Forensic Science International: Genetics Supplement Series xxx (xxxx) xxx–xxx

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Forensic Science International: Genetics Supplement Series

journal homepage: www.elsevier.com/locate/fsigss

DNA analysis from human skeletal remains in forensic casework

⁎
Dragana Zgonjanina,b, , Mirjana Antovc, Rashed Alghafrid, Stojan Petkovića,b,
Radenko Vukovića,b, Goran Stojiljkovića,b, Danka Toljića
a
Institute of Forensic Medicine, Clinical Center of Vojvodina, Novi Sad, Serbia
b
Faculty of Medicine, University of Novi Sad, Novi Sad, Serbia
c
Faculty of Technology, University of Novi Sad, Novi Sad, Serbia
d
General Department of Forensic Sciences and Criminology, Dubai Police G.H.Q., Dubai, United Arab Emirates

A R T I C L E I N F O A B S T R A C T

Keywords: To assess our laboratory’s success with skeletal remains and provide a benchmark for the forensic community
Forensic identification involved in identification of these remains, we retrospectively examined our ability to develop DNA profiles from
Skeletal remains the remains analyzed in our laboratory in the last 7 years. Between January 2009 and December 2016, 70 DNA
DNA extraction extractions were completed on skeletal remains from routine casework. 92% of skeletal remains analyzed were
Degraded DNA
samples submitted for body identifications by law enforcement and only 8% were samples submitted to answer
Bones
family identity or historical questions. Overall, the ability to obtain a full or partial profile primarily reflects the
difference in the average age and the condition of the samples in these two categories and thus, difference in the
quantity and quality of the DNA. We describe here the approximate age and type of remains we have received,
whether a full, partial, or no profile was obtained, as well as the condition of the samples.

1. Introduction 2. Material and methods

Typically with missing person cases, only skeletal remains are
available as an evidentiary source of DNA [1–4]. DNA analysis from
unidentified human remains involves many steps, but extraction and
optimized recovery of DNA is paramount in this process. Numerous
challenges exist with extracting DNA from bone; the structure and
chemical composition of bone make extracting and amplifying DNA
difficult and the environmental conditions from which the bone is re-
covered can dramatically alter the preservation state of the bone ma-
terial and consequently the integrity and availability of the DNA.
Our laboratory focuses exclusively on STR DNA from bone from the
beginning of our work since 2003, a powerful tool in missing person
cases. The present study describes our work on the genetic identifica-
tion of 70 skeletal remains from routine casework in the last 7 years. Of
particular interest was our work on the identification of skeletal re-
mains from several cases of criminal burning, where the intent was to
destroy the body [5] which helped to identify the victim of the murder
that shook the public and cases of remains recovered from water.
2.1. Samples and DNA extraction
This study analyzed 70 bones and teeth: 64 submitted for routine
casework body identifications by law enforcement and 6 submitted to
answer family identity. We analyzed the following samples: 55 femurs,
4 skulls, 3 tibias, 3 ribs and 5 tooth samples.
All bones were cleaned from the remnant soft tissue and all soil
traces. The cut bone fragments were washed and air dried [4]. The
resulting sample was pulverized into fine powder in mill MM 301
(Retsch).
Extraction of 40 DNA samples was carried out by the phenol
chloroform isoamyl alcohol (PCIA) organic extraction method, whereas
the extraction of 30 DNA samples was carried out by PrepFiler® BTA
Forensic DNA Extraction Kit (Applied Biosystems). In addition, both
extraction methods were carried out on 4 samples.
When genomic DNA was obtained using PCIA organic extraction
method, 2.5 g of bone powder was extracted according to Zgonjanin
et al. [5]. Extraction of DNA using PrepFiler® BTA Forensic DNA Ex-
traction Kit (Applied Biosystems) was performed using 50 mg of pow-

⁎
Corresponding author at: Institute of Forensic Medicine, Clinical Center of Vojvodina, Hajduk Veljkova 1, 21000, Novi Sad, Serbia.
E-mail address: dragana.zgonjanin@forensicns.com (D. Zgonjanin).

http://dx.doi.org/10.1016/j.fsigss.2017.09.117
Received 20 August 2017; Received in revised form 17 September 2017; Accepted 19 September 2017
1875-1768/ © 2017 Elsevier B.V. All rights reserved.

Please cite this article as: Zgonjanin, D., Forensic Science International: Genetics Supplement Series (2017),
http://dx.doi.org/10.1016/j.fsigss.2017.09.117
D. Zgonjanin et al. Forensic Science International: Genetics Supplement Series xxx (xxxx) xxx–xxx

Table 1
Nuclear DNA quantity; the efficiency of autosomal STR) typing (AmpFℓSTR® Identifiler® and AmpFℓSTR® NGM™ Amplifiction Kit) expressed as the number of successfully typed STRs;
efficiency of Y-STR typing (AmpFℓSTR® Yfiler®), expressed as the number of successfully typed Y-STRs; and efficiency of mtDNA typing (HVI and HVII) in bones and teeth from 70 cases
human identification cases.

Bone sample Q/Casework Quantity (ng/μL) Efficiency of DNA typing by

AmpFℓSTR® Identifiler AmpFℓSTR® NGM™ AmpFℓSTR® Yfiler®

Femur m (Case #1)a 1.05 16/16 16/16 17/17

Femur m (Case #2)a 1.28 16/16 16/16 17/17
Skull m (Case #3)a 0.62 16/16 16/16 17/17
Femur m (Case #4)a 9.20 16/16 16/16 17/17
Femur m (Case #5)a 0.29 16/16 16/16 17/17
Femur m (Case #6)a 1.02 16/16 16/16 17/17
Femur w (Case #7)a 234.95 16/16 16/16 –
Femur w (Case #8)a 0.47 16/16 16/16 –
Femur m (Case #9)a 93.73 16/16 16/16 17/17
Tibia m (Case #10)a 0.69 16/16 16/16 17/17
Femur w (Case #11)a 1.18 16/16 16/16 –
Femur w (Case #12)a 4.10 16/16 16/16 –
Femur w (Case #13)a 0.92 16/16 16/16 –
Femur w(Case #14)a 0.32 16/16 16/16 –
Femur m (Case #15)a 0.44 16/16 16/16 17/17
Femur m (Case #16)a 0.59 16/16 16/16 17/17
Femur m (Case #17)a 207.40 16/16 16/16 17/17
Rib m (Case #18)a 1.78 16/16 16/16 17/17
Femur m (Case #19)a 12.11 16/16 16/16 17/17
Femur w (Case# 20)a 0.69 10/16 13/16 –
Tooth w (Case #21)a 1.75 16/16 16/16 –
Femur m (Case #22a 1.14 16/16 16/16 17/17
Tooth w (Case #23)a 0.001 – – –
Femur m (Case #24)a 1.24 16/16 16/16 17/17
Femur w (Case #25)a 1.33 16/16 16/16 mtDNA HV1,2
Femur m (Case #26)a 373.76 16/16 16/16 17/17
Femur w (Case #27)a 0.35 16/16 16/16 –
Femur w (Case #28)a 185.26 16/16 16/16 –
Femur m (Case #29)a 2.11 16/16 16/16 17/17
Femur m (Case #30)a 0.039 16/16 16/16 17/17
Femur m (Case #31)a 0.53 16/16 16/16 17/17
Femur m (Case #32)a 0.098 16/16 16/16 17/17
Femur m (Case #33)a 0.13 16/16 16/16 17/17
Femur m (Case #34)a 0.0002 – – –
Tibia w (Case #35)a 0.164 16/16 16/16 –

Bone sample Q/Casework Quantity (ng/μL) Efficiency of DNA typing by

AmpFℓSTR® Identifiler AmpFℓSTR® NGM™ AmpFℓSTR® Yfiler®

Femur m (Case #36)a 0.019 16/16 16/16 17/17

Rib m (Case #37)a 0.180 16/16 16/16 17/17
Femur m (Case #38)a 0.555 16/16 16/16 17/17
Skull m (Case #39)a 0 – – –
Femur w (Case #40)a,b 70.48 16/16 16/16 –
Femur m (Case #41)a,b 150.61 16/16 16/16 17/17
Tooth w (Case #42)a,b 0.041 16/16 16/16 –
Femur w (Case #43)a,b 0.011 16/16 16/16 –
Femur m (Case #44)b 0.055 16/16 16/16 17/17
Femur m (Case #45)b 1.82 16/16 16/16 17/17
Femur w (Case #46)b 0.018 16/16 16/16 –
Tooth m (Case #47)b 0.117 16/16 16/16 17/17
Femur m (Case #48)b 0.902 16/16 16/16 17/17
Femur w (Case #49)b 0.045 16/16 16/16 –
Femur m (Case #50)b 0.087 16/16 16/16 17/17
Femur w (Case #51)b 14.61 16/16 16/16 –
Femur m (Case #52)b 35.36 16/16 16/16 17/17
Femur w (Case #53)b 0.133 16/16 16/16 –
Femur w (Case #54)b 2.115 16/16 16/16 –
Rib m (Case #55)b 0.280 16/16 16/16 17/17
Femur w (Case #56)b 0.209 10/16 14/16 –
Tibia m (Case #57)b 0.075 16/16 16/16 17/17
Skull m (Case #58)b 0.162 16/16 16/16 17/17
Tooth m (Case #59)b 0.098 16/16 16/16 17/17
Femur m (Case #60)b 7.582 16/16 16/16 17/17
Femur m (Case #61)b 0.351 16/16 16/16 17/17
Femur w (Case #62)b 0.922 16/16 16/16 –
Femur w (Case #63)b 2.111 16/16 16/16 –
Femur m (Case #64)b 0.390 16/16 16/16 17/17
(continued on next page)

2
D. Zgonjanin et al.
Table 1 (continued)
Bone sample Q/Casework Quantity (ng/μL) Efficiency of DNA typing by
AmpFℓSTR® Identifiler
Femur w (Case #65)b 0.533 16/16
Skull m (Case #66)b 0.145 16/16
Femur w (Case #67)b 2.92 16/16
Femur m (Case #68)b 79.99 16/16
Femur m (Case #69)b 76.07 16/16
Femur w (Case #70)b 0.060 16/16
DNA Extraction Kit; m-man; w-woman.
a
PCIA organic extraction method.
b
PrepFiler® BTA Forensic.
dered bone following the protocol recommended by the manufacturer.
Attempts were made to improve the DNA recovery in some challenging
bone samples by overnight incubation, up to 18 h, of bone samples in
lysis buffer solution component during lysis step. Moreover, another
modification was made by adding 3 μl 1 M DTT in addition to 300 μl
PrepFiler® Lysis Buffer with incubation at 56 °C and 900 rpm for
45 min.
Family reference samples accompanying the skeletal remains are
usually obtained from buccal swabs or dried blood samples using the
QIAamp DNA Micro Kit (QIAGEN).
2.2. PCR amplification and typing
DNA was quantified with an ABI Prism® 7000 Sequence Detection
System (Applied Biosystems) using Quantifiler™ Human DNA
Quantification kit. Amplifications were performed on the GeneAmp
PCR System 9700 Gold Plate (Applied Biosystems) using the
AmpFℓSTR® Identifiler® (Applied Biosystems), AmpFℓSTR® NGM™
(Applied Biosystems) and AmpFℓSTR® Yfiler® (Applied Biosystems)
following the manufacturers’ protocols. Amplified product are sepa-
rated and detected on ABI Prism® 310 Genetic Analyzer (Applied
Biosystems) and ABI 3500 Genetic Analyzer (Applied Biosystems).
3. Results
3.1. Sample types
Seventy DNA extractions were completed on skeletal remains from
routine casework and the results are presented in Table 1. Full profiles
were obtained on 65 samples (92.8%), partial profiles were obtained for
2 samples (2.8%), and no profiles were obtained for 3 samples (4.3%).
Femurs were the most common samples, and if available, these were
used in all cases due to an overall high rate of obtaining profiles.
In two bones (Case #20 and #56), with partial profiles, the loci that
were not amplified were primarily the longest loci D2S1338, D18S51,
and FGA. With this bone samples in 13 out of 16 (Case #20) and 14 out
of 16 (Case #56) loci available in the AmpFℓSTR® NGM™, including 5
loci in the extended European Standard Set (ESS) were successfully
amplified, while applying AmpFℓSTR® Identifiler® kit obtained only 10
out of 16 loci in both cases. Typing of low-level DNA samples from
casework with new ESS amplification kits also showed better results in
comparison with older amplification kits [6,7].
As for the exposure of examined samples of skeletal remains to
different environmental influences samples can be categorized into
three larger groups: burned bodies (21.9%), remains recovered from the
water (28.1%) and remains recovered from the fields (50.0%). In this
lab, nine extraction attempts of skeletal remains from nine cases of
criminal burning, where the intent was to destroy the body, were suc-
cessful. Cases of remains recovered from water resulted in full profiles.
When comparing genetic profiles, we matched 66 of the 70 skeletal
remains analyzed to accompanying reference sample with high
3
Forensic Science International: Genetics Supplement Series xxx (xxxx) xxx–xxx
AmpFℓSTR® NGM™ AmpFℓSTR® Yfiler®
16/16 –
16/16 17/17
16/16 –
16/16 17/17
16/16 17/17
16/16 –
confidence of correct identification for all 66 victims (probability from
99.9% to 99.999999%).
3.2. Body identification cases versus historical cases
Of the 70 skeletal remains from which DNA was extracted in our
laboratory, 64 (91.43%) were submitted for body identification by law
enforcement and 6 (8.57%) for questions of historical interest or by
private individuals. Overall, body identification cases were more likely
to yield a full profile whereas historical cases were more likely to result
in partial profiles. Most samples in historical cases were greater than 50
years of age, whereas most of the samples in body identifications were
less than 15 years of age.
In addition, most of the samples in body identifications were less
than 1 year of age (40 samples, 62.5%), 18 samples (28.1%) were from
1 up to 5 years of age, 5 samples (7.8%) were from 5 up to 10 years of
age, whereas the only one sample (1.5%) was from 10 to less than 15
years of age.
4. Discussion
Our results showed that the PrepFiler® BTA Forensic DNA Extraction
Kit can yield both DNA quantity and STR profiles comparable or greater
to that of the standard organic extraction method.
Simple modifications to extraction techniques can dramatically
improve DNA typing success and provide conclusive, reliable profiles
using different amplification kits even when working with difficult
samples. Our experience has shown, in accordance with the experiences
of others [6,7], that the AmpFℓSTR® NGM™ with the ESS loci is more
tolerant to common inhibitors, which enabled us to overcome the
challenges associated with processing compromised skeletal remains.
5. Conclusion
The age and condition of the sample are, in the most cases, corre-
lated with success in developing a DNA profile but it was shown that is
not a general rule [5–7].
Advanced extraction and purification techniques were found to be
essential tools for obtaining sufficient DNA from bones and teeth ske-
letal remains from routine casework in our laboratory. Extraction and
purification methods using the PrepFiler® BTA Forensic DNA Extraction
Kit, together with more sensitive and robust new amplification kits with
the ESS loci allowed us to overcome the challenges associated with
processing compromised skeletal remains and ultimately obtain STR
DNA profiles in 96% of the bones and teeth.
Conflict of interest statement
None.
D. Zgonjanin et al.
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