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Forensic genetic analysis of bone remain samples
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 b
Human Genetics Laboratory, Department of Pathology, Faculty of Medicine, Ramathibodi Hospital, Mahidol University, Bangkok 10400, Thailand 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
Article history: Received 14 July 2017 Received in revised form 16 October 2017 Accepted 28 December 2017 Available online 8 January 2018 Keywords: Forensic genetics DNA typing Bone remain Degraded DNA
A B S T R A C T
DNA typing from degraded human remains is still challenging forensic DNA scientists not only in the prospective of DNA purification but also in the interpretation of established DNA profiles and data manipulation, especially in mass fatalities. In this report, we presented DNA typing protocol to 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 overall STR amplification efficacy. A total of 80 bone samples were subjected to DNA isolation using the modified DNA IQTM System (Promega, USA) for bone extraction following with STR analysis using the AmpFLSTR Identifiler1 (Thermo Fisher Scientific, USA). In low destruction group, complete STR profiles were observed as 84.4% whereas partial profiles and non-amplified were found as 9.4% and 6.2%, 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 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: [email protected] (B. Rerkamnuaychoke). https://doi.org/10.1016/j.forsciint.2017.12.045 0379-0738/© 2018 Elsevier B.V. All rights reserved.
belongings; the matching of fingerprints (provided pre-mortem 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
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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 decomposition, 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, decontaminating 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 decontamination 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 phenolchloroform 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 decalcified 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 Technologies). 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
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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 amplification 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
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(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 occasionally 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 demonstrated that YM-100 combined with LCN could improve the amplification 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 RealTime 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.
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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 HGU2 HGU3 HGU4 HGU5 HGU6 HGU7 HGU8 HGU9 HGU10 HGU11 HGU12 HGU13 HGU14 HGU15 HGU16 HGU17 HGU18 HGU19 HGU20 HGU21 HGU22 HGU23 HGU24 HGU25 HGU26 HGU27 HGU28 HGU29 HGU30 HGU31 HGU32 HGU33 HGU34 HGU35 HGU36 HGU37 HGU38 HGU39 HGU40 HGU41 HGU42 HGU43 HGU44 HGU45 HGU46 HGU47 HGU48 HGU49 HGU50 HGU51 HGU52 HGU53 HGU54 HGU55 HGU56 HGU57 HGU58 HGU59 HGU60 HGU61 HGU62 HGU63 HGU64 HGU65 HGU66 HGU67 HGU68 HGU69 HGU70 HGU71 HGU72 HGU73 HGU74 HGU75 HGU76
Right femur Right femur Right femur Left femur Right femur Left femur Right femur Right femur Left femur Right femur Right femur Right femur Right femur Right femur Right femur Left femur Left femur Left femur Femur Femur Right humerus Right humerus Left humerus Rib Rib Right fibula Right fibula Right radius Left tibia Left pelvis Left ulna Cervical thorasis Left femur Femur Femur Left femur Femur Femur Burned lumbar vertebra Lumbar vertebra Lumbar vertebra 9th vertebra Right rib Left fibula Right tibia Right tibia Left pelvis Cervical thorasis Skull fragment Left tibia Burned lumbar spine Skull fragment Bone fragment Bone fragment Bone fragment Bone fragment Burned bone fragment Burned bone fragment Burned bone fragment Burned bone fragment Burned bone fragment Burned bone fragment Burned bone fragment Burned bone fragment Burned bone fragment Burned bone fragment Burned and immerge bone Burned and immerge bone Burned and immerge bone Burned and immerge bone Burned and immerge bone Burned and immerge bone Burned and immerge bone Burned and immerge bone Burned and immerge bone Burned and immerge bone
Low Low Low Low Low Low Low Low Low Low Low Low Low Low Low Low Low Low Low Low Low Low Low Low Low Low Low Low Low Low Low Low Medium Medium Medium Medium Medium Medium Medium Medium Medium Medium Medium Medium Medium Medium Medium Medium High High High High High High High High High High High High High High High High High High High High High High High High High High High High
0.2 0.0036 0.124 0.311 0.0371 0.106 0.133 0.829 10.27 0.291 4.6 0.703 29.28 3.09 41.58 0.164 0.0631 2.34 0.53 0.228 4.2 0.703 6.77 5.33 0.412 0.0135 0.531 0.185 0.0229 0.112 1.56 0.291 0.0222 0.0139 0.142 Undetected 0.006 0.0128 0.0881 0.0201 0.0032 0.0063 Undetected Undetected 0.639 0.174 0.0362 Undetected Undetected Undetected Undetected Undetected Undetected Undetected Undetected Undetected Undetected Undetected Undetected Undetected Undetected Undetected Undetected Undetected Undetected Undetected Undetected Undetected Undetected Undetected Undetected Undetected Undetected Undetected Undetected Undetected
Complete Partial Complete Complete Complete Complete Complete Complete Complete Complete Complete Complete Complete Complete Complete Complete NA Complete Complete Complete Complete Complete Complete Complete Complete NA Partial Complete Partial Complete Complete Complete Partial Complete Complete NA NA Partial Partial Partial Partial NA NA NA Complete Complete Complete NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA
fragment fragment fragment fragment fragment fragment fragment fragment fragment fragment
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Table 1 (Continued) Sample name
Bone type
HGU77 HGU78 HGU79 HGU80
Burned Burned Burned Burned
and and and and
immerge immerge immerge immerge
bone bone bone bone
fragment fragment fragment fragment
Degree of decomposing
DNA amount (ng/ml)
DNA profile
High High High High
Undetected Undetected Undetected Undetected
NA NA NA 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 destruction
Anatomical futures
Physical futures
Result
Low (32 from 80 samples)
Nearly to complete bone with known originated organ
White, creamy or light brown surface with unburned trace and not immersed in water
CP1 = 84.4% PP2 = 9.4% NA3 = 6.2% CP1 = 31.2% PP2 = 31.2% NA3 = 37.5% NA3 = 100%
Medium (16 from 80 Incomplete bone fragment with known Creamy or dark brown surface with a few burned trace or buried or immersed bone samples) originated organ High (32 from 80 samples)
Cannot classify bone type and the originated organ
Completely burned (charcoal like), long time buried or immersed in water (sometime 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
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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.
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Table 3 Types of bone remains and amplification results. Sample
Femur Humerus Vertebra Rib Spine Fibular Radius Tibia Pelvis Ulna Skull Unidentified fragments Total
STR profiles obtained Complete
Partial
None
20 (62.5%) 3 (9.4%) 1 (3.1%) 2 (6.3%) – – 1 (3.1%) 2 (6.3%) 2 (6.3%) 1 (3.1%) – – 32
3 – 3 – – 1 – 1
3 (7.5%) – 2 (5%) 1 (2.5%) 1 (2.5%) 2 (5%) – 1 (2.5%) – – 2 (5%) 28 (70%) 40
– – – 8
(37.5%) (37.5%)
(12.5%) (12.5%)
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.
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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.
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Contents lists available at ScienceDirect
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 b
Human Genetics Laboratory, Department of Pathology, Faculty of Medicine, Ramathibodi Hospital, Mahidol University, Bangkok 10400, Thailand 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
Article history: Received 14 July 2017 Received in revised form 16 October 2017 Accepted 28 December 2017 Available online 8 January 2018 Keywords: Forensic genetics DNA typing Bone remain Degraded DNA
A B S T R A C T
DNA typing from degraded human remains is still challenging forensic DNA scientists not only in the prospective of DNA purification but also in the interpretation of established DNA profiles and data manipulation, especially in mass fatalities. In this report, we presented DNA typing protocol to 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 overall STR amplification efficacy. A total of 80 bone samples were subjected to DNA isolation using the modified DNA IQTM System (Promega, USA) for bone extraction following with STR analysis using the AmpFLSTR Identifiler1 (Thermo Fisher Scientific, USA). In low destruction group, complete STR profiles were observed as 84.4% whereas partial profiles and non-amplified were found as 9.4% and 6.2%, 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 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: [email protected] (B. Rerkamnuaychoke). https://doi.org/10.1016/j.forsciint.2017.12.045 0379-0738/© 2018 Elsevier B.V. All rights reserved.
belongings; the matching of fingerprints (provided pre-mortem 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
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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 decomposition, 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, decontaminating 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 decontamination 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 phenolchloroform 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 decalcified 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 Technologies). 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
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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 amplification 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
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(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 occasionally 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 demonstrated that YM-100 combined with LCN could improve the amplification 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 RealTime 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.
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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 HGU2 HGU3 HGU4 HGU5 HGU6 HGU7 HGU8 HGU9 HGU10 HGU11 HGU12 HGU13 HGU14 HGU15 HGU16 HGU17 HGU18 HGU19 HGU20 HGU21 HGU22 HGU23 HGU24 HGU25 HGU26 HGU27 HGU28 HGU29 HGU30 HGU31 HGU32 HGU33 HGU34 HGU35 HGU36 HGU37 HGU38 HGU39 HGU40 HGU41 HGU42 HGU43 HGU44 HGU45 HGU46 HGU47 HGU48 HGU49 HGU50 HGU51 HGU52 HGU53 HGU54 HGU55 HGU56 HGU57 HGU58 HGU59 HGU60 HGU61 HGU62 HGU63 HGU64 HGU65 HGU66 HGU67 HGU68 HGU69 HGU70 HGU71 HGU72 HGU73 HGU74 HGU75 HGU76
Right femur Right femur Right femur Left femur Right femur Left femur Right femur Right femur Left femur Right femur Right femur Right femur Right femur Right femur Right femur Left femur Left femur Left femur Femur Femur Right humerus Right humerus Left humerus Rib Rib Right fibula Right fibula Right radius Left tibia Left pelvis Left ulna Cervical thorasis Left femur Femur Femur Left femur Femur Femur Burned lumbar vertebra Lumbar vertebra Lumbar vertebra 9th vertebra Right rib Left fibula Right tibia Right tibia Left pelvis Cervical thorasis Skull fragment Left tibia Burned lumbar spine Skull fragment Bone fragment Bone fragment Bone fragment Bone fragment Burned bone fragment Burned bone fragment Burned bone fragment Burned bone fragment Burned bone fragment Burned bone fragment Burned bone fragment Burned bone fragment Burned bone fragment Burned bone fragment Burned and immerge bone Burned and immerge bone Burned and immerge bone Burned and immerge bone Burned and immerge bone Burned and immerge bone Burned and immerge bone Burned and immerge bone Burned and immerge bone Burned and immerge bone
Low Low Low Low Low Low Low Low Low Low Low Low Low Low Low Low Low Low Low Low Low Low Low Low Low Low Low Low Low Low Low Low Medium Medium Medium Medium Medium Medium Medium Medium Medium Medium Medium Medium Medium Medium Medium Medium High High High High High High High High High High High High High High High High High High High High High High High High High High High High
0.2 0.0036 0.124 0.311 0.0371 0.106 0.133 0.829 10.27 0.291 4.6 0.703 29.28 3.09 41.58 0.164 0.0631 2.34 0.53 0.228 4.2 0.703 6.77 5.33 0.412 0.0135 0.531 0.185 0.0229 0.112 1.56 0.291 0.0222 0.0139 0.142 Undetected 0.006 0.0128 0.0881 0.0201 0.0032 0.0063 Undetected Undetected 0.639 0.174 0.0362 Undetected Undetected Undetected Undetected Undetected Undetected Undetected Undetected Undetected Undetected Undetected Undetected Undetected Undetected Undetected Undetected Undetected Undetected Undetected Undetected Undetected Undetected Undetected Undetected Undetected Undetected Undetected Undetected Undetected
Complete Partial Complete Complete Complete Complete Complete Complete Complete Complete Complete Complete Complete Complete Complete Complete NA Complete Complete Complete Complete Complete Complete Complete Complete NA Partial Complete Partial Complete Complete Complete Partial Complete Complete NA NA Partial Partial Partial Partial NA NA NA Complete Complete Complete NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA
fragment fragment fragment fragment fragment fragment fragment fragment fragment fragment
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Table 1 (Continued) Sample name
Bone type
HGU77 HGU78 HGU79 HGU80
Burned Burned Burned Burned
and and and and
immerge immerge immerge immerge
bone bone bone bone
fragment fragment fragment fragment
Degree of decomposing
DNA amount (ng/ml)
DNA profile
High High High High
Undetected Undetected Undetected Undetected
NA NA NA 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 destruction
Anatomical futures
Physical futures
Result
Low (32 from 80 samples)
Nearly to complete bone with known originated organ
White, creamy or light brown surface with unburned trace and not immersed in water
CP1 = 84.4% PP2 = 9.4% NA3 = 6.2% CP1 = 31.2% PP2 = 31.2% NA3 = 37.5% NA3 = 100%
Medium (16 from 80 Incomplete bone fragment with known Creamy or dark brown surface with a few burned trace or buried or immersed bone samples) originated organ High (32 from 80 samples)
Cannot classify bone type and the originated organ
Completely burned (charcoal like), long time buried or immersed in water (sometime 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
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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.
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Table 3 Types of bone remains and amplification results. Sample
Femur Humerus Vertebra Rib Spine Fibular Radius Tibia Pelvis Ulna Skull Unidentified fragments Total
STR profiles obtained Complete
Partial
None
20 (62.5%) 3 (9.4%) 1 (3.1%) 2 (6.3%) – – 1 (3.1%) 2 (6.3%) 2 (6.3%) 1 (3.1%) – – 32
3 – 3 – – 1 – 1
3 (7.5%) – 2 (5%) 1 (2.5%) 1 (2.5%) 2 (5%) – 1 (2.5%) – – 2 (5%) 28 (70%) 40
– – – 8
(37.5%) (37.5%)
(12.5%) (12.5%)
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.
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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.
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