Egyptian Journal of Forensic Sciences (2015) 5, 30–35

H O S T E D BY Contents lists available at ScienceDirect

Egyptian Journal of Forensic Sciences

journal homepage: www.ejfs.org

ORIGINAL ARTICLE

Evaluation of techniques for human bone

decalcification and amplification using sixteen
STR markers
Ajay Balayan, Abhilasha Kapoor, Garima Chaudhary, Anupuma Raina *

DNA Profiling Lab., Department of Forensic Medicine, All India Institute of Medical Sciences, New Delhi, India

Received 30 July 2013; revised 21 March 2014; accepted 5 May 2014

Available online 14 June 2014

KEYWORDS Abstract Efficient DNA extraction procedures, as well as accurate DNA amplification, are critical
Decalcification; steps involved in the process of successful DNA analysis of skeletal samples. Unfortunately, at pre-
Sternum bone; sent there is no infallible method to recover DNA from highly degraded samples due to variations in
Short tandem repeats; DNA yield from larger bone fragments, which may be attributed to heterogeneity within bones. We
Multiplex PCR; evaluated two different protocols for bone decalcification in the DNA extraction procedure for
Genotyping bones. This study is important for analysis of challenging forensic samples.
ª 2014 Hosting by Elsevier B.V. on behalf of The International Association of Law and Forensic Sciences
(IALFS).

1. Introduction commonly available biological samples. Bone is a complex,

Since inception of the DNA fingerprinting technique by
Jeffreys et al. in 1985 it has become a powerful tool in medi-
co-legal cases. The development and validation of new tech-
nology for detection of DNA polymorphisms have been very
rapid. Over the last twenty years DNA profiling has become
an important method for forensic human identification, in par-
ticular by introducing the study of microsatellite regions –
Short Tandem Repeat (STR) loci – in criminal cases as well
as in civil cases.1
In cases like missing personal identification, mass disaster
and ancient DNA investigation, bone and teeth are the most
* Corresponding author. Tel.: +91 9868397147.
E-mail address: anupumaraina@gmail.com (A. Raina).
Peer review under responsibility of The International Association of
Law and Forensic Sciences (IALFS).
http://dx.doi.org/10.1016/j.ejfs.2014.05.002
2090-536X ª 2014 Hosting by Elsevier B.V. on behalf of The International Association of Law and Forensic Sciences (IALFS).
highly organized and specialized connective tissue. The major-
ity of DNA in the bone is located in the osteocytes; a micro-
gram quantity of DNA could potentially be extracted from a
gram of bone.2,3
We extract DNA using different decalcification protocols
for the sternum bones, which are more than 20 years old. This
method consists of separation of DNA from proteins and
waste material by using a phenol–chloroform mixture.
Moreover, the recovery of information from these degraded
samples is enhanced by the use of STR (Short Tandem
Repeats) typing by multiplex PCR.4
2. Materials and methods
The present study was conducted on ten sternum bone sam-
ples. All the samples were cleaned thoroughly using sandpaper
Evaluation of techniques for human bone decalcification and amplification 31

0.5 M EDTA 0.25 M EDTA 2.2. DNA purification

4° C 37° C 4° C 37° C
DNA from dried bone powder samples was purified using a
Nanosep centrifugal device. DNA was purified by employing
centrifugation for 10 min at 10,000 rpm for 3 times to get a
better yield.

2.3. Agarose gel electrophoresis

Extracted DNA from bone powder was allowed to run on 1%

agarose gel with 1· TBE buffer to check the quality of the
extracted DNA (Fig. 1). Quantification was performed using
an automatic UV spectrophotometer (Table 3).

Table 2 Loci amplified with AmpFlSTR Identifiler PCR

Amplification Kit, the range of PCR products expressed in base
pair and the corresponding dyes used.
Figure 1 The agarose gel picture of extracted DNA from one Locus Range of PCR product sizes (bp) Dye label
sample. D8S1179 123–169 6-FAM
D21S11 185–240
to remove potential contamination. Bones were crushed to D7S820 255–291
bone powder for further processing of the samples. CSF1PO 305–341
D3S1358 112–140 VIC
2.1. Decalcification TH01 163–202
D13S317 217–245
D16S539 252–292
We used two different methods for decalcification of bone D2S1338 307–359
using two different concentrations of the ethylene diamine D19S433 102–135 NED
tetra-acetic acid (EDTA) buffer (0.5 M EDTA and 0.25 M TPOX 222–250
EDTA) and two temperatures (37 C and 4 C) for decalcifica- D18S51 262–346
tion of the bone powder. These samples were incubated at two Amelogenin 106/112 PET
different temperatures (37 C and 4 C) for 7 days with daily D5S818 134–172
changes of EDTA buffer DNA extraction. FGA 215–355
DNA extraction was performed using the organic extrac-
tion method proposed by Sambrook et al. After 7 days the
tubes were centrifuged, the supernatant was discarded and
the remaining decalcified pellet was extracted using the organic
extraction method.5
Table 3 Concentration of different bone samples using UV
spectrophotometer.
Table 1 Genotype profile of sample analyzed.
Samples Conc./Temp 260 nm 280 nm Ratio Quantity
Marker 0.5 M 0.5 M 0.25 M 0.25 M
EDTA EDTA EDTA EDTA Bone 1 0.5 M (37 C) 7.1 29.0 1.5 235
37 C 4 C 37 C 4 C 0.5 M (4 C) 5.7 3.7 1.6 288
* * 0.25 M (37 C) 4.0 2.4 1.6 200
D8S1179 12, 13 12, 13
* * 0.25 M (4 C) 0.5 0.3 1.5 29
D21S11 29, 31.2 29, 31.2
* * * Bone 2 0.5 M (37 C) 2.1 1.1 1.7 109
D7S820 8, 12
* * 0.5 M (4 C) 2.7 1.4 1.6 135
CSF1P0 12 12
* * 0.25 M (37 C) 2.4 1.5 1.7 115
D3S1358 15, 16 15, 16
0.25 M (4 C) 1.8 1.5 1.5 120
THO1 6, 9.3 6, 9.3 6, 9.3 6, 9.3
* * Bone 3 0.5 M (37 C) 2.2 1.4 1.7 144
D13S317 10 10
* * 0.5 M (4 C) 2.7 1.4 1.5 165
D16S539 9, 12 9, 12
* * 0.25 M (37 C) 3.5 2.2 1.8 179
D2S1338 23 23
* * 0.25 M (4 C) 2.0 1.8 1.5 190
D19S433 12, 15 12, 15
* * Bone 4 0.5 M (37 C) 5.0 2.5 1.9 251
vWA 15, 17 17
* * 0.5 M (4 C) 5.7 3.9 1.4 286
TPOX 8, 11 8, 11
* * * 0.25 M (37 C) 7.2 8.1 1.7 186
D18S51 15, 17
0.25 M (4 C) 3.7 1.9 1.9 189
Amelogenin X, X X, X X, X X, X
* * Bone 5 0.5 M (37 C) 0.9 0.5 1.9 496
D5S818 11 11
* * 0.5 M (4 C) 1.3 0.7 1.8 65
FGA 21, 23 21, 23
0.25 M (37 C) 2.9 3.4 0.8 147
*
No amplification at the particular locus. 0.25 M (4 C) 1.81 0.96 1.2 90
32 A. Balayan et al.

16 PCR amplifications were performed in reaction volumes of

14
12
10 0.5 M(37)
8 0.5 M(4)
6 0.25 M(37)
0.25 M(4)
4
2 2.5. Sample preparation
0
1 2 3 4 5  Samples were prepared for electrophoresis on the 3130
Figure 2 Graph showing number of alleles amplified on the Y
axis and number of samples on the X axis.
2.4. DNA amplification
DNA amplification was performed using an AmpFlSTR
Identifiler PCR Amplification Kit (Applied Biosystems),
genotyping was done using a genetic Analyzer ABI 3130
(Applied Biosystems, Foster City, USA) and analysis was done
using the Gene Mapper ID 3.2 software. Negative and positive
controls were used for quality management.
The Amplification Kit is an STR multiplex assay that ampli-
fies 15 tetranucleotide repeat loci and the Amelogenin gender
determining marker in a single PCR amplification (Table 2).
25 ll using 10.5 ll of the PCR Reaction Mix, 0.5 ll of Ampli-
Taq Gold DNA polymerase, 5.5 ll of AmpFlSTR Identifiler
Primer Set. After being vortexed for 5 s, 15 ll of the master
mix was dispensed in each PCR tube and 10 ll of the DNA
sample having a concentration of 0.125 ng/ll was added. Ther-
mal cycling was performed using the following conditions:
95 C for 11 min, 94 C for 1 min, 59 C for 1 min, 72 C for
1 min, 28 cycles, 60 C for 7 min, 4 C forever.
Genetic Analyzer from Applied Biosystems, using 8.75 ll
of Hi-Di formamide, 0.25 ll of the LIZ Size Standard and
1 ll of PCR products.
 The reaction plate was heated in a thermal cycler for 5 min
at 95 C and then cooled to 4 C to ensure that the
denaturation process had occurred.
 Denatured samples were run in a genetic analyzer using
POP-4 (Polymer for electrophoresis) which provides a siev-
ing matrix for the separation of DNA.
3. Results
Bone powder decalcified with 0.5 M EDTA and incubated at
37 C (Fig. 3) and 4 C (Fig. 4) shows amplification on all 16
loci using a multiplex PCR kit whereas bone powder decalci-

Figure 3 The electropherogram showing the DNA profile using 0.5 M EDTA at 37 C decalcification protocol.
Evaluation of techniques for human bone decalcification and amplification 33

Figure 4 The electropherogram showing the DNA profile using 0.5 M EDTA at 4 C decalcification protocol.

Figure 5 The electropherogram showing the DNA profile using 0.25 M EDTA at 4 C decalcification protocol.
34
Figure 6 The electropherogram showing the DNA profile using 0.25 M EDTA at 37 C decalcification protocol.
fied with 0.25 M EDTA and incubated at 37 C (Fig. 6) and
4 C (Fig. 5) shows partial amplification, and hence is unable
to amplify large DNA fragments.
4. Discussion
The aim of this study was to compare the effects of two differ-
ent decalcification techniques. We used the decalcification
agent EDTA in two different concentrations (0.5 M and
0.25 M) at two different temperatures (37 C and 4 C). The
results unequivocally show that 0.5 M concentration of EDTA
gives better results than the 0.25 M EDTA (Table 1) (Fig. 2).
Various attempts have been made earlier to standardize the
decalcification procedure of bone, viz., three commercially
available decalcifying agents including one EDTA-based and
two hydrochloric acid-based solutions (S/P decal, RBD).
Vers-enate showed good results as compared to S/P decal,
RBD.6 Similar findings were observed by Walsh et al., who
compared mRNA ISH, using nitric acid, formic acid and
EDTA, of which EDTA was found to be the better decalcify-
ing agent.7
At present, EDTA as a decalcifying agent is widely accept-
ed. However, several modifications have been made such as
decalcification in EDTA using a microwave oven,8 addition
of ammonium hydroxide to the EDTA,9 electrolyte decalcifica-
tion10 etc. But our study demonstrated the use of only EDTA
it was possible to extract ample amounts of DNA from the
bone sample.
A. Balayan et al.
This study also demonstrated that the multiplex PCR is a
reliable method for the DNA analysis of the postdecalcifica-
tion material. The sample may also be used for STR typing
using multiplex PCR to perform DNA profiling of human
bone powder. Here we conclude that the multiplex PCR
approach may constitute a good option, especially when given
a large amount of sample material (e.g., mass graves) or when
given time-windows are narrow.
However, due to the inherent risk of creating artifacts
using PCR, it is advisable to be cautious and to confirm
results by analysing proper DNA extracts parallel to STR
typing by using multiplex PCR before arriving at a final
conclusion.
Funding
We hereby acknowledge AIIMS, New Delhi and investigating
agencies for funding this study.
Conflict of interest
None declared.
Ethical approval
Necessary ethical approval was obtained from the Institute
Ethics Committee.
Evaluation of techniques for human bone decalcification and amplification
References
1. Jeffreys AJ, Brookfield JFY, Semeonoff R. Positive identification
of an immigration test-case using human DNA fingerprints.
Nature 1985;317:818–9.
2. Butler JM. Forensic DNA typing: biology, technology, and genetics
of STR markers. 2nd ed. New York: Elsevier Academic Press;
2005.
3. Hochmeister MN, Budowle B, Borer UV, Eggmann U, Comey R,
Dirnhofer R. Typing of deoxyribonucleic acid. (DNA) extracted
from compact bone from human remains. J Forensic Sci
1991;36:1649–61.
4. Collins MJ, Nielsen-Marsh CM, Hiller J, Smith CI, Roberts JP,
Prigodich RV. The survival of organic matter in bone: a review.
Archaeometry 2002;44:383–94.
5. Sambrook J, Fritch EF, Maniatis T. Molecular cloning. A
laboratory manual. 2nd ed. New York (NY): Cold Spring Har-
bour Laboratory Press; 1989.
35
6. Arber JM, Weiss LM, Chang KL, Battifora H, Arber DA. The
effect of decalcification on in situ hybridization. Mod Pathol
1997;10:1009–14.
7. Walsh L, Freemont AJ, Hoyland JA. The effect of tissue
decalcification on mRNA retention within bone for in situ
hybridization studies. Int J Exp Pathol 1993;74:237–41.
8. Hellstrom S, Nilsson M. The microwave oven in temporal bone
research. Acta Otolaryngol Suppl 1992;494:15–8.
9. Sanderson C, Radley K, Mayton L. Ethylenediaminetetraacetic
acid in ammonium hydroxide for reducing decalcification time.
Biotech Histochem 1995;70:12–8.
10. Loyson SA, Rademakers LH, Joling P, Vroom TM, Vandentweel
JG. Immuno-histochemical analysis of decalcified paraffin embed-
ded human bone marrow biopsies with emphasis on MHC class I
and CD34 expression. Histopathology 1997;31:412–9.