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Detection of bone glue treatment as a major source of contamination in ancient DNA analyses.

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AMERICAN JOURNAL OF PHYSICAL ANTHROPOLOGY 118:117–120 (2002)
Detection of Bone Glue Treatment as a Major Source of
Contamination in Ancient DNA Analyses
Graeme J. Nicholson,1 Jürgen Tomiuk,2 Alfred Czarnetzki,2 Lutz Bachmann,3* and Carsten M. Pusch2
1
Institute of Organic Chemistry, University of Tübingen, D72076 Tübingen, Germany
Department of Human Genetics, University of Tübingen, D72074 Tübingen, Germany
3
Natural History Museums and Botanical garden, Zoological Museum, University of Oslo, N-0562 Oslo, Norway
2
KEY WORDS
amino acid; paleogenetics; racemization; skeletal remains
ABSTRACT
Paleogenetic investigations of ancient
DNA extracted from fossil material is for many reasons
susceptible to falsification by the presence of more recent
contamination from several sources. Gelatine-based bone
glue that has been used extensively for nearly two centuries by curators to preserve hard tissues contributes nonauthentic DNA to paleontological material. This fact has
been frequently neglected and is barely mentioned in the
The vast majority of ancient DNA analyses employ the polymerase chain reaction (PCR). While the
enormous sensitivity of the method allows the amplification of minute amounts of authentic ancient
DNA, even traces of contaminating nonauthentic
DNA will inevitably lead to artifacts. Consequently,
numerous control experiments are required to verify
PCR-based results in ancient DNA analyses (Handt
et al., 1994; Cooper and Poinar, 2000). Contaminating nucleic acids may stem from various sources.
These include human DNA, derived from the persons performing the genetic experiments or from
people who previously handled the specimen, and
from edaphon DNA, primarily derived from bacterial or fungal growth. Another source of contamination is seen in the numerous substances used for
hard-tissue conservation (Rixon, 1976; Brommelle et
al., 1984; Horie, 1987; Collins, 1995). From the early
19th century up to the present, gelatine-based glue
has been widely used, because it is cheap, easily
available, and very effective (Lepper and Lewis,
1941; Shelton and Johnson, 1995). Although some
authors have expressed suspicions that such conservation techniques may cause severe complications if
not hindrance for the analysis of old biomolecules
(Horie, 1987; Hall et al., 1993; Cooper, 1994; Shelton
and Johnson, 1995), this source of experimental pitfalls is underestimated.
Bone glue is produced from bones, hides, sinews,
ligaments, and gristle which are degreased, demineralized with acid, and swollen by a prolonged treatment (up to 20 weeks) with aqueous alkali (usually
Ca(OH)2) at ambient temperature, before extraction
and partial hydrolysis of the remaining collagen
©
2002 WILEY-LISS, INC.
literature. Now paleogeneticists, curators, and conservators are faced with the problem that treatment of samples
with adhesives and consolidants for conservatory purposes
has seldom been recorded. Here, we show that racemization
of amino acids, and in particular serine, is an excellent
indicator for the treatment of paleontological samples with
glue. Am J Phys Anthropol 118:117–120, 2002.
©
2002 Wiley-Liss, Inc.
with hot water. The resulting viscous and colloidal
solution penetrates the material to be conserved and
hardens therein. DNA, however, is coextracted in
this procedure. Thus bone glue is a rich source of
nonauthentic DNA for paleontological samples.
Moreover, due to the processing of collagen for glue
production (i.e., extreme pH), such DNA is expected
to be severely degraded as is the authentic fossil
DNA. Thus, it is unlikely that both sorts of DNA can
be distinguished by physico-chemical properties.
Glues that were produced decades ago most probably contain even more impurities than today’s products. Therefore, it is of importance for paleogeneticists to know whether or not a find has been treated
with natural hardeners (Cooper, 1994). In addition,
curators and conservators would also appreciate a
simple method for determining previous glue treatment, since many substances used for conservation
of hard tissues are not compatible and will destroy
the material rather than conserve it (Howie, 1984;
Collins, 1995; Stoneking, 1995). We propose the extent of racemization of serine as the principal parameter for detection of previous treatment with
bone glue.
*Correspondence to: Lutz Bachmann, Natural History Museums
and Botanical Garden, Zoological Museum, University of Oslo, Sars
Gate 1, N-0562 Oslo, Norway. E-mail: bachmann@nhm.uio.no
Received 21 September 2000; accepted 3 December 2001.
DOI 10.1002/ajpa.10061
Published online in Wiley InterScience (www.interscience.wiley.
com).
118
G.J. NICHOLSON ET AL.
METHODS AND RESULTS
The condition used for the swelling and hydrolysis
of the collagen affects the degree of racemization of
the constituent amino acids, in particular those susceptible to racemization under basic conditions, i.e.,
those with electron-withdrawing groups (e.g., ⫺OH,
⫺COOH) close to the center of chirality (Neuberger,
1948; Bada and Schröder, 1975), leading in particular to elevated levels of D-aspartic acid and D-serine.
Phenylalanine is also affected, although to a much
lesser degree.
We determined the [D]/[L] ratio of serine, alanine,
leucine, aspartic acid, glutamic acid, and phenylalanine in 64 bone samples, 14 of which are known to
have been treated at least once, and 50 of which had
not been treated with glue. The age of samples
ranged from 2 years (modern control) to ⬃500 000
years B.P., and they originated from a variety of
species including pig, cow, horse, bear, rhinoceros,
mammoth, reindeer, Neandertals, and anatomically
modern humans. Additionally, three different glue
samples were examined: two were obtained from a
local drugstore, and a third, of vintage origin, has
already been used for several years in an osteological collection.
The DNA that can be extracted from commercially
available glue can easily be detected by a simple
experiment. Granulate pills of glue were inoculated
into large-size slots of an ethidium bromide-stained
1.5% (w/v) agarose gel (NuSieve/SeaKem, 3:1) and
electrophoresed in 1 ⫻ TBE at 8 V/cm. Subsequently, the DNA can be extracted from the gel slice
by standard methods (e.g., Pusch, 1997). Rough dotblot hybridization experiments of radioactively labeled DNA extracted from an old bone glue sample
to various genomic DNAs spotted onto a nylon membrane reveal in this example a mixture of nucleic
acids originating from, e.g., cow, pig, and even human (Fig. 1). This result sufficiently answers the
question of whether or not DNA can survive the glue
manufacturing process. In particular, there might
be a surprising quantity of human DNA in the vintage glue sample used for the dot-blot hybridization
experiment. However, it is impossible to identify the
source of human DNA detected in the vintage glue
sample, because the origin of the glue is unclear.
Nevertheless, in the context of ancient DNA analysis, it is important to note that bone glue might
contribute nonauthentic DNA to paleontological material. Glues that were produced decades ago most
probably contain even more impurities than today’s
products.
Approximately 1 mg of pulverized bone sample
was hydrolyzed in 200 ␮l 6 N DCl in D2O (24 hr/
110°C), esterified with 200 ␮l 1.5 N DCl in CH3OD
(15 min/110° C), and trifluoroacetylated with 100 ␮l
trifluoroacetic anhydride (TFAA) (10 min/110°C).
Insoluble inorganic salts from the bone were largely
removed by decanting the TFAA solution containing
the dissolved amino-acid derivatives into a fresh vial
Fig. 1. Dot-blot hybridization experiment of radioactively labeled DNA extracted from an old bone glue sample to various
genomic DNAs spotted onto a nylon membrane. Spotted DNA
samples are as follows (positive hybridization is indicated in
bold). A: 1, soil (sample 1); 2, soil (sample 2); 3, Phage ␾X174; 4,
pig; 5, Lumbricus; 6, Drosophila; 7, cyprinid fish; 8, dog; A9,
mouse. B: 1, soil (sample 3); 2, soil (sample 4); 3, E. coli; 4,
Diphyllobotrium; 5, Musca; 6, starfish; 7, frog; 8, cow; 9, Tropaeolum. C: 1, soil (sample 5); 2, baltic amber; 3, Lawrist 4 cosmid
vector; 4, Sepia; C5, plasmid pUC19; 6, clupeid fish; 7, cat; 8, rat;
9, human (blood).
before evaporating off excess TFAA. The amino-acid
derivatives, dissolved in approximately 10 ␮l toluene, were separated by enantioselective gas chromatography on a Chirasil-Val capillary and detected by
mass spectrometric selective ion monitoring, using
the following ions: m/z 138 (serine), m/z 140 (alanine), m/z 182 (leucine), m/z 156 (aspartic acid), m/z
214 (glutamic acid), and m/z 162 (phenylalanine).
The [D]/[L] ratio of each amino acid was calculated
directly from the respective peak areas. This general
technique for racemization control was described in
more detail elsewhere (Gerhardt and Nicholson,
1994).
The results are summarized in Figure 2. The
[D]/[L]-serine values of the glue samples are in the
range of 169 –201 ⫻ 10⫺3 and are similar to those of
bone samples treated with glue that range from
106 –202 ⫻ 10⫺3 (mean, 151.3 ⫻ 10⫺3 ⫾ 29.7 ⫻ 10⫺3;
median, 155.5 ⫻ 10⫺3; 0.1 quantile, 110.2 ⫻ 10⫺3;
0.9 quantile, 184.3 ⫻ 10⫺3). The [D]/[L]-serine values of untreated bone fragments, on the other hand,
are significantly lower, ranging from 0 –98 ⫻ 10⫺3
(mean, 28.8 ⫻ 10⫺3 ⫾ 23.9 ⫻ 10⫺3; median, 22.7 ⫻
10⫺3; 0.1 quantile, 2.3 ⫻ 10⫺3; 0.9 quantile, 72.3 ⫻
10⫺3). The [D]/[L]-serine values of glue-treated and
untreated bone samples are neither normally nor
Poisson-distributed. Thus, the values of the two
groups cannot be simply summarized by the mean
and standard deviation. This is not unexpected,
since each sample could have experienced widely
different environmental conditions such as time,
temperature, pH, or moisture content, all of which
will affect the [D]/[L]-serine values.
In accordance with the guidelines of Poinar et al.
(1996), the bone material used for amino-acid race-
DETECTION OF BONE GLUE TREATMENT
Fig. 2. Distribution of subsurface [D]/[L]-serine values of 64
bone samples. Bar charts illustrate number of cases of [D]/[L]serine that fall into the indicated 10 ⫻ 10⫺3 intervals. Values are
not normally distributed, and thus the mean (⽧), median (䊐),
minimum-maximum (whiskers), and 0.1 and 0.9 quantiles (boxed)
are depicted. A: Fifty bone samples that were never treated with
bone glue. Additional measurements of surface samples derived
from 13 untreated bones (indicated by open bars) are summarized
in the small bar chart. B: Fourteen bone samples that were
treated at least once with bone glue. Arrows indicate [D]/[L]serine values of three glue samples. *Glue sample used for hybridization experiment shown in Figure 1.
mization was originally taken from the middle of the
compacta in order to avoid any influence of surface
contaminants on the values measured. However, the
point in question in this study (presence or not of
contamination with glue) differs from that of Poinar
et al. (1996). Since subsurface sampling is invasive
and causes visible damage to the specimen, the
question arises as to whether surface sampling,
which can be carried out with almost no visible
damage, is sufficient in order to identify previous
glue treatment of the fossil.
Samples taken at different depths (0 –3 mm) from
a glue-treated bone specimen showed similar values
for [D]/[L]-serine, with a slight tendency to lower
values with increasing depth. Since these measurements were made on a well-preserved bone specimen with optically intact compacta, this is also an
indication of the high degree of penetration of bone
glue into the compacta. Analogous measurements on
an untreated bone specimen displayed the opposite
119
tendency. Here, the values of [D]/[L]-serine from the
surface were significantly lower than those from
subsurface samples. Therefore, additional measurements were performed on surface samples from 13 of
the untreated bones previously measured with subsurface sampling. In all cases, the [D]/[L]-serine values of these surface samples were lower than corresponding values from the middle of the compacta,
and ranged from 0 –32.6 ⫻ 10⫺3 (mean, 19.2 ⫻
10⫺3 ⫾ 5.8 ⫻ 10⫺3; median, 18.1 ⫻ 10⫺3; 0.1 quantile, 14.2 ⫻ 10⫺3; 0.9 quantile, 24.2 ⫻ 10⫺3).
The data indicate that reliable differentiation between glue-treated and untreated bone samples is
possible. The [D]/[L]-serine values from untreated
samples and samples treated with bone glue fall
within two nonoverlapping intervals. The highest
value of an untreated sample with subsurface sampling (98 ⫻ 10⫺3) is still lower than the smallest
(106 ⫻ 10⫺3) of a sample treated with bone glue.
Differentiation is even more reliable with surface
sampling: here, the maximum value determined for
an untreated bone specimen was 32 ⫻ 10⫺3. Thus, a
[D]/[L]-serine value of 100 ⫻ 10⫺3 can be used as a
threshold. [D]/[L]-serine values exceeding this
threshold indicate that the material has almost certainly been treated at least once with bone glue. This
holds true, regardless of the age of the bones or from
which species the material originated.
As stated earlier, elevated levels of D-phenylalanine can also be expected, although to a much lower
degree. [D]/[L]-phenylalanine values of the 48 untreated samples taken from the compacta range
from 1.3–109 ⫻ 10⫺3 (mean, 15.1 ⫻ 10⫺3 ⫾ 24.9 ⫻
10⫺3; median, 5.7 ⫻ 10⫺3; 0.1 quantile, 3.5 ⫻ 10⫺3; 0.9
quantile, 30.2 ⫻ 10⫺3), and those of 11 untreated ones
taken from the surface range from 1.2–30.4 ⫻ 10⫺3
(mean, 8.8 ⫻ 10⫺3 ⫾ 8.9 ⫻ 10⫺3; median, 4 ⫻ 10⫺3;
0.1 quantile, 2.7 ⫻ 10⫺3; 0.9 quantile, 22 ⫻ 10⫺3).
The [D]/[L]-phenylalanine values of 14 glue treated
samples taken from the compacta range from 13.8 –
20.8 ⫻ 10⫺3 (mean, 18. ⫻ 10⫺3 ⫾ 2.2 ⫻ 10⫺3; median, 18.9 ⫻ 10⫺3; 0.1 quantile, 14.8 ⫻ 10⫺3; 0.9
quantile, 20.3 ⫻ 10⫺3). The three glue samples have
[D]/[L]-phenylalanine values of 27.5, 29, and 41 ⫻
10⫺3, respectively. It is evident that the [D]/[L]phenylalanine ratio is less powerful for the identification of glue treatment than the [D]/[L]-serine ratio, since the intervals of [D]/[L]-phenylalanine
values from glue-treated and untreated samples
overlap.
CONCLUSIONS
Amino-acid racemization offers a valuable means
of assessing the risk of glue-based DNA contamination of fossil bones. The approach has two major
advantages. 1) Amino-acid racemization is a standard procedure in ancient DNA research; most scientists interested in studying fossil DNA extracted
from bone material apply the technique prior to
their genetic experiments in order to estimate DNA
survival. Our procedure is a quick extension of this.
120
G.J. NICHOLSON ET AL.
2) The amount of sample required is small (⬃1 mg).
It is sufficient and in fact preferable to sample from
the bone surface, thus reducing the destruction of
valuable material to a minimum.
Samples showing [D]/[L]-serine values higher
than 100 ⫻ 10⫺3 should not be used for analyses of
ancient DNA, at least when corresponding untreated material is available. If it is inevitable to
analyze DNA from glue-treated bones, one should be
aware of artifacts caused by glue-based DNA contaminations. Control experiments performed to
prove the authenticity of the obtained data must
take into account that glue may contaminate the
sample with DNA from a variety of species, including humans. Therefore, the use of species-specific
primers alone in order to obtain sequence data that
make phylogenetic sense is not sufficient to avoid
artifacts.
The degree of racemization of amino acids has
already been proposed as an indicator for the presence of amplifiable authentic DNA in ancient samples (Poinar et al., 1996; Cooper, 1997; Krings et al.,
1997). Racemization of aspartate is the principal
criterion considered, and a value smaller than 0.08
for [D]/[L]-aspartic acid was proposed for the presence of DNA endogeneous to a find (Poinar et al.,
1996). In the case of glue-treated samples, we face
the problem that although values measured are always beyond this threshold, they may nevertheless
contain authentic DNA (i.e., the proportion of extraneous to endogenous DNA cannot be reliably estimated). Accordingly, contamination from more recent times can allegedly be recognized by [D]/[L]
ratios of alanine and leucine being higher than those
of aspartic acid (Poinar et al., 1996; Cooper, 1997;
Krings et al., 1997). However, this was never observed in any of the samples that had been treated
with glue. Therefore, we suggest extending the list
of indicators for the presence of amplifiable authentic DNA in ancient samples to include the degree of
racemization of serine, in order to take into account
glue treatment of the sample.
However, bone glue is only one (albeit frequently
used) agent to preserve bone material (Howie, 1984).
A variety of substances, including shellac, casein,
dammar, bee’s wax derivatives, vegetable-based
tannins, and fungicidal and insecticidal agents, may
have been used in the conservation of a specific find
in question (Cooper, 1994). Many of these may complicate ancient DNA analyses, either as a source of
contamination with proteins or nucleic acids, or by
inhibiting enzyme-based DNA modifications such as
the polymerase chain reaction (Horie, 1987; Hall et
al., 1993; Shelton and Johnson, 1995). The poor re-
cording of treatment of samples with adhesives and
consolidants for conservatory purposes makes it necessary to develop methods to identify such treatments, since the investigation of curated and conserved samples will become more and more important
in ancient DNA research.
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