Journal of Archaeological Science 32 (2005) 785e793
http://www.elsevier.com/locate/jas
Biochemical and physical correlates of DNA
contamination in archaeological human bones and
teeth excavated at Matera, Italy
M. Thomas P. Gilberta,*, Lars Rudbeckb, Eske Willersleva,c,
Anders J. Hansenc,d, Colin Smithe,1, Kirsty E.H. Penkmane,2,
Kurt Prangenberge,f, Christina M. Nielsen-Marshe, Miranda E. Jansg,
Paul Arthurh, Niels Lynnerupi, Gordon Turner-Walkerj,3, Martin Biddlek,
Birthe Kjølbye-Biddlek, Matthew J. Collinse,g,2
a
Henry Wellcome Ancient Biomolecules Centre, Department of Zoology, University of Oxford,
South Parks Road, Oxford OX1 3PS, UK
b
Research Laboratory, Institute of Forensic Medicine, University of Copenhagen, Frederik V Vej 11,
DK-2100 Copenhagen, Denmark
c
Department of Evolutionary Biology, Zoological Institute, University of Copenhagen, 5 Universitetsparken,
DK-2100 Copenhagen Ø, Denmark
d
Department of Forensic Genetics, University of Copenhagen, Frederik V’s Vej 11, DK-2100 Copenhagen, Denmark
e
Fossil Fuels and Environmental Geochemistry, NRG, Drummond Building, University of Newcastle,
Newcastle upon Tyne NE1 7RU, UK
f
Institut für Geowissenschaften, Sigwartstrasse 10, 72076 Tübingen, Germany
g
Institute for Geo and Bioarchaeology, Vrije Universiteit Amsterdam, De Boelelaan 1085, 1081 HV Amsterdam, Holland
h
Dipartimento di Beni Culturali, Via D. Birago, 64, University of Lecce, 73100 Lecce, Italy
i
Laboratory of Biological Anthropology, Institute of Forensic Medicine, University of Copenhagen,
Frederik V Vej 11, DK-2100 Copenhagen, Denmark
j
Institutt for arkeologi og kulturhistorie, NTNU, Vitenskapsmuseet, 7491 Trondheim, Norway
k
Hertford College, University of Oxford, Oxford OX1 3BW, UK
Received 28 October 2004; received in revised form 29 October 2004
Abstract
The majority of ancient DNA studies on human specimens have utilised teeth and bone as a source of genetic material. In this
study the levels of endogenous contamination (i.e. present within the sample prior to sampling for the DNA analysis) are assessed
within human bone and teeth specimens sampled from the cemetery of Santa Lucia alle Malve, Matera, Italy. This site is of
exceptional interest, because the samples have been assayed for 18 measures of biochemical and physical preservation, and it is the
only one identified in a study of more than 107 animal and 154 human bones from 43 sites across Europe, where a significant
number of human bones was well preserved. The findings demonstrate several important issues: (a) although teeth are more resilient
* Corresponding author. Ecology and Evolutionary Biology, The University of Arizona, 1041 East Lowell Street, Tucson, AZ 85721, USA. Tel.:
C1 520 621 4881; fax: C1 520 621 9190.
E-mail address: [email protected] (M. Thomas P. Gilbert).
1
Present address: Department of Palaeobiology, Museo Nacional de Ciencias Naturales (CSIC), C/José Gutiérrez Abascal, 228006 Madrid, Spain.
2
Present address: BioArch, The King’s Manor, University of York, York YO1 7EP, UK.
3
Present address: Institute for Cultural Heritage Conservation, National Yunlin University of Science and Technology, 123 University Road Sec.
3, Touliu, Yunlin 640, Taiwan.
0305-4403/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.jas.2004.12.008
786
M. Thomas P. Gilbert et al. / Journal of Archaeological Science 32 (2005) 785e793
to contamination than bone, both are readily contaminated (presumably through handling or washing), and (b) once contaminated
in this way, both are difficult (if not impossible) to decontaminate. Furthermore, although assessed on bone samples, several of the
specific biochemical and physical characteristics that describe overall sample preservation, levels of microbial attack and related
increases in sample porosity directly correlate with the presence of observable contamination in both bone and teeth samples from
individual samples. While we can only speculate on the cause of this relationship, we posit that they provide useful guides for the
assessment of whether samples are likely to be contaminated or not.
Ó 2005 Elsevier Ltd. All rights reserved.
Keywords: Ancient DNA; Biopreservation; Bone; Contamination; Diagenesis; Human; Teeth
1. Introduction
Bones and teeth are normally the longest lasting
physical evidence of human or animal presence at an
archaeological site, and are also the most widely used
sources of samples for ancient DNA (aDNA) studies.
Post hoc explanations of their suitability as a source
of ancient DNA have identified retarded rates of
decomposition, arising from adsorption of DNA to
hydroxyapatite [30], low water content [22], ‘mummification’ of individual cells [4,5] and physical exclusion of
microbes and external contaminants [22]. Recently, an
awareness of sample handling as a source of contamination has led researchers to investigate teeth as an
aDNA source. One hypothetical benefit is protection
conferred by enamel [32]. Additionally, although histological studies identify higher numbers of DNA
containing cells per unit area of bone than teeth [12],
several studies have reported better DNA yields in teeth
than bone [29,32].
Richards et al. [36] have argued convincingly that
contamination, not DNA preservation, is the greatest
problem facing the field, although the two are evidently
linked e sparse, damaged endogenous DNA is less likely
to be amplified than modern contamination. Although it
is known that teeth and bone may become contaminated
prior to aDNA extraction [36], current techniques used
to decontaminate specimens e the application of bleach,
exposure to UV light, and grinding or shot-blasting e
reflect a belief that contamination is concentrated in the
outer surface of the material. Protocols designed to limit
contamination stress the prevention of contact between
samples and previously amplified DNA (amplicons)
[11,18]. Nevertheless, even when strict protocols are
followed contaminants are frequently observed. For
example, human DNA has been reported from cave bear
[21], 500-year-old pig samples [36] and 109 out of 168
relatively recent fox teeth [43]. More seriously, several
studies report significant numbers of human remains
contaminated with multiple human sequences [19,27].
Obviously, decontamination methods are not 100%
efficient, and contamination remains a serious threat to
the validity of ancient DNA studies, particularly on
human templates.
In this study we have investigated the presence and
persistence of contamination in teeth and femur samples
collected from human skeletons excavated at the
cemetery of Santa Lucia alle Malve, Matera, Italy [8].
Bone samples from the specimens have been assayed for
18 measures of biochemical and physical preservation,
allowing these parameters to be correlated with modern
DNA contamination in both bones and teeth.
2. Materials and methods
Twenty-six teeth and eight femur samples were taken
from 13 individuals excavated at the cemetery above the
cave-church of Santa Lucia alle Malve, Matera, Italy [8]
(Table 1). The pH of the soil at the site ranges from 8.0
to 8.3. Carbon-14 dating indicates that the samples date
to approximately the late 14th century (Clare Owen,
Oxford RLAHA, pers. comm.). In a study that
investigated more than 107 animal and 154 human
bones from 41 sites across Europe (spanning four
climatic regimes e Mediterranean, Continental, Maritime (coastal) and Subarctic, and dating from 250 to
5950 Y.B.P. [24]), this site was the only one in which
a significant number of human bones (7 of 14) were well
preserved [23e25,38] (Table 1). Additionally, as part of
this study the extent of the combined asparagines/
aspartic acid (Asx) racemization was determined in one
tooth from each individual using the extraction method
of Poinar et al. [35] and the analytical method of
Kaufman and Manley [26]. Furthermore, all have been
stored together, and have undergone similar amounts of
human handling post-excavation.
2.1. Ancient DNA extraction and amplification
DNA was extracted from samples following strict
ancient DNA protocols in order to prevent sample
contamination with previously amplified DNA [15].
Importantly, several different extractions (2e3) were
performed per individual from distinct parts of the body
(i.e. different teeth plus femur) to help identify both the
‘endogenous’ DNA sequence and any contaminants.
One extraction blank was used for every four samples to
Table 1
Bone biochemical and physical preservation, and contamination of teeth and bone samples
Sample
Skeleton
Tissue
Contaminated
Histology
Wedl
Linear
long
Budded
Lamellar
Cracking
Collagen
C:N
N
IRSF
C:P
Bulk
density
Skeletal
density
Total
Hg
Hg !
100 nm
Hg ! 100
nme6 mm
Hg O
6 mm
Asx
TG457
TG456
TG458
TG460
TG425
TG453
TG447
TG448
TG449
TG450
TG446
TG461
TG423
TG435
TG436
TG451
TG439
TG426
TG438
TG455
TG454
TG445
TG443
TG452
TG497
TG498
TG499
TG500
TG501
TG502
TG503
TG505
p-value
(teeth)
SLM-1
SLM-1
SLM-3
SLM-3
SLM-4
SLM-4
SLM-5
SLM-5
SLM-6
SLM-6
SLM-7
SLM-7
SLM-8
SLM-8
SLM-9
SLM-9
SLM-10
SLM-10
SLM-11
SLM-11
SLM-12
SLM-13
SLM-14
SLM-14
SLM-5
SLM-6
SLM-7
SLM-8
SLM-9
SLM-10
SLM-11
SLM-12
Tooth
Tooth
Tooth
Tooth
Tooth
Tooth
Tooth
Tooth
Tooth
Tooth
Tooth
Tooth
Tooth
Tooth
Tooth
Tooth
Tooth
Tooth
Tooth
Tooth
Tooth
Tooth
Tooth
Tooth
Femur
Femur
Femur
Femur
Femur
Femur
Femur
Femur
?
?
N
N
N
N
N
N
N
N
Y
Y
Y
Y
Y
N
Y
?
Y
Y
Y
Y
Y
Y
N
Y
Y
Y
Y
Y
?
Y
4
4
5
5
5
5
4
4
2
2
3
3
2
2
5
5
3
3
4
4
2
1
2
2
4
2
3
2
5
3
4
2
0.01
1
1
0
0
0
0
0
0
0
0
1
1
1
1
0
0
1
1
1
1
0
1
1
1
0
0
1
1
0
1
1
1
!0.01
1
1
0
0
0
0
1
1
1
1
0
0
0
0
0
0
0
0
0
0
1
1
0
0
1
1
0
0
0
0
0
0
0.06
0
0
0
0
0
0
1
1
1
1
1
1
1
1
0
0
1
1
0
0
1
0
1
1
1
1
1
1
0
1
0
1
0.16
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
18
18
46
46
7
7
17
17
11
11
9
9
0
0
0
0
0
0
0
0
53
0
0
0
17
11
9
0
0
0
0
0
0.07
20.2
20.2
20.9
20.9
20.8
20.8
20.4
20.4
15.2
15.2
13.1
13.1
9.5
9.5
21.2
21.2
13.1
13.1
22.2
22.2
13.9
9
7.8
7.8
20.4
15.2
13.1
9.5
21.2
13.1
22.2
7.8
!0.01
3.3
3.3
3.2
3.2
3.3
3.3
3.3
3.3
3.3
3.3
3.3
3.3
3.4
3.4
3.2
3.2
3.3
3.3
3.2
3.2
3.3
3.3
3.3
3.3
3.3
3.3
3.3
3.4
3.2
3.3
3.2
3.3
0.44
3.3
3.3
3.7
3.7
3.9
3.9
3.4
3.4
2.3
2.3
2.6
2.6
1.9
1.9
3.9
3.9
2.8
2.8
3.8
3.8
2
1.8
2
2
3.4
2.3
2.6
1.9
3.9
2.8
3.8
2
0.02
3.3
3.3
3
3
3.3
3.3
3.5
3.5
3.8
3.8
3.2
3.2
3.8
3.8
2.9
2.9
3
3
2.9
2.9
3.7
3.2
3.5
3.5
3.5
3.8
3.2
3.8
2.9
3
2.9
3.5
0.79
1.14
1.14
0.68
0.68
1.03
1.03
0.85
0.85
0.28
0.28
0.29
0.29
0.21
0.21
0.53
0.53
0.34
0.34
0.66
0.66
0.23
0.31
0.31
0.31
0.85
0.28
0.29
0.21
0.53
0.34
0.66
0.31
!0.01
2.1
2.1
2
2
1.6
1.6
1.8
1.8
1.5
1.5
1.6
1.6
1.4
1.4
1.9
1.9
1.6
1.6
1.8
1.8
1.5
0.8
1.4
1.4
1.8
1.5
1.6
1.4
1.9
1.6
1.8
1.4
0.01
2.6
2.6
2.2
2.2
2
2
2.5
2.5
2.2
2.2
2.2
2.2
2.3
2.3
2.1
2.1
2.2
2.2
2.1
2.1
2.3
1.1
2.3
2.3
2.5
2.2
2.2
2.3
2.1
2.2
2.1
2.3
0.20
0.187
0.187
0.085
0.085
0.154
0.154
0.237
0.237
0.312
0.312
0.268
0.268
0.375
0.375
0.083
0.083
0.221
0.221
0.123
0.123
0.334
0.229
0.387
0.387
0.237
0.312
0.268
0.375
0.083
0.221
0.123
0.387
0.07
0.133
0.133
0.024
0.024
0.05
0.05
0.059
0.059
0.07
0.07
0.077
0.077
0.089
0.089
0.023
0.023
0.045
0.045
0.031
0.031
0.08
0.039
0.07
0.07
0.059
0.07
0.077
0.089
0.023
0.0449
0.031
0.07
0.82
0.055
0.055
0.021
0.021
0.034
0.034
0.097
0.097
0.194
0.194
0.163
0.163
0.266
0.266
0.032
0.032
0.159
0.159
0.065
0.065
0.223
0.167
0.24
0.24
0.097
0.194
0.163
0.266
0.032
0.1592
0.065
0.24
!0.01
ÿ0.001
ÿ0.001
0.04
0.04
0.07
0.07
0.081
0.081
0.048
0.048
0.028
0.028
0.02
0.02
0.028
0.028
0.017
0.017
0.027
0.027
0.031
0.023
0.077
0.077
0.081
0.048
0.028
0.02
0.028
0.0171
0.027
0.077
0.25
0.06
0.07
0.09
0.08
0.04
0.05
0.05
0.05
0.06
0.06
0.07
0.07
0.08
0.06
0.06
0.06
0.06
0.07
0.05
0.05
0.06
0.06
0.06
0.07
n/a
n/a
n/a
n/a
n/a
n/a
n/a
n/a
0.75
Bone samples taken from each specimen have been assessed for various measures of biochemical preservation (with the exception of Asx which was directly assessed on teeth specimens). For
methodological details see Smith et al. [39], Kars and Kars [25]. Measurements are as follows: Histology, histological assessment of bones from each individual where 0 = minimum, 5 = maximum
preservation; Wedl, evidence for presence (1) or absence (0) or Wedl fungal tunneling; Linear Long, presence (1) or absence (0) of linear longitudinal fungal attack; Budded, presence (1) or absence (0) of
budded fungal attack; Lamellar, presence (1) or absence (0) of lamellar fungal attack; Cracking, evidence of bone microscopic cracking, measured as the percentage of osteons with at least one radial crack;
Collagen, percentage collagen remaining in specimen; C:N, bone collagen carbon to nitrogen ratio; N, total bone nitrogen content (%); IRSF, bone infra-red splitting factor, measuring phosphate oxygen
bond stretch thus how perfect the crystal lattice is; C:P, carbonate to phosphate ratio; Bulk Density, bulk density (g cmÿ3) of the total bone, including all pores (density in a vacuum on 0); Skeletal Density,
skeletal density (g cmÿ3) (density after all pores have been filled); Total Hg, total pore volume as measured by mercury porosimetry; Hg ! 100 nm, pore volume in the pore range of less than 100 nm as
measured by mercury porosimetry; Hg ! 100 nm, pore volume in the range of 100 nme6 mm as measured by mercury porosimetry; Hg O 6 mm, pore volume in the pore range of greater than 6 mm as
measured by mercury porosimetry; Asx, combined aspartic acid and asparagine amino acid D:L enantiomer ratios. The 0.034% racemization induced by hydrolysis (6 M HCl, 6 h at 110 C [35] was
subtracted from all the reported data.) Student’s unpaired t-test (performed on teeth where contamination state could be determined) was used to investigate whether any correlation exists between
contamination and measures of biochemical and physical preservation.
788
M. Thomas P. Gilbert et al. / Journal of Archaeological Science 32 (2005) 785e793
monitor contaminants entering during the DNA extraction. DNA extractions from bone used 0.2 g bone
powder, collected as in Barnes et al. [3]. Unless
otherwise stated, all DNA was extracted from teeth
following Gilbert et al. [15]. All PCRs were performed
on each sample at least twice, using the polymerase
enzyme Platinum Taq Hi-Fidelity (invitrogen), and incorporating one PCR blank to every three samples.
PCRs for human mitochondrial DNA (mtDNA) used
primers L16209-H16356 [19] following Gilbert et al. [15].
We have previously demonstrated that these primers are
exceedingly sensitive to low levels of template DNA,
thus are unlikely to generate false negative data [16].
DNA extracts that did not yield PCR products were
screened for the presence of PCR inhibitors (a common
phenomena in aDNA studies [33]) through ‘spiking’
PCRs containing amplifiable DNA with an equal
volume (1 ml) of the potential inhibitor, and monitoring
any reduction in PCR success [15]. All amplified human
PCR products were cloned. Up to 12 colonies were
sequenced per cloned PCR (325 total, average 10.2
clones per PCR), thus providing between 16 and 35
cloned sequences across all independent extracts of each
individual skeleton (average 25 per skeleton). For
further details refer to supplemental information.
Endogenous and contaminant samples were identified as described in the supplemental information. In
brief, cloned PCR products were sequenced and
compared with the Cambridge Reference Sequence [2]
to identify sample-specific motifs, both within the clones
from each extract, and between different extracts from
each individual. Under an assumption that samples
contain authentic, uncontaminated DNA, it is expected
that all cloned sequences will yield the same sequence
(Fig. 1A). However, due to small amounts of postmortem DNA damage (for example, hydrolytic deamination of cytosine to uracil, providing isolated C/T
and G/A mutations, or hydrolytic deamination of
adenine to hypoxanthine, providing isolated A/G and
T/C mutations), aDNA sequences often exhibit small
amounts of variation around a consensus sequence
(Fig. 1B). If however the sample is uncontaminated, all
sequences will contain shared motifs that identify them
as originating from one original source of DNA
(Fig. 1A and B). However, should a sample be
contaminated with non-endogenous sources of DNA,
in most situations sequences that do not share motifs
will be observed among the clones (Fig. 1C).
This method has two potential weaknesses. Firstly, if
a sample contains no endogenous DNA, but has been
contaminated with one source of exogenous DNA
(which possibly due to the time-lapse since contamination may even contain some evidence of damage-driven
miscoding lesions), the results will appear authentic.
However, as all samples investigated in this study have
been handled by multiple individuals, there is no
plausible reason why only one DNA source will be
represented among contaminant sequences. A more
problematic issue arises when samples have been
contaminated by mtDNA sequences that are identical,
or very similar (e.g. differing by 1e2 bp) to that of the
sample. Based on the diversity observed among Western
European mtDNA sequences (c.f. [37]), this is unlikely
to be an issue unless the sequence is exceedingly
common, as in the case of basal haplogroup H
sequences (those that are identical to, or differ by
a single step from, the Cambridge Reference Sequence
(CRS) over the region of interest, and observed at
frequencies of up to 60% in Western Europe [41]). In
such cases the lack of distinct motifs in the sequence
makes it exceedingly difficult to distinguish contaminant
sequences from original, damaged sequences (Fig. 1D).
Therefore, such samples were left out of the analysis to
prevent misidentification of contaminated samples. For
further details on this method and why we believe it to
enable us to differentiate between authentic and
contaminant DNA sequences, we refer readers to the
arguments presented elsewhere [16] and to the information presented in the supplemental information.
2.2. Assessment of bone preservation correlates
with contamination
Human bones suffer common patterns of alteration
with microscopic focal destruction resulting in areas of
dense mineralization surrounding pores with diameters
at 600 nm and 1.2 mm microbial alteration (‘‘m’’
porosity, 600 nme1.5 mm [24,42]). We believe that this
diagenetic feature is a characteristic of the rapid
putrefaction of interred corpses by blood borne gut
bacteria as suggested by Bell et al. [5]. In order to
simplify inter-sample variation, the mid-shaft of the
femur (as a large and commonly preserved element) was
assessed to determine the extent of diagenetic alteration.
At the Matera site, only half of the sampled individuals
had the putrefaction alteration which characterized 74%
of all human skeletons in the European study. The
remaining femurs were remarkably well preserved,
partly it would appear because of secondary mineral
precipitation. Matera was the only site of 50 studied
with a significant number of well-preserved human
bones [25]. We still remain ignorant of why Matera is so
unusual in this respect, the burial in limestone cuts, and
extreme seasonal variation in temperature and moisture,
may both be implicated. The detailed record of bone
preservation of the Matera samples, the dipolar pattern
of preservation and the similar treatment of the bones
since excavation presents an ideal opportunity to
investigate correlations between contamination and
preservation indices.
Unpublished data shows that bone is more prone
to secondary contamination than dentine (M.T.P.G,
M. Thomas P. Gilbert et al. / Journal of Archaeological Science 32 (2005) 785e793
A
B
C
D
E
F
G
H
1.A
201
210
220
230
240
250
260
caagcaagtacagcaatcaaccctcaactatcacacatcaactgcaactccaaagccacccctc
......................t................................t........
......................t................................t........
......................t................................t........
......................t................................t........
......................t................................t........
......................t................................t........
......................t................................t........
......................t................................t........
A
B
C
D
E
F
G
H
1.B
201
210
220
230
240
250
260
caagcaagtacagcaatcaaccctcaactatcacacatcaactgcaactccaaagccacccctc
......................t...g............................t........
......................t................................t........
......................t................................t........
..........t....g......t................................t........
....a.................t................................t........
.......................................................t........
......................t................................t........
......................t................................t........
A
B
C
D
E
F
G
H
1.C
201
210
220
230
240
250
260
caagcaagtacagcaatcaaccctcaactatcacacatcaactgcaactccaaagccacccctc
......................t...........g....................t........
......................t................................t........
........c.......................................................
......................t................................t......c.
........c.......................................................
......................t................................t........
........c.......................................................
........c.......................................................
A
B
C
D
E
F
G
H
1.D
201
210
220
230
240
250
260
caagcaagtacagcaatcaaccctcaactatcacacatcaactgcaactccaaagccacccctc
.......................................................t........
......................t.........................................
.......................................................t........
................................................................
................................................................
.......................................................t........
................................................................
.....................................g..........................
789
Fig. 1. The identification of contaminated sequences from a cloned PCR product. (A) Cloned PCR products from uncontaminated, undamaged
DNA extracts show no sequence variation. (B) Uncontaminated, but damaged PCR products show some sequence variation, but also conserved
sequence motifs among the clones. (C) Cloned PCR products containing 1 old, damaged DNA source, plus a modern, contaminant source of DNA.
(D) In some situations it is not possible to differentiate whether variation among cloned sequences is due to contamination or post mortem damage.
For full details on figure refer to main text. For each decision applied to samples in this study refer to supplemental information.
unpublished data), as the enamel partially protects the
dentine from allochthnaous DNA. We, therefore, chose
to attempt to extract DNA from the dentine, in addition
to extractions from the mid-shaft femur samples,
reasoning that although we were unable to conduct the
same suite of analyses on the dentine, the femur data
should give an insight into the deterioration of the whole
skeleton.
results are unlikely to derive from contaminants in the
extraction or PCR processes. Furthermore, the extent of
racemization of aspartic acid and alanine in the samples
was below the threshold over which DNA is unlikely to
survive (following [35]).
3. Results
Nine Matera teeth samples contained only one DNA
sequence, and appear to be uncontaminated, while 12
teeth extracts contained multiple sequences and were
identified as contaminated (Table 1). The majority of
All extraction and PCR blanks were consistently
negative throughout the study, indicating that the
3.1. Preservation and contamination of
Matera teeth and bones
790
M. Thomas P. Gilbert et al. / Journal of Archaeological Science 32 (2005) 785e793
contaminated samples appear to contain more than two
sources of DNA, indicating either contamination on
multiple occasions, or that the samples have been
exposed to multiple sources of DNA in one go (for
example, through washing in dirty water). Additionally
in most cases, the multiple teeth and bone specimens
from the same skeleton share at least some of the
contaminant sequences. A further three teeth could not
be accurately identified as contaminated, as it could not
be resolved whether observed sequence variation was
due to damage-driven miscoding lesions or the presence
of multiple DNA sources. DNA could not be amplified
from the remaining two teeth e one each from SLM-12
and SLM-13 (data not shown). For full sequence details
see supplemental information.
There are several clear patterns between DNA
contamination in teeth and the preservation state
determined for bone samples from the same individuals.
For p-values calculated on the comparisons of biochemical and physical measurements between contaminated and uncontaminated samples (using Student’s
unpaired t-test) refer to Table 1. In particular, all
uncontaminated samples (except for teeth from skeleton
SLM-6) lack evidence of organic degradation (Histology
( p = 0.01), collagen content ( p ! 0.01), nitrogen content ( p = 0.02)), but have high mineral carbonate:phosphate ratios, indicative of secondary carbonate
precipitation ( p ! 0.01). Similarly, they all lack evidence of fungal attack (identified through a lack of Wedl
tunneling, p ! 0.01). Most interesting, the presence of
human contaminant DNA in teeth correlates with the
increased porosity in the bones of the same individual
( p ! 0.01), the pores having a diameter that contains
the range of those indicative of microbial alteration (socalled ‘m’ porosity ranging from 600 nm to 1.5 mm [42]).
Despite the rigorous bleach and UV cleaning methods, all Matera bone samples contained multiple DNA
sequences, with the exception of samples Tg497, extracted from skeletons SLM-5. Preservation data [38] on
this sample identifies it as exhibiting similar values for
histology, porosity, collagen content and mineralogy as
modern bone, with no evidence of microbial alteration
(Table 1). Perhaps the most intriguing aspect of this
sample is its very high carbonate:phosphate ratios in the
mineral, indicating the presence of secondary carbonate.
Amino acid racemization (AAR) values tended to be
lower in uncontaminated samples with the exception of
skeleton SLM-03, although all values were tightly ranged
(0.07e0.13). However even in this sample there was no
evidence from D:L ratios of Ala, Glx or Ser of measurable (peptidoglycan derived) microbial biomass (c.f. [34],
data not shown). The bone from skeleton SLM-03 had
the lowest porosity of any bone from Matera, so it is
possible that the elevated AAR value is due to the lower
rate of leaching of soluble (highly racemized gelatin), as
speculated by Collins et al. [10].
4. Discussion
The results of this study highlight a correlation
between sample bone and tooth contamination, and
overall bone preservation and porosity e in particular
that characteristic of microbial attack [42]. It has
previously been speculated [42] that putrefaction of
intact inhumed corpses is the dominant factor controlling ‘m’ porosity linked to microbial attack. Consequently (butchered) animal bone is less likely to putrefy,
thus likely to have lower ‘m’ porosity and more likely to
have potential for reliable aDNA recovery than buried
human remains. Furthermore the data indicate that as
previously hypothesized (c.f. [13]), bone is more
susceptible to contamination than teeth.
The degree and persistence of contamination must be
largely attributed to the ultra-structural organization of
bones and teeth. Fresh human bone is porous (Fig. 2),
and mercury porosimetry demonstrates that the minimum total inter-connected porosity is never less than
8% of bone volume, the majority of which is derived
from the Haversian canals, and 1e1.5% of which has
been attributed to canaliculi/osteocytes [42]. As porosity
increases post mortem, from both chemical decomposition of the collagen, and microbial destruction of the
collagen/apatite composite [42], it is likely that successive hydration cycles would allow liquid-borne contaminants deep inside specimens. Furthermore, we note that
human bone porosity is greater than that in most other
mammals, due to the higher levels of secondary
modeling experienced. This suggests that archaeological
human bone samples face greater contamination risks
Circumferential Lamellae
Concentric Lamellae
Cement Line
Osteoblast
Blood Vessel
Bone Lining Cell
Cannaliculi
Nucleus
Haversian Canal
Resorption Cavity
Osteoclast
Osteocyte
Fig. 2. Histological structure of compact bone.
M. Thomas P. Gilbert et al. / Journal of Archaeological Science 32 (2005) 785e793
(due to fluid uptake) than associated-animal remains,
thus implies that the use of such remains as a ‘contamination test’ (e.g. [11]) may be inadequate. Similar
problems are also likely to affect teeth e although tooth
dentine is devoid of Haversian canals, and the enamel
encasing tooth crowns is ideal for preventing permeation
of contaminants [13], tooth roots are surprisingly
porous (Fig. 3). In particular, the pulp cavity is directly
connected to the exterior of the root by numerous dental
tubules [12]. Therefore, once a tooth is washed or the
root is directly handled, the impervious nature of enamel
is of no help in limiting contamination, which may
explain the multiple sources of DNA found in most
teeth in this study.
In view of the correlation observed between bone
porosity, and tooth and bone contamination in such
well-preserved samples, it seems plausible that widespread contamination may be present in archaeological
specimens that were excavated in the past. For example,
in many cases freshly excavated samples were cleaned in
dirty water that was not changed between samples. In
addition, one tool of choice to aid cleansing was old and
used human toothbrushes. Thus these would have
provided an immediate source of human and bacterial
DNA. However it is surprising that in our experiments,
soaking samples in bleach does not degrade contaminant DNA. One possibility is that bleach is prevented
from fully penetrating samples by deposits (potentially
deriving from sediment in water used to wash samples)
that are laid down within the porous regions of the
samples as samples dry over time. Alternatively some
drying may seal pore interconnectivity through collapsing collagen bundles or drying fronts of other dissolved
species. Indirect evidence for this comes from one
measure of preservation that inversely correlates with
contamination e ‘cracking’ e which Hedges [20] has
Enamel
Dental Tubules
Crown
Pulp cavity
Jaw Bone
Dentine
Supporting Ligaments
Cementum
Root
Accessory Canal
Root End Opening
Fig. 3. Histological structure of a human tooth. Dental tubules are
shown larger than actual size.
791
hypothesized results from bone swelling, due to mineral
deposition within Haversian canals.
If the above hypothesis is true, then it is possible that
the disruption of internal blockages through techniques
which powder the samples prior to cleansing warrant
further investigation. An alternative approach to consider is partial demineralization of the bone or teeth with
EDTA prior to the bleaching. This may increase both
accessibility to contaminant DNA through the action of
EDTA removing diagenetic calcium and iron salts, and
the action of bleach destroying collapsed regions of
collagen. A final method that is also worth investigating
is the application of bleach to bone under a vacuum.
4.1. Prescreening samples for contamination
Why a correlation between bone preservation indices
and tooth contamination exists is a difficult question.
We speculate that the fundamental biochemical and
structural similarities between teeth and bone [12] result
in similar patterns of degradation. However, regardless
of the explanation, the data clearly indicate that
correlations exist, and these provide an important aid
to identifying suitable specimens for aDNA analysis.
Such indices would save considerable time, while
limiting destructive sampling, but may help critically
assess previous ancient human DNA results. Overall
these will be of most use if they help provide a
taphonomic context in which DNA preservation and
contamination can be understood.
Porosity, in particular that caused by microbial
alteration of the bone, appears to be the most helpful
measure. Whilst this is not the only factor, techniques
such as microscopy or mercury porosimetry which assay
this [42] will provide a useful guide to the contamination
load of a sample (other factors that will presumably
destroy DNA but not lead to microbial porosity include
accelerated destruction of the organic phase as observed
at Apigliano, Italy [38]). An alternative technique now
routinely used on ancient samples prior to DNA
analysis that may also be of use is amino acid
racemization analyses ([35], though see [10]) though
whether a significant correlation exists between racemization and porosity requires further investigation.
However, given the presence of microbial destruction
even in bones with apparently exceptional preservation
from Matera, perhaps the best approach is to select for
bones which have not derived from interred corpses. We
note in passing that because animals are rarely interred
as intact corpses they display much less evidence of
porosity attributed to putrefaction [24]. In addition to
a potential associated impact on that may reduce the
overall rate of DNA degradation, this may explain why
they are proving a more promising substrate for aDNA
study than human material.
792
M. Thomas P. Gilbert et al. / Journal of Archaeological Science 32 (2005) 785e793
5. Conclusions
The results of this study highlight serious inadequacies in techniques used to decontaminate archaeological
samples (in particular those that rely on bone) from
contaminant DNA, and have implications for any
aDNA study where contamination is a risk. Importantly,
this also includes studies of pathogen DNA in ancient
samples, which may be at risk from DNA originating
from environmental organisms [14], as well as studies on
animal bones that have been treated with preservatives
and glues containing animal DNA [31]. Furthermore, the
results suggest that, if such contamination persists within
‘cleaned’ samples, even the adoption of authenticity
criteria (e.g. [11]) such as independent replication cannot
guarantee the retrieval of authentic DNA sequences.
The simplest remedy for human DNA contamination
is the controlled excavation of human samples, preventing direct handling and washing of samples that are to
be used for aDNA analyses. Unfortunately as most
samples cannot be freshly excavated, and many important specimens were collected in the past, the majority is
likely to be already contaminated. Thus further studies
are required to investigate the possibility of new
decontamination techniques on such specimens. In
addition other sources of aDNA that may be more
resilient to contamination such as hair need to be
investigated [16].
The findings of this study also suggest that a critical
reader must consider three key points before gauging
the reliability of results from previous human aDNA
studies. Firstly, the preservation of the samples. For
example, Clisson et al. [9] report human DNA retrieval
from a O2000 year old specimen from Kazakhstan. The
excellent preservation reported suggests that contamination is not an issue. Secondly, sequence authenticity.
For example, although not well preserved, the distinct
nature of Neanderthal sequences helps suggest authenticity of results [28]. Thirdly, and most importantly,
knowledge of the history of the sample handling prior to
the analysis is critical. For example, in the important
early Australian ‘Mungo man’ study [1], although the
researchers took full anti-contamination precautions, it
was not mentioned that the sample had been excavated
in the 1970s and handled numerous times since (thus
likely to be contaminated), contained ‘negligible organic
preservation’ [17], and is considered too fragmentary to
sex reliably [7,40] e in short, of very poor preservation.
Without such information, it is very difficult to comment
objectively on the reliability of results.
Acknowledgements
The impetus behind this work was a series of incomprehensible aDNA results [15] on human bone from
Repton, England [6]. We thank Dott.ssa Maria Luisa
Nava (Archaeological Superintendent of Basilicata)
permission to sample, and Dott.sse Brunella Bruno and
Erminia Lapadula for assistance in sampling. M.T.P.G.
thanks the Wellcome Trust for funding his research
(061610/Z/00/Z). E.W. and A.J.H. thank Villum Rassmussen Fonden for funding their research. Diagenetic
parameters of the bone samples were analyzed as part of
the ‘Degradation of Bone as an Indicator in the Deterioration of the European Archaeological Property’
project funded by the EU (ENV4-CT98-0712).
Appendix A. Supplementary information
Supplementary information for this manuscript can
be downloaded at doi: 10.1016/j.jas.2004.12.008.
References
[1] G. Adcock, E. Dennis, S. Easteal, G. Huttley, L. Jermelin,
W. Peacock, A. Thorne, Mitochondrial DNA sequences in
ancient Australians: implications for modern human origins,
Proc. Natl. Acad. Sci. USA 98 (2001) 537e542.
[2] S. Anderson, A. Bankier, B. Arrell, M. de Bruijn, A. Coulson,
J. Drouin, I. Eperon, D. Nierlich, B. Roe, F. Sanger, P. Schreier,
A. Smith, R. Staden, I. Young, Sequence and organisation of the
human mitochondrial genome, Nature 290 (1981) 457e465.
[3] I. Barnes, P. Matheus, B. Shapiro, D. Jensen, A. Cooper,
Dynamics of mammal population extinctions in Eastern Beringia
during the last glaciation, Science 295 (2002) 2267e2270.
[4] L.S. Bell, G. Cox, J. Sealy, Determining isotopic life history
trajectories using bone density fractionation and stable isotope
measurements: a new approach, Am. J. Phys. Anthropol. 116
(2001) 66e79.
[5] L.S. Bell, M.F. Skinner, S. Jones, The speed of post mortem
change to the human skeleton and its taphonomic significance,
Forensic Sci. Int. 82 (1996) 129e140.
[6] M. Biddle, B. Kjølbye-Biddle, Repton and the ‘‘great heathen
army’’, 873e4, in: J. Graham-Campbell (Ed.), Vikings and the
Danelaw: Select Papers from the Proceedings of the Thirteenth
Viking Congress, Oxbow Books, Oxford, 2001, pp. 45e60.
[7] P. Brown, Australian pleistocene variation and the sex of Lake
Mungo 3, J. Hum. Evol. 38 (2000) 743e749.
[8] Excavations at Matera were directed by Paul Arthur. See B. Bruno,
Archeologia nei Sassi di Matera, in: S. Patitucci Uggeri (Ed.), Scavi
Medievali in Italia 1996e1999, Herder, Rome, 2001, pp. 137e148.
[9] I. Clisson, C. Keyser, H.P. Francfort, E. Crubézy, Z. Samashev,
B. Ludes, Genetic analysis of human remains from a double
inhumation in a frozen kurgan in Kazakhstan (Berel site, Early
3rd Century BC), Int. J. Legal Med. 116 (2002) 304e308.
[10] M.J. Collins, E.R. Waite, A.C.T. van Duin, Predicting protein
decomposition: the case of aspartic-acid racemization kinetics,
Philos. Trans. R. Soc. Lond. B 354 (1999) 51e64.
[11] A. Cooper, H. Poinar, Ancient DNA: do it right or not at all,
Science 289 (2000) 1139.
[12] J. Currey, Bones: structure and mechanics, Princeton University
Press, Princeton, 2002.
[13] M. Drancourt, G. Aboudharam, M. Signoli, O. Dutour,
D. Raoult, Detection of 400-year-old Yersinia pestis DNA in
M. Thomas P. Gilbert et al. / Journal of Archaeological Science 32 (2005) 785e793
[14]
[15]
[16]
[17]
[18]
[19]
[20]
[21]
[22]
[23]
[24]
[25]
[26]
[27]
[28]
[29]
human dental pulp: an approach to the diagnosis of ancient
septicemia, Proc. Natl. Acad. Sci. USA 95 (1998) 12637e12640.
M.T.P. Gilbert, J. Cuccui, W. White, N. Lynnerup, R.W. Titball,
A. Cooper, M.B. Prentice, Absence of Y. pestis-specific DNA in
human teeth from five European excavations of putative plague
victims, Microbiology 150 (2004) 341e354.
M.T.P. Gilbert, E. Willerslev, A.J. Hansen, L. Rudbeck,
I. Barnes, N. Lynnerup, A. Cooper, Distribution patterns of post
mortem damage in human mitochondrial DNA, Am. J. Hum.
Genet. 72 (2003) 32e47.
M.T.P. Gilbert, A.S. Wilson, M. Bunce, A.J. Hansen,
E. Willerslev, B. Shapiro, T.F.G. Higham, M.P. Richards,
T.C. O’Connell, D.J. Tobin, R.C. Janaway, A. Cooper, Ancient
mitochondrial DNA from hair, Curr. Biol. 14 (2004) R463e
R464.
R. Gillespie, R.G. Roberts, On the reliability of age estimates for
human remains at LakeMungo, J. Hum. Evol. 38 (2000) 727e732.
O. Handt, M. Höss, M. Krings, S. Pääbo, Ancient DNA:
methodological challenges, Experientia 50 (1994) 524e529.
O. Handt, M. Krings, R. Ward, S. Pääbo, The retrieval of ancient
human DNA sequences, Am. J. Hum. Genet. 59 (1996) 368e376.
R.E.M. Hedges, Bone diagenesis: an overview of processes,
Archaeometry 44 (2002) 319e328.
M. Hofreiter, D. Serre, H.N. Poinar, M. Kuch, S. Pääbo, Ancient
DNA, Nat. Rev. Genet. 2 (2001) 353e358.
S. Hummel, B. Hermann, General aspects of sample preparation,
in: B. Hermann, S. Hummel (Eds.), Ancient DNA, Springer
Verlag, New York, 1994, pp. 59e68.
M.M.E. Jans, H. Kars, C.M. Nielsen-Marsh, C.I. Smith,
A.G. Nord, P. Arthur, N. Earl, In situ preservation of
archaeological bone: a histological study within a multidisciplinary approach, Archaeometry 44 (2002) 343e352.
M.M.E. Jans, C.M. Nielsen-Marsh, C.I. Smith, M.J. Collins,
H. Kars, Characterisation of microbial attack on archaeological
bone, J. Archeol. Sci. 31 (2004) 87e95.
E.A.K. Kars, H. Kars, The degradation of bone as an indicator
for the deterioration of the European archaeological property,
Final Report, Rijksdienst voor het Oudheidkundig Bodemonderzoek, Amersfoort, 2003.
D.S. Kaufman, W.F. Manley, A new procedure for determining
DL amino acid ratios in fossils using reverse phase liquid
chromatography, Quatern. Sci. Rev. 17 (1998) 987e1000.
C.J. Kolman, N. Tuross, Ancient DNA analysis of human
populations, Am. J. Phys. Anthropol. 111 (2000) 5e23.
M. Krings, A. Stone, R. Schmitz, H. Krainitzki, M. Stoneking,
S. Pääbo, Neanderthal DNA sequences and the origin of modern
humans, Cell 90 (1997) 19e30.
G. Kurosaki, T. Matsushita, S. Ueda, Individual DNA identification from ancient human remains, Am. J. Hum. Genet. 53
(1993) 638e643.
793
[30] T. Lindahl, Instability and decay of the primary structure of
DNA, Nature 362 (1993) 709e715.
[31] G.J. Nicholson, J. Tomiuk, A. Czarnetzki, L. Bachmann,
C. Pusch, Detection of bone glue treatment as a major source
of contamination in ancient DNA analyses, Am. J. Phys.
Anthropol. 118 (2002) 117e120.
[32] H. Oota, N. Saitou, T. Matsushita, S. Ueda, A genetic study of
2000 year old human remains from Japan using mitochondrial
DNA sequences, Am. J. Phys. Anthropol. 98 (1995) 133e145.
[33] S. Pääbo, R. Higuchi, A. Wilson, Ancient DNA and the
polymerase chain reaction, J. Biol. Chem. 264 (1989) 9709e9712.
[34] A.G.U. Pedersen, T.R. Thomsen, B.A. Lomstein, N.O.G.
Jorgensen, Bacterial influence on amino acid enantiomerization in
a coastal marine sediment, Limnol. Oceanogr. 46 (2001) 1358e1369.
[35] H. Poinar, M. Höss, J. Bada, S. Pääbo, Amino acid racemization
and the preservation of ancient DNA, Science 272 (1996) 864e866.
[36] M. Richards, B. Sykes, R. Hedges, Authenticating DNA
extracted from ancient skeletal remains, J. Archeol. Sci. 22
(1995) 291e299.
[37] M. Richards, V. Macaulay, E. Hickey, E. Vega, B. Sykes,
V. Guida, C. Rengo, D. Sellitto, F. Cruciani, T. Kivisild,
R. Villems, M. Thomas, S. Rychkov, O. Rychkov, Y. Rychkov,
M. Gölge, D. Dimitrov, E. Hill, D. Bradley, V. Romano, F. Calı̀,
G. Vona, A. Demaine, S. Papiha, C. Triantaphyllidis,
G. Stefanescu, J. Hatina, M. Belledi, A. Di Rienzo,
A. Novelletto, A. Oppenheim, S. Nørby, S. Santachiara-Benerecetti, R. Scozzari, A. Torroni, H.-J. Bandelt, Tracing European
founder lineages in the Near Eastern mtDNA pool, Am. J. Hum.
Genet. 67 (2000) 1251e1276.
[38] Smith, Modelling diagenesis in archaeological bone, PhD thesis,
University of Newcastle, 2002.
[39] C.I. Smith, C.M. Nielsen-Marsh, M.M.E. Jans, P. Arthur,
A.G. Nord, M.J. Collins, The strange case of Apligliano:early
‘fossilization’ of medieval bone in southern Italy, Archaeometry
44 (2002) 405e416.
[40] A. Thorne, D. Curnoe, Sex and significance of Lake Mungo 3:
reply to Brown ‘‘Australian Pleistocene variation and the sex of
Lake Mungo 3’’, J. Hum. Evol. 39 (2000) 587e600.
[41] A. Torroni, H.-J. Bandelt, L. D’Urbano, P. Lahermo, P. Moral,
D. Sellitto, C. Rengo, P. Forster, M.-L. Savontaus, B. BonnéTamir, R. Scozzari, MtDNA analysis reveals a major late
paleolithic population expansion from southwestern to northeastern Europe, Am. J. Hum. Genet. 62 (1998) 1137e1152.
[42] G. Turner-Walker, C.M. Nielsen-Marsh, U. Syversen, H. Kars,
M.J. Collins, Sub-micron spongiform porosity is the major ultrastructural alteration occurring in archaeological bone, Int. J.
Osteoarcheol. 12 (2002) 407e414.
[43] P. Wandeler, S. Smith, P.A. Morin, R.A. Pettifor, S.M. Funk,
Patterns of nuclear DNA degeneration over time e a case study in
historic teeth samples, Mol. Ecol. 12 (2003) 1087e1093.