA in human bones from Neolithic and Bronze Age sites in Greece and Crete

Attempts were made to detect ancient DNA (aDNA) in samples of 88 human skeletons from eight Neolithic and Bronze Age sites in Greece and Crete. Ancient DNA was absent in specimens from Nea Nikomedia, Lerna, Karaviádena (Zakro), Antron Grave Circle A and Mycenae Grave Circle A. For each of three skeletons from Antron Grave Circle B that were sampled, polymerase chain reactions (PCRs) gave products for nuclear but not mitochondrial DNA, but amplicon yield was low and inconsistent with replicate PCRs failing to give reproducible results. With specimens from Mycenae Grave Circle B, evidence for mitochondrial aDNA was obtained for four of the 22 skeletons that were studied, and at Kouphovouno evidence for mitochondrial and/or nuclear aDNA was obtained with eight of the 20 skeletons that were examined. We conclude that, although aDNA might be present in some Eastern Mediterranean skeletons from later centuries of the Bronze Age, it is not commonly found in material from this period and is likely to be absent from older material.     

DNA of Mycobacterium leprae detected by PCR in ancient bone

We report the extraction and amplification of DNA of Mycobacterium leprae, from ancient skeletal material. The significance of the extraction of ancient bacterial DNA is discussed, as are future directions of research into ancient disease.     

Aspartic acid racemization variability in ancient human remains: implications in the prediction of ancient DNA recovery

The extent of racemization of aspartic acid (Asp) – expressed as d/l ratio – has been used as a marker of biomolecular degradation in ancient remains. However, Asp racemization rate is highly variable, and depends on biochemical and geochemical factors. In this paper we aim to determine to which extent the fraction analyzed and the kind of sample used may influence the d/l Asp ratios. Other factors, such as burial site and sample preservation conditions, are also considered.     

Accurate sex identification of ancient human remains using DNA shotgun sequencing

Accurate identification of the biological sex of ancient remains is vital for critically testing hypotheses about social structure in prehistoric societies. However, morphological methods are imprecise for juvenile individuals and fragmentary remains, and molecular methods that rely on particular sex-specific marker loci such as the amelogenin gene suffer from allelic dropout and sensitivity to modern contamination. Analyzing shotgun sequencing data from 14 present-day humans of known biological sex and 16 ancient individuals from a time span of 100 to ∼70,000 years ago, we show that even relatively sparse shotgun sequencing (about 100,000 human sequences) can be used to reliably identify chromosomal sex simply by considering the ratio of sequences aligning to the X and Y chromosomes, and highlight two examples where the genetic assignments indicate morphological misassignment. Furthermore, we show that accurate sex identification of highly degraded remains can be performed in the presence of substantial amounts of present-day contamination by utilizing the signature of cytosine deamination, a characteristic feature of ancient DNA.     

古陶瓷科学技术研究在科技考古中的三例应用

利用多元统计分析方法研究了从杭州古中河南段采集的月白釉瓷片,浙江临安天目山地区发现的黑釉瓷片和景德镇历代青花瓷片的胎、釉化学组成和青花色料的变化规律。从而讨论了杭州古中河南段采集的瓷片可能就是凤凰山下万松林附近的修内司官窑制品;指山临安天目山地区新发现的窑群和黑釉瓷才是名副其实的天目窑和天目瓷;发现了元代以及明初洪武和永乐朝青花瓷所用的料可能是来自西域的进口料,而宣德青花瓷所用的色料多数可能是国产的粘土矿。     

The molecular palaeoecology of geese: identification of archaeological goose remains using ancient DNA analysis

The remains of six species of geese are commonly recovered from archaeological sites in Britain dating from the Saxon and later periods. However, identification of this material to species level is hampered by a lack of morphological variation and a large overlap in size. To address this issue we obtained DNA sequence data for a section of the mitochondrial cytochrome b gene from modern samples of each species, and successfully identified several DNA markers for Branta species. No markers were found within the cytochrome b gene for the genus Anser. Ancient DNA techniques were then used to recover DNA from goose bones excavated from two archaeological sites. The DNA sequences enabled identification of Barnacle goose (Branta leucopsis) from one site and confirmed the presence of Anser species at another. © 1998 John Wiley & Sons, Ltd.     

A flock of sheep,goats and cattle: ancient DNA analysis reveals complexities of historical parchment manufacture

Parchments comprise one of the most common and valuable sources of archaeological and historical data. Previous studies have shown that parchment also preserves genetic data. These data could be valuable for population studies, to understand past animal husbandry, the development of breeds and varieties and to comment on the provenance of parchments. To improve our understanding of DNA contained in parchments, we analysed genetic data, including both mitochondrial and autosomal loci, from 18th to 19th century English parchments which stable isotope analysis had indicated were well-preserved. DNA results were unexpected. All but one of the parchments produced multiple sequences matching several different species. Ion beam analysis ruled out surface treatments of the parchments (including ink and animal glues) as the origin of these multiple sequences. Our results suggest that the DNA content of parchment is more complex than previous research has suggested and that multiple stages of parchment manufacture, treatment and storage are preserved in parchment DNA extracts.     

Aspects of ancient Greek trade re-evaluated with amphora DNA evidence

Ancient DNA trapped in the matrices of ceramic transport jars from Mediterranean shipwrecks can reveal the goods traded in the earliest markets. Scholars generally assume that the amphora cargoes of 5th-3rd century B.C. Greek shipwrecks contained wine, or to a much lesser extent olive oil. Remnant DNA inside empty amphoras allows us to test that assumption. We show that short ∼100 nucleotides of ancient DNA can be isolated and analyzed from inside the empty jars from either small amounts of physical scrapings or material captured with non-destructive swabs. Our study material is previously inaccessible Classical/Hellenistic Greek shipwreck amphoras archived at the Ministry of Culture and Tourism Ephorate of Underwater Antiquities in Athens, Greece. Collected DNA samples reveal various combinations of olive, grape, Lamiaceae herbs (mint, rosemary, thyme, oregano, sage), juniper, and terebinth/mastic (genus Pistacia). General DNA targeting analyses also reveal the presence of pine (Pinus), and DNA from Fabaceae (Legume family); Zingiberaceae (Ginger family); and Juglandaceae (Walnut family). Our results demonstrate that amphoras were much more than wine containers. DNA shows that these transport jars contained a wide range of goods, bringing into question long-standing assumptions about amphora use in ancient Greece. Ancient DNA investigations open new research avenues, and will allow accurate reconstruction of ancient diet, medicinal compounds, value-added products, goods brought to market, and food preservation methods.     

Wild or domesticated: DNA analysis of ancient water buffalo remains from north China

Recent zooarchaeological studies on water buffalo (Bubalus sp.) remains from China and south Asia question the traditional view that water buffalo were first domesticated in Neolithic China over 7000 years ago. The results from several recent population genetic studies of modern domesticated buffalo (Bubalus bubalis) are not consistent with each other, placing the original center of buffalo's domestication in south Asia, southeast Asia, or China. This paper reports a study using an ancient DNA approach to analyze water buffalo remains from Neolithic sites in north China to investigate their affinities with modern domesticated water buffalo, and to shed light on the origin of modern domesticated water buffalo in China.A 169 bp fragment of D-loop mitochondrial DNA was successfully amplified and verified for 13 of 24 bone samples obtained from seven archaeological sites along the Wei River valley in Shaanxi Province, China. The bone samples which yielded positive DNA can be dated to 8000–3600 cal. BP. The phylogenetic analysis of the obtained DNA sequences along with modern water buffalo sequences indicated that the ancient water buffalos were not the direct ancestor of modern domesticated water buffalo. However, the phylogenetic analysis, along with BLAST searches of these ancient DNA sequences, did demonstrate their relatedness to water buffalo more so than to any other bovid species, confirming the existence of indigenous wild (but now extinct) water buffalo species (B. mephistopheles) in ancient China.The DNA analysis of these ancient remains failed to establish direct links between modern domesticated water buffalo (B. bubalis) and indigenous water buffalo (B. mephistopheles) from ancient China. If further DNA studies of more ancient remains from other regions of China confirm the observation of solely indigenous water buffalo species in ancient China, it would suggest modern water buffalo might not have been first domesticated in China.     

Fossil dogs and wolves from Palaeolithic sites in Belgium,the Ukraine and Russia: osteometry,ancient DNA and stable isotopes

Using multivariate techniques, several skulls of fossil large canids from sites in Belgium, Ukraine and Russia were examined to look for possible evidence of the presence of Palaeolithic dogs. Reference groups constituted of prehistoric dogs, and recent wolves and dogs. The fossil large canid from Goyet (Belgium), dated at c. 31,700 BP is clearly different from the recent wolves, resembling most closely the prehistoric dogs. Thus it is identified as a Palaeolithic dog, suggesting that dog domestication had already started during the Aurignacian. The Epigravettian Mezin 5490 (Ukraine) and Mezhirich (Ukraine) skulls are also identified as being Palaeolithic dogs. Selected Belgian specimens were analyzed for mtDNA and stable isotopes. All fossil samples yielded unique DNA sequences, indicating that the ancient Belgian large canids carried a substantial amount of genetic diversity. Furthermore, there is little evidence for phylogeographic structure in the Pleistocene large canids, as they do not form a homogenous genetic group. Although considerable variation occurs in the fossil canid isotope signatures between sites, the Belgian fossil large canids preyed in general on horse and large bovids.     

Essentials in the use of mycolic acid biomarkers for tuberculosis detection: response to “High-throughput mass spectrometric analysis of 1400-year-old mycolic acids as biomarkers for ancient tuberculosis infection” by

High molecular weight long-chain mycolic acids are key structural components of the cell envelope of Mycobacterium tuberculosis and they are established as biomarkers for the identification of both ancient and modern tuberculosis. Mycolic acids from M. tuberculosis have a characteristic profile, reflecting contributions from five major distinct homologous series of mycolate structural types. Diagnosis of tuberculosis in archaeological material, using mycolic acid biomarkers, depends on objective recognition of the key characteristic mycolic acid components. A recent article in this journal claimed that tuberculosis could be confirmed in ancient bones by high throughput mass spectrometric analysis of mycolic acids. Scrutiny of the data presented reveals no convincing evidence for the presence of mycolic acids, characteristic of the M. tuberculosis complex, in the skeletal remains examined. This communication reviews the essential criteria necessary for positive tuberculosis diagnosis, using mycolic acids.