Forensic Science International 152 (2005) 109–114

www.elsevier.com/locate/forsciint

Identification of DNA of human origin based on amplification

of human-specific mitochondrial cytochrome b region
Hirokazu Matsuda a,*, Yasuhisa Seo a, Eiji Kakizaki a, Shuji Kozawa b,
Eri Muraoka a, Nobuhiro Yukawa a
a
Department of Legal Medicine, Miyazaki Medical College, 5200 Kihara, Kiyotake-cho, Miyazaki 889-1692, Japan
b
Department of Legal Medicine, Yamaguchi University School of Medicine, Minami-kogushi 1-1-1,
Ube city, Yamaguchi 755-8505, Japan
Received 3 March 2004; received in revised form 26 July 2004; accepted 28 July 2004
Available online 29 September 2004

Abstract

Species-specific differences in a non-polymorphic region of the mitochondrial cytochrome b gene appear to be large enough
to allow human-specific amplification of forensic DNA samples. We therefore developed a PCR-based method using newly
designed primers to amplify a 157-bp portion of the human mitochondrial cytochrome b gene. The forward and reverse primers
were designed to hybridize to regions of the human mitochondrial cytochrome b gene with sequences differing from those of
chimpanzee by 26% (7 bp/27 bp) and 26% (6 bp/23 bp), respectively. Using this primer pair, we successfully amplified DNA
extracted from blood samples of 48 healthy adults. All these human samples produced a single band of the expected size on
agarose gel electrophoresis, and the sequence of the single band was shown to be identical to that of the target region (157 bp) by
sequence analysis. On the other hand, no visible bands were amplified from DNA extracted from blood samples of animals
including non-human primates (chimpanzee, gorilla, Japanese monkey, crab-eating monkey) and other species (cow, pig, dog,
goat, rat, chicken and tuna). Thus, DNA producing a single band following PCR amplification using this primer pair can be
reasonably interpreted as being of human origin. In addition, aged biological specimens comprising bloodstains, hair shafts and
bones were successfully identified as being of human origin, illustrating the applicability of the present method to forensic
specimens.
# 2004 Elsevier Ireland Ltd. All rights reserved.

Keywords: Human identification; Species determination; Mitochondrial DNA; Cytochrome b gene; Forensic casework

1. Introduction quality including highly degraded specimens, old bone

PCR-based typing of short tandem repeats (STR) usually
produces human-specific results [1–4] and is routinely used
as an established method for forensic practice. Not infre-
quently, however, STR typing fails in samples of poor
* Corresponding author. Tel.: +81 985 85 0991;
fax: +81 985 85 6406.
E-mail address: hmatsuda@med.miyazaki-u.ac.jp
(H. Matsuda).
fragments or hair shafts without roots. For these types of
samples, mitochondrial DNA (mtDNA) analysis has been
successfully introduced [5–11].
The cytochrome b (cyt b) gene is located in the mito-
chondrial genome. Nucleotide sequences of human and
animal cyt b genes are known to be species-specific and
have been used for species determination in phylogenetic
and forensic investigations [12–14]. In these investigations,
amplification of the cyt b gene using primer pairs targeting
highly conserved regions was followed by sequence analysis

0379-0738/$ – see front matter # 2004 Elsevier Ireland Ltd. All rights reserved.
doi:10.1016/j.forsciint.2004.07.019
110 H. Matsuda et al. / Forensic Science International 152 (2005) 109–114
to perform species determination. This approach is very
efficient as DNA samples of human as well as non-human
origin can be positively identified to the species level.
However, confirmation of the human origin of samples is
likely to be useful in routine forensic cases.
In this study, we designed a new primer pair for use
in a PCR-based method for identifying DNA as being of
human origin. Animal DNA, including that of higher pri-
mates, was not amplified by this method; only human DNA
was amplified to produce visible bands on agarose gel
electrophoresis.
2. Materials and methods
2.1. Specimens
Human peripheral blood samples were obtained from 48
healthy adults who gave written informed consent (30 males
and 18 females). Aged samples were obtained from both
bloodstains on cotton gauze and hair shafts that had been
stored at room temperature for 20 years of 10 identified
donors (5 males and 5 females). Human aged bone samples
were obtained from 10 unidentified skeletonized individuals
on which post mortem examinations had been performed in
our laboratory and which had been stored at room tempera-
ture for 25–30 years.
Animal blood samples were obtained from three different
individuals from each species, with the exception that only
one individual was sampled for gorilla. Samples from the
following species were studied: Pan troglodytes (chimpan-
zee), Gorilla gorilla (gorilla), Macaca fuscate (Japanese
monkey), M. fascicularis (crab-eating monkey), Bos taurus
(cow), Sus scrofa (pig), Canis familiaris (dog), Capra hircus
(goat), Rattus norvegicus (rat), Gallus gallus (chicken) and
Thunnus thynnus thynnus (tuna).
This study was approved by the Miyazaki Medical
College of Medicine Ethics Committee on Genetic Study.
2.2. DNA extraction
The amount of sample used for DNA extraction
depended on the type of material: blood, 500 ml; blood-
stains on cotton gauze, 5 mm
5 mm area; hair shaft,
10 mm; and bone powder, 20 mg. Bone samples were
powdered and decalcified by soaking in 1 M EDTA for
36 h [15]. DNA extraction from blood samples was per-
formed using a DNA Extractor WB Kit (Wako Pure
Chemical Industries, Osaka, Japan), and extraction from
bloodstains, hair shafts and bone samples was carried out
using a DNA Extractor FM Kit (Wako Pure Chemical
Industries Ltd.) according to the respective manufacturer’s
instructions.
Extracted DNA was quantified spectrophotometrically
and diluted to a concentration of 2 ng/ml for use in PCR
amplification.
2.3. Direct (one-step) amplification of a target region
within cyt b gene
A portion (157 bp) of human cyt b gene was amplified
from extracted DNA using the following primer pair (Fig. 1):
L15674, 50 -TAGCAATAATCCCCATCCTCCATATAT-30 ;
H15782, 50 -ACTTGTCCAATGATGGTAAAAGG-30 . The
numbering system follows that used for human mtDNA
by Anderson [16]. The reaction mixture (50 ml) contained
2 ng of template DNA, 0.1 mM of each primer (L15674,
H15782), 200 mM of dNTPs, 5 ml of 10
standard reac-
tion buffer, 0.25 U Tag DNA polymerase (TaKaRa Ex Taq,
TaKaRa Shuzo Co. Ltd., Shiga, Japan). PCR was per-
formed in a thermal cycler (GeneAmp PCR system
9700; Perkin Elmer Corporation, Norwalk, CT, U.S.A.)
for 40 cycles of 30 s at 96 8C, 40 s at 59 8C, and 1 min
at 72 8C.
2.4. Two-step amplification of the target region within
cyt b gene
In two-step amplification, the entire cyt b gene was
amplified by primary PCR using the primer pair L14734/
H15863 (see below), and then the human-specific region
within the cyt b gene was amplified by nested PCR using the
primer pair L15674/15782.
2.4.1. Amplification of the entire cyt b gene (primary PCR)
Amplification of the entire cyt b gene was performed on
DNA extracted from human and animal blood samples using
the following primer pair, which was designed to hybridize
to portions with high homology in humans and animals
(Fig. 1): L14734, 50 -AACCATCGTTGTATTTCAACT-30 ;
H15863, 50 -ATTTGAGTATTTTGTTTTC-30 . L14734 is
located immediately adjacent to the 50 -end of the cyt b
gene in the 30 -region of the tRNAGlu gene and H15863 is
located at the 30 -end of the cyt b gene. The ingredients of the
reaction mixture were the same as those used for the direct
(one-step) amplification of the human-specific region.
PCR was performed for 35 cycles of 30 s at 96 8C, 40 s
at 50 8C, and 1 min at 72 8C. Amplification products were
visualized by electrophoresing aliquots (10 ml) on a 3%
agarose gel.
2.4.2. Amplification of human-specific region (nested PCR)
Aliquots of primary PCR products were diluted 100-fold
with 1
TE buffer and subjected to nested PCR with the
primer pair (L15674/H15782), which was used for the direct
(one-step) amplification of the human-specific region. The
reaction mixture (50 ml) contained 1 ml of the diluted pri-
mary PCR product, 0.1 mM of each primer (L15674,
H15782), 200 mM of dNTPs, 1
standard reaction buffer,
and 0.25 U Tag DNA polymerase. Amplification was carried
out for 20 cycles of 30 s at 96 8C, 40 s at 59 8C, and 1 min at
72 8C.
 H. Matsuda et al. / Forensic Science International 152 (2005) 109–114 111

Fig. 1. Schematic representation of the human mitochondrial cyt b gene and the amplification strategy for human identification. (a) Positions
and orientations of the primers used in this study are indicated by arrows. ( ) The entire cyt b gene amplified by the L14734/H15863 primer
pair. ( ) The regions targeted for human-specific amplification using the L15674/H15782 primer pair. (b) Comparison of the human and
various animal nucleotide sequences of regions targeted by primers L15674 and H15782. GenBank accession numbers are given in
parenthesis.

2.5. Amplification of conservative region as a 3. Results and discussion

positive control
As a positive control, a conservative region of the cyt
b gene was amplified from serially diluted DNA extracted
from human and animal blood samples using PCR condi-
tions identical to those of the direct amplification and
the following primer pair [13]: L14816, 50 -CCATCCAA-
CATCTCAGCATGATGAAA-30 ; H15173, 50 -CCCCTCA-
GAATGATATTTGTCCTCA-30 .
2.6. Cycle sequencing
PCR products of the one-step amplification and primary
and nested PCR products of the two-step amplification were
electrophoresed on a 1% agarose gel, and the respective
fragments were recovered from the gel. DNA sequencing
was performed using fluorescent dideoxynucleotides
(ABI PRISM BigDye Terminator Cycle Sequencing
Ready Reaction Kit; PE Applied Biosystems, Foster City,
CA, U.S.A.) and an ABI PRISM 310 Genetic Analyzer
(PE Applied Biosystems). Sequence comparisons were per-
formed using Sequence Navigator Software, Version 1.0.1
(Perkin Elmer).
3.1. Development of primer pair L15674/H15782
The nucleotide sequences of the mitochondrial cyt b gene
of human and animals, including primates, were available
from the GenBank database. By targeting portions of the
gene that differ between human (GenBank access number
NC001807) and chimpanzee (NC001643), we synthesized a
primer pair (L15674/H15782) with the target of human-
specific amplification. The primers were designed to hybri-
dize to portions of the human mitochondrial cyt b gene with
sequence differences of 26% (7 bp/27 bp) from that of
chimpanzee for the forward primer and 26% (6 bp/23 bp)
for the reverse primer.
To determine whether this primer pair could certainly
amplify human DNA, we performed direct amplification
using this primer pair on human DNA extracted from blood
samples of 48 healthy subjects. No negative amplification
was observed, and all amplifications produced a single band
with the approximate size of 160 bp.
Sequencing of these amplified products produced
sequences identical to the Anderson reference sequence in
46 of 48 subjects. The remaining two subjects had single A
112 H. Matsuda et al. / Forensic Science International 152 (2005) 109–114
to G transitions, one at 15,746 and the other at 15,758.
Although neither of these transitions fell within the binding
sites of the present primer pair (L15674/H15782), some
single base pair substitutions within either of the primer
binding sites were found in a search of the MITOMAP
database (http://www.mitomap.org/).
3.2. Human specificity and amplification sensitivity
For the present method (direct amplification), specificity
for human DNA was evaluated using DNA extracted from
human and animal blood as template DNA in amplifications
with the primer pair (L15674/H15782). On agarose gel
electrophoresis, DNA of animals produced no visible bands,
whereas human DNA showed a single band (data not
shown), indicating that human specificity of the present
method was sufficiently high to distinguish human DNA
from that of animals, including primates.
For this method (50 ml reaction volume), the minimum
amount of DNA required for successful amplification was
found to be 1 pg based on serial dilutions for extractions
from fresh human blood (Fig. 2).
3.3. Interpretation of results and a positive control
Like the present method, some earlier reports on species
identification have also utilized simple positive/negative
results (the presence or absence of a band, or difference
in migratory position of band). The primer pairs used in
these reports either amplified DNA from higher primates
[17] or were used to investigate only a few species of animals
or primates [18–22]. By contrast, the present method uses a
primer pair that amplifies human DNA only, and therefore,
positive results (the presence of a single band) can be
reasonably interpreted as indicating the presence of human
DNA.
Negative results (the absence of a visible band) are,
however, difficult to interpret because there are many
possible causes for negative results. The presence of non-
human DNA only, or undetectably small amounts of human
DNA produce negative results. However, problems with
amplification of human DNA, including PCR inhibition
and degradation of DNA may produce negative results.
Fig. 2. Detection limit of the portion of human-specific cyt b gene using four-fold serial dilutions of human DNA. M: HaeIII digest marker; lane
1, 1024 pg; lane 2, 256 pg; lane 3, 64 pg; lane 4, 16 pg; lane 5, 4 pg; lane 6, 1 pg; lane 7, 0.25 pg; NC, negative control (no template DNA was
added).
Point substitutions in the binding sites of the present primer
pair, though uncommon, may also cause amplification
failure.
For samples producing negative results, it is necessary to
conduct further analyses to confirm the presence or absence
of any DNA of human or animal origin. Confirming the
presence of DNA, regardless of origin, would greatly reduce
the probability that problems with amplification were
responsible for the negative result and would serve as a
positive control of the present method. This type of check
can be realized through the use of a primer pair that can
amplify any DNA under the same PCR conditions used in the
present method. A promising candidate is the primer pair
[13], which targets a very conservative region of the cyt b
gene with the goal of achieving broad range (species-wide)
amplification. In our evaluation of this primer pair on DNA
extracted from fresh human and animal blood, we found that
it worked well under the PCR conditions of the present
method and that the minimum amount of DNA required was
0.5 pg for human DNA and 0.5–1 pg for DNA for the animal
species. Bands of the expected size (approximately 360 bp)
were visible on agarose gel electrophoresis (data not shown)
for all animals tested in this study.
3.4. Further evaluation of human specificity
The entire cyt b gene was amplified (primary PCR) using
a primer pair (L14734/H15863) designed to hybridize to the
high homology regions. Using the amplified entire cyt b gene
as a template DNA, specificity for human DNA was further
evaluated by nested PCR using the primer pair L15674/
H15782.
3.4.1. Primary PCR
As shown in Fig. 3a, amplification of DNA extracted
from human and various animals gave PCR products with
the expected size of approximately 1.2 kbp, with the excep-
tion of no products for chicken and tuna. Sequencing of
animal PCR products revealed that the sequences of the
regions corresponding to the target region for primers for
human-specific amplification (157 bp, 15674–15782) were
identical to sequences for the corresponding animals in
GenBank.
 H. Matsuda et al. / Forensic Science International 152 (2005) 109–114 113

Fig. 3. Amplification products of the primary and nested PCR. Amplified products were electrophoresed on 3% agarose gel and stained with
ethidium bromide. (a) Primary PCR with outer primers (L14734/H15863). M: HaeIII digest marker (TaKaRa); lane 1, amplified product from
human DNA; lane 2, chimpanzee; lane 3, gorilla; lane 4, Japanese monkey; lane 5, crab-eating monkey; lane 6, pig; lane 7, cow; lane 8, dog; lane
9, goat; lane 10, chicken; lane 11, rat; lane 12, tuna; NC, negative control (no template DNA was added). (b) Nested PCR with inner primers
(L15674/H15782). PCR products were amplified from the products in corresponding lanes of (a).

In both chicken and tuna, the target 1.2 kbp PCR products
were not amplified, though high molecular weight DNA was
shown on an agarose gel of the extracted DNA. Primer pair
L14734 and H15863 used for amplification of the entire cyt b
gene showed several substitutions compared to the chicken
(NC001323) and tuna (NC004901) mtDNA sequences. Such
substitutions may explain the failure to amplify the cyt b
gene of these two species. Another explanation may be the
divergent order of genes on the mtDNA [12,23].
3.4.2. Nested PCR
In order to alleviate saturation, primary PCR products of
the entire amplified cyt b genes of human and various
animals except for chicken and tuna were diluted 100-fold
prior to use in nested PCR. As shown in Fig. 3b, human DNA
alone produced a clear single band of the expected size of
approximately 160 bp. The sequence of the single band was
shown to be identical to that of the target region (157 bp,
15674–15782) by sequence analysis, confirming human-
specific amplification of the primer pair.
3.5. Forensic biological specimens
Forensic biological specimens often undergo degrada-
tion, including DNA fragmentation. DNA extracted from
these types of samples is typically of low molecular weight
and may show band loss in PCR-based human identification
methods. We therefore investigated the applicability of the
present method (direct amplification of the human-specific
region within cyt b gene) using aged biological specimens,
including bloodstains and hair shafts stored at room tem-
perature for 20 years and bone samples stored at room
temperature for 25–30 years. As might be expected, DNA
extracted from the aged specimens, with the exception of
some bloodstains, was generally of low molecular weight.
Despite degradation, all samples produced the single
expected band by the present method (Fig. 4a–c), and the
sequences were confirmed to be those of the human-specific
target region. The relatively small size (157 bp) of the target
region may contribute to the success in amplifying DNA
from aged specimens.

Fig. 4. Detection of the portion of human-specific cyt b gene from aged forensic biological specimens. (a) Bloodstain samples stored at room
temperature for 20 years. M: HaeIII digest marker; lanes 1–5, male subjects; lanes 6–10, female subjects; NC, negative control (no template DNA
was added). (b) Hair shaft samples stored at room temperature for 20 years. Hair shaft samples were obtained from the same donors shown in the
corresponding lanes of (a). (c) Bone samples stored at room temperature for 25–30 years. Lanes 1–10, one unidentified individual, each.
114 H. Matsuda et al. / Forensic Science International 152 (2005) 109–114
In this study, we have determined that for the species
tested, amplified products are detected only for DNA of
human origin. Positive results verify the human origin of
samples. Negative results are inconclusive, however. In the
case of negative amplification, employment of another cyt b
gene primer pair as a positive control [13], and if necessary,
subsequent sequence analysis may be necessary to achieve
conclusive results [12–14].
Acknowledgments
The authors express their gratitude to Dr. Satoru
Miyaishi, Department of Legal Medicine, Okayama Uni-
versity Graduate School of Medicine and Dentistry,
Okayama, Japan, for providing the invaluable animal sam-
ples. This work was supported in part by a Grant-in-Aid for
General Scientific Research C (No. 15590581) from the
Japan Society for the Promotion of Science.
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