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.     

古代人骨的分子考古学研究现状与展望

本文重点讨论分子生物学在考古学研究中的应用。根据对人类学研究的回顾与展望,在以研究人类的起源和进化为首要任务的人类学研究领域,由于现代分子生物学理论和方法的应用,为人类学的发展提供了科学可信的研究方法和具发展前景的研究方向。     

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.     

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.     

A first prehistoric case of tuberculosis from Britain

A likely case of tuberculosis in an Iron Age human burial from Dorset, England is described. Osteological examination and biomolecular study support the diagnosis. A radiocarbon determination indicates a date range for the burial of BC 400–230. This case represents the earliest reported case of tuberculosis from Britain, and indicates that the disease was present here prior to the Roman invasion. Copyright © 2003 John Wiley & Sons, Ltd.     

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.