Forensic Science International 284 (2018) 167–175

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Forensic Science International

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

Original Research Article

Forensic genetic analysis of bone remain samples

T. Siriboonpiputtanaa , T. Rinthachaia , J. Shotivaranona , V. Peonimb ,
B. Rerkamnuaychokea,*
a
Human Genetics Laboratory, Department of Pathology, Faculty of Medicine, Ramathibodi Hospital, Mahidol University, Bangkok 10400, Thailand
b
Forensic Medical Laboratory, Department of Pathology, Faculty of Medicine, Ramathibodi Hospital, Mahidol University, Bangkok 10400, Thailand

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

Article history:
Received 14 July 2017 DNA typing from degraded human remains is still challenging forensic DNA scientists not only in the
Received in revised form 16 October 2017 prospective of DNA purification but also in the interpretation of established DNA profiles and data
Accepted 28 December 2017 manipulation, especially in mass fatalities. In this report, we presented DNA typing protocol to
Available online 8 January 2018 investigate many skeletal remains in different degrees of decomposing. In addition, we established the
grading system aiming for prior determination of the association between levels of decomposing and
Keywords: overall STR amplification efficacy. A total of 80 bone samples were subjected to DNA isolation using the
Forensic genetics modified DNA IQTM System (Promega, USA) for bone extraction following with STR analysis using the
DNA typing
AmpFLSTR Identifiler1 (Thermo Fisher Scientific, USA). In low destruction group, complete STR profiles
Bone remain
were observed as 84.4% whereas partial profiles and non-amplified were found as 9.4% and 6.2%,
Degraded DNA
respectively. Moreover, in medium destruction group, both complete and partial STR profiles were
observed as 31.2% while 37.5% of this group was unable to amplify. Nevertheless, we could not purify DNA
and were unable to generate STR profile in any sample from the high destroyed bone samples. Compact
bones such as femur and humerus have high successful amplification rate superior than loose/spongy
bones. Furthermore, costal cartilage could be a designate specimen for DNA isolation in a case of the body
that was discovered approximately to 3 days after death which enabled to isolate high quality and
quantity of DNA, reduce time and cost, and do not require special tools such as freezer mill.
© 2018 Elsevier B.V. All rights reserved.

1. Introduction belongings; the matching of fingerprints (provided pre-mortem

Human identification strategies of decomposing samples are
still challenging forensic pathologists, forensic odontologists and
forensic geneticists in our world today. In some extreme conditions
such as natural disasters (e.g. earth quakes, tsunami, volcanoes,
and avalanches) and human made catastrophes (e.g. wars,
terrorists, political crisis, plane clashes, and bombings), the degree
of destroyed/decomposed samples largely depends on the specific
type of natural casualty and the difference in tactics of the criminal
to hide/destroy the crime scene evidences [1,2]. Many methods
have been used to identify human remains depending on the
circumstances and the state of remains. The common human
identification methods are including; the victim data giving by
living witnesses or deceased relatives such as direct facial and
special feature recognitions, tattoo, scar or mark and their
* Corresponding author.
E-mail address: budsaba.rer@mahidol.ac.th (B. Rerkamnuaychoke).
inked prints are available) or matching of dental profiles (provided
pre-mortem dental records are available) [3]. Consequently, these
techniques as described above are required for the comparison
between huge and informative ante-mortem (AM) data and the
post-mortem (PM) data of the remains. However, in most mass
casualty cases and missing person identification, AM information
is not available or less informative for several victims to generate
the match. Moreover, in extremely disasters such as 9/11 and
MH37, the physical appearances of several victims were extremely
destroyed and the organs of victims were not intact to their bodies.
Thus, using those data to identify human remains in case of mass
catastrophes and decomposed samples are very difficult and still
stimulating forensic scientists.
DNA typing methods have been used in forensic laboratories
worldwide for human identification as well in mass fatalities (e.g.
9/11 World Trade Center Attack, USA; 2001, India ocean Tsunami;
2004). The stunning roles of DNA typing technologies for human
identification in extreme mass catastrophes and high degree of
decomposing samples are including the test is not restricted to any

https://doi.org/10.1016/j.forsciint.2017.12.045
0379-0738/© 2018 Elsevier B.V. All rights reserved.
168 T. Siriboonpiputtana et al. / Forensic Science International 284 (2018) 167–175
particular one to one body landmark comparison (e.g. friction ridge
details in fingerprints) and DNA profile matching can be conducted
to associate separated remains or body parts. In addition, DNA
typing techniques are able to identify human remained samples in
a variety degree of destroyed status. Moreover, low amount of
samples is required and many types of sample (blood, hair, nail,
tissue, bone etc.) could be selected as a source for DNA isolation
depending on forensic scenarios. DNA typing is a very rapid test
and can be performed and analyzed by automatic machines.
Furthermore, the applications of population DNA database and
biostatistics calculation for missing person scenario help forensic
geneticists have more confidence to generate and report the DNA
matching results [1].
DNA as genetic material encodes many crucial proteins to drive
human being through a life. Human remains such as body fluid,
bone, teeth, and hair are the source of DNA. Recently, many reports
have been focused on the development of a powerful method to
isolate a tiny amount of DNA molecules from remain samples. In
several forensic cases, human remains including bone samples
have been found on the ground in different degree of decomposi-
tion, embedded in soil for some years or immersed in water or sea.
Many factors that commonly inhibit PCR amplification of DNA
isolated from remained samples are following, the originate
environment of the remains, hydroxyapatite and contamination
from another organism (e.g. bacteria or fungi). Three common
sources of contamination in DNA extraction from bone samples are
including co-extracted surface contamination, contamination
during laboratory practice, and PCR carryover. Therefore, the
preliminary cleaning processes of decomposing samples, decon-
taminating and removing PCR inhibitor are necessary to perform
the successful PCR amplification and generate the complete DNA
profile. Various methods to remove PCR inhibitors and decontam-
ination have been reported such as surface washing, surface
removing by physical methods, interior part extraction, surface
washing with acid, irradiating with UV light, exposing to high
concentrated ethanol, exposing to a bleach (NaOCl), and the
combinations of those above techniques. The criteria to choose a
decontamination method are largely depending on the laboratory
personal experiences, the nature of contaminations, time, cost and
previous reports [2]. Moreover, laboratory experiences, specialized
equipment such as cutting, sanding and powdering tools as well as
freezer mill are still necessary to generate the appropriate amount
of started bone material for efficient DNA isolation. In addition, to
avoid contamination occurred, the scientists should have a
separate area for dealing with bone, teeth and decomposed
samples from other laboratory function [2]. Several effective
methods to extract DNA from decomposed human samples have
been reported in various publications. Those are including phenol-
chloroform extraction [4–8], silica-based extraction [7,9–13],
chelex extraction [14–16], and commercial kits as well as QIAamp1
DNA mini Kit (QIAGEN) and DNA IQTM (Promega, USA). In this
report, we proposed routinely effective DNA extraction and
amplification protocol for human identification from decomposed
bone samples registered from 2007 to 2014.
2. Materials and methods
2.1. Samples
A total of 80 human skeletal samples in variety degrees of
decomposing were included in this study. Samples were grouped
into three categories based on the degrees of decomposing; high
(cannot identify bone origin), medium (bone fragments of known
origin) and low (complete bones) degree. This criteria was also
including the physical examination such as colors, burned marks,
water or soil immerged bone.
2.2. Pretreatment and decontamination
Bone samples were carried out under the sterile condition using
groves, mask and separated working areas (biosafety cabinets).
Bone surface material was removed by using surgical blade and
washed with sterile water for three times and finally washed with
95% ethanol. The cleaned bones were dried in an incubator at 56  C
for overnight.
2.3. Bone powdering
Bone samples were divided to approximately 0.5–1.0 cm long
by using electrical bone surgery instrument. Bone powder was
generated by using Freezer/Mill1, model 6750 (Spex/Mill, Spex,
Metuchen, NJ) and weighted to 0.5–2.0 g in 15 ml centrifuge tube.
To prevent contamination, before cutting the next bone sample,
the electrical bone surgery instrument was cleaned with 70%
ethanol and subsequently decontaminated with UV treatment.
Additionally, the surgical blade was detached and washed with
water, 70% ethanol, treated with UV and autoclaved prior used.
2.4. DNA extraction
DNA extraction was applied from the bone extraction protocol
with DNA IQTM System (Promega, USA) as following, add decal-
cified 0.5–2.0 g bone powder in 4 ml of bone incubation buffer
(10 mM Tris pH 8.0, 100 mM NaCl, 50 mM EDTA, 0.5% SDS, DW to
200 ml), incubate at 56  C for overnight, and remove the remaining
bone powder by centrifugation at 4000 rpm for 10 min. Then,
follow modified DNA IQ procedure, November 2001 (split lab) to
isolate DNA from the supernatant.
2.5. DNA quantification
The extracted DNA quantity was measured by the QuantifilerTM
Human DNA Quantification Kit (Life Technologies), and worked
with ABI7500 Fast Real-Time PCR (Life Technologies).
2.6. DNA purification and concentration
Some extracted DNA solution with quantity lower than
0.1 ng/ml were passed through YM-100 MICROCON1 (Millipore,
USA) to concentrate the DNA amount and to remove non-requisite
materials in DNA solution that can impact on PCR amplification
process.
2.7. PCR amplification and genotyping
The 28-cycle standard multiplex STR analysis recommended
from the AmpFlSTR Identifiler PCR Amplification kit (Life
Technologies) was performed on samples with DNA concentration
higher than or equal to 0.1 ng/ml. The low copy number (LCN)
protocol with 32-cycle combined with double amount of AmpliTaq
Gold1 (Life Technologies) was performed on samples with DNA
amount lower than 0.1 ng/ml. A total 25 ml of PCR reaction was
amplified on GeneAmp1 PCR System 9700 thermal cycler (Life
Technologies). Genotyping was performed with 3130 Genetic
Analyzer and analyzed by GeneMapper1 software (Life Technolo-
gies).
2.8. Analysis of data
The acceptance criteria of DNA profiles were concordant to the
standard operating procedure (SOP) obtained from various DNA
laboratories in Thailand (Thailand Tsunami Victim Identification;
TTVI) and used at the Information Management Center (IMC) at
 T. Siriboonpiputtana et al. / Forensic Science International 284 (2018) 167–175
Phuket province, Thailand during December 2004 to February
2006. The acceptance criteria were following, minimum RFU
thresholds for allele discrimination of heterozygous peaks were
3:1 and 4.5:1 for homozygous peaks, maximum peak imbalance
was 50%, maximum signal that demonstrated exceeds fluorescent
saturation point should not be represented, all control materials
(positive control DNA, extraction bank and amplification bank)
should have correct results, all of the internal lane standard should
be properly labeled and no artificial peaks presented, mixture
profiles should not be present, artificial peaks (pull-up, spike peak,
stutter) should not be observed, and the minimum number of
common locus acceptance were eight. However, incomplete
profiles with less than eight loci may be accepted based on other
factors such as statistical significance, and the present of rare
alleles.
2.9. Quality control
The laboratory has participated in proficiency testing with the
College of American Pathologists (CAP) and achieved ISO/IEC 17025
accreditation since 2013.
3. Result
The total of 80 human bone remained samples were graded into
three groups (Table 1) based on the degree of decomposing as
described in the previous section. To determine the optimal
amount of bone powder that could provide a finest DNA yield after
extraction with the proposed protocol, DNA isolation was
performed on different amount of bone powder including 0.5,
0.75, 1.0, 1.5, and 2.0 g, respectively. The optimal weight of bone
powder that was able to give the highest yield of DNA (0.9 ng/ml)
and sufficient to generate a complete DNA profile after amplifica-
tion with the AmpFLSTR Identifiler1 kit was 0.75 g (Figs. 1 and 2).
This finding indicated that the proposed protocol represents as a
very highly sensitive method for the isolation of DNA from
decomposed bone specimen which requires very low amount of
started bone powder. However, the obtained result was only
performed on the complete left femur bone that was found in dry
condition (Fig. 3). In this study, using our proposed protocol, we
were able to isolate DNA from 80 bones which range from
undetected to 41.58 ng/ml (Table 1). The effectiveness of the
proposed method to provide the complete/minimum core STR
profile after amplified with the AmpFLSTR Identifiler1 kit
depended on the type of bones and the degree of decomposing.
Here, we were able to categorize bone samples in three groups
based on the degree of decomposing and their physical futures
(Table 2). Firstly, the low destroyed group that presented almost
complete bone features and classifiable bone type. This group had
white/gray surface color with no burn trace. Secondly, the medium
destroyed bone fragment which could determine the origin (e.g.
femur bone fragment). This group had a variety in colors such as
cream, dark brown and burning trace that could be observed in
some fragments. Finally, the highly destroyed group which was
referred as bone fragments was not able to classify the origin of
bones. In this group, some bone fragments had very dark brown to
black color. In addition, bone surfaces were sometimes completely
burnt (charcoal like), represented the limestone-like, and barnacle
surfaces (immersed bones). Although, this grading system was
strictly used only in our laboratory, we highly recommended that a
laboratory should establish a standard guideline for categorizing
the degree of decomposing of bone remain samples to reduce cost,
time, and laboratory processes. Moreover, our grading system has
been represented as a very helpful tool for forensic scientists in our
laboratory to decide appropriate further molecular genetic tools
169
(e.g. mtDNA sequencing, mini-STR, SNP-based genotyping) to
dealing with the critical and extremely destroyed samples such as
in strong violent crimes with cremated and sunk bones occasion-
ally found. Furthermore, we observed that long compact bone
samples such as femur and tibia were highly preserved the genetic
material than flat and spongy bones such as vertebrae or ribs
(Table 3). To gather, we demonstrated that our proposed DNA
extraction protocol is very sufficient to isolate DNA from several
degrees of decomposed bone samples and the efficacy of this
method could affect by the type of bone remains as well as the
degree of decomposing.
The amplification efficacy of STR analysis is affected by several
factors in PCR reaction including started DNA quantity and quality,
size of amplified products contained in each commercial kit, and
PCR inhibitors (Fig. 4). In this work, the amplification efficacy of the
AmpFLSTR Identifiler1 kit on the isolated DNA samples was shown
in Fig. 4. To enhance the amplification efficacy, we performed the
YM-100 MICROCON1 (centrifuge at 2400 rpm 12 min followed by
4500 rpm 5 min) to concentrate the isolated DNA. Furthermore, we
conducted the low copy number technique (LCN) (32 PCR cycles
and twice that of Taq Polymerase) for PCR in cases with DNA
concentration lower than 50 pg. The obtained results demonstrat-
ed that YM-100 combined with LCN could improve the amplifica-
tion efficacy of the low amount and prone to have DNA
contamination which frequently found in skeletal remain samples.
Besides, LCN issue is still challenging many forensic DNA
laboratories to standardize and validate the appropriate results
in forensic casework.
3.1. Case report
Interestingly, we had the great experience for dealing with
human decomposed samples, ribs that contained costal cartilage
(Fig. 5). Samples were collected from a murder scene which the
bodied was found in the river. The time of death was approximately
2–3 days before the body was discovered. We used our routine
established DNA extraction method as described in this report to
isolate genomic DNA from a small piece of costal cartilage. In brief,
decomposed tissues were removed from the rib sample followed
with pre-cleaning in sterile water for 3 times and final wash with
70% ethanol. The sample was dried in 56  C incubator for overnight.
A slice piece of costal cartilage was generated by using surgical
blade and incubated in 1 ml bone incubation buffer at 56  C in
1.5 ml microcentrifuge tube until the sample was completely
resolved. We used the DNA IQTM System (Promega, USA) to isolate
DNA from the supernatant. Purified DNA was measured as
0.412 ng/ml by using the QuantifilerTM Human DNA Quantification
Kit (Applied Biosystems, USA) and worked with ABI7500 Fast Real-
Time RCR. Multiplex PCR was performed with the AmpF/STR
Identifiler PCR Amplification kit (PE Applied Biosystems, Foster
City, CA, USA) in 25 ml final volume and amplified on GeneAmp1
PCR System 9700 thermal cycler (Applied Biosystems, USA).
Genotyping was performed with 3130 Genetic Analyzer (Applied
Biosystems, USA) and analyzed by GeneMapper1 software.
Complete profiles of all 15 autosomal STRs and gender-specific
amelogenin X and Y were obtained by using our proposed
technique (Fig. 6). To conclude, we demonstrated that the costal
cartilage could be a designate specimen for DNA isolation from
human decomposed body especially in a case that the body was
discovered approximately 3 days after death. Furthermore, DNA
isolation from costal cartilage was simple, fast, less expensive, no
freezer mill required. Costal cartilage sample is an alternative
source for isolating high quality and quantity genomic DNA from
decomposed samples.
170 T. Siriboonpiputtana et al. / Forensic Science International 284 (2018) 167–175

Table 1
Bone types, the degree of decomposing, extracted DNA amounts, and the amplification efficacies after amplified with the AmpFLSTR Identifiler1 kit.

Sample name Bone type Degree of decomposing DNA amount (ng/ml) DNA profile
HGU1 Right femur Low 0.2 Complete
HGU2 Right femur Low 0.0036 Partial
HGU3 Right femur Low 0.124 Complete
HGU4 Left femur Low 0.311 Complete
HGU5 Right femur Low 0.0371 Complete
HGU6 Left femur Low 0.106 Complete
HGU7 Right femur Low 0.133 Complete
HGU8 Right femur Low 0.829 Complete
HGU9 Left femur Low 10.27 Complete
HGU10 Right femur Low 0.291 Complete
HGU11 Right femur Low 4.6 Complete
HGU12 Right femur Low 0.703 Complete
HGU13 Right femur Low 29.28 Complete
HGU14 Right femur Low 3.09 Complete
HGU15 Right femur Low 41.58 Complete
HGU16 Left femur Low 0.164 Complete
HGU17 Left femur Low 0.0631 NA
HGU18 Left femur Low 2.34 Complete
HGU19 Femur Low 0.53 Complete
HGU20 Femur Low 0.228 Complete
HGU21 Right humerus Low 4.2 Complete
HGU22 Right humerus Low 0.703 Complete
HGU23 Left humerus Low 6.77 Complete
HGU24 Rib Low 5.33 Complete
HGU25 Rib Low 0.412 Complete
HGU26 Right fibula Low 0.0135 NA
HGU27 Right fibula Low 0.531 Partial
HGU28 Right radius Low 0.185 Complete
HGU29 Left tibia Low 0.0229 Partial
HGU30 Left pelvis Low 0.112 Complete
HGU31 Left ulna Low 1.56 Complete
HGU32 Cervical thorasis Low 0.291 Complete
HGU33 Left femur Medium 0.0222 Partial
HGU34 Femur Medium 0.0139 Complete
HGU35 Femur Medium 0.142 Complete
HGU36 Left femur Medium Undetected NA
HGU37 Femur Medium 0.006 NA
HGU38 Femur Medium 0.0128 Partial
HGU39 Burned lumbar vertebra Medium 0.0881 Partial
HGU40 Lumbar vertebra Medium 0.0201 Partial
HGU41 Lumbar vertebra Medium 0.0032 Partial
HGU42 9th vertebra Medium 0.0063 NA
HGU43 Right rib Medium Undetected NA
HGU44 Left fibula Medium Undetected NA
HGU45 Right tibia Medium 0.639 Complete
HGU46 Right tibia Medium 0.174 Complete
HGU47 Left pelvis Medium 0.0362 Complete
HGU48 Cervical thorasis Medium Undetected NA
HGU49 Skull fragment High Undetected NA
HGU50 Left tibia High Undetected NA
HGU51 Burned lumbar spine High Undetected NA
HGU52 Skull fragment High Undetected NA
HGU53 Bone fragment High Undetected NA
HGU54 Bone fragment High Undetected NA
HGU55 Bone fragment High Undetected NA
HGU56 Bone fragment High Undetected NA
HGU57 Burned bone fragment High Undetected NA
HGU58 Burned bone fragment High Undetected NA
HGU59 Burned bone fragment High Undetected NA
HGU60 Burned bone fragment High Undetected NA
HGU61 Burned bone fragment High Undetected NA
HGU62 Burned bone fragment High Undetected NA
HGU63 Burned bone fragment High Undetected NA
HGU64 Burned bone fragment High Undetected NA
HGU65 Burned bone fragment High Undetected NA
HGU66 Burned bone fragment High Undetected NA
HGU67 Burned and immerge bone fragment High Undetected NA
HGU68 Burned and immerge bone fragment High Undetected NA
HGU69 Burned and immerge bone fragment High Undetected NA
HGU70 Burned and immerge bone fragment High Undetected NA
HGU71 Burned and immerge bone fragment High Undetected NA
HGU72 Burned and immerge bone fragment High Undetected NA
HGU73 Burned and immerge bone fragment High Undetected NA
HGU74 Burned and immerge bone fragment High Undetected NA
HGU75 Burned and immerge bone fragment High Undetected NA
HGU76 Burned and immerge bone fragment High Undetected NA
 T. Siriboonpiputtana et al. / Forensic Science International 284 (2018) 167–175 171

Table 1 (Continued)
Sample name Bone type Degree of decomposing DNA amount (ng/ml) DNA profile
HGU77 Burned and immerge bone fragment High Undetected NA
HGU78 Burned and immerge bone fragment High Undetected NA
HGU79 Burned and immerge bone fragment High Undetected NA
HGU80 Burned and immerge bone fragment High Undetected NA

Fig. 1. The relationship between bone amount (g) and extracted DNA (ng/ml) by using the proposed protocol (in triplicate).

Table 2
The grading criteria of bone samples and the respective amplification efficacies of each bone group after amplification with the AmpFLSTR Identifiler1 kit.

Degree of Anatomical futures Physical futures Result

destruction
Low (32 from 80 Nearly to complete bone with known White, creamy or light brown surface with unburned trace and not immersed in water CP1 = 84.4%
samples) originated organ PP2 = 9.4%
NA3 = 6.2%
Medium (16 from 80 Incomplete bone fragment with known Creamy or dark brown surface with a few burned trace or buried or immersed bone CP1 = 31.2%
samples) originated organ PP2 = 31.2%
NA3 = 37.5%
High (32 from 80 Cannot classify bone type and the Completely burned (charcoal like), long time buried or immersed in water (sometime NA3 = 100%
samples) originated organ present limestone like and barnacle surface)

CP1 = complete profile, PP2 = partial profile and NA3 = non-amplified DNA using the AmpFLSTR Identifiler1 kit.

4. Discussion
DNA recovery and STR analysis from decomposed samples have
become a useful tool for human identification in several forensic
caseworks. The successful rate for DNA analysis from human
degraded bone samples was affected by several factors including
period of decomposing, evidence surrounding environment, DNA
isolation protocol, laboratory special equipment and experiences.
In this report, we proposed our routine effective DNA isolation and
amplification protocol for DNA profiling of human decomposed
bone samples. Similar to previous publications, we observed that
long compact bones such as femur and humerus well preserved the
genetic material better than flat and spongy bones such as skull,
vertebrae, and rib [3,11]. Thus, in this manner, forensic scientists
might consider compact bones as the first option for STR analysis.
Although we enabled to isolate and generate the complete STR
profile from very low amount of started bone powder (0.75 g), this
validation process was only conducted by using the dried bone
which categorized as low destroyed group. The characterization of
degree of decomposing of skeletal remains helped us predict the
achievement of STR analysis and provided several advantages such
as reduce time, cost as well as guide us to further/optional DNA
analysis techniques (e.g. mtDNA sequencing and mini STR
analysis).
Prospective the decision of appropriate DNA isolation technique
for skeletal remain samples was generally based on several factors
including laboratory core equipments (e.g. freezer mill, electric
surgical blade, and biosafety cabinet with UV) and reagents as well
as experiences. Thus, forensic laboratories are responsible to
validate their own protocol for DNA analysis of human degraded
samples.
Recently, several techniques have been proposed to improve the
successful rate in the amplification of samples originally with low
template DNA (extracted DNA measured lower than 100 pg). In this
report, we first performed the YM-100 MICROCON1 (Millipore,
USA) which recognized as a method of choice to concentrate and
remove PCR inhibitors from DNA solution [14,15]. In addition, we
performed the special 32-cycle PCR combined with double DNA
polymerase enzyme to amplify particular samples which could not
amplify by using the standard 28-cycle PCR (recommended for
AmpFlSTR Identifiler PCR Amplification kit). Although several
studies were recommended to use a 34-cycles PCR to amplify low
template DNA [15,17–19], we found that over-amplification as well
as imbalance peaks often occurred when PCR over than 32 cycles.
Besides LCN results in the difficulty to generate complete STR
profiles, it is critical for forensic laboratories to establish a practical
interpretation guideline to deal with the extreme degraded
samples [20,21].
We further applied our DNA isolation protocol to determine the
appropriate specimens for DNA isolation from human degraded
samples which frequently observed in forensic scenarios in
Thailand such as in a case that the body was found in the river.
We found that costal cartilage represented as a good sample for
DNA isolation in a case which the body was detected about 3 days
after death. The superior advantages of using costal cartilage
172 T. Siriboonpiputtana et al. / Forensic Science International 284 (2018) 167–175

Fig. 2. Electropherogram from the amplification of 0.75 g bone powder with the AmpFLSTR Identifiler1 kit.

Fig. 3. Dried left femur bone and a cutting site (4 cm  2 cm) which was used in the validation of bone amount process.
 T. Siriboonpiputtana et al. / Forensic Science International 284 (2018) 167–175 173

Table 3
Types of bone remains and amplification results.

Sample STR profiles obtained

Complete Partial None

Femur 20 (62.5%) 3 (37.5%) 3 (7.5%)
Humerus 3 (9.4%) – –
Vertebra 1 (3.1%) 3 (37.5%) 2 (5%)
Rib 2 (6.3%) – 1 (2.5%)
Spine – – 1 (2.5%)
Fibular – 1 (12.5%) 2 (5%)
Radius 1 (3.1%) – –
Tibia 2 (6.3%) 1 (12.5%) 1 (2.5%)
Pelvis 2 (6.3%) –
Ulna 1 (3.1%) – –
Skull – – 2 (5%)
Unidentified fragments – – 28 (70%)
Total 32 8 40

Fig. 4. The amplification efficacies of 16 genetic markers (AmpFLSTR Identifiler1 kit) from a total of 39 amplifiable skeletal remains. There were complete (40%) and partial
(10%) profiles.

Fig. 5. Sample preparation for DNA isolation from costal cartilage; A: remove bone intact tissue and wash with the same protocol as described above, B: slice off the costal
cartilage with sterile surgical blade, C: collect one piece of the 3rd or 4th or the inner slice for extracting DNA, D: transfer the slice of costal cartilage to a microcentrifuge tube.
174 T. Siriboonpiputtana et al. / Forensic Science International 284 (2018) 167–175
Fig. 6. Electropherogram from the amplification of DNA isolated from costal cartilage using the AmpFLSTR Identifiler1 kit.
compared to compact bones for DNA profiling were including
faster, less expensive, and no special equipment required.
Acknowledgments
We gratefully recognize and express our sincere thanks to
Dr. Thamrong Chirajariyavej, the former head of Toxicology and
Forensic Autopsy Units, Department of Pathology Ramathibodi
Hospital, for his kindly teaching us about human identification
strategies. We would like to thank Miss Ubonrat Jomsawat for
technical guidance. We also would like to thank members of
Forensic Medical Laboratory for collaboration and valuable data.
Finally, we would like to thank our friends, the Forensic
DNA Network in Thailand for sharing the information and
knowledge.
References
[1] B. Budowle, F.R. Bieber, A.J. Eisenberg, Forensic aspects of mass disasters:
strategic considerations for DNA-based human identification, Legal Med. 7 (4)
(2005) 230–243.
[2] M. Prinz, A. Carracedo, W.R. Mayr, N. Morling, T.J. Parsons, A. Sajantila, et al.,
DNA Commission of the International Society for Forensic Genetics (ISFG):
recommendations regarding the role of forensic genetics for disaster victim
identification (DVI), Forensic Sci. Int. Genet. 1 (1) (2007) 3–12.
[3] S. Andelinovic, D. Sutlovic, I. Erceg Ivkosic, V. Skaro, A. Ivkosic, F. Paic, et al.,
Twelve-year experience in identification of skeletal remains from mass graves,
Croat. Med. J. 46 (4) (2005) 530–539.
[4] R. Barnett, G. Larson, A phenol-chloroform protocol for extracting DNA from
ancient samples, Methods Mol. Biol. 840 (2012) 13–19.
[5] M.N. Hochmeister, B. Budowle, U.V. Borer, U. Eggmann, C.T. Comey, R.
Dirnhofer, Typing of deoxyribonucleic acid (DNA) extracted from compact
bone from human remains, J. Forensic Sci. 36 (6) (1991) 1649–1661.
[6] J. Jakubowska, A. Maciejewska, R. Pawlowski, Comparison of three methods of
DNA extraction from human bones with different degrees of degradation, Int. J.
Legal Med. 126 (1) (2012) 173–178.
 T. Siriboonpiputtana et al. / Forensic Science International 284 (2018) 167–175
[7] J. Davoren, D. Vanek, R. Konjhodzic, J. Crews, E. Huffine, T.J. Parsons, Highly
effective DNA extraction method for nuclear short tandem repeat testing of
skeletal remains from mass graves, Croat. Med. J. 48 (4) (2007) 478–485.
[8] O.M. Loreille, T.M. Diegoli, J.A. Irwin, M.D. Coble, T.J. Parsons, High efficiency
DNA extraction from bone by total demineralization, Forensic Sci. Int. Genet. 1
(2) (2007) 191–195.
[9] C. Rucinski, A.L. Malaver, E.J. Yunis, J.J. Yunis, Comparison of two methods for
isolating DNA from human skeletal remains for STR analysis, J. Forensic Sci. 57
(3) (2012) 706–712.
[10] N. Rohland, M. Hofreiter, Comparison and optimization of ancient DNA
extraction, BioTechniques 42 (3) (2007) 343–352.
[11] A. Milos, A. Selmanovic, L. Smajlovic, R.L. Huel, C. Katzmarzyk, A. Rizvic, et al.,
Success rates of nuclear short tandem repeat typing from different skeletal
elements, Croat. Med. J. 48 (4) (2007) 486–493.
[12] D.Y. Yang, B. Eng, J.S. Waye, J.C. Dudar, S.R. Saunders, Technical note: improved
DNA extraction from ancient bones using silica-based spin columns, Am. J.
Phys. Anthropol. 105 (4) (1998) 539–543.
[13] H.Y. Lee, M.J. Park, N.Y. Kim, J.E. Sim, W.I. Yang, K.J. Shin, Simple and highly
effective DNA extraction methods from old skeletal remains using silica
columns, Forensic Sci. Int. Genet. 4 (5) (2010) 275–280.
[14] J. Sewell, I. Quinones, C. Ames, B. Multaney, S. Curtis, H. Seeboruth, et al.,
Recovery of DNA and fingerprints from touched documents, Forensic Sci. Int.
Genet. 2 (4) (2008) 281–285.
175
[15] L. Forster, J. Thomson, S. Kutranov, Direct comparison of post-28-cycle PCR
purification and modified capillary electrophoresis methods with the 34-cycle
“low copy number” (LCN) method for analysis of trace forensic DNA samples,
Forensic Sci. Int. Genet. 2 (4) (2008) 318–328.
[16] Y.M. Coulson-Thomas, A.L. Norton, V.J. Coulson-Thomas, R. Florencio-Silva, N.
Ali, S. Elmrghni, et al., DNA and bone structure preservation in medieval
human skeletons, Forensic Sci. Int. 251 (2015) 186–194.
[17] P. Gill, J. Whitaker, C. Flaxman, N. Brown, J. Buckleton, An investigation of the
rigor of interpretation rules for STRs derived from less than 100 pg of DNA,
Forensic Sci. Int. 112 (1) (2000) 17–40.
[18] J.P. Whitaker, E.A. Cotton, P. Gill, A comparison of the characteristics of profiles
produced with the AMPFlSTR SGM Plus multiplex system for both standard
and low copy number (LCN) STR DNA analysis, Forensic Sci. Int. 123 (2–3)
(2001) 215–223.
[19] P. Gill, Application of low copy number DNA profiling, Croat. Med. J. 42 (3)
(2001) 229–232.
[20] S. Petricevic, J. Whitaker, J. Buckleton, S. Vintiner, J. Patel, P. Simon, et al.,
Validation and development of interpretation guidelines for low copy number
(LCN) DNA profiling in New Zealand using the AmpFlSTR SGM Plus multiplex,
Forensic Sci. Int. Genet. 4 (5) (2010) 305–310.
[21] J. Buckleton, Validation issues around DNA typing of low level DNA, Forensic
Sci. Int. Genet. 3 (4) (2009) 255–260.