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Comparison of two DNA extraction methods: PrepFiler® BTA and modified PCI-silica based for DNA analysis from bone
Journal Pre-proof COMPARISON OF TWO DNA EXTRACTION METHODS: PREPFILER® BTA AND MODIFIED PCI-SILICA BASED FOR DNA ANALYSIS FROM BONE Laila Hasap, Wilaiwan Chotigeat, Jintana Pradutkanchana, Watee Asawutmangkul, Thitika Kitpipit, Phuvadol Thanakiatkrai
PII:
S1875-1768(19)30084-8
DOI:
https://doi.org/10.1016/j.fsigss.2019.10.132
Reference:
FSIGSS 1765
To appear in:
Forensic Science International: Genetics Supplement Series
Received Date:
14 September 2019
Revised Date:
8 October 2019
Accepted Date:
10 October 2019
Please cite this article as: Hasap L, Chotigeat W, Pradutkanchana J, Asawutmangkul W, Kitpipit T, Thanakiatkrai P, COMPARISON OF TWO DNA EXTRACTION METHODS: PREPFILER® BTA AND MODIFIED PCI-SILICA BASED FOR DNA ANALYSIS FROM BONE, Forensic Science International: Genetics Supplement Series (2019), doi: https://doi.org/10.1016/j.fsigss.2019.10.132
This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier.
Page 1 of 4 COMPARISON OF TWO DNA EXTRACTION METHODS: PREPFILER® BTA AND MODIFIED PCI-SILICA BASED FOR DNA ANALYSIS FROM BONE Laila Hasapa, Wilaiwan Chotigeata, Jintana Pradutkanchanab, Watee Asawutmangkulc, Thitika Kitpipitd, Phuvadol Thanakiatkraid a
Department of Molecular Biotechnology and Bioinformatics, Faculty of Science, Prince of Songkla University, Thailand Forensic Medicine and Toxicology Unit, Department of Pathology, Faculty of Medicine, Prince of Songkla University, Thailand c Institute of Forensic Medicine, Royal Thai Police, Thailand d Forensic Science Program, Department of Applied Science, Faculty of Science, Prince of Songkla University, Thailand b
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* Corresponding author: [email protected] Tel : +66-88-074-7074. Fax : +66-74-446-681. Address: 15 Karnjanavanich Road, Department of Applied Science, Faculty of Science, Prince of Songkla University, Hat Yai, Songkhla, Thailand 90110
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Abstract Bones are important evidence often found in crime scene associated with mass grave disaster and missing persons. Substantial fragmentation and intermixing of bones limit the use of morphological observation. Human individualization using DNA analysis can complement but the challenges are the PCR inhibitors contained and low amount of DNA in degraded bone. The selection of effective DNA extraction method that minimizes PCR inhibitors and maximizes DNA recovery thus is crucial. This study aimed to compare the efficiency of the currently modified PCI-silica and the latest forensic commercial kit developed for challenging bone sample (PrepFiler® BTA). The results showed that PrepFiler® BTA provided significantly higher median DNA concentration (p < 0.05) and 95.3% probability of obtaining more alleles compared to modified PCI-silica based method. The efficiency of PCR inhibitors removal showed that both extraction methods were comparable at minimizing PCR inhibitors. Our study could serve as another guideline for selection of most suitable method for DNA analysis from bone.
1.) Introduction
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Keywords: DNA extraction, bone, PrepFilerBTA, phenol-chloroform, silica
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Bones are one important evidence often found in crime scenes related to mass disaster, terrorism, human trafficking and missing persons. Morphological observation by forensic anthropologists can be used to identify age, sex, stature, and race for human individualization [1], but it is not accurate when highly fragmented and intermixed remains are found. DNA analysis can overcome these problems but the challenge is bones contain high amounts of PCR inhibitors, which could be co-purified during DNA extraction process and, sometimes only low amount and highly degraded DNA are found, especially in forensic samples [2]. An effective DNA extraction method, thus, simultaneously minimizes PCR inhibitors and maximizes DNA recovery. Modified organic extraction combined with silica purification has been shown to improve DNA profiles from bones [3]. Additionally, the commercial PrepFiler® BTA kit (Thermo Fisher Scientific, USA) has been developed specifically for challenging bone samples and proven to improve the resulting STR profiles [4, 5]. Therefore, we aimed to compare these two DNA extraction methods by assessing of the amount of DNA recovered, the presence of PCR inhibitors, and the number of alleles detected in the STR profiles of both fresh and casework bone samples. 2.) Materials and methods Five fresh tibia and eighteen casework bones were obtained from the Department of Anatomy, Faculty of Science, Prince of Songkla University, and Forensic Medicine and Toxicology Unit, Department of Pathology, Faculty of Medicine, Prince of Songkla University. DNA was extracted from the samples using the PrepFiler® BTA extraction kit according to the manufacturer’s protocol and the modified PCI-silica based method with QIAamp® DNA Investigator kit purification
Page 2 of 4 (Qiagen, Germany) [3]. DNA from the two extraction methods were quantified using the Quantifiler® Human DNA Quantification Kit. STR typing of casework samples was performed using the QIAGEN® Investigator® IDplex Plus kit.
3.) Results and discussion
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3.1 DNA concentrations and number of alleles detected For fresh samples, median DNA concentrations were 59.5 ng/µl using PrepFiler® BTA and 0.620 ng/µl using modified PCIsilica (Fig 1a). A similar vast difference in DNA concentration was seen in casework samples, for which PrepFiler® BTA yielded median DNA concentration of 0.230 ng/µl and modified PCI-silica yielded 0.010 ng/µl (Fig 1b). The difference in means of DNA concentration between these two methods calculated using “Bayesian Estimation Supersedes the t-test” [6] (with the 95% credible interval) were 62.8 (28.7-100) ng/µl for fresh samples and 0.148 (0.002 – 0.296) ng/µl for casework samples, indicating a credible difference between the two methods.
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STR typing was also performed in casework samples. DNA extracted using PrepFiler® BTA resulted in a median of 30 alleles while DNA extracted using modified PCI-silica resulted in a median of only eight alleles (Fig 1b). Statistically, the difference in means of number of alleles between the two methods was 8.74 alleles. The probability of superiority (the chance that a sample picked at random from one group will have high number of allele than the other group) were 95.3% for PrepFiler® BTA vs modified PCI-silica based.
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3.2 Residual PCR inhibitors PCR inhibitors were also monitored using the threshold cycle (Ct) of synthetic internal PCR control (IPC) of the Quantifiler® kit. The results showed that median IPC Ct of PrepFiler® BTA and modified PCI-silica were 26.07 and 26.14, respectively. These values were not credibly different from the median of 26.05 of the negative controls (Fig 2), indicating that both PrepFiler® BTA and modified PCI-silica were efficient at minimizing carry-over PCR inhibitors. 4.) Conclusion
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The efficiency of PrepFiler® BTA and modified PCI-silica based method was compared in this study. Assessment of DNA recovery and number of alleles found showed that PrepFiler® BTA achieved better results for both fresh and casework samples. No difference was seen in terms of inhibitor removal. Our results suggest that PrepFiler® BTA should be used for casework bone samples.
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Acknowledgement We acknowledge Pol.Maj. Sukanya Phetpeng and Mr. Kosal Puriwatanakul for technical help. Conflict of interest None Role of funding This work was supported by Thailand's Education Hub for ASEAN Countries Scholarship 2015 and Graduate Studies Research Grant, Prince of Songkla University, Thailand.
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References 1. Zietkiewicz, E., et al., Current genetic methodologies in the identification of disaster victims and in forensic analysis. J Appl Genet, 2012. 53(1): p. 41-60. 2. Rohland, N. and M. Hofreiter, Ancient DNA extraction from bones and teeth. Nat Protoc, 2007. 2(7): p. 1756-1762. 3. Desmyter, S., et al., Organic extraction of bone lysates improves DNA purification with silica beads. Forensic Sci Int, 2017. 273: p. 96-101. 4. Barbaro, A., P. Cormaci, and G. Falcone, Validation of BTA™ lysis buffer for DNA extraction from challenged forensic samples. Forensic Sci Int Genet Suppl Ser, 2011. 3(1): p. e61-e62. 5. Zupanic Pajnic, I., et al., Highly efficient automated extraction of DNA from old and contemporary skeletal remains. J Forensic Leg Med, 2016. 37: p. 78-86. 6. Kruschke, J.K., Bayesian estimation supersedes the t test. J Exp Psychol Gen, 2013. 142(2): p. 573-603.
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Fig 1 Comparison of the median DNA concentrations and number of alleles obtained using the PrepFiler ® BTA and modified PCI-silica from (a) fresh samples (n = 5) and (b) casework samples (n = 18).
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Fig 2 The IPC Ct of DNA extracted using PrepFiler® BTA and modified PCI-silica (n = 18) compared to those of the negative controls. Each dot represents one sample. The solid color lines show the median IPC Ct. The boxes show the 95% Bayesian credible intervals. Non-overlapping intervals indicate credible difference.
PII:
S1875-1768(19)30084-8
DOI:
https://doi.org/10.1016/j.fsigss.2019.10.132
Reference:
FSIGSS 1765
To appear in:
Forensic Science International: Genetics Supplement Series
Received Date:
14 September 2019
Revised Date:
8 October 2019
Accepted Date:
10 October 2019
Please cite this article as: Hasap L, Chotigeat W, Pradutkanchana J, Asawutmangkul W, Kitpipit T, Thanakiatkrai P, COMPARISON OF TWO DNA EXTRACTION METHODS: PREPFILER® BTA AND MODIFIED PCI-SILICA BASED FOR DNA ANALYSIS FROM BONE, Forensic Science International: Genetics Supplement Series (2019), doi: https://doi.org/10.1016/j.fsigss.2019.10.132
This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier.
Page 1 of 4 COMPARISON OF TWO DNA EXTRACTION METHODS: PREPFILER® BTA AND MODIFIED PCI-SILICA BASED FOR DNA ANALYSIS FROM BONE Laila Hasapa, Wilaiwan Chotigeata, Jintana Pradutkanchanab, Watee Asawutmangkulc, Thitika Kitpipitd, Phuvadol Thanakiatkraid a
Department of Molecular Biotechnology and Bioinformatics, Faculty of Science, Prince of Songkla University, Thailand Forensic Medicine and Toxicology Unit, Department of Pathology, Faculty of Medicine, Prince of Songkla University, Thailand c Institute of Forensic Medicine, Royal Thai Police, Thailand d Forensic Science Program, Department of Applied Science, Faculty of Science, Prince of Songkla University, Thailand b
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* Corresponding author: [email protected] Tel : +66-88-074-7074. Fax : +66-74-446-681. Address: 15 Karnjanavanich Road, Department of Applied Science, Faculty of Science, Prince of Songkla University, Hat Yai, Songkhla, Thailand 90110
lP
re
-p
Abstract Bones are important evidence often found in crime scene associated with mass grave disaster and missing persons. Substantial fragmentation and intermixing of bones limit the use of morphological observation. Human individualization using DNA analysis can complement but the challenges are the PCR inhibitors contained and low amount of DNA in degraded bone. The selection of effective DNA extraction method that minimizes PCR inhibitors and maximizes DNA recovery thus is crucial. This study aimed to compare the efficiency of the currently modified PCI-silica and the latest forensic commercial kit developed for challenging bone sample (PrepFiler® BTA). The results showed that PrepFiler® BTA provided significantly higher median DNA concentration (p < 0.05) and 95.3% probability of obtaining more alleles compared to modified PCI-silica based method. The efficiency of PCR inhibitors removal showed that both extraction methods were comparable at minimizing PCR inhibitors. Our study could serve as another guideline for selection of most suitable method for DNA analysis from bone.
1.) Introduction
ur na
Keywords: DNA extraction, bone, PrepFilerBTA, phenol-chloroform, silica
Jo
Bones are one important evidence often found in crime scenes related to mass disaster, terrorism, human trafficking and missing persons. Morphological observation by forensic anthropologists can be used to identify age, sex, stature, and race for human individualization [1], but it is not accurate when highly fragmented and intermixed remains are found. DNA analysis can overcome these problems but the challenge is bones contain high amounts of PCR inhibitors, which could be co-purified during DNA extraction process and, sometimes only low amount and highly degraded DNA are found, especially in forensic samples [2]. An effective DNA extraction method, thus, simultaneously minimizes PCR inhibitors and maximizes DNA recovery. Modified organic extraction combined with silica purification has been shown to improve DNA profiles from bones [3]. Additionally, the commercial PrepFiler® BTA kit (Thermo Fisher Scientific, USA) has been developed specifically for challenging bone samples and proven to improve the resulting STR profiles [4, 5]. Therefore, we aimed to compare these two DNA extraction methods by assessing of the amount of DNA recovered, the presence of PCR inhibitors, and the number of alleles detected in the STR profiles of both fresh and casework bone samples. 2.) Materials and methods Five fresh tibia and eighteen casework bones were obtained from the Department of Anatomy, Faculty of Science, Prince of Songkla University, and Forensic Medicine and Toxicology Unit, Department of Pathology, Faculty of Medicine, Prince of Songkla University. DNA was extracted from the samples using the PrepFiler® BTA extraction kit according to the manufacturer’s protocol and the modified PCI-silica based method with QIAamp® DNA Investigator kit purification
Page 2 of 4 (Qiagen, Germany) [3]. DNA from the two extraction methods were quantified using the Quantifiler® Human DNA Quantification Kit. STR typing of casework samples was performed using the QIAGEN® Investigator® IDplex Plus kit.
3.) Results and discussion
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3.1 DNA concentrations and number of alleles detected For fresh samples, median DNA concentrations were 59.5 ng/µl using PrepFiler® BTA and 0.620 ng/µl using modified PCIsilica (Fig 1a). A similar vast difference in DNA concentration was seen in casework samples, for which PrepFiler® BTA yielded median DNA concentration of 0.230 ng/µl and modified PCI-silica yielded 0.010 ng/µl (Fig 1b). The difference in means of DNA concentration between these two methods calculated using “Bayesian Estimation Supersedes the t-test” [6] (with the 95% credible interval) were 62.8 (28.7-100) ng/µl for fresh samples and 0.148 (0.002 – 0.296) ng/µl for casework samples, indicating a credible difference between the two methods.
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STR typing was also performed in casework samples. DNA extracted using PrepFiler® BTA resulted in a median of 30 alleles while DNA extracted using modified PCI-silica resulted in a median of only eight alleles (Fig 1b). Statistically, the difference in means of number of alleles between the two methods was 8.74 alleles. The probability of superiority (the chance that a sample picked at random from one group will have high number of allele than the other group) were 95.3% for PrepFiler® BTA vs modified PCI-silica based.
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3.2 Residual PCR inhibitors PCR inhibitors were also monitored using the threshold cycle (Ct) of synthetic internal PCR control (IPC) of the Quantifiler® kit. The results showed that median IPC Ct of PrepFiler® BTA and modified PCI-silica were 26.07 and 26.14, respectively. These values were not credibly different from the median of 26.05 of the negative controls (Fig 2), indicating that both PrepFiler® BTA and modified PCI-silica were efficient at minimizing carry-over PCR inhibitors. 4.) Conclusion
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The efficiency of PrepFiler® BTA and modified PCI-silica based method was compared in this study. Assessment of DNA recovery and number of alleles found showed that PrepFiler® BTA achieved better results for both fresh and casework samples. No difference was seen in terms of inhibitor removal. Our results suggest that PrepFiler® BTA should be used for casework bone samples.
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Acknowledgement We acknowledge Pol.Maj. Sukanya Phetpeng and Mr. Kosal Puriwatanakul for technical help. Conflict of interest None Role of funding This work was supported by Thailand's Education Hub for ASEAN Countries Scholarship 2015 and Graduate Studies Research Grant, Prince of Songkla University, Thailand.
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References 1. Zietkiewicz, E., et al., Current genetic methodologies in the identification of disaster victims and in forensic analysis. J Appl Genet, 2012. 53(1): p. 41-60. 2. Rohland, N. and M. Hofreiter, Ancient DNA extraction from bones and teeth. Nat Protoc, 2007. 2(7): p. 1756-1762. 3. Desmyter, S., et al., Organic extraction of bone lysates improves DNA purification with silica beads. Forensic Sci Int, 2017. 273: p. 96-101. 4. Barbaro, A., P. Cormaci, and G. Falcone, Validation of BTA™ lysis buffer for DNA extraction from challenged forensic samples. Forensic Sci Int Genet Suppl Ser, 2011. 3(1): p. e61-e62. 5. Zupanic Pajnic, I., et al., Highly efficient automated extraction of DNA from old and contemporary skeletal remains. J Forensic Leg Med, 2016. 37: p. 78-86. 6. Kruschke, J.K., Bayesian estimation supersedes the t test. J Exp Psychol Gen, 2013. 142(2): p. 573-603.
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Fig 1 Comparison of the median DNA concentrations and number of alleles obtained using the PrepFiler ® BTA and modified PCI-silica from (a) fresh samples (n = 5) and (b) casework samples (n = 18).
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Fig 2 The IPC Ct of DNA extracted using PrepFiler® BTA and modified PCI-silica (n = 18) compared to those of the negative controls. Each dot represents one sample. The solid color lines show the median IPC Ct. The boxes show the 95% Bayesian credible intervals. Non-overlapping intervals indicate credible difference.