- Email: [email protected]
DNA analysis from human skeletal remains in forensic casework
Forensic Science International: Genetics Supplement Series xxx (xxxx) xxx–xxx
Contents lists available at ScienceDirect
Forensic Science International: Genetics Supplement Series journal homepage: www.elsevier.com/locate/fsigss
DNA analysis from human skeletal remains in forensic casework ⁎
Dragana Zgonjanina,b, , Mirjana Antovc, Rashed Alghafrid, Stojan Petkovića,b, Radenko Vukovića,b, Goran Stojiljkovića,b, Danka Toljića a
Institute of Forensic Medicine, Clinical Center of Vojvodina, Novi Sad, Serbia Faculty of Medicine, University of Novi Sad, Novi Sad, Serbia Faculty of Technology, University of Novi Sad, Novi Sad, Serbia d General Department of Forensic Sciences and Criminology, Dubai Police G.H.Q., Dubai, United Arab Emirates b c
A R T I C L E I N F O
A B S T R A C T
Keywords: Forensic identification Skeletal remains DNA extraction Degraded DNA Bones
To assess our laboratory’s success with skeletal remains and provide a benchmark for the forensic community involved in identification of these remains, we retrospectively examined our ability to develop DNA profiles from the remains analyzed in our laboratory in the last 7 years. Between January 2009 and December 2016, 70 DNA extractions were completed on skeletal remains from routine casework. 92% of skeletal remains analyzed were samples submitted for body identifications by law enforcement and only 8% were samples submitted to answer family identity or historical questions. Overall, the ability to obtain a full or partial profile primarily reflects the difference in the average age and the condition of the samples in these two categories and thus, difference in the quantity and quality of the DNA. We describe here the approximate age and type of remains we have received, whether a full, partial, or no profile was obtained, as well as the condition of the samples.
1. Introduction
2. Material and methods
Typically with missing person cases, only skeletal remains are available as an evidentiary source of DNA [1–4]. DNA analysis from unidentified human remains involves many steps, but extraction and optimized recovery of DNA is paramount in this process. Numerous challenges exist with extracting DNA from bone; the structure and chemical composition of bone make extracting and amplifying DNA difficult and the environmental conditions from which the bone is recovered can dramatically alter the preservation state of the bone material and consequently the integrity and availability of the DNA. Our laboratory focuses exclusively on STR DNA from bone from the beginning of our work since 2003, a powerful tool in missing person cases. The present study describes our work on the genetic identification of 70 skeletal remains from routine casework in the last 7 years. Of particular interest was our work on the identification of skeletal remains from several cases of criminal burning, where the intent was to destroy the body [5] which helped to identify the victim of the murder that shook the public and cases of remains recovered from water.
2.1. Samples and DNA extraction
⁎
This study analyzed 70 bones and teeth: 64 submitted for routine casework body identifications by law enforcement and 6 submitted to answer family identity. We analyzed the following samples: 55 femurs, 4 skulls, 3 tibias, 3 ribs and 5 tooth samples. All bones were cleaned from the remnant soft tissue and all soil traces. The cut bone fragments were washed and air dried [4]. The resulting sample was pulverized into fine powder in mill MM 301 (Retsch). Extraction of 40 DNA samples was carried out by the phenol chloroform isoamyl alcohol (PCIA) organic extraction method, whereas the extraction of 30 DNA samples was carried out by PrepFiler® BTA Forensic DNA Extraction Kit (Applied Biosystems). In addition, both extraction methods were carried out on 4 samples. When genomic DNA was obtained using PCIA organic extraction method, 2.5 g of bone powder was extracted according to Zgonjanin et al. [5]. Extraction of DNA using PrepFiler® BTA Forensic DNA Extraction Kit (Applied Biosystems) was performed using 50 mg of pow-
Corresponding author at: Institute of Forensic Medicine, Clinical Center of Vojvodina, Hajduk Veljkova 1, 21000, Novi Sad, Serbia. E-mail address: [email protected] (D. Zgonjanin).
http://dx.doi.org/10.1016/j.fsigss.2017.09.117 Received 20 August 2017; Received in revised form 17 September 2017; Accepted 19 September 2017 1875-1768/ © 2017 Elsevier B.V. All rights reserved.
Please cite this article as: Zgonjanin, D., Forensic Science International: Genetics Supplement Series (2017), http://dx.doi.org/10.1016/j.fsigss.2017.09.117
Forensic Science International: Genetics Supplement Series xxx (xxxx) xxx–xxx
D. Zgonjanin et al.
Table 1 Nuclear DNA quantity; the efficiency of autosomal STR) typing (AmpFℓSTR® Identifiler® and AmpFℓSTR® NGM™ Amplifiction Kit) expressed as the number of successfully typed STRs; efficiency of Y-STR typing (AmpFℓSTR® Yfiler®), expressed as the number of successfully typed Y-STRs; and efficiency of mtDNA typing (HVI and HVII) in bones and teeth from 70 cases human identification cases. Bone sample Q/Casework
Quantity (ng/μL)
Efficiency of DNA typing by AmpFℓSTR® Identifiler
AmpFℓSTR® NGM™
AmpFℓSTR® Yfiler®
16/16 16/16 16/16 16/16 16/16 16/16 16/16 16/16 16/16 16/16 16/16 16/16 16/16 16/16 16/16 16/16 16/16 16/16 16/16 13/16 16/16 16/16 – 16/16 16/16 16/16 16/16 16/16 16/16 16/16 16/16 16/16 16/16 – 16/16
17/17 17/17 17/17 17/17 17/17 17/17 – – 17/17 17/17 – – – – 17/17 17/17 17/17 17/17 17/17 – – 17/17 – 17/17 mtDNA HV1,2 17/17 – – 17/17 17/17 17/17 17/17 17/17 – –
AmpFℓSTR® Identifiler
AmpFℓSTR® NGM™
AmpFℓSTR® Yfiler®
16/16 16/16 16/16 – 16/16 16/16 16/16 16/16 16/16 16/16 16/16 16/16 16/16 16/16 16/16 16/16 16/16 16/16 16/16 16/16 10/16 16/16 16/16 16/16 16/16 16/16 16/16 16/16 16/16
16/16 16/16 16/16 – 16/16 16/16 16/16 16/16 16/16 16/16 16/16 16/16 16/16 16/16 16/16 16/16 16/16 16/16 16/16 16/16 14/16 16/16 16/16 16/16 16/16 16/16 16/16 16/16 16/16
Femur m (Case #1)a Femur m (Case #2)a Skull m (Case #3)a Femur m (Case #4)a Femur m (Case #5)a Femur m (Case #6)a Femur w (Case #7)a Femur w (Case #8)a Femur m (Case #9)a Tibia m (Case #10)a Femur w (Case #11)a Femur w (Case #12)a Femur w (Case #13)a Femur w(Case #14)a Femur m (Case #15)a Femur m (Case #16)a Femur m (Case #17)a Rib m (Case #18)a Femur m (Case #19)a Femur w (Case# 20)a Tooth w (Case #21)a Femur m (Case #22a Tooth w (Case #23)a Femur m (Case #24)a Femur w (Case #25)a Femur m (Case #26)a Femur w (Case #27)a Femur w (Case #28)a Femur m (Case #29)a Femur m (Case #30)a Femur m (Case #31)a Femur m (Case #32)a Femur m (Case #33)a Femur m (Case #34)a Tibia w (Case #35)a
1.05 1.28 0.62 9.20 0.29 1.02 234.95 0.47 93.73 0.69 1.18 4.10 0.92 0.32 0.44 0.59 207.40 1.78 12.11 0.69 1.75 1.14 0.001 1.24 1.33 373.76 0.35 185.26 2.11 0.039 0.53 0.098 0.13 0.0002 0.164
16/16 16/16 16/16 16/16 16/16 16/16 16/16 16/16 16/16 16/16 16/16 16/16 16/16 16/16 16/16 16/16 16/16 16/16 16/16 10/16 16/16 16/16 – 16/16 16/16 16/16 16/16 16/16 16/16 16/16 16/16 16/16 16/16 – 16/16
Bone sample Q/Casework
Quantity (ng/μL)
Efficiency of DNA typing by
Femur m (Case #36)a Rib m (Case #37)a Femur m (Case #38)a Skull m (Case #39)a Femur w (Case #40)a,b Femur m (Case #41)a,b Tooth w (Case #42)a,b Femur w (Case #43)a,b Femur m (Case #44)b Femur m (Case #45)b Femur w (Case #46)b Tooth m (Case #47)b Femur m (Case #48)b Femur w (Case #49)b Femur m (Case #50)b Femur w (Case #51)b Femur m (Case #52)b Femur w (Case #53)b Femur w (Case #54)b Rib m (Case #55)b Femur w (Case #56)b Tibia m (Case #57)b Skull m (Case #58)b Tooth m (Case #59)b Femur m (Case #60)b Femur m (Case #61)b Femur w (Case #62)b Femur w (Case #63)b Femur m (Case #64)b
0.019 0.180 0.555 0 70.48 150.61 0.041 0.011 0.055 1.82 0.018 0.117 0.902 0.045 0.087 14.61 35.36 0.133 2.115 0.280 0.209 0.075 0.162 0.098 7.582 0.351 0.922 2.111 0.390
2
17/17 17/17 17/17 – – 17/17 – – 17/17 17/17 – 17/17 17/17 – 17/17 – 17/17 – – 17/17 – 17/17 17/17 17/17 17/17 17/17 – – 17/17 (continued on next page)
Forensic Science International: Genetics Supplement Series xxx (xxxx) xxx–xxx
D. Zgonjanin et al.
Table 1 (continued) Bone sample Q/Casework
Femur w (Case #65)b Skull m (Case #66)b Femur w (Case #67)b Femur m (Case #68)b Femur m (Case #69)b Femur w (Case #70)b
Quantity (ng/μL)
0.533 0.145 2.92 79.99 76.07 0.060
Efficiency of DNA typing by AmpFℓSTR® Identifiler
AmpFℓSTR® NGM™
AmpFℓSTR® Yfiler®
16/16 16/16 16/16 16/16 16/16 16/16
16/16 16/16 16/16 16/16 16/16 16/16
– 17/17 – 17/17 17/17 –
DNA Extraction Kit; m-man; w-woman. a PCIA organic extraction method. b PrepFiler® BTA Forensic.
confidence of correct identification for all 66 victims (probability from 99.9% to 99.999999%).
dered bone following the protocol recommended by the manufacturer. Attempts were made to improve the DNA recovery in some challenging bone samples by overnight incubation, up to 18 h, of bone samples in lysis buffer solution component during lysis step. Moreover, another modification was made by adding 3 μl 1 M DTT in addition to 300 μl PrepFiler® Lysis Buffer with incubation at 56 °C and 900 rpm for 45 min. Family reference samples accompanying the skeletal remains are usually obtained from buccal swabs or dried blood samples using the QIAamp DNA Micro Kit (QIAGEN).
3.2. Body identification cases versus historical cases Of the 70 skeletal remains from which DNA was extracted in our laboratory, 64 (91.43%) were submitted for body identification by law enforcement and 6 (8.57%) for questions of historical interest or by private individuals. Overall, body identification cases were more likely to yield a full profile whereas historical cases were more likely to result in partial profiles. Most samples in historical cases were greater than 50 years of age, whereas most of the samples in body identifications were less than 15 years of age. In addition, most of the samples in body identifications were less than 1 year of age (40 samples, 62.5%), 18 samples (28.1%) were from 1 up to 5 years of age, 5 samples (7.8%) were from 5 up to 10 years of age, whereas the only one sample (1.5%) was from 10 to less than 15 years of age.
2.2. PCR amplification and typing DNA was quantified with an ABI Prism® 7000 Sequence Detection System (Applied Biosystems) using Quantifiler™ Human DNA Quantification kit. Amplifications were performed on the GeneAmp PCR System 9700 Gold Plate (Applied Biosystems) using the AmpFℓSTR® Identifiler® (Applied Biosystems), AmpFℓSTR® NGM™ (Applied Biosystems) and AmpFℓSTR® Yfiler® (Applied Biosystems) following the manufacturers’ protocols. Amplified product are separated and detected on ABI Prism® 310 Genetic Analyzer (Applied Biosystems) and ABI 3500 Genetic Analyzer (Applied Biosystems).
4. Discussion Our results showed that the PrepFiler® BTA Forensic DNA Extraction Kit can yield both DNA quantity and STR profiles comparable or greater to that of the standard organic extraction method. Simple modifications to extraction techniques can dramatically improve DNA typing success and provide conclusive, reliable profiles using different amplification kits even when working with difficult samples. Our experience has shown, in accordance with the experiences of others [6,7], that the AmpFℓSTR® NGM™ with the ESS loci is more tolerant to common inhibitors, which enabled us to overcome the challenges associated with processing compromised skeletal remains.
3. Results 3.1. Sample types Seventy DNA extractions were completed on skeletal remains from routine casework and the results are presented in Table 1. Full profiles were obtained on 65 samples (92.8%), partial profiles were obtained for 2 samples (2.8%), and no profiles were obtained for 3 samples (4.3%). Femurs were the most common samples, and if available, these were used in all cases due to an overall high rate of obtaining profiles. In two bones (Case #20 and #56), with partial profiles, the loci that were not amplified were primarily the longest loci D2S1338, D18S51, and FGA. With this bone samples in 13 out of 16 (Case #20) and 14 out of 16 (Case #56) loci available in the AmpFℓSTR® NGM™, including 5 loci in the extended European Standard Set (ESS) were successfully amplified, while applying AmpFℓSTR® Identifiler® kit obtained only 10 out of 16 loci in both cases. Typing of low-level DNA samples from casework with new ESS amplification kits also showed better results in comparison with older amplification kits [6,7]. As for the exposure of examined samples of skeletal remains to different environmental influences samples can be categorized into three larger groups: burned bodies (21.9%), remains recovered from the water (28.1%) and remains recovered from the fields (50.0%). In this lab, nine extraction attempts of skeletal remains from nine cases of criminal burning, where the intent was to destroy the body, were successful. Cases of remains recovered from water resulted in full profiles. When comparing genetic profiles, we matched 66 of the 70 skeletal remains analyzed to accompanying reference sample with high
5. Conclusion The age and condition of the sample are, in the most cases, correlated with success in developing a DNA profile but it was shown that is not a general rule [5–7]. Advanced extraction and purification techniques were found to be essential tools for obtaining sufficient DNA from bones and teeth skeletal remains from routine casework in our laboratory. Extraction and purification methods using the PrepFiler® BTA Forensic DNA Extraction Kit, together with more sensitive and robust new amplification kits with the ESS loci allowed us to overcome the challenges associated with processing compromised skeletal remains and ultimately obtain STR DNA profiles in 96% of the bones and teeth.
Conflict of interest statement None. 3
Forensic Science International: Genetics Supplement Series xxx (xxxx) xxx–xxx
D. Zgonjanin et al.
Forensic Sci. 38 (1993) 542–553. [5] D. Zgonjanin, S. Petković, M. Maletin, et al., Case report: DNA identification of burned skeletal remains, Forensic Sci. Int.: Genet. Suppl. Ser. 5 (2015) e444–e446. [6] V.C. Tucker, A.J. Hopwood, C.J. Sprecher, et al., Developmental validation of the PowerPlex® ESI 16 and PowerPlex® ESI 17 systems: STR multiplex for the new european standard, Forensic Sci. Int. Genet. 5 (2011) 436–448. [7] L. Albinsson, L. Noren, R. Hedell, Swedish population data and concordance for the kits PowerPlex® ESX 16 system, PowerPlex® ESI 16 system AmpFlSTR® NGM™, AmpFlSTR® SGM™ plus and investigator ESSplex, Forensic Sci. Int. Genet. 5 (2011) e89–92.
References [1] E. Hagelberg, B. Sykes, R. Hedges, Ancient bone DNA amplified, Nature 342 (1989) 485. [2] E. Hagelberg, I.C. Gray, A.J. Jeffreys, Identification of the skeletal remains of a murder victim by DNA analysis, Nature 352 (1991) 427–429. [3] P. Gill, P.L. Ivanov, C. Kimpton, et al., Identification of the remains of the Romanov family by DNA analysis, Nat. Genet. 6 (1994) 130–135. [4] M.M. Holland, D.L. Fisher, L.G. Mitchell, et al., Mitochondrial DNA sequence analysis of human skeletal remains: identification of remains from the Vietnam War, J.
4
Contents lists available at ScienceDirect
Forensic Science International: Genetics Supplement Series journal homepage: www.elsevier.com/locate/fsigss
DNA analysis from human skeletal remains in forensic casework ⁎
Dragana Zgonjanina,b, , Mirjana Antovc, Rashed Alghafrid, Stojan Petkovića,b, Radenko Vukovića,b, Goran Stojiljkovića,b, Danka Toljića a
Institute of Forensic Medicine, Clinical Center of Vojvodina, Novi Sad, Serbia Faculty of Medicine, University of Novi Sad, Novi Sad, Serbia Faculty of Technology, University of Novi Sad, Novi Sad, Serbia d General Department of Forensic Sciences and Criminology, Dubai Police G.H.Q., Dubai, United Arab Emirates b c
A R T I C L E I N F O
A B S T R A C T
Keywords: Forensic identification Skeletal remains DNA extraction Degraded DNA Bones
To assess our laboratory’s success with skeletal remains and provide a benchmark for the forensic community involved in identification of these remains, we retrospectively examined our ability to develop DNA profiles from the remains analyzed in our laboratory in the last 7 years. Between January 2009 and December 2016, 70 DNA extractions were completed on skeletal remains from routine casework. 92% of skeletal remains analyzed were samples submitted for body identifications by law enforcement and only 8% were samples submitted to answer family identity or historical questions. Overall, the ability to obtain a full or partial profile primarily reflects the difference in the average age and the condition of the samples in these two categories and thus, difference in the quantity and quality of the DNA. We describe here the approximate age and type of remains we have received, whether a full, partial, or no profile was obtained, as well as the condition of the samples.
1. Introduction
2. Material and methods
Typically with missing person cases, only skeletal remains are available as an evidentiary source of DNA [1–4]. DNA analysis from unidentified human remains involves many steps, but extraction and optimized recovery of DNA is paramount in this process. Numerous challenges exist with extracting DNA from bone; the structure and chemical composition of bone make extracting and amplifying DNA difficult and the environmental conditions from which the bone is recovered can dramatically alter the preservation state of the bone material and consequently the integrity and availability of the DNA. Our laboratory focuses exclusively on STR DNA from bone from the beginning of our work since 2003, a powerful tool in missing person cases. The present study describes our work on the genetic identification of 70 skeletal remains from routine casework in the last 7 years. Of particular interest was our work on the identification of skeletal remains from several cases of criminal burning, where the intent was to destroy the body [5] which helped to identify the victim of the murder that shook the public and cases of remains recovered from water.
2.1. Samples and DNA extraction
⁎
This study analyzed 70 bones and teeth: 64 submitted for routine casework body identifications by law enforcement and 6 submitted to answer family identity. We analyzed the following samples: 55 femurs, 4 skulls, 3 tibias, 3 ribs and 5 tooth samples. All bones were cleaned from the remnant soft tissue and all soil traces. The cut bone fragments were washed and air dried [4]. The resulting sample was pulverized into fine powder in mill MM 301 (Retsch). Extraction of 40 DNA samples was carried out by the phenol chloroform isoamyl alcohol (PCIA) organic extraction method, whereas the extraction of 30 DNA samples was carried out by PrepFiler® BTA Forensic DNA Extraction Kit (Applied Biosystems). In addition, both extraction methods were carried out on 4 samples. When genomic DNA was obtained using PCIA organic extraction method, 2.5 g of bone powder was extracted according to Zgonjanin et al. [5]. Extraction of DNA using PrepFiler® BTA Forensic DNA Extraction Kit (Applied Biosystems) was performed using 50 mg of pow-
Corresponding author at: Institute of Forensic Medicine, Clinical Center of Vojvodina, Hajduk Veljkova 1, 21000, Novi Sad, Serbia. E-mail address: [email protected] (D. Zgonjanin).
http://dx.doi.org/10.1016/j.fsigss.2017.09.117 Received 20 August 2017; Received in revised form 17 September 2017; Accepted 19 September 2017 1875-1768/ © 2017 Elsevier B.V. All rights reserved.
Please cite this article as: Zgonjanin, D., Forensic Science International: Genetics Supplement Series (2017), http://dx.doi.org/10.1016/j.fsigss.2017.09.117
Forensic Science International: Genetics Supplement Series xxx (xxxx) xxx–xxx
D. Zgonjanin et al.
Table 1 Nuclear DNA quantity; the efficiency of autosomal STR) typing (AmpFℓSTR® Identifiler® and AmpFℓSTR® NGM™ Amplifiction Kit) expressed as the number of successfully typed STRs; efficiency of Y-STR typing (AmpFℓSTR® Yfiler®), expressed as the number of successfully typed Y-STRs; and efficiency of mtDNA typing (HVI and HVII) in bones and teeth from 70 cases human identification cases. Bone sample Q/Casework
Quantity (ng/μL)
Efficiency of DNA typing by AmpFℓSTR® Identifiler
AmpFℓSTR® NGM™
AmpFℓSTR® Yfiler®
16/16 16/16 16/16 16/16 16/16 16/16 16/16 16/16 16/16 16/16 16/16 16/16 16/16 16/16 16/16 16/16 16/16 16/16 16/16 13/16 16/16 16/16 – 16/16 16/16 16/16 16/16 16/16 16/16 16/16 16/16 16/16 16/16 – 16/16
17/17 17/17 17/17 17/17 17/17 17/17 – – 17/17 17/17 – – – – 17/17 17/17 17/17 17/17 17/17 – – 17/17 – 17/17 mtDNA HV1,2 17/17 – – 17/17 17/17 17/17 17/17 17/17 – –
AmpFℓSTR® Identifiler
AmpFℓSTR® NGM™
AmpFℓSTR® Yfiler®
16/16 16/16 16/16 – 16/16 16/16 16/16 16/16 16/16 16/16 16/16 16/16 16/16 16/16 16/16 16/16 16/16 16/16 16/16 16/16 10/16 16/16 16/16 16/16 16/16 16/16 16/16 16/16 16/16
16/16 16/16 16/16 – 16/16 16/16 16/16 16/16 16/16 16/16 16/16 16/16 16/16 16/16 16/16 16/16 16/16 16/16 16/16 16/16 14/16 16/16 16/16 16/16 16/16 16/16 16/16 16/16 16/16
Femur m (Case #1)a Femur m (Case #2)a Skull m (Case #3)a Femur m (Case #4)a Femur m (Case #5)a Femur m (Case #6)a Femur w (Case #7)a Femur w (Case #8)a Femur m (Case #9)a Tibia m (Case #10)a Femur w (Case #11)a Femur w (Case #12)a Femur w (Case #13)a Femur w(Case #14)a Femur m (Case #15)a Femur m (Case #16)a Femur m (Case #17)a Rib m (Case #18)a Femur m (Case #19)a Femur w (Case# 20)a Tooth w (Case #21)a Femur m (Case #22a Tooth w (Case #23)a Femur m (Case #24)a Femur w (Case #25)a Femur m (Case #26)a Femur w (Case #27)a Femur w (Case #28)a Femur m (Case #29)a Femur m (Case #30)a Femur m (Case #31)a Femur m (Case #32)a Femur m (Case #33)a Femur m (Case #34)a Tibia w (Case #35)a
1.05 1.28 0.62 9.20 0.29 1.02 234.95 0.47 93.73 0.69 1.18 4.10 0.92 0.32 0.44 0.59 207.40 1.78 12.11 0.69 1.75 1.14 0.001 1.24 1.33 373.76 0.35 185.26 2.11 0.039 0.53 0.098 0.13 0.0002 0.164
16/16 16/16 16/16 16/16 16/16 16/16 16/16 16/16 16/16 16/16 16/16 16/16 16/16 16/16 16/16 16/16 16/16 16/16 16/16 10/16 16/16 16/16 – 16/16 16/16 16/16 16/16 16/16 16/16 16/16 16/16 16/16 16/16 – 16/16
Bone sample Q/Casework
Quantity (ng/μL)
Efficiency of DNA typing by
Femur m (Case #36)a Rib m (Case #37)a Femur m (Case #38)a Skull m (Case #39)a Femur w (Case #40)a,b Femur m (Case #41)a,b Tooth w (Case #42)a,b Femur w (Case #43)a,b Femur m (Case #44)b Femur m (Case #45)b Femur w (Case #46)b Tooth m (Case #47)b Femur m (Case #48)b Femur w (Case #49)b Femur m (Case #50)b Femur w (Case #51)b Femur m (Case #52)b Femur w (Case #53)b Femur w (Case #54)b Rib m (Case #55)b Femur w (Case #56)b Tibia m (Case #57)b Skull m (Case #58)b Tooth m (Case #59)b Femur m (Case #60)b Femur m (Case #61)b Femur w (Case #62)b Femur w (Case #63)b Femur m (Case #64)b
0.019 0.180 0.555 0 70.48 150.61 0.041 0.011 0.055 1.82 0.018 0.117 0.902 0.045 0.087 14.61 35.36 0.133 2.115 0.280 0.209 0.075 0.162 0.098 7.582 0.351 0.922 2.111 0.390
2
17/17 17/17 17/17 – – 17/17 – – 17/17 17/17 – 17/17 17/17 – 17/17 – 17/17 – – 17/17 – 17/17 17/17 17/17 17/17 17/17 – – 17/17 (continued on next page)
Forensic Science International: Genetics Supplement Series xxx (xxxx) xxx–xxx
D. Zgonjanin et al.
Table 1 (continued) Bone sample Q/Casework
Femur w (Case #65)b Skull m (Case #66)b Femur w (Case #67)b Femur m (Case #68)b Femur m (Case #69)b Femur w (Case #70)b
Quantity (ng/μL)
0.533 0.145 2.92 79.99 76.07 0.060
Efficiency of DNA typing by AmpFℓSTR® Identifiler
AmpFℓSTR® NGM™
AmpFℓSTR® Yfiler®
16/16 16/16 16/16 16/16 16/16 16/16
16/16 16/16 16/16 16/16 16/16 16/16
– 17/17 – 17/17 17/17 –
DNA Extraction Kit; m-man; w-woman. a PCIA organic extraction method. b PrepFiler® BTA Forensic.
confidence of correct identification for all 66 victims (probability from 99.9% to 99.999999%).
dered bone following the protocol recommended by the manufacturer. Attempts were made to improve the DNA recovery in some challenging bone samples by overnight incubation, up to 18 h, of bone samples in lysis buffer solution component during lysis step. Moreover, another modification was made by adding 3 μl 1 M DTT in addition to 300 μl PrepFiler® Lysis Buffer with incubation at 56 °C and 900 rpm for 45 min. Family reference samples accompanying the skeletal remains are usually obtained from buccal swabs or dried blood samples using the QIAamp DNA Micro Kit (QIAGEN).
3.2. Body identification cases versus historical cases Of the 70 skeletal remains from which DNA was extracted in our laboratory, 64 (91.43%) were submitted for body identification by law enforcement and 6 (8.57%) for questions of historical interest or by private individuals. Overall, body identification cases were more likely to yield a full profile whereas historical cases were more likely to result in partial profiles. Most samples in historical cases were greater than 50 years of age, whereas most of the samples in body identifications were less than 15 years of age. In addition, most of the samples in body identifications were less than 1 year of age (40 samples, 62.5%), 18 samples (28.1%) were from 1 up to 5 years of age, 5 samples (7.8%) were from 5 up to 10 years of age, whereas the only one sample (1.5%) was from 10 to less than 15 years of age.
2.2. PCR amplification and typing DNA was quantified with an ABI Prism® 7000 Sequence Detection System (Applied Biosystems) using Quantifiler™ Human DNA Quantification kit. Amplifications were performed on the GeneAmp PCR System 9700 Gold Plate (Applied Biosystems) using the AmpFℓSTR® Identifiler® (Applied Biosystems), AmpFℓSTR® NGM™ (Applied Biosystems) and AmpFℓSTR® Yfiler® (Applied Biosystems) following the manufacturers’ protocols. Amplified product are separated and detected on ABI Prism® 310 Genetic Analyzer (Applied Biosystems) and ABI 3500 Genetic Analyzer (Applied Biosystems).
4. Discussion Our results showed that the PrepFiler® BTA Forensic DNA Extraction Kit can yield both DNA quantity and STR profiles comparable or greater to that of the standard organic extraction method. Simple modifications to extraction techniques can dramatically improve DNA typing success and provide conclusive, reliable profiles using different amplification kits even when working with difficult samples. Our experience has shown, in accordance with the experiences of others [6,7], that the AmpFℓSTR® NGM™ with the ESS loci is more tolerant to common inhibitors, which enabled us to overcome the challenges associated with processing compromised skeletal remains.
3. Results 3.1. Sample types Seventy DNA extractions were completed on skeletal remains from routine casework and the results are presented in Table 1. Full profiles were obtained on 65 samples (92.8%), partial profiles were obtained for 2 samples (2.8%), and no profiles were obtained for 3 samples (4.3%). Femurs were the most common samples, and if available, these were used in all cases due to an overall high rate of obtaining profiles. In two bones (Case #20 and #56), with partial profiles, the loci that were not amplified were primarily the longest loci D2S1338, D18S51, and FGA. With this bone samples in 13 out of 16 (Case #20) and 14 out of 16 (Case #56) loci available in the AmpFℓSTR® NGM™, including 5 loci in the extended European Standard Set (ESS) were successfully amplified, while applying AmpFℓSTR® Identifiler® kit obtained only 10 out of 16 loci in both cases. Typing of low-level DNA samples from casework with new ESS amplification kits also showed better results in comparison with older amplification kits [6,7]. As for the exposure of examined samples of skeletal remains to different environmental influences samples can be categorized into three larger groups: burned bodies (21.9%), remains recovered from the water (28.1%) and remains recovered from the fields (50.0%). In this lab, nine extraction attempts of skeletal remains from nine cases of criminal burning, where the intent was to destroy the body, were successful. Cases of remains recovered from water resulted in full profiles. When comparing genetic profiles, we matched 66 of the 70 skeletal remains analyzed to accompanying reference sample with high
5. Conclusion The age and condition of the sample are, in the most cases, correlated with success in developing a DNA profile but it was shown that is not a general rule [5–7]. Advanced extraction and purification techniques were found to be essential tools for obtaining sufficient DNA from bones and teeth skeletal remains from routine casework in our laboratory. Extraction and purification methods using the PrepFiler® BTA Forensic DNA Extraction Kit, together with more sensitive and robust new amplification kits with the ESS loci allowed us to overcome the challenges associated with processing compromised skeletal remains and ultimately obtain STR DNA profiles in 96% of the bones and teeth.
Conflict of interest statement None. 3
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Forensic Sci. 38 (1993) 542–553. [5] D. Zgonjanin, S. Petković, M. Maletin, et al., Case report: DNA identification of burned skeletal remains, Forensic Sci. Int.: Genet. Suppl. Ser. 5 (2015) e444–e446. [6] V.C. Tucker, A.J. Hopwood, C.J. Sprecher, et al., Developmental validation of the PowerPlex® ESI 16 and PowerPlex® ESI 17 systems: STR multiplex for the new european standard, Forensic Sci. Int. Genet. 5 (2011) 436–448. [7] L. Albinsson, L. Noren, R. Hedell, Swedish population data and concordance for the kits PowerPlex® ESX 16 system, PowerPlex® ESI 16 system AmpFlSTR® NGM™, AmpFlSTR® SGM™ plus and investigator ESSplex, Forensic Sci. Int. Genet. 5 (2011) e89–92.
References [1] E. Hagelberg, B. Sykes, R. Hedges, Ancient bone DNA amplified, Nature 342 (1989) 485. [2] E. Hagelberg, I.C. Gray, A.J. Jeffreys, Identification of the skeletal remains of a murder victim by DNA analysis, Nature 352 (1991) 427–429. [3] P. Gill, P.L. Ivanov, C. Kimpton, et al., Identification of the remains of the Romanov family by DNA analysis, Nat. Genet. 6 (1994) 130–135. [4] M.M. Holland, D.L. Fisher, L.G. Mitchell, et al., Mitochondrial DNA sequence analysis of human skeletal remains: identification of remains from the Vietnam War, J.
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