The survival of PCR-amplifiable DNA in cow leather

We have investigated the survival of PCR-amplifiable mitochondrial and nuclear DNA in a small number of modern and medieval bovine leather samples. The results of this preliminary investigation demonstrate that, while no nuclear DNA can be PCR-amplified from any of the specimens, mitochondrial DNA can be amplified from all samples. To investigate this contrasting pattern of DNA survival further, we have vegetable-tanned cow skin in our own laboratory, and directly assayed the survival of PCR-amplifiable mitochondrial and nuclear DNA at each step of the process. The results indicate that nuclear DNA is reduced to sub-amplifiable levels as a result of the tanning baths, whereas amplifiable mitochondrial DNA survives the complete process. Our results suggest that old and archaeological bovine leather may represent a useful source of genetic information, although this information will most likely be limited to that which can be gained from mitochondrial DNA.     

Molecular markers for the discrimination of Triticum turgidum L. subsp. dicoccum (Schrank ex Schübl.) Thell. and Triticum timopheevii (Zhuk.) Zhuk. subsp. timopheevii

The persistent uncertainty on the classification of the “new” glume wheat found in Neolithic and Bronze Age sites from Greece and other European settlements might be resolved only through analysis of its ancient DNA. Tools able to discriminate among different Triticum species on the basis of scarce, very damaged DNA, are therefore essential. While current attempts concentrate on DNA fragments sequencing and comparison, in some instances PCR-based selective amplification techniques might offer a cheaper and quicker alternative. The purpose of this research was therefore the identification of species-specific primers, able to distinguish caryopses of Triticum timopheevii subsp. timopheevii from those of Triticum turgidum subsp. dicoccum. Primers and their working conditions were defined and optimized using DNA from modern accessions. The ribosomal primers ITS1 tim and ITS2 tim, and the nuclear primer acetyl-coenzyme A tim clearly discriminated the sequences of Triticum timopheevii from other species. Finally, Neolithic charred wheat grains found in the sites of Sammardenchia (Pozzuolo del Friuli, Udine) and La Marmotta (Lago di Bracciano, Roma), belonging to the “new” wheat type or to emmer, were tested with the three selected primers. However, the results were not conclusive, because the samples analysed were apparently too degraded to yield useful DNA.     

Ancient DNA (aDNA) studies of man and microbes: general similarities,specific differences

The study of ancient DNA (aDNA) using molecular methods is an increasingly valuable tactic in bioarchaeology. While this method must be carefully undertaken to ensure that the molecules detected are representative of the ancient sample and not modern contaminates, there is a danger that a ‘one size fits all’ approach to validation will lead to misinterpretation and/or missed opportunities of valuable findings. When comparing human and pathogen aDNA, there are many shared technical means that can ensure best practice. However, there are a number of assumptions that should not be used for both scenarios. We discuss these aspects in reference to a recent article published by this journal and highlight some of the latest advances in molecular detection of ancient pathogen DNA that can further improve this endeavour. Copyright © 2009 John Wiley & Sons, Ltd.     

Amplification and sequencing of Trichuris trichiura ancient DNA extracted from archaeological sediments

Despite high prevalence of Trichuris trichiura infection, PCR-based analysis on T. trichiura from archaeological samples has not been established so far. In the present study, we sought to perform PCR-based amplification of T. trichiura aDNA using the sediments from medieval tomb of Korea. The presence of Trichuris eggs were first detected by microscopic observation; then confirmed by PCR-based aDNA analysis. Obtained sequence showed 100% homology to that of the small subunit ribosomal RNA (SSUrRNA) gene of T. trichiura but distinct from that of other Trichuris species. PCR-based aDNA analysis in this study can serve as effective method to confirm the presence of T. trichiura eggs in the soils or coprolites collected from archaeological sites.     

Ultrastructure and DNA sequence analysis of single Concentricystis cells from Alta Val Tiberina Holocene sediment

We successfully combined ultrastructure and chloroplast DNA analysis for single specimens of Concentricystis isolated from Holocene sediment by a palynology method based on filtration, progressive sieving and percoll-gradient sedimentation to concentrate fossil cells. We compared our method with the traditional harsh chemical treatment commonly used to isolate fossil pollen and spores. With our method, the cytoplasm of Concentricystis was sufficiently preserved to distinguish several cell structures by light and electron microscopy. DNA analysis of Concentricystis involved PCR amplification using specific primers for a spacer region of the chloroplast genome. Significant homologies were found with the Angiosperm chloroplast genome by BLAST DNA search for PCR product sequences.     

Variable nucleotide tandem repeat (VNTR) typing of two palaeopathological cases of lepromatous leprosy from Mediaeval England

Using ancient DNA methods, we have examined in detail two archaeological cases of leprosy from Mediaeval England. The first was a child skeleton with rhino-maxillary changes typical of lepromatous leprosy (LL). The second case was the skeleton of a male adult who showed both typical rhino-maxillary changes and osteitis/periostitis on the leg and foot bones. Bone powder was sampled from both cases and DNA extracts were prepared. These were subjected to a series of polymerase chain reactions (PCRs) specific for regions on the Mycobacterium leprae genome. The repetitive element RLEP was used for confirmation of M. leprae DNA and then three polymorphic regions were successfully amplified and sequenced to determine the number of variable nucleotide tandem repeats (vntr) at these loci. These were the microsatellite regions ML2344 and ML2172 and the minisatellite region ML0058. Genotyping data from the strains preserved within the skeletal remains were compared with those obtained for a reference strain of M. leprae. Variation at these three loci was found between both burials and the reference strain, indicating that vntr typing of LL cases from the archaeological record is a useful way of confirming disease and an additional means of authenticating aDNA data. This demonstrates the feasibility of targeting multiple loci for phylogenetic studies of leprosy strains from archival sources.     

A Mediaeval Case of Lepromatous Leprosy from 13–14th Century Orkney, Scotland

Erosion in the 1960s resulted in exposure of human skeletal remains from a Norse Christian cemetery at Newark Bay, Orkney, Scotland. One set of remains showed osteological evidence of advanced lepromatous leprosy, but the absence of bones from the lower limbs precluded definitive diagnosis. The aim of the present study was to determine whether Mycobacterium leprae could be detected in bone extracts, as a means of confirming the diagnosis of leprosy. Bone samples were examined from the suspected leprosy case and from a second contemporary burial thought to be free of disease. DNA was amplified by polymerase chain reaction (PCR) using primers specific for a repetitive element (RLEP) characteristic of M. leprae. Additional PCR tests specific for Mycobacterium tuberculosis and for amelogenin (a human gene suitable for sex determination) were also applied to the samples. M. leprae DNA was detected only in the skull sample from the suspected leprosy case. The DNA sequence was identical to that found in present day isolates of M. leprae. Positive results were obtained only using a PCR reaction designed to amplify relatively short stretches of DNA (<175 bp), suggesting the microbial DNA had undergone extensive fragmentation. There was no evidence of M. tuberculosis DNA in bones from the leprosy suspect or control individual. The ability to recover ancient samples of DNA provides an opportunity to study long-term evolutionary changes that may affect the epidemiology of microbial pathogens.     

Mycobacterium leprae genotype amplified from an archaeological case of lepromatous leprosy in Central Asia

We have amplified Mycobacterium leprae DNA from the skeleton of an adult human female exhibiting signs of lepromatous leprosy (LL). The remains were excavated from the site of Devkesken 6 on the Ustyurt plateau of Uzbekistan and date to between the 1st and 4th centuries AD. Recovered DNA was fragmented but of sufficient quality and quantity to allow a series of biomolecular genotyping methods to be applied. These methods included variable nucleotide tandem repeat (VNTR) typing of two microsatellite and one minisatellite regions and also single-nucleotide polymorphism (SNP) typing for nine informative loci.Genotyping showed that the causative strain of M. leprae exhibited a SNP-type 3 profile, characteristic of cases associated geographically with Europe and North Africa. Further SNP sub-typing was performed and the data obtained from the Uzbek leper was compared with the same loci amplified from a case of LL recovered from Blackfriars, Ipswich, UK dating to between the 13th and 16th centuries AD. Unique group 3 subtypes were found in both the Uzbek case and Ipswich 1914. These appear to be ancestral to recent type 3 strains. Mycolic acid analysis confirmed the presence of M. leprae in the Uzbek samples. Phylogenetically informative SNPs and other polymorphic loci will contribute to the study of human migrations, as well as the origin and spread of leprosy.     

An improved PCR method for endogenous DNA retrieval in contaminated Neandertal samples based on the use of blocking primers

Neandertal skeletal remains are usually contaminated with modern human DNA derived from handling and washing of the specimens during excavation. Despite the fact that the distinct Neandertal haplotypes allow the design of specific primer pairs, for instance in most of the mitochondrial DNA (mtDNA) hypervariable region 1 (HVR1), the human contaminants can often outnumber the endogenous DNA, thus preventing a successful retrieval of Neandertal sequences. We have developed a novel PCR method, based on the use of blocking primers that preferentially bind to modern human contaminant DNA and block their amplification, and greatly improve the efficiency of Neandertal DNA retrieval. We tested the method in four El Sidrón Neandertal samples (two teeth and two bone fragments) with different contamination levels and taphonomic conditions, and we have been able to significantly increase the Neandertal yield from figures around 25.23% (5–69.6%) up to 90.18% (75.3–100%).     

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.