Download Species Determination using Species-discriminating PCR

Ancient Biomolecules, 2002 Vol. 4 (1), pp. 19–23
Species Determination using Species-discriminating PCRRFLP of Ancient DNA from Prehistoric Skeletal Remains
J. BURGERa,b,*, R. SCHOONc, B. ZEIKEa, S. HUMMELa and B. HERRMANNa
a
Historical Anthropology and Human Ecology, University of Goettingen, Goettingen, Germany; bInstitute of Anthropology, University of Mainz,
Mainz, Germany; cInstitute of Archaeology, Kiel, Germany
(Received 12 September 2000; In final form 10 December 2000)
Interspecific sequence polymorphisms in the mitochondrial cytochrome b gene were analyzed by PCR-RFLP to
determine the species origin of Bronze Age animal
skeletal remains. Existing techniques were refined by
targeted primer design focusing on a DNA fragment
shorter than 200 bp, an approach allowing us to identify
up to six animal species at the same time. Possible
contaminants, such as human DNA, were reliably ruled
out. For routine applications in archaeometry, food or
material analyses, PCR-RFLP may thus provide a simple
alternative to sequencing of PCR products, allowing
discrimination between species, even if the template
DNA is degraded or contains traces of DNA from various
species.
costly procedure. We have therefore developed a
timesaving and less expensive PCR-RFLP method (in
the literature the term CAPS [cleaved amplified
polymorphic site] is used synonymously to PCRRFLP), which allows reliable species identification
from prehistoric animal bone samples, even those
containing only degraded DNA.
Keywords: Ancient DNA; Animal bone; CAPS; DNA extraction;
DNA mixtures; PCR-RFLP; Species determination
Figure 1 shows the result of amplification of modern
DNA from seven different species with varying
amounts of target DNA and varying annealing
temperatures, using primers CB7u and CB7l.
Template DNA from cattle, goat, and sheep, whose
sequences have an almost complete match with the
primers, was amplified under all conditions and
produced strong bands. The differing amplification
success for all other species can be explained by the
alignment of the respective sequences with the two
primers, as shown in Fig. 2. Within the 30 pentamer,
the sequence of Cervus elaphus has no mismatch with
CB7u (there are two mismatches outside the 30
pentamer), but there is one mismatch at the third
position of CB7l (three outside). CB7u matches
almost completely but CB7l does not, resulting in
irregularities in the amplification. Although the C.
elaphus specimen produced a band at 608C with
6.8 ng of DNA, there was no band at 608C and 34 ng
of DNA. It is obvious that, under less stringent
INTRODUCTION
Determination of the species origin of (pre)historic
objects is one of the common tasks of ancient DNA
(aDNA) analysis. Usually, short fragments of conserved regions of mitochondrial DNA (mtDNA) are
PCR amplified and then sequenced (e.g. Loreille et al.,
1997). If a more detailed phylogenetic analysis of the
obtained sequence is required or if the specimen
contains extremely degraded DNA, the PCR products must be cloned before sequencing (Handt et al.,
1996). However, for routine applications in archaeozoology or food analysis, this is a laborious and
RESULTS
Primer Characteristics and Species Discrimination
*Corresponding author. Address: Institute of Anthropology, Johannes Gutenberg-University, SB II 02, D-55099 Mainz, Germany. Tel.:
þ49-6131-39-2 4489. Fax: þ49-6131-39-2 5132. E-mail: [email protected]
ISSN 1358-6122 print/ISSN 1607-8411 online q 2002 Taylor & Francis Ltd
DOI: 10.1080/13586120290018491
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J. BURGER et al.
FIGURE 1 Amplification of 195 bp fragments of various species
under varying conditions, using primers CB7u and CB7l. The
annealing temperature and amount of target DNA is shown for
each PCR.
conditions (548C), although there is a mismatch with
CB71 at the third position, C. elaphus template DNA
was always amplified, though less efficiently.
Within the 30 pentamer, the sequence of Capreolus
capreolus shows one mismatch with CB7u at the
second position (two outside) and one mismatch
with CB7l at the third position (one outside). Under
stringent conditions (608C), there was no amplification product visible for the roe deer. At 548C, a band
appeared only with the larger amount of DNA target
(34 ng), but even here, the amplification was not
efficient.
Within the 30 pentamer, the human cytochrome b
sequence has two mismatches with CB7u at positions
one and two (five outside) and one mismatch with
CB7l at the third position (four outside). The 30
mismatch with CB7u and the numerous mismatches
outside the 30 pentamer did not result in a
significantly different amplification behavior compared with the roe deer. Homo sapiens, too, amplified
only under non-stringent conditions and with a high
copy number of targets, but less efficiently than roe
deer DNA.
FIGURE 3 Restriction profiles of the 195 bp cytochrome b PCR
fragments obtained after treatment with Tsp509, showing
interspecific polymorphism between O. aries, B. taurus, C. hircus,
C. capreolus and C. elaphus. The ancient samples DoT 926 and DoT
904 show the fragment lengths of 105 and 77 bp characteristic for
O. aries. DoT 1491 shows characteristic fragment lengths of 114 and
68 bp, as expected for B. taurus. DoT 1584a and DoT 1584b show a
fragment of 182 bp, characteristic for C. hircus. The modern control
of roe deer (C. capreolus, pos.) shows the expected band size of
162 bp, and deer (C. elaphus, pos.) shows the characteristic bands at
54 and 108 bp. ExA and ExB indicate two independent DNA
extractions.
Species Determination by PCR-RFLP
The 195 bp PCR amplified fragment was digested
with the restriction endonuclease Tsp509. Figure 3
shows the RFLP pattern for five of the specimens
from the Lichtenstein Cave compared with various
modern species as controls. Evidently, all species can
be distinguished from one another and all specimens, including those derived from prehistoric
remains, can be attributed to a given species: DoT
926 and DoT 904 show fragment lengths of 105 and
77 bp, characteristic for Ovis aries. (cf. Table II) DoT
1491 shows characteristic fragment lengths of 114 and
68 bp, as expected for Bos taurus. DoT 1584a and DoT
1584b each show a fragment of 182 bp, characteristic
for Capra hircus. All results were obtained from two
independent DNA extractions (ExA, ExB) and
FIGURE 2 Alignment of the CB7u and CB7l primer annealing sequences for seven species (O. aries, sheep; O. aries musimon [subspecies],
mouflon; C. hircus, goat; B. taurus, cattle; C. elaphus, deer; C. capreolus, roe deer, H. sapiens, human).
SPECIES DETERMINATION BY PCR-RFLP
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TABLE I Comparison of the results of morphological and genetic
species determination
Sample
DoT 926
DoT 904
DoT 7
DoT 1491
DoT 1285
DoT 1584a
DoT 1584b
DoT 1584c
DoT 1566
DoT 1468
DoT 1477
Morphological species
Genetic species
Ovis aries
Ovis aries
Ovis aries
Bos taurus
Bos taurus
Capra hircus
Capra hircus
Capra hircus
Capra hircus
Capra hircus or Ovis aries
Capra hircus or Ovis aries
Ovis aries
Ovis aries
Ovis aries
Bos taurus
–*
Capra hircus
Capra hircus
Capra hircus
Capra hircus
Capra hircus
–*
* No DNA available.
confirmed several times by independent PCRs, as well
as by DNA sequencing (results not shown).
In Table I, the morphological species determination of all 11 specimens is compared with the
results of the genetic analysis. Two of the examined
bones did not contain ancient DNA. One previously
unidentified bone (DoT 1468) was attributed to the
species C. hircus. In all other cases, the species
established by morphology was identical to the
genetic species.
Mixtures of Modern DNA
In addition to the ancient DNA results, experiments
with small amounts of mixed modern DNA were
carried out. Figure 4 shows the RFLP pattern of
mixtures with varying proportions of two to three
species, and demonstrates that species can be
identified specifically when template mixtures from
two species are analyzed up to a mixture ratio of 59:1.
Mixtures of three species are detectable as well.
However, when minimizing target DNA, bands tend
to fade away on the agarose gel. In general, shorter
fragments are less intense than longer fragments,
irrespective of the amount of target DNA.
DISCUSSION
Primer Design
Human DNA is the most common source of
contamination when working with ancient DNA.
For this reason, amplification of human DNA should
be avoided by use of maximally discriminating
primers when working with animal material. The
data presented here show that one mismatch within
the 30 pentamer is not sufficient to exclude a
sequence from amplification (see Fig. 1, C. elaphus ).
However, one mismatch in each primer near the 30
end is sufficient to exclude a sequence either under
stringent conditions or with little target in the
reaction (see Fig. 1, C. capreolus ). Many additional
FIGURE 4 Restriction cleavage patterns of DNA mixtures with
varying proportions of target DNA.
mismatches outside the 30 pentamer alter this
situation only minimally (see Fig. 1, H. sapiens ). In
the final analysis, the experiments show that, when
using universal primers, it is necessary to carry out a
sequence alignment with the desired species and to
perform a PCR under varying conditions (temperature, target number). This is the only way to gain a
more detailed knowledge about the annealing
properties and thus amplification characteristics of
primers, necessary when dealing with highly
degraded material in general.
Identifying the Species of Origin
Our data clearly show that it is possible to identify
the species of origin from circa 3000-year-old bone
specimens by using RFLPs of a 195 bp PCR amplified
fragment and at the same time to exclude possible
(human) contamination. If the locus and the
restriction enzyme are chosen properly, this procedure can be applied to other species or populations
as well. Certainly, the principle of PCR-RFLP is not
new and is widely used, for example in the field of
food analysis (e.g. Meyer et al., 1995; Plath et al., 1997;
Carrera et al., 2000). However, fulfilling each of the
four criteria mentioned in Materials and Methods
(see below) (presence of a RFLP specific for the
considered species, fragment length of less than
200 bp, exclusion of contaminating sequences, and a
restriction site within at least one of the primers)
optimizes and refines existing methods, making
them applicable to analysis of truly old and highly
degraded DNA, but at the same time makes the
experimental design slightly more laborious. In
difficult cases, it is possible to combine several
enzymes and thus to accumulate restriction sites
(data not shown). Here, enzymes should be added
one at a time to the PCR product, not simultaneously.
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J. BURGER et al.
Using PCR-RFLP analysis, we were additionally
able to identify DNA from up to three species within
template mixtures. This result is of particular interest
when foodstuffs containing mixtures of components
are to be analyzed. The same is true for the
examination of (pre)historic artifacts, such as glues,
binders, consumer goods, or food incrustations in
containers, which usually consist of several components. Figure 4 shows the possibility of identifying
mixtures, even in proportions of 59:1. Note here that
the detection limit can certainly be improved by
using PAGE instead of an agarose gel. This might
also reduce the observed phenomenon of shading of
shorter bands on agarose gels. Reliable analysis of
DNA mixtures may also be valuable when frequent
contaminants, such as cattle, cannot be excluded by
discriminative primer design. While direct sequencing of coamplified endogenous DNA and contaminant DNA would lead to multiple sequences, PCRRFLP is able to separate the two signals. Thus, PCRRFLP is a rapid alternative method of species
determination from highly degraded DNA, even
when multiple organisms contribute to a given
sample, and can be applied when the exact sequence
information of a specimen is not required.
MATERIALS AND METHODS
Lichtenstein Cave in Osterode, Harz, Germany, is a
Bronze Age archeological site dating to 900– 700 BC.
In addition to numerous human skeletons, the cave
contains bones from some autochthonous animal
species. Earlier analysis showed that the state of
DNA preservation in the bones is excellent, mainly
due to the low temperature prevailing in the cave
since prehistoric times (Burger et al., 1999). Eleven
animal bone samples were chosen for aDNA
analysis. With a high degree of certainty, three of
them were morphologically attributed to the species
Ovis aries (sheep), two to Bos taurus (cattle) and four
to C. hircus (goat). In two cases, the bone fragments of
sheep and goat could not clearly be distinguished.
All samples underwent the standard procedures
for aDNA work as previously described (Burger et al.,
2000).
water (Ampuwa, Fresenius) was added. As the
extraction procedure was automated the volumes
of reagents dispensed may have varied between
runs. Five hundred microliters of Proteinase K
was added and the mixture incubated for 1 h
at 588C with shaking. Three milliliters of
phenol/chloroform/isoamyl alcohol (25:24:1, pH
7.5 – 8.0) was added and the mixture was further
incubated at room temperature for 6 min with
shaking. The phases were allowed to separate by
incubating at room temperature for 8 min without
shaking and the organic phase and interphase, if
present, were discarded. Chloroform (4.5 ml, 100%)
was added to the aqueous phase and the mixture
incubated for 6 min at room temperature while
shaking. The phases were again allowed to separate
by incubating at room temperature for 8 min without
shaking and the organic phase was discarded.
Ninety microliters of sodium acetate (pH 4.5) and
3.2 ml of 100% isopropanol were added followed by
incubation for 2 min with shaking. Five microliters of
Glasmilk (Dianova) was added and the suspension
was shaken for another 10 min. To obtain a pellet, the
solution was filtered through Precipitette filters
(Applied Biosystems) or centrifuged for 3 min at
5000 rpm. The pellet was washed with 80% ethanol
and eluted into 50 ml sterile distilled water
(Ampuwa, Fresenius). Five to ten microliters of
extract were used for PCR amplification or the
extract was stored at 2208C. Glasmilk was not
removed prior to amplification.
Primer Design
Primers were designed to fulfill the following criteria
(see also Fig. 2): primers ought to have a maximum
match with the sequences of the targeted animal
species, in this case sheep, goat, and cattle; primers
ought to have a maximum mismatch with the
corresponding human sequence; primers should
amplify a sequence of less than 200 bp; the sequence
amplified should contain a recognizable RFLP
between sheep, cattle, goat, and possible other
species, here deer and roe deer; and at least one
primer should contain a restriction site serving as a
reaction control.
DNA Extraction
PCR
Tooth or bone samples were roughly ground with a
pestle and mortar, then finely powdered in a Retsch
mill. Bone/tooth powder (0.3 g) was incubated in
1.5 ml of 0.5 M EDTA (pH 8.3) for 20 h while rotating.
The suspension was centrifuged for 4 min at
4000 rpm. The supernatant was transferred to a
fresh tube or to an automated nucleic acids
extraction system (Nucleic Acid Extractor 341A,
Applied Biosystems) and 1.6 ml sterile distilled
A 195 bp segment of the mitochondrial cytochrome b
gene was amplified using the primers CB7u: 50 GCGTACGCAATCTTACGATCAA-30 and CB7l: 50 CTGGCCTCCAATTCATGTGAG-30 . The PCRs were
carried out in 50 ml of 60 mM KCl; 12 mM Tris –HCl;
2.5 mM MgCl2; 150 mM dNTPs; 0.18 mM each primer;
and 2 U AmpliTaq Gold (PE Applied Biosystems).
The temperature profile was 948C for 1 min, 54 or
608C for 1 min, and 728C for 1 min, for 32 cycles.
SPECIES DETERMINATION BY PCR-RFLP
TABLE II Fragment lengths for various species after digestion of
the 195 bp PCR product with restriction enzyme Tsp509
Species
Fragment lengths (bp)
Ovis aries (sheep)
Capra hircus (goat)
Bos taurus (cattle)
Cervus elaphus (deer)
Capreolus capreolus (roe deer)
Homo sapiens (human)
13, 75, 105
13, 182
3, 68, 114
13, 20, 54, 108
13, 20, 162
12, 182
Known amounts of modern DNA (3.4, 6.8, 34, 68 ng)
were amplified at 54 and at 608C. Five microliters of
ancient DNA extracts from the bones was used for
PCR amplification without separation of the nucleic
acids from the Glasmilk. Modern DNA in mixtures
of various proportions (1:1 to 59:1 and 1:1:1)
consisting of two to three species (sheep, goat, cattle)
were also used in PCRs.
Restriction Endonuclease Digestion
Seven microliters of PCR product, 12 U restriction
enzyme Tsp509 (New England Biolabs) and 2 ml
buffer (New England Biolabs) were incubated for 2 h
at 658C. Fragments were separated in 2.5% agarose
gels for 2 h. Expected fragment lengths are shown in
Table II.
23
Acknowledgments
We thank Stephan Flindt for providing samples and
Helen Fletcher and Ekkehard May for assistance. We
thank the German Ministry for Education and
Research for financial support.
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