The American Journal of Forensic Medicine and Pathology 23(3):268 –271, 2002.
, 2002. ©2002 by Lippincott Williams & Wilkins, Inc., Philadelphia
Chelating Resin-Based Extraction of DNA from Dental Pulp and
Sex Determination from Incinerated Teeth with Y-Chromosomal
Alphoid Repeat and Short Tandem Repeats
Tsukasa Tsuchimochi, D.D.S., Mineo Iwasa, Ph.D., Yoshitaka Maeno, Ph.D.,
Hiroyoshi Koyama, M.D., Ph.D., Hiroyuki Inoue, Ph.D., Ichiro Isobe, M.D., Ph.D.,
Ryoji Matoba, M.D., Ph.D., Motoo Yokoi, D.D.S., Ph.D., and Masataka Nagao, M.D., Ph.D.
A procedure utilizing Chelex 100, chelating resin, was adapted
to extract DNA from dental pulp. The procedure was simple
and rapid, involved no organic solvents, and did not require
multiple tube transfers. The extraction of DNA from dental
pulp using this method was as efficient, or more so, than using
proteinase K and phenol-chloroform extraction. In this study,
the Chelex method was used with amplification and typing at
Y-chromosomal loci to determine the effects of temperature on
the sex determination of the teeth. The extracted teeth were
incinerated in a dental furnace for 2 minutes at 100°C, 200°C,
300°C, 400°C, and 500°C. After the isolation of DNA from the
dental pulp by the Chelex method, alphoid repeats, and short
tandem repeats, the human Y chromosome (DYZ3), DYS19,
SYS389, DYS390, and DYS393 could be amplified and typed
in all samples incinerated at up to 300°C for 2 minutes. The
DYS389 locus in some samples could not be amplified at
300°C for 2 minutes. An autopsy case is described in which
genotypings of DYS19, DYS390, and DYS393 from dental
pulp obtained from a burned body were needed. The data
presented in this report suggest that Chelex 100 – based DNA
extraction, amplification, and typing are possible in burned
teeth in forensic autopsy cases.
Key Words: Alphoid repeats of human Y chromosome
(DYZ3)—DYS19 —DYS389 —DYS390 —Incinerated teeth—
Dental pulp—Sex determination.
Manuscript received September 19, 2001; accepted January 7, 2002.
From the Department of Oral surgery (T.T., M.Y.), Nagoya City
University Hospital, and Department of Legal Medicine (M.I., Y.M.,
H.K., I.I., M.N.), Nagoya City University Medical School, Nagoya, and
the National Research Institute of Police Science (H.I.), Kashiwa, and
Department of Legal Medicine (R.M.), Osaka University Medical
School, Suita, and Division of Physical Therapy, Department of Care
and Rehabilitation (M.I.), Faculty of Care and Rehabilitation, Seijoh
University, Tukai-city 476-8588, Japan.
Address correspondence and reprint requests to Mineo Iwasa,
Department of Legal Medicine, Nagoya City University Medical
School, Kawasumi 1, Mizuho-ku, Nagoya 467– 8601, Japan; email:
iwasami@med.nagoya-cu.ac.jp.
268
Polymerase chain reaction (PCR) is a method of am-
plifying small quantities of relatively short target se-
quences of DNA using sequence-specific oligonucleo-
tide primers and thermostable Taq DNA polymerase (1).
PCR does not require native high molecular weight DNA
to amplify the target sequence. Only the target sequence
itself needs to be intact. Thus, PCR can amplify partially
degraded and/or denatured DNA. PCR has a great po-
tential benefit for the analysis of forensic case work
samples, of which the amount and molecular weight of
DNA may be less than optimal for analysis by other
methods.
The teeth are fever durable and are used for personal
identification in forensic medicine (2). In the case of few
teeth or missing dental records, there is not enough
information to identify the person. The dental pulp enclosed
by the hard tissue is not influenced by fever, unlike the
buccal mucous membrane, saliva, and calculus.
We extracted DNA from the dental pulp by the Chelex
method from experimentally incinerated teeth and am-
plified it with PCR and typed on the polymorphic loci.
Moreover, we describe a case of personal identification
from dental information and the genotyping results of
DYS19, DYS390, and DYS393.
MATERIALS AND METHODS
Sample Treatments
Teeth with remaining dental pulp were obtained from
patients, with their permission, who had such conditions
as pericoronitis, difficult dentition, and impacted teeth.
The extracted teeth were dried at room temperature and
were incinerated in a dental furnace (KDF-HR7, Denken,
Japan) at 100°C, 200°C, 300°C, 400°C, and 500°C for 2
minutes. The incinerated teeth were broken open with a
 SEX DETERMINATION OF BURNED TEETH 269

TABLE 1. Polymerase chain reaction primer

sequences used in this study
Locus Primer sequence (5⬘–3⬘)
DYZ3 Forward ATG ATA GAA ACG GAA ATA TG
Reverse AGT AGA ATG CAA AGG GCT C
DYS19 Forward CTA CTG AGT TTC TGT TAT AGT
Reverse ATG GCA TGA TGT GAG GAC A
DYS389 Forward CCA ACT CTC ATC TGT TAT TAT CTA T
Reverse TCT TAT CTC CAC CCA CCA GA
DYS390 Forward TAT ATT TTT ACA CAT TTT TGG GCC
Reverse TGA CAG TAA AAT GAA CAC ATT GC
DYS393 Forward GTG GTC TTC TAC TTG TGT CAA TAV
Reverse AAC TCA AGT CCA AAA AAT GAG G

hammer, and the dental pulp was manually recovered
with a pair of forceps.
DNA Extraction from Teeth
DNA was extracted from the powdered pulp by the
following procedure (3,4): 1 ml of sterile distilled water
was pipetted into a sterile 1.5-ml microcentrifuge tube,
and approximately 50 mg of powdered pulp was added
and mixed gently. The tube was incubated at room tem-
perature for 30 minutes with gentle inversion and cen-
trifuged for 5 minutes at 10,000 g, and the supernatant
was discarded. Five percent Chelex 100 in water was
added to the final volume of 150 ␮l, and 50 ␮l of
proteinase K (2 mg/ml water) was added. The tube was
incubated at 56°C for 30 minutes. Then, the tube was
vortex-mixed at high speed for 10 seconds and incubated
in a boiling water bath for 8 minutes. The tube was
vortex-mixed at high speed for 10 seconds and centri-
fuged at 10,000 g for 5 minutes. The supernatant was
used for PCR amplification.
Polymerase Chain Reaction Procedure
The PCR was carried out in 10 ␮l of reaction volumes,
using 3 or 5 ␮l of extracted DNA, 1 U of Taq DNA
polymerase (Takara Co. Ltd., Otsu, Japan), 200 mmol/L
each of deoxynucleotide, 1 mmol/L each of primer, and
2 ␮l of reaction buffer (500 mmol/L KCl, 100 mmol/L
Tris-HCl pH 8.0, 1% Triton X-100, 0.1% gelatin). The
PCR primers and tested reaction condition are shown in
Tables 1 and 2, respectively. The forward primer for
each primer pair was directly labeled with infrared flu-
orophore (5). The amplification products were processed
by the LI-COR DNA sequencer (LI-COR, Inc., Lincoln,
NE, USA) in sequence-format denaturing gels with stan-
dard electrophoretic conditions. The genotype classifica-
FIG. 1. Appearance of body found in a burned car. (A)
Charred body seen from the left side. (B) Extracted
burned teeth.
tion was obtained by side-to-side comparisons with a
homemade allelic ladder. The nomenclature adopted in
this study reflects the number of repeat units and com-
plies with ISFH recommendations (6).
Case Profile and Autopsy Findings
In July 1997, a male burned body, 50 to 60 years old,
was found in a burned car left in a dam site. The body
was entirely burned (Fig. 1). Autopsy findings showed
neither remarkable injuries nor lethal diseases. The car-
bon monoxide hemoglobin concentration was estimated
to be approximately 56%, and ethanol was not detected
in the heart blood. The charred body was identified by
comparison of antemortem dental records with postmor-
tem data.
RESULTS
Aliquots of DNA prepared from the dental pulp by the
Chelex method were amplified. A comparative study by
the heating process at various temperatures was per-
formed, using a total of 46 teeth. Sometimes we could
not recover the dental pulp from teeth incinerated at
500°C or higher (Fig. 2), because the pulp began to
carbonize at those temperatures. Table 3 shows the re-
sults of DNA amplification and typing of the dental pulp
exposed to various high temperatures for as long as 2
minutes. Despite the limited number of tooth samples,
DYS389 locus at 300°C, all samples could be amplified
and typed at up to 300°C for 2 minutes. DYZ3 and
DYS390 were fairly resistant to the effects of burning.

TABLE 2. Summary of the polymerase chain reaction amplification conditions

Predenaturation Denaturation Annealing Extension Cycles
DYZ3 94°C, 5 min 94°C, 1 min 52°C, 1 min 72°C, 2 min 30
DYS19 94°C, 5 min 94°C, 1 min 56°C, 1 min 72°C, 2 min 30
DYS389 94°C, 5 min 94°C, 1 min 55°C, 1 min 72°C, 2 min 30
DYS390 94°C, 5 min 94°C, 1 min 55°C, 1 min 72°C, 2 min 30
DYS393 94°C, 5 min 94°C, 1 min 55°C, 1 min 72°C, 2 min 30

Am J Forensic Med Pathol, Vol. 23, No. 3, September 2002

270 T. TSUCHIMOCHI, ET AL.

FIG. 3. Y-short tandem repeat genotyping of DNA from

the autopsy case. M, allelic ladder; T, polymerase chain
reaction product of DNA obtained from the dental pulp of
the cadaver.

In the autopsy case, DNA could be extracted using the

Chelex method, amplified, and genotyped as allele 17 in
DYS19, allele 15 in DYS393, and allele 24 in DYS390
(Fig. 3).

FIG. 2. Appearance of teeth incinerated experimentally in
a dental furnace for 2 minutes at various temperatures.
DISCUSSION
Chelex-based extracted DNA can be amplified by
PCR and typed. Because of the relative simplicity of the
Chelex extraction, it is anticipated that this procedure
will make the use of PCR in forensic analysis easier. The
reduction of the number of steps in sample preparation,
in which the sample is transferred, will also help reduce
the chance of operator-introduced DNA contamination
of the sample mix. The dental pulp is enclosed by hard
tissues, enamel, cement, and dentine, which means that
the dental pulp is not contaminated easily. The Chelex-
based extraction of DNA from dental pulp is suitable to
obtain DNA of superior quality for PCR amplification
and genotyping.
It should be noted that the teeth incinerated at 400°C
for 2 minutes did not yield any PCR products. These
findings agree with the results of Garcia et al. (7), in
which they amplified short tandem repeats on the auto-
somal chromosomes. The fact that the dental pulp is
enclosed by hard tissues has the advantage of preventing
heat conduction. With the exception of the alphoid repeat
and DYS390 locus, complete negative results were ob-
tained after incineration at 400°C for 2 minutes.

TABLE 3. Y-chromosomal alphoid repeat and short tandem repeat analysis of dental pulp from teeth incinerated
Control 100°C 200°C 300°C 400°C 500°C
DYZ3 7/7 7/7 7/7 7/7 2/6 0/6
DYS19 7/7 7/7 7/7 7/7 0/6 0/6
DYS389 7/7 7/7 7/7 5/7 1/6 0/6
DYS390 7/7 7/7 7/7 7/7 0/6 0/6
DYS393 7/7 7/7 7/7 7/7 0/6 0/6
Typed number/sample number.

Am J Forensic Med Pathol, Vol. 23, No. 3, September 2002

 SEX DETERMINATION OF BURNED TEETH
Dental identification is traditionally based on a com-
parison of antemortem with postmortem dental charts
and radiographs. In cases where antemortem morpho-
logic data can not be obtained, DNA typing has a re-
markable validity comparable to personal identification.
The data described here suggest that Chelex-based DNA
extraction, amplification, and typing are possible in
burned teeth in forensic autopsy cases.
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