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Author's personal copy
Journal of Archaeological Science 37 (2010) 2831e2841
Contents lists available at ScienceDirect
Journal of Archaeological Science
journal homepage: http://www.elsevier.com/locate/jas
Assessing the effects of conservation treatments on short sequences
of DNA in vitro
Julie A. Eklund a, *,1, Mark G. Thomas b, c
a
Institute of Archaeology, 31e34 Gordon Square, University College London, London WC1H 0PY, United Kingdom
Research Department of Genetics, Evolution, and Environment, University College London, Gower Street, London, WC1E 6BT, United Kingdom
c
Department of Evolutionary Biology, Evolutionary Biology Centre, Uppsala University, Norbyvagen 18D, Uppsala, SE-752 36, Sweden
b
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 30 November 2009
Received in revised form
4 June 2010
Accepted 18 June 2010
Little is known about what effects conservation treatments used to preserve human and animal hard and
soft tissues have on DNA preservation. We have developed a method to assess quantitatively the extent
of lesions or strand breakage caused by conservation treatments on short sequences of DNA in vitro. The
method developed enables the determination of the percentage of DNA preserved following exposure to
a conservation treatment solution relative to control samples, thereby allowing the direct comparison of
treatments based upon their preserving/damaging effects on a DNA sequence. Forty-three chemicals
commonly used in the preparation and/or conservation of human and/or animal remains were examined.
We found that the majority were damaging, in particular and as expected, acidic treatments and
treatments carried out at elevated temperatures. A few, primarily organic solvents, were not damaging.
The approach we have adopted can be applied to screen other treatments either used in the past or for
future conservation applications as they are developed to assess their effects on DNA. How these results
should be interpreted in terms of conservation and sampling is also discussed.
Ó 2010 Elsevier Ltd. All rights reserved.
Keywords:
Ancient DNA
Conservation treatment
DNA damage
DNA preservation
Use of collections
1. Introduction
Several studies have set out to address the effects of chemical
treatments on the extraction and/or utility of DNA from museum
collection materials for molecular genetic research (see for
example: Crisan and Mattson, 1993; Fukatsu, 1999; Post et al., 1993;
Srinivasan et al., 2002; Vink et al., 2005). The majority of such
studies have focused on the effects of fixatives used for soft tissue
or arthropod preservation with the objectives of either optimising
methods of DNA extraction from specimens, or enabling the shortterm preservation of high molecular weight DNA in freshly
collected material. The methods used in these studies typically
consisted of: (1) treating a specimen or selecting a previously
treated specimen, (2) DNA extraction, (3) visualising extracted DNA
on an agarose gel, (4) PCR amplification of the extracted DNA, and
(5) visualising amplified DNA on an agarose gel.
* Corresponding author. Tel.: þ44 7939 021 038; fax: þ44 1865 275885.
E-mail addresses: julie.eklund@ouce.ox.ac.uk, eklundjulie@yahoo.com (J.A.
Eklund), m.thomas@ucl.ac.uk (M.G. Thomas).
1
Present address: School of Geography and the Environment, Oxford University
Centre for the Environment, Dyson Perrins Building, South Parks Road, University of
Oxford, Oxford OX1 3QY, United Kingdom.
0305-4403/$ e see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.jas.2010.06.019
Although the methods used in previous studies as outlined
above may enable the identification of general DNA degradation,
they are of limited utility from a museum collection conservation
point of view, as the results typically were neither quantitative nor
sequence specific, and most importantly could not identify damage
due solely to conservation treatment. In reviewing these previous
studies, it was difficult to identify damage resulting exclusively
from conservation treatment due to a number of uncontrolled
variables, such as the efficiency of DNA extraction and PCR amplification protocols, and the potential inhibitory effects of residual
chemicals used in the treatment of specimens. There was some
uncertainty regarding the initial quantity of DNA in fresh specimens, as well as the preservation of specimen DNA prior to treatment and DNA extraction. In particular, the use of freshly collected
specimens as sample material made it difficult to compare the
results of many previous studies, as different specimens or tissues
may have responded differently to conservation treatment and
storage environments, and some DNA damage may have been due
to treatments failing to retard natural deterioration processes
rather than their actively damaging DNA. In some studies, the use of
PCR amplification may have compensated for any damage sustained from treatment, as PCR is rarely quantitative. Also, for the
majority of previous studies the reporting of the chemicals tested,
as well as the exposure time and concentration of treatments used,
Author's personal copy
2832
J.A. Eklund, M.G. Thomas / Journal of Archaeological Science 37 (2010) 2831e2841
was inadequate. This made it difficult to assess the impact of
individual treatments or to compare the results of different studies
with any degree of consistency. Therefore, we consider the results
of previous studies and the approaches used to be inappropriate to
study the effects of conservation treatments on short sequences of
DNA, as are typically recovered in ancient DNA research.
Here we present a new approach to screen chemicals used in the
past in preparation or conservation treatments on human and
animal hard and soft tissues, to measure the degree of strand
breakage induced in short sequences of DNA in vitro. We use fluorescently labelled PCR products, rather than the endogenous DNA
contained in archaeological or other specimen material, to assay for
DNA damage. This was done because the initial condition and
amount of DNA in each sample could be controlled, enabling the
quantification of DNA damage for each treatment. Such an
approach also eliminates the need to consider potential sources of
DNA loss due to sampling and extraction procedures, as well as the
risk of contamination in PCR amplification. PCR products used were
approximately 100e200 bp in length, to reflect fragmentation of
DNA expected in either archaeological remains (300 bp) (Poinar
et al., 2006) or specimens held in collections for an extended
period of time (500 bp) (Pääbo et al., 1989).
Four fluorescently-labelled PCR products were generated for use
in this study following Thomas et al. (1999), two of which were
around 100 bp in length, YAP (99 bp) and TAT (112 bp), and another
two around 200 bp in length, M9 (214 bp) and SRY 4064 (225 bp). A
“test” solution and a “reference” solution were made with the PCR
products, with each solution containing one sequence roughly
100 bp in length and one sequence roughly 200 bp in length. The
two sequences included in the test solution contained different
amounts of guanine to cytosine (GC) bonds. This was done in order
to investigate how chemical damage may be sequence specific, as
a higher GC content is thought to improve DNA stability
(Yakovchuk et al., 2006) and may enhance durability against
chemical attack. Conservation treatment solution was added to
samples of the test solution. Then, following the designated
treatment period, the reference solution was subsequently added
to this mixture. Following fragment analysis using an automated
DNA sequencer (ABI PRISMÒ 3100 Genetic Analyzer), the ratio of
the peak heights between the two 100 bp sequences and the two
200 bp sequences in each sample was calculated in order to assess
treatment-induced damage to the DNA in the test solution. A key
feature of this experimental design is that any damage resulting
from sample preparation procedures following chemical treatment
is controlled for, as both the test and reference solution sequences
should be affected equally. Data generated were assessed to identify those treatments considered safe to use for conservation
applications.
It is acknowledged that naked DNA, or DNA suspended in water,
which is in direct contact with conservation treatment solutions, as
tested here, may be affected by treatment differently than DNA in
a specimen. DNA in a specimen may receive some degree of
protection by encapsulation within tissues, as it has been reported
that DNA adsorbed onto hydroxyapatite exhibits a two-fold
decrease in depurination (Lindahl, 1993). Additionally, DNA may
also benefit from a conservation treatment penetration gradient,
particularly from surface treatments (i.e. surface DNA may be more
exposed to and affected by treatment than interior DNA). Therefore,
the results of this study are best used to assess the effects of
treatments relative to each other, rather than as absolute values of
the potential damage to DNA in a specimen if exposed to a particular treatment.
Research into the history of preparation and conservation
treatments administered to human and animal hard and soft
tissues across a range of disciplines identified approximately 475
chemicals that have been used since the 1880s. These chemicals
have been used for many different applications, and include: acid
preparation, adhesive, barrier coat, bleaching agent, chelating
agent, cleaning agent, consolidant, degreasing agent, dry soft tissue
preservative, drying agent, finishing materials, fungicide,
moulding/casting materials, packing material, pesticide, photographic aid, sealant, skeleton preparation, solvent, and wet soft
tissue preservative. Based on frequency of use and the level of
published detail available to enable replication of the treatments,
a selection of 43 chemicals commonly used in the past was chosen
for testing. Based upon the degree of DNA preserved in treated
samples compared to control samples in deionised water purified
by reverse osmosis (USF ELGA Option 7/15), as commonly used in
conservation treatment, the suitability of treatments for use in
conservation was determined.
2. Materials and methods
2.1. PCR products
The PCR conditions and primers used for this research are
modifications of those published by Thomas et al. (1999). Primers
were labelled with the ABI dye NED. TAT and M9 PCR amplifications
were performed separately in final reaction volumes of 25 ml,
consisting of 1 ml of gel purified PCR products from a previous PCR
as template DNA, 250 mM of dNTPs, 0.1 units of SuperTaqÔ polymerase (Enzyme Technologies Limited), 2.5 ml of the 10 buffer
supplied with the SuperTaqÔ, and 0.64 mM of each primer. YAPand SRY 4064 amplifications were performed separately as above,
except 0.32 mM of each primer was used.
Cycling parameters for amplification were as published, with
modifications. For the TAT and M9 amplification, the annealing
temperature was 55 C. For the YAP- and SRY 4064 amplifications,
pre-incubation was for 4 min, followed by 40 cycles, and the
annealing temperature was 59 C, followed by 1 min 40 s extension. TAT and M9 amplifications were performed in a DNA Engine
DYADÒ Peltier Thermal Cycler. YAP- and SRY 4064 amplifications
were performed in a BiometraÒ UNO II Peltier Thermal Cycler.
The resulting PCR products for each sequence were then pooled
and purified using a Vivaspin 30K molecular weight cut-off
(MWCO) centrifugal ultrafiltration column (Vivascience, UK).
Columns were washed three times with TE buffer (10 mM TriseHCl
pH 8.0, 1 mM EDTA), and retained DNA was resuspended in 500 ml
TE buffer.
2.2. Test solution and reference solution
A two-fold serial dilution was set up for each of the PCR product
solutions, which was analysed using capillary electrophoresis on an
ABI PRISMÒ 3100 Genetic Analyzer under the same conditions and
settings future test samples would be analysed (see below). Data
were analysed using ABI PRISMÒ GeneScanÒ Analysis software
version 3.7 for Windows (Applied Biosystems), and PCR products
were diluted to have their peak heights fall within the detection
limits of the equipment; each peak was high enough to read
without exceeding detection limits and being truncated. Milli-QÒ
water (resistivity at 25 C ¼ 18 MU $ cm) was used to dilute the PCR
products. Diluted PCR products were mixed to create two solutions.
A “test” solution was made, consisting of TAT (112 bp) and SRY 4064
(225 bp) PCR products, to which treatment solutions were added. A
“reference” solution was also made, consisting of YAP- (99 bp) and
M9 (214 bp) PCR products, which was added to the combined test
and chemical treatment solutions after the experimental treatment
period had ended. Xylene cyanole blue was added to both solutions
at a concentration of 0.02% to enable better visibility of the aqueous
Author's personal copy
J.A. Eklund, M.G. Thomas / Journal of Archaeological Science 37 (2010) 2831e2841
phase (which would contain the DNA) during ether extraction of
the organic solvent-based treatments (see below). Although xylene
cyanole blue was only necessary in the ether extracted test solution, it was added to both the test solution and reference solution at
the same concentration to control for any possible effect on DNA
preservation.
For each sample, 15 ml of test solution was loaded into a 96-well
PCR plate. Treatment solutions were added in 30 ml volumes at the
appropriate exposure time (see below). Organic solvent-based
treatment samples had an additional 30 ml of water added at the
outset to maintain equal water volumes across all samples. In
addition to 43 treatments tested, two water controls and two
mineral oil negative controls were run in each treatment set, as
well as an additional 8 treatment controls and 8 mineral oil
samples. Four replicate sets of each treatment were run, and each
sample was run on an ABI PRISMÒ 3100 Genetic Analyzer twice to
ensure consistency across samples for each treatment.
2.3. Conservation treatments
Forty-three chemicals commonly used for the preparation and/
or conservation of human and/or animal remains in the past were
tested in this study. To imitate the conditions of conservation
treatment, the published concentration, exposure time and
temperature of treatment administration were replicated as faithfully as possible. If several references were found for each treatment, the published conditions with the highest concentration
were used. Control samples and aqueous treatment solutions were
mixed using deionised water purified by reverse osmosis (resistivity at 25 C ¼ 10 to >15 MU $ cm), as would be used in conservation treatment. Chemicals tested, their published conditions and
experimental conditions are listed in Table 1.
Aqueous treatments were prepared at a concentration 50%
higher than that published (referred to as the "Experimental
concentration" in Table 1), so that mixing with the PCR products
would dilute the treatment concentration to the appropriate level.
Further adjustments were required for particularly viscous treatment solutions, such as gum arabic, as volumes could not be
accurately measured using pipettes, potentially resulting in the
addition of excess treatment. Cellulose nitrate was diluted to
facilitate pipetting, and therefore was not administered at the
concentration suggested in the literature. In order to assess the
effects of chemicals dissolved in organic solvents, organic solvents
were tested separately at full strength.
Treatment exposure times reported in the literature were
replicated as closely as possible, however some modifications to
published exposure times were necessary to streamline sample
processing, and several references did not provide specific exposure times. A maximum of one week treatment time was set for
experimental purposes. To keep to a viable laboratory schedule for
mass processing of all samples, the end time of the experiment
was set for 12.00 on the seventh day, with the addition of treatment solutions to samples timed so all experiments ended
simultaneously. For the purposes of this study, if treatment
instructions were vague or not provided, exposure times were as
follows: “a few days” ¼ 1 week, “a few hours” ¼ overnight (18 h),
“simmer” ¼ 6 h/80 C (Mooney et al., 1982, p. 125), organic solvent
bone immersion and all soft tissue surface treatments ¼ 2 h, bone
surface treatments ¼ 1 h. To keep to a workable schedule, one
treatment with a maximum published exposure time of 12 h was
left overnight (approximately 18 h). If a treatment was to be
repeated, the sum total of the exposure time was used in the
experiment. Published instructions for sodium perborate treatment included heating to boiling and then cooling overnight to
room temperature (Roche 1954 translated in Hangay and Dingley,
2833
1985, pp. 342e343). This was replicated by heating sample tubes
to 100 C in a thermal cycler for 1 min, and allowing them to cool
to room temperature overnight.
Some treatments selected for testing were undertaken at
elevated temperatures. Heated samples were maintained at the
required temperature in incubation ovens at either 37 C or 80 C.
For each heated treatment, unheated replicate samples of the same
treatment were also set up. This was done to account for the effects
of the chemical treatment and of heating the treatment separately.
To ensure contact between the test solution and treatment solutions, unheated samples were continuously mixed on a Vortex
Genie 2Ò at low speed for the duration of the prescribed exposure
time (heated treatments were not mixed). Experimental exposure
time refers to the time the treatment was in contact with the DNA
on a mixer or a heated environment, but does not include any time
lapse prior to the addition of the reference solution to all of the
samples (in this particular experiment, approximately 8 h). At the
end of the treatment exposure time, heated samples were removed
from the incubation ovens and all aqueous samples were stored at
4 C whilst all organic solvent-based samples underwent ether
extraction to remove the organic treatment phase. Ether extraction
was carried out by adding 170 ml of ether to the sample, mixing, and
pipetting off the organic solvent phase three times, followed by air
drying for 10 min. During the exposure time, evaporation occurred
with both organic solvent-based treatments and heated treatments. Although ParafilmÒ was used to cover the lids of the heated
samples, this had a minimal effect in preventing evaporation.
However, this replicates the conditions of a normal treatment
where some evaporation would be expected. It is also worth noting
that heating DNA in the dry state compared to heating DNA in
solution should result in less damage, as hydrolytic damage would
not occur (Greer and Zamenhof, 1962). Therefore, damage observed
in heated samples where evaporation took place may be somewhat
underrepresented. Milli-QÒ water was added to heated treatments
to compensate for any loss by evaporation. Finally, 15 ml of reference
solution was added to all samples. Sample precipitation can
proceed directly from this step, or samples can be frozen prior to
precipitation to minimise further DNA damage from treatment
solutions or residues.
2.4. DNA precipitation
DNA was precipitated by adding 20 ml of 0.8 M NaCl and 170 ml of
chilled 99% isopropanol to each tube (note: some conservation
treatments were not soluble in isopropanol resulting in coprecipitation with DNA; discussion of this is included in the
Supplemental material). Samples were then mixed and incubated
at room temperature for 10 min. Samples were centrifuged at
4000 rpm for 40 min, followed by centrifugation at 300 rpm for
15 s with the plate inverted to remove the supernatant. To each
well 150 ml of chilled 70% ethanol was added, and samples were
centrifuged at 4000 rpm for 20 min, followed by centrifugation at
300 rpm for 15 s with the plate inverted to remove the supernatant.
Samples were dried by heating to 65 C for 5 min. To redissolve the
DNA, 10 ml TE buffer was added to each sample, which were then
frozen until needed.
To prepare for analysis, the samples were thawed, mixed and
centrifuged briefly to spin down. Next, 1 ml of sample was transferred to a clean 96-well plate with each well containing 10 ml of an
internal size standard mixed with deionised formamide (1:90
GeneScanÔe500 ROXÔ:Hi-DiÔ formamide (both Applied Biosystems)). The plate was heated to 96 C for 4 min and snap-cooled
on ice. Samples were run under denaturing conditions using
capillary electrophoresis on an ABI PRISMÒ 3100 POP-6 polymer in
a 50 cm capillary array on an ABI PRISMÒ 3100 Genetic Analyzer, at
N/A
3 dayse3
weeks or
longer
24 h
100%
25%
9% w/v
30%
Acetone [67e64e1]
Acrylic dispersion/emulsion
in water
Aluminium potassium
sulfate (alum)
[10043e67e1
(anhydrous);
7784e24e9
(dodecahydrate)] in
water
Ammonium hydroxide
[1336e21e6] in water
1 weekþ
N/A
100%
100%
w24.4e24.7%
Benzene [71e43e2]
Carbon tetrachloride
[56e23e5]
Cellulose nitrate
[9004e70e0] in
ethanol:ether (1:1)
Few hours
100%
10% v/v
Chloroform [67e66e3]
Enzyme active detergent in
water
30 mine8 h/day,
3e5 days
N/A
1:1 Ethanol:ether
N/A
N/A
3% w/v
Arsenic trioxide
[1327e53e3] in water
N/A
100%
Harris (1959, p. 223)
Mooney et al. (1982, p. 125)
75e80 C
Hornaday (1912, p. 346)
Hornaday 1912, p. 346
Rowley (1925, pp. 211e213)
Hangay and Dingley (1985, pp.
345e347)
Solvent: cited in: Bather (1908, p. 87),
Nichols and Orr (1932, pp. 49e50);
British Museum (Natural History)
1934, p. 5; Camp and Hanna (1937,
pp. 31e32); North et al. (1941, p. 77);
Satyamurti (1967, p. 16); Wagstaffe
and Fidler (1968, pp. 284, 285); Rixon
(1976, p. 9); Koob (1982, p. 33);
Lindsay (1987, p. 460) (referred to
Rixon, 1976); Green (2001, p. 83)
(referred to Rixon, 1976)
Hornaday (1912, p. 348)
Hildebrand (1968 in Williams, 1999,
p. 71)
Hornaday (1912, p. 103)
Storch (2003, p. 4)
Harris (1959, p. 223)
Satyamurti (1967, p. 16)
Reference
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
Published
temperature
w9.9% w/v
100%
< 24.7%
100%
100%
Room
temperature
Room
temperature
Room
temperature
and 80 C
<Overnight
(18 h)
40 h
Room
temperature
Room
temperature
Room
temperature
Room
temperature
Room
temperature
Room
temperature
Room
temperature
Room
temperature
Room
temperature
Room
temperature
Experimental
temperature
1h
1h
1 week
1 week
2h
2h
100%
4.5% w/v
24 h
Overnight
(18 h)
1 week
Overnight
(18 h)
1h
Experimental
exposure
time
35%
13.5% w/v
37.5% v/v
100%
22.5% v/v
Experimental
concentration
(%)
Aldrich, 99%, Prod: 22, 762e5,
Lot: S05309e302, CAS:
1327e53e3, EC: 215e481e4
BDH Laboratory Supplies, AnalaR,
99.7%, Prod: 100514E, Lot:
K28372065 146, EC: 200e753e7
Prolabo Normapur AR 99.8%,
Prod: 22 521.293, Lot: L067, EC:
200e262e8
HMG (nitrocellulose) Heat and
Waterproof Adhesive, Lot:
405064 (evaporated off solvent
and redissolved in ether/ethanol)
Ethanol as below; and ether: BDH
AnalaR, Prod: 10094 6B, Lot:
331K19890127
BDH, AnalaR, Prod: 100775A, Lot:
K32079841 323, EC: 200e663e8
Sainsbury’s basics biological
powder, 5e15% phosphate, less
than 5% oxygen-based bleaching
agent, non-ionic surfactant, ionic
surfactant, contains perfume,
enzymes and optical brightener
BDH, GPR 35% Ammonia solution,
sp gr. 0.880, Prod: 27141, Lot:
5970160J, 35% NH3
Alfa Aesar, Technical Grade 100%,
Prod: 036285, Lot: D15J08, EC:
211e047e3
BDH Laboratory Supplies, AnalaR,
100% Acetic Acid, Prod: 10001CU,
Lot: K31738717 307, EC:
200e580e7
BDH Laboratory Supply, AnalaR,
99.5%, Prod: 100034Q, Lot:
K28042206, EC: 200e662e2
Conservation resources, Primal
WS24 (Acrysol WS24)
J.M. Loveridge plc, BN A631
Chemical source information
2834
Amyl acetate [628e63e7]
Few hours
N/A
15%
Acetic acid [64e19e7] in
water
Published
exposure
time
Published
concentration
(%)
Chemical tested [CAS
number]
Table 1
Chemicals tested in the screening experiment, their published and experimental conditions, as well as source information.
Author's personal copy
J.A. Eklund, M.G. Thomas / Journal of Archaeological Science 37 (2010) 2831e2841
Potassium carbonate
(potash) [584e08e7] in
water
Poly(vinyl) acetate (PVAC)
[9003e20e7] in water
Poly(vinyl) acetate/poly
(vinyl) alcohol emulsion
(PVAC/PVAL) in water
Poly(vinyl) butyral resin
[63148e65e2] in
acetone:IMS (1:1)
1:1 Acetone:IMS
Linseed oil [8001e26e1] in
turpentine
Mercury (II) chloride
[7487e94e7] in
alcohol:water (1:1)
Methylmethacrylate/
ethylacrylate resin
[80e62e6] in acetone
Oxalic acid [144e62e7
(anhydrous);
6153e56e6 (dihydrate)]
in water
Pepsin [9001e75e6] in
water
Industrial methylated spirit
(IMS) [64e17e5]
Kerosene [8008e20e6]
N/A
N/A
N/A
3% w/v
N/A
N/A
Simmer
N/A
N/A
Ryder (1968, p. 23)
See poly(vinyl) butyral resin
Croucher and Woolley (1982, p. 28)
Lewis and Redfield (1970, pp. 7e8)
Toombs and Rixon (1950. p. 141)
Mahoney (1973, p.442); Hangay and
Dingley (1985, p. 344) note that
pepsin and trypsin are used under
similar conditions
37 C
20% w/v
Up to 48 h
1% w/v
Hamilton (1999/2001, pp. 314e315)
Cannon (1997, p. 36)
Hornaday (1912, pp. 150e151)
Hornaday (1912, p. 255)
Satyamurti (1967, p. 16)
Plenderleith (1962, p. 148)
Harris (1959, p. 223)
Camp and Hanna (1937, pp. 16e17)
Anderson (1932, pp. 410e411)
Solvent: cited in: Bather (1908, pp.
82e83) (referred to Reid, no date);
Jackson (1926, pp. 117e118)
(referred to Back 1924); Rixon (1976,
p. 11); Croucher and Woolley (1982,
pp. 46, 47); Lindsay (1987, p. 460)
(also referred to Bather (1908);
Whybrow and Lindsay (1990, p. 501)
Godfrey et al. (2002, p. 530)
Anderson (1932, pp. 410e411)
N/A
N/A
N/A
10% w/v
N/A
20%
Overnight
30%
N/A
6e12 h
N/A
Saturated
solution
N/A
N/A
1/3 dilution
N/A
N/A
100%
50%
5 s, 4e5 times
N/A
N/A
1h
N/A
20 Volumes
hydrogen
peroxide
100%
93% v/v
N/A
N/A
24 h, repeat
5% w/v
Ethylene diaminetetracetic
acid, disodium salt
(EDTA) [139e33e3
(anhydrous);
6381e92e6 (dihydrate)]
in water
Gasoline [8006e61e9] in
alcohol/turpentine
Gum arabic [9000e01e5]
in water
Hydrogen peroxide
[7722e84e1]
N/A
N/A
50%
6 weeks (fresh
solution each
week)
100%
Ethyl acetate [141e78e6]
24 h, repeat
6%
Ethanol [64e17e5]
4.5% w/v
20% w/v
30% v/v
49.5% v/v
1.5% w/v
15% w/v
6h
1h
1h
Overnight
(18 h)
Overnight
(18 h)
48 h
1 week
Overnight
(18 h)
2h
w7.5% w/v
30% v/v
2h
1h
50% v/v
100%
25 s
1h
9% v/v
100%
1h
48 h
1 week
2h
48 h
75% w/v
93% v/v
7.5% w/v
100%
99% v/v
Room
temperature
Room
temperature
and 80 C
Room
temperature
Room
temperature
Room
temperature
Room
temperature
and 37 C
Room
temperature
Room
temperature
Room
temperature
Room
temperature
Room
temperature
Room
temperature
Room
temperature
Room
temperature
Room
temperature
Room
temperature
Room
temperature
Room
temperature
J.A. Eklund, M.G. Thomas / Journal of Archaeological Science 37 (2010) 2831e2841
(continued on next page)
BDH Laboratory Supplies, AnalaR,
Potassium carbonate anhydrous,
Prod: 101964H, Lot: A919836
636, EEC: 209e529e3
Acetone and IMS as above
Conservation Resources, Mowital
B30H
Sigma (Pepsin A/800e2,500
units/mg protein, EC 3.4.23.1),
1:10,000, P-7000, Lot: 103K0099,
From Porcine stomach mucosa
[9001e75e6], EC: 232e629e3
Brian Clegg Washable P.V.A.
Adhesive
Elmer’s Glue-All, Elmer’s Products
Inc.
No data available
Conservation Resources, Paraloid
B-72
Superwarm Fuel, Parasene,
Kerosene, Paraffin
Homebase boiled linseed oil,
Prod: K17015
Aldrich, Prod: 215465e5G, Batch:
03706DO
Bell’s Hydrogen Peroxide Solution
with stabilizer, 9% (30 volumes),
Prod: BN5278F1
Hayman Ltd., Batch: 6/D/339/26
BDH Chemicals Ltd., Prod: 33001
97 Octane, unleaded
GPR [VWR] International Ltd.,
Prod: 280254D, Lot: 0965T02265
340, EC: 205e358e3
VWR International, BDH AnalaR,
Prod: 10107, EC: 200e578e6
VWR International Ltd., AnalaR
99.5%, Prod: 101084H, Lot:
K33783169 440, EC: 205e500e4
Author's personal copy
2835
6%
Sodium hypochlorite
(bleach) [7681e52e9] in
water
Sodium perborate
[7632e04e4] in water
N/A
N/A
N/A
N/A
N/A
Simmer
From
boiling
N/A
N/A
Harris (1959, p. 223)
Hornaday (1912, pp. 287, 288)
Anderson (1932, pp. 410, 411)
Solvent: cited in: North et al. (1941.
p. 78); Cornwall (1956, pp. 213, 214,
216, 217); Satyamurti (1967, p. 16);
Mahoney (1973, p. 449); Thurmond
(1974, p. 195); Rixon (1976, pp. 10,
11); Leigh (1978, p. 33); Howie (1979,
p. 280); Gehlert (1980, p. 8); Koob
(1984, p. 100); Snow and Weisser
(1984, p. 142); Horie (1987, p. 96)
(referred to Unwin, 1951); Payton
(1992, p. 23); Johnson (1994, p. 227)
(referred to Brothwell, 1981, p. 229);
Sease (1994, p. 51); Kres and Lovell
(1995, p. 510); Shelton and Johnson
(1995, p. 66); Green (2001, p. 83)
(referred to Rixon, 1976); Storch
(2003, p. 4); Davidson (2004, p. 55)
Mahoney (1973, p. 445)
Harris (1951, p. 97)
Roche 1954 translated by Hangay and
Dingley (1985, pp. 342e343)
Stephens 1979 in Krogman and Isçan
(1986, p. 42)
Stephens 1979 in Krogman and Isçan
(1986, p. 42)
Harris (1959, pp. 223e224)
up to 50 C
Prod, product number; Lot, lot number; EC, European Commission number.
100%
100%
24 h,
repeat
Several
days
Few hours
1%
White spirit, (naptha)
[8052e41e3]
Xylene [1330e20e7]
N/A
100%
Trichloroethylene
[79e01e6]
Turpentine [8006e64e2]
N/A
100%
Toluene [108e88e3]
6h
0.1% w/v
Sodium sulfide
[1313e82e2] in water
(saline solution)
3e4 h repeated
for several
days
3e4 h repeated
for several
days
Overnight
Hornaday (1912, pp. 285e288)
Hangay and Dingley (1985, p. 347)
80 C
N/A
Reference
Published
temperature
100%
100%
100%
100%
100%
0.15% w/v
15% w/v
9% v/v
1.5% w/v
1.35% w/v
15% w/v
15% w/v
Experimental
concentration
(%)
<Overnight
(18 h)
1 week
48 h
2h
2h
6h
Overnight
(18 h)
28 h
28 h
24 h
5 min
24 h
Experimental
exposure
time
Room
temperature
Room
temperature
Room
temperature
Room
temperature
Room
temperature
Room
temperature
and heated to
100 C for 1 min
in thermal cycler,
then allowed to
cool to room
temperature
Room
temperature
and 80 C
Room
temperature
Room
temperature
and 37 C
Room
temperature
Room
temperature
and 80 C
Room
temperature
Experimental
temperature
Fluka Chemika, Prod: 91129, Lot:
431827/142202, EC: 2011674
Hilton Banks Ltd., spirits of
turpentine, EC: 232e350e7
Prolabo, Prod: 28 963.368, Lot:
K243, EC: 232e443e2
BDH Laboratory Supplies, GPR,
99.0%, Prod: 305756G, Lot:
K31502911 250, EC: 215e535e7
Alfa Aesar, Johnson Matthey
GmbH & Co., hydrate:
27610e45e3, Prod: 011664, Lot:
C11Q49, EC: 215e211e5
BDH Laboratory Supplies, GPR,
Prod: 30454EC, EC: 203e625e9
BDH Laboratory Supplies, AnalaR,
min 99%, Prod: 102524X, Lot:
B868650 124, EC: 215e185e5
BDH Laboratory Supplies, GPR,
12% available chlorine, Prod:
301696S Lot: K31848623 317
BDH Limited, GPR, Min. assay
96.0%, Prod: 30196, Lot:
3367530M
VWR International Ltd., GPR,
Prod: 301235Q, Lot: K34042132
BDH Laboratory Supplies, GPR,
Prod: 301214L, Lot: A217831 016
Super cook bicarbonate of soda
Chemical source information
2836
10% w/v
1%
24 h
0.9% w/v
Sodium hydroxide
[1310e73e2] in water
5 min
10% w/v
Sodium carbonate (washing
soda) [497e19e8] in
water
Sodium chloride (salt)
[7647e14e5] in water
12e24 h
10% w/v
Sodium bicarbonate
[144e55e8] in water
Published
exposure
time
Published
concentration
(%)
Chemical tested [CAS
number]
Table 1 (continued)
Author's personal copy
J.A. Eklund, M.G. Thomas / Journal of Archaeological Science 37 (2010) 2831e2841
Author's personal copy
J.A. Eklund, M.G. Thomas / Journal of Archaeological Science 37 (2010) 2831e2841
2837
Fig. 1. Electropherograms of: (A) typical water control sample after 7 days, and (B) typical mineral oil negative control sample after 7 days. Note the reduction in TAT and SRY 4064
peak heights in the mineral oil electropherogram, indicative of damage.
60 C, for 4000 s, using filter set D. Data was analysed using ABI
PRISMÒ GeneScanÒ Analysis software version 3.7 for Windows
(Applied Biosystems).
2.5. Data analysis
Analysed samples had the following data recorded: minute, size,
peak height, peak area and data point. Size, as well as the peak
pattern, was used to identify each DNA sequence (confirmed by
minute and data point if necessary).
Some of the DNA sequences used in this study appear in electropherograms as multiple peaks (YAP- ¼ 5 peaks, M9 and SRY
4064 ¼ 2 peaks each). As TAT only produced a single peak, data for
this peak was used for comparison with the all of the YAP-peaks.
Damage was assessed in the first instance by visual inspection of
electropherograms by identifying irregularities in peak
morphology (such as split peaks) and cases where treated DNA
peaks were reduced or were not detectable (see Fig. 1).
GeneScan electropherogram peak height, rather than peak area,
was used as a proxy for the amount of each specific DNA sequence,
as it has been found to be a more reliable measurement (Baudhuin
et al., 2005). Based on fluorescence, the relative concentration of
the DNA sequences in each sample is ascertained rather than the
absolute concentration. Therefore, the ratio between the test and
reference solution peak heights for each sample was calculated and
compared to the peak height ratios of the deionised water control
samples as a means of measuring DNA damage. Ratios were
calculated by dividing the test peak height by the reference peak
height for both the 100 bp (TAT/YAP-) and 200 bp (SRY 4064/M9)
sequence pairs. For each of the sample runs, 7 peak height ratios
were calculated per sample using all of the peaks present. A smaller
test peak height occurs due to a lower concentration of a test
solution sequence, and results in a lower ratio, thereby indicating
damaged DNA. A higher ratio indicates better preserved DNA.
Peak height ratios were used to calculate the percentage of DNA
preservation to assess quantitatively the effect of each treatment
tested. Mean peak height ratios were calculated for each set of
treatment samples and control samples. Using the deionised water
control peak height ratio as a baseline (theoretically representing
100% DNA preservation, although it is acknowledged that some
damage would result from exposure to water, this effect was equal
across all samples), both positive and negative preservation effects
due to conservation chemical treatment could be estimated.
Treated sample means were divided by the water control sample
means, and the value obtained expressed as a percentage. To assess
any differences in preservation between the two different
sequences and between runs, the average and standard deviation of
the values for both the first and second runs of all the TAT/YAP- and
SRY 4064/M9 peaks were calculated. Finally, the “overall DNA
percent preservation” was calculated, represented by the average
and standard deviation of all calculated values for each treatment.
All percentage of DNA preserved data are presented in the
Supplemental material.
3. Results
The results of this research were used to identify which of those
treatments tested should be considered safe or unsafe for use in
a conservation context, guided by the principles of conservation
that treatments should minimise damage, have the least adverse
effect, preserve maximum information possible and not affect
future analyses. Results of the overall mean percentage of DNA
preserved are summarised in Table 2.
3.1. Safe treatments
The two PCR products used in the test solution were found to
behave differently, and we propose that this is due to differences in
GC content. In general, the TAT sequence e with a lower GC content
of 36% e was adversely affected by more treatments than the SRY
4064 sequence with a higher GC content of 47%. A final list of
treatments considered safe for use for conservation purposes was
compiled based on the most conservative interpretation of the
experimental results of this study. Using the overall percent DNA
preserved calculations (the average of all ratios) for treatments
with data for both the TAT and SRY 4064 sequences, and the criteria
Author's personal copy
2838
J.A. Eklund, M.G. Thomas / Journal of Archaeological Science 37 (2010) 2831e2841
Table 2
Tested treatments ranked by overall mean DNA preservation.
Treatment
Overall mean
preservation (%)
Overall standard
deviation (%)
Standard deviation
lower limit (%)
Potassium carbonate e 3% w/v in water, 6 h
Industrial methylated spirit (IMS) e 100% (67% in solution), 25 s
Trichloroethylene e 100% (67% in solution), 2 h
Sodium chloride e 0.9% w/v in water, 24 h at 37 C
Water (control) e 100%, 1 week
Toluene e 100% (67% in solution), 2 h
Amyl acetate e 100% (67% in solution), 2 h
Acetone e 100% (67% in solution), 18 h
Chloroform e 100% (67% in solution), 18 h
Xylene e 100% (67% in solution), 18 h
Sodium chloride e 0.9% w/v in water, 24 h
Arsenic trioxide e 3% w/v in water, 2 h
1:1 acetone:IMS e 100% (67% in solution), 1 h
1:1 ethanol:ether e 100% (67% in solution), 1 h
Ethyl acetate e 100% (67% in solution), 2 h
Acrylic dispersion e 25% v/v in water, 18 h
Ethanol e 66% v/v in water, 48 h
Kerosene e 100% (67% in solution), 1 h
Cellulose nitrate e <25% w/v in 1:1 ethanol:ether (<17% in solution), 1 h
PVAC e 33% v/v in water, 18 h
Methylmethacrylate/ethylacrylate e 30% v/v in acetone (20% in solution), 18 h
Carbon tetrachloride e 100% (67% in solution), 1 week
Sodium bicarbonate e 10% w/v in water, 24 h
White spirit e 100% (67% in solution), 1 week
Benzene e 100% (67% in solution), 1 week
Poly(vinyl) butyral resin e 20% w/v 1:1 acetone:IMS (13% in solution), 1 h
Sodium carbonate e 10% w/v in water, 5 min
Ammonium hydroxide e 23% ammonia solution in water, 24 h
Linseed oil e 50% v/v in turpentine (33% in solution), 2 h
PVAC/PVAL e 20% v/v in water, 18 h
Gasoline e 93% v/v in alcohol/turpentine (62% in solution), 48 h
Sodium hydroxide e 1% w/v in water, 28 h
Ethylene diaminetetracetic acid, (EDTA) disodium salt e 5% w/v in water, 1 week
Detergent (enzyme active) e 7% w/v in water, 40 h
Hydrogen peroxide e 6% in water (20 volumes), 1 h
Gum arabic e 50% w/v in water, 1 h
Acetic acid e 15% v/v in water, 1 h
Mercury (II) chloride e 7.5% w/v in 1:1 ethanol:water (5% in solution), 2 h
Pepsin e 1% w/v in water, 48 h
Sodium bicarbonate e 10% w/v in water, 24 h at 80 C
Mineral oil e 100% (67% in solution), 1 week
Potassium carbonate e 3% w/v in water, 6 h at 80 C
Pepsin e 1% w/v in water, 48 h at 37 C
Sodium sulfide e 0.1% w/v in 0.9% sodium chloride in water, 6 h at 80 C
Sodium sulfide e 0.1% w/v in 0.9% sodium chloride in water, 6 h
Turpentine e 100% (67% in solution), 48 h
Aluminium potassium sulfate e 9% w/v in water, 1 week
Detergent (enzyme active) e 7% w/v in water, 40 h at 80 C
Oxalic acid e 10% w/v in water, 1 week
Sodium hypochlorite e 6% v/v in water, 28 h
Sodium perborate e 10% w/v in water, 18 h
Sodium perborate e 10% w/v in water, 18 h, heated to100 C for 1 min and
allowed to cool to room temperature
102.3
100.4
100.4
100.4
100.0
99.1
97.7
97.5
96.6
96.3
96.3
96.2
96.0
95.5
94.4
93.1
91.7
88.6
88.6
88.1
87.9
84.4
82.7
82.7
81.6
81.5
79.9
79.6
76.7
75.7
75.3
73.8
70.9
64.3
62.3
56.2
39.8
31.4
27.3
25.1
24.8
24.8
21.5
13.9
8.9
8.6
ND
ND
ND
ND
ND
ND
71.2
8.4
8.9
11.7
0.0
8.1
8.7
8.4
9.0
10.0
25.4
11.5
9.9
10.9
10.2
38.3
11.9
9.2
12.3
14.6
6.5
9.5
66.9
10.5
8.4
20.9
44.7
10.6
18.9
14.0
8.7
14.9
44.7
49.7
12.9
22.5
17.2
17.0
9.1
8.7
3.3
10.6
8.4
4.3
3.3
2.8
ND
ND
ND
ND
ND
ND
31.1
92.0
91.5
88.7
100.0
91.0
89.0
89.1
87.7
86.4
70.8
84.7
86.1
84.6
84.2
54.8
79.8
79.4
76.3
73.5
81.5
74.8
15.8
72.2
73.2
60.6
35.2
68.9
57.8
61.7
66.6
58.9
26.2
14.7
49.4
33.7
22.6
14.4
18.2
16.4
21.5
14.2
13.1
9.6
5.6
5.8
Treatments shaded in grey are unsafe treatments; unshaded treatments are safe treatments. All treatments were carried out at room temperature unless noted otherwise.
As treatments were added to DNA suspended in water, concentrations of organic-soluble treatments noted as “in solution” are adjusted to take the final solution concentration
into account even if solutions were not miscible. “ND” means no data was obtained, as peaks were too irregular or not detected and for the purposes of this experiment are
considered unsafe.
of an average DNA preservation of greater than 90% with a standard
deviation less than 12% (this corresponds with the standard deviation calculated for the water control samples, and therefore
considered an acceptable degree of variation) and a lower limit of
DNA preservation of 80%, the list is, effectively, those treatments
safe for use on both sequences tested.
Safe treatments include: industrial methylated spirits (IMS),
trichloroethylene, sodium chloride (heated to 37 C), toluene, amyl
acetate, acetone, chloroform, xylene, arsenic trioxide, 1:1 acetone:industrial methylated spirits, 1:1 ethanol:ether, and ethyl
acetate. It should be noted that both chloroform and xylene evaporated overnight, therefore these treatments can only currently be
deemed safe for short treatment periods, and their effects if used
for the full exposure time remain unknown. Of those treatments
deemed safe to use, some evidence was found that amyl acetate,
IMS, sodium chloride, trichloroethylene and xylene may have
a positively preserving effect, but the effects were not consistent for
both DNA sequences tested in the study, so further research into
treatments that may enhance DNA preservation is required.
3.2. Unsafe treatments
Treatments considered unsafe were those that either resulted in
less than 90% overall mean DNA preserved or had a standard
Author's personal copy
J.A. Eklund, M.G. Thomas / Journal of Archaeological Science 37 (2010) 2831e2841
deviation greater than 12%, or had a standard deviation lower limit
of less than 80%. Although many treatments tested were found to
be only slightly damaging compared to the deionised water control,
a few treatments were particularly damaging to DNA. Sodium
sulfide and turpentine resulted in almost complete destruction of
both the TAT and SRY 4064 sequences. Pepsin, mineral oil, mercury
(II) chloride, acetic acid and treatments heated to 80 C or higher
were only slightly less damaging, but their ratios were still well
below that of the water control and the majority of other treatments. That the most damaging treatments would include acetic
acid and heat was not surprising, as it is known that DNA is
susceptible to damage by both acids and heat (Lindahl, 1993). In
general, heated treatments were more damaging than their
unheated replicates. Also, organic solvents alone were typically less
damaging than organic solvents acting as a carrier for another
material. For example, acetone was found to have 97.5 8.4% DNA
preserved, whereas methylmethacrylate/ethylacrylate 30% v/v in
acetone was found to have 87.9 6.5% DNA preserved.
4. Discussion
The method developed for this study is intended to assess the
degree of in vitro DNA strand breakage due to a particular treatment,
enabling treatments to be classified as to their effects on DNA e
whether a treatment is damaging, preserving or has a similar effect
to deionised water. By knowing the degree of strand breakage caused
by preparation and conservation treatments in direct contact with
DNA, the results of this study should allow conservators to choose
treatments that cause minimal damage to DNA and assist sampling
of collection materials for research by avoiding specimens treated
with chemicals known to be damaging.
A conservative definition as to what constitutes a safe treatment for conservation applications is used here, for several
reasons. Firstly, the principles of conservation dictate that treatments should have the least damaging effect and not interfere
with future analyses. Secondly, it is not possible to predict the
state of preservation of a particular DNA target sequence in any
given specimen, and it was found that different sequences may
suffer different degrees of damage from a specific treatment,
thought to be due to higher GC bond content potentially providing
greater resistance to damage (Yakovchuk et al., 2006). Thirdly, it is
not possible to be certain of the efficiency of extraction and
amplification procedures on an individual sample, so the lower
limit of DNA preservation in a specimen that will be viable for
research purposes is difficult to characterise. Taking these factors
into account, only those treatments having an effect similar to
deionised water were defined as ‘safe’ to use for the purposes of
this study. As a result, the list of treatments considered safe is
limiting, as it largely consists of organic solvents. The use of
organic solvents alone on hard and soft tissues is uncommon
(perhaps with the exception of degreasing); organic solvents
typically serve as a liquid medium for another material, such as
a consolidant or pesticide. Additionally, organic solvents are
usually a second choice for many conservators on health and
safety grounds, thereby making aqueous treatments preferred in
practice. Although arsenic trioxide was found to be safe for DNA, it
is also typically avoided on health and safety grounds. Sodium
chloride was also found to be safe but has limited applications in
preparation and conservation treatments. Although it may not be
possible to select only safe treatments for any given conservation
application, by ranking chemicals in order of least to most
damaging, those found to cause less damage than others can be
chosen for use. It should be noted that many treatments considered
unsafe were only slightly more damaging to the sequences tested
than deionised water and resulted in the preservation of
2839
approximately 70% or more of the DNA present whilst maintaining
a standard deviation of 12% or less. Some of these treatments may be
preferred for use over other chemicals found to be more damaging.
This category of somewhat damaging treatment materials includes:
ethanol, kerosene, methylmethacrylate/ethylacrylate, carbon tetrachloride, white spirit, benzene, ammonium hydroxide and gasoline.
Some of these materials may not be considered appropriate for
conservation purposes, such as kerosene and gasoline, and others
may be avoided on health and safety grounds, such as carbon
tetrachloride and benzene. However, others may currently be in use
and have been widely used in the recent past, such as ethanol and
methylmethacrylate/ethylacrylate. Testing of additional treatments
at different concentrations and exposure times is recommended to
increase our understanding of how preparation and conservation
treatments affect DNA.
By consulting conservation records when selecting specimens
for DNA sampling, it may be useful to check for a history of treatments containing chemicals found to be damaging in this study.
Although many factors may contribute to the preservation of DNA
in collection material, DNA extraction and amplification may be
more successful using samples from specimens treated with less
damaging compounds or not treated at all. Including sampling
prior to chemical intervention into conservation treatment strategies is of great importance, particularly as biochemical analytical
techniques are developing rapidly, and it is almost impossible to
determine future research needs that may affect any individual
specimen. In some instances it may be advisable to use multiple
different treatments to address a single preservation problem, with
the hope that at least one treatment will be compatible with future
research objectives.
5. Conclusion
The method developed for this study was used to screen 43
chemicals commonly used in the past to prepare and/or conserve
human and/or animal remains for their effects on DNA in vitro. The
results of this study indicate that the majority of treatments used in
the past are at least somewhat damaging to DNA. Several treatments, mostly organic solvents, were found to affect the sequences
tested to a degree similar to deionised water, resulting in over 90%
DNA preservation, and were deemed ‘safe’ for use in conservation
applications. Results of this research may be used in several ways.
Conservators can use this information to guide the development of
future conservation treatments by using less damaging materials
when possible. Museum managers and researchers may find it
useful to select specimens treated in the past using less damaging
materials when sampling for DNA studies. However, the results of
this study should not be used to restrict access to collections by
assuming that DNA analysis will be unsuccessful with specimens
treated with the more damaging treatments identified. A number
of factors other than treatment history influence the success of DNA
extraction and amplification from collection materials, such as
inhibition, contamination and the efficiency of extraction and
amplification methods, to name a few.
The method developed for this experiment is only intended as
a tool to identify treatments that damage raw DNA. However, it is
acknowledged that the effects of treatments on DNA in vitro are not
necessarily directly comparable to the effects of treatments on DNA
in various hard and soft tissues which may have a buffering effect,
and that multiple treatments, re-treatment and aged treatments
may present additional problems associated with DNA extraction
and analysis. Whether or not treatments considered in this study as
unsafe were damaging enough to prevent DNA extraction and
amplification, or if their use would inhibit PCR amplification
remains unknown. It is possible that with an efficient DNA
Author's personal copy
2840
J.A. Eklund, M.G. Thomas / Journal of Archaeological Science 37 (2010) 2831e2841
extraction method, PCR amplification could be successful even if
conservation treatment caused a considerable degree of damage to
DNA in a specimen. Such issues require further research. Future use
of the method presented here could be used to investigate such
issues by attempting to PCR amplify treated samples, and it is
suggested that a combination of methods should be employed to
gain the best understanding possible as to the role conservation
treatments may play in DNA deterioration or preservation and their
impact on research results. The method developed may also be
useful for research into sequence specific damage and/or repair
mechanisms, avoiding many of the difficulties associated with DNA
extraction, amplification and contamination. Additional methods to
assess the effects of treatments on DNA in both hard and soft
tissues require further development to facilitate better conservation of our natural and cultural heritage in collections and thereby
uphold the principles of conservation.
Acknowledgements
This research was undertaken as part of PhD research at the
Institute of Archaeology, University College London, which was
made possible by funding from the Overseas Research Students
Awards Scheme and the Institute of Archaeology. Many thanks are
due to Sandra Bond and James Hales for materials and access to
facilities, as well as Dr. Ian Barnes, Abigail Jones and Beth Caldwell
for training and advice. Thanks also to the anonymous reviewers for
their supportive and helpful comments.
Appendix. Supplementary material
Supplementary data associated with this article can be found in
the online version, at doi:10.1016/j.jas.2010.06.019.
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