- Email: [email protected]
Evaluation of five DNA extraction systems for recovery of DNA from bone
Forensic Science International: Genetics Supplement Series 4 (2013) e174–e175
Contents lists available at ScienceDirect
Forensic Science International: Genetics Supplement Series journal homepage: www.elsevier.com/locate/FSIGSS
Evaluation of five DNA extraction systems for recovery of DNA from bone S. Iyavoo a,b, S. Hadi a, W. Goodwin a,* a b
School of Forensic and Investigative Sciences, University of Central Lancashire, Preston, UK Forensic Division, Department of Chemistry Malaysia (Kimia), Ministry of Science, Technology and Innovation, Malaysia
A R T I C L E I N F O
A B S T R A C T
Article history: Received 4 September 2013 Accepted 2 October 2013
Five DNA extraction systems were assessed for their DNA extraction efficiency on samples of fresh pig bone. Four commercially available silica-based extraction kits (ChargeSwitch1 gDNA Plant Kit (Life Technologies), DNA IQTM System Kit (Promega), DNeasy1 Blood & Tissue Kit (Qiagen) and PrepFiler1 BTA Forensic DNA Extraction Kit (Life Technologies)) and a conventional phenol-chloroform method were tested in this study. Extracted DNA samples were quantitated with GoTaq1 qPCR Master Mix (Promega) using an Applied Biosystems1 7500 Real-Time PCR System and the extracts were amplified using an inhouse multiplex system. The phenol-chloroform extraction produced higher yields of DNA than the silica-based extraction methods. Among the silica-based extractions ChargeSwitch1 gDNA Plant Kit recovered the highest amounts of DNA. However, all methods produced DNA that could be amplified and none of the extracts contained any detectable inhibition. ß 2013 Elsevier Ireland Ltd. All rights reserved.
Keywords: DNA extraction Bone Silica-based extraction Forensic
1. Introduction Isolation of DNA from skeletal human remains can be problematic. In addition to DNA degradation, which is enhanced by high temperature and humidity, there are often potent PCR inhibitors present within the samples. It is therefore important to extract the maximum amount of available DNA whilst removing any PCR inhibitors that may be present [1]. Phenol-chloroformbased extraction methods have been used for extracting DNA from bone for many years [2]. However, the use of phenol-chloroform method has reduced because of the toxicity of the chemicals used; in contrast silica-based methods have gained popularity due to their relative ease of use, their ability to remove many PCR inhibitors and also the potential to automate some steps in the extraction process [3,4]. However, all extraction methods are still in use in forensic laboratories. 2. Materials and methods 2.1. Sample collection and preparation Fresh pig bone samples (femur and rib) were purchased from a butcher. Samples were stored at 20 8C and prior to extraction any
* Corresponding author. Tel.: +44 1772 894254; fax: +44 1772 894981. E-mail addresses: [email protected] (S. Iyavoo), [email protected] (S. Hadi), [email protected] (W. Goodwin). 1875-1768/$ – see front matter ß 2013 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.fsigss.2013.10.090
soft tissue on the bones was removed. The bones were then soaked in bleach for 15 min, rinsed with water and then dried. Portions of each bone were prepared weighing between 1 and 2 g; after cutting any bone marrow was removed before pulverization under liquid nitrogen. 2.2. DNA extraction DNA extraction was carried out using ChargeSwitch1 gDNA Plant Kit, DNA IQTM System Kit, DNeasy1 Blood & Tissue Kit and PrepFiler1 BTA Forensic DNA Extraction Kit and a conventional phenol-chloroform method, according to the manufacturer’s instructions and published methods. Approximately 50 mg of pulverized bone samples were used in each extraction. Extractions were carried out in triplicate for each method and the final elution volume was set at 100 ml in each extraction. 2.3. DNA quantification and amplification The extracted DNA samples were quantified with GoTaq1 qPCR Master Mix using an Applied Biosystems1 7500 Real-Time PCR System. A 70 bp amplicon was targeted in reactions comprising 6.25 ml 2 GoTaq qPCR Master Mix, 4.75 ml H2O, 0.5 ml primers and 1.00 ml template DNA. The thermal cycler conditions were: 95 8C for 2 min, followed by 40 cycles of 95 8C for 15 s and 60 8C for 1 min. The DNA concentration for each sample was estimated in ng/ml. All DNA extracts were amplified using an in-house
S. Iyavoo et al. / Forensic Science International: Genetics Supplement Series 4 (2013) e174–e175
DNA concentraon (ng/μl)
120 Femur
100
Rib
80 60 40 20 0
Extracon methods Fig. 1. Comparison of DNA yield from fresh pig bone samples (femur and rib) using different extraction methods: phenol-chloroform, DNeasy1 Blood and Tissue kit, Chargeswitch1 gDNA Plant kit, PrepFiler1 BTA Forensic DNA Extraction Kit and DNA IQTM System Kit. The final volume of all extraction methods was set at 100 ml.
e175
All of the tested methods have proven to be suitable in that amplifiable DNA has been extracted. However, the samples tested so far are fresh bones which contain abundant DNA. When dealing with more challenging samples the increased recovery of DNA seen with the phenol-chloroform method may become more important. Although phenol-chloroform method produces high DNA yield in this study, previous studies show that silica-based extraction methods can be more efficient at removing inhibitors from bone samples [7]. Also, when using with environmentally challenged samples the ability to remove PCR inhibitors will become more relevant [1]. Further studies will be undertaken using degraded bone samples and also bone samples that have been shown to contain high levels of PCR inhibitors. This will allow the most appropriate method for extracting DNA from samples that have been exposed to environmental insult and are likely to be problematic. Role of funding This study was funded by a grant from Public Service Department of Malaysia.
Conflict of interest multiplex [5] and inhibition was tested for using internal amplification controls [6].
None. Acknowledgements
3. Results DNA was successfully extracted from bone samples using all tested extraction methods. The comparison study shows that the phenol-chloroform extraction method yields more DNA than the silica-based extraction methods (Fig. 1). Although phenol-chloroform method produced the highest DNA yields, all samples could be successfully amplified using an in-house multiplex that targets four amplicons that range in size between 70 bp and 384 bp [5]. No inhibition was detected in any of the samples when amplifying two internal amplification controls [6]. 4. Discussion The main aim of this research was to assess the capability of five DNA extraction systems on bone samples. The ultimate aim of the extraction process is to obtain the maximum amount of DNA from the samples submitted. At the same time, it is also important to get a pure DNA extraction by eliminating inhibitors which can reduce the efficiency of the amplification process, thus selection of extraction techniques is very important for samples containing PCR inhibitors.
We thank the Director General, Section Head and the staff at the Forensic DNA Section, Forensic Division, Department of Chemistry (Kimia), Ministry of Science, Technology and Innovation in Malaysia. References [1] R. Alaeddini, Forensic implications of PCR inhibition – a review, Foren. Sci. Int. Genet. 6 (2012) 297–305. [2] J. Jakubowska, A. Maciejewska, R. Pawlowski, Comparison of three methods of DNA extraction from human bones with different degrees of degradation, Int. J. Legal Med. 126 (2012) 173–178. [3] T.Y. Wang, L. Wang, J.H. Zhang, W.H. Dong, A simplified universal genomic DNA extraction protocol suitable for PCR, Genet. Mol. Res. 10 (2011) 519–525. [4] S. Witt, J. Neumann, H. Zierdt, G. Gebel, C. Roescheisen, Establishing a novel automated magnetic bead-based method for the extraction of DNA from a variety of forensic samples, Foren. Sci. Int. Genet. 6 (2012) 539–547. [5] M.S. Nazir, J.A. Smith, W. Goodwin, DNA degradation in post-mortem soft muscle tissues in relation to accumulated degree-days (ADD), Foren. Sci. Int. Genet. Suppl. Ser. 3 (2011) e536–e537. [6] N. Zahra, S. Hadi, J.A. Smith, A. Iyengar, W. Goodwin, Development of internal amplification controls for DNA profiling with the AmpFlSTR1 SGM Plus1 kit, Electrophoresis 32 (2011) 1371–1378. [7] J. Davoren, D. Vanek, R. Konjhodzic, J. Crews, E. Huffine, T.J. Parsons, Highly effective DNA extraction method for nuclear short tandem repeat testing of skeletal remains from mass graves, Croat. Med. J. 48 (2007) 478–485.
Contents lists available at ScienceDirect
Forensic Science International: Genetics Supplement Series journal homepage: www.elsevier.com/locate/FSIGSS
Evaluation of five DNA extraction systems for recovery of DNA from bone S. Iyavoo a,b, S. Hadi a, W. Goodwin a,* a b
School of Forensic and Investigative Sciences, University of Central Lancashire, Preston, UK Forensic Division, Department of Chemistry Malaysia (Kimia), Ministry of Science, Technology and Innovation, Malaysia
A R T I C L E I N F O
A B S T R A C T
Article history: Received 4 September 2013 Accepted 2 October 2013
Five DNA extraction systems were assessed for their DNA extraction efficiency on samples of fresh pig bone. Four commercially available silica-based extraction kits (ChargeSwitch1 gDNA Plant Kit (Life Technologies), DNA IQTM System Kit (Promega), DNeasy1 Blood & Tissue Kit (Qiagen) and PrepFiler1 BTA Forensic DNA Extraction Kit (Life Technologies)) and a conventional phenol-chloroform method were tested in this study. Extracted DNA samples were quantitated with GoTaq1 qPCR Master Mix (Promega) using an Applied Biosystems1 7500 Real-Time PCR System and the extracts were amplified using an inhouse multiplex system. The phenol-chloroform extraction produced higher yields of DNA than the silica-based extraction methods. Among the silica-based extractions ChargeSwitch1 gDNA Plant Kit recovered the highest amounts of DNA. However, all methods produced DNA that could be amplified and none of the extracts contained any detectable inhibition. ß 2013 Elsevier Ireland Ltd. All rights reserved.
Keywords: DNA extraction Bone Silica-based extraction Forensic
1. Introduction Isolation of DNA from skeletal human remains can be problematic. In addition to DNA degradation, which is enhanced by high temperature and humidity, there are often potent PCR inhibitors present within the samples. It is therefore important to extract the maximum amount of available DNA whilst removing any PCR inhibitors that may be present [1]. Phenol-chloroformbased extraction methods have been used for extracting DNA from bone for many years [2]. However, the use of phenol-chloroform method has reduced because of the toxicity of the chemicals used; in contrast silica-based methods have gained popularity due to their relative ease of use, their ability to remove many PCR inhibitors and also the potential to automate some steps in the extraction process [3,4]. However, all extraction methods are still in use in forensic laboratories. 2. Materials and methods 2.1. Sample collection and preparation Fresh pig bone samples (femur and rib) were purchased from a butcher. Samples were stored at 20 8C and prior to extraction any
* Corresponding author. Tel.: +44 1772 894254; fax: +44 1772 894981. E-mail addresses: [email protected] (S. Iyavoo), [email protected] (S. Hadi), [email protected] (W. Goodwin). 1875-1768/$ – see front matter ß 2013 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.fsigss.2013.10.090
soft tissue on the bones was removed. The bones were then soaked in bleach for 15 min, rinsed with water and then dried. Portions of each bone were prepared weighing between 1 and 2 g; after cutting any bone marrow was removed before pulverization under liquid nitrogen. 2.2. DNA extraction DNA extraction was carried out using ChargeSwitch1 gDNA Plant Kit, DNA IQTM System Kit, DNeasy1 Blood & Tissue Kit and PrepFiler1 BTA Forensic DNA Extraction Kit and a conventional phenol-chloroform method, according to the manufacturer’s instructions and published methods. Approximately 50 mg of pulverized bone samples were used in each extraction. Extractions were carried out in triplicate for each method and the final elution volume was set at 100 ml in each extraction. 2.3. DNA quantification and amplification The extracted DNA samples were quantified with GoTaq1 qPCR Master Mix using an Applied Biosystems1 7500 Real-Time PCR System. A 70 bp amplicon was targeted in reactions comprising 6.25 ml 2 GoTaq qPCR Master Mix, 4.75 ml H2O, 0.5 ml primers and 1.00 ml template DNA. The thermal cycler conditions were: 95 8C for 2 min, followed by 40 cycles of 95 8C for 15 s and 60 8C for 1 min. The DNA concentration for each sample was estimated in ng/ml. All DNA extracts were amplified using an in-house
S. Iyavoo et al. / Forensic Science International: Genetics Supplement Series 4 (2013) e174–e175
DNA concentraon (ng/μl)
120 Femur
100
Rib
80 60 40 20 0
Extracon methods Fig. 1. Comparison of DNA yield from fresh pig bone samples (femur and rib) using different extraction methods: phenol-chloroform, DNeasy1 Blood and Tissue kit, Chargeswitch1 gDNA Plant kit, PrepFiler1 BTA Forensic DNA Extraction Kit and DNA IQTM System Kit. The final volume of all extraction methods was set at 100 ml.
e175
All of the tested methods have proven to be suitable in that amplifiable DNA has been extracted. However, the samples tested so far are fresh bones which contain abundant DNA. When dealing with more challenging samples the increased recovery of DNA seen with the phenol-chloroform method may become more important. Although phenol-chloroform method produces high DNA yield in this study, previous studies show that silica-based extraction methods can be more efficient at removing inhibitors from bone samples [7]. Also, when using with environmentally challenged samples the ability to remove PCR inhibitors will become more relevant [1]. Further studies will be undertaken using degraded bone samples and also bone samples that have been shown to contain high levels of PCR inhibitors. This will allow the most appropriate method for extracting DNA from samples that have been exposed to environmental insult and are likely to be problematic. Role of funding This study was funded by a grant from Public Service Department of Malaysia.
Conflict of interest multiplex [5] and inhibition was tested for using internal amplification controls [6].
None. Acknowledgements
3. Results DNA was successfully extracted from bone samples using all tested extraction methods. The comparison study shows that the phenol-chloroform extraction method yields more DNA than the silica-based extraction methods (Fig. 1). Although phenol-chloroform method produced the highest DNA yields, all samples could be successfully amplified using an in-house multiplex that targets four amplicons that range in size between 70 bp and 384 bp [5]. No inhibition was detected in any of the samples when amplifying two internal amplification controls [6]. 4. Discussion The main aim of this research was to assess the capability of five DNA extraction systems on bone samples. The ultimate aim of the extraction process is to obtain the maximum amount of DNA from the samples submitted. At the same time, it is also important to get a pure DNA extraction by eliminating inhibitors which can reduce the efficiency of the amplification process, thus selection of extraction techniques is very important for samples containing PCR inhibitors.
We thank the Director General, Section Head and the staff at the Forensic DNA Section, Forensic Division, Department of Chemistry (Kimia), Ministry of Science, Technology and Innovation in Malaysia. References [1] R. Alaeddini, Forensic implications of PCR inhibition – a review, Foren. Sci. Int. Genet. 6 (2012) 297–305. [2] J. Jakubowska, A. Maciejewska, R. Pawlowski, Comparison of three methods of DNA extraction from human bones with different degrees of degradation, Int. J. Legal Med. 126 (2012) 173–178. [3] T.Y. Wang, L. Wang, J.H. Zhang, W.H. Dong, A simplified universal genomic DNA extraction protocol suitable for PCR, Genet. Mol. Res. 10 (2011) 519–525. [4] S. Witt, J. Neumann, H. Zierdt, G. Gebel, C. Roescheisen, Establishing a novel automated magnetic bead-based method for the extraction of DNA from a variety of forensic samples, Foren. Sci. Int. Genet. 6 (2012) 539–547. [5] M.S. Nazir, J.A. Smith, W. Goodwin, DNA degradation in post-mortem soft muscle tissues in relation to accumulated degree-days (ADD), Foren. Sci. Int. Genet. Suppl. Ser. 3 (2011) e536–e537. [6] N. Zahra, S. Hadi, J.A. Smith, A. Iyengar, W. Goodwin, Development of internal amplification controls for DNA profiling with the AmpFlSTR1 SGM Plus1 kit, Electrophoresis 32 (2011) 1371–1378. [7] J. Davoren, D. Vanek, R. Konjhodzic, J. Crews, E. Huffine, T.J. Parsons, Highly effective DNA extraction method for nuclear short tandem repeat testing of skeletal remains from mass graves, Croat. Med. J. 48 (2007) 478–485.