- Email: [email protected]
An innovative DNA extraction method: Water versus commercial buffers
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
An innovative DNA extraction method: Water versus commercial buffers ⁎
P. Mármola,1, B. Gómeza,1, C. Gomesa,b, , C. Romeroa, C. Baeza-Richera,b, A. Hernández-Corderoa, M. Martín-Arrebolaa, R. Rosell-Herreraa, A.M. López-Parraa,b, S. Palomo-Díeza,b, E. Labajo-Gonzálezc, B Perea-Pérezc, C. López-Matayoshia,b, E. Arroyo-Pardoa,b a
Laboratory of Forensic and Population Genetics, Legal Medicine, Psychiatry and Pathology Department, Medicine School, Complutense University of Madrid (UCM), Pza Ramón y Cajal s/n, 28040, Madrid, Spain b Grupo de Ciencias Forenses: Genética y Toxicología forense, Instituto de Investigación Sanitaria del Hospital Clínico San Carlos (IdISSC), Madrid, Spain c Medicine School, Complutense University of Madrid, Madrid, Spain
A R T I C LE I N FO
A B S T R A C T
Keywords: DNA-extraction Commercial buffer Water mtDNA STRs
The main objective of DNA extraction is to obtain good quality genetic material in order to carry out its amplification, and corresponding analysis. Most laboratories tend to resort to commercial extraction buffers, which allow a simple and rigorous DNA extraction, with limited handling of the sample, but with high financial cost. As an alternative, we proposed to use water as reagent to extract DNA from blood spots, collected on Whatman® FTA® Cards (Sigma-Aldrich®). We propose to take advantage of hypotonic solutions, in order to obtain a better efficiency in the extraction of genetic material (DNA). On this sense, 219 extractions were performed with Whatman® FTA® Cards (SigmaAldrich®) stained with blood, based on Garcia-Palacios et al., extraction method, with some modifications. Extractions were performed with two types of water (sterile (Lonza™) and distilled (Milli-Q, MilliporeSigma®), as well as, with a commercial extraction kit (Prep-n-Go™, ThermoFisher™ Scientific, Foster City, USA). To evaluate and compare each reagent effectiveness, we amplified DNA extract with mitochondrial DNA Hypervariable regions I and II, and with nuclear DNA markers (autosomal STRs and X-InDels). Our preliminary results suggest that distilled water allows an extraction as effective, or even better, as that obtained with the commercial buffer. These preliminary results offer the possibility for laboratories to choose an inexpensive protocol for DNA extraction, avoiding expensive commercial buffers. Further research is needed to evaluate the performance of each method in blood samples, but also in other biological samples.
1. Introduction The analysed samples in the field of forensic genetics usually present particular characteristics, like low template DNA, or inhibitors, for example, compelling the extraction process to be specific and directed to those samples. Therefore, to achieve an optimal DNA analysis, it is necessary to carry out an efficient and specific DNA extraction, since it constitutes a crucial point for forensic genetics research. The main purpose of DNA extractions is to obtain, from a biological sample and through the use of different solutions and physical and mechanical processes, cell lysis to extract the genetic material to allow its subsequent amplification [1]. Most laboratories tend to resort to expensive commercial kits, such as the Prep-n-Go™ buffer (ThermoFisher™ Scientific, Foster City, USA).
Designed for saliva extraction on different supports, this commercial buffer has also been tested on blood samples and the results have been satisfactory and previously published [2,3]. While on the one hand it has the advantage of reduced sample handling and extraction time, it has the important disadvantages of not reporting on the reagents that compose it, as well as, a very high monetary cost. Consequently, we proposed to use water as reagent to extract DNA from blood spots, by taking advantage of different water solutions, in order to obtain a better efficiency in the extraction of genetic material, and at the same time, to recommend a cheaper alternative method for DNA extraction.
⁎
Corresponding author at: Laboratory of Forensic and Population Genetics, Legal Medicine, Psychiatry and Pathology Department, Medicine School, Complutense University of Madrid, Avda. Complutense s/n 28040, Madrid, Spain. E-mail address: [email protected] (C. Gomes). 1 Both authors contributed equally to this study. https://doi.org/10.1016/j.fsigss.2019.09.111 Received 6 September 2019; Accepted 29 September 2019 1875-1768/ © 2019 Elsevier B.V. All rights reserved.
Please cite this article as: P. Mármol, et al., Forensic Science International: Genetics Supplement Series, https://doi.org/10.1016/j.fsigss.2019.09.111
Forensic Science International: Genetics Supplement Series xxx (xxxx) xxx–xxx
P. Mármol, et al.
2. Materials and Methods
analysing mtDNA, after a positive band was observed on the agarose gel.
2.1. DNA Extraction 4. Discussion DNA extractions were performed with Whatman® FTA® cards (SigmaAldrich®) stained with blood from 73 independent voluntary donors, after signed an informed consent. To perform the DNA extraction, form each stained Whatman® FTA® card, approximately 1 mm diameter were cut into a previously sterilized 1,5 mL tube. Additionally, bloodless FTA cards were used as a negative control. From each volunteer’s blood, three extractions were performed (a total of 219 analysis) - with sterile (Lonza™, Switzerland), and distilled (Milli-Q™ Advantage A10, MerkGroup, Germany) waters, and with the commercial extraction kit Prep-n-Go™(ThermoFisher™ Scientific, Foster City, USA), according to Garcia-Palacios et al., [2], with some modifications.
Our preliminary results indicate that it is possible to perform a DNA extraction from a FTA card stained with blood, both with sterile and distilled water. Indeed, these results suggest distilled water allows an extraction as effective, or even better as that obtained with the commercial buffer. To give an explanation to these results, it is necessary to understand the functionality of the FTA cards used in this experiment. When coming into contact with the blood sample, FTA cards smooth the cells and attach the DNA to the matrix card, allowing its preservation [7]. So, FTA cards perform a first extraction by causing the membrane lysis of the cells. Distilled and sterile water have both an ionic charge that allows them to interact with the DNA, causing the release of the genetic material from the matrix of the FTA card. The difference between them is based on the presence of a lower and higher ion concentration, respectively. In the case of distilled water, it corresponds to an ultra-hypotonic solution, with lower ions concentration compared to sterile water. So, the interaction between distilled water and DNA is stronger, and extraction from FTA card more effective. It is not possible to have access to the commercial buffer chemistry. However, since mtDNA is the most abundant genetic material in the intracellular environment, it is possible that most of the negative results may be due to a DNA inhibiting effect. We recommend a decrease in extract volume to amplify, when extraction has been performed with this buffer. Regarding auSTRs, all partial profiles were due to loci dropout in larger amplicon markers. In this case, the commercial buffer seems to be more efficient, since no partial profile was observed. Concerning X-Indels results, all amplifications produced positive results, probably due to its short length fragments, being impossible to differentiate between the three extractions methodologies efficiency.
2.2. DNA Amplification To evaluate and compare each reagents effectiveness, we amplified the extracts with nuclear DNA markers (autosomal STRs –auSTRs-, with AmpFLSTR NGM Select, ThermoFisher Scientific®, Foster City, USA, according to users’ manual, and X-chromosomal Insertion-Delection polymorphisms -X-InDels-, according to Pereira et al., [4]), using 5 μl of extract volume for each amplification. In the case of mitochondrial DNA (mtDNA), hypervariable regions I and II were amplified, according to [5], method C, evaluating DNA extraction and amplification with an agarose gel (1 g/50 mL TBE 1%). Then, 20 random samples were chosen from DNA amplifications (mitochondrial and each nuclear DNA). Considering mtDNA results, samples were chosen if a band could be detected on the agarose gel, being purified with ExoSAP-IT PCR product Cleanup reagent (ThermoFisher Scientific®, Foster City, USA). Sequences’ haplotypes were defined relative to the revised Cambridge reference mtDNA sequence [6], by running Mutation Surveyor® (Softgenetics) software, v 5.1.2. Considering nuclear DNA, capillary electrophoresis and detection were achieved in a 3130xl Genetic Analyzer (ThermoFisher™ Scientific, Foster City, USA). Electropherogram analysis and allele assignment were performed with GeneMapper v 4.0 (ThermoFisher Scientific®, Foster City, USA).
5. Conclusion These preliminary results offer the possibility for laboratories to choose an inexpensive protocol for DNA extraction from FTA cards stained with blood.
3. Results
Declaration of Competing Interest
MtDNA, auSTRs and X-InDels results, considering the three extraction reagents (sterile and distilled waters, and the commercial buffer), are presented on Table 1. In general, distilled water presented better results, with more positive results than the others extraction reagents. The commercial buffer was the reagent with worse performance, with the highest rate of negative results. Commercial buffer also showed five negative results
None. Acknowledgement This work was supported by [PR41/17-21018] project, funded by
Table 1 Mitochondrial (mtDNA), Autosomal (auSTRs) and X-chromosomal Insertion-Delection polymorphisms (X-InDels) results, considering the three analysed extraction reagents (Sterile water (Lonza™), Distilled water (Milli-Q, MilliporeSigma®), and a commercial buffer (Prep-n-Go™ (ThermoFisher™ Scientific, Foster City, USA)). Considering mtDNA results, the 20 random samples were chosen from those with positive agarose gel results. Considering nuclear amplifications, the 20 random analysed samples were chosen from the 73 amplifications. Partial results were observed taking into account all positive results from each analysis. Sterile water
Positive results Partial nuclear profiles, considering positive results Negative results
Distilled water
mtDNA agarose gel N = 73
Sequenced mtDNA N = 20
au-STRs
X-Indels
N = 20
30 –
20 –
43
0
Commercial extraction buffer
Sequenced mtDNA N = 20
au-STRs
X-Indels
N = 20
mtDNA agarose gel N = 73
N = 20
16 8
20 0
46 –
20 –
4
0
27
0
2
Sequenced mtDNA N = 20
au-STRs
X-Indels
N = 20
mtDNA agarose gel N = 73
N = 20
N = 20
15 5
20 0
20 –
15 –
15 0
20 0
5
0
53
5
5
0
Forensic Science International: Genetics Supplement Series xxx (xxxx) xxx–xxx
P. Mármol, et al.
Banco Santander-Universidad Complutense de Madrid, Spain (https:// www.bancosantander.es/es/universidades)
(2017) 265–266. [4] R. Pereira, V. Pereira, I. Gomes, et al., A method for the analysis of 32 X chromosome insertion deletion polymorphisms in a single PCR, Int. J. Legal Med. 126 (1) (2012) 97–105. [5] C. Gomes, A. Alonso, D. Marquina, et al., Inhibiting inhibitors: “inhibiting inhibitors”: preliminary results of a new “DNA extraction-amplification” disinhibition technique in critical human samples, Forensic Sci. Int. Genet. Suppl. Ser. 6 (2017) 197–199. [6] R.M. Andrews, I. Kubacka, P.F. Chinnery, et al., Reanalysis and revision of the Cambridge reference sequence for human mitochondrial DNA, Nat. Genet. 23 (2) (1999) 147. [7] GE Healthcare Life Sciences, Instructions for Blood Collection using WhatmanTM FTATM Blood Collection kit, (2013).
References [1] A. Dhaliwal, A comprehensive review of DNA extraction and purification kits cited in the literature, Labome (2013). [2] M.P. Garcia-Palacios, J. Martínez-Gómez, C. Gomes, et al., A new strategy for a “direct” amplification of forensic samples, Forensic Sci. Int. Genet. Suppl. Ser. 6 (2017) 560–561. [3] C. Gomes, J. Martínez-Gómez, L. Díez-Juárez, et al., Prep-n- goTM: a new and fast extraction method for forensic blood samples, Forensic Sci. Int. Genet. Suppl. Ser. 6
3
Contents lists available at ScienceDirect
Forensic Science International: Genetics Supplement Series journal homepage: www.elsevier.com/locate/fsigss
An innovative DNA extraction method: Water versus commercial buffers ⁎
P. Mármola,1, B. Gómeza,1, C. Gomesa,b, , C. Romeroa, C. Baeza-Richera,b, A. Hernández-Corderoa, M. Martín-Arrebolaa, R. Rosell-Herreraa, A.M. López-Parraa,b, S. Palomo-Díeza,b, E. Labajo-Gonzálezc, B Perea-Pérezc, C. López-Matayoshia,b, E. Arroyo-Pardoa,b a
Laboratory of Forensic and Population Genetics, Legal Medicine, Psychiatry and Pathology Department, Medicine School, Complutense University of Madrid (UCM), Pza Ramón y Cajal s/n, 28040, Madrid, Spain b Grupo de Ciencias Forenses: Genética y Toxicología forense, Instituto de Investigación Sanitaria del Hospital Clínico San Carlos (IdISSC), Madrid, Spain c Medicine School, Complutense University of Madrid, Madrid, Spain
A R T I C LE I N FO
A B S T R A C T
Keywords: DNA-extraction Commercial buffer Water mtDNA STRs
The main objective of DNA extraction is to obtain good quality genetic material in order to carry out its amplification, and corresponding analysis. Most laboratories tend to resort to commercial extraction buffers, which allow a simple and rigorous DNA extraction, with limited handling of the sample, but with high financial cost. As an alternative, we proposed to use water as reagent to extract DNA from blood spots, collected on Whatman® FTA® Cards (Sigma-Aldrich®). We propose to take advantage of hypotonic solutions, in order to obtain a better efficiency in the extraction of genetic material (DNA). On this sense, 219 extractions were performed with Whatman® FTA® Cards (SigmaAldrich®) stained with blood, based on Garcia-Palacios et al., extraction method, with some modifications. Extractions were performed with two types of water (sterile (Lonza™) and distilled (Milli-Q, MilliporeSigma®), as well as, with a commercial extraction kit (Prep-n-Go™, ThermoFisher™ Scientific, Foster City, USA). To evaluate and compare each reagent effectiveness, we amplified DNA extract with mitochondrial DNA Hypervariable regions I and II, and with nuclear DNA markers (autosomal STRs and X-InDels). Our preliminary results suggest that distilled water allows an extraction as effective, or even better, as that obtained with the commercial buffer. These preliminary results offer the possibility for laboratories to choose an inexpensive protocol for DNA extraction, avoiding expensive commercial buffers. Further research is needed to evaluate the performance of each method in blood samples, but also in other biological samples.
1. Introduction The analysed samples in the field of forensic genetics usually present particular characteristics, like low template DNA, or inhibitors, for example, compelling the extraction process to be specific and directed to those samples. Therefore, to achieve an optimal DNA analysis, it is necessary to carry out an efficient and specific DNA extraction, since it constitutes a crucial point for forensic genetics research. The main purpose of DNA extractions is to obtain, from a biological sample and through the use of different solutions and physical and mechanical processes, cell lysis to extract the genetic material to allow its subsequent amplification [1]. Most laboratories tend to resort to expensive commercial kits, such as the Prep-n-Go™ buffer (ThermoFisher™ Scientific, Foster City, USA).
Designed for saliva extraction on different supports, this commercial buffer has also been tested on blood samples and the results have been satisfactory and previously published [2,3]. While on the one hand it has the advantage of reduced sample handling and extraction time, it has the important disadvantages of not reporting on the reagents that compose it, as well as, a very high monetary cost. Consequently, we proposed to use water as reagent to extract DNA from blood spots, by taking advantage of different water solutions, in order to obtain a better efficiency in the extraction of genetic material, and at the same time, to recommend a cheaper alternative method for DNA extraction.
⁎
Corresponding author at: Laboratory of Forensic and Population Genetics, Legal Medicine, Psychiatry and Pathology Department, Medicine School, Complutense University of Madrid, Avda. Complutense s/n 28040, Madrid, Spain. E-mail address: [email protected] (C. Gomes). 1 Both authors contributed equally to this study. https://doi.org/10.1016/j.fsigss.2019.09.111 Received 6 September 2019; Accepted 29 September 2019 1875-1768/ © 2019 Elsevier B.V. All rights reserved.
Please cite this article as: P. Mármol, et al., Forensic Science International: Genetics Supplement Series, https://doi.org/10.1016/j.fsigss.2019.09.111
Forensic Science International: Genetics Supplement Series xxx (xxxx) xxx–xxx
P. Mármol, et al.
2. Materials and Methods
analysing mtDNA, after a positive band was observed on the agarose gel.
2.1. DNA Extraction 4. Discussion DNA extractions were performed with Whatman® FTA® cards (SigmaAldrich®) stained with blood from 73 independent voluntary donors, after signed an informed consent. To perform the DNA extraction, form each stained Whatman® FTA® card, approximately 1 mm diameter were cut into a previously sterilized 1,5 mL tube. Additionally, bloodless FTA cards were used as a negative control. From each volunteer’s blood, three extractions were performed (a total of 219 analysis) - with sterile (Lonza™, Switzerland), and distilled (Milli-Q™ Advantage A10, MerkGroup, Germany) waters, and with the commercial extraction kit Prep-n-Go™(ThermoFisher™ Scientific, Foster City, USA), according to Garcia-Palacios et al., [2], with some modifications.
Our preliminary results indicate that it is possible to perform a DNA extraction from a FTA card stained with blood, both with sterile and distilled water. Indeed, these results suggest distilled water allows an extraction as effective, or even better as that obtained with the commercial buffer. To give an explanation to these results, it is necessary to understand the functionality of the FTA cards used in this experiment. When coming into contact with the blood sample, FTA cards smooth the cells and attach the DNA to the matrix card, allowing its preservation [7]. So, FTA cards perform a first extraction by causing the membrane lysis of the cells. Distilled and sterile water have both an ionic charge that allows them to interact with the DNA, causing the release of the genetic material from the matrix of the FTA card. The difference between them is based on the presence of a lower and higher ion concentration, respectively. In the case of distilled water, it corresponds to an ultra-hypotonic solution, with lower ions concentration compared to sterile water. So, the interaction between distilled water and DNA is stronger, and extraction from FTA card more effective. It is not possible to have access to the commercial buffer chemistry. However, since mtDNA is the most abundant genetic material in the intracellular environment, it is possible that most of the negative results may be due to a DNA inhibiting effect. We recommend a decrease in extract volume to amplify, when extraction has been performed with this buffer. Regarding auSTRs, all partial profiles were due to loci dropout in larger amplicon markers. In this case, the commercial buffer seems to be more efficient, since no partial profile was observed. Concerning X-Indels results, all amplifications produced positive results, probably due to its short length fragments, being impossible to differentiate between the three extractions methodologies efficiency.
2.2. DNA Amplification To evaluate and compare each reagents effectiveness, we amplified the extracts with nuclear DNA markers (autosomal STRs –auSTRs-, with AmpFLSTR NGM Select, ThermoFisher Scientific®, Foster City, USA, according to users’ manual, and X-chromosomal Insertion-Delection polymorphisms -X-InDels-, according to Pereira et al., [4]), using 5 μl of extract volume for each amplification. In the case of mitochondrial DNA (mtDNA), hypervariable regions I and II were amplified, according to [5], method C, evaluating DNA extraction and amplification with an agarose gel (1 g/50 mL TBE 1%). Then, 20 random samples were chosen from DNA amplifications (mitochondrial and each nuclear DNA). Considering mtDNA results, samples were chosen if a band could be detected on the agarose gel, being purified with ExoSAP-IT PCR product Cleanup reagent (ThermoFisher Scientific®, Foster City, USA). Sequences’ haplotypes were defined relative to the revised Cambridge reference mtDNA sequence [6], by running Mutation Surveyor® (Softgenetics) software, v 5.1.2. Considering nuclear DNA, capillary electrophoresis and detection were achieved in a 3130xl Genetic Analyzer (ThermoFisher™ Scientific, Foster City, USA). Electropherogram analysis and allele assignment were performed with GeneMapper v 4.0 (ThermoFisher Scientific®, Foster City, USA).
5. Conclusion These preliminary results offer the possibility for laboratories to choose an inexpensive protocol for DNA extraction from FTA cards stained with blood.
3. Results
Declaration of Competing Interest
MtDNA, auSTRs and X-InDels results, considering the three extraction reagents (sterile and distilled waters, and the commercial buffer), are presented on Table 1. In general, distilled water presented better results, with more positive results than the others extraction reagents. The commercial buffer was the reagent with worse performance, with the highest rate of negative results. Commercial buffer also showed five negative results
None. Acknowledgement This work was supported by [PR41/17-21018] project, funded by
Table 1 Mitochondrial (mtDNA), Autosomal (auSTRs) and X-chromosomal Insertion-Delection polymorphisms (X-InDels) results, considering the three analysed extraction reagents (Sterile water (Lonza™), Distilled water (Milli-Q, MilliporeSigma®), and a commercial buffer (Prep-n-Go™ (ThermoFisher™ Scientific, Foster City, USA)). Considering mtDNA results, the 20 random samples were chosen from those with positive agarose gel results. Considering nuclear amplifications, the 20 random analysed samples were chosen from the 73 amplifications. Partial results were observed taking into account all positive results from each analysis. Sterile water
Positive results Partial nuclear profiles, considering positive results Negative results
Distilled water
mtDNA agarose gel N = 73
Sequenced mtDNA N = 20
au-STRs
X-Indels
N = 20
30 –
20 –
43
0
Commercial extraction buffer
Sequenced mtDNA N = 20
au-STRs
X-Indels
N = 20
mtDNA agarose gel N = 73
N = 20
16 8
20 0
46 –
20 –
4
0
27
0
2
Sequenced mtDNA N = 20
au-STRs
X-Indels
N = 20
mtDNA agarose gel N = 73
N = 20
N = 20
15 5
20 0
20 –
15 –
15 0
20 0
5
0
53
5
5
0
Forensic Science International: Genetics Supplement Series xxx (xxxx) xxx–xxx
P. Mármol, et al.
Banco Santander-Universidad Complutense de Madrid, Spain (https:// www.bancosantander.es/es/universidades)
(2017) 265–266. [4] R. Pereira, V. Pereira, I. Gomes, et al., A method for the analysis of 32 X chromosome insertion deletion polymorphisms in a single PCR, Int. J. Legal Med. 126 (1) (2012) 97–105. [5] C. Gomes, A. Alonso, D. Marquina, et al., Inhibiting inhibitors: “inhibiting inhibitors”: preliminary results of a new “DNA extraction-amplification” disinhibition technique in critical human samples, Forensic Sci. Int. Genet. Suppl. Ser. 6 (2017) 197–199. [6] R.M. Andrews, I. Kubacka, P.F. Chinnery, et al., Reanalysis and revision of the Cambridge reference sequence for human mitochondrial DNA, Nat. Genet. 23 (2) (1999) 147. [7] GE Healthcare Life Sciences, Instructions for Blood Collection using WhatmanTM FTATM Blood Collection kit, (2013).
References [1] A. Dhaliwal, A comprehensive review of DNA extraction and purification kits cited in the literature, Labome (2013). [2] M.P. Garcia-Palacios, J. Martínez-Gómez, C. Gomes, et al., A new strategy for a “direct” amplification of forensic samples, Forensic Sci. Int. Genet. Suppl. Ser. 6 (2017) 560–561. [3] C. Gomes, J. Martínez-Gómez, L. Díez-Juárez, et al., Prep-n- goTM: a new and fast extraction method for forensic blood samples, Forensic Sci. Int. Genet. Suppl. Ser. 6
3