DNA fromMycobacterium tuberculosisIdentified in Mediaeval Human Skeletal Remains Using Polymerase Chain Reaction

Journal of Archaeological Science (1996) 23, 789–798

DNA from Mycobacterium tuberculosis Identified in Mediaeval Human Skeletal Remains Using Polymerase Chain Reaction G. Michael Taylor* Division of Biochemistry and Genetics, Institute of Child Health, 30 Guilford Street, London WC1N 1EH, U.K.

Mary Crossey Department of Medicine, St Mary’s Hospital, Paddington, London W2 1NY, U.K.

John Saldanha National Institute of Biological Standards and Control, South Mimms, Herts, U.K.

Tony Waldron Institute of Archaeology, University College, 31–34 Gordon Square, London WC1H 0PY, U.K. (Received 31 August 1995, manuscript accepted 26 October 1995) A polymerase chain reaction (PCR) method has been used to confirm the presence of tuberculosis bacterial DNA in extracts of three human bone specimens with lesions suggestive of this disease. A fused wrist bone and a lumbar vertebra from the mediaeval period were found to be positive and an adjacent lumbar vertebra was weakly positive. A control vertebra from the same period (1350–1538 ) from an individual with bone lesions characteristic of syphilis was negative. Against a background of partially degraded DNA, nested PCR was found to be superior for detecting tuberculosis bacterial sequences compared with reamplification of first-round products or modification of other PCR stages. DNA survival in bone was assessed using PCR for a human-specific region of the Alu repetitive short interspersed nucleotide elements (SINES) and the ability to amplify a single copy gene. Genomic DNA was found in all bones examined but the single copy gene did not amplify from the vertebra weakly positive for tuberculosis or the control. This may represent relatively poor DNA preservation in these samples. These findings corroborate the use of PCR as an aid in the study of infectious disease in ancient bone samples and stress the importance of assessing native DNA survival. ? 1996 Academic Press Limited Keywords: ANCIENT DNA, TUBERCULOSIS, POLYMERASE CHAIN REACTION.

30% of modern cases. The central nervous system, adrenal glands, middle ear, kidney and skeleton are all susceptible to infection although involvement of some tissues, for example kidney and bone, may only occur some months or years after the primary infection (Smith, Starke & Marquis, 1992). Except in unusual circumstances, such as with mummified remains, soft tissue lesions leave no trace for the archaeologist to find, but diagnostic lesions on bones have been seen from neolithic Europe and in many other parts of the world (Ryan, 1994). In modern series, the skeleton is usually involved in less than 10% of cases with spinal involvement in up to 50% of these cases, followed next in frequency by other load-bearing

Introduction uberculosis is a disease of considerable antiquity, which in some cases, may give rise to characteristic skeletal changes. Lesions suggestive of tuberculosis have been recognized in the archaeological record for many years and have been described in some detail (Ortner & Putschar, 1981). Disease in man is usually the result of infection with either Mycobacterium tuberculosis or with M. bovis. Infection may spread from a primary focus in the abdomen or lungs to other parts of the body via blood or lymph. Non-respiratory tuberculosis occurs in up to

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*Author for correspondence.

789 0305-4403/96/050789+10 $18.00/0

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Figure 1. Fused wrist bones, Royal Mint site, London. Dated between 1350 and 1538.

joints such as the hip and knee (Smith, Starke & Marquis, 1992). The skull and non load-bearing bones are less frequently affected. There is speculation that the tuberculosis bacterium has remained almost unchanged at the genetic level for many thousands of years (Kapur, Whittam & Musser, 1994), but it is difficult to say whether pathogenicity to man has changed over time. It has been suggested that human tuberculosis may have arisen originally from the species infecting cattle, M. bovis (Manchester, 1984) which may have passed into early farming and herding communities where man was in close contact with domesticated animals. It seems likely, therefore, that tuberculosis could have become prevalent from the neolithic period onwards, once a sedentary lifestyle was adopted and various animal species domesticated. Risk of infection in man would increase due to settlement density, standards of hygiene, methods of animal husbandry and ingestion of untreated dairy products. There was a great increase in the incidence of the disease in western Europe following the plagues of the early middle ages and it reached epidemic proportions during periods of urbanization and the industrial revolution of the 18th and 19th centuries. For the palaeopathologist, the diagnosis is usually dependent upon the skeletal evidence. Unambiguous diagnosis may be difficult in poorly preserved specimens, or in those exhibiting pseudopathology or damaged during excavation. The differential diagnoses to be considered, particularly with tuberculous spondylitis are syphilis, brucellosis and pyogenic osteomyelitis (Ortner & Putschar, 1981; Stirland & Waldron, 1990). The latter two conditions may be associated with new bone formation, allowing them to be distinguished from tuberculosis. Tumours, granulomas and leukaemic deposits invading the vertebrae may mimic the

lytic erosions due to cold abscess formation in the vertebral bodies seen in tuberculosis. The polymerase chain reaction (PCR) is a means of amplifying DNA sequences using specific nucleotide primers and a thermostable DNA polymerase enzyme (Mullis & Faloona, 1987). It is becoming an increasingly important part of the science of molecular archaeology and is a useful addition to the diagnosis of infectious disease in animal and human remains. It has been successfully used to confirm disease in museum exhibits (Marshall et al., 1994), in soft tissue remains (Salo et al., 1994) and in bone (Spigelman & Lemma, 1993; Rafi et al., 1994). A positive result with PCR can confirm the presence of a bacterial DNA sequence in ancient material but a negative result cannot rule out the possibility of a prior, healed infection or poor DNA preservation in the sample. To resolve some of these alternatives, we have used a sensitive nested PCR method to examine mediaeval bone samples with lesions suggestive of tuberculosis for evidence of the causative bacterial DNA sequences. We have also assessed the preservation of human genomic DNA in the same bone samples using PCR to amplify the Alu family of short interspersed nucleotide DNA repeat elements (SINES) as well as a single copy gene.

Methods Bone samples The specimens studied for tuberculosis DNA included a fused wrist and two adjacent lumbar vertebrae. The wrist (Figure 1) came from the skeleton of a male aged at least 45 years at the time of his death. The specimen comprised the left third metacarpal, the underlying captitate and the trapezium, trapezoid and triquetral

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Figure 2. Two lumbar vertebrae, L1 and L2. Royal Mint site, London. Dated between 1350 and 1538. Sampling sites (arrowed) can be seen distal to the lytic lesions.

bones. It was clear that the second and fourth metacarpals had at one time been fused to the carpus but had been broken off some time after death. There was a small amount of reactive new bone formed on all surfaces of the carpals. The shaft of the metacarpal and the head were normal and there were no signs of infectious disease elsewhere in the skeleton. The two lumbar vertebrae (L1 and L2, Figure 2) came from the skeleton of a male aged between 15 and 25 years at death. The most obvious pathological feature was the presence of large lytic lesions in the bodies of both with a minimum of reactive new bone formation. The posterior elements of the vertebrae were normal but there was some marginal osteophyte around one of the two. There was no evidence of pathology elsewhere in the skeleton. A lumbar vertebra from an individual with skeletal lesions suggestive of syphilis served as a control. All specimens came from the large graveyard overlying part of the Black Death cemetery on the site of the old Royal Mint, London. The cemetery was associated with the Abbey of St Mary Graces, the last Cistercian foundation in England which was founded in 1350 and lasted until the dissolution in 1538. Extraction of DNA from bone specimens Bone samples were removed by sterile scalpel blade, weighed, washed in sterile saline to remove surface contamination and when necessary, immersed in liquid

nitrogen to make them friable. They were then ground in a clean pestle and mortar to produce a bone powder. Approximately 200–300 mg of powder was incubated in 1 ml of 4  guanidinium thiocyanate (Sigma-G6639) buffer containing 50 m Tris-HCl pH 7·0, and 20 m EDTA for up to 12 h at 55)C to extract DNA. After centrifugation at 10,000 g for 10 min, the supernatant was transferred to a separate set of tubes and 500 ìl of phenol:chloroform:isoamyl alcohol (25:24:1, SigmaP3803) was added to remove protein. After centrifugation the upper, aqueous phase was pipetted into another set of tubes and 0·5 ml of DNA-bind (silica diatoms, Sigma D-5384 suspended in guanidinium buffer) was added. Thereafter the procedure of Carter and Milton (1993), was followed to wash the resin and elute any DNA. Briefly, this consisted of washing the diatoms in 50% ethanol containing 200 m Tris-HCl, 10 m EDTA at pH 7·4, followed by washing in 1 ml of acetone and air drying. DNA bound to the diatoms was eluted in 100 ìl DNAse-free water at 65)C. This was stored at "20)C until assay. An extraction blank, i.e. omitting bone powder, was carried through the same procedure to ensure no contamination of buffers had taken place. Where possible, two extracts were prepared on separate occasions from bone samples to compare and confirm results of the PCR methods. PCR methods Avoidance of contamination. Stringent precautions were taken to avoid contamination during extraction of

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bone specimens and in the PCR reaction. These included the use of DNAse and RNAse-free Molecular Biology Grade chemicals and autoclaving of water and buffer solutions when appropriate. Disposable test tubes and pipette tips were also autoclaved before use. Extraction of bone samples was performed at a separate work station to the PCR amplification and electrophoresis. The surfaces of bone samples were washed prior to extraction in sterile saline to remove any DNA acquired from handling. Disposable gloves were worn during subsequent procedures and changed frequently. An extraction blank was taken through the same procedure and PCR was performed on the silica diatom water eluate. A further PCR blank was always included in each assay. This consisted of all PCR components but with water in place of the DNA template. PCR was performed in a laboratory not previously used for either tuberculosis or other microbiology research. Positive control DNA was amplified on the minimum number of occasions required to establish the methods. Pipettes dedicated only to PCR were used to dispense liquids and pipette tips containing a filter were used to avoid contamination of the pipettes by aerosol vapour from PCR products. As many physical barriers as possible were placed between PCR reagents and possible sources of contamination. Tuberculosis PCR. The target for amplification by PCR was the repetitive element in the tuberculosis genome known as insertion sequence 6110 (IS 6110). The sequence of this mobile, multicopy gene was first reported in 1990 (Thierry et al., 1990a). The sequence of PCR primers used (IS-1 and IS-2) were as previously reported (Walker et al., 1992). These primers amplify a 123 bp region of IS 6110 from nucleotides 762–884 inclusive and they were a generous gift from Dr Rory Shaw, St Mary’s Hospital, Paddington, London, who also provided a positive control, M. tuberculosis H37Rv DNA, for optimizing the method. The latter was used at a final concentration of 13·80 ng/25 ìl in PCR reactions. To increase sensitivity and specificity of the tuberculosis PCR a second set of primers was designed and synthesized ‘‘in-house’’. The sequences of these primers was IS-3; 5*-TTC-GGA-CCA-CCA-GCACCT-AA-3* and IS-4; 5*-TCG-GTG-ACA-AAGGCC-ACG-TA-3*. These bind internally (with some overlap) to IS-1 and IS-2 and amplify a 92 bp product. Human Genomic DNA PCR. To assess the preservation of DNA in ancient bone samples as well as the extraction process and removal of PCR inhibitors, we amplified various ‘‘housekeeper’’ human genes. The main gene chosen for this was the Alu family of repetitive SINE (short interspersed repeat DNA’s) elements. A number of PCR primers have been described for Alu amplification (Nelson, Ledbetter & Corbo, 1989) and several of these were evaluated. These primers were designed to amplify regions between the Alu sequences and give rise to thousands of

products which are apparent as a ‘‘streak’’ after gel electrophoresis of PCR products. Primers evaluated included: (1) 517 with the sequence 5*-AGC-TCCGCG-GAT-CTc/t-g/aGC-TCA-CTG-CAA-3*; (2) TC-65, 5*-AAG-TCG-CGG-CCG-CTT-GCA-GTGAGC-CGA-GAT-3*; (3) Alu 5*, 5*-GGA-TTA-CAGGCG-TGA-GCC-AC-3* and (4) Alu 3* with the sequence 5-GAT-CGC-GCC-ACT-GCA-CTC-C-3* (Tagle & Collins, 1992). Primers 517 and TC 65 were used singly as they will amplify between inverted Alu sequences (Nelson, Ledbetter & Corbo, 1989). The positive control in the Alu PCR was partially-purified human genomic DNA which was prepared from white blood cells by a standard molecular biology method (Davis, Dibner & Battey, 1986) and diluted 1/10 to give 2 ng/5 ìl. A further human gene, known as MPV-17, served as an additional housekeeper to check for human DNA in bone extracts. This is present as a single copy in the human genome (Karasawa et al., 1993) and we had PCR primers which amplify a 450 bp length product. The sequence of the two primers was 5*-CGC-AAG-TGT-TAA-TTT-GTT-CCT-3* and 5*-TTT-GTC-TCC-AAC-TGT-TGG-TAA-3*. DNA amplification (a) Tuberculosis. The amplification reaction used 25 pmol of each primer in a volume of 1·25 ìl. The final concentrations of the buffer components were 10 m Tris-HCl, pH 8·3, 50 m KCl and 1 m magnesium chloride (PCR-II kit, Sigma Chemical Company, Poole, Dorset, U.K.) with 200 ì of each deoxynucleotide (Pharmacia, Uppsala, Sweden) and 1·5 units (0·3 ìl) of a thermostable DNA polymerase (Bioline, Finchley, London, U.K.). Bone extract or positive control tuberculosis DNA was added in a volume of 5 ìl and the volume was then made up to 25 ìl with high performance liquid chromatography (HPLC) grade water. Blank tubes, with water in place of the DNA sample, were always included in all amplifications. The reaction was overlaid with 40 ìl mineral oil to prevent evaporation (Sigma-M5904). The PCR conditions for IS-6110 DNA amplification were an initial denaturation at 94)C for 4 min after which the polymerase was added (‘‘hot start’’) and this was followed by 35 cycles of denaturation at 94)C for 40 s, primer annealing at 68)C for 1 min and extension for 1 min at 72)C. In the nested PCR an aliquot of the first round products (0·25 ìl, 1%) was transferred to a second tube with fresh reaction components, DNA polymerase and primers IS-3 and IS-4. The annealing temperature for these primers was 58)C. In preliminary experiments the number of cycles in the second round of amplification was investigated over the range 15–35 after first round amplification with 25 cycles. Optimum amplification was found after a further 25 cycles and this was used in subsequent experiments with nested primers. These steps were performed in an Omnigene Thermal Cycler (Hybaid Ltd., Teddington, Middlesex, U.K.).

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(b) Alu and MPV-17. PCR for Alu consisted of 35 cycles of denaturation at 94)C for 30 s, annealing at 58)C for 30 s followed with 60 s elongation at 72)C. An increment of 5 s per cycle was added to the elongation time to allow for synthesis of longer products. Primers 517 and TC 65 used alone and primers 5* and 3* used together gave an expected ‘‘streak’’ of products with partially-purified genomic DNA as template. For the MPV-17 gene primers, the annealing temperature was 56)C, otherwise conditions were as for tuberculosis PCR. In initial experiments in all PCR methods, the concentration of magnesium in the reactions was optimized over the range 1·0–3·0 m in 0·5 m increments. All other buffer components were as for the tuberculosis method. Band-stab PCR On some gels after electrophoresis, the expected product was just visible but was not of sufficient intensity to be photographed. This was particularly the case after first round amplification with tuberculosis primers on the diseased bone samples. To overcome this problem the gel was washed in distilled water, blotted with Whatman filter paper and under UV illumination a sterile needle was used to ‘‘stab’’ the relevant product band. The needle was then used to inoculate a further PCR tube containing all the reagents, less DNA template. This tube was subjected to a further 20–30 cycles of amplification with fresh polymerase enzyme and the products run on agarose gel. Gel electrophoresis Amplification products were run in a 3% agarose gel (Sigma-A9539) using a Mini Sub-Cell DNA apparatus with a model 200/2.0 power source, both from Bio-Rad Laboratories, Hemel Hempstead, Herts., U.K. Eight microlitres of PCR product were loaded into preformed wells in the gel, together with 4 ìl of loading buffer. This comprised 50% glycerol, 1#TAE buffer (1·6  Tris, 0·8  sodium acetate, and 40 m EDTA, adjusted to pH 7·4 with acetic acid) and 1% Orange G (Sigma O-3756) as a dye front marker. At least one lane of the gel contained a 100 bp DNA ladder (GibcoBRL, U.K.) to size the products. The gel was overlaid with 1#TAE electrophoresis buffer and run for 3 h at 45 volts. DNA products were visualized by staining the gel in 200 mls TAE buffer with 10 ìl of ethidium bromide (Sigma-E7637, 10 mg/ml in TAE buffer) for 30 min at room temperature. DNA bands visualized under ultraviolet light were photographed with a Polaroid camera. DNA sequencing PCR products from samples which were positive by nested PCR were used to inoculate several further reactions and reamplified for a further 25 cycles. The

products were separated by electrophoresis on a 3% agarose gel. After ethidium bromide staining the bands of expected size (92 bp) were cut from the gel with a sterile scalpel blade and the freeze-squeeze technique (Horton & Pease, 1991) was used to recover the product from the agarose. DNA in these extracts was pooled, precipitated with an equal volume of isopropanol and centrifuged for 15 min at 10,000#g. The small visible pellet was washed with 75% ethanol, air dried and the residue taken up in a final volume of 25 ìl of autoclaved DNAse-free water and stored at "20)C. PCR products (approximately 200 fmoles) were directly sequenced using the Stratagene Cyclist= Exo- pfu DNA sequencing kit (Stratagene, U.S.A.) and 35 S dATP. The sequencing cycles consisted of 95)C for 1 min , followed by 30 cycles of 95)C for 30 s, 63)C for 20 s and 72)C for 20 s with a final cycle of 95)C for 30 s, 63)C for 20 s and 72)C for 3 min. PCR products from the wrist bone and the vertebra L1 were sequenced with primers IS-3 and IS-4. In later sequencing reactions, the annealing temperature was increased to 67)C and the number of cycles reduced to 25 in order to eliminate cross-banding.

Results Optimization of PCR methods (1) Tuberculosis. The optimum Mg2+ concentration was found to be 1·0 m and this was used in subsequent experiments. Additional bands of larger size than the expected 123 bp product were seen at higher Mg2+ concentrations. These probably reflect the two first-round primers binding to different copies of the insertion sequence and amplifying the DNA between them. In initial experiments it was also shown that the primers did not amplify any products when human genomic DNA was used as a template (data not shown). Using the nested primers and H37RV DNA as template the predicted band of 92 bp was seen after gel electrophoresis. The magnesium ion concentration was optimal at 1·5 m. (2) Alu and MPV-17 PCR for human DNA. Various primers were tried to optimize the Alu PCR method. These included primers 5* and 3* used together on human genomic DNA at various Mg2+ concentration and primers 517 and TC 65 used alone. The latter two primers bind to the same region of the Alu consensus sequence but amplify in opposite directions (Nelson, Ledbetter & Corbo, 1989). With all these approaches a ‘‘streak’’ of products is the correct result, due to the large number of copies of Alu in the human genome and the high molecular weight nature of the products. As the Alu PCR methods appeared comparable, Alu PCR with primer 517 was chosen to assess DNA survival in the bone samples. The PCR method for the single copy gene MPV-17 was found to be optimal at a

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Figure 3. Tuberculosis PCR. Gel electrophoresis on 3% agarose gel of reamplified first round products (5 ìl), using original primers IS-1 and IS-2. Lanes 1 and 2: two extracts from fused wrist. Lanes 3 and 4: two extracts from L1. Lanes 6 and 8: two extracts from L2. Lane 7: positive control. Lanes 10 and 11: band-stab PCR products from fused wrist extract (lanes 1 and 2). Lane 12: band-stab of first round positive control. Lane 13: reamplified water blank. DNA standards (100 bp ladders) are present in lanes 5 and 9. Arrow shows position of putative 123 bp product.

2+

Mg concentration of 2 m. No PCR product was found at concentrations below 1·5 m (data not shown). PCR results Tuberculosis. In preliminary experiments the tuberculosis PCR produced a complex pattern of products when applied to the bone extracts. The wrist and lumbar vertebrae showed indistinct bands of around 120 bp, 250 bp and some fainter, higher molecular weight products (not shown). The impression gained was that there was an indistinct product present at or near the expected size (123 bp) but this was too faint to convince or to photograph well. Reamplification of first round products with fresh reaction components did not product much improvement and resulted in ‘‘streaking’’ of the positive control and some extracts. This is seen in Figure 3, lanes 3, 4 and 7. The positive control run in lane 7 had previously produced a strong band of 123 bp on first round amplification. A band stab of the supposed tuberculosis PCR product just

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Figure 4. Tuberculosis PCR. Gel electrophoresis on 3% agarose of products after 50 cycles of amplification with primers IS-1 and IS-2. Cycling parameters were modified to minimize 72)C elongation step. Lanes 1 and 2; two extracts from fused wrist. Lane 3: L1. Lane 4: L2. Lane 5: control mediaeval vertebra. Lanes 6, 9 and 10: extracted blanks. Lane 11: water blank. Lane 12: positive control DNA. DNA 100 bp ladders are present in lanes 7 and 13. Arrow shows position of expected 123 bp product.

detected on first round amplification from the wrist and lumbar vertebra 1 all resulted in a single band of expected size (Figure 3, lanes 10, 11). Further improvement of first round PCR was found by reducing the 72)C extension step to 20 s and increasing cycle number to 50. This produced bands of correct size in one of the two wrist extracts and in the lumbar vertebrae. Resolution of product from primers and low molecular weight DNA fragments remained poor, however. This is seen in Figure 4. Two extracts of the control vertebra, the extracted blanks and the water blank were all negative. At this stage it was decided to design internal ‘‘nested’’ primers for the tuberculosis product. The results of nested PCR with these new primers is seen in Figure 5. Bands of correct size were now clearly seen in both wrist extracts (lanes 1 and 2) as well as in the lumbar vertebrae (lanes 3 and 4). Both extracts prepared from the control vertebra were again negative for tuberculosis as were the extraction blanks, prepared at the same time, and the water blank. Two extracts prepared from the fused wrist were amplified on three separate occasions using nested PCR with the same result. Similarly, the results from L1 and

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Figure 5. Tuberculosis nested PCR. Gel electrophoresis on 3% agarose after 25 cycles of amplification with IS-1 and IS-2 followed by 25 cycles of second round amplification with primers IS-3 and IS-4. Lanes 1 and 2: two extracts from fused wrist. Lane 3: L1. Lane 4: L2. Lane 5: control vertebra. Lanes 6–8: extracted blanks. Lane 9: water blank (first round, reamplified). Lane 10: water blank (second round). Lane 11: positive control. Lane 12: nil. Lane 13: DNA 100 bp ladder. Lanes 14–15: nil. Arrow shows position of 92 bp product.

L2 were confirmed in two separate experiments. L1 was consistently shown to be more strongly positive than L2. Human DNA The PCR for human sequences using the Alu primer 517 is shown in Figure 6. Intense ‘‘streaks’’ of PCR products were seen in lanes 1–3 from samples of the fused wrist and both lumbar vertebrae. Two extracts from the control vertebra both showed fewer, discrete products after Alu PCR. For comparison, the pattern obtained from modern DNA is shown in lane 10. Extraction and water blanks were all negative. Amplification of MPV-17, a single copy gene, in bone extracts To establish the integrity of the DNA which had been detected by the Alu PCR experiments, all the bone extracts were probed for the presence of a single copy gene known as MPV-17. The results of this experiment are seen in Figure 7. Two lanes showed bands of product (450 bp) on the left of the gel. That in lane 1 is

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Figure 6. Alu PCR with primer 517. Gel electrophoresis on 3% agarose after 35 cycles of amplification. Lane 1: fused wrist extract. Lane 2: L1. Lane 3: L2. Lane 4: nil. Lanes 5 and 6: two extracts from control mediaeval vertebra. Lane 7: extracted blank. Lane 8: water blank. Lane 9: nil. Lane 10: positive control human genomic DNA (2 ng). Lane 11: DNA 100 bp ladder. Lanes 12–15: nil.

from the fused wrist sample. Lane 2 is from the lumbar vertebra (L1) which was also more strongly positive for tuberculosis on nested PCR. Lane 3 is the extract from the adjacent lumbar vertebra (L2) which was weakly positive for tuberculosis. It now seems this might be due to poor DNA recovery from this sample as no product was seen. A very faint band was present in one of the two extracts from the control vertebra (lane 4). Extraction and water blanks run in lanes 6 and 8 were negative. The results from the MPV-17 amplification matched very closely the findings of the Alu experiments, with better DNA survival in the fused wrist and L1 and poorer survival in L2 and in the control vertebra.

DNA sequencing The two 92 base products (from the wrist and vertebra L1) were directly sequenced using the two internal PCR primers, IS-3 and IS-4 to sequence both strands. In both cases, sequences were obtained starting 10–20 bases downstream from the sequencing primer and extending to within 5–10 bases of the end of the DNA template. The sequence of both products was identical

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Figure 7. MPV-17 PCR. Gel electrophoresis on 3% agarose after 35 cycles of amplification. Lane 1: fused wrist extract. Lane 2: L1. Lane 3: L2. Lanes 4 and 5: two extracts from control mediaeval vertebra. Lane 6: extracted blank. Lane 7: positive control human genomic DNA (2 ng). Lane 8: water blank. Lane 9: nil. Lane 10: DNA 100 bp ladder. Lanes 11–15: nil. Arrow shows position of expected 450 bp product.

to the published sequence of M. tuberculosis (Thierry et al., 1990a) with no insertions or deletions.

Discussion We have applied and refined some PCR methods for assessing the preservation of DNA remaining in human bone, as well as for a specific bacterial sequence (IS-6110) which would confirm previous infection with tuberculosis. The results confirm the diagnosis of tuberculosis in the three bones which date from the mediaeval period. The fact that one of the lumbar vertebrae was only weakly positive by PCR could reflect poor DNA preservation or unlucky choice of sampling area. The use of nested PCR (Figure 5) was far more satisfactory for amplification of tuberculosis DNA than increasing cycle numbers or reamplfying with first round primers (Figures 3 & 4). The use of nested primers also gives some confidence that the band amplified was in fact from IS-6110, as four probes (two outer and two inner) were able to hybridize under stringent conditions of a ‘‘hot start’’ PCR. The findings of the PCR were reproducible and all controls, including examining a vertebra from a

probable case of syphilis were always negative. For these reasons, and given the nature of the bone lesions, we consider it very unlikely that our findings were due to modern contamination. Sequencing of the PCR products amplified from both the wrist and L1 confirmed that they were identical with the published sequence of IS-6110. The conservation of this region of IS-6110 is consistent with an earlier report of the sequence amplified from a pre-Columbian mummy (Salo et al., 1994) and with the general lack of nucleotide variation in various genetic loci of contemporary strains of M. tuberculosis (Kapur, Whittam & Musser, 1994). The necessity for nested PCR to amplify ancient DNA is in agreement with the experience of Salo and colleagues who used this approach (Salo et al., 1994). The first round primers we used were first used by Eisenach and co-workers (Eisenach et al., 1990). The second round primers were designed in-house and generated a slightly smaller product (92 bp) than those described by Salo (97 bp). We anticipate the sensitivity of the two methods would be broadly similar. The presence of tuberculosis DNA in bones from Turkey, Borneo and Scotland has also been reported in preliminary form (Spigelman & Lemma, 1993). The two samples from the fused wrist were taken from the base of the third metacarpal and from reactive bone formation around the triquetral bone. The samples from the lumbar vertebrae were removed from areas in the vertebral bodies posterior to the lytic lesions. Interestingly, although these were adjacent vertebrae, tuberculosis DNA and indeed human genomic DNA appeared to be better preserved in L1. Whilst we were able to confirm the diagnosis of tuberculosis in these two cases, a negative result would not necessarily have ruled this out, as bacteria may have ceased to exist before the death of the sufferer. It is also possible that bones without obvious lesions may produce a positive result in persons exposed to the bacterium but without obvious clinical disease. The PCR methods will sometimes detect the bacterium in clinical samples from apparently well relatives of those with tuberculosis (Walker et al., 1992). We have not as yet established the limits of detection of our tuberculosis PCR but some indication may be inferred from previous papers. Based on these reports, where nested PCR for tuberculosis was used, we feel our method would enable the detection of DNA in fg amounts (Miyazaki et al., 1993; Thierry et al., 1990b; Eisenach et al., 1990). Some workers maintain that if first round PCR is optimized there is little need for nested PCR. In the present study, we found the use of inner primers was superior for amplifying the specific product against a background of degraded DNA. Band-stab PCR is a reasonable alternative as it amplifies only a particular band after purification on the gel but is always open to the criticism of contamination by diffusion from the positive control.

DNA from Mycobacterium tuberculosis 797

The specificity of the tuberculosis PCR method for IS-6110 has been confirmed by others (Eisenach et al., 1990; Thierry et al., 1990b). It will detect all strains of M. tuberculosis, M. bovis, M. microti as well as M. simae and M. africanum. The last three species are of academic interest only, and can be virtually excluded as causes of skeletal tuberculosis in mediaeval England. We cannot say whether the disease in these two individuals was caused by the human or bovine form of tuberculosis, although this may eventually be possible using techniques such as random amplified polymorphic DNA analysis or restriction fragment length polymorphism (Linton et al., 1995). Preliminary experiments in an independent laboratory indicate the presence of tuberculosis DNA from other regions of the genome in the extracts from these bones but have not as yet been able to type the organism. The PCR method we have used will not detect the other species of Mycobacteria such as M. kanasaii, M. marinum, M. scrofulaceum, M. gordonae, M. avium, M. fortuitum, M. chelonei and M. intracellulare and M. paratuberculosis. From what we know of modern cases of tuberculous spondylitis, the disease is only very rarely the result of atypical mycobacterial infection in man (Miller et al., 1994). The Alu PCR is a very useful candidate for assessing human DNA. Firstly, there are nearly a million copies of Alu in the human genome (Nelson, Ledbetter & Corbo, 1989). Secondly, using a primer for the 31 bp conserved region (e.g. primer 517) has the advantage that human-specific sequences can be amplified against a complex background, including DNA from other species. This is particularly useful in the case of human vertebrae which we found inevitably contained traces of soil. Even with washing before extraction, we found it was impossible to remove all traces of soil once it was in the spongy matrix. Fewer PCR products were noted after Alu PCR of the control vertebral extract (Figure 6). This appeared to correlate with failure to convincingly amplify the single copy gene in the same extracts. The amplification of a housekeeper gene is useful not only in determining DNA survival but also in ruling out the presence of PCR inhibitors in bone extracts which might mask a positive result for the sequence of interest. In Alu PCR experiments with primer 517, about 2 ng of modern DNA was added as a positive control. This always produced a strong signal. The level of DNA from the bone extracts never quite matched this level of intensity but was of a similar order. This would suggest a recovery of around 40 ng of human DNA in total from each bone extract. This would approximate to between 40–120 ng DNA per g of bone powder extracted. We emphasize this is only an estimate as the Alu PCR is not a quantitative method. Amplification from Alu consensus sequences might be a useful first step in looking for other sequences of interest, for example X or Y chromosome or mutations in specific genes. The latter could conceivably find applications in the study of population migrations.

The single-copy MPV-17 gene amplified from two of the Royal Mint site bones, being detected in the wrist lesion and in L1. It was not seen in L2. This would agree with the results of the Alu PCR which in some gel runs generally showed a fainter streak of products after amplification. The MPV-17 gene was also not detected in two separate extracts prepared from the control vertebra of comparable age. This was also taken from a burial from the Royal Mint site and was from an individual with skeletal lesions characteristic of syphilis. During the course of this study we became aware of a paper by Brown, O’Donoghue & Brown (1995). This group extracted and amplified DNA from human cremated bones from an early Bronze age site at Bedd Branwen, Wales. Interestingly, they used identical extraction methods with silica diatoms as well as PCR for a region within the Alu consensus sequence to confirm survival of human DNA. In early experiments we also used the same primer pair but abandoned these for the use of primers which amplify DNA between the repeated elements, as this appeared to provide more information on DNA preservation. Broadly, their experience was very similar to ours in terms of quantity of DNA in their specimens. The polymerase chain reaction is finding many applications in the field of molecular archaeology and is beginning to make a real contribution to our understanding of past peoples, their diseases and population movements in antiquity (Hagelberg et al., 1994). DNA can be remarkably tenacious in bone samples, lasting tens of thousands of years given a suitable environment. For example Hoss & Paabo (1993) have recovered and sequenced DNA from Quagga, a Pleistocene horse dating back 25,000 years. For the paleopathologist, interpretation of bone lesions provides some evidence for disease but it is often not possible to make a definitive diagnosis. Interpretation of the lesions is also dependent on bone preservation and post-mortem events such as the ravages of colonizing bacteria, insects and other animals. In this study we have been able to confirm the diagnosis of tuberculosis in these bone samples, thus enabling us confidently to eliminate other conditions such as those caused, for example, by staphylococcal or fungal infections or tumours. It is likely that the use of PCR will enable a wider range of bacteria and viral infections to be diagnosed in ancient human remains and thus add to our understanding of diseases in the past.

Acknowledgements This study was undertaken by GMT and MC in partial fulfilment for the final year of the Diploma in Field Archaeology, Birkbeck College, University of London. Our grateful thanks to our course tutor, A. J. Legge, Reader in Archaeology, Centre for Extramural Studies, 26 Russell Square, London WC1.

798 G. M. Taylor et al.

We are also grateful to Paul Rutland, Department of Molecular Genetics, Institute of Child Health, London, who synthesized several of the primers used in this study and provided much helpful advice. We thank also Karen Johnstone, Molecular Medicine Unit, Institute of Child Health, who donated various Alu primers for us to evaluate.

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