1
Recovery and Amplification of Ancient DNA from Herculaneum Victims
2
Killed by the 79 AD Vesuvius Hot Surges
3
4
Fabio Maria GUARINO1*, Claudio BUCCELLI2, Vincenzo GRAZIANO2, Pietro
5
LA PORTA1, Marcello MEZZASALMA1, Gaetano ODIERNA1, Mariano
6
PATERNOSTER2 , Pierpaolo PETRONE3
7
8
1
Department of Biology, University of Naples Federico II, Via Cinthia 21, 80126
9
Naples, Italy.
2
10
Department of Advanced Medical Sciences, Section of Legal Medicine,
11
University of Naples Federico II, Azienda Ospedaliera Universitaria, Via Pansini,
12
5 - 80131 Naples, Italy.
13
3
Department of Advanced Biomedical Sciences, Section of Legal Medicine,
14
Laboratory of Human Osteobiology and Forensic Anthropology, University of
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Naples Federico II, Via Pansini, 5 - 80131 Naples, Italy.
16
17
*Corresponding author:
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Dr. Fabio Maria Guarino
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Tel. +39-081-679211, Fax. +39 0816 79033.
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E-mail address:[email protected]
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1
1
Abstract: In thermally damaged archaeological bones the quantity, quality, and
2
amplifiability of DNA are very much reliant on both the extent of heating and the
3
environmental conditions of the burial context. In this paper we tested the
4
possibility of extracting and amplifying ancient DNA (aDNA) from human bone
5
remains of Herculaneum victims of the 79 AD eruption of Vesuvius, using a
6
combination of histochemical and molecular methods. Long bone samples with
7
variable degrees of chromatic and morphological alterations consistent with
8
exposure to temperatures of about 300°C were taken from four specimens. Using
9
histochemical stains, bone cryostat sections from three individuals revealed DNA
10
within osteocyte lacunae but only for one sample DNA suitable for PCR
11
amplification was obtained, namely from reactions with primer pair for X and Y
12
amelogenin (AMEL) loci. The relative sequence differed from the homologous
13
trait of AMELX deposited in GenBank for six bases, probably due to degradation
14
processes following death.
15
Our data are indicative that also archaeological bones exposed to high
16
temperatures up at about 300°C should be considered for DNA analysis, given the
17
favorable conditions of corpse burial and skeleton preservation, such as those
18
occurred for the 79 AD victims in Herculaneum and Pompeii.
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20
Keywords: ancient DNA, DNA amplification. archaeological bones, 79 AD
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eruption of Vesuvius, Herculaneum
22
2
1
1.
2
The study of ancient DNA (aDNA) presents many challenges. Indeed, DNA
3
extraction and amplification is hampered by degradation processes following the
4
death of an individual, limiting the amount of extractable and amplifiable DNA,
5
and by enzymatic inhibitors that are co-extracted with aDNA (Pääbo 1989;
6
Lindahl 1993; Hoss et al. 1996; Kaestle and Horsburgh 2002). In addition,
7
taphonomic condition, abrupt changes in macroenvironmental conditions, such as
8
those occurring during the excavation of remains and their transfer to collections,
9
and the risk of contamination by foreign DNA can also limit DNA extraction and
10
amplification of aDNA (O’ Rourke et al. 2000; Gilbert et al. 2005a; Pruvost et al.
11
2007, Elsner et al. 2015). Preservation and amplification of aDNA are also
12
hindered in thermally damaged archaeological remains (Brown and Brown 2011),
13
including partially charred skeletons such as those of victims of volcanic eruption.
14
The possibility to successfully analyze aDNA from thermally damaged
15
archaeological bones (for example as consequence of cooking) was highlighted in
16
the study by Cottoni et al. (2009). However, this study concludes that heavily
17
burnt or cremated bones, exposed to temperature above 170° C, are unlikely to
18
produce authentic DNA sequences.
19
The first analysis of aDNA yielded from archaeological remains of human victims
20
of the Vesuvius eruption in AD 79 was performed on thirteen skeletons from the
21
House of Julius Polybius in Pompeii (Cipollaro et al. 1998). These studies proved
22
that the success of aDNA extraction and amplification was closely related to the
23
degree of bone preservation of the samples, evaluated under a light microscope.
24
Indeed, it was possible to extract and amplify DNA only from well preserved,
Introduction
3
1
non-diagenized bones. In particular, the amelogenin gene and Y-specific alphoid
2
repeat
3
unambiguously sexed, thus implementing anthropometric observations. In a
4
further study, after preliminary histological analysis, the STR (short tandem
5
repeats) and vWF (von Willenbrandt factor) loci for individual identification were
6
also analyzed (Cipollaro et al. 1999). A favorable aDNA preservation due to
7
burial conditions was also showed by Bailey et al. (1999), who extracted DNA
8
from a young Barbary macaque from the Sarno Baths in Pompeii. More recently,
9
analysis of mitochondrial and nuclear DNA fragments was performed on five
10
equids discovered in Pompeii within the House of the Chaste Lovers and on an
11
equid found in the archaeological site of Herculaneum, thus allowing the
12
taxonomical status of these equids to be tested (Di Bernardo et al. 2004a, 2004b).
13
The human and equine Pompeian remains were also successfully investigated to
14
prove the presence of ancient DNA within the osteocytic lacunae by means of
15
histochemical methods (Guarino et al. 2000; 2006). Finally, a new molecular
16
study on the thirteen skeletons found in the House of Polybius in Pompeii
17
revealed that six individuals were genetically closely related (Di Bernardo et al.
18
2009).
sequences
were
successfully
amplified
and
individuals
were
19
Except for the above mentioned work by Di Bernardo et al. (2004b), to our
20
knowledge only one other study, largely preliminary, concerning aDNA analysis
21
from 79 AD victims at Herculaneum has been carried out (Geraci et al. 2002).
22
Nevertheless, the latter study showed greater difficulty in recovering and
23
analyzing DNA from Herculaneum skeletal remains when compared with
4
1
molecular analyses on Pompeii bone remains, likely due to exposure to higher
2
thermal stress in the former (Mastrolorenzo et al. 2001a; 2010).
3
The present paper takes its cue from a bio-anthropological and volcanological
4
multidisciplinary study (Mastrolorenzo et al. 2010) aiming to further investigate
5
the cause of death in Pompeii and the other Roman towns buried by the 79 AD
6
Vesuvius Plinian eruption (Mastrolorenzo et al. 2001a). Using histochemical
7
techniques, Mastrolorenzo et al. (2010) showed the presence of DNA within
8
osteocytic lacunae of bone samples experimentally heated up to 300 °C. Here we
9
test the possibility of extracting and amplifying aDNA from human bone remains
10
of Herculaneum victims showing macro-microscopic evidence of exposure to
11
temperatures of about 300 °C, using a combination of histological and molecular
12
methods.
13
2.
Materials and Methods
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2.1
Sampling
15
The study of the human skeletal remains which formed the focus of this work was
16
approved by the Ethics Committee for Biomedical Activities of the University of
17
Naples, Hospital University of Naples Federico II (Protocol 154/10, 9.08.10). The
18
Archaeological Superintendency of Pompeii granted permission for field
19
investigation and study of the human skeletal materials unearthed by one of the
20
authors (PP) in the 1997-99 excavations of the waterfront chambers at
21
Herculaneum archaeological area (Province of Naples, southern Italy) (Figure 1).
22
23
We analyzed several human skeletal remains unearthed from the volcanic
surge deposit on the ancient beach of the Herculaneum archeological site.
5
1
The skeletons were belonging to some of the 300 human victims instantly
2
killed by the 79 AD eruption’s hot first surge (Mastrolorenzo et al. 2001). Skeletal
3
remains were carefully handled by one of the authors (PP), and stored in a
4
hermetically sealed boxes until laboratory analyses. In particular, in order to
5
examine bone samples that were most likely exposed to temperatures up to 300 °C
6
at the 79 AD first pyroclastic surge emplacement, we selected four long bones
7
with colors ranging from bright brown to dark brown (about 300° C) and showing
8
bone macro and microfractures typically induced by heat exposure (Shipman et al.
9
1984; Holden et al. 1995; Mastrolorenzo et al. 2010) (Table 1). Sex and age at
10
death were assessed according to standard diagnostic procedure (Ferembach et al.
11
1980; Buikstra et al. 1994). From each of the selected samples, a cross slice
12
(about 400 mg and 4 mm thick) was taken through the mid-shaft using a hacksaw,
13
also recording the condition of the bone according to Stiner et al. (1995) as
14
follows: heavy, when the bone could be sectioned without cracking; friable, when
15
the bone cracked and crumbled owing to cutting. These slices were used both for
16
histological and molecular analysis (see below).
17
2.2
18
In order to eliminate contaminating DNA from the bone surface, the bone slices
19
were first scraped with a brush to remove the outer layers, subsequently immersed
20
in 5.0% sodium hypochlorite for about 15 min, and finally exposed to UV light
21
for 10 min at 20 cm distance (Cipollaro et al. 1998). Sample pretreatment as well
22
as the successive laboratory steps (DNA extraction and PCR) were performed by
23
a single operator (LPP) in a completely sterile room with standard precautions,
24
including the use of workstations decontaminated by means of UV irradiation and
Sample pretreatment and contamination control
6
1
bleach, ultrapure reagents, sterile disposables, full-body personal protective
2
clothing (Kemp and Smith, 2005; Gamba et al. 2011). PCR amplification did not
3
take place in the same room as pre-PCR handling such as DNA extraction and
4
PCR setup (Gilbert et al. 2005a; Elsner et al. 2015).
5
The bone slices were decalcified in 0.5 M EDTA (ethylenediamine tetraacetic
6
acid), pH 7.4, from 7 to 13 days depending on their size and hardness. All the
7
decalcified slices were transversally cut in two parts and used for the histological
8
analysis and DNA extraction as reported below.
9
2.3
Histological analysis
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Decalcified bone slices were embedded in autoclaved Tissue-Tek O.C.T.
11
compound (Sakura, USA), frozen at – 22 °C, and cross-sectioned by means of a
12
cryostat. About 20 non-serial sections, 10 microns thick, were collected on slides
13
and stained with Mayer’s hemallum or 4’,6’-diamidino-2-phenylindole (DAPI,
14
Sigma) in order to explore the persistence of nuclei within osteocyte lacunae
15
according to Guarino et al. (2000; 2006). The stained sections were observed
16
under a Leica Motic BA340 transmitted light microscope or under a Leica DM100
17
fluorescence microscope, both equipped with a Nikon Coolpix 5000 digital
18
camera.
19
2.4 DNA extraction and authenticity
20
Decalcified bone slices were minced in very small pieces by means of forceps
21
(method A) and incubated at 37 °C over night in lysis buffer (TrisHCl 50 mM,
22
NaCl 100 mM, EDTA 10 mM, sodium dodecyl sulphate (SDS) 1%, Proteinase K
23
1%) (Sokolov 2000). After addition of 1/10 vol/vol of a KCl saturated solution
24
and centrifugation at 14.000 g per 10 min, DNA was extracted with phenol-
7
1
chloroform-isoamyl alcohol 25:24:1. DNA was precipitated in absolute ethanol
2
(EtOH) exsiccated and dissolved in 20 µl of 1 x TE (tris10 mM EDTA 1 mM) .
3
For DNA extraction we also used about 100 serial cryostat sections (see
4
histological analysis), collected in 500 µl of 70% EtOH (method B) and stored
5
until DNA extraction which was performed as above reported.
6
In addition, we used laboratory operator’s (LPP) DNA previously extracted
7
from his blood cells in a distinct laboratory. Negative controls (both reagents and
8
products, both for method A and B) were used in each step of molecular analysis.
9
To verify the ancient DNA authenticity we performed multiple extractions and
10
PCR in two different laboratories, each one with own instruments and reagents
11
(Gilbert et al. 2005a; Elsner et al. 2015).
12
All extracted DNAs were visualized with agarose gel (2%) electrophoresis,
13
using ethidium bromide (5 µg/ml). Where Maillard products (see O’Rourke et al.
14
2000) were visible, DNA was eluted from a gel trait ranging from about 50 bp to
15
1000 bp, using the "squeeze and freeze" method (Tauz and Renz 1983).
16
DNA concentration and purity were assessed using NanoDrop 1000 (Thermo
17
Scientific, USA) taking into considerations the methodological limits of this
18
procedure (Rohland and Hofreiter 2007).
19
2.5 PCR amplification and sequencing
20
In order to minimize the possibility of amplifying segments of foreign bacterial
21
DNA we performed PCR reactions for specific regions the single-copy nuclear
22
gene of amelogenin (AMEL) X (106 bp) and Y (112 bp) , useful for determining
23
the gender of human remains (Cipollaro et al. 1998). Primers used for PCR
24
amplification of AMEL were as follows (Mannucci et al., 1994): 5’
8
1
CCCTGGGCTCTGTAAAGAATAGTG
3’(forward)
2
TCAGAGCTTAAACTGGGAAGCTG
3
conducted in a total volume of 25 µl, using a Thermal Cycler GeneAmp PCR
4
system 3400 (Applied Biosystem, USA) with the following conditions: initial
5
denaturation at 95 °C for 5 min followed by 36 cycles composed by 94 °C for 45 s
6
(denaturation), 58 °C for 45 s (primer annealing), 72 °C for 1 min (extension),
7
and a final elongation step at 72 ° C for 7 min. After purification with the Wizard
8
SV Gel and PCR Clean-Up System (Promega, USA) kit, PCR products were re-
9
amplified using the same conditions described above.
3’(reverse).
PCR
and
amplification
5’
was
10
Amplicons were sequenced on an automated sequencer ABI 377 (Applied
11
Biosystems) using the BIGDYE TERMINATOR v3.1 kit (ABI, Foster City, CA).
12
The sequences obtained were imported into BioEdit 7.0.5.3 (Hall 1999) and
13
aligned with ClustalW (Thomson et al. 1994).
14
3.
Results
15
3.1
Histological analysis
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All bone samples showed a good state of histological preservation (Figure 2).
17
Analysis of the bone sections showed large areas with typical microstructure of
18
human bone, albeit partly broken up owing to microcracking, typical of exposure
19
to high temperatures close to 300 °C (Mastrolorenzo et al. 2010) and enhanced by
20
the decalcification process. Signs of diagenesis due to microbial activity such as
21
destructive microscopic foci, MDF, or other form of bioerosion (Jans et al. 2004;
22
White and Booth 2014) were not found. Several osteocytes stained with hemalum
23
as well as several DAPI-positive lacunae were observed in bone sections from
24
EF10.Q2A, EF12.27 and EF10.35 (Figure 2), but not from EF12.20.
9
1
3.2
2
DNA extractions and multiple PCR gave consistent results within and between the
3
dedicate laboratories where experimental procedures were carried out. Agarose
4
electrophoretic gel of DNA from decalcified and fragmented Herculaneum bones
5
(method A) showed an evident green band of Maillard products close to the front
6
buffer. These products sensibly affected spectrophotometric quantifications of
7
extracted DNA, overvaluing it (Table 2). In fact, after elution from gel bands by
8
means of "freeze and squeeze", for all samples the DNA values were lower and
9
with a ratio of absorbance at 260/280 nm close to 1.8, which is generally accepted
10
for DNA purity (Table 2). In contrast, DNA extracted from cryostat sections
11
(method B) did not show Maillard products and had acceptable purity values at
12
260/280 nm (Table 3). Although we obtained low DNA yields from all samples
13
and from both methods (method A plus " freeze and squeeze" and method B),
14
however it was enough to perform PCR reactions. Nevertheless, only the DNA
15
extracted from EF10.Q2A by means of the method B was successfully amplified
16
by PCR (Figure 3). The amplicons of EF10.Q2A were about 100 bp, as evidenced
17
by 2% agarose gel electrophoresis. AMEL sequences of the EF10.Q2A samples
18
obtained in both laboratories (see methods) were of 106 bp, differing from the
19
homologous trait of AMELX deposited in GenBank (accession number:
20
NG012040) for six bases: three C/T and one G/A transitions, one T/G and one
21
G/T transversions (Figure 4).
22
The rate of point mutations, excluding primers, calculated for the AMEL segment
23
was about 10%.
24
4.
Molecular analysis
Discussion
10
1
Site and laboratory analysis of the skeletons of the 79 AD human victims
2
provided evidence that people sheltering in the waterfront chambers on the ancient
3
beach of Herculaneum were engulfed by the eruption’s hot first surge at about
4
500°C. The thermal shock caused instant death and sudden immobilization of the
5
corpses in suspended actions (life-like stance) by cadaveric spasm, a kind of
6
instantaneous rigor mortis associated with instant violent death, typical of mass
7
disasters. Shortly after, the high temperature of the ash surge caused the rapid
8
vaporization of the flesh of the victims bodies, bone cracking and blackening
9
(Mastrolorenzo et al. 2001a, 2001b; 2010; Petrone 2011).
10
Nevertheless, even if it could be argued that the endogenous DNA is
11
completely or largely degraded in bones exposed to such a high temperature, the
12
present study shows the feasibility of molecular anthropological studies on those
13
bones from the 79 AD victims which show evidence of exposure to lower
14
temperatures of about 300 °C. This temperature confirms the taphonomic
15
evidence, indicating that heat effects locally might show some variability, mostly
16
related to the density of corpses sheltered within each chamber: the fewer the
17
people per chamber, the greater the effects of high temperature on victims (i.e.
18
heat-induced skull fractures, teeth fractures, bone fractures and blackening, spine
19
extension, hands and feet hyperflexion) and vice versa (Mastrolorenzo et al.
20
2001a; 2001b). In general, the heat of the ash was sufficient to vaporize most of
21
the organic matter. Hence the initial violent vaporization caused a rapid drop in
22
ash temperature, as confirmed by paleomagnetic data (Incoronato et al. 1998).
23
This could also explain why the most evident effects of the heat are limited to
11
1
teeth and those parts of the skeleton least protected by fat and tissue, a process
2
which is indeed particularly evident in less crowded chambers.
3
Our experimental evidence shows that also the archaeological bones clearly
4
altered by high temperatures deserve to be considered for DNA analysis,
5
reinforcing what was already pointed out by other studies (Zhang and Wu 2005;
6
Ottoni et al. 2009). Interestingly, the study of Ottoni et al. (2009) showed that in
7
moderately heated (up to 140 °C) archaeological bone, DNA preservation is not
8
related to the presence of intact collagen fibrils. A possible alternative explanation
9
is that moderate heating inactivates nucleases and PCR inhibitors, destroys cell
10
walls and partially degrades collagens. As a consequence, there is both a greater
11
ability of the bone crystals to bind DNA fragments and more DNA fragments
12
available for absorption, resulting in greater preservation of DNA (Ottoni et al.
13
2009). The crucial role of the hydroxyapatite crystals in DNA preservation within
14
bone tissue has been shown by several studies (Gotherstrom et al. 2002; Salamon
15
et al. 2005) and bone recrystallization begins when bone is exposed to
16
temperatures equal to or higher than 600 °C (Holden et al. 1995; Quatrehomme et
17
al. 1998), as recently detected on some bones of the 79 AD victims unearthed in
18
Oplontis, a suburban village closed to Pompeii (Mastrolorenzo et al. 2010).
19
To date, a wide variety of methods (Rohland and Hofreiter 2007) have been
20
developed to solve the difficulties associated with recovery and analysis of aDNA,
21
such as low amounts of DNA obtained, degraded templates, PCR inhibitors, etc.
22
In this study we successfully experimented for the first time a method that allows
23
DNA extraction from osteocytes, placing cryostat sections of decalcified bone in
24
70% EtOH and then in proteinase K plus SDS digestion solution. This procedure
12
1
offers a double advantage. Firstly, PCR inhibition by-products of sugar reduction
2
such as Maillard products (O’Rourke et al. 2000) were not observed. Probably,
3
the low thickness of the sections (12 µm) allows to remove most of the PCR
4
inhibitors during their storage in ethanol prior to the next steps for DNA
5
extraction. Interestingly, Maillard products were observed in the gel
6
electrophoresis of genomic DNA obtained by mean of the mincing procedure
7
(method A). Furthermore, it should be noted that the EF10.EQ2A sample did not
8
give amplification products using method A also after gel elution. This is
9
probably because PCR inhibitors were not totally removed. In addition, although
10
the EF10.35, EF12.20, EF12.27 samples gave measurable DNA by method B
11
(cryostat method), they were not successfully amplified probably because their
12
DNA was more fragmented than that of EF10.Q2A.
13
Secondly, cryostat method allows to select the bone samples to process for
14
molecular analysis on the basis of DNA detection in the osteocytic lacunae by
15
histochemical staining.
16
In our study, only for the EF10.Q2A sample, yields and quality of aDNA
17
were suitable for direct amplification and sequencing of short single-locus
18
sequences, such as AMEL. The length (106 bp) of obtained amplicons was
19
consistent with target AMEL fragment and their nucleotide sequence differed
20
from that deposited in GenBank for six bases. This finding allowed us to attribute
21
the EF10.Q2A sample to a female solving the uncertain sex determination based
22
on anthropometric analysis. On the other hand, if the EF10.Q2A sample was of a
23
male, the direct sequencing of AMEL would have produced unreadable
24
chromatograms owing to the presence of two sequences of different length, 106
13
1
bp (AMELX) and 112 bp (AMELY). The difference in six bases of our trait of
2
sequence AMELX from that deposited in GenBank is mainly due to pyrimidine
3
transitions. This kind of point mutations are commonly observed in nuclear
4
sequences of aDNA, because they are supposed to be more prone than purines to
5
damages by oxidation (Hansen et al. 2001; Gilbert et al. 2003, 2005b; Binladen et
6
al. 2006). Furthermore, even if referred to one datum, our AMELX sequence
7
displays a rate of point mutations threefold than that observed in nuclear genes of
8
aDNA by Binladen et al. (2006). This higher rate could be explained as a
9
consequence of the changes induced by the high temperatures, also considering
10
the diagenetic alterations that occurred post-mortem. Further studies on
11
Herculaneum skeletal remains or other archaeological bones with similar heat
12
alteration could support our hypothesis.
13
It is generally accepted by aDNA researchers that the criteria list of Cooper
14
and Poinar (2000) as the basis to test the authenticity and reliability of data
15
(Gilbert et al. 2005a) should be used. In our study we didn’t consider necessary to
16
clone amplicons because our chromatograms didn't show any ambiguous
17
nucleotide positions. On the other hand, as showed by Winters et al. (2011) in
18
their study on aDNA, the consensus sequence of clones doesn’t differ from the
19
direct sequence.
20
To conclude, the main goal of the present paper was to demonstrate PCR
21
amplifiability of DNA recovered from archaeological skeletal remains of the
22
victims killed by the first hot pyroclastic surge at Herculaneum. The results
23
achieved in the present study are the first successful attempt to analyze
24
Herculaneum archaeological human bones at molecular level by means of
14
1
amplification of single copy gene sequences. Our data, although based only on a
2
positive sample, indicate that even ancient bones showing signs of thermal
3
exposure up to 300 °C, as the case of the 79 AD victims skeletons, should be
4
considered for DNA analysis. This opens up the chance to carry out systematic
5
studies about the genetic characteristics of the ancient population of Herculaneum,
6
as also could be done for that of Pompeii.
7
Acknowledgments
8
We thank the Superintendence of Pompeii for granting a permit for field
9
investigation and for the study of the human skeletal material. We also thank
10
Mark Walters for reviewing the English version of the manuscript.
11
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Figure Legends
2
Figure 1. A) Location of Herculaneum archaeological site. B) The suburban area and
3
the 12 chambers on the seashore. C) and D) Taphonomic context of skeletal
4
remains analyzed in the present study: human victims within the chambers 12 (C)
5
and 10 (D).
6
Figure 2. Cryostat sections of humerus from EF10.Q2A stained with hemalum (A) and
7
with DAPI (B). Note in A) some osteocytes stained with hemalum and in B) several
8
DAPI-positive lacunae. Scale bar: 40 µm
9
Figure 3. 2% agarose gel electrophoresis of amplicons from method B (cryostat
10
sections). From left to right, lane 1: EF10.Q2A; lane 2: EF10.35; lane 3: EF12.27;
11
lane 4: EF12.20; lane 5: modern human; 6: blank; lane L: Ladder 100 bp (New
12
England Biolabs Inc, UK).
13
Figure 4. Comparison and alignment by ClustalW of canonical AMELX and EQ2A
14
DNA sequences. C→T and G→A transitions are evidenced by dark grey (light
15
blue); T→G and A→T transversions, in light grey (yellow). In EQ2A lower cases
16
indicate primer base pairs for AMEL.
17
22
1
2
Figure 1. A) Location of Herculaneum archaeological site. B) The suburban area and
3
the 12 chambers on the seashore. C) and D) Taphonomic context of skeletal remains
4
analyzed in the present study: human victims within the chambers 12 (C) and 10 (D).
5
23
1
2
Figure 2. Cryostat sections of humerus from EF10.Q2A stained with hemalum (A) and
3
with DAPI (B). Note in A) some osteocytes stained with hemalum and in B) several
4
DAPI-positive lacunae. Scale bar: 40 µm
5
24
1
2
Figure 3. 2% agarose gel electrophoresis of amplicons from method B (cryostat
3
sections). From left to right, lane 1: EF10.Q2A; lane 2: EF10.35; lane 3: EF12.27;
4
lane 4: EF12.20; lane 5: modern human; 6: blank; lane L: Ladder 100 bp (New
5
England Biolabs Inc, UK).
6
25
1
2
Figure 4. Comparison and alignment by ClustalW of canonical AMELX and EQ2A
3
DNA sequences. C→T and G→A transitions are evidenced by dark grey (light
4
blue); T→G and A→T transversions, in light grey (yellow). In EQ2A lower cases
5
indicate primer base pairs for AMEL.
26
1
Table Legends
2
Table 1. Human skeletal remains used in the present work.
3
Table 2. DNA yields obtained from minced bone (method A), and the relative ratio of
4
absorbance at 260 nm and 280 nm. Further details are in the Material and Methods section.
5
NA: not applicable (value below the instrument sensibility); NP: not performed. * Note
6
Modern sample was processed in a separate way, at a laboratory different from those of
7
ancient samples.
8
Table 3. DNA yields obtained from cryostat sections (method B) and the relative ratio of
9
absorbance at 260 nm and 280 nm. Further details are in the Material and Methods.
10
11
12
27
1
2
Table 1
3
Human skeletal remains used in the present work.
Code
4
5
Sex
Bone
Macroscopic appearance
EF10.Q2A
F?
Tibia dx
Orange-reddish, heavy
EF10.35
M
Fibula sx
Orange-reddish, heavy
EF12.20
M
Femur dx
Orange-reddish, heavy
EF12.27
M
Humerus dx
Orange-reddish, heavy
Sex was determined on the basis of anthropometric analysis.
Abbreviations: EF, Herculaneum remains; F?, probably, a female
6
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1
Table 2.
2
DNA yields obtained from minced bone (method A), and the relative ratio of
3
absorbance at 260 nm and 280 nm. Further details are in the Material and
4
Methods. NA: not applicable (value below the instrument sensibility); NP: not
5
performed. * Note Modern sample was processed in a separate way, at a
6
laboratory different from those of ancient samples.
Sample
before gel elution
ng/µL
A260/280
after gel elution
ng/µL
A260/280
EF10.Q2A
EF10.35
EF12.20
EF12.27
Mod human*
Neg control
153.5
49.0
135.8
49.8
210.5
NA
10.0
4.6
7.9
6.4
NP
NP
1.46
1.35
1.35
1.26
1.85
NA
1.75
1.75
1.85
1.70
NP
NP
7
8
29
1
Table 3.
2
DNA yields obtained from cryostat sections
3
(method B) and the relative ratio of
4
absorbance at 260 nm and 280 nm. Further
5
details are in the Material and Methods.
Sample
EF10.Q2A
EF10.35
EF12.20
EF12.27
ng/µL
17.5
4.5
6.5
6.9
A260/280
1.73
1.74
1.70
1.78
6
30