Journal of Pathology
J Pathol 2000; 192: 554±559.
DOI: 10.1002 /1096-9896(2000)9999 : 9999<: : AID-PATH768>3.0.CO;2-C
Original Paper
An assessment of the long-term preservation of the
DNA of a bacterial pathogen in ethanol-preserved
archival material
Ian Barnes1,2{, John Holton1, Dino Vaira3, Mark Spigelman1 and Mark G. Thomas2
1
Department of Bacteriology, The Windeyer Institute of Medical Sciences, The Windeyer Building, 46 Cleveland Street, London W1P 6DB, UK
The Centre for Genetic Anthropology, Department of Biology, University College London, Darwin Building, Gower Street, London WC1E 6BT, UK
3
First Medical Clinic, University of Bologna, via Masserrenti 9, 40138 Bologna, Italy
2
{
Current address and
correspondence:
Institute of Biological
Anthropology, University of
Oxford, 58 Banbury Road,
Oxford OX2 6QS, UK
Received: 22 November 1999
Revised: 22 June 2000
Accepted: 10 July 2000
Published online:
23 October 2000
Abstract
To examine the potential for DNA recovery from spirit-preserved medical material, a set of
specimens from the Hunterian Collection of the Royal College of Surgeons was investigated.
Using a range of DNA extraction techniques and the PCR, no replicable positive ampli®cations
were made from this material of either human or Helicobacter DNA. Experiments with modern
stomach biopsies of H. pylori-positive patients suggest that the bacterial DNA is typically present
in a much lower concentration (103-fold) than that of the host. The potential for recovery of this
organism from spirit specimens is therefore low. The absence of DNA in this material is probably
due to several factors, chie¯y the incomplete ®xation of the specimen by the ethanol storage ¯uid.
Studies such as this demonstrate the need for a good understanding of specimen history when
working with archival material. Copyright # 2000 John Wiley & Sons, Ltd.
Keywords:
Helicobacter; ancient DNA; PCR; museum; spirit specimen
Introduction
World-wide, museums house a vast number of spiritpreserved specimens which include a variety of animal
taxa, examples of a range of pathological conditions,
and specimens from species which are now extinct or
heavily endangered. These museum collections have
considerable potential for evolutionary and population
genetics surveys, and have been exploited for this type
of research (e.g. Beebee et al. [1]). Work has thus far
focused primarily on formaldehyde-preserved specimens, although DNA recovery can be problematic
[2,3].
Spirit-preserved specimens which demonstrate a
clearly diagnosable pathological condition could be
used to investigate long-term changes in the population
genetics of disease-causing micro-organisms and
viruses, to investigate changes in the aetiology of
diseases, and to explore changes in the distribution of
different subspecies of the micro-organisms over time.
In this study, we investigate the possibility for survival
of DNA from the bacterial pathogen Helicobacter
pylori in a set of ethanol-preserved specimens from
the18th century. These specimens are housed in the
Hunterian Museum at the Royal College of Surgeons
in Lincolns Inn Fields, London.
H. pylori is now a well-recognized gastroduodenal
pathogen and a class I carcinogen. It is the cause of
gastritis and of the majority of cases of peptic ulcer
disease (PUD) and through long-term carriage, is
thought to increase the risk of gastric adenocarcinoma
and lymphoma. The complete genome sequence of the
Copyright # 2000 John Wiley & Sons, Ltd.
organism was ®rst published in 1997 [4] and has been
shown to carry a pathogenicity island, for which the
marker is cagA. The presence of this marker identi®es
type I strains, which are correlated with more severe
gastrointestinal disease than type II strains lacking
cagA [5].
Materials and methods
Modern samples
Eight biopsy samples and clinical details were obtained
(by DV) from patients who had been diagnosed as
being positive for H. pylori infection on the basis of
culture, histology, biopsy urease test, and 13C-urea
breath test [6]. All patients had given informed consent
to biopsy sampling. Gastric biopsy samples were
obtained from the antrum of each patient and stored
in 70% ethanol for a period of 1±2 weeks prior to
DNA extraction.
Museum samples
Six specimens from the Hunterian Collection (Museum
Reference Numbers P1017, P1018, P1019, P1020,
P1029, and P1030) were subsampled for DNA extraction. These specimens are documented in the museum
records as follows: P1017±P1020 were simple gastric
and duodenal ulcers; P1029 and P1030 were gastric
cancers.
The following is a summary of the archive specimens
used for our study: P1017: portion of pylorus with a
DNA preservation in long-term ethanol-preserved material
super®cial ulcer; P1018: stomach of a 22-year-old lady
showing a perforated ulcer; P1019: stomach of a
nobleman with a small perforated pyloric ulcer,
opposite another ulcer which was still super®cial;
P1020: stomach with a perforating ulcer of the posterior wall; P1029: a woman's stomach with a cancer
growing anteriorly below the cardia, histologically
described as columnar-celled type. There was a 25year history of symptoms of gastric disease; P1030:
pylorus with a soft lobulated mass nearly ®lling the
stomach.
The specimens were stored in glass containers ®lled
with ethanol-based preservative. The original composition of this preservative is unknown, although contemporary accounts from the period would suggest the
use of spirit of wine, likely to have been a crude
distillate of around 40% (v/v) concentration [7]. The
specimens originally were sealed with a bitumen and
gutta percha coated pig's bladder and capped with a
lead sheet and further bitumen. These seals had been
broken at some point in the past to allow for
replacement of the preservative.
The two specimens with stomach cancer (P1029 and
P1030) were sampled twice, with samples taken from
the stomach mucosa and from an adjacent region of
stomach cancer. The other four specimens, diagnosed
as simple gastric or duodenal ulcers, were sampled only
from the mucosa. All samples were stored at room
temperature in ethanol aliquoted from the parent
specimen until DNA extraction.
All sampling of the museum specimens was undertaken at the Royal College of Surgeons' pathology
department. Each specimen was biopsied using full
aseptic surgical techniques, with all instruments used
being of a sterile disposable type. For negative control,
we also sampled three specimens from the collection of
female genitalia which were affected by carcinoma of
the cervix.
All DNA extractions from museum samples, and the
set-up of subsequent PCR ampli®cations, were conducted in a laboratory which is dedicated to ancient
DNA analysis and which is physically isolated from
post-PCR ampli®cation facilities. All equipment and
surfaces were regularly decontaminated with a 10%
sodium hypochlorite solution. Tubes and non-UVsensitive solutions were irradiated with UV at 254 nm
prior to use and all glass and metal objects used were
baked at >400uC for a minimum of 3 days before use.
Histological analysis of the museum samples
Histological sections of the specimens were prepared
by standard histopathological techniques. Sections
were cut and stained with haematoxylin and eosin
(H&E) or Giemsa stains and examined microscopically.
DNA extraction
DNA extractions of the modern stomach biopsy
material were carried out by an overnight digestion of
Copyright # 2000 John Wiley & Sons, Ltd.
555
the tissue in a buffer containing 10 mM Tris±HCl
(pH 8.0), 0.5% SDS, and 100 mg of proteinase K
(Sigma), followed by a standard phenol±chloroform
procedure with isopropanol precipitation. DNA was
resuspended in TE buffer (10 mM Tris±HCl, 1 mM
EDTA, pH 9.0).
Extraction of DNA from the museum samples was
carried out on 20 mg subsamples from which the bulk
of the ethanol was removed either by brief (10 min) air
drying or by centrifugation for 1 min over a coarsely
meshed ®lter. Extractions were carried out using four
different techniques: (i) a standard phenol±chloroform
procedure with isopropanol precipitation as given
above for the modern samples, but with resuspension
in 100 ml of TE; (ii) the same procedure as above but
with further puri®cation of DNA by three 200-ml
washes with ultrapure water in a 30 kD MWCO
microconcentrator (Pro-Mem) and concentration to a
®nal volume of 50±150 ml; (iii) the silica-based extraction method of Hoss and Paabo [8] modi®ed by predigestion of the sample with proteinase K as above and
by elution of the ®nal product in 50 ml of TE buffer;
(iv) as (iii) but without the initial pre-digestion phase,
using guanidine isothiocyanate as a lysis agent. In all
cases, a negative extraction control, containing no
tissue, was carried through all phases of the extraction.
PCR ampli®cation
Twenty-one primers were designed (see Table 1) and
used to generate 12 different PCR products from four
different regions of the genome: ¯aA, which encodes
one of the ¯agella apparatus proteins; vacA, which
encodes the vacuolating cytotoxin protein; IS605, a
multicopy insertion sequence; and the 16S ribosomal
RNA. The primers were designed to ful®l the following
criteria: they are capable of amplifying from all
cultures of H. pylori for which sequence data are
available for the region of interest; they amplify a
range of fragment sizes including several under 100 bp
in length; they show high speci®city for the genus
Helicobacter as determined by comparison with database sequences; and they generate products which
incorporate known regions of variability between
different H. pylori cultures. In addition to the H.
pylori primers, we also used two pairs of human
mitochondrial DNA primers: PMT1 and PMT2, which
amplify a 277 bp segment of the mtDNA control
region; and RVM1 and RVM2 [10], which amplify a
111 bp region between the COII and tRNALys genes.
All primers used in this study were optimized for
maximum ef®ciency by testing a range of magnesium
ion and primer concentrations and thermal cycler
conditions.
Ampli®cations were carried out in 20 ml reaction
volumes consisting of 1±10 ml of the DNA extract,
200 mM of dNTPs, 10 mM Tris±HCl (pH 9.0), 0.1%
Triton X-100, 0.01% gelatin, 50 mM KCl, 1.5 mM
MgCl2, 0.2 mg/ml bovine serum albumin, 0.2 units of
Taq polymerase (HT Biotech) enzyme bound to 3.5 mM
J Pathol 2000; 192: 554±559.
556
I. Barnes et al.
Table 1. PCR primers used in this study. Primers above the double rule are those designed by us to amplify a region
of the H. pylori genome; the position of the 3k end of the primer is relative to the H. pylori whole genome sequence
[4]. Primers below the double bar amplify a region of the human mitochondrial genome and their position is given
relative to the reference sequence [9]
Primer name
Primer sequence (5k±3k)
3k position
Pairs with
(reverse primer only)
vacA-1-F
vacA-2-R
vacA-3-R
vacA-4-F
vacA-5-R
vacA-6-R
IS605-1-F
IS605-2-R
IS605-3-F
IS605-4-R
¯aA-1-F
¯aA-2-R
¯aA-3-F
¯aA-4-R
16S-1-F
16S-2-R
16S-5-F
16S-6-R
16S-8-R
16S-11-F
16S-12-R
RVM1
RVM2
PMT1
PMT2
HVM1
HVM4
AAACCCCAGATAAACCCGATAAA
CCGCCTTTGACCCAATAATG
CAATCCCAGCCTCCATCAATC
GCTGCTGTAGGAACGGTCTCA
TATCGGGTTTATCTGGGGTTTTAT
TGAATGCGCCAAACTTTATCG
CATTATCATTGCGATGGAAAGC
AAATCTCTTATCACGCCAAACTCTAT
TGCAAGGGGATTTGAACAACTTTA
TATATCGCTTTCCATCGCAATGAT
AAAATCGGTCAGGTTCGTATCG
CATCATTCACACCATCCACTTGTT
AACAAGTGGATGGTGTGAATGATG
GCGACTAACCTTCCGTCTGAGT
GACACACTGGAACTGAGACACG
CTACGGATTTTACCCCTACACC
AGGGCTTAGTCTCTCCAGTAATGC
CCCCGTCTATTCCTTTGAGTTTTA
ACTTAACCCAACATCTCACGACAC
GCGTGGAGGATGAAGGTTTTAGG
TTTACGCCCAGTGATTCCGAGTA
AGGGCCCGTATTTACCCTATAG
ATTTAGTTGGGGCATTTCACTG
CTCACCCATCAACAACCGCTAT
GGGAGCAGAAGGGATTTGACTG
CTAACCTGAATCGGAGGACAAC
GCATACCGCCAAAAGATAAAA
938654
938775
938738
938588
938629
938648
454567
454478
454629
454584
637084
637853
637876
638087
1512319
1511993
1511814
1511768
1511596
1512226
1512123
8248
8359
16067
16344
15749
398
±
vacA-1-F
vacA-1-F
±
vacA-4-F
vacA-4-F
±
IS605-1-F
±
IS605-3-F
±
¯aA-1-F
±
¯aA-3-F
±
16S-1-F
±
16S-5-F
16S-5-F
±
16S-11-F
±
RVM1
±
PMT1
±
HVM4
TaqStart MAb (Clontech), and between 0.2 and 0.5 mM
of each primer. Cycling parameters for all H. pylori
primer pairs and for the mtDNA primer pair RVM1
and RVM2 were pre-incubation for 1 min at 92uC,
followed by 40 cycles of 50 s at 92uC for denaturation,
1 min at 52uC for annealing, 1 min at 72uC for
extension, and then a ®nal incubation step of 72uC
for 6 min. For the mtDNA primer pair PMT1 and
PMT2, the same cycling parameters were used as
above, except annealing was carried out at 55uC.
Between one and three ampli®cation blanks were
performed for each batch of PCRs. Ampli®cation
products were resolved by electrophoresis on 2%
agarose gels. Using series of 1/10 dilutions of template
DNA of known concentration, we determined that
PMT1/2 were PCR-sensitive to between 10 and 100
molecules of the DNA template. Further ampli®cations of total human DNA with the primer pair RVM1
and RVM2 (Table 2) suggest that they have an
equivalent ef®ciency.
In order to be considered a genuine ampli®cation
derived from the archival material, rather than a
contaminant from a modern source, positive PCR
amplicons had to ful®l the following criteria: (i) extract
blanks and PCR negative controls should be devoid of
PCR products of the expected size; (ii) amplicons
should be consistently obtained from repeated ampliCopyright # 2000 John Wiley & Sons, Ltd.
Product size
(base pairs)
163
127
85
101
136
92
94
256
369
93
265
148
133
297
1259
®cations from the same extracted template DNA, and
from further extractions of the same sample.
Results
Histological examination
Histological sections are shown in Figures 1A and 1B.
Good preservation of the histological structure of the
specimens can be seen clearly. In the sections, one can
see the structure of the gastric glands and there
appears to be a dense cellular in®ltrate in the lamina
propria consistent with an in¯ammatory in®ltrate
(Figures 1A and 1B ).
Proportions of H. pylori and mitochondrial DNA
in modern biopsy samples
In order to derive the relative concentration of
ampli®able H. pylori DNA to ampli®able human
mtDNA in a typical infected stomach, DNA extracted
from the eight biopsy samples was serially diluted, tenfold, to a maximum dilution of one in one billion
(1 : 109). The dilutions were then PCR-ampli®ed using
two different human mtDNA primer pairs (PMT1/
PMT2 and RVM1/RVM2) along with H. pylori
primers of equivalent size (16S-5-F/16S-8-R and
vacA-1-F/vacA-3-R, respectively). The results, sumJ Pathol 2000; 192: 554±559.
DNA preservation in long-term ethanol-preserved material
557
Table 2. Results of PCR ampli®cations of 1/10 serial dilutions of stomach biopsy template DNA, using two sets of
human mitochondrial genome and H. pylori primers. The numbers given refer to the log10 of the lowest template
concentration at which a product was visible on a 2% agarose gel. These values were con®rmed by replication
Patient
No.
PMT1/PMT2
16S-5-F/16S-8-R
Absolute
difference
RVM1/RVM2
vacA-1-F/vacA-3-R
Absolute
difference
1
2
3
4
5
6
7
8
x4
x8
x7
x6
x8
x7
x7
x6
x1
x3
x4
x3
x2
x4
x1
x3
3
5
3
3
6
3
6
3
x6
x9
x6
x6
x5
x7
x8
x6
x3
x3
x4
x3
x2
x4
x3
x2
3
6
2
3
3
3
5
4
Meant1SE
x6.6t1.2
x2.6t1.1
4.0t1.3
x6.6t1.2
x3.0t0.7
3.6t1.2
marized in Table 2, demonstrate that there is a
consistent relationship between the minimum ampli®able dilution of template for both sets of primers.
Ampli®able human mtDNA is present at a concentration which is at least 103 times higher than the
concentration of H. pylori. This gives a baseline ®gure
for the amount of human mtDNA which needs to be
recovered (i.e. 104 times the minimum PCR-detectable
amount) before the recovery of any H. pylori is likely,
assuming similar rates of degradation for the DNA of
both species. Unless a positive PCR ampli®cation can
be achieved using DNA extracted from museum
samples diluted by a factor of 1 in 104, then the
ampli®cation of H. pylori DNA would be unlikely.
Recovery of DNA from the museum samples
Each of the six tissue samples was extracted using all
four of the techniques outlined above on three separate
occasions. PCR ampli®cation of these DNA extracts
was then attempted using all 12 H. pylori primer pairs
and the two human mtDNA primer pairs, giving a
total of 1008 ampli®cation reactions. No products were
observed with any of the ampli®cations made using the
H. pylori primers, although PCR ampli®cations were
successful for a positive control of stomach biopsy
DNA run concurrently in all PCRs. `Spiking' of this
A
positive control with the same volume of a museum
extract did not lead to any reduction in ampli®cation
ef®ciency, suggesting that enzymatic inhibition of the
PCR process by unknown substances in the archival
material did not occur.
No positive ampli®cations were made with the
human mtDNA primers PMT1/PMT2, but positive
results were obtained on four occasions with RVM1/
RVM2. However, these positive ampli®cations were
rejected on the basis of the criteria which are outlined
above, due to the presence of positive ampli®cations in
the PCR negative controls on two occasions and
because in all cases the results could not be reliably
repeated.
Discussion
Given that ethanol is widely used in molecular biology
during DNA handling procedures, and that more
recently, ethanol-preserved material is routinely used
as a source of DNA, these results seem surprising.
There are four possible explanations for these results.
Firstly, an insuf®cient number of samples may have
been tested to allow for the vagaries of preservation of
biomolecules in this type of material. We would reject
this suggestion on the basis that spirit storage, unlike
B
Figure 1. (A, B) Light microscope images of the museum material. For details see text
Copyright # 2000 John Wiley & Sons, Ltd.
J Pathol 2000; 192: 554±559.
558
the soil environment, is a fairly consistent and
homogeneous matrix for biomolecular preservation.
Although variation in storage conditions may occur,
we feel that any variation is likely to be of limited
effect. Additionally, the recovery of bacterial pathogen
DNA would seem unlikely even under signi®cantly
better preservation conditions, given that no mtDNA
was recovered from any of the samples and that the
DNA of any bacterial pathogen is likely to be present
at signi®cantly lower concentration than mtDNA.
Secondly, the methodologies used may not have
been suitable for the recovery of DNA from these
specimens. We would refute this suggestion on the
basis that we have employed four different extraction
protocols, all of which have been previously used to
recover DNA from archaeological and archival material; we have employed a range of different PCR
primers for both H. pylori and human mtDNA which
have been optimized to give their maximum sensitivity;
and a range of amplicon sizes have been targeted,
including several below 150 base pairs, in order to
allow for the potentially small size of highly degraded
fragments found in ancient specimens [11].
Thirdly, DNA may be present in the specimens, but
cannot be PCR-ampli®ed due to extensive base
modi®cation. It is known that Taq polymerase does
not recognize some chemical modi®cations of nucleotide bases and chain extension will therefore not occur
[11]. While some proportion of nucleotide bases are
likely to be chemically modi®ed over long time periods
[12], there is no evidence to suggest that this modi®cation is more likely in ethanol preservation than other
environments and we would therefore reject this
suggestion.
Finally, an absence of DNA in the specimens may
have been caused by complete degradation of the
DNA. Of the possible explanations for the lack of
recovery of DNA, we would suggest this to be the most
likely. For modern samples, formalin ®xation is
generally used to generate cross-links between the
proteins before ethanol storage. Ethanol treatment
alone will not ®x the tissue, but will simply lead to
some degree of protein denaturation and tissue
dehydration [13].
For the Hunterian specimens, poor penetration of
the ethanol would leave the interior of the specimen in
a biologically active state. In these conditions, it seems
likely that enzymatic activity could proceed for a
considerable period. This would include both human
and bacterial nucleases, potentially leading to a
considerable loss of DNA. Furthermore, cell wall
disruption due to protein denaturation and ethanolinduced leaching of lipid components would allow
DNA to migrate from the specimen into the surrounding preservative medium. Unfortunately, the ethanol
preservative in these specimens is known to have been
changed on a number of occasions (exact details are
undocumented), as the presence of a large quantity of
lipid suspended in the media makes it dif®cult to see
the specimen and thereby reduces its ef®ciency as a
Copyright # 2000 John Wiley & Sons, Ltd.
I. Barnes et al.
teaching aid. Much of the DNA in these specimens
may therefore have been ¯ushed away.
Although the specimens were originally sealed by the
use of bitumen and gutta percha-soaked pig bladder,
subsequent openings will have allowed oxygen to enter
into the storage jar, leading to a reduction in the pH of
the preservation environment and a corresponding
increase in the rate of DNA strand cleavage due to
acid hydrolysis [14]. Chemically and biologically
mediated degradation of the DNA, coupled with
incomplete ®xation of the component macromolecules,
therefore seems to be the most likely explanation for
the absence of DNA in these samples.
In conclusion, the results presented in this paper are
contrary to the general expectation of good DNA
preservation in long-term ethanol-preserved material.
These results suggest that ethanol may not be a
suitable all-purpose tissue storage medium where the
possibility of DNA recovery in the long term is
considered to be desirable. We would suggest that
museum curators should consider alternative media or
freezing for at least a subsection of samples destined
for possible genetic analysis. Equally, molecular biologists should be aware of the potential for poor nucleic
acid recovery from ethanol-preserved specimens before
embarking on research involving this material. Even
where molecular preservation is good, the DNA of
pathogenic bacteria is likely to constitute only a small
proportion of all DNA present. These results further
demonstrate the need for a good understanding of the
relevant biochemistry and a knowledge of specimen
history before undertaking the recovery of biomolecules from archival material.
Acknowledgements
We would like to thank The Board of Trustees of the Hunterian
Museum for providing access to the collection, as well as
Elizabeth Allen, curator of the Hunterian Museum, and
Martyn Cooke, Department of Pathology, Royal College of
Surgeons, for their assistance. We would also like to thank the
late Sir Reginald Murley, past Chairman of Trustees of the
Hunterian Museum, for his interest and vision in actively
supporting our research. MGT is supported by the Melford
Charitable Trust and a Nuf®eld Foundation Grant (NUFNAL). This work was supported by a grant from the Wellcome
Trust, to whom we give our grateful appreciation.
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