AMERICAN JOURNAL OF PHYSICAL ANTHROPOLOGY 142:321–327 (2010)
Technical Note: PCR Analysis of Minimum Target
Amount of Ancient DNA
Daniela Woide,1 Albert Zink,2 and Stefan Thalhammer1*
1
Helmholtz Zentrum Munich, Institute of Radiation Protection, 85764 Neuherberg, Germany
2
EURAC Research, Institute for Mummies and the Iceman, 39100 Bolzano, Italy
KEY WORDS ancient DNA; DNA extraction; laser microdissection; LV-PCR; SPATS
ABSTRACT The study of ancient DNA plays an
important role in archaeological and palaeontological
research as well as in pathology and forensics. Here,
we present a new tool for ancient DNA analysis, which
overcomes contamination problems, DNA degradation,
and the negative effects of PCR inhibitors while reduc-
ing the amount of starting target material in the pico-
gram range. Ancient bone samples from four Egyptian
mummies were examined by combining laser microdis-
section, conventional DNA extraction, and low-volume
PCR. Initially, several bone particles (osteons) in the
micrometer range were extracted by laser microdissec-
Molecular archaeology, which was first described in
the early 1980s, is a particularly promising emerging
archaeometric discipline for dealing with molecular bio-
logical analyses of human remains (Pääbo, 1985). For
instance, sex determination of human findings can easily
be defined using small amounts of remains such as
bones and teeth via i.e., polymerase chain reaction
(PCR) (Hummel and Herrmann, 1991; Faerman et al.,
1995, 1997). For a significant genetic analysis usually
1–2 g of bone or tooth material are adequate. This mate-
rial is purified and pulverized, and ancient DNA (aDNA)
is then chemically extracted. Here, the coextraction of
humic acids, these are organic compounds originating
from the soil, being present in soil buried bones and
teeth, and having the same chemical characteristics as
DNA can cause a problem inhibiting enzymatic reactions
like PCR (Goodyear et al., 1994). Various ancient DNA
extraction methods are currently in use, which rely on
different principles like spin column, alcohol precipita-
tion, or silica binding. All of these methods aim to maxi-
mize DNA yields, while minimizing the coextraction of
PCR inhibitors. No single method has been shown to
outbalance the others therefore no standardized proce-
dure exists so far.
Microdissection techniques performed on tissue struc-
tures from histological preparations enable the precise
manipulation and isolation of genetic material in the
range of several micrometers (Greulich and Leitz, 1994;
Thalhammer et al., 2004). These techniques can be
combined with subsequent analysis of DNA in the
microdissectants. Several dissection techniques such as
extraction via glass needle (Weimer et al., 2001), laser
capture (Simone et al., 1998), laser pressure catapulting
(Thalhammer et al., 2003), laser impulse (Kirschner
and Plaschke-Schluetter, 2007), or via gravity effects
(Di Martino et al., 2004) are commonly in use. Recently,
a novel technique based on the combination of laser
C 2010
V WILEY-LISS, INC.
tion. Subsequently, ancient DNA amplification was
performed to verify our extraction method. Ameloge-
nin and b-actin gene specific fragments were ampli-
fied via low-volume PCR in a total reaction volume of
1 ll. Results of microdissected mummy DNA samples
were compared to mummy DNA, which was extracted
using a standard DNA extraction method based on
pulverization of bone material. Our results highlight
the combination of laser microdissection and low-vol-
ume PCR as a promising new technique in ancient
DNA analysis. Am J Phys Anthropol 142:321–327,
2010. V 2010 Wiley-Liss, Inc.
C
microdissection and low-pressure technology was intro-
duced by Woide et al. (2009). This technique enables
gently controlled extraction and horizontal transfer of a
smallest amount of isolated material. Based on this new
approach, we present the preparation and analysis of
very small ancient paraffine-embedded bone tissue sam-
ples employing laser microdissection and subsequent
conventional DNA extraction. The advantages of this
technique include a) minimization of contamination dur-
ing handling of specimens, b) sample extraction exclu-
sively from internal bone parts with a minimization of
degradation effects and coextraction of inhibiting sub-
stances, and c) a reduction in the amount of starting ma-
terial down to single osteon islets allowing maximum
preservation of ancient material.
The reduction in target material requires an addi-
tional enhancement of analysis sensitivity, which can be
achieved by reducing the reaction volume of PCR reac-
tions (Gaines et al., 2002; Kricka and Wilding, 2003;
Additional Supporting Information may be found in the online
version of this article.
Grant sponsor: Deutsche Forschungsgemeinschaft (DFG); Grant
number: SFB 486; Grant sponsor: Excellence Cluster ‘‘Nanosystems
Initiative Munich’’ (NIM).
*Correspondence to: Dr. Stefan Thalhammer, Helmholtz Zentrum
Munich, Institute of Radiation Protection, Ingolstädter Landstrasse
1, 85764 Neuherberg, Germany.
E-mail: stefan.thalhammer@helmholtz-muenchen.de
Received 3 September 2009; accepted 10 December 2009
DOI 10.1002/ajpa.21268
Published online 12 March 2010 in Wiley InterScience
(www.interscience.wiley.com).
322 D. WOIDE ET AL.
Leclair et al., 2003; Schmidt et al., 2006). It is believed
that this enhanced sensitivity may result from the better
contact between primer or polymerase molecules and the
DNA because the overall amount of DNA is less diluted
than in a higher volume (Proff et al., 2006; Schmidt et
al., 2006). Thus, using low-volume PCR (LV-PCR) tech-
nology in combination with the laser-based DNA extrac-
tion method, offers the opportunity to further reduce the
amount of DNA starting material needed while retaining
sensitive genetic analysis.
MATERIALS AND METHODS
Mummy bone tissue material
Bone samples from four ancient Egyptian mummies of
the ‘‘Mummy Collection of Munich’’ were examined and
derived from an independent laboratory after anthropo-
logical and archaeological study. The mummies originally
originated from the so-called ‘‘Tombs of the Nobles,’’ the
huge necropolis of Thebes-West that had been mainly
built during the New Kingdom (NK) (c. 1550–1070 BC)
and which was used during the Third Intermediate
Period (TIP) until the Late Period (LP) (ca. 500 BC).
According to the collection records, the long bones origi-
nated from mummies found in four different tombs of
the necropolis of Thebes-West. The four specimens were
all long bone samples and comprised of a fibula (Mummy
1), a distal part of a tibia (Mummy 2), a distal part of a
humerus (Mummy 3) and a tibia diaphysis (Mummy 4).
Initial sample preparation
Bones were first cleaned with sodium hypochlorite
(0.5% solution) and subsequently the outer surface was
removed mechanically with appropriate sterile tools.
DNA extraction of pulverized mummy sample
material
Bone particles of the four bone samples were pulver-
ized using a mixer mill (MM200, Retsch, Haan,
Germany). Pulverized bone specimens were subjected to
conventional pathological DNA extraction as described
previously (Zink et al., 2003). Amounts of 50 pg respec-
tively 100 pg of extracted mummy DNA material were
used for PCR experiments.
DNA extraction of paraffin-embedded laser
microdissected mummy sample material
A tissue block from each of the four bone samples was
removed and subsequently rehydrated as previously
described in detail (Parsche and Nerlich, 1997). To avoid
external contamination, several tissue samples were
taken exclusively from the inner parts of the bones in a
nested way using sterile blades (Fig. 1A). For paraffin-
embedding procedures, decalcification of bone particles
was achieved by 0.1 M EDTA-solution, pH 7.4, followed
by postfixation with 4% buffered formaldehyde. Sections
of 3–5 lm in size were placed on a 2-lm thin polyethyl-
ene-naphtalate (PEN) laser supporting membrane,
mounted on object slides (MicroDissect GmbH, Herborn,
Germany). Deparaffinization was achieved by xylol incu-
bation for 30 min and subsequent decreasing alcohol
series (100% EtOH for 5 min, 90% EtOH for 2 min
American Journal of Physical Anthropology
and 70% EtOH for 2 min) at room temperature. Sample
material, fixed on ultra thin 2-lm PEN-carrier
membrane-coated slides, was then used for laser micro-
dissection.
Laser microdissection was performed as described pre-
viously (Thalhammer et al., 2004; Woide et al., 2009) by
using a modified UVA-Laser System (Axio Observer.Z1,
Carl Zeiss GmbH, Jena, Germany). Here, single osteon
areas were isolated in the range of 350 lm in diameter.
For extraction and transfer of microdissected material
we used the recently developed low-pressure sample
transfer system SPATS (Woide et al., 2009). Osteon bone
particles were released directly into a 0.5-ml reaction
tube containing 112.5 ll of total lysis solution for subse-
quent DNA extraction using the First-DNA All-tissue
DNA kit (Gen-ial, Troisdorf, Germany) according to the
manufacturer’s protocol. Amounts of 60 pg of the
extracted mummy DNA material were used for PCR
experiments.
Amplification of human amelogenin and b-actin
gene fragments via LV-PCR
Extracted mummy DNA samples, microdissected as
well as conventionally extracted ones, were analyzed via
low-volume polymerase chain reaction (LV-PCR) using
chemically structured object slides (AmpliGridTM, Beck-
man Coulter Advalytix, Munich, Germany) and a corre-
sponding thermocycler (AmpliSpeed, Beckman Coulter
Advalytix, Munich, Germany). After drying up of 1 ll of
DNA sample solution at 308C on top of the reaction sites
of the slide, 1 ll PCR reaction mix containing 13 Qiagen
master mix plus 13 Qiagen Q-Solution (QIAGEN1 Fast
Cycling PCR kit, Qiagen GmbH, Hilden, Germany), and
1 lM of each primer (Metabion GmbH, Martinsried, Ger-
many) was added and immediately covered with 5 ll of
sealing solution (Beckman Coulter Advalytix, Munich,
Germany) to prevent evaporation and external contami-
nation. Amplification of a 297 bp segment of the human
multicopy gene b-actin was performed with primers
b-actin up (50 -TCA-CCC-ACA-CTG-TGC-CCC-ATC-TAC-
GA-30 ) and b-actin down (50 -CAG-CGG-AAC-CGC-
TCA-TTG-CCA-ATG-G-30 ) (Taylor et al., 1997). For mo-
lecular sex determination a segment of the amelogenin
gene was amplified. Amplifications of a 106 bp segment
from the X-chromosome and a 112 bp fragment of
the Y-chromosome were performed using primers Amel1
(50 -CCC-TGG-GCT-CTG-TAA-AGA-ATA-GTG-30 ) and
Amel2 (50 -ATC-AGA-GCT-TAA-ACT-GGG-AAG-CTG-30 )
(Shadrach et al., 2004). b-actin and amelogenin specific
PCR conditions were 5 min initial denaturation at 958C,
40 cycles of 948C for 30 s, 608C for 30 s, and 728C for
30 s, and final extension at 728C for 1 min. PCR prod-
ucts were analyzed on polyacrylamide gels (CleanGel
HyRes, ETC GmbH, Kirchentellinsfurt, Germany) after
DNA silver staining (DNA silver staining kit, GE
Healthcare, Uppsala, Sweden). Blank, negative, and pos-
itive controls were included in every reaction batch and
all experiments were done in multiplicates.
Sequencing of PCR products
Sequencing was performed on PCR products of sample
Mummy 4, exemplary for all of four mummy samples.
Amplification of b-actin and amelogenin fragments was
performed in 0.2-ml PCR-tubes (Eppendorf, Hamburg,
Germany) on a normal PCR thermocycler (Cyclone 25,
 PCR ANALYSIS OF MINIMUM TARGET AMOUNT OF ADNA 323

Fig. 1. Preparation and collection of smallestamounts of target material from ancient bone tissue. (A) For sample extraction,
the outer surface was removed from bone material and tiny tissue blocks were prepared out of the inner bone tissue parts (see
arrow). (B) Isolation of an osteon bone particle, diameter 300 lm, via laser microdissection. (C) Release of the low-pressure trans-
ferred particle into a 0.2-ll droplet of lysis solution; the inset to figure C shows the isolated bone particle adsorbed to the adsorption
head of the novel transfer system SPATS. The isolated particle can be tracked along the entire isolation and transfer process (see
arrows). [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]

PeqLab Biotechnologie GmbH, Erlangen, Germany) DNA extraction

using the QIAGEN1 Fast Cycling PCR kit (Qiagen
GmbH, Hilden, Germany). Twenty microliter total PCR
reaction mix contained 60 pg mummy DNA, 13 Qiagen
master mix, 13 Qiagen Q-Solution, and 1 lM of each
primer Amel1 and Amel2, or primer b-actin up and b-
actin down, respectively (for primer sequences and PCR
protocol see previous paragraph). For purification and
sequencing analysis, PCR products were sent to
GENEART AG sequencing service (Regensburg, Ger-
many).
General precautions to prevent contamination
During our experiments, numerous precautions were
taken to minimize the risk of contamination, which were
summarized by Pääbo et al. (2004). To further exclude
possible cross-contaminations, DNA typing reactions
were performed by using selected STR markers
D7S1824, D9S302, D10S2325, and also the AmpF/STR1
SEfilerTM PCR amplification kit (Applied Biosystems
GmbH, Darmstadt, Germany). Typing of extracted
mummy material as well as genomic material of involved
scientists, archaeologist/excavator, technical assistance
staff, laboratory personnel, and all people who had been
knowingly in contact with any kind of mummy material
(including bone particles, paraffin-embedded tissue
blocks, tissue slides, and mummy DNA extracts) and
also with laboratory equipment (e.g., laboratory working
places and laboratory working tools like LMD micro-
scope, DNA extraction accessory, and the PCR thermocy-
clers) was accomplished (available Supporting Informa-
tion). Extraction blanks were always included in every
procedure and PCR blanks and negative controls, con-
taining no DNA template, were performed in each PCR
reaction.
RESULTS
DNA of four mummy samples was extracted in two
independent approaches, where a novel laser microdis-
section-based method was highlighted compared to an
conventionally used extraction technique. Main results
concerning target DNA amount as well as PCR amplifi-
cation are summarized in Table 1.
In our laboratories, strict precautions were taken dur-
ing all molecular genetic analysis minimizing the hazard
of amplifying contaminating modern DNA in ancient
specimens, and controls performed at all steps were con-
sistently negative. Based on the results of DNA typing
analysis (available as Supporting Information), we were
able to widely exclude contamination and cross-contami-
nation of the ancient bone samples.
Ancient DNA could successfully be extracted out of
pulverized mummy bone material via DNA precipitation
using guanidine isothiocyanate solution and diatoma-
ceous earth (Zink et al., 2003). DNA extraction using 1 g
of pulverized bone tissue material of each mummy sam-
ple revealed DNA amounts of 2.1–10.0 ng ll21.
Nested isolation of paraffin-embedded blocks, prepared
on PEN-membrane-coated object slides, was performed
and several single 150–350 lm osteon islets could suc-
cessfully be isolated out of mummy bone tissue material
via laser microdissection (Fig. 1B,C). Osteons, about
50–500 lm in diameter, are composed of concentric
layers of mineralized matrix surrounding a Haversian
canal, which is 20–150 lm in diameter. Here, sample tis-
sue material was taken exclusively from the inner parts
of the bones (Fig. 1A) for subsequent conventional DNA
extraction. Numerous blank extraction controls (without
sample material) were processed in parallel and were
consistently negative. Because of the fact that osteons
consist to the greatest extent of concentric layers of min-
eralized matrix, and that the Haversian canal enclosed
forms just a minor part, the DNA amount extracted
from microdissected mummy osteon material was

60 pg of DNA present in 1 ll of extracted mummy sam-
ple material.
Amplification of b-actin and amelogenin gene
fragments using low-volume PCR
DNA amplification experiments were performed using
low-volume PCR in 1-ll total reaction volume covered
with 5 ll of mineral oil. The reliability of this method
was 90–95%, apart from one to two reaction batches out
of about 20. These had been accidentally destroyed by
bubbling reaction mixes, which might be due to evapo-
rating gas bubbles present in the aqueous master mix.
This was either caused by too extensive mixing of
reagents or by inferior surface chemistry of the slides.

American Journal of Physical Anthropology

324 D. WOIDE ET AL.
TABLE 1. Summary of the compared DNA extraction methods and results of PCR amplification
Sample Preparation Analysis
Fragment amplification
Starting amount of DNA concentration via low-volume
sample material after extraction PCR on chemically
for DNA extraction procedures structured object slides
Laser-based Powder-based
extraction method extraction method
(
60 pg of target (50–100 pg of target
DNA present in DNA present in
1-ll total 1-ll total
Laser-based Powder-based Laser-based Powder-based reaction volume) reaction volume)
extraction extraction extraction extraction Amelogenin b-actin Amelogenin b-actin
method method method method 106/112 bp 297 bp 106/112 bp 297 bp
Mummy1 Several 150–350 lm 1 g of bone
60 pg ll21 2.1–10.0 ng ll21 female 1 – 6(2:4)
Mummy2 osteon islets powder female 1 female –
Mummy3 male 1 – –
Mummy4 male 1 male 2(1:5)

The preparation part shows a comparison of the standard extraction method and our novel microdissection-based one in relation to
the extraction amounts of the ancient material. The analysis part presents a summary of the obtained aDNA PCR results according
to the applied extraction method.

Fig. 2. Polyacrylamid gel electrophoresis of isolated and via LV-PCR amplified mummy DNA. Four different mummy samples
were examined. PCR was performed on fragments of the human gene b-actin (297 bp) and the sex specific amelogenin gene (female
106 bp, male 106/112 bp). (A) Principle of VRC PCR: 1-ll reaction mix (1) is placed on a chemically modified substrate (3) and cov-
ered by 5 ll of mineral oil (2) to prevent evaporation; a hydrophilic reaction center (4, 40 lm in diameter) is enclosed by a hydropho-
bic ring, holding the reaction mix in place (5); the surrounding hydrophobic area holds the cover oil in place (6). (B–E): LV-PCR
results of mummy samples Mummy 2 (B), Mummy 4 (C), Mummy 3 (D), and Mummy 1 (E). M: 100 bp molecular length standard
(Peqlab). Amelogenin PCR (LMD DNA extraction method): (B) Lane 1,
60-pg female Mummy 2 DNA; (C) Lane 8,
60-pg male
Mummy 4 DNA; (D) Lane 2,
60 pg male Mummy 3 DNA; (E) Lane 5,
60-pg female Mummy 1 DNA. Amelogenin PCR (conven-
tional DNA extraction method): (B) Lane 3, 50-pg female Mummy 2 DNA; (C) Lane 7, 100-pg male Mummy 4 DNA; (D) Lane 1,
100-pg male Mummy 3 DNA; (E) Lane 4, 100-pg female Mummy 1 DNA. Amelogenin PCR (100-pg male and female human refer-
ence DNA, positive control): (B) Lane 516; (C) Lane 213; (D) Lane 718; (E) Lane 112. Amelogenin PCR (PEN carrier membrane,
lysis buffer, PCR master mix and H2O control as negative controls): (B) Lanes 2, 7, 4, 8; (C) Lanes 6, 5, 4, 1; (D) Lanes 3, 4, 5, 6;
(E) Lanes 6, 8, 7, 3. b-actin PCR: (B–E) Lane 9,
60-pg mummy DNA (LMD DNA extraction method). b-actin PCR: (B–E) Lane 10,
negative control (PCR master mix). [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.
com.]

Amplification was performed on 106/112 bp fragments of
the amelogenin gene for gender determination, as well
as on a larger fragment of the human b-actin gene com-
prising 297 bp. DNA was extracted from each of the four
mummy samples at least twice in separate preparations.
Each mummy DNA extract was tested at minimum of
five to eight times via PCR for amplifiable b-actin and
amelogenin gene fragments. Amplified gene fragments
were rated as reproducible and authentic only after a)
five PCR reactions showed consistent fragment determi-
nation, b) this result could be reproduced in another
extract from the same mummy sample and c) all controls
were negative.
LV-PCR amplification of pulverized samples. When
amplifying small gene fragments of 106/112 bp of the
amelogenin gene, two out of the four mummy samples
revealed a successful amplification of sex specific frag-
ments (Table 1). While samples Mummy 3 and Mummy
1 showed no positive PCR products (Fig. 2D,E), sample
Mummy 2 revealed female-specific 106-bp fragments
(Fig. 2B) and sample Mummy 4 revealed male-specific
fragments of 106-bp respectively 112 bp in size (Fig. 2C).
Amplification of a 297-bp fragment of the b-actin gene
was less successful. Despite repeated efforts, samples
Mummy 1 and Mummy 4 revealed inconstant positive
amplification products, while Mummy 2 and Mummy
3 revealed no positive amplification product at all (Table
1). Sample Mummy 1 showed an amplification rate of
about 2:4, revealing a positive b-actin PCR product in
two out of six reactions. The amplification rate of sample
Mummy 4 was about 1:5, having just one positive PCR
product in six reaction batches. All extraction and PCR
negative controls including lysis mix, PEN supporting
membrane, H2O control, and the PCR master mix con-
taining no DNA, were consistently negative (Fig. 2B–E).
LV-PCR amplification of microdissected samples.
Microdissected samples Mummy 1–4 were tested for
the existence of amplifiable nuclear DNA, in respect to
isolated osteon islets, comprising a very minute tissue
amount of just a few micrometers (corresponding to

American Journal of Physical Anthropology

 PCR ANALYSIS OF MINIMUM TARGET AMOUNT OF ADNA
1–5 lg) compared to standard amounts of 1–2 g of bone
tissue material. All of four mummy samples revealed
successful amplification products of 106/112 bp male and
female amelogenin fragments (Table 1). While samples
Mummy 2 and Mummy 1 showed female 106-bp seg-
ments (Fig. 2B,E) samples, Mummy 4 and Mummy 3
produced male segments of 106 and 112 bp (Fig. 2C,D).
Concerning the larger 297 bp segment of the human
b-actin gene, in each approach human b-actin fragments
could successfully and constantly be amplified and
detected in each of the microdissected samples
Mummy1–4 (Fig. 2B–E; Table 1). All extraction and PCR
negative controls like lysis mix, PEN supporting mem-
brane, H2O control, and PCR master mix, containing no
DNA, were consistently negative in all cases (Fig. 2B–E).
Sequencing of PCR products, originating from a
microdissected mummy sample
Sequencing analysis was performed with amelogenin
and b-actin PCR products, amplified from the microdis-
sected male mummy sample Mummy 4, exemplarily for
all of four samples. Sequencing results were aligned to
human nucleotide sequences, recalled from the human
genomic database of NCBI (www.ncbi.nlm.nih.gov).
Sequencing 106/112 bp amelogenin PCR products
showed sequence identities to amelogenin loci of 90% for
the Y-fragmental sequence and 97% for the X-fragmental
sequence (results not shown). Sequencing the 297 bp b-
actin PCR product showed sequence identities to the b-
actin data of 96–98% (results not shown).
DISCUSSION
To enhance our analysis sensitivity, all aDNA PCR
analyses were performed by virtual reaction chamber
low-volume PCR, using 1-ll total reaction volume. One
can easily imagine that there is a much higher impact
probability between reactants and DNA template
enclosed in small volume assays (1 ll) than in macro-
scopic samples like e.g., in 25-ll standard reaction vol-
umes. Using this low-volume PCR method further
enabled us to significantly reduce the starting amounts
of extracted ancient DNA sample material needed for
PCR reactions. This is an important fact regarding the
upcoming technology of lab-on-a-chip systems dealing
with reaction volumes in the micro- and nanoscopic
range. As plastics have been identified as potential sources
of bioactive environmental contaminants, it will be an
advising step to depart from plastic PCR-tubes to analy-
sis devices fabricated of modified glass substrates.
Moreover, it was also reported that processing agents
from laboratory plastic ware can be leaking into biologi-
cal media and solvents, particularly during storage
(McDonald et al., 2008). Because of these criteria, PCR-
tubes, even when declared as ‘‘sterile,’’ are assumed to
contain leaching agents and facilitate contamination of
PCR reactions when heated. However, as reported sev-
eral times, working with aDNA originating and
extracted from soil buried bones and teeth is by itself
associated with problems like low DNA quantity, high
DNA degradation, the presence of PCR inhibitors, and
most importantly DNA contamination (Primorac et al.,
1996; Alonso et al., 2001).
Extraneous contamination can arise from adjacent
treatments such as the handling of the remains by exca-
vators and laboratory staff, airborne contaminants, and
325
contaminants present within laboratory reagents or on
consumable items (Tuross, 1994). Soil type and microbial
community characteristics can adversely affect DNA
recovery (Zhou et al., 1996). The total DNA extraction
from soil entails coextraction of humic substances,
mainly humic acid, as well as DNA originating from
external organisms like bacteria and fungi (Tsai and
Olson, 1992; Tebbe and Vahjen, 1993; Kreader, 1996;
Zhou et al., 1996). These interferences can provide false-
negative results in case of PCR inhibition (Zhou et al.,
1996; Zipper et al., 2003) or false-positive ones (like non-
specific products) in case of contamination, greatly
reducing the information, which can be attained.
The expansive variety of DNA extraction techniques
being employed [as reported by Anderung et al. (2008)
and Rohland and Hofreiter (2007)], highlights that all of
these procedures are affected by problems and limita-
tions concerning DNA quality, quantity, and coextracted
PCR inhibitors as mentioned above. As an effort to mini-
mize these interfering troubles as efficiently as possible,
in the present study the surface of bone tissue sample
fragments was carefully decontaminated. First by chemi-
cal cleaning with DNA-degrading sodium hypochlorite
which was followed by a mechanical removal of the outer
layers of the bone fragments before sample processing
procedures. However, postmortem contaminations can
hardly be removed completely as reported by Gilbert et
al. (2005, 2006). Nevertheless, there seems to be a signif-
icant correlation between the sector of extraction, outer
versus inner sectors of bones, and the amount of DNA
present in resulting samples (Kaiser et al. 2008). The
outer sectors, adjacent to the soil, were shown to have
higher DNA concentrations than the inner ones, due to
the presence of bacteria and fungi DNA. Furthermore, it
was shown that the localization within the bone piece
influences its degree of degradation. This is the result of
varying degrees of protection against destructive envi-
ronmental influences like temperature, humidity, pH,
and the geochemical properties of the soil including pres-
ence of microorganisms, UV irradiation, and radioiso-
topes (Burger et al., 1999). Since the outer surface is sig-
nificantly more influenced by the direct contact to the
environmental soil, DNA is best preserved in the inner
part of bones (Kaiser et al., 2008). Based on laser micro-
dissection of internal bone areas, the present study thus
introduces a promising method for contamination-
reduced bone tissue particle isolation and subsequent
aDNA extraction. With respect to extracted aDNA qual-
ity, the grade of DNA degradation, and interfering de-
structive factors, our approach significantly excelled the
traditional method. While the current DNA extraction
operating procedure uses whole bone tissue particles for
pulverization and subsequent DNA extraction, our
method concentrates on internal bone areas including
osteon systems. The focus of bone particle isolation was
on tissue areas containing these osteon systems to iso-
late fragments with the highest probability of preserved
vascular cell material. Thus, several osteon areas were
microdissected exclusively from the inner parts of the
bones where surface contamination and inhibitor influ-
ences were least possible to occur. Despite higher DNA
recovery and extraction rates in the classical extraction
methods, the DNA quality is supposed to be considerably
worse. This is most likely caused by present interfering
substances and less authentic DNA provided by the pul-
verization method. For the higher DNA amount is little
informative about the source of DNA and provides a
American Journal of Physical Anthropology
326 D. WOIDE ET AL.
mixture of desired mummy DNA and DNA of bacterial
and fungal origin.
The 106/112 bp amelogenin X- and Y-chromosomal
fragments as well as a 297-bp fragment of the human
multicopy gene b-actin were amplified by PCR. In ampli-
fication reactions, particular attention was paid to the
quality of extracted DNA, including possible effects of
degradation or PCR inhibitors. In microdissection-based
mummy samples amplification of amelogenin gene as
well as b-actin gene fragments resulted in reproducible,
constantly positive PCR products using only about 60 pg
of DNA starting material (Fig. 2B–E). In contrast, PCR
results of samples based on pulverized bone tissue
showed just 50% success in amplifying amelogenin gene
fragments (Mummy 2 and Mummy 4; Fig. 2B,C) and no
reliable positive amplification products for b-actin frag-
ments using 100 pg of DNA starting material. These
lower yields of PCR products may be due to poorer DNA
quality, consistence, or state because of degradation
effects. Taking into account that also primer dimeriza-
tion occurred during PCR, the failing amplification may
more likely be attributed to DNA degradation than to
present inhibiting substances. Moreover, especially the
inconsistent amplification of 297 bp b-actin fragments is
in agreement with a general amplification limit dis-
cussed for aDNA. Propagating amplification fragments
longer than 200 bp are very unlikely due to degradation
processes. Here, the amplification success correlates neg-
atively with the length of the amplicon. This is supposed
to be the result of contaminating outer bone layers,
where extensive DNA degradation could have occurred.
However, this larger b-actin fragment could be amplified
reliably in microdissection-based samples, where the an-
alyzed bone segments originated from the more pre-
served inner part of bone particles.
Furthermore, sequencing of amelogenin and b-actin
PCR products was performed on one male microdissec-
tion-based mummy sample (Mummy 4). This enabled us
to confirm authentic PCR products and eliminate possi-
ble false positives, which may indeed show the right
lengths of the wanted amelogenin and b-actin fragments
while presenting a different sequence of DNA base pairs.
Analysis of the sequencing data for both the 106/112 bp
X- and Y-chromosomal amelogenin fragments and the
297 bp b-actin fragment definitively identified them as
human amelogenin and b-actin sequences once aligned
to gene sequences from the human genomic database of
NCBI (data not shown). Furthermore, sequencing results
were also important to invalidate the possibly occurring
phenomenon of jumping PCR, induced by damaged tem-
plate DNA and resulting in the production of a chimeric
sequence, as first reported by Pääbo et al. in 1990. Thus,
via sequencing, the authenticity of the amplified DNA
could be confirmed. Both, the successful amplification of
a fragment significantly longer than 200 bp and the suc-
cessful sequence alignment results of b-actin gene frag-
ments highlight the advantage of laser microdissection
for ancient material extraction.
Overall, we always obtained higher yields of PCR
products by using laser microdissected samples than by
amplifying samples being extracted via the classical
method. This may be attributed to a better DNA quality
present in microdissected samples due to less exposure
to degradation effects. These results implicate that isola-
tion of tiny target tissue material by laser microdissec-
tion combined with standard kit DNA extraction meth-
ods is sufficient for aDNA extraction and successful
American Journal of Physical Anthropology
amplification. Combined with low-volume PCR using pla-
nar microdevices, our method enables the amplification
of minute amount of laser-microdissected material in
extremely small reaction volumes.
The presented nanotechnological approach provides an
adequate tool for reliable and highly sensitive DNA anal-
ysis, ensuring the optimum use of limited evidence mate-
rial. This approach could adapt the preparative and
extraction procedures to the low amount of preserved an-
cient DNA, thus offering the possibility to increase the
amount of extracted, less degraded and less contami-
nated, authentic genetic material and decreasing the
effect of destructive factors.
ACKNOWLEDGMENTS
Special thanks go to the Medizinisch Genetisches Zen-
trum (MGZ) Munich for offering the opportunity to per-
form all typing analyses and fruitful discussion. S. Thal-
hammer would like to dedicate this article to Dr. Sabine
Schüssler.
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