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ORIGINAL ARTICLE
DNA Profiling Lab., Department of Forensic Medicine, All India Institute of Medical Sciences, New Delhi, India
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).
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.
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 A. Balayan et al.
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 This study also demonstrated that the multiplex PCR is a
4 C (Fig. 5) shows partial amplification, and hence is unable reliable method for the DNA analysis of the postdecalcifica-
to amplify large DNA fragments. tion material. The sample may also be used for STR typing
using multiplex PCR to perform DNA profiling of human
4. Discussion bone powder. Here we conclude that the multiplex PCR
approach may constitute a good option, especially when given
The aim of this study was to compare the effects of two differ- a large amount of sample material (e.g., mass graves) or when
ent decalcification techniques. We used the decalcification given time-windows are narrow.
agent EDTA in two different concentrations (0.5 M and However, due to the inherent risk of creating artifacts
0.25 M) at two different temperatures (37 C and 4 C). The using PCR, it is advisable to be cautious and to confirm
results unequivocally show that 0.5 M concentration of EDTA results by analysing proper DNA extracts parallel to STR
gives better results than the 0.25 M EDTA (Table 1) (Fig. 2). typing by using multiplex PCR before arriving at a final
Various attempts have been made earlier to standardize the conclusion.
decalcification procedure of bone, viz., three commercially
available decalcifying agents including one EDTA-based and Funding
two hydrochloric acid-based solutions (S/P decal, RBD).
Vers-enate showed good results as compared to S/P decal, We hereby acknowledge AIIMS, New Delhi and investigating
RBD.6 Similar findings were observed by Walsh et al., who agencies for funding this study.
compared mRNA ISH, using nitric acid, formic acid and
EDTA, of which EDTA was found to be the better decalcify- Conflict of interest
ing agent.7
At present, EDTA as a decalcifying agent is widely accept- None declared.
ed. However, several modifications have been made such as
decalcification in EDTA using a microwave oven,8 addition
Ethical approval
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 Necessary ethical approval was obtained from the Institute
bone sample. Ethics Committee.
Evaluation of techniques for human bone decalcification and amplification 35
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