Forensic Science International

113 (2000) 7985

www.elsevier.com / locate / forsciint

The personal identification of many samples recovered from under

the sea
a,
b
a
c
c
Masahiro Mukaida *, Hiroko Kimura , Yuzo Takada , Tomoo Masuda , Yasuko Nakata
a

Department of Forensic Medicine, National Defense Medical College, 3 -2 Namiki, Tokorozawa, Saitama 359 -8513, Japan
b
Department of Forensic Medicine, Juntendo University School of Medicine, Homgoz-1 -1, Tokyo 113 -8421, Japan
c
Biochemistry Section, Second Division, Aeromedical Laboratory, Japan Air Self Defense Force, Sakae-cho 1 -2 -10, Tachikawa,
Tokyo 190 -0003, Japan

Abstract
An automatic and rapid DNA typing system was employed for personal identification, using fragmentary tissue samples
from victims in an airplane accident. Two victims were crushed into small pieces, and 33 samples suspected to belong to
them were recovered from under the sea. From each sample, 10 mg was used for testing. The parents bloods of two
presumptive victims were also examined. DNA extraction from samples was performed by the NaI method, and the obtained
DNA samples were analyzed with the ABI PRISM system. Among 33 samples, 31 samples were identified to be human
tissues, possibly from two victims. The other two samples seemed to be parts of marine animals. ABO blood group, STR
polymorphism, and mitochondrial DNA polymorphism typing were possible in every examined human sample. Two victims
fragmentary tissues were identified by determining ABO genotype, STR type and mitochondrial DNA type. The system we
employed enabled an accurate typing of many fragmentary samples in a short time, thus contributing to the fast and secure
identification of many victims in such cases as big air accidents. 2000 Elsevier Science Ireland Ltd. All rights reserved.
Keywords: Identification; PCRSSP; TaqMan PCR; Mitochondrial DNA; STR-marker; Fragmentary tissue

1. Introduction
We often have to perform personal identification
using many kinds of specimens, for example fragments of tissue, pieces of hair and / or based on
bloodstains. Test samples are available in various
forms: some are not necessarily fresh and some have
also greatly changed due to putrefaction. When
samples have changed either chemically or biologically, it is often difficult to determine the blood type
using antiserum and enzyme polymorphism using the

enzymatic activity. Consequently, a DNA analysis is

performed for identification in many cases [18].
In airplane crashes or explosion accidents, the
limbs and organs of the victims are crushed into
small pieces [9,10]. In such cases with many samples
of fragmentary tissue, a definite identification is
required within a short time. To achieve this goal, we
thus examined whether DNA extraction and analyses
from small samples were possible or not. DNA
extracts from samples were identified by DNA
polymorphic markers.

2. Case
*Corresponding author. Fax.: 181-42-996-5198.
E-mail address: mmukaida@cb3.so-net.ne.jp (M. Mukaida).

Two F-4 fighters collided during a training flight

0379-0738 / 00 / $ see front matter 2000 Elsevier Science Ireland Ltd. All rights reserved.
PII: S0379-0738( 00 )00219-X

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M. Mukaida et al. / Forensic Science International 113 (2000) 79 85

over the ocean. Two persons (one crew member from

each aircraft) became missing. Tissue fragments,
apparently from the missing crew, were recovered
from the ocean 2 days after the accident.

3. Materials and methods

3.1. Sample source

In the test, we analyzed 33 samples (Nos. 537)
(about 10 mg) from the recovered tissue fragments
(25000 g). We used hair specimens collected from
their brushes and blood specimens (Nos. 14) which
were obtained from the crews parents (F1; M1 and
F2; M2) as reference samples for identification. In
addition, we also used some samples obtained from
volunteers as a normal control; these included blood
stains (2 samples) which adhered to gauze and were
left at room temperature for 1 day, fresh venous
blood (6 samples) and one strand of hair (2 samples).

3.2. DNA extraction

We performed DNA extraction on 10 mg of tissue
fragments (33 samples), a blood stain adhering to
one gauze thread measuring 5 mm in length (2
samples), a strand of hair measuring 5 mm in length
(2 samples) and 50 ml whole blood (8 samples). The
samples were all deposited separately into micro
tubes. We then performed DNA extraction on the
samples. We added TE into the tubes and a total
volume of 90 ml. Five ml SDS (20%) and 5 ml

Proteinase K solution (100 mg / ml) was also added

and mixed with the solution. The hydrolysis of the
samples was performed by heating for 10 min at
608C. We next added 200 ml of NaI solution (6 M
NaI, 13 mM EDTA, 26 mM TrisHCl pH 8.0) into
the mixtures, and the solutions were all well mixed.
The solution materials were heated at 608C for 10
min, and then centrifuged at 15 000 rpm for 10 min.
After that, 300 ml isopropanol were added. The
recovered DNA was washed in 70% ethanol, after
that it was dried up and dissolved in 40 ml TE. The
hair samples were washed in detergent as a pretreatment. Next, they were dehydrated in ethanol, and
dried afterwards. The samples were deposited into
microtubes, while 100 ml of 0.1 M DTT in TE was
added, and then the samples were heated at 608C for
30 min. Thereafter, the samples were treated similarly to the DNA extraction from the blood samples.

3.3. ABO blood group typing

For genotyping the ABO blood group, the presence of an exon arrangement which encoded A
transferase (a13GalNAc transferase) or B transferase (a13Gal transferase) was estimated by TaqMan PCR [1115]. The primers and TaqMan probes
used for PCR amplification with sequence-specific
primers (PCRSSP) are shown in Table 1. The
arrangement of the primers and the TaqMan probes
for the ABO Blood group genotyping was the
following; A and B allele was detected by the set of
ABFABR primers and a TaqMan probe of ABO1,
OFOR primers and ABO1 probe were used for the
detection of O-allele, also AOFABOR primers and

Table 1
Primers and TaqMan probe
Marker

Primer and probe

Sequence

ABO

Primer ABF
ABR
OF
OR
AOF
BF
ABOR
Probe ABO1
ABO2
Primer F16055
R16437

GAA GGA TGT CCT CGT GGT G

TAC TTC TTG ATG GCA AAC ACA GTT A
CAG TAG GAA GGA TGT CCT CGT GGT A
TTA ACC CAA TGG TGG TGT TCT GG
GGA CGA GGG CGA TTT CTA CTA CC
AGG ACG AGG GCG ATT TCT ACT ACA
TGT TCA GGT GGC TCT CGT CGT
FAMTTG GCT GGC TCC CAT TGT CTG GGA GTAMRA
FAMCGG TGC AAG AGG TGC AGC GGCTAMRA
CCC AAG TAT TGA CTC ACC CAT
CCC GGA GCG AGG AGA GTA GCA

mtDNA

M. Mukaida et al. / Forensic Science International 113 (2000) 79 85

ABO2 probe were prepared for the detection of A

and O allele, AOFABOR primers and ABO2 probe
were for the detection of B allele. The fragments of
DNA were amplified by PCR with PCRSSP. The
amount of PCR amplified fragment was directly
measured with the intensity of fluorescence released
from the TaqMan probe by the nick-translation of
Taq enzyme. The degree of the amplification of the
fragment DNA at each cycle was measured in realtime using the ABI PRISM 7700 Sequence Detection
System. A sample was determined to be positive
after establishing the threshold value of the negative
value, after the rate of product obtained in the one
PCR cycle was fixed.

3.4. Analysis of mitochondrial DNA

A pair of forward and reverse primers for the PCR
was prepared and a 403bp PCR fragment was then
amplified from the D-loop region of mitochondrial
DNA (mtDNA) by the PCR with a thermal cycler.
The sequence of primers for PCR of mtDNA is
shown in Table 1. The numbering system and a
standard sequence of mtDNA were following a
previously reported method [16]. The labeling for the
sequencing was performed using the BigDye Terminator Cycle Sequencing Ready Reaction kit (PE
Biosystems). An analysis of the sequence was performed with ABI PRISM 377XL DNA Sequencer.

3.5. Analysis of STR-marker

STR markers were analyzed by using the
AmpFlSTR Profiler kit (PE Biosystems). Nine STR
markers (D3S1358, vWA, FGA, TH01, TPOX,
CSFIPO, D5S818, D13S317 and D7S820) and XY
were analyzed with Multiplex-PCR according to a
protocol provided by PE Biosystems. An ABI
PRISM 377XL DNA Sequencer was used for the
analysis.

81

150300 ng. The obtained DNA was 7502500 ng

from a blood stain which adhered to a piece of 5 mm
gauze thread, 1.52.5 mg from 50 ml fresh blood and
also over 400 ng of DNA from a 10 mg tissue
fragment.

4.2. ABO blood group typing

The TaqMan PCR data are shown in Fig. 1. The
normal control brought an adequate result (Fig. 1).
The genotypes of both pairs of parents were F1; OO,
M1; BO and F2; AB, M2; OO. The genotype of crew
member 1 was thus determined to be OO or BO and
the genotype of crew No. 2 was found to be AO or
BO. All samples, except for No. 9 and No. 10
showed productive signals of TaqMan PCR. Sample
No. 25 did not show good results at the 1st trial. The
result of the blood group typing is shown in Table 3.
The results of the blood group typing showed that
crew member 1 was BO type and crew member 2
was AO type.

4.3. Sequence of mtDNA

The base sequence of a D-loop of mtDNA for the
each sample was determined by direct sequence
PCR. There was a substitution of T16189C in the
D-loop from the mother of crew member 2 as shown
in Table 2. Samples No. 9 and No. 10 did not show
any productive PCR signal. All samples, except for
No. 9 and No. 10, demonstrated the PCR fragment of
mtDNA. The results are shown in Table 3.

4.4. STR-marker
The amplification product was not completely
obtained from samples No. 9 and No. 10. Similarly,
no good results were obtained from samples No. 16
and No. 25. The other samples and normal control
samples respectively brought an adequate result. The
results are shown in Fig. 2 and Table 3.

4. Results
5. Discussion

4.1. Recovered quantity of DNA

The quantity of DNA extracted from one 5 mm
strand of hair by the NaI extraction method was

A total of 35 of the 37 samples could be sufficiently distinguished. A histological inspection was

carried out on 2 samples for which a sufficient DNA

M. Mukaida et al. / Forensic Science International 113 (2000) 79 85

82

Fig. 1. Visualization of the decision for the TaqMan PCR (s) positive, (d) negative, the threshold level was 0.05. (1) Father of crew
member 1, (2) Mother of crew member 1, (3) Father of crew member 2, (4) Mother of crew member 2, (516) The samples recovered from
under the sea.

analysis could not be performed. These two samples

(No. 9; 5000 g and No. 10; 150 g) had to be
excluded from our result because they showed a
different histological pattern from one of the crews
(the results are not shown in this report).
When the methods of ABO genotyping, mtDNA
typing and the STR analysis were compared, an

amplification product suitable for the analysis was

easily obtained from mtDNA by the PCR of the
D-loop region. Therefore, depending on the condition of the sample, performing ABO genotyping by
TaqMan PCR may thus be difficult.
One sample (No. 25) did not show any data for
STR marker, while another sample (No. 16) showed

Table 2
Result of the sequence of mtDNA from the crews parents a
Base No.

Father 1

Mother 1

Crew 1

Father 2

Mother 2

Crew 2

16111
16182
16183
16189
16223
16234
16243
16291
16319
16325
16362

C
A
A
T
T
C
T
C
G
T
C

C
A
A
T
T
C
T
C
G
C
C

C
A
A
T
T
C
T
C
G
C
C

C
A
A
T
T
C
T
C
G
C
C

T
C
C
C
C
T
C
T
A
T
T

T
C
C
C
C
T
C
T
A
T
T

(1) Father of crew member 1, (2) Mother of crew member 1, (3) Father of crew member 2, (4) Mother of crew member 2.

M. Mukaida et al. / Forensic Science International 113 (2000) 79 85

83

Table 3
Results of DNA analysis a
Sample
No.

ABO
Genotype

mtDNA
16189 b

1
2
3
4
7
8
9c
10 c
11
16
17
18
19
20
25
26
27
28
29
30

OO
BO
AB
OO
BO
AO

AO
BO
AO
AO
AO
BO
AO d
BO
BO
BO
AO
AO

T
T
T
C
T
C

C
T
C
C
C
T
C
T
T
T
C
C

STR type

Identification e

D3S1358

vWA

FGA

Amelogenin

TH01

TPOX

CSF1PO

D5S818

D13S317

D7S820

17,
14,17
15,18
15,15
17,
15,18
,
,
15,18
,
15,18
15,18
15,18
17,
,
17,
17,
17,
15,18
15,18

14,15
15,21
18,19
14,19
14,15
18,19
,
,
18,19
14,15
18,19
18,19
18,19
14,15
,
14,15
14,15
14,15
18,19
18,19

22,25
20,21
21,23
23,24
20,22
23,24
,
,
23,24
,
23,24
23,24
23,24
20,22
,
20,22
20,22
20,22
23,24
23,24

X,Y
X,
X,Y
X,
X,Y
X,Y
,
,
X,Y
,
X,Y
X,Y
X,Y
X,Y
,
X,Y
X,Y
X,Y
X,Y
X,Y

07,09
07,09
06,09
07,07
07,09
06,07
,
,
06,07
07,09
06,07
06,07
06,07
07,09
,
07,09
07,09
07,09
06,07
06,07

11,
08,11
08,
09,09
08,11
08,09
,
,
08,09
,
08,09
08,09
08,09
08,11
,
08,11
08,11
08,11
08,09
08,09

10,11
10,12
11,13
10,12
11,12
11,12
,
,
11,12
,
11,12
11,12
11,12
11,12
,
11,12
11,12
11,12
11,12
11,12

09,12
09,13
09,13
12,
09,
12,13
,
,
12,13
,
12,13
12,13
12,13
09,
,
09,
09,
09,
12,13
12,13

10,11
08,11
08,11
09,13
08,11
09,11
,
,
09,11
,
09,11
09,11
09,11
08,11
,
08,11
08,11
08,11
09,11
09,11

08,
08,
11,12
11,12
08,
12,
,
,
12,
,
12,
12,
12,
08,
,
08,
08,
08,
12,
12,

Ref. 1
Ref. 2
Ref. 3
Ref. 4
Crew 1
Crew 2

Crew
Crew
Crew
Crew
Crew
Crew
Crew
Crew
Crew
Crew
Crew
Crew

2
1
2
2
2
1
2
1
1
1
2
2

Sample number 14: reference samples; sample number 530: samples recovered from under the sea.
The substitution of T16189C is shown only for the purpose of identifying crew member 2.
c
: no fragments detected.
d
No ABO genotype data were obtained at the 1st trial.
e
Ref. 1: Father of crew member 1, Ref. 2: Mother of crew member 1, Ref. 3: Father of crew member 2, Ref. 4: Mother of crew member
2, Crew 1: Crew member 1, Crew 2: Crew member 2. Samples No. 9 and No. 10 were decided to be samples from an animal based on the
histological findings.
b

incomplete results. One reason for the failure of PCR

amplification may be related to the degeneration of
the sample or pollution of the sample due to various
chemicals or biological substances which could have
led to a degradation of the DNA. The AmpFlSTR
Profiler kit, which we used for the analyses, consists
of a combination of multiple primers. For this
reason, the optimal reaction condition may thus be
greatly narrowed, and thus result in the failure of
PCR.
When human specimens such as blood stains,
epidermis samples or hairs are detected after various
crimes and accidents, such specimens become important evidence for the presence of individuals at
the scene. In addition, many people also die due to
explosions and airplane crashes, and the number of
tissue samples is therefore large in such cases.
Identification based on dental records and body

features is only possible when the bodies are in good

condition. Dental records and an X-ray film are used
as reference samples for such identification. In
addition, the identification of organs and tissue
fragments becomes difficult, when detached parts of
the body are severely crushed. In addition, identification based on morphological features is also not
possible after severe postmortem changes, since
identification marks decrease remarkably in such
cases. When the materials putrefy, the blood group
phenotype becomes almost inadequate for individual
identification. As a result, the use of markers for the
ABO blood group and / or the Rh blood group as
markers for individual identification becomes almost
impossible. In many cases, one of the control
samples for individual identification are mucous cells
which adhere to either a toothbrush, a strand of hair
or strand of facial hair [5]. In addition, genetic

84

M. Mukaida et al. / Forensic Science International 113 (2000) 79 85

Fig. 2. Electropherogram of STR markers, Father 1; Father of crew member 1, Mother 1: Mother of crew member 1, Crew 1: Crew member
1, Father 2: Father of crew member 2, Mother 2: Mother of crew member 2, Crew 2: Crew member 2. This figure is a part (Amelogenin,
TH01, TPOX and CSF1PO) of an electropherogram of STR markers.

markers of surviving family members can also be

used. When inspecting a number of tissue fragments
and blood stains, not only an accurate identification
but also the ability to analyze small specimens and
finish such identification within a short time is
necessary.
DNA analyses using polymorphism with short
genomic DNA and polymorphism of mtDNA are
very effective for identifying decomposed samples
[1721]. Especially, the recovery of mtDNA samples
and identification based on mtDNA typing is possible using small samples due to high mtDNA copy
numbers [22].
For fresh samples and samples with only slight
changes, automated identification by analyzing the
DNA type of HLADRB allele and / or the ABO
allele is possible by utilizing the TaqMan PCR
within a short time [15] and this inspection method is
considered to be an effective method. In addition, the
utilization of an STR marker is recommended in

order to distinguish a large number of individuals at

one time. It is necessary to effectively combine
inspection methods in order to identify victims
within a short time when analyzing a large number
of samples.

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