Spiking of Contemporary Human Template DNA with
Ancient DNA Extracts Induces Mutations Under PCR and Generates
Nonauthentic Mitochondrial Sequences
Carsten M. Pusch* and Lutz Bachmann *Institute of Anthropology and Human Genetics, Division of Molecular Genetics, University of Tübingen, Tübingen, Germany;
Department of Zoology, Natural History Museums and Botanical Garden, University of Oslo, Oslo, Norway
Proof of authenticity is the greatest challenge in palaeogenetic research, and many safeguards have become standard
routine in laboratories specialized on ancient DNA research. Here we describe an as-yet unknown source of artifacts that
will require special attention in the future. We show that ancient DNA extracts on their own can have an inhibitory and
mutagenic effect under PCR. We have spiked PCR reactions including known human test DNA with 14 selected ancient
DNA extracts from human and nonhuman sources. We find that the ancient DNA extracts inhibit the amplification of
large fragments to different degrees, suggesting that the usual control against contaminations, i.e., the absence of long
amplifiable fragments, is not sufficient. But even more important, we find that the extracts induce mutations in
a nonrandom fashion. We have amplified a 148-bp stretch of the mitochondrial HVRI from contemporary human
template DNA in spiked PCR reactions. Subsequent analysis of 547 sequences from cloned amplicons revealed that the
vast majority (76.97%) differed from the correct sequence by single nucleotide substitutions and/or indels. In total, 34
positions of a 103-bp alignment are affected, and most mutations occur repeatedly in independent PCR amplifications.
Several of the induced mutations occur at positions that have previously been detected in studies of ancient hominid
sequences, including the Neandertal sequences. Our data imply that PCR-induced mutations are likely to be an intrinsic
and general problem of PCR amplifications of ancient templates. Therefore, ancient DNA sequences should be
considered with caution, at least as long as the molecular basis for the extract-induced mutations is not understood.
Introduction
The retrieval of nucleotide sequences from ancient
DNA templates requires technical skills and extensive
expertise. The major shortcoming of ancient DNA research
is that authentic sequences can only be determined in an
indirect way. This is particularly true for samples from
extinct taxa, i.e., when no high-quality DNA sample is
available. To overcome the problem, short overlapping
PCR products are cloned, and the authentic sequence is
subsequently deduced as the consensus of many individual
sequences that are derived from several independent
amplifications (Cooper and Poinar, 2000; Hofreiter et al.
2001a,b). Such cloned PCR products are usually heterogeneous due to contamination with contemporary DNA,
PCR artifacts, and assumed postmortem damage of
authentic DNA molecules.
Contamination with modern DNA has plagued
ancient DNA research, and this is a matter of particular
concern when working with hominid samples. It might be
impossible to prove authenticity of the obtained data
simply because of the expected very high level of
sequence similarity between authentic ancient DNA from
a hominid fossil and contaminating contemporary DNA.
Even minute amounts of contaminating DNA might
question the results obtained by amplification of ancient
DNA using the extremely sensitive polymerase chain
reaction (PCR). However, it must be stressed that
contamination is a general problem in ancient DNA
research and affects the analysis of samples from any
taxon (Pusch et al. 2000). Usually, PCR protocols are
Key words: ancient DNA extracts, mitochondrial DNA, mutation,
PCR errors, PCR inhibition
E-mail: bachmann@nhm.uio.no.
Mol. Biol. Evol. 21(5):957–964. 2004
DOI:10.1093/molbev/msh107
Advance Access publication March 10, 2004
optimized using DNA from closely related taxa and
carryover contaminations might occur.
PCR artifacts such as PCR jumping, allelic dropouts,
regular polymerase errors, and assumed postmortem
damage of authentic DNA molecules are also great
obstacles in the retrieval of authentic sequence data from
ancient DNA (Pääbo 1989; Willerslev et al. 1999; Hansen
et al. 2001; Gilbert et al. 2003a). Strand breaks decrease
the average molecular weight of nucleic acids (Pääbo
1989). Oxidative and hydrolytic damage may lead to
incorporation of incorrect nucleotides or block strand
elongation during PCR (Willerslev et al. 1999). The
conversion of adenine to hypoxanthine and cytosine to
uracil, respectively, are considered common forms of
DNA damage (Lindahl 1996; Gilbert et al. 2003a).
Region- and position-specific damage rates add to the
problem (Gilbert et al. 2003a). Unfortunately, DNA
survival and postmortem damage of DNA of a particular
sample are unpredictable and difficult to assess. The
indirect determination of ancient nucleotide sequences is
accompanied by very high standards that must be
safeguarded to ‘‘guarantee’’ authenticity of the results
(summarized by Cooper and Poinar 2000).
Here, we show that one important aspect is neglected in
the discussion on the authenticity of PCR-based results, i.e.,
the mutagenic effect of ancient DNA extracts themselves.
We have noticed such mutagenic effects of ancient DNA
extracts in another study (Pusch et al., in preparation). Here,
we present evidence that artificial (i.e., nonauthentic) HVRI
sequences can be obtained from modern human DNA
templates when spiked with ancient DNA extracts. The
results indicate that the authenticity of PCR-generated
ancient DNA sequences must be reconsidered and illustrate
the need for developing standards for an assessment of the
mutagenic effect of ancient DNA extracts prior to analysis.
Molecular Biology and Evolution vol. 21 no. 5 Ó Society for Molecular Biology and Evolution 2004; all rights reserved.
958 Pusch and Bachmann
Material and Methods
DNA Extractions
The contemporary human genomic DNA was extracted according to the instructions of the QIAamp DNA
Blood Kit (Qiagen, Valencia, Calif.). Ancient DNA
extracts were prepared according to a slightly modified
‘‘silica-based protocol’’ (Höss and Pääbo 1993). Bone
powder was added to 1 ml extraction buffer (5 M
guanidinium thiocyanate, 0.1 M Tris–HCl, 0.02 M EDTA,
1.3% Triton-X-100 pH 7.4) and incubated at 608C for at
least several hours. After centrifugation for 5 min at 5.000
r.p.m., 500 ll of the supernatant was added to 500 ll
extraction buffer and 40 ll silica suspension (Boom et al.
1990). Following incubation at room temperature for 10
min, the silica was washed twice with 5 M guanidinium
thiocyanate, 0.1 M Tris–HCl, pH 7.4 and twice with 70%
ethanol, and the DNA was eluted with water from the airdried pellet.
For some initial control experiments, the ‘‘voltageinduced release method’’ (Bachmann et al. 2000) and the
‘‘mix-and-clean method’’ (Scholz and Pusch 1997) were
used. In short, the voltage-induced release method is based
on an electrophoretic separation using 2% w/v SeparideÔ
(Gibco BRL Life Technologies, Carlsbad, Calif.) gels in 1
X Tris-borate-EDTA (TBE) buffer. A maximum of 25–30
mg ground sample is loaded into a slot along with ;25 ll
of 1.5 X loading buffer (15% glycerol, 3% SDS, 150 mM
dithiothreitol, and 50 mM Tris–HCl, pH 7.5). After gentle
stirring, electrophoresis is carried out at 7 V/cm until the
xylenecyanol/bromophenol blue dye loaded into empty
lanes moved into the gel. The DNA is subsequently
isolated from the gel following Pusch’s method (1997).
For the mix-and-clean method up to 0.2 g, the ground
sample is vigorously vortexed for 1 min at 608C in 500 ll
8% sucrose, 5% Triton-X-100, 10 mM EDTA, and 5 mM
Tris–HCl, pH 8.0 buffer. After adding 500 ll phenol, the
mixture is incubated for 5 h at room temperature on
a shaker. Following an extraction of the aqueous phase
with 1 vol chloroform the DNA is precipitated with 0.7 vol
propan-2-ol and 20 lg glycogen. The air-dried DNA pellet
is resuspended in 8%, 0.1% Triton-X-100, 5 mM EDTA,
and 1.2 M NaCl, pH 8.0. The precipitation and
resuspension of DNA is repeated twice before the DNA
is finally dissolved in TE buffer.
PCR Amplifications for Subsequent Sequencing
Human mitochondrial HVRI (16147–16294) was
amplified for 30 cycles. Each cycle consisted of 30-s
denaturation at 948C, 30-s annealing between 558C and
638C (5 cycles at 638C, 5 cycles at 598C, and 20 cycles at
558C), and 30-s extension at 728C. A final extension step
of 10 min at 728C terminated the program. The PCR mix
(50 ll) consisted of 0.2 mM of each dNTP, 0.2 lM of each
primer, 1 U Ampli-Taq polymerase (Applied Biosystems,
Foster City, Calif.), and 50 ng contemporary human DNA
in 30 mM MgCl2, 500 mM KCl, and 100 mM Tris, pH 8.9.
To test the effect of various ancient DNA extracts on
PCR fidelity, 1 ll water was replaced by 1 ll ancient
DNA extract. The primers used for amplification were:
L16170
59-CCACCTGTAGTACATAAAAACCCA-39
and H16271 59-GTGGGTAGGTTTGTTGGTATCCTA39 for the full-size products, L16170 and H16245
59-TGAGGGGTGGCTTTGGAGTTG-39 for the short left
side (sls) products, and L16182 59-TACATAAAAACCCAATCCACATCAAA-39 and H16271 for the short right
side (srs) products.
PCR Amplifications for Length-Inhibition Analysis
To study the inhibitory effect of ancient DNA extracts
on strand elongation during PCR the human mitochondrial
region encompassing the ATPase 6, cytochrome oxidase
subunit 3, tRNA-Gly, and NADH dehydrogenase subunit 3
(Anderson et al. 1981) was targeted. One reverse primer (H
strand) was used in combination with seven forward
primers (L strand) in order to amplify fragments of
increasing length. The PCR mixture was as the one
described earlier. The PCR protocol for cycling consisted
of 30 cycles with 30 s denaturation at 948C, 30 s annealing
between 558C and 608C (5 cycles at 608C, 5 cycles at 588C,
and 20 cycles at 558C), and 30-s extension at 728C. A final
10-min extension at 728C terminated the program. The
following primers were used for amplification: H10152 59CTATGTAGCCGTTGAGTTGTG-39, L9952 59-GACTATTTCTGTATGTCTCCATC-39 (201 bp), L9747
59-ACTTCGAGTCTCCCTTCACCA-39 (406 bp), L9501 59TGAGCCTTTTACCACTCCAGC-39 (652 bp), L9301 59CCATGTGATTTCACTTCCACTC-39 (852 bp), L9081
59-CCTTCCCTCTACACTTATCATC-39 (1072 bp),
L8901 59-AGCCCACTTCTTACCACAAGG-39 (1252 bp),
and L8650 59-CTAATCACCACCCAACAATGAC-39
(1503 bp).
Plasmid Cloning and DNA Sequencing
The obtained PCR products were cleaned by means
of the Concert Gel Extraction System (Invitrogen) and
subsequently cloned using a TA cloning kit (Invitrogen).
Plasmid DNA from positive colonies was prepared by
means of the Plasmid Mini Kit (Qiagen) and subsequently
sequenced using BigDye chemistry (Applied Biosystems).
Results
The nucleotide sequence of a 148-bp stretch of the
mitochondrial HVRI (16147–16294) of a contemporary
human male was sequenced and found to be identical to
the Cambridge Reference Sequence (CRS) published by
Anderson et al. (1981). The same HVRI stretch was then
repeatedly amplified from the same contemporary DNA
template but spiked with a series of 14 selected ancient
DNA extracts listed in table 1. In most cases, a significant
inhibitory effect of the ancient DNA extract on the Taq
polymerase activity was observed (table 1).
Inhibitory Effect of Ancient DNA Extracts
To quantify the inhibitory effect, 201-, 406-, 652-,
852-p, 1,072-, 1,252-, and 1,503-bp stretches of the human
mitochondrial region encompassing the ATPase 6, cytochrome oxidase subunit 3, tRNA-Gly, and NADH
PCR-Induced Mutations 959
Table 1
Samples Used for Spiking PCR Amplifications of a 148-bp Stretch of the Mitochondrial HVRI (16147–16294) of
a Contemporary Human Template DNA and the Observed Interim Consensus Sequences
Sample, Origin
Nonhominid Samples
Columba spp, Germany
Cervus elaphus, Germany
Equus spp, Spain
Falco spp, Egypt
Equus spp, Germany
Tissue
Age (Years)
Required
Dilution*
Bone
Bone
Bone
Muscle
Tooth
;60
220
2,000
4,300
12,000
0
1:10
1:1,000
0
1:10
Sequoia spp, USA
Wood
25–40 Myr
1:1,000
Alnus glutinosa, Germany
Leafs
,50 Myr
1:10,000
Rodent, Germany
Theropod, USA
Coprolite
Bone
40–55 Myr
Jurassic
0
0
Hominid Samples
Homo sapiens, Germany
H. sapiens, Germany
Bone
Tooth
2,300
5,200
1:100
1:10,000
H. sapiens, Germany
Bone
5,200
1:100
H. erectus, Germany
Australopithecus africanus,
South Africa
Bone
Bone
310,000
2.6–2.8 Myr
0
1:10,000
Observed Interim Consensus Sequences
(Number of Clones)
CRS (9), II (6), III (3), IV (4), IX (3), XI (9)
CRS (9), XI (5), XVIII (9), XIX (5), XXVI (18)
CRS (2), XI (8), XIX (5), XXVI (26)
CRS (21), III (2)
CRS (4),3(8), XV (12), XVI (8), XVII (3),
XXII (4), XXV (3), sls-a (11), sls-b (3), sls-c (4),
sls-d (5), sls-e (4)
CRS (7), IX (10), XXI (2), XXVI (6), XXVII (3),
XXVIII (4)
CRS (7), VII (3), IX (4), XXII (4), XXIV (10),
XXVI (3)
CRS (13), V (8), VI (8), VIII (4)
CRS (12), IX (4), XI (2), XII (2), XIV (9), XX (3),
XXV (6)
CRS (9), XXIII (3), XXVI (17)
CRS (7),3(8), XIX (11), XXIV (5), XXVI (10),
srs-a (16)
I (6), IX (4), XIX (11), XXIII (5), XXVI (6),
srs-b (17)
CRS (16),3(4), XIII (2)
CRS (10), IX (4), XVIII (9), XIX (7), XXIII (10),
XXVII (5)
Hypothetical
Consensus
Sequence**
B
G
J
A (¼CRS)
E
D
H
A (¼CRS)
C
J
H
H
A (¼CRS)
F
* Dilutions in order to restore full polymerase activity were determined by comparing the amount of ethidium–bromide-stained PCR products after gel electrophoresis.
Required dilutions vary between extractions.
** Derived according to the majority rule. The hypothetical consensus sequences are displayed in table 4.
dehydrogenase subunit 3 were targeted after spiking the
contemporary human template DNA with diluted ancient
DNA extracts. It turned out that the inhibitory effect of
ancient DNA extracts does not only affect the yield of
PCR products but, in some instances, also the length of the
fragment that can be amplified. For example, figure 1
shows a dilution of 1:100 of the extract from an
approximately 12,000-year-old tooth of Equus spp is
necessary in order to successfully amplify the 201-bp
fragment from a 50-ng modern human template DNA.
Spiking with a 1:1,000,000 dilution of the Equus spp
extract does still not allow the successful amplification of
an 852-bp fragment. All fragments amplified equally from
the modern DNA template in the control experiments
without spiking (fig. 1).
Mutagenic Effect of Ancient DNA Extracts
After determining the required dilutions of the 14
ancient DNA extracts used for the spiking experiments
(table 1), the obtained PCR products of the targeted 148-bp
stretch of the mitochondrial HVRI (16147–16294) were
cloned and sequenced. A total of 547 HVRI sequences
were subsequently analyzed. Only 23.03% (126 sequences) were identical to CRS and were, therefore, correct.
The vast majority of sequences (76.97%) differed from
CRS by single nucleotide substitutions and/or insertions/
deletions (two examples are shown in fig. 2). Some of
these mutations were clone specific, whereas others were
shared by several or all clones of a particular amplicon,
indicating that mutations were introduced at different
stages of the PCR. After eliminating all clone-specific
alterations, leaving only mutations that were shared by at
least two clones of a particular amplicon, the interim
consensus sequences were collected for each amplicon. It
was not our intention to derive the hypothetical authentic
sequences of the amplicons, but to comprehensively assess
the observed sequence diversity. The deduced 35 interim
consensus sequences were subsequently aligned to CRS
(table 2). To improve the alignment, three gaps had to be
assumed, extending the alignment to 103 positions (PCR
primers excluded). The interim consensus sequences that
are most different from CRS (XXVII and XXVIII) differ
from CRS by as many as 18 positions. Interim consensus
sequence XXVI that represents 86 sequences (15.72%)
still differs from CRS at 16 positions.
The observed sequence alterations are not randomly
distributed, i.e., interim consensus sequences of amplicons
generated through independent amplifications spiked with
different ancient DNA extracts share similar nucleotide
substitutions. In total, 34 positions are affected by 38
mutations of which 25 are transitions, 6 are transversions,
and 7 are insertions/deletions. Twenty of these mutations
occur in less than 10%, 7 in 10%–30%, and 11 in more
than 30% of all clones analyzed (table 3).
Many of the observed mutations affect positions that
are variable in human populations and were previously
detected in either contemporary or prehistoric human
samples. The C!T transition at position 16223 (19
interim consensus sequences in table 2) that was detected
960 Pusch and Bachmann
Myr), and Carcharocles angustidens (tooth) from the USA
(23–28 Myr)) were pretreated with DNaseI prior to the
spiking experiments. A total of 67 sequences were
deduced and the majority of them (71.6%) were altered.
Many mutations were identical to those observed previously and were grouped into 14 different interim
consensus sequences (fig. 3).
Discussion
FIG. 1.—Amplification of seven PCR products (201, 406, 652, 852,
1,072, 1,252, and 1,503 bp) of the human mitochondrial region
encompassing the ATPase 6, cytochrome oxidase subunit 3, tRNA-Gly
and NADH dehydrogenase subunit 3 using modern human DNA that was
spiked with various dilutions of a DNA extract from an approximately
12,000-year-old tooth of Equus spp.
in the Pagglici 12 remains of a 24,000-year-old anatomically modern human from the Pagglici cave in southern
Italy (Caramelli et al. 2003) and the T!C transition at
position 16224 (interim consensus sequences II and XIV
in table 2) that was detected in the Tyrolean iceman (Handt
et al. 1994) are two examples. Interim consensus sequence
XXVI (table 2), which represents 15.7% of all analyzed
sequences, differs from the closest human match in
GenBank by nine substitutions and three gaps but shares
7 out of 11 mutations that are in combination characteristic
for the Neandertal sequence AF011222 (Krings et al.
1997). All 7 mutations belong to those that occur in more
than 30% of the analyzed clones. Furthermore, eight
positions were previously identified as mutation hotspots
that are likely to be affected by postmortem damage
(Gilbert et al. 2003b).
To test if the observed sequence alterations were
caused by the DNA molecules in the ancient DNA
extracts, six ancient DNA extracts (i.e., Falco spp, rodent
coprolite, Sequoia spp, and Homo erectus listed in table 1,
and Araucaria spp (wood) from Madagascar (190–230
We present experimental evidence that ancient DNA
extracts may have a strong inhibitory effect on PCR
amplifications (table 1). In spiking experiments with
ancient DNA extracts of various age and origin, 3 out of
14 extracts required a 1:10,000 dilution in order to restore
full Taq polymerase activity for a targeted 148-bp
amplicon. In our collection of ancient DNA extracts, the
extent of the inhibitory effect varied considerably, e.g.,
five samples showed no inhibitory effect at all. The
inhibitory potential of ancient DNA extracts on PCR
amplifications was unpredictable and could not be related
to the age of a sample, i.e., the inhibitory effect had to be
determined experimentally for each particular extract.
Our experiments show that failure of amplification of
large fragments does not necessarily indicate absence of
high molecular weight template DNA in the PCR mixture
(in our inhibition studies by spiking experiments all PCR
mixtures contained high molecular weight human template
DNA). This argument is frequently applied in the literature
(e.g., Caramelli et al. 2003, Poinar et al. 2003) in order to
support lack of contamination in ancient DNA extracts
according to the ‘‘appropriate molecular behavior’’
criterion of Cooper and Poinar (2000), i.e., large 500–
1,000 base pair products are unusual.
We also present experimental evidence that ancient
DNA extracts may induce mutations under PCR amplifications. The strength of the ‘‘spiking contemporary human
DNA templates with ancient DNA extracts approach’’ is
that properties of ancient DNA extracts can be studied in
a modern DNA experimental setup. Doing so, the
shortcomings of ancient DNA experiments can be avoided.
The nucleotide sequence of the modern PCR template used
in our experiments can be determined unambiguously, i.e.,
it is identical to the human mtDNA determined by
Anderson et al. (1981) when amplified without spiking.
The approach allows us to conclude that the frequently
observed sequence alterations in sequences from spiked
amplifications must have occurred under PCR.
Analysis of 547 sequences from cloned amplicons of
a 148-bp stretch (16147–16294) of the mitochondrial
HVRI indicates that extract-induced mutations do not
occur at random. In total, 34 positions of a 103-bp
alignment (PCR primers excluded) are affected, the
majority of those repeatedly in independent PCR amplifications. However, this does not mean that extractinduced mutations can predictably be reproduced. It is
more that particular positions of the target sequence have
a high likelihood of being altered.
It is certainly difficult to identify the cause of the
observed sequence heterogeneity. Intuitively, one might
suspect human DNA in the ancient DNA extracts used for
PCR-Induced Mutations 961
FIG. 2.—Examples of altered human sequences of cloned PCR products of a 148-bp stretch of mitochondrial HVRI (16147–16294) generated in
spiking experiments with ancient DNA extracts. The upper sequence is altered at 6 positions and the lower one at 18 positions. The correct nucleotides
of the human PCR template (identical to CRS) are indicated at the respective positions under the sequence.
spiking (note: in this context it is irrelevant whether such
human DNA is authentic for a particular find or results
from contamination). Such DNA might serve as a proper
template and differ in sequence from targeted human DNA
of a known sequence. Alternatively, such DNA might be
degenerated and, therefore, not serve as proper template
but cause sequence variation in the obtained amplicons
through the process of PCR jumping (Pääbo 1989). We
believe that this line of argument has to be rejected for the
following reasons. First, the majority of DNA extracts
used for spiking had to be diluted prior to PCR
amplification (table 1). Only 1 ll diluted extract was then
added to 50 ng modern human template DNA; i.e., the
number of modern human template DNA molecules in the
Table 2
Observed Interim Consensus Sequences of Cloned PCR Products of a 148-bp Stretch of Mitochondrial HVRI
(16147–16294) of a Contemporary Human Amplified Under the Presence of Various Ancient DNA extracts (table 1)
962 Pusch and Bachmann
Table 3
Frequency of Mutations Listed in Table 2 Among Cloned PCR Products of a 148-bp Stretch of Mitochondrial HVRI
(16147–16294) of Spiked Contemporary Human DNA
mtDNA
Position
16172a
16182a,b
16183a,c
16188
16189a,c
16191
16192a
16192a
16193
16193.1
16196
16211
16213
16216
16216
16217
16221
16222
16222.1c
Observed
Mutation
T!C
A!C
A!C
C!T
T!C
C!gap
C!gap
C!T
C!gap
gap!C
G!C
C!T
G!A
A!T
A!G
T!C
C!T
C!T
gap!C
Number of
Clones Affected
Percentage of
Clones Affected
mtDNA
Position
Observed
Mutation
Number of
Clones Affected
Percentage of
Clones Affected
3
2
340
131
373
5
13
6
59
142
4
17
302
302
4
302
3
30
281
0.6%
0.4%
62.1%
23.9%
68.2%
0.9%
2.4%
1.1%
10.8%
25.9%
0.7%
3.1%
55.2%
55.2%
0.7%
55.2%
0.5%
5.5%
51.4%
16223a
16224
16230a,c
16234a,c
16236
16244c
16244
16245
16247
16253
16255
16256c
16258d
16260
16261
16261
16262c
16263.1c
16269
C!T
C!T
A!G
C!T
C!T
G!A
G!C
C!T
A!G
A!G
G!A
C!A
A!G
C!T
C!T
C!gap
C!T
gap!A
A!G
317
15
225
225
3
233
3
103
114
94
85
4
254
3
3
8
12
12
4
57.9%
2.7%
41.1%
41.1%
0.5%
42.6%
0.5%
19.8%
21.9%
18.1%
16.3%
0.8%
48.8%
0.6%
0.6%
1.5%
2.3%
2.3%
0.8%
a
Mutation hotspots according to Gilbert et al. (2003b).
Mutation also found in Neandertal sequences AF254446, AF282971, AY149291.
c
Mutation also found in Neandertal sequences AF011222, AF254446, AF282971, AY149291.
d
Mutation also found in Neandertal sequences AF011222, AF282971.
b
PCR mixture therefore exceeded by far putative human
DNA molecules from the spiking extracts by several
orders of magnitudes. Second, human DNA from other
sources than the targeted modern human DNA can hardly
explain why 11 mutations were found in more than 30% of
the analyzed clones (table 3). Third, human DNA from
other sources than the targeted modern human DNA can
hardly explain that a combination of, e.g., 16 mutations
(interim consensus sequence XXVI in table 2) occurs in 86
clones, i.e., 15.7% of all sequences, generated in seven
different spiking experiments. Fourth, human DNA from
other sources than the targeted modern human DNA can
hardly explain why many interim consensus sequences
occur that do not match any HVRI sequences deposited in
sequence databases. Finally, spiking experiments using six
ancient DNA extracts pretreated with DNaseI (67
sequences) also yielded mutated sequences with similar
nucleotide alterations. Accordingly, DNA could be
excluded as the mutagenic agent.
We speculate that coextracted chemicals other than
DNA might induce the observed sequence alterations.
Such coextracted mutagenic substances may accumulate in
the samples during diagenesis. Although diagenetic processes are barely understood, we know that specimens
accumulate postmortem multivalent metal ions. For
example, concentrations of manganese, barium, strontium,
iron, and uranium can be enriched up to 5,000 times
compared with contemporary samples (Bischoff 1981,
Velasco-Vazquez et al. 1997, Kohn et al. 1999). In
particular, manganese is known for its mutagenic effect,
and molecular biologists use this effect when using
mutagenesis kits. In preliminary studies, we noticed
substantially elevated manganese concentrations for the
samples used here (data not shown). In contrast,
manganese was not detected in 87 modern controls. If
coextracted manganese is the reason for the observed
mutations, one would, at least at first glance, expect to see
a predictive relationship between manganese concentration
FIG. 3.—Observed consensus sequences of cloned PCR products of a 148-bp stretch of mitochondrial HVRI (16147–16294) of a contemporary
human amplified under the presence of six ancient DNA extracts digested with DNaseI prior to amplification. Dashes indicate gaps introduced in order
to improve the alignment.
PCR-Induced Mutations 963
Table 4
Deduced Hypothetical Consensus Sequences of Cloned PCR Products of a 148-bp Stretch of Mitochondrial HVRI
(16147–16294) of a Contemporary Human Amplified Under the Presence of Various Ancient DNA Extracts (table 1)
(CRS is the correct sequence)
and reliability of sequences. However, there is no such
relationship, and, even worse, there is no reason to expect
such a simple correlation. This is because the chemistry of
manganese is very complex. The element occurs with
a variety of valence stages that differ in their biological
activity. However, manganese measurements refer to the
total amount of manganese and cannot differentiate
valence stages. In addition, the mutagenic effect of
manganese also depends on the concentration of other
ions. Svetlov and Cooper (1998) and Kunichika et al.
(2002), for example, noticed that the mutagenic effect of
manganese changed with changing concentration of
magnesium. Other reasons may be added (see, e.g.,
Fletcher et al. 1994, Pelletier et al. 1996). Nevertheless,
Pusch et al. (in preparation) showed in other experiments
that a G!A transition occurs frequently at cDNA position
1,138 of the human FGFR3 gene in PCR amplifications
under the presence of 0.25 mM MnCl2. The same
diagnostic mutation also occurs when contemporary
human template DNA is spiked with an ancient DNA
extract from a Pleistocene Ursus spelaeus bone. Future
research will have to address this topic.
If coextracted chemicals such as multivalent metal
ions were the reason for the observed sequence alterations,
they might be removable by extraction methods, which are
based on different extraction principles than the default
silica-based protocol. In preliminary experiments, we also
tested two other extraction methods, i.e., the ‘‘voltageinduced release method’’ (Bachmann et al. 2000) and the
‘‘mix-and-clean method’’ (Scholz and Pusch 1997).
However, DNA extracted according to these alternative
methods also yielded altered sequences in spiking experiments (data not shown). Future studies will have to address
why hypothetical coextracted mutagenic substances cannot
be removed by currently applied methods.
The data presented here have implications for ancient
DNA research and may challenge the authenticity of
a number of PCR-generated sequences from ancient
samples. For some reasons it is, however, impossible to
assess how frequently sequence alterations induced by
ancient DNA extracts may have affected published
sequences. Many papers report only the deduced consensus sequences and neither the sequences of the individual
clones nor information on the observed sequence variation.
Furthermore, as can be seen from table 1, different ancient
DNA extracts induce sequence alterations to a different
extent. For some extracts, the majority of sequences were
identical to CRS and the obtained mutations occurred at
very low frequencies. In those cases, extract-induced
sequence alterations would not make it into a consensus
sequence when applying the majority rule (hypothetical
consensus sequence A in tables 1 and 4). In other
instances, the majority of sequences were altered and the
deduced consensus sequences wrong (hypothetical consensus sequences B–J in tables 1 and 4). It might also be
a matter of chance which mutations are considered to be
authentic, because usually the clones used for analysis are
selected at random.
Intuitively, one might feel that applying the rule to
clone and sequence short overlapping PCR products from
several independent amplifications in order to deduce
a consensus sequence may prevent from pitfalls. According
to our data, this assumption is not justified. In the data set
presented here 11 alterations occurred in more than 30% of
the analyzed clones and some of those would make it into
a consensus sequence in an ancient DNA approach when
following the guidelines for ancient DNA. Independent
replication of the experiments in other laboratories to verify
deduced consensus sequences of ancient samples are of no
help since such mutations can, as shown, repeatedly occur
in independent amplifications. As stated earlier, particular
positions have a high likelihood of being altered.
Our data imply that an unknown number of published
ancient DNA sequences have to be considered with
caution. There is an urgent need to identify the causes
for the observed mutations in the spiking experiments and
to develop standards for an assessment of the mutagenic
effect of ancient DNA extracts as a mandatory criterion for
authenticity. Although to a different extent in different
studies, it is likely that extract-induced mutations are an
intrinsic and a general problem of PCR amplifications of
ancient templates.
Acknowledgments
We are grateful to M. Broghammer for excellent
technical assistance. This work was supported in part by
964 Pusch and Bachmann
grants from the Research Council of Norway (National
Centre for Biosystematics, 146515/420), the German
Federal Ministry of Education, Science, Research and
Technology (Fö. 01KS9602), and the Interdisciplinary
Center of Clinical Research (IZKF-Q3) Tübingen.
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Diethard Tautz, Associate Editor
Accepted February 7, 2004