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ISSN: 0301-4460 (print), 1464-5033 (electronic)

Ann Hum Biol, Early Online: 1–10

! 2014 Informa UK Ltd. DOI: 10.3109/03014460.2014.954614


Methodological strategies to assess the degree of bone preservation for

ancient DNA studies
Gabriele Scorrano1, Federica Valentini2, Cristina Martı́nez-Labarga1, Mario Federico Rolfo3,
Antonella Fiammenghi4*, Domenico Lo Vetro5, Fabio Martini5, Antonella Casoli6, Giovanni Ferraris7,
Giuseppe Palleschi2, Antonio Palleschi2, and Olga Rickards1
Centro di Antropologia molecolare per lo studio del DNA antico, Dipartimento di Biologia, 2Dipartimento di Scienze e Tecnologie Chimiche,
Università degli Studi di Roma Tor Vergata, via della Ricerca Scientifica, Roma, Italy, 3Dipartimento di Beni Culturali, Musica e Spettacolo, Facoltà di
Lettere e Filosofia, Università di Roma ‘Tor Vergata’, Roma, Italy, 4Soprintendenza per i Beni Archeologici di Salerno, Avellino e Benevento, Salerno,
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Italy, 5Museo e Istituto Fiorentino di Preistoria, Università di Firenze, Firenze, Italy, 6Dipartimento di Chimica, Università degli Studi di Parma, Parco
Area delle Scienze, Parma, Italy, and 7Istituto per lo Studio dei Materiali Nanostrutturati-CNR, c/o Dipartimento di Chimica, ‘Sapienza’ Università di
Roma, Roma, Italy

Abstract Keywords
Background: Archaeological bones contain only small amounts of DNA due to post-mortem Amino-acid racemization, crystallinity, Early
DNA degradation and the changes endogenous DNA is subjected to during diagenesis. An Neolithic, roman imperial age, upper
important step before undertaking such time-consuming and costly analyses as ancient DNA paleolithic
investigation is to predict the presence of DNA in ancient samples. To date, the leading
For personal use only.

screening method has been amino acid racemization; however, other analytical techniques can History
also be used to assess the degree of bone preservation.
Aim: The aim of the present study was to relate the presence of DNA with bone preservation Received 1 April 2014
in order to select samples potentially suitable for ancient DNA analysis. Revised 4 July 2014
Subjects and methods: Bones collected from several archaeological sites, different locations Accepted 14 July 2014
(cave, rockshelter or sub divo) and diachronic periods were selected for analytical and Published online 18 September 2014
spectroscopic analysis in order to correlate bone tissue preservation with the presence of DNA.
Different techniques were combined to assess the degree of preservation of organic and
inorganic components.
Results: As determined by different analytical methods, preservation of the inorganic
component was best associated with the presence of DNA.
Conclusion: Evaluation of the bone preservation state may be an efficient step to predict the
presence of DNA in ancient samples prior to aDNA analysis.

Introduction Since the aDNA is in bone, diagenetic processes that alter

the bone structure also alter the chemical microstructure that
Archaeological bone remains provide a valuable source of
protects aDNA (Sosa et al., 2013). Normally, aDNA and
information about the past, but only a few samples contain
collagen are physically protected against external attack by an
analysable ancient DNA (aDNA). As the bones come into
inorganic component (Nielsen-Marsh et al., 2000a). Indeed,
contact with sediments, water and micro-organisms, their
DNA has a strong affinity for the mineral phase by virtue of
organic and inorganic composition is altered by partial or
the attraction between the negative charges of the phosphate
complete dissolution, precipitation and re-crystallization
groups of the DNA and the positive charges of Ca2+ of the
(Reiche et al., 2003). Hence, the state of bone preservation
hydroxyapatite (Okazaki et al., 2001). This binding confers
is highly variable and chiefly influenced by direct environ-
the DNA more resistance to degradation (Paget et al., 1992).
mental conditions.
Poinar et al. (1996) reported that DNA survival could be
predicted by measuring the extent of aspartic acid racemiza-
tion. Since the kinetics of aspartic amino acid racemization
*Antonella Fiammenghi, who promoted the Velia Project, unfortunately are believed to be similar to the rate of DNA depurination,
died before the submission of this article. they could represent a valuable measure of the likelihood of
Correspondence: Gabriele Scorrano, Centro di Antropologia molecolare DNA survival (Bada et al., 1994). Later analyses on 91 bone
per lo studio del DNA antico, Dipartimento di Biologia, Università degli
and teeth samples demonstrated, however, that aspartic acid
Studi di Roma Tor Vergata, via della Ricerca Scientifica n. 1, 00173
Roma, Italy. Tel: +39 06 72594350. Fax: +39 06 2023500. Email: racemization is not a useful screening technique for aDNA preservation in archaeological remains (Collins et al., 2009).
2 G. Scorrano et al. Ann Hum Biol, Early Online: 1–10

Therefore, the relation between aDNA and bone preservation BP (Alessio et al., 1966). The five single burials (Romito 3, 4,
is incompletely understood. 7, 8 and 9) were in the cave: Romito 3 and 4 were dated to the
Diagenetic parameters are evaluated to quantify post- early 14th millennia cal BP and Romito 7 and 8 to the
mortem alterations. This can be done with the combined late 14th millennia cal BP. Romito 9, the oldest burial, is
use of a variety of analytical techniques, including related to an evolved Epigravettian layer between 18 890
Fourier transform infrared spectroscopy (FT-IR), Brunauer, and 18 100 cal BP.
Emmett and Teller (BET) specific surface area and porosity Velia (Salerno, 40 130 N, 15 170 E) (Figure 1) was
measurements, collagen yield analysis and amino acid an important Imperial Roman port in southern Italy
racemization. (Fiammenghi, 2003).
The aim of the present study was to assess the state of
bone preservation and to relate it with the survival of DNA. Materials and methods
To do this and gain a general understanding of the degree
of bone tissue preservation, bones collected from several Samples
archaeological sites were selected for the analysis of samples Table 1 reports the characteristics of the archaeological
from different locations (cave, rockshelter or sub divo) and samples. Fragments of long bones were collected with a drill
diachronic periods. The sites were two Italian pre-historic and properly prepared for subsequent analyses. A modern
cave sites/rockshelters, Mora Cavorso in Latium (Early control from an individual who died 30 years ago (provided
Neolithic 7319 cal BP) and Romito in Calabria (Late Upper by the Department of Legal Medicine, Rome University Tor
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Paleolithic 18 000 to 13 000 cal BP) and one Roman Imperial Vergata) was analysed for comparison.
Age coastal site, the necropolis of Velia in the Salerno area Collagen quantification, preservation of the inorganic
(2150 to 1750 BP) (Figure 1). component and DNA analysis were performed in all the
The Mora Cavorso cave is located near Jenne (Rome; 41 samples. Two samples from each site were selected for amino
87 N and 13 170 E) in the Simbruini Mountains Natural Park
acid racemization analysis. In order to gain more complete
at 715 m altitude (Figure 1). The cave exhibits a pre-cave and information about bone diagenesis in the pre-historic samples
lower and upper chambers where numerous human bones, (eight from Romito, two from Mora Cavorso and two modern
fauna and archaeological materials have been found (Rolfo controls), porosimetric analysis (Barrett et al., 1951; Brunauer
et al., 2012). Radiocarbon dates were obtained on one sample et al., 1938; Lowell et al., 2004) was also performed.
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recovered in the lower chamber and one in the upper chamber.

The dates (7319 ± 79 cal BP and 7153 ± 119 cal BP, respect-
Precautions and criteria of authentication for the
ively) ascribe the level of human remains and the archaeo-
study of ancient DNA
logical materials to the Early Neolithic (Rolfo et al., 2010).
The Romito Cave (Cosenza, 39 540 N, 15 550 E) is Archaeological bone samples contain only small amounts of
located in southern Italy, 275 m altitude, 25 km inland from DNA that are often damaged. This makes precautions
the Tyrrhenian coast of Calabria in the Lao Valley (Figure 1) necessary to minimize the risk of amplifying and typing
(Colonese et al., 2007). Nine burials were found during modern contaminating DNA molecules. To maximize the
excavations carried out in 1965–1967 and 2000–2007 (Fabbri probability of extracting and sequencing authentic DNA from
et al., 1989; Graziosi 1971; Martini 2002; Martini et al., the ancient bone samples, we complied with the following
2004). Two were double burials (Romito 1 and 2, Romito 5 criteria (Cooper & Poinar 2000; De Angelis et al., 2013;
and 6) and were found in the rockshelter, directly in front of Pääbo et al., 2004): the work areas were kept physically
the cave entrance, dating back between 13 290 and 12 860 cal isolated and dedicated only to the study of ancient DNA; the
archaeologists and the laboratory operators were genetically
analysed to detect any source of contamination on the bone
material (Table A2 in the Appendix). Negative control
amplification and extraction was performed; at least two
independent extractions of each sample were performed and
at least two independent amplifications for each investigated
region were carried out on each extract. Because aDNA is
fragmented, small fragments of 100–150 bp were amplified
(list of primers given in Table A1 in the Appendix) so that the
amplification efficiency could be inversely correlated with the
length of amplification. Cloning of amplicons and sequencing
of multiple clones were carried out to detect heterogeneity in
the amplification products due to contamination, DNA
damage or jumping PCR (Hofreiter et al., 2001; Pääbo
et al., 1990). The faunal remains associated with the
archaeological human bone samples were analysed (list of
primers given in Table A1 in the Appendix). The presence of
a faunal DNA template in associated faunal remains is an
Figure 1. Geographical location of the archaeological sites: (A) Mora essential supporting element and its analysis can also be used
Cavorso Cave, Rome; (B) Romito, Cosenza; (C) Velia, Salerno. to control for contamination by exogenous human DNA.
DOI: 10.3109/03014460.2014.954614 Chemical parameters for ancient DNA analysis 3
Table 1. Collagen ratio; inorganic component and DNA analysis results.

DNA molecules/mL DNA molecules/mL DNA 1: present

Lab code % Collagen SF C/P CaCO3 (67 bp) (129 bp) l 0: absent
Velia (Central Italy) Imperial Roman Age
T. 51 13.00 3.23 0.30 53% 56 370 44 450 0.0038 1
T. 70 45.03 3.33 0.55 53% 18 420 12 420 0.0063 1
T. 71 74.74 3.25 0.68 53% 24 870 16 359 0.0067 1
T. 78 28.85 3.90 0.28 53% 0 0 0
T. 85 36.96 3.72 0.36 53% 9 526 6 670 0.0057 1
T. 99 47.28 3.23 0.30 53% 13 645 9 600 0.0057 1
T. 100 63.00 3.18 0.29 53% 25 475 20 560 0.0034 1
T. 102 39.26 3.46 0.34 53% 18 560 9 650 0.010 1
T. 118 43.27 3.38 0.30 53% 96 0 0
T. 119 41.11 3.90 0.23 53% 0 0 0
T. 136 82.50 3.16 0.27 53% 19 400 14 549 0.0046 1
T. 137 80.50 2.87 0.43 53% 23 000 18 560 0.0034 1
T. 140 10.42 3.82 0.26 53% 86 0 0
T. 177 10.75 3.65 0.41 53% 0 0 0
T. 182 59.04 3.30 0.81 43% 13 760 9 580 0.0058 1
T. 203 79.50 3.21 0.21 53% 23 450 18 430 0.0039 1
T. 337 60.00 3.15 0.30 43% 15 870 12 450 0.0039 1
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T. 351 37.66 3.33 0.45 53% 22 450 17 240 0.0043 1

T. 361 39.61 3.66 0.45 53% 131 0 0
T. 362 30.45 3.11 0.39 43% 21 030 16 340 0.0041 1
T. 364 45.00 4.10 0.15 53% 118 0 0
T. 389 20.83 3.85 0.28 53% 0 0 0
T. 392 66.50 3.12 0.38 53% 22 400 17 450 0.0040 1
T. 393 30.85 3.22 0.41 53% 19 450 14 870 0.0043 1
T. 400 66.25 3.32 0.45 53% 20 500 13 340 0.0069 1
T. 401 60.54 2.86 0.72 43% 42 390 32 430 0.0043 1
T. 402 26.44 3.33 0.40 53% 32 840 24 730 0.0046 1
T. 403 69.51 2.99 0.61 43% 21 003 15 324 0.0051 1
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T. 428 67.26 3.31 0.36 53% 32 402 24 650 0.0044 1

T. 429 32.93 3.62 0.42 53% 0 0 0
T. 443 69.51 3.16 0.39 53% 24 090 18 450 0.0043 1
T. 444 56.45 3.28 0.57 53% 18 432 13 750 0.0047 1
T. 449 41.11 3.70 0.37 43% 0 0 0
T. 450 36.29 3.64 0.43 53% 29 183 22 320 0.0043 1
Mora Cavorso (Central Italy) Early Neolithic
10*VI A 35.33 3.21 0.82 43% 5 000 3 670 0.0050 1
SS 5 44.67 3.02 0.49 43% 4 800 3 420 0.0055 1
100 V 34.31 3.08 0.64 43% 8 349 6 140 0.0049 1
67 63.60 2.93 0.77 43% 3 798 2 766 0.0051 1
117 29.44 3.19 0.23 43% 9 893 7 370 0.0047 1
SR 61/4 4.06 4.15 0.10 43% 0 0 0
10 V D 4.77 4.10 0.13 43% 0 0 0
10 V v 55.84 3.08 0.48 43% 1 200 800 0.0065 1
92 B 51.57 2.98 0.52 43% 1 132 787 0.0059 1
92 A 31.98 2.95 0.46 43% 965 660 0.0061 1
Romito (Southern Italy) Upper Paleolithic
1 N/A 4.00 0.41 53% 0 0 0
2 19.35 3.50 0.52 53% 0 0 0
3 41.94 3.27 0.37 53% 1 080 750 0.0059 1
4 16.94 3.52 0.35 53% 1 500 1 050 0.0057 1
5 66.94 3.29 0.76 43% 1 400 980 0.0057 1
6 18.55 3.45 0.40 53% 2 500 1 800 0.0053 1
7 43.55 3.43 0.39 53% 2 700 1 980 0.0050 1
8 100.00 2.95 0.32 53% 1 200 830 0.0059 1
9 36.29 3.30 0.32 53% 942 680 0.0052 1
30 years ago
Modern 100.00 2.77 0.62 53%
Modern deproteinated N/A 3.040 0.33 53%

Real-Time qPCRs (with a probe-based method) were amplifiable DNA were sequenced for HVS-I and HVS-II
performed to quantify mtDNA molecules; separate PCRs regions. The DNA quantification results were used to estimate
targeting different regions of the D-loop were carried out in the probability of a nucleotide being damaged (l) using the
order to sequence the products and to further authenticate the formula proposed by Deagle et al. (2006). This parameter was
results. Forty-three samples which showed the presence of then used as a further variable to account for when describing
4 G. Scorrano et al. Ann Hum Biol, Early Online: 1–10

the degree of preservation of the samples. The details of the Deventer, the Netherlands) and treated with 0.2 mL of
chemistry related to the sequencing process of the aDNA trifluoroacetic anhydride (Aldrich, Steinheim, Germany) for
analysis are reported in the Appendix. 1 hour at 60  C. After cooling, the tube was opened and
the solvent evaporated. Derivatization led to the formation
Analysis of organic component (% protein) of N-trifluoroacetyl-O-2-propyl esters. The residue was
recovered in 0.2 mL of dichloromethane and used for gas
Collagen extraction followed the modified Longin protocol
chromatography-mass spectrometry (GC/MS) injection.
(Brown et al., 1988). The values obtained from the ancient
A standard mixture of amino acids (Sigma, St Louis, MO)
samples were compared with the modern bovine data, where
in 6 N HCl at a concentration of 100 mg/mL each was
the collagen yield was assumed to be equal to 100%.
prepared and derivatized with the same procedure as
described above. This was further used as a reference for
Analysis of inorganic component
gas chromatographic retention times and mass spectra of the
Bone powder was mixed with 50 mg of KBr and pressed amino-acid derivatives.
into 7-mm diameter pellets with a mini-press. Pellet concen- Amino acid analysis was carried out on a HP-6890 Series
tration of bone ranged from 0.4–1.4%. The tablets were gas chromatograph coupled to an HP-5973 mass selective
analysed by FT-IR spectroscopy using the instrument param- detector (Hewlett-Packard, Palo Alto, CA). Chromatographic
eters reported in Surovell and Sitner (2001). For the inorganic separations were accomplished using a Chirasil-L-Val (25 m
component analyses, absorbance peaks of 565 (v4 PO3 4 ),  0.25 mm i.d  0.12 mm d.f.) fused-silica column
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605 (v4 PO3 3
4 ), 1035 (v3 PO4 ) and 1415 cm (v3 CO2 3 ) (Chrompack Italia, Milan, Italy) according to the following
were selected for FT-IR spectroscopy; the valley between temperature programme: initial 50  C held for 1 minute, then
peaks at 565 and 605 cm1, 595 cm1, was also studied to increased to 145  C at 4  C/minute, held at 145  C for 1
calculate the splitting factor (SF) (Featherstone et al., 1984; minute, then increased to 170  C at 10  C/minute and held
Lee-Thorp & van der Merwe, 1991; Nielsen-Marsh & at 170  C for 1 minute. The injector was kept at 280  C, while
Hedges, 2000). The SF corresponds to a generalized the helium gas flow was 0.66 mL/min.
‘‘degree of hydroxyapatite crystallinity’’. The FT-IR spectra The injection (1 mL) was split (split ratio of 20:1). The MS
showed the presence of calcite (CaCO3), a non-apatite conditions were as follows: interface temperature ¼ 190  C;
mineral structure, with a peak at 710 cm1, while the peak ion source temperature ¼ 240  C; and electron impact ¼70 eV.
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at 1096 cm1 was characteristic of francolite (F-apatite) The mass spectrometer was operated in selected ion moni-
(Shemesh 1990). To better understand the degree of bone toring (SIM) mode to tentatively measure the corresponding
preservation, we also calculated the C/P ratio (which is 0.5 D/L ratios. The following target ions were selected: mass/
in modern bone). The C/P ratio indicates diagenetic loss or charge (m/z) 140 for alanine (Ala); m/z 168 for norvaline and
acquisition of carbonate from bone to environment and norleucine; m/z 184 for aspartic acid (Asp); and m/z 198 for
conversely. glutamic acid (Glu).

Preparation of modern deproteinated bone Porosity study by nitrogen physisorption

Modern human bone and modern deproteinated bone were Textural characterization [BET specific surface area as
analysed to assess the extent of diagenetic alteration in the described by Brunauer et al. (1938), BJH pore size distribu-
archaeological bone samples. The modern deproteinated bone tion as described by Barrett et al. (1951) and total pore
was prepared according to Nielsen-Marsh and Hedges (1999). volume as described by Lowell and colleagues (2004)] was
obtained from the N2 adsorption-desorption isotherms volu-
Amino acid racemization metrically determined at 77 kelvins (K) by using an ASAP
2010 analyser (Micromeritics, Norcross, GA).
In living organisms, all amino acids are present in the
Before measurement, the sample tube, containing 1 g of
L-enantiomeric form; as soon as the individual dies, the
bone powder, was sealed in the degas part of the ASAP 2010
L-amino acids start to convert to the D-form through
analyser and preheated under vacuum by means of a rotary
racemization. To test for biochemical preservation as indirect
vacuum pump (ultimate vacuum 5*103 torr) at 423 K for 2
evidence for DNA survival, we estimated the degree of amino
hours. The sample tube was then transferred to the analysis
acid racemization in each sample.
part of the apparatus and immersed in a N2 liquid bath. After
About 5 mg of each sample were dissolved in 2 mL of 6 N
calibration with helium (Rivoira, 5.5 IP) of the ‘dead space’ of
HCl, transferred into a screw-capped tube under a nitrogen
the sample tube (i.e. the space inside the sample tube not
atmosphere and hydrolysed for 5 hours in an oil bath at
occupied by the powder), nitrogen (Rivoira, 5.5 IP) was
100  C. During hydrolysis, the proteins were transformed into
introduced into the sample tube and the measurement started.
amino acids. The hydrolysed samples were spiked with 50 mg
of D-L norleucine (10 mL of a 5 g/L solution) and 5 mg of D-L
Statistical analyses
norvaline (10 mL of a 0.5 g/L solution) per 1 mg of weighted
sample and completely evaporated. The residue was dissolved Past software v. 2.08 b (Hammer 2001) was used to analyse
in 3 mL of 2 N HCl in 2-propanol (Acros Organics, Morris the effect of different ages and different parameters (Kruskal–
Plains, NJ) and kept in a screw-capped tube for 1 hour at Wallis test) and the correlation between organic and inorganic
90  C. After the solvent was evaporated, the residue was component preservation. The DNA results were defined as a
dissolved in 2 mL of dichloromethane (Baker, HPLC grade, dichotomic variable: 0 when endogenous DNA was absent
DOI: 10.3109/03014460.2014.954614 Chemical parameters for ancient DNA analysis 5

and 1 when present. The Mann-Whitney test was applied to Marsh & Hedges 2000); however, the ratio can also rise if a
study correlations between the presence or absence of sample absorbs calcite from the surrounding environment
DNA and the state of human bone preservation: organic (Smith et al., 2007). In our analysis, the samples that
and inorganic phases. Logistic regression was also employed presented a peak at 710 cm1, with an amount of CaCO3
to determine which variables were better associated with 43%, had C/P values 40.7. The relationship between SF and
the presence of amplifiable DNA molecules (IBM SPSS the C/P ratio (r ¼ 0.56; p50.001) implied that variations in
Statistics 19). the C/P ratio are a fundamental part of increasing crystallin-
ity. When the SF of the three different sites (Figure 2(a)) was
Results and discussion compared, the Kruskal-Wallis test indicated no significant
difference among them (H ¼ 4.33; p ¼ 0.11). This was
DNA and inorganic component analysis
probably due to the climatic conditions. Because Velia is a
MtDNA copy number estimates were determined by Real sub divo site, the bones were subjected to climate variations
Time qPCR for each amplicon size (Appendix). qPCR results, that accelerated inorganic diagenesis. The samples from Mora
of short fragments (67 bp), showed the presence of DNA with Cavorso and some from Romito were recovered from caves
more than 900 molecules/mL in 39 of 53 samples (denoted as with high humidity but constant temperature, which prevented
dichotomic variable 1 in Table 1). Four samples showed 100 thermal shock that could have increased bone diagenesis.
molecules/mL and the other 10 samples 0 molecules/mL Because SF increases during the diagenesis of bones,
(denoted as dichotomic variable 0 in Table 1). The quality of values of 4 could suggest a post-burial growth in crystal size
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the aDNA was highlighted by differences in the amplification or selective dissolution of more soluble but less ordered
efficiency for the two length fragments, with greater crystals. In fact, the samples with no amplifiable DNA
efficiency for the smaller than the larger fragment showed SF values 43.6 and only two samples, Romito 2 and
(Table 1). The DNA damage parameter (l), proposed by T.118 (Velia), had SF values 53.5. When the SF values
Deagle et al. (2006), was also calculated (Table 1). The DNA between samples showing the presence/absence of aDNA
damage estimate (l) varied between 0.0034 and 0.010 in the were compared, the Mann–Whitney test (U ¼ 14;
Velia samples, between 0.0047 and 0.0065 in the Mora p ¼ 1.83*107) indicated a significant difference between
Cavorso samples and between 0.0050–0.0059 in the Romito amplifiable and non-amplifiable aDNA. This is probably
samples. The mean fragment size in each sample was related to the fact that DNA molecules bind to hydroxyapatite,
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estimated using the formula: 1/l. The mean amplicon protecting the DNA from environmental attack (Sosa et al.,
fragment size was 214 bp for Velia, 185 bp for Mora 2013).
Cavorso and 181 bp for Romito. These results indicated that
the aDNA was in good condition, with a probable average
DNA and organic component analysis
fragment size greater than 150 bp, as proposed by Deagle
et al. (2006). Moreover, when the damage parameter (l) of the For the organic component, the amount of collagen was
three different sites was compared, the Kruskal-Wallis test gravimetrically measured (Table 1). The modern controls had
indicated no significant difference among them (H ¼ 4.92; a range of collagen yield between 0 (for the modern
p ¼ 0.085), suggesting that the environmental conditions deproteinated control) and 100% (for the modern control).
rather than the different ages probably played a key role in The collagen yield values indicated a loss of collagen in
DNA diagenesis. Since the preservation of aDNA is chiefly almost all samples, with protein content values 520%,
influenced by climatic conditions and hot climates in highlighting strong diagenesis of collagen. However, some
particular damage aDNA (Bollongino et al., 2008), the samples (Table 1) with a collagen yield close to 20% showed
caves’ micro-environments most likely provided the proper good SF values, which could suggest only partial dissolution
conditions for preserving the aDNA. Complete HVS-I and of the organic component in spite of the inorganic component.
HVS-II mtDNA sequences were obtained for 20 samples A statistically significant correlation was found between
(46.51% of the processed sequences); nine consensus collagen yield and SF (r ¼ 0.50; p50.001). This implies that,
sequences from the Velia site, which were totally confirmed as collagenous material is lost from the bone, its re-organiza-
by cloning, are reported in Table A2 (in the Appendix). No tion within the microstructure affects bone crystallinity
matches were found between the ancient sequences and those (Hedges & Millard 1995; Nielsen-Marsh & Hedges 2000).
of either the archaeologists or the laboratory staff (Table A2 Although the mean collagen percentage was better in the
in the Appendix). samples from Velia (Figure 2(b)), the Kruskal–Wallis test
Table 1 reports the results of FT-IR spectroscopy, collagen showed no significant differences among the three different
yield analysis and the presence or absence of aDNA in the sites (H ¼ 3.22; p ¼ 0.20). This is probably due to the different
ancient samples as compared to the modern bone samples. environmental conditions of the three sites. Although Velia is
For the inorganic component, all samples showed the later than the other two, the Mora Cavorso and Romito caves
presence of francolite (F-apatite), which is the first form of have almost constant internal temperatures, which might also
re-crystallization of hydroxyapatite. Table 1 reports the whole have favoured slow degradation not only of the inorganic but
range of SF (2.86–4.15). The C/P ratio ranged between 0.1– also of the organic component.
0.82 (Table 1). The C/P ratio was generally 0.5 and The Mann-Whitney test of the collagen ratio values
decreased to 0.1 as degradation increased. Low C/P values between samples showing the presence/absence of aDNA
will indicate diagenetic loss of carbonate, possibly during indicated a significant difference between amplifiable and
rearrangement or dissolution of the inorganic phase (Nielsen- non-amplifiable aDNA (U ¼ 97; p ¼ 4.00*104). The samples
6 G. Scorrano et al. Ann Hum Biol, Early Online: 1–10

Figure 2. (a) Mean and standard deviation of

SF results; (b) Mean and standard deviation
of collagen ratio of samples from three burial
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with amplifiable DNA showed a range of collagen percentage Table 2. Racemization values: D/L Asp; Ala; Glu enantiomeric ratio of
430%. Only five samples (T.118, T.119, T.361, T.364 and total and of collagen.
T.449) from Velia had collagen values 430% and failed to
yield DNA. Conversely, five samples (T.51, T.402 from Velia, Racemization
117 from Mora Cavorso, Romito 4 and Romito 6) showed Lab code D/L - Ala D/L - Asp D/L - Glu
collagen ratio values 530%, but they yielded aDNA, perhaps T.85
because DNA molecules are trapped by mineralizing collagen Total 0.0048 ± 0.0002 0.0440 ± 0.0020 0.0080 ± 0.0000
fibrils during bone resorption and formation (Sosa et al., Collagen 0.0018 ± 0.0001 0.0225 ± 0.0019 0.0029 ± 0.0006
2013) and being so packed probably makes them more
Total 0.0032 ± 0.0001 0.0540 ± 0.0020 0.0051 ± 0.0001
resistant to hydrolysis or microbial attack. Collagen 0.0009 ± 0.0002 0.0148 ± 0.0026 0.0011 ± 0.0002
To complete the analysis of the organic component, two SS 5
samples from each site underwent amino acid racemization Total 0.0068 ± 0.0002 0.0520 ± 0.0030 0.0130 ± 0.001
Collagen 0.0036 ± 0.0009 0.0158 ± 0.0011 0.0029 ± 0.0006
analysis. We calculated the rate of racemization in both total 92 B
protein of bone and collagen extract (Table 2) (Verzijl et al., Total 0.0065 ± 0.0004 0.0490 ± 0.0020 0.0110 ± 0.0009
2000). In order to determine the degree of contamination Collagen 0.0014 ± 0.0004 0.0150 ± 0.0009 0.0026 ± 0.0008
from exogenous proteins, three amino acids were studied. 4
Total 0.0075 ± 0.0002 0.0600 ± 0.020 0.0099 ± 0.0001
Their speed of racemization was: D/L Asp 4 D/L Glu 4 D/L Collagen 0.0023 ± 0.0007 0.0220 ± 0.0036 0.0031 ± 0.0009
Ala (Ohtani et al., 2004). 5
Table 2 shows the results of amino acid racemization Total 0.0077 ± 0.0003 0.0900 ± 0.0004 0.0130 ± 0.0008
(AAR) analysis of total protein and collagen extract. For the Collagen 0.0017 ± 0.0003 0.0260 ± 0.0077 0.0022 ± 0.0004
total protein AAR analysis, the speed of the three amino acids
was adequate, demonstrating that bone decontamination was
successful. The D/L Asp values were 50.1 (Poinar et al., preserved bone collagenous protein (Sosa et al., 2013).
1996) for all samples and on both analyses (total protein and Contrary to our hypothesis and the literature data (Sosa
collagen extract) (Table 2). et al., 2013), we observed no correlation between the collagen
Only SS 5 from Mora Cavorso had a higher D/L ratio for Ala ratio and AAR (p40.05), probably due to the scarcity of the
than for Glu on collagen extract analysis; this could be taken as samples.
evidence for contamination by more recent amino acids. The Considering both the organic (ratio of collagen) and
AAR analysis in both studies was in agreement with well- inorganic (SF) phases, logistic regression was also calculated
DOI: 10.3109/03014460.2014.954614 Chemical parameters for ancient DNA analysis 7
Table 3. Textural analysis.

BET specific area Pore volume

Lab code (m2/g) (mL/g)
Modern 1 0.0048
SS 5 54 0.12
92 B 38 0.098
2 42 0.16
3 49 0.17
4 66 0.15
5 55 0.11
6 27 0.076
9 65 0.17
Modern deproteinated 145 0.37

to determine which parameters were associated with the

presence of DNA. The model based on the SF and the
collagen percentage was statistically significant (p ¼ 0.004).
Our data seem to suggest that, of the two variables, the SF is Figure 3. Pore size distribution of the ancient samples and the modern
Ann Hum Biol Downloaded from by Tulane University on 09/29/14

inversely associated with the presence of DNA (r ¼ 0.79) controls, expressed as the variation of cumulative pore volume (V) as a
function of pore diameter (D).
with respect to collagen yields (r ¼ 0.52).

The extent of diagenetic alteration can influence the shift
Pre-historic samples were chosen for porosity analysis in of pore size distribution to larger pores (Hedges & Millard,
order to better describe the state of preservation in these 1995; Nielsen-Marsh, 1997). Because large pores in archaeo-
bones. Bone porosity, as measured by nitrogen adsorption, is a logical bone are much more susceptible to the action of water
useful indicator of the state of bone preservation. and micro-organisms, accelerating mineral dissolution and
Modern bone is reported to contain 12% porosity, mainly collagen degradation, we assessed the pore size distribution
For personal use only.

contained in micropores (Nielsen-Marsh et al., 2000b). The (BJH method) of the samples (Figure 3). The effect of
modern sample exhibited a volume per gram of pores as low deproteination on modern bone is striking when compared
as 0.0048 mLg1 and a BET surface area of 1 m2/g (Table 3). with the literature (Smith et al., 2007). As regards the porosity
For the archaeological bones, textural analysis based on range determined by nitrogen adsorption, all the pre-historic
nitrogen adsorption revealed alterations in the micropores, samples showed intermediate values between the modern and
defined as a pore diameter ranging between 0.01–0.1 mm the modern deproteinated controls, suggesting a proper state
according to Turner-Walker et al. (2002). of preservation. The 92 B (Mora Cavorso) and 6 (Romito)
Table 3 reports the textural parameters (BET total surface bone samples showed the same pore size distribution and the
area and pore volume) of the bone samples from Mora best state of preservation.
Cavorso and Romito as compared with the modern and the
modern deproteinated controls. As compared with the modern
untreated sample, the bone without proteins showed a sharp Conclusion
increase in both surface and pore volume, 145 m2/g and Several studies on different parameters of bone preservation
0.37 mL/g, respectively. In the deproteinated bone, the have been carried out to understand the diagenetic process
porosity increased because the whole bone matrix, especially (Hedges & Millard 1995; Nielsen-Marsh 1997; Nielsen-
the organic component, was completely destroyed by the Marsh & Hedges 1999). To date, few have correlated bone
hydrazine hydrate treatment responsible for the conversion of preservation with the presence of DNA and only Sosa et al.
micropores in macropores. In contrast, the archaeological (2013) described the relationship between the preservation of
samples showed intermediate porosity values between those the organic/inorganic component and DNA survival in ancient
obtained for the modern and the modern deproteinated samples. The study of aDNA is limited by DNA degradation
controls (Table 3). This could indicate a suitable degree of and contamination by contemporary exogenous DNA in
preservation of bone tissue in both sites, which appears to be almost all ancient remains. Therefore, we tried to obtain a
unrelated to the age of the sample. clearer picture of the relationships between the state of human
Porosity data should reflect the degree of diagenetic bone preservation and the presence of aDNA. On this basis,
alteration, irrespective of protein content (% collagen) and SF, we propose an approach to selecting ancient bone samples
because porosity should depend on both the organic and suitable for aDNA analysis.
inorganic preservation of bone tissue (Nielsen-Marsh & We employed several multidisciplinary methods to
Hedges 1999). Unexpectedly, we observed only a negative describe the natural diagenetic states of bone preservation
correlation between porosity and the percentage of collagen: observed in bone tissue samples from several different
BET/ratio of collagen (r ¼ 0.67; p ¼ 0.035) and pore archaeological sites. The archaeological bone samples were
volume/ratio of collagen (r ¼ 0.73; p ¼ 0.016). No correl- found to contain both well-preserved organic and inorganic
ation was found between porosity and SF (p40.05), probably components and to yield amplifiable DNA. Moreover, our
due to the scarcity of samples. data show that the SF is the parameter best associated with the
8 G. Scorrano et al. Ann Hum Biol, Early Online: 1–10

presence of DNA. Therefore, since SF calculation is rapid, cytosine deamination in ancient DNA. Nucleic Acids Res 23:
inexpensive and requires a small quantity of sample, its Lee-Thorp JA, van der Merwe NJ. 1991. Aspects of the
evaluation constitutes an efficient step to take before chemistry of modern and fossil biological apatites. J Archaeol Sci
proceeding with time-consuming and costly aDNA analyses. 18:343–354.
Loreille O, Vigne JD, Hardy C, Callou C, Treinen-Claustre F,
Dennebouy N, Monnerot M. 1997. First distinction of sheep and
goat archaeological bones by the means of their fossil mtDNA.
Declaration of interest J Archaeol Sci 24:33–37.
The authors report no conflicts of interest. The authors alone are Lowell S, Shields JE, Thomas MA, Thommes M. 2004. Characterization
responsible for the content and writing of this article. of porous solids and powders: surface area, pore size and density, vol.
This study was partially supported by MIUR-PRIN: Grant number 16. New York: Springer.
2010EL8TXP_001 allotted to O.R. We wish to thank Kenneth Britsch for Martini F, Bisconti M, Casciarri S, Fabbri PF, Leonini V, Lo Vetro D,
his assistance with the English revision of the manuscript. Mallegni F, et al. 2004. La nuova sepoltura epigravettiana ‘Romito 7’
a Papasidero. In: I.I.P.P., editor. Atti della XXXVII Riunione
Scientifica I.I.P.P., ‘Preistoria e Protostoria della Calabria’. Firenze:
Istituto Italiano di Preistoria e Protostoria. p 101–111.
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Appendix: Ancient DNA protocols applied in the activation at 95  C for 20 seconds, followed by 40 cycles at 95  C for 3
seconds and 60  C for 30 seconds. The starting quantity of aDNA was
present work determined by comparing it to a standard curve of a known amount of
modern DNA. Five-fold serial dilutions, from 270 to 2.70*106
Extraction and analysis of ancient DNA were carried out at the molecules/mL, for the two modern DNA fragments, were used to make
aDNA Laboratory, Departmental Center of Molecular Anthropology the standards.
for Ancient DNA Studies, University of Rome, Tor Vergata, in
Villa Mondragone, Monte Porzio Catone, Rome (
antico/Facilities.htm). The laboratory has state-of-the-art facilities for mtDNA amplification and sequencing
aDNA studies, including a contamination-resistant facility, maintained at The hypervariable regions HVS-I and HVS-II (each &400 bp long) of
positive pressure, UV-irradiated and HEPA-filtered, with restricted
Ann Hum Biol Downloaded from by Tulane University on 09/29/14

the control region D-loops of the mtDNA were sequenced through short
access to minimize potential contamination with extant human DNA. (80–140 bp) and overlapping fragments. Enzymatic amplification of
these traits was performed using a PCR assay. The reaction mixture
Ancient DNA extraction contained: DNA template 2–3 mL, 1X PCR Gold Buffer (Applied
Biosystems), 2.5 mM MgCl2 (Applied Biosystems), 1 mM dNTPs
To maximize the potential for success and minimize the potential for (Invitrogen), 80 nM Primers L (Eurofins MWC Operons), 80 nM
inhibition/contamination, a silica-based approach was used (Yang et al., Primers H (Eurofins MWC Operons), 0.1 mg/mL BSA (New England
1998). This method has the advantage that it is more specific for DNA BioLabs Inc.), 1 U AmpliTaq Gold (Applied Biosystems), ddH2O up
and less likely to co-purify PCR inhibitors. However, since silica to 25 mL.
particles are powerful PCR inhibitors, care must be taken to ensure that Cycle conditions were initial denaturation at 94  C for 15 minutes,
the final extract is free of residual silica particles. DNA was extracted 38–55 cycles of 30 seconds at 92–94  C, 30 seconds at 53–58  C and 30
using a QIAquickÔ PCR Purification kit (Qiagen). The kit provides spin seconds at 72  C, followed by a final extension at 72  C for 10 minutes.
For personal use only.

columns, buffers and collection tubes for silica-membrane-based puri- Table A1 lists the primers covering the HVS-I and HVS-II regions.
fication of PCR products from 100 bp to 10 kb. This protocol is ideally PCR products were visualized by electrophoresis on 1.5% agarose gel
suited for aDNA samples analysis since the DNA templates are highly stained with GelStar (Cambrex, Rockland, ME). Positive amplification
degraded and the target regions for amplification are generally quite products were purified with ExoSAP-IT (USB Affymetrix).
small (100–250 bp). Dye labelling was performed with both forward and reverse primers
Extraction was performed on ancient bones. The procedure was (used for the amplification fragments; Table A1) to obtain overlapped
divided into four different steps as follows. After pre-treatment with sequences of both strands using Big Dye Terminator chemistry (v1.1,
sterile surgical blades and overnight UV irradiation to remove potential Applied Biosystems) following the manufacturer’s procedure. Cycle
contaminants on the outer surface of the bone samples, &500 mg of conditions provide 25 cycles at 96  C for 10 seconds, at 50  C for 5
bone was pulverized using a mortar and pestle. Lysis and digestion: 2 mL seconds and at 64  C for 4 minutes. The products were purified
of extraction buffer (0.5 M EDTA pH 8, 10% SDS and 20 mg/mL using CENTRI-SEP Columns (Princeton separations) and/or standard
proteinase K) were added to the powder. The solution was incubated in a ethanol precipitation technique and submitted to capillary electrophor-
shaking water bath at 55  C overnight and then at 37  C for 24 hours. esis on an ABI Prism 3130 Avant (Applied Biosystems). The results of
DNA extraction: the extraction solution was centrifuged for 15 minutes some of the ancient samples are shown in Table A2.
at 4000 rpm and the supernatant was transferred to a new 50 mL FalconÕ
tube. Five volumes of PB (QIAquickÔ PCR purification kit, Qiagen,
containing guanidine hydrocloride and isopropanol) were added to the
solution. The solution was filtered using a QIAquickÔ column by PCR product cloning
centrifugation at 12 800 rpm for 2 minutes until the entire volume was All samples were cloned. Cloning was performed by using a TOPO TA
filtered. The DNA entrapped in the filter was washed by adding 750 mL cloning kit (Invitrogen, Carlsbad, CA) according to the supplier’s
PE (QIAquickÔ PCR purification kit, containing ethanol) and protocol. Selected colonies of the positive transformants were individu-
centrifuged at 12 800 rpm for 2 minutes. Then, 100 mL of TE buffer ally suspended in 20 mL of the PCR product and then analysed
(QIAquickÔ PCR purification kit, containing 10 mM Tris-Cl, 1 mM and sequenced. At least 15 clones corresponding to each amplified
EDTA, pH 8.0) were added into the filter tube to elute the DNA and the region were sequenced to obtain a statistically significant consensus
DNA solution was centrifuged at 12 800 rpm for 2 min. sequence.

DNA quantification DNA analysis in faunal remains

The number of molecules of mitochondrial DNA (mtDNA) was To authenticate the results, sheep faunal remains were analysed.
quantified using a 7500 Fast real-time PCR System (Applied Cytochrome B (Loreille et al., 1997) and D-loop (Cai et al., 2007) of
Biosystems). Two different fragment lengths were amplified from the mtDNA were analysed.
HVS-I: the shorter 67 bp delimited by primers F16453 (50 CCGGGCCC The reaction mixture contained: DNA template 2–3 mL, 1X PCR
ATAACACTTG 30 ) and R16497 (50 ACCCTGAAGTAGGAACCAGAT Gold Buffer (Applied Biosystems), 2.5 mM MgCl2 (Applied
GT 30 ) and the longer 129 bp delimited by primers F16453 and R16560 Biosystems), 1 mM dNTPs (Invitrogen), 80 nM Primers L (Eurofins
(50 AGACCTGTGATCCATCGTGATG 30 ). All quantitative PCR MWC Operons), 80 nM Primers H (Eurofins MWC Operons), 0.1 mg/
(qPCR) assays were carried out in a 25 mL reaction volume containing mL BSA (New England BioLabs Inc.), 1 U AmpliTaq Gold (Applied
12.5 mL TaqManÕ (Applied Biosystems), 0.5 mL Probe (6-FAM- Biosystems), ddH2O up to 25 mL.
TAGCTAAAGTGAACTGTATCC-MGB, Applied Biosystems), for- For L15496 and H15661, the cycle conditions were initial denatur-
ward and reverse primers (100 mM), 2.5 mL of DNA extract and ation at 94  C for 15 minutes, 38–55 cycles at 92–94  C for 30 seconds,
ddH2O up to 25 mL. Thermocycling conditions were initial enzyme at 53  C for 30 seconds and at 72  C for 30 seconds, followed by a final
10 G. Scorrano et al. Ann Hum Biol, Early Online: 1–10

Table A1. Primers for amplification reactions of HVS-I and HVS-II of mtDNA in human samples and faunal remains.

Region Primer forward (L) Primer reverse (H)

Ann Hum Biol Downloaded from by Tulane University on 09/29/14

Table A2 mtDNA sequences of archaeologists, laboratory staff and some samples from the Velia site. mtDNA positions were numbered according to
the rCRS (Andrews et al., 1999).

Samples HVS-I sequence HVS-II sequence

Archaeologists and laboratory staff
1 16126C; 16153A; 16294T; 16296T 73G; 150T; 199C; 263G
2 CRS 152C; 263G
3 16183G, 16189C; 16215G; 16223T; 16278T; 16295T; 16365T 73G; 153G; 195C; 225A; 226C; 263G
4 16129A; 16182C; 16183C; 16189C; 16223T; 16249C; 16359C; 16362C 73G; 195C; 204C
For personal use only.

5 16192T; 16239T; 16256T; 16270T; 16291T; 16385G 73G; 263G

6 16193T; 16224C; 16311C 73G; 263G
7 16336A 263G
Sample analysed
Velia a CRS 263G
Velia b CRS 263G
Velia c 16093C; 16224C; 16311C 73G; 152C; 263G
Velia d 16126C; 16289G; 16294T; 16296T 73G; 263G
Velia e 16126C; 16294T; 16296T 73G; 263G
Velia f 16145A; 16176G; 16223T; 16367G 73G; 152C; 263G
Velia g 16252G; 16336A; 16390A 73G; 263G
Velia h 16069T; 16126C; 16261T 73G; 263G
Velia i 16129A 263G

extension at 72  C for 10 minutes. For the cytochrome B fragment Since the animal bones yielded the expected sequences (sheep DNA
(L15690 and H15784), the cycle conditions were 38–55 cycles at from sheep bone), it is reasonable to expect that the human DNA is
92–94  C for 30 seconds, at 55  C for 1 minute and at 72  C similarly preserved in human bones from the same site rather than
for 10 seconds. Table A1 lists the primers used for each fragment derived from contamination.

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