DNA Extraction - archeologia.unifg.it

CASE REPORT 

DNA Extraction 

An Anthropologic Aspect of Bone Remains From Sixth- to 

Seventh-Century AD Bone Remains 

Nunzio Di Nunno, MD, PhD,* Sandro Sublimi Saponetti, BSc,‡ Vito Scattarella, BSc,‡ 

Patrizia Emanuel, BSc,‡ Stefania Lonero Baldassarra, BSc,† Giuliano Volpe, BSc,§ 

and Cosimo Di Nunno, MD† 

Abstract: In the archeological site of the early Christian Episcopal 

complex of Saint Peter, in Canosa di Puglia (Bari, Italy), during the 

operations of archaeological excavations, tombs were discovered. 

They were dated between the sixth and seventh centuries AD with 

carbon 14 methodology. Five skeletons were found in the 5 tombs: 

28A: male individual, 43 years old. The height was 170 cm; the 

biomass was 65.7 kg. The analysis of the bones indicated several 

noteworthy pathologies, such as a number of hypoplasia lines of the 

enamel, the presence of Schmorl hernias on the first 2 lumbar 

vertebrae, and the outcome of subacromial impingement syndrome. 

28E was a male individual, with a biologic age of death of between 

44 and 60 years. The height was 177 cm. He had a posttraumatic 

fracture callus of the medial third of the clavicle, with an oblique 

fracture rima. 29B was a female individual, 44–49 years old. The 

height was 158.8 cm; the biomass was 64.8 kg. There was Wells 

bursitis on the ischial tuberosity on both sides. 29E was a male 

individual, 45–50 years old. The height was 169.47 cm; the biomass 

was 70.8 kg. The third and the fourth vertebrae showed Baastrup 

syndrome (compression of the vertebral spine). There were radiologic 

signs of deformity on the higher edge of the acetabula and 

results of frequent sprains of the ankles. 31A was a male individual, 

47–54 years old. The height was 178.65 cm; the biomass was 81 kg. 

The vertebral index showed a heavy overloading in the thoracic 

lumbar region. There were bony formations under the periosteum on 

both on the higher and medium facets of the first metatarsus and on 

the higher and lateral facets of the fifth metatarsus on both sides. 

As the topography indicates, these small ossifications coincided with 

the contact points between the back of the foot and parts of the upper 

shoe. From the osseous remains, in particular from the teeth (central 

Manuscript received December 19, 2005; accepted January 5, 2006. 

From the *Dipartimento di Scienze Pedagogiche, Psicologiche e Didattiche, 

Università degli Studi del Salento, Lecce, Italy; †Sezione di Medicina 

Legale, Di.M.I.M.P., ‡Sezione di Antropologia, Dipartimento di Zoologia, 

Università degli Studi di Bari, Bari, Italy; and the §Dipartimento di Scienze 

Umane, Università degli Studi di Foggia, Foggia, Italy. 

Reprints: Nunzio Di Nunno, MD, PhD, Dipartimento di Scienze Pedagogiche, 

Psicologiche e Didattiche, Università degli Studi del Salento, 

Via Mario Stampacchia 45, 73100 Lecce, Italy. E-mail: forem@ateneo. 

unile.it. 

Copyright © 2007 by Lippincott Williams & Wilkins 

ISSN: 0195-7910/07/2804-0333 

DOI: 10.1097/PAF.0b013e3181405f35 

incisors), the DNA was extracted and typed to identify potential 

family ties among all the subjects. The extraction technique used 

came from the DNA Promega technique, partially modified by the 

authors. Stay times of the sample in the extraction buffer were 

increased and were increased the polymerase chain reaction (PCR) 

cycles. 

Key Words: ancient bone remains, DNA extraction, DNA 

fingerprint, anthropology 

(Am J Forensic Med Pathol 2007;28: 333–341) 

As of 2001, systematic archeological digs have been carried 

out in the area of the hill at San Pietro in Canosa, in 

the suburbs to the southeast of the town. 1–5 These archeological 

investigations are being conducted jointly by the Universities 

of Foggia and Bari and by the Soprintendenza per i Beni 

archeologici della Puglia (Apulian Superintendency of Archeological 

Heritage). During the digs, many tombs have 

come to light, containing bone remains. These have recently 

started to be analyzed from the anthropological standpoint, 

performing DNA typing. First, a brief geographic and historical 

outline of the town of Canosa: It is situated in the south 

of Italy, in the Apulian region, approximately 400 km to the 

southeast of Rome, and is one of the most important archeological 

centers in Apulia and in south Italy in general. The 

town was already important in pre-Roman times, when 

Canosa was one of the richest and most powerful Daunian 

settlements, and it emerged even more during the Roman era. 

In the late part of the era, the town became the seat of the 

governors of the provincia Apulia et Calabria; thus, the 

regional capital. It very early assumed a leading function 

from the religious point of view, too, thanks to the presence 

of a large Christian community guided by powerful bishops, 

involved in important councils and diplomatic activities. The 

greatest moment for the Canosa church was in the sixth 

century AD, when it was presided over by the famous bishop 

Sabino, who is traditionally held to have governed the diocese 

for over 50 years (AD 514-566) and who set in motion the 

construction of a great number of holy buildings. 

The American Journal of Forensic Medicine and Pathology Volume 28, Number 4, December 2007 333

Di Nunno et al The American Journal of Forensic Medicine and Pathology Volume 28, Number 4, December 2007 

MATERIALS AND METHODS 

Anthropological Study 

An anthropological study was made of 5 adult individuals 

found in 3 tombs (nos. 28, 29, 31) at the Paleo-Christian 

archeological dig at San Pietro, in Canosa (Figs. 1–4). The 

skeletal remains were in a mediocre state of preservation and 

various parts were missing, especially in 2 of the tombs that 

showed taphonomic conditions referable to secondary deposits. 

The morphometric examinations were made according to 

the indications specified by Martin and Saller. 6 Determination 

of age and sex was based on the methodologies reported 

by Ferembach et al, 7 and Lovejoy et al. 8 The calculations of 

stature and biomass were made according to the methods 

established by Trotter and Gleser 9 and Ruff et al, 10 respectively. 

Assessment of the markers of nutritional and/or disease 

stress on the dental apparatus, in terms of periodontal 

disease and caries, was based on the indications by Brothwell 

11 ; tartar deposits and the degree of dental wear were 

FIGURE 1. The Paleo-Christian archeological dig at San Pietro, in Canosa. 

334 

analyzed following the indications given by Dobney and 

Brothwell 12 and Molnar, 13 respectively. The study of hypoplasia 

lines on the enamel was performed according to 

Goodman et al. 14 The investigation of skeletal markers of 

biomechanical stress, in terms of assessment of syndesmopathy, 

enthesopathy, supernumerary articular faces, and degenerative 

joint disease was carried out according to the methods 

suggested by Kennedy, 15 Lai and Lovell, 16 Robb and Mallegni, 

17 and Rogers et al 18 ; assessment of alterations of the 

spinal column was made following the indications by Borgognini 

Tarli and Repetto. 19 Geometrical techniques were 

applied on the transverse sections of the humerus and femur 

according to Larsen, 20 Capasso et al, 21 Ledger et al, 22 Stock 

and Pfeiffer, 23 and Tracey et al. 24 

DNA Typing 

The present study was conducted on 5 samples typed 

with short tandem repeat (STR) belonging to the CODIS 

system: CSF1PO, FGA, TH01, TPOX, vWA, D3S1358, 

© 2007 Lippincott Williams & Wilkins

The American Journal of Forensic Medicine and Pathology Volume 28, Number 4, December 2007 Ancient Bone Remains 

FIGURE 2. Particulars of the dig. 

FIGURE 3. The Paleo-Christian archeological dig at San Pietro, 

in Canosa, the tombs. 

D5S818, D7S820, D8S1179, D13S317, D16S539, D18S51, 

and D21S11. For samples with teeth, the teeth were treated as 

follows. They were first extracted from the dental arch and 

then washed first in water and then in a 10% ethanol solution 

to eliminate any bacterial contamination. Subsequently, a 

longitudinal incision was made of each tooth, using the 

laboratory drill KAVO EWL 3–36 V � 25.000/min 50 W, 

equipped with a diamond-point mill disk Mesinger 94 off HP 

220. The hemidental elements, with the root canal exposed, 

presented brownish residues of dental pulp; these were removed 

from the root canals using an odontic curette, ASA 

Stainless 1807-12, and were collected in 1.5-mL Eppendorf 

test tubes. As a preliminary step in the skeletal investigations, 

the femur shafts were cleaned with a brush and detergent, to 

remove fragments of earth and any tree roots adhering to the 

bone. After abundant washing to remove any residual detergent, 

the femur shafts were left to dry at room temperature. 

Later, electromechanical scraping of their surfaces was performed, 

using a Dremel. The bone structure was drilled and 

a window was made, from which a piece of compact bone 

8 � 3 cm in size was taken. The piece of bone was first 

broken into smaller pieces about 1 � 2 cm, using the Dremel. 

These pieces were placed in a Falcon test tube and repeatedly 

washed in deionized water until the washing water was 

perfectly clear. They were then washed in ethanol 90°C and 

then in ethanol 95°C. After completely drying the samples, 

they were reduced to powder. DNA extraction from the 

dental pulp samples was performed following a modified 

protocol of the Promega Kit “SV Total RNA Isolation System,” 

suitable for DNA extraction from samples containing 

only a small number of nucleated cells to begin with. The 

same method was used for the bone samples. The protocol 

was partially modified by lengthening the incubation time of 

the cell lysis step: each sample of dental pulp was placed, 

overnight, at room temperature, in a single microtube containing 

350 L of SV RNA Lysis Buffer. 

DNA extraction from the bone powder was performed 

using the commercial kit Geneclean for Ancient DNA 

Q-Biogene, following a protocol that we modified only as 

regards the first step: a quantity of 100–500 mg of bone 

powder is added to a 1.5-mL Eppendorf test tube containing 

1 mL of DeHybernation Solution. The microtube is left to 

incubate at a temperature of 45°C–60°C overnight and agitated 

occasionally. This step may sometimes need to be 

protracted for 24 hours. Amplification was obtained using a 

thermal cycler-DNA Gene Amp polymerase chain reaction 

(PCR) system 9700 (Applied Biosystems, Foster City, CA) and 

the AmpFlSTR Identifiler PCR Amplification Kit (Applied Biosystems). 

25,26 Analysis of the amplified allele fragments was 

performed by capillary electrophoresis using the ABI Prism 310 

Genetic Analyzer (Applied Biosystems) and the 310 Genetic 

Analyzer performance optimized polymer 4 (POP-4; Applied 

Biosystems), 10� Genetic Analyzer Buffer with EDTA, allele 

ladders, and GeneScan-500 (internal standard). 27–30 

Data Analysis of the DNA Typing 

The results obtained for the 5 samples were used to 

make a comparison with the haplotypes of present-day subjects 

belonging to the Apulian population, to assess genotypical 

and allelic homologies and differences. For this purpose, 

the genotypes of all the systems analyzed were inserted in the 

computer program S.H.S. Hardware software systems that 

© 2007 Lippincott Williams & Wilkins 335


FIGURE 4. A, Tomb T28. B, Tomb T29. 

C, Tomb T31. 

allows estimation of the frequency of the haplotype (genotype 

combinations) of each sample from the Apulian population 

(reference population). Many cases of alleles no longer 

present in the reference population, at least in the samples 

submitted to statistical analysis, were found (Table 1); the 

calculation was therefore based only on the frequency of the 

alleles present in the Apulian population. For all the samples, 

the differences between the haplotypes of the 5 individuals 

and of the present-day Apulian population were statistically 

significant (Table 2). 

RESULTS 

Tomb 28 

This is a multiple tomb in which 7 adults were found: 

6 male adults and 1 male child. The identification of the 

single individuals was made by exploiting morphometric, 

336 

articular, sex, and bone robustness compatibilities, as well as 

pathologic signs. 

Individual 28A 

This male subject was aged 40–43 years. He appears to 

have had a robust constitution, with a well-developed muscular 

frame. The estimated stature is 170 cm and the body 

biomass, 66 kg. Examination of the teeth showed severe 

periodontosis, moderate dental wear, and absence of caries 

but 2 odontogenic abscesses. There was a condition of weight 

overload of the cervical vertebrae, as well as Schmorl hernias 

of the lumbar vertebrae. The femurs showed Poirier facets 

and flattening of the subtrochanteric shaft sections, and the 

tibias showed lateral accessory facets. There was moderate 

osteoarthritic degeneration in the form of pitting. From the 

pathologic standpoint, phlogosis of the os zygomaticum (Fig. 



TABLE 1. Data Analysis of the DNA Typing 

D8S1179 D21S11 D7S820 CSF1PO D19S433 vWA TPOX FGA 

Bone Canosa T28A 8–9 26–31 15–15 11–13 – 13–17 8–10 – 

Bone Canosa T28E 13–18 31.2–33 11–11 12–13 10–10 14–19 6–7 27–27 

Bone Canosa T29B 12–13 26–26 7–7 �6–�6 – – 6–9 – 

Bone Canosa T29E 12–13 26–26 7–7 �6–�6 – – – – 

Bone Canosa T31A 9–10 33–36 8–9 6–7 16–17 12–15 7–7 – 

Lab. anthropology personnel 15–15 28–28 8–10 11–13 14–14.2 16–19 8–10 20–22 

Lab anthropology personnel 13–14 29–32.2 9–9 10–11 14–14 16–20 8–8 23.2–24.2 

Lab forensic genetics personnel 12–14 29–30 8–12 10–10 13–14 17–18 8–11 21–22 


Lab forensic genetics personnel 11–13 29–32.2 10–11 10–12 13–14 17–17 8–9 20–25 


D18S51 D3S1358 TH01 D13S317 D16S539 D2S1338 D5S818 Amel 

Bone Canosa T28A 15–20 16–16 6–6 14–15 9–9 – 7–8 X-Y 

Bone Canosa T28E – 20–20 4–8 9–9 5–8 16–17 10–10 X-Y 

Bone Canosa T29B 15–17 – 6–9.3 8–12 9–12 19–21 9–12 X-Y 

Bone Canosa T29E 15–17 – 6–9 �8–10 �8–9 16–18 9–12 X-Y 

Bone Canosa T31A – 13–17 9.3–9.3 8–13 10–15 24–24 13–16 X-Y 

Lab anthropology personnel 14–16 15–17 6–7 12–12 11–11 17–19 9–12 X-X 

Lab anthropology personnel 13–16 15–16 8–9.3 9–12 11–12 17–18 11–12 X-Y 

Lab forensic genetics personnel 15–16 15–17 8–9.3 8–11 9–11 20–24 11–12 X-Y 

Lab forensic genetics personnel 15–16 15–17 6–9.3 8–12 9–12 20–23 9–12 X-Y 

Lab forensic genetics personnel 13–18 16–17 6–10 8–11 11–13 24–25 12–12 X-X 

Lab forensic genetics personnel 16–16 15–17 6–9 9–12 9–11 17–19 12–12 X-Y 

TABLE 2. Frequency of Haplotypes With Respect to the 

Apulian Population 

Sample Haplotype Frequency 

T28A 3.89/10 22 

T28E 3.65/10 23 

T29B 8.8/10 19 

T29E 2.87/10 16 

T31A 3.54/10 18 

5), loss of substance in a half-moon shape was evident on the 

clavicula, corresponding to the area of insertion of the conoid 

ligament, together with a bone alteration of the acromial joint 

facet. These lesions are compatible with a past acromionclavicular 

dislocation. The person had tibial periostitis (Fig. 6). 

Individual 28E 

This subject was judged to be a male aged between 44 

and 60 years. His stature was calculated as 177 cm. There 

were many lines of enamel hypoplasia, which had formed 

between the ages of 1 and 6 years (Fig. 7). Assessment of the 

postcranial indexes revealed a condition of eurybrachia of 

both humeri and of mesocnemia of both tibias; the bone 

robustness index was average in both humeri. The vertebral 

load index showed weight overload in the lumbar and cervical 

tracts and mild overload in the thoracic tract. Vertebral 

osteolysis (Fig. 8). There was disk herniation of the 12th and 

10th thoracic vertebrae and the second lumbar vertebra. The 

FIGURE 5. Phlogosis of the os zygomaticum. 

few musculoskeletal markers available indicate intense muscular 

activity. Osteoarthrosis is present in the form of moderate 

lipping and pitting. The patellas show the incisura and 

the fossa of the lateral vastus, both tibias have a kneeling 

facet. The left astragalus presents the os trigonum. On the left 

clavicula (Fig. 9), a bone callous due to fracture of the third 

clavicular median along an oblique line was observed, with 

shortening of the segment due to raising of the sternal stump 

over the acromial stump. The fracture was not compound and 

could have been due to falling on the palm of the hand or on 

the shoulder, with the arm abducted. 



FIGURE 6. Tibial periostitis. 

FIGURE 7. Enamel hypoplasia. 

Tomb 29 

This is a multiple tomb in which 5 individuals were 

found: 3 adults, 1 male, and 2 females, as well as 1 young 

female and 1 child. The identification of the single individuals 

was made by exploiting morphometric, articular, sex, 

and bone robustness compatibilities. 

Individual 29B 

This is a female subject aged between 50 and 65 years. 

This was the only case in which diagnosis of the sex from the 

bone remains was discordant with the DNA result. There was 

complete fusion between the manubrium and the body of the 

sternum. The stature was calculated to be 159 cm, and the 

body biomass, 65 kg. Assessment of the postcranial indexes 

showed flattening of the diaphyses of the right humerus, left 

ulna, and both femurs. The index of robustness of the bones 

showed a moderate mean score. The lumbar and thoracic 

vertebrae showed a load within normal limits, while it was 

not possible to estimate the cervical load; herniation of the 

first, third, and fourth lumbar vertebrae was observed (Fig. 

338 

FIGURE 8. Verteblal osteolysis. 

FIGURE 9. On the left clavicula, a bone callous due to fracture 

of the third clavicular median along an oblique line. 

10). Analysis of the degree of enthesopathies showed moderate 

muscular activity. There was osteoarthritis in the form 

of mild lipping of the upper limbs, moderate lipping of the 

lower limbs, and mild pitting. Osteochondritis dissecans 

(O.D.) was observed at the level of the hip joint in both 

acetabular cavities, while the ischiatic tuberosities presented 

traces of Wells bursitis. Humeral osteolysis (Fig. 11). 

Individual 29E 

This individual was diagnosed as being of male sex and 

aged approximately 45 years. His stature was calculated to be 

169 cm, and body biomass, 71 kg. Dental examination revealed 

mild dental wear, the presence of 2 caries, an odontogenic 

abscess, and a large amount of tartar. Outcome of 

sinusitis (Fig. 12). The medial and lateral incisors were spade 

shaped. The axial skeleton showed moderate vertebral load in 

the cervical tract and the presence of herniation of the last 

part of the thoracic tract. The third and fourth lumbar vertebrae 

presented Baastrup syndrome (Fig. 13). The sacrum 



FIGURE 10. Herniation of the first, third, and fourth lumbar 

vertebrae was observed. 

presents a bridge vertebra. Examination of the musculoskeletal 

markers of the postcranial skeleton reveals strong tendon 

impression on all the segments examined. There were signs 

of moderate osteoarthrosis, in terms of moderate lipping, mild 

pitting, but no eburnation. Poirier facet, Charles facet, and 

Allen fossa were present on the left femur; the right femur 

showed only Allen fossa and Poirier facet; both tibias presented 

accessory lateral and medial facets. Accessory facets 

were also present on both astragals. The right patella presented 

the fossa of the lateral vastus muscle. There was 

evident syndesmopathy in virtually all the ligament insertion 

areas. The iliac bone showed a rough surface bilaterally in the 

right region above the acetabulum. These roughened, wrinkled 

surfaces were more extensive and evident on the right, with the 

presence of bone neoformations of syndesmopathic type in the 

hip joint capsule insertion zone. The superior part of the anterior 

sacroiliac ligament was completely ossified. The posterior face 

of the ischiatic tuberosity presented clear signs of Wells bursitis. 

The acetabulum had a marginal bone formation bilaterally, with 

extension of the superior joint surface and the presence of O.D. 

cavitation. 

Tomb 31 

This tomb contained 2 individuals of the male sex, 1 

adult and 1 adolescent. 

FIGURE 11. Humeral osteolysis. 

FIGURE 12. Outcome of sinusitis. 

Individual 31A 

This male subject was aged approximately 54 years. 

His stature was 179 cm, and body biomass, 81 kg. Dental 

examination showed the presence of 2 caries and an abscess 

extending to the left maxilla in the malar zone; there was also 

severe periodontitis, moderate tooth wear, and lines of hypoplasia 

attributable to formation between the ages of 2 years 

and 6 and a half years. Assessment of the postcranial indexes 



FIGURE 13. The third and fourth lumbar vertebrae presented 

Baastrup syndrome. 

FIGURE 14. There are bone neoformations of the feet and 

subperiosteal formations of the superior medial face of the 

first metatarsal. 

demonstrated a condition of eurybrachia of the humeri, platolenia 

of the ulnas, eurymeria of the left femur, and eurycnemia 

of the tibias; a moderate robustness index of both 

radiuses, the right femur, and both tibias; and high robustness 

of the left clavicle. The pillar index was zero in the right 

femur and weak in the left. The vertebral load index showed 

TABLE 3. Distribution of Skeletal Alterations in the Study Sample 

Tomb Sex Age 

Endocr. 

Alterations 

Vertebr. 

Ipervasc 

Vertebr. 

Cistic 

General 

Ipervasc 

Long 

Bones 

Periostitis 

a moderate load in the cervical region and overload in the 

thoracolumbar region. There were bone neoformations of the 

feet and subperiosteal formations of the superior medial face 

of the first metatarsal and the superior lateral face of the fifth 

metatarsal (Fig. 14). These small bone formations, due to 

continual, mild irritant stimuli, were located in correspondence 

with the points of contact between the back of the foot 

and parts of the soles of the shoes worn at the time, such as 

sandals. The observation of enthesopathic traces demonstrates 

intense muscular activity, as also evinced from the 

syndesmopathic traces and osteoarticular degenerations. Both 

femurs presented an extensive Poirier facet and Charles facet; 

both tibias had a kneeling facet. Accessory facets were 

present on the astragals. The hip bones showed the calcar of 

the iliac crest. 

Concluding Pathology Notes 

The paleopathological investigation of the 5 individuals 

revealed a whole series of skeletal alterations, involving the 

skull, ribs, vertebral column, and long bones, attributable to 

inflammatory foci (Table 3), whose distribution and morphology 

could likely indicate a specific nature, probably of tubercular 

origin. This finding is of particular interest because it 

can be generalized to other individuals found in the same 

cemetery area. 

CONCLUSIONS 

Genetic and anthropologic investigations have yielded 

a series of new data on the bone remains found in the area 

around San Pietro in Canosa di Puglia. From the genetic 

standpoint, we can state that significant differences were 

observed between the STR sequences analyzed in the presentday 

inhabitants of the Apulian region and their medieval 

ancestors. These findings may be justified by the fact that, 

during the Middle Ages, there were strong gene flows from 

the Germanic and Asian peoples (Goths, Lombards, Avars) 

that may have conditioned the genome of the native populations. 

In the subsequent centuries, these gene pools may have 

been diluted, thus strongly altering the gene status of the 

Italian population. From the anthropologic standpoint, it must 

be borne in mind that part of the cemetery in the archeological 

site at San Pietro shows selective conditions, in terms of 

alterations indicating inflammatory disease, likely of a specific 

nature and probably of tubercular origin. 

Ribs 

Phlogosis/Alterations Hyperostosis 

Periartitis 

Humerus 

Alterat. 

Sternum 

Alterations 

Turbinate 

Bones 

Phlogosis 

e28 A M 40–43 x x x x x x n.r. 

28 E M 44–60 n.r. x x x x x n.r. x n.r. n.r. 

29 B F 44–65 x x x x n.r. x x 

29 E M 45–49 x x x x x x 

31 A M 47–54 x x x x x x x x n.r. 

340 



ACKNOWLEDGMENTS 

The authors would like to thank M. V. C. Pragnell, BA, 

for help with translation of the text. 

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