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Simplified, rapid DNA extraction protocol for STR typing from bones
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
Simplified, rapid DNA extraction protocol for STR typing from bones Cheng Ho Phuaa, Laila Hasapb, Phuvadol Thanakiatkraia, Thitika Kitpipita,* a b
Forensic Science Programme, Department of Applied Science, Faculty of Science, Prince of Songkla University, Thailand Department of Molecular Biotechnology and Bioinformatics, Faculty of Science, Prince of Songkla University, Thailand
A R T I C LE I N FO
A B S T R A C T
Keywords: Bone extraction DNA Rapid
Bones are one of the body parts most tolerant towards various environmental conditions, especially as unidentified remains found in several circumstances: mass disaster, terrorism, war, and plague. Typically, the most complex and time-consuming part of the STR typing process is DNA extraction, with many different methods developed, such as organic extraction, total demineralization (TD), and silica-based column extraction. These protocols may take more than a day to complete. In such events where time is of the essence for human identification of the remains, a simple and fast but effective method is required. This study aimed to develop a simplified and rapid DNA extraction protocol from bone samples. Two bone types (fresh femur and tibia) were incubated in a buffer of SLS, EDTA, proteinase K, and DTT. We also evaluated extraction from different amounts of bone powder. Comparison was done for the developed method against standard total demineralisation protocol. Quantity of DNA extracted was slightly lower for the developed protocol compared to TD, however this did not affect STR typing and profile interpretability. DNA extraction was also possible from merely 0.1 g of bone. The developed method used significantly shorter incubation time and processing steps. Our final protocol only required two hours of incubation and 0.1 g of bone powder. STR typing results gave full profiles. The rapid protocol could be used when there is an urgent need of STR typing for human identification or when the sample amount is limited with a significant reduction in cost and time for investigation.
1. Introduction
2. Materials and methods
Bones tend to last longer than other biological materials in extreme conditions such as mass disaster, plague, terrorism, and etc [1,2]. They are often found as remains and may be fragmented and intermixed, which makes it difficult for morphological identification by forensic anthropologists. Therefore, DNA-based analysis methods are often necessary. Being enduring biological material, DNA entrapped within cells in the bone matrix are also difficult to be isolated. Extraction of DNA is normally required for analysis from bone samples in which there have been many different methods with varying approach, efficiency, and application: organic extraction method [3], Hi-Flow silica-based column [3], and proprietary PrepFiler® BTA [4], and total demineralization [5]. The extracted DNA vary in terms of quantity and purity, as well as the type and amount of carry-over PCR inhibitors [6]. Additionally, these methods are tedious, time-consuming, and laborious. Therefore, we aimed to develop a simplified and rapid DNA extraction protocol from bone samples.
Fresh femur and tibia (n = 5 each) were used. The bone powder from a cryogenic grinder was incubated in SLS, EDTA, proteinase K, DTT at 56 °C for two hours. The mixture was then centrifuged at 10k rpm for three minutes. The supernatant was aliquoted and then diluted 1:50 with sterile water whereby this dilution was then used for subsequent DNA analysis. For comparison, the same samples were DNA extracted using the total demineralization protocol (TD) [5]. DNA from both rapid extraction and TD were quantified using Quantifiler® Trio Human DNA Quantification kit (Thermo Fisher Scientific, USA). STR typing was performed using QIAGEN® Investigator® IDplex Plus kit (Qiagen, Germany) in half-volume reaction with two μL of DNA added. Fragment analysis was performed using ABI PRISM® 310 Genetic Analyzer and data analysis was done using GeneMapper 4.2.1. 3. Results and discussion 3.1. Comparison of DNA quantity Table 1 shows that the developed extraction method yielded slightly
⁎
Corresponding author. E-mail address: [email protected] (T. Kitpipit).
https://doi.org/10.1016/j.fsigss.2019.10.108 Received 14 September 2019; Accepted 9 October 2019 1875-1768/ © 2019 Published by Elsevier B.V.
Please cite this article as: Cheng Ho Phua, et al., Forensic Science International: Genetics Supplement Series, https://doi.org/10.1016/j.fsigss.2019.10.108
Forensic Science International: Genetics Supplement Series xxx (xxxx) xxx–xxx
C.H. Phua, et al.
Table 1 DNA concentrations obtained from DNA extracted using the developed method and TD (n = 5 per condition). No data is available for DNA extracted from 0.1 g of bone powder using TD as the amount is unfeasible for the protocol. DNA concentration (ng/μL) Method Developed
Bone Type Femur Tibia
Total Demineralisation
Femur Tibia
Bone amount (g) 0.1 0.5 0.1 0.5 0.5
Med 33.88 54.59 30.22 51.02 64.54 66.33
Min 26.63 35 24.69 47.94 55.93 40.3
Q1 28.06 50.51 25.71 50.17 60.29 59.47
Q3 38.76 56.12 38.24 56.53 93.92 66.51
Max 85.78 148.96 68.69 116.23 171.16 102.91
profiles from fresh bone samples. The rapid protocol allows fast, simple and high efficiency extraction of DNA from bone samples even from small amounts of samples. Role of funding This work was supported by Thailand’s Education Hub for ASEAN Countries Scholarship 2018, Prince of Songkla University, Thailand (Grant no. SCI6201021S).
Fig. 1. Peak heights and numbers of alleles obtained from DNA extracts of bone powder using the developed method and total demineralization.
Declaration of Competing Interest lower but comparable result in terms of quantity of DNA when compared to TD described by Loreille et al. [5]. Even when only 0.1 g of bone powder was used, sufficient DNA concentrations for further STR profiling were obtained (> 1 ng/μL). We attributed the success of the rapid protocol to the buffer used, which helped to break down the bone matrix and thus released DNA within. SLS functions to lysis the cells by disrupting the cell membrane, EDTA targets and chelates calcium presented, proteinase K denatures the structural proteins, and DTT reduces the disulphide bonds in bones.
None Acknowledgement We acknowledge the Forensic Medicine and Toxicology Unit, Department of Pathology, Faculty of Medicine, Prince of Songkla University, Thailand and the Department of Biotechnology, Faculty of Science, Prince of Songkla University, Thailand. References
3.2. Comparison of STR typing from DNA extracted with the developed method and TD
[1] D. Marjanović, et al., Identification of skeletal remains of Communist Armed Forces victims during and after World War II: combined Y-chromosome (STR) and MiniSTR approach, Croat. Med. J. 50 (2009) 296–304 http://www.ncbi.nlm.nih.gov/ pubmed/19480024. [2] M.M. Holland, C. Cave, C. Holland, T.W. Bille, Development of a quality, high throughput DNA analysis procedure for skeletal samples to assist with the identification of victims from the World Trade Center attacks, Croat. Med. J. 44 (2003) 264–272, https://doi.org/10.1371/journal.pone.0126935. [3] P.L. Marshall, et al., A high volume extraction and purification method for recovering DNA from human bone, Forensic Sci. Int. Genet. 12 (2014) 155–160, https://doi.org/ 10.1016/j.fsigen.2014.06.011. [4] A. Barbaro, P. Cormaci, G. Falcone, Validation of BTA™ lysis buffer for DNA extraction from challenged forensic samples, Forensic Sci. Int. Genet. Suppl. Ser. 3 (2011) e61–e62, https://doi.org/10.1016/j.fsigss.2011.08.030. [5] O.M. Loreille, et al., High efficiency DNA extraction from bone by total demineralization, Forensic Sci. Int. Genet. 1 (2007) 191–195, https://doi.org/10.1016/j. fsigen.2007.02.006. [6] J. Jakubowska, A. Maciejewska, R. Pawłowski, Comparison of three methods of DNA extraction from human bones with different degrees of degradation, Int. J. Legal Med. 126 (2012) 173–178, https://doi.org/10.1007/s00414-011-0590-5.
DNA from two of the five samples that were extracted were selected from both tibia and femur for each extraction method. Full profiles were obtained for both methods but with differences in median peak heights. This did not affect profile interpretability, but the developed method used significantly shorter incubation time and processing steps. This suggests that it has the potential to be used for cases where immediate results are needed or when there is only a small amount of bone sample acquired. Future experiment could include actual casework samples for real-life applications (Fig. 1). 4. Conclusion A simplified and rapid DNA extraction protocol from bone samples was successfully developed. Our final protocol only required two hours of incubation and 0.1 g of bone powder. STR typing results gave full
2
Contents lists available at ScienceDirect
Forensic Science International: Genetics Supplement Series journal homepage: www.elsevier.com/locate/fsigss
Simplified, rapid DNA extraction protocol for STR typing from bones Cheng Ho Phuaa, Laila Hasapb, Phuvadol Thanakiatkraia, Thitika Kitpipita,* a b
Forensic Science Programme, Department of Applied Science, Faculty of Science, Prince of Songkla University, Thailand Department of Molecular Biotechnology and Bioinformatics, Faculty of Science, Prince of Songkla University, Thailand
A R T I C LE I N FO
A B S T R A C T
Keywords: Bone extraction DNA Rapid
Bones are one of the body parts most tolerant towards various environmental conditions, especially as unidentified remains found in several circumstances: mass disaster, terrorism, war, and plague. Typically, the most complex and time-consuming part of the STR typing process is DNA extraction, with many different methods developed, such as organic extraction, total demineralization (TD), and silica-based column extraction. These protocols may take more than a day to complete. In such events where time is of the essence for human identification of the remains, a simple and fast but effective method is required. This study aimed to develop a simplified and rapid DNA extraction protocol from bone samples. Two bone types (fresh femur and tibia) were incubated in a buffer of SLS, EDTA, proteinase K, and DTT. We also evaluated extraction from different amounts of bone powder. Comparison was done for the developed method against standard total demineralisation protocol. Quantity of DNA extracted was slightly lower for the developed protocol compared to TD, however this did not affect STR typing and profile interpretability. DNA extraction was also possible from merely 0.1 g of bone. The developed method used significantly shorter incubation time and processing steps. Our final protocol only required two hours of incubation and 0.1 g of bone powder. STR typing results gave full profiles. The rapid protocol could be used when there is an urgent need of STR typing for human identification or when the sample amount is limited with a significant reduction in cost and time for investigation.
1. Introduction
2. Materials and methods
Bones tend to last longer than other biological materials in extreme conditions such as mass disaster, plague, terrorism, and etc [1,2]. They are often found as remains and may be fragmented and intermixed, which makes it difficult for morphological identification by forensic anthropologists. Therefore, DNA-based analysis methods are often necessary. Being enduring biological material, DNA entrapped within cells in the bone matrix are also difficult to be isolated. Extraction of DNA is normally required for analysis from bone samples in which there have been many different methods with varying approach, efficiency, and application: organic extraction method [3], Hi-Flow silica-based column [3], and proprietary PrepFiler® BTA [4], and total demineralization [5]. The extracted DNA vary in terms of quantity and purity, as well as the type and amount of carry-over PCR inhibitors [6]. Additionally, these methods are tedious, time-consuming, and laborious. Therefore, we aimed to develop a simplified and rapid DNA extraction protocol from bone samples.
Fresh femur and tibia (n = 5 each) were used. The bone powder from a cryogenic grinder was incubated in SLS, EDTA, proteinase K, DTT at 56 °C for two hours. The mixture was then centrifuged at 10k rpm for three minutes. The supernatant was aliquoted and then diluted 1:50 with sterile water whereby this dilution was then used for subsequent DNA analysis. For comparison, the same samples were DNA extracted using the total demineralization protocol (TD) [5]. DNA from both rapid extraction and TD were quantified using Quantifiler® Trio Human DNA Quantification kit (Thermo Fisher Scientific, USA). STR typing was performed using QIAGEN® Investigator® IDplex Plus kit (Qiagen, Germany) in half-volume reaction with two μL of DNA added. Fragment analysis was performed using ABI PRISM® 310 Genetic Analyzer and data analysis was done using GeneMapper 4.2.1. 3. Results and discussion 3.1. Comparison of DNA quantity Table 1 shows that the developed extraction method yielded slightly
⁎
Corresponding author. E-mail address: [email protected] (T. Kitpipit).
https://doi.org/10.1016/j.fsigss.2019.10.108 Received 14 September 2019; Accepted 9 October 2019 1875-1768/ © 2019 Published by Elsevier B.V.
Please cite this article as: Cheng Ho Phua, et al., Forensic Science International: Genetics Supplement Series, https://doi.org/10.1016/j.fsigss.2019.10.108
Forensic Science International: Genetics Supplement Series xxx (xxxx) xxx–xxx
C.H. Phua, et al.
Table 1 DNA concentrations obtained from DNA extracted using the developed method and TD (n = 5 per condition). No data is available for DNA extracted from 0.1 g of bone powder using TD as the amount is unfeasible for the protocol. DNA concentration (ng/μL) Method Developed
Bone Type Femur Tibia
Total Demineralisation
Femur Tibia
Bone amount (g) 0.1 0.5 0.1 0.5 0.5
Med 33.88 54.59 30.22 51.02 64.54 66.33
Min 26.63 35 24.69 47.94 55.93 40.3
Q1 28.06 50.51 25.71 50.17 60.29 59.47
Q3 38.76 56.12 38.24 56.53 93.92 66.51
Max 85.78 148.96 68.69 116.23 171.16 102.91
profiles from fresh bone samples. The rapid protocol allows fast, simple and high efficiency extraction of DNA from bone samples even from small amounts of samples. Role of funding This work was supported by Thailand’s Education Hub for ASEAN Countries Scholarship 2018, Prince of Songkla University, Thailand (Grant no. SCI6201021S).
Fig. 1. Peak heights and numbers of alleles obtained from DNA extracts of bone powder using the developed method and total demineralization.
Declaration of Competing Interest lower but comparable result in terms of quantity of DNA when compared to TD described by Loreille et al. [5]. Even when only 0.1 g of bone powder was used, sufficient DNA concentrations for further STR profiling were obtained (> 1 ng/μL). We attributed the success of the rapid protocol to the buffer used, which helped to break down the bone matrix and thus released DNA within. SLS functions to lysis the cells by disrupting the cell membrane, EDTA targets and chelates calcium presented, proteinase K denatures the structural proteins, and DTT reduces the disulphide bonds in bones.
None Acknowledgement We acknowledge the Forensic Medicine and Toxicology Unit, Department of Pathology, Faculty of Medicine, Prince of Songkla University, Thailand and the Department of Biotechnology, Faculty of Science, Prince of Songkla University, Thailand. References
3.2. Comparison of STR typing from DNA extracted with the developed method and TD
[1] D. Marjanović, et al., Identification of skeletal remains of Communist Armed Forces victims during and after World War II: combined Y-chromosome (STR) and MiniSTR approach, Croat. Med. J. 50 (2009) 296–304 http://www.ncbi.nlm.nih.gov/ pubmed/19480024. [2] M.M. Holland, C. Cave, C. Holland, T.W. Bille, Development of a quality, high throughput DNA analysis procedure for skeletal samples to assist with the identification of victims from the World Trade Center attacks, Croat. Med. J. 44 (2003) 264–272, https://doi.org/10.1371/journal.pone.0126935. [3] P.L. Marshall, et al., A high volume extraction and purification method for recovering DNA from human bone, Forensic Sci. Int. Genet. 12 (2014) 155–160, https://doi.org/ 10.1016/j.fsigen.2014.06.011. [4] A. Barbaro, P. Cormaci, G. Falcone, Validation of BTA™ lysis buffer for DNA extraction from challenged forensic samples, Forensic Sci. Int. Genet. Suppl. Ser. 3 (2011) e61–e62, https://doi.org/10.1016/j.fsigss.2011.08.030. [5] O.M. Loreille, et al., High efficiency DNA extraction from bone by total demineralization, Forensic Sci. Int. Genet. 1 (2007) 191–195, https://doi.org/10.1016/j. fsigen.2007.02.006. [6] J. Jakubowska, A. Maciejewska, R. Pawłowski, Comparison of three methods of DNA extraction from human bones with different degrees of degradation, Int. J. Legal Med. 126 (2012) 173–178, https://doi.org/10.1007/s00414-011-0590-5.
DNA from two of the five samples that were extracted were selected from both tibia and femur for each extraction method. Full profiles were obtained for both methods but with differences in median peak heights. This did not affect profile interpretability, but the developed method used significantly shorter incubation time and processing steps. This suggests that it has the potential to be used for cases where immediate results are needed or when there is only a small amount of bone sample acquired. Future experiment could include actual casework samples for real-life applications (Fig. 1). 4. Conclusion A simplified and rapid DNA extraction protocol from bone samples was successfully developed. Our final protocol only required two hours of incubation and 0.1 g of bone powder. STR typing results gave full
2