Forensic Science International: Genetics 9 (2014) 81–84
Contents lists available at ScienceDirect
Forensic Science International: Genetics journal homepage: www.elsevier.com/locate/fsig
Efficiency of a novel forensic room-temperature DNA storage medium Christophe Frippiat *, Fabrice Noel National Institute of Criminalistics and Criminology, Chausse´e de Vilvoorde 100, 1120 Brussels, Belgium
A R T I C L E I N F O
A B S T R A C T
Article history: Received 28 February 2013 Received in revised form 9 October 2013 Accepted 30 November 2013
The success of forensic genetics has led to considerable numbers of DNA samples that must be stored. Thus, the ability to preserve the integrity of forensic samples is essential. The possibility of retesting these samples after many years should be guaranteed. DNA storage typically requires the use of freezers. Recently, a new method that enables DNA to be stored at room temperature was developed. This technology is based on the principles of anhydrobiosis and thus permits room-temperature storage of DNA. This study evaluates the ability of this technology to preserve DNA samples mimicking true mixture casework samples for long periods of time. Mixed human DNA from 2 or 3 persons and at low concentrations was dried and stored for a period ranging from 6 months to 2 years in the presence of a desiccant. The quality of the stored DNA was evaluated based on quantitative peak height results from Short Tandem Repeat (STR) genotyping and the number of observed alleles. Furthermore, we determined whether this matrix has a potential inhibitory or enhancing effect on the PCR genotyping reactions. In our previous work, we demonstrated the considerable potential of this new technology. The present study complements our previous work. Our results show that after 2 years of aging at room temperature, there is a decrease in the number of observed alleles and in the peak height of these alleles. ß 2013 Elsevier Ireland Ltd. All rights reserved.
Keywords: DNA Storage Anhydrobiosis Long term Matrix
1. Introduction The success of forensic genetics has highlighted the importance of effective storage methods for extracted DNA. Numerous forensic samples recovered from crime scenes are degraded or damaged, leading to a reduced likelihood of obtaining meaningful results. This observation has led to the design of new amplification methods for low quantities of low quality DNA by targeting mini STRs. It is also obvious that such DNA evidence should be preserved in a way that reduces the potential for damage to occur. Poor storage methods may compromise samples and negatively impact results, as testing is not always performed immediately. Moreover, retesting is a vital component of forensic science. Such casework DNA is typically frozen at 20 8C or 80 8C; however, with increasing numbers of samples, freezing becomes costly and not without risk of failure. Although DNA is routinely frozen, little is known about potential DNA degradation following freezing/thawing cycles. Several studies have suggested that repeated freezing/thawing impairs the quality of the DNA [1–3], while others do not report such freezing/thawing degradation of DNA [2,4]. Moreover, some authors claim that degradation can even be observed during storage at 20 8C [2].
* Corresponding author. Tel.: +32 02240 05 29; fax: +32 02240 05 01. E-mail address: [email protected] (C. Frippiat). 1872-4973/$ – see front matter ß 2013 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.fsigen.2013.11.009
Bonnet et al. investigated dry storage of solid-state DNA [5,6]. Their studies showed that both humidity and oxygen have detrimental effects on DNA. The authors also suggested that in absence of water and oxygen, DNA has a very long lifetime. Many sample storage studies have considered dry storage to be a solution [7], although DNA damage can occur even at low moisture levels [8]. Nevertheless, the absence of water and oxygen is rarely attained without the use of costly equipment. To address these problems, Morin and co-worker tested the use of trehalose as a method for storing DNA in an easy and safe manner at room temperature [9]. Trehalose is one of the major compounds that accumulates during anhydrobiosis [10,11]. Trehalose, in addition to other compounds, replaces water and interacts with macromolecules during dehydration. The work of Smith and Morin showed that drying DNA in the presence of trehalose is an acceptable alternative to freezing [9]. DNA could be stored and remain amplifiable for up to 1 year [9]. Molina and Anchordoquy, however, investigated the storage of plasmid DNA lyophilized in trehalose [12] and detected single-strand breaks after 2 weeks at 20 8C. This observation was confirmed by Colotte et al. [6]. Trehalose is also known to retain the activities of dry enzymes for days [13] and has been used to preserve various biological materials, including vaccines [14]. Biomatrica Inc. has developed a sample matrix, based on anhydrobiosis, to address the need to stabilize and prevent the degradation of DNA. The samples are dried in a synthetic matrix
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and can be stored at room temperature. This matrix, also called Qiasafe, is a synthetic polymer that mimics the anhydrobiosis process. The protective effect appears to be based on its ability to form glass through minor groove interactions, thus stabilizing DNA [15]. GenVault produced a similar product called GenTegra. Biomatrica Inc. has performed internal studies using their matrix and accelerated aging studies by heating DNA samples. Heating of DNA is known to induce damage [16]. This strategy allows samples to undergo virtual years of aging in only a few weeks. According to Biomatrica Inc., the matrix can protect DNA for approximately 30 accelerated years [17]; however, this artificial aging does not perfectly mimic natural aging. An early study underlined the high potential of this technology. Using the Qiagen (Qiasafe) conservation matrix, DNA was stored for up to 3 weeks at room temperature without obvious degradation or loss [17]. Our previous study proved that medium to low quantities (150 pg/mL) of DNA can be safely stored for up to 6 months at room temperature using this method [18]. We also showed that multiple cycles of dehydration/hydration had no detrimental effects on the DNA. We observed that the conservation efficiency appeared to be dependent on the extraction method. DNA extracted with phenol/ chloroform could be stored in the GenTegra matrix for more than 6 months without any degradation. By contrast, DNA extracted using magnetic beads could not be safely stored over the same period of time [18]. Lee et al. evaluated the storage of low DNA quantities for 1 year at room temperature [15]. This second study confirmed that samples stored in a matrix permitted a much higher recovery compared with samples stored in a freezer [18]. Lee et al., however, observed a loss of DNA stored both at 20 8C and in the storage matrix, but this loss was lower in the latter case. This group concluded that storage in the Qiagen matrix is better than freezing at 20 8C. These results demonstrate the potential of this technology, but the effects of long-term studies and true aging should also be studied to confirm its efficiency. Additionally, a great portion of the samples treated in forensic laboratories is obtained from skin contact. The quantities of DNA obtained from such skin samples are low. Moreover, the DNA obtained is often a mixture of DNA from several persons. Here, we studied the potential boosting effect of the Qiasafe matrix on PCR amplification. Finally, we stored low quantities of DNA mimicking casework samples, obtained from a mixture of two or three persons, for 6 months, followed by rehydration and dehydration for a supplemental storage period to reach a conservation period of 2 years in the matrix at room temperature (natural aging). 2. Materials and methods 2.1. Samples Samples mimicking casework mixtures were obtained from proficiency tests. One table, which includes the type of sample, the origin of the sample and the storage condition used is presented in the supplemental data section (Table S1). 2.2. DNA extraction DNA was extracted using a standard organic extraction protocol with phenol/chloroform/isoamyl alcohol, followed by ethanol precipitation. First, cells were lysed for 6 h at 56 8C in 1 vol. of HOMO PK (0.1 M NaCl, 0.01 M EDTA, 0.5% SDS, 0.01 M Tris–HCl (pH 8)), to which 0.2 mg of Proteinase K (Promega, Madison, USA) and
0.02 vol. of b-mercaptoethanol were added. After 6 h, 0.2 mg of Protease K was added for a second incubation of 2 h at 56 8C. One volume of phenol/chloroform/isoamyl alcohol was added to the lysate and gently mixed for 10 min, followed by centrifugation. The aqueous phase was preserved. Another volume of phenol/chloroform/isoamyl alcohol was added to the aqueous phase, gently mixed for 10 min and centrifuged. The aqueous phase was again preserved, and two volumes of ether were added to the upper layer, followed by gentle mixing and centrifugation. The organic phase was removed. The remaining ether in the lower phase was removed by evaporation at 37 8C for 1 h 30 min. The DNA was precipitated using ethanol and resuspended in T10E0.1. Epithelial or semen fractions (Table S1) were prepared by differential lysis using Proteinase K, followed by a standard organic extraction with phenol/chloroform and an ethanol precipitation. 2.3. DNA quantification The nuclear DNA in the extracts was quantified using the Quantifiler system (Applied Biosystems, Carlsbad, USA) and an Applied Biosystems 7500 Real-Time thermal cycler, according to the manufacturer’s specifications. Each quantification was performed in duplicate. The average concentration was considered for the experiments. 2.4. PCR amplification with the ESI kit The ESI kit (Promega, Madison, USA) was used according to the manufacturer’s recommendations. Ten mL of sample per reaction, in a total reaction volume of 25 mL, were amplified on a C1000 Thermal Cycler (Bio-Rad, Nazareth, Belgium). Each sample was amplified once per experiment. Samples were analyzed on a 3500xL Genetic Analyzer (Applied Biosystems, USA) using a 36 cm capillary array (Applied Biosystems, USA), POP-4 polymer (Applied Biosystems, USA) and 1 genetic analysis buffer with EDTA (Applied Biosystems, Carlsbad, USA). Injection conditions were as follows: 15 kV and 8 s or 12 s. For the PCR-enhancing study, the samples were analyzed on a 3130xL Genetic Analyzer (Applied Biosystems, USA) using a 36 cm capillary array (Applied Biosystems, USA), POP-4 polymer (Applied Biosystems, USA), 1 genetic analysis buffer with EDTA (Applied Biosystems, USA), a 5 s injection and 15 kV electrophoresis. Two mL of each PCR product were loaded with 10 mL of formamide (Applied Biosystems, USA). The Genemapper IDx 1.2 software (Applied Biosystems, USA) was used for analysis. The detection threshold was fixed at 50 RFU for the samples analyzed on the 3130xL Genetic Analyzer. For the 3500xL Genetic Analyzer, the detection threshold was calculated based on the following formula: (average background + 10 STD) and was dependent on the color, as follows: 70 RFU for green; 60 RFU for yellow; 50 RFU for blue and 60 RFU for red. A stochastic threshold of 135 RFU was calculated for analysis on the 3500xL Genetic Analyzer. The following formula was used: ((average background + 3 STD) 3 + (average background + 3STD) DPHR)). DPHR (the peak height ratio between two alleles of a heterozygous system) was fixed at 0.6. 2.5. Application of DNA to the conservation matrix The Qiasafe (Qiagen) conservation matrix was used according to the manufacturer’s specifications. Twenty microliters of diluted DNA extract were applied to a tube coated with the matrix, mixed gently by pipetting and allowed to dry overnight (16 h) under a chemical flow hood with a constant air humidity of 18%. The humidity was measured using a hygrometer (Oregon Scientific, Portland, USA). Then, tubes were closed and placed in the aluminum bags in which the Qiasafe tubes were delivered. These
C. Frippiat, F. Noel / Forensic Science International: Genetics 9 (2014) 81–84
83 6 months
bags were sealed with both zippers and tape. Each bag contained 6 g of desiccant (silica gel) combined with an orange gel humidity indicator (Merck, Belgium). One part of each sample dilution was also stored at 20 8C for the same duration. Hydration of the dried samples mixed with the matrix was achieved via incubation in 20 mL of MilliQ water for 30 min at room temperature. Samples were mixed two times during the 30 min period by pipetting up and down.
2 years
160
% of the freezed sample
140
2.6. Statistics
120 100 80 60 40 20
Comparisons between conditions were performed using t-tests.
0
3. Results
In our previous work, we demonstrated that under some conditions, there is no loss of DNA after 6 months of storage using the Qiasafe matrix [18]. Moreover, the intensity of the PCR signal obtained with Qiasafe-stored DNA reached 90.71 3.97% of the reference DNA stored at 20 8C. In a similar investigation, Lee et al. observed that, despite a loss of DNA, drying on the Qiasafe matrix offered better conservation of the DNA than did freezing [2]. These results could be due to an enhancing effect of the matrix on STR amplification, masking a potential loss of DNA. Consequently, as reported by Lee et al., we searched for a potential boosting effect on STR profiling by amplifying 150 pg of DNA diluted in water or diluted in water added to half of the Qiasafe matrix contained in one manufacturer microtube. No significant difference was observed (data not shown), thus indicating that the matrix has no enhancing or inhibitory effect on the STR amplification. This observation confirms the results of our previous study [18] and those of Lee et al. [2]. 3.2. Natural long-term storage The DNA mimicking casework samples obtained from proficiency tests was extracted according to a standard organic extraction protocol using phenol/chloroform/isoamyl alcohol, followed by ethanol precipitation. These samples were mixtures of DNA from two or three persons (see supplemental data Table S1). Three hundred picograms of DNA in a total volume of 20 mL were stored in the Qiasafe matrix for a period of 6 months at 20 8C in a sealed aluminum bag containing 6 g of desiccant and protected from light. These bags were sealed with a hermetic zipper. To be sure that they were hermetically sealed, we then sealed the bags twice more using tape. Additionally, the desiccant used contained an indicator. The normal color of this indicator is yellow/orange, and it becomes green if humidity is present. For all samples tested in this study, the indicator remained yellow. The same quantity issued from the same dilution was stored at 20 8C for 6 months as a reference. After recovery, the STR profile was established using half of the stored sample. Our results showed no difference between the frozen references and the DNA stored in Qiasafe. The average intensity of the Qiasafe-stored samples reached 102.89 18.86% of the reference signal (Fig. 1), and no significant difference was observed relative to the number of alleles detected (n = 17). The percent of the reference allele number was 108.86 17.95% (Fig. 2). As for the casework samples, we simulated the situation in which a second set of testing would be requested. Samples used for the 6-month storage study were then dehydrated and stored at room temperature in the conditions described above to attain a total storage time of 2 years. References samples were also stored
Fig. 1. Intensity of the signal obtained after ESI STR profiling from 150 pg of mixed DNA samples stored in Qiasafe at room temperature for 6 months (gray columns) or stored at room temperature for 6 months, followed by dehydration and a second storage at room temperature to reach 2 years of aging (black columns). Results are expressed as a percentage of the respective samples stored at 20 8C. For sample 5, an abnormal value was obtained for 2 years of aging, suggesting a technical error. Thus, we did not report this value in the graph. 6 months % of the reference allleles
3.1. PCR enhancing effect
2 years
180 160 140 120 100 80 60 40 20 0
Fig. 2. Allele count after ESI STR profiling from 150 pg of mixed DNA samples stored in Qiasafe at room temperature for 6 months (gray columns) or stored at room temperature for 6 months, followed by dehydration and a second storage at room temperature to reach 2 years of aging (dark columns). The results are expressed as the percent of detected alleles compared to the 20 8C reference profile in each sample.
for 2 years at 20 8C. Then, matrix samples were rehydrated, and the STRs were established using the ESI 17 kit from Promega. As previously described, we observed the intensity of the signal and the number of obtained alleles. In this situation, the signal obtained from the Qiasafe DNA decreased to 71.75 21.67% (p < 0.05) compared to the 20 8C reference samples. Fig. 1 illustrates the conservation of DNA samples in the Qiasafe matrix. Figs. 1 and 2 of the supplemental data section show the same results as the average intensity of the signal in RFU of the 6 months and 2 years of room-temperature aging, respectively. Concerning the number of alleles, all samples stored on the matrix, with the exception of three, demonstrated a loss of observed alleles (n = 16), with an average loss of 28.25 28.07% of the allele number (Fig. 2). This difference was significant according to a t-test (p < 0.05). In terms of the intensity of the signal, the difference between the frozen references and the Qiasafe DNA appeared to be sample-dependent. 4. Discussion DNA amplification is one of the most important and powerful techniques used in forensic science, but this technique is dependent on the quality of the DNA. As such, DNA should be stored in the most optimal conditions possible to allow for retesting in cases that have been reopened. Our previous study has shown that DNA stored on a conservation matrix such as Qiasafe
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could be rehydrated at least three times without affecting the recovery and the STR profiling. We also demonstrated that samples extracted by a classical phenol/organic method could be stored for up to 6 months without any visible degradation. In a complementary study, Lee et al. [15] investigated the storage of various quantities of DNA for a maximum of 52 weeks and concluded that the matrix is more suitable than 20 8C freezing for DNA storage. These two studies, however, suffer from certain limitations. Neither study examined the long-term storage of low quantities of mixed samples. In reality, numerous DNA samples obtained from small traces are often at low concentrations. Our present work addresses these limitations. Our results show that the Qiasafe matrix does not affect STR profiling. We also prove that low concentrations of mixed DNA can be stored on the matrix for shorter periods of time (e.g., 6 months at room temperature). Concerning long-term storage, we observed a significant reduction in the number of alleles after 2 years of storage at room temperature, compared to the frozen samples. In light of these results, long-term storage experiments, on the order of decades, should be performed. One way to simulate decades of aging is by accelerated aging using heating. This method has been used by several groups [15,19]. For example, Lee et al. [15] and the manufacturers of Qiasafe and Genetegra used this heataccelerated aging process [20]. Nevertheless, this experimental design suffers from two main weaknesses. First, accelerated aging does not perfectly mimic natural aging. Second, the use of higher storage temperatures does not permit a direct comparison of DNA stored on a matrix with frozen references. Some of these accelerated aging experiments used inappropriate reference samples. The controls of the accelerated aging experiments were only stored at 20 8C during the incubation of the tested samples at high temperatures. To truly compare the freezing and the matrix storage methods, a comparison of samples stored on the matrix should be made with samples stored at 20 8C for the same duration. In conclusion, we show here that conservation on a matrix appears to be less efficient than classical freezing at 20 8C for low quantities of DNA. To achieve the same efficiency as freezing, some optimization will likely be required, including dehydration and storage in an atmosphere with very low humidity. Another potential source of DNA degradation should also be investigated. Anchordoquy and Molina, in their review, discussed the presence of trace amounts of metals in the buffer as a potential cause of degradation [12]. Such traces of metals can generate a Fenton reaction, leading to DNA degradation. Because it seems that DNA is better preserved when stored at higher concentrations [19], increasing the DNA concentration prior to matrix storage should also be tested. Interlaboratory experiments involving the storage of DNA using this matrix could also be of great value, as they would permit the comparison of storage protocols, as well as the investigation of the potential origins of DNA degradation (e.g., purity of DNA). These new data, combined with our previous results [18] and results from other groups [15,19], suggest that Qiasafe is useful for the conservation of DNA at room temperature for medium to high DNA quantities. In regard to low DNA quantities, as suggested above, some optimization will be required.
Nevertheless, this technology could be used for the safe and low-cost transportation of DNA samples. Acknowledgments This study was supported by the Federal Ministry of Justice of Belgium. The authors would like to thank Dr. S. Desmyter from the National Institute of Criminalistics and Criminology for his help with this work. Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:10.1016/j.fsigen.2013.11.009. References [1] D.L. Davis, E.P. O’Brien, C.M. Bentzley, Analysis of the degradation of oligonucleotide strands during the freezing/thawing processes using MALDI-MS, Anal. Chem. 72 (2000) 5092–5096. [2] S.B. Lee, C.A. Crouse, M.C. Kline, Optimizing storage and handling of DNA extracts, Forensic Sci. Rev. 2 (2010) 131–144. [3] V.N. Lyscov, Y. Moshkovsky, DNA cryolysis, Biochim. Biophys. Acta 190 (1969) 101–110. [4] K. Shikama, Effect of freezing and thawing on the stability of double helix of DNA, Nature 207 (1965) 529–530. [5] J. Bonnet, M. Colotte, D. Coudy, V. Couallier, J. Portier, B. Morin, S. Tuffet, Chain and conformation stability of solid-state DNA: implications for room temperature storage, Nucleic Acids Res. 38 (2010) 1531–1546. [6] M. Colotte, D. Coudy, S. Tuffet, J. Bonnet, Adverse effect of air exposure on the stability of DNA stored at room temperature, Biopreserv. Biobank. 9 (2011) 47–50. [7] S. Arsenault, K. McWeeny, D. Bilodeau, D. Gaudet, Establishment of a sustainable biobank, Genet. Eng. Biotechnol. News 28 (2008). [8] T. Lindahl, Instability and decay of the primary structure of DNA, Nature 362 (1993) 709–715. [9] S. Smith, P.A. Morin, Optimal storage conditions for highly dilute DNA samples: a role for trehalose as a preserving agent, J. Forensic Sci. 50 (2005) 1101–1108. [10] M. Sakurai, T. Furuki, K. Akao, D. Tanaka, Y. Nakahara, T. Kikawada, M. Watanabe, T. Okuda, Vitrification is essential for anhydrobiosis in an African chironomid, Polypedilum vanderplanki, Proc. Natl. Acad. Sci. U. S. A. 105 (2008) 5093–5098. [11] D.A. Wharton, C.J. Marshall, How do terrestrial Antarctic organisms survive in their harsh environment? J. Biol. 8 (2009) 29. [12] T.J. Anchordoquy, M.C. Molina, Preservation of DNA, Cell Preserv. Technol. 5 (2007) 180–188. [13] M. Uritani, M. Takai, K. Yoshinaga, Protective effect of disaccharides on restriction endonucleases during drying under vacuum, J. Biochem. 117 (1995) 774–779. [14] J.H. Crowe, Trehalose as a ‘‘chemical chaperone’’: fact and fantasy, Adv. Exp. Med. Biol. 594 (2007) 143–158. [15] S.B. Lee, K.C. Clabaugh, B. Silva, K.O. Odigie, M.D. Coble, O. Loreille, M. Scheible, R.M. Fourney, J. Stevens, G.R. Carmody, T.J. Parsons, A. Pozder, A.J. Eisenberg, B. Budowle, T. Ahmad, R.W. Miller, C.A. Crouse, Assessing a novel room temperature DNA storage medium for forensic biological samples, Forensic Sci. Int. Genet. 6 (2012) 31–40. [16] V.I. Bruskov, L.V. Malakhova, Z.K. Masalimov, A.V. Chernikov, Heat-induced formation of reactive oxygen species and 8-oxoguanine, a biomarker of damage to DNA, Nucleic Acids Res. 30 (2002) 1354–1363. [17] E. Wan, M. Akana, J. Pons, J. Chen, S. Musone, P.Y. Kwok, W. Liao, Green technologies for room temperature nucleic acid storage, Curr. Issues Mol. Biol. 12 (2010) 135–142. [18] C. Frippiat, S. Zorbo, D. Leonard, A. Marcotte, M. Chaput, C. Aelbrecht, F. Noel, Evaluation of novel forensic DNA storage methodologies, Forensic Sci. Int. Genet. 5 (2011) 386–392. [19] S.E. Howlett, H.S. Castillo, L.J. Gioeni, J.M. Robertson, J. Donfack, Evaluation of DNAstable for DNA storage at ambient temperature, Forensic Sci. Int. Genet. (2013), http://dx.doi.org/10.1016/j.fsigen.2013.09.003. [20] M. Hogan, GenVault’s ambient temperature technologies for biospecimen managament, P3G Meeting, 2009 http://p3g.org/sites/default/files/site/default/files/ Hogan_Michael_GenVault_Luxembourg_092009.pdf (accessed 23.09.13).
Contents lists available at ScienceDirect
Forensic Science International: Genetics journal homepage: www.elsevier.com/locate/fsig
Efficiency of a novel forensic room-temperature DNA storage medium Christophe Frippiat *, Fabrice Noel National Institute of Criminalistics and Criminology, Chausse´e de Vilvoorde 100, 1120 Brussels, Belgium
A R T I C L E I N F O
A B S T R A C T
Article history: Received 28 February 2013 Received in revised form 9 October 2013 Accepted 30 November 2013
The success of forensic genetics has led to considerable numbers of DNA samples that must be stored. Thus, the ability to preserve the integrity of forensic samples is essential. The possibility of retesting these samples after many years should be guaranteed. DNA storage typically requires the use of freezers. Recently, a new method that enables DNA to be stored at room temperature was developed. This technology is based on the principles of anhydrobiosis and thus permits room-temperature storage of DNA. This study evaluates the ability of this technology to preserve DNA samples mimicking true mixture casework samples for long periods of time. Mixed human DNA from 2 or 3 persons and at low concentrations was dried and stored for a period ranging from 6 months to 2 years in the presence of a desiccant. The quality of the stored DNA was evaluated based on quantitative peak height results from Short Tandem Repeat (STR) genotyping and the number of observed alleles. Furthermore, we determined whether this matrix has a potential inhibitory or enhancing effect on the PCR genotyping reactions. In our previous work, we demonstrated the considerable potential of this new technology. The present study complements our previous work. Our results show that after 2 years of aging at room temperature, there is a decrease in the number of observed alleles and in the peak height of these alleles. ß 2013 Elsevier Ireland Ltd. All rights reserved.
Keywords: DNA Storage Anhydrobiosis Long term Matrix
1. Introduction The success of forensic genetics has highlighted the importance of effective storage methods for extracted DNA. Numerous forensic samples recovered from crime scenes are degraded or damaged, leading to a reduced likelihood of obtaining meaningful results. This observation has led to the design of new amplification methods for low quantities of low quality DNA by targeting mini STRs. It is also obvious that such DNA evidence should be preserved in a way that reduces the potential for damage to occur. Poor storage methods may compromise samples and negatively impact results, as testing is not always performed immediately. Moreover, retesting is a vital component of forensic science. Such casework DNA is typically frozen at 20 8C or 80 8C; however, with increasing numbers of samples, freezing becomes costly and not without risk of failure. Although DNA is routinely frozen, little is known about potential DNA degradation following freezing/thawing cycles. Several studies have suggested that repeated freezing/thawing impairs the quality of the DNA [1–3], while others do not report such freezing/thawing degradation of DNA [2,4]. Moreover, some authors claim that degradation can even be observed during storage at 20 8C [2].
* Corresponding author. Tel.: +32 02240 05 29; fax: +32 02240 05 01. E-mail address: [email protected] (C. Frippiat). 1872-4973/$ – see front matter ß 2013 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.fsigen.2013.11.009
Bonnet et al. investigated dry storage of solid-state DNA [5,6]. Their studies showed that both humidity and oxygen have detrimental effects on DNA. The authors also suggested that in absence of water and oxygen, DNA has a very long lifetime. Many sample storage studies have considered dry storage to be a solution [7], although DNA damage can occur even at low moisture levels [8]. Nevertheless, the absence of water and oxygen is rarely attained without the use of costly equipment. To address these problems, Morin and co-worker tested the use of trehalose as a method for storing DNA in an easy and safe manner at room temperature [9]. Trehalose is one of the major compounds that accumulates during anhydrobiosis [10,11]. Trehalose, in addition to other compounds, replaces water and interacts with macromolecules during dehydration. The work of Smith and Morin showed that drying DNA in the presence of trehalose is an acceptable alternative to freezing [9]. DNA could be stored and remain amplifiable for up to 1 year [9]. Molina and Anchordoquy, however, investigated the storage of plasmid DNA lyophilized in trehalose [12] and detected single-strand breaks after 2 weeks at 20 8C. This observation was confirmed by Colotte et al. [6]. Trehalose is also known to retain the activities of dry enzymes for days [13] and has been used to preserve various biological materials, including vaccines [14]. Biomatrica Inc. has developed a sample matrix, based on anhydrobiosis, to address the need to stabilize and prevent the degradation of DNA. The samples are dried in a synthetic matrix
82
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and can be stored at room temperature. This matrix, also called Qiasafe, is a synthetic polymer that mimics the anhydrobiosis process. The protective effect appears to be based on its ability to form glass through minor groove interactions, thus stabilizing DNA [15]. GenVault produced a similar product called GenTegra. Biomatrica Inc. has performed internal studies using their matrix and accelerated aging studies by heating DNA samples. Heating of DNA is known to induce damage [16]. This strategy allows samples to undergo virtual years of aging in only a few weeks. According to Biomatrica Inc., the matrix can protect DNA for approximately 30 accelerated years [17]; however, this artificial aging does not perfectly mimic natural aging. An early study underlined the high potential of this technology. Using the Qiagen (Qiasafe) conservation matrix, DNA was stored for up to 3 weeks at room temperature without obvious degradation or loss [17]. Our previous study proved that medium to low quantities (150 pg/mL) of DNA can be safely stored for up to 6 months at room temperature using this method [18]. We also showed that multiple cycles of dehydration/hydration had no detrimental effects on the DNA. We observed that the conservation efficiency appeared to be dependent on the extraction method. DNA extracted with phenol/ chloroform could be stored in the GenTegra matrix for more than 6 months without any degradation. By contrast, DNA extracted using magnetic beads could not be safely stored over the same period of time [18]. Lee et al. evaluated the storage of low DNA quantities for 1 year at room temperature [15]. This second study confirmed that samples stored in a matrix permitted a much higher recovery compared with samples stored in a freezer [18]. Lee et al., however, observed a loss of DNA stored both at 20 8C and in the storage matrix, but this loss was lower in the latter case. This group concluded that storage in the Qiagen matrix is better than freezing at 20 8C. These results demonstrate the potential of this technology, but the effects of long-term studies and true aging should also be studied to confirm its efficiency. Additionally, a great portion of the samples treated in forensic laboratories is obtained from skin contact. The quantities of DNA obtained from such skin samples are low. Moreover, the DNA obtained is often a mixture of DNA from several persons. Here, we studied the potential boosting effect of the Qiasafe matrix on PCR amplification. Finally, we stored low quantities of DNA mimicking casework samples, obtained from a mixture of two or three persons, for 6 months, followed by rehydration and dehydration for a supplemental storage period to reach a conservation period of 2 years in the matrix at room temperature (natural aging). 2. Materials and methods 2.1. Samples Samples mimicking casework mixtures were obtained from proficiency tests. One table, which includes the type of sample, the origin of the sample and the storage condition used is presented in the supplemental data section (Table S1). 2.2. DNA extraction DNA was extracted using a standard organic extraction protocol with phenol/chloroform/isoamyl alcohol, followed by ethanol precipitation. First, cells were lysed for 6 h at 56 8C in 1 vol. of HOMO PK (0.1 M NaCl, 0.01 M EDTA, 0.5% SDS, 0.01 M Tris–HCl (pH 8)), to which 0.2 mg of Proteinase K (Promega, Madison, USA) and
0.02 vol. of b-mercaptoethanol were added. After 6 h, 0.2 mg of Protease K was added for a second incubation of 2 h at 56 8C. One volume of phenol/chloroform/isoamyl alcohol was added to the lysate and gently mixed for 10 min, followed by centrifugation. The aqueous phase was preserved. Another volume of phenol/chloroform/isoamyl alcohol was added to the aqueous phase, gently mixed for 10 min and centrifuged. The aqueous phase was again preserved, and two volumes of ether were added to the upper layer, followed by gentle mixing and centrifugation. The organic phase was removed. The remaining ether in the lower phase was removed by evaporation at 37 8C for 1 h 30 min. The DNA was precipitated using ethanol and resuspended in T10E0.1. Epithelial or semen fractions (Table S1) were prepared by differential lysis using Proteinase K, followed by a standard organic extraction with phenol/chloroform and an ethanol precipitation. 2.3. DNA quantification The nuclear DNA in the extracts was quantified using the Quantifiler system (Applied Biosystems, Carlsbad, USA) and an Applied Biosystems 7500 Real-Time thermal cycler, according to the manufacturer’s specifications. Each quantification was performed in duplicate. The average concentration was considered for the experiments. 2.4. PCR amplification with the ESI kit The ESI kit (Promega, Madison, USA) was used according to the manufacturer’s recommendations. Ten mL of sample per reaction, in a total reaction volume of 25 mL, were amplified on a C1000 Thermal Cycler (Bio-Rad, Nazareth, Belgium). Each sample was amplified once per experiment. Samples were analyzed on a 3500xL Genetic Analyzer (Applied Biosystems, USA) using a 36 cm capillary array (Applied Biosystems, USA), POP-4 polymer (Applied Biosystems, USA) and 1 genetic analysis buffer with EDTA (Applied Biosystems, Carlsbad, USA). Injection conditions were as follows: 15 kV and 8 s or 12 s. For the PCR-enhancing study, the samples were analyzed on a 3130xL Genetic Analyzer (Applied Biosystems, USA) using a 36 cm capillary array (Applied Biosystems, USA), POP-4 polymer (Applied Biosystems, USA), 1 genetic analysis buffer with EDTA (Applied Biosystems, USA), a 5 s injection and 15 kV electrophoresis. Two mL of each PCR product were loaded with 10 mL of formamide (Applied Biosystems, USA). The Genemapper IDx 1.2 software (Applied Biosystems, USA) was used for analysis. The detection threshold was fixed at 50 RFU for the samples analyzed on the 3130xL Genetic Analyzer. For the 3500xL Genetic Analyzer, the detection threshold was calculated based on the following formula: (average background + 10 STD) and was dependent on the color, as follows: 70 RFU for green; 60 RFU for yellow; 50 RFU for blue and 60 RFU for red. A stochastic threshold of 135 RFU was calculated for analysis on the 3500xL Genetic Analyzer. The following formula was used: ((average background + 3 STD) 3 + (average background + 3STD) DPHR)). DPHR (the peak height ratio between two alleles of a heterozygous system) was fixed at 0.6. 2.5. Application of DNA to the conservation matrix The Qiasafe (Qiagen) conservation matrix was used according to the manufacturer’s specifications. Twenty microliters of diluted DNA extract were applied to a tube coated with the matrix, mixed gently by pipetting and allowed to dry overnight (16 h) under a chemical flow hood with a constant air humidity of 18%. The humidity was measured using a hygrometer (Oregon Scientific, Portland, USA). Then, tubes were closed and placed in the aluminum bags in which the Qiasafe tubes were delivered. These
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83 6 months
bags were sealed with both zippers and tape. Each bag contained 6 g of desiccant (silica gel) combined with an orange gel humidity indicator (Merck, Belgium). One part of each sample dilution was also stored at 20 8C for the same duration. Hydration of the dried samples mixed with the matrix was achieved via incubation in 20 mL of MilliQ water for 30 min at room temperature. Samples were mixed two times during the 30 min period by pipetting up and down.
2 years
160
% of the freezed sample
140
2.6. Statistics
120 100 80 60 40 20
Comparisons between conditions were performed using t-tests.
0
3. Results
In our previous work, we demonstrated that under some conditions, there is no loss of DNA after 6 months of storage using the Qiasafe matrix [18]. Moreover, the intensity of the PCR signal obtained with Qiasafe-stored DNA reached 90.71 3.97% of the reference DNA stored at 20 8C. In a similar investigation, Lee et al. observed that, despite a loss of DNA, drying on the Qiasafe matrix offered better conservation of the DNA than did freezing [2]. These results could be due to an enhancing effect of the matrix on STR amplification, masking a potential loss of DNA. Consequently, as reported by Lee et al., we searched for a potential boosting effect on STR profiling by amplifying 150 pg of DNA diluted in water or diluted in water added to half of the Qiasafe matrix contained in one manufacturer microtube. No significant difference was observed (data not shown), thus indicating that the matrix has no enhancing or inhibitory effect on the STR amplification. This observation confirms the results of our previous study [18] and those of Lee et al. [2]. 3.2. Natural long-term storage The DNA mimicking casework samples obtained from proficiency tests was extracted according to a standard organic extraction protocol using phenol/chloroform/isoamyl alcohol, followed by ethanol precipitation. These samples were mixtures of DNA from two or three persons (see supplemental data Table S1). Three hundred picograms of DNA in a total volume of 20 mL were stored in the Qiasafe matrix for a period of 6 months at 20 8C in a sealed aluminum bag containing 6 g of desiccant and protected from light. These bags were sealed with a hermetic zipper. To be sure that they were hermetically sealed, we then sealed the bags twice more using tape. Additionally, the desiccant used contained an indicator. The normal color of this indicator is yellow/orange, and it becomes green if humidity is present. For all samples tested in this study, the indicator remained yellow. The same quantity issued from the same dilution was stored at 20 8C for 6 months as a reference. After recovery, the STR profile was established using half of the stored sample. Our results showed no difference between the frozen references and the DNA stored in Qiasafe. The average intensity of the Qiasafe-stored samples reached 102.89 18.86% of the reference signal (Fig. 1), and no significant difference was observed relative to the number of alleles detected (n = 17). The percent of the reference allele number was 108.86 17.95% (Fig. 2). As for the casework samples, we simulated the situation in which a second set of testing would be requested. Samples used for the 6-month storage study were then dehydrated and stored at room temperature in the conditions described above to attain a total storage time of 2 years. References samples were also stored
Fig. 1. Intensity of the signal obtained after ESI STR profiling from 150 pg of mixed DNA samples stored in Qiasafe at room temperature for 6 months (gray columns) or stored at room temperature for 6 months, followed by dehydration and a second storage at room temperature to reach 2 years of aging (black columns). Results are expressed as a percentage of the respective samples stored at 20 8C. For sample 5, an abnormal value was obtained for 2 years of aging, suggesting a technical error. Thus, we did not report this value in the graph. 6 months % of the reference allleles
3.1. PCR enhancing effect
2 years
180 160 140 120 100 80 60 40 20 0
Fig. 2. Allele count after ESI STR profiling from 150 pg of mixed DNA samples stored in Qiasafe at room temperature for 6 months (gray columns) or stored at room temperature for 6 months, followed by dehydration and a second storage at room temperature to reach 2 years of aging (dark columns). The results are expressed as the percent of detected alleles compared to the 20 8C reference profile in each sample.
for 2 years at 20 8C. Then, matrix samples were rehydrated, and the STRs were established using the ESI 17 kit from Promega. As previously described, we observed the intensity of the signal and the number of obtained alleles. In this situation, the signal obtained from the Qiasafe DNA decreased to 71.75 21.67% (p < 0.05) compared to the 20 8C reference samples. Fig. 1 illustrates the conservation of DNA samples in the Qiasafe matrix. Figs. 1 and 2 of the supplemental data section show the same results as the average intensity of the signal in RFU of the 6 months and 2 years of room-temperature aging, respectively. Concerning the number of alleles, all samples stored on the matrix, with the exception of three, demonstrated a loss of observed alleles (n = 16), with an average loss of 28.25 28.07% of the allele number (Fig. 2). This difference was significant according to a t-test (p < 0.05). In terms of the intensity of the signal, the difference between the frozen references and the Qiasafe DNA appeared to be sample-dependent. 4. Discussion DNA amplification is one of the most important and powerful techniques used in forensic science, but this technique is dependent on the quality of the DNA. As such, DNA should be stored in the most optimal conditions possible to allow for retesting in cases that have been reopened. Our previous study has shown that DNA stored on a conservation matrix such as Qiasafe
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could be rehydrated at least three times without affecting the recovery and the STR profiling. We also demonstrated that samples extracted by a classical phenol/organic method could be stored for up to 6 months without any visible degradation. In a complementary study, Lee et al. [15] investigated the storage of various quantities of DNA for a maximum of 52 weeks and concluded that the matrix is more suitable than 20 8C freezing for DNA storage. These two studies, however, suffer from certain limitations. Neither study examined the long-term storage of low quantities of mixed samples. In reality, numerous DNA samples obtained from small traces are often at low concentrations. Our present work addresses these limitations. Our results show that the Qiasafe matrix does not affect STR profiling. We also prove that low concentrations of mixed DNA can be stored on the matrix for shorter periods of time (e.g., 6 months at room temperature). Concerning long-term storage, we observed a significant reduction in the number of alleles after 2 years of storage at room temperature, compared to the frozen samples. In light of these results, long-term storage experiments, on the order of decades, should be performed. One way to simulate decades of aging is by accelerated aging using heating. This method has been used by several groups [15,19]. For example, Lee et al. [15] and the manufacturers of Qiasafe and Genetegra used this heataccelerated aging process [20]. Nevertheless, this experimental design suffers from two main weaknesses. First, accelerated aging does not perfectly mimic natural aging. Second, the use of higher storage temperatures does not permit a direct comparison of DNA stored on a matrix with frozen references. Some of these accelerated aging experiments used inappropriate reference samples. The controls of the accelerated aging experiments were only stored at 20 8C during the incubation of the tested samples at high temperatures. To truly compare the freezing and the matrix storage methods, a comparison of samples stored on the matrix should be made with samples stored at 20 8C for the same duration. In conclusion, we show here that conservation on a matrix appears to be less efficient than classical freezing at 20 8C for low quantities of DNA. To achieve the same efficiency as freezing, some optimization will likely be required, including dehydration and storage in an atmosphere with very low humidity. Another potential source of DNA degradation should also be investigated. Anchordoquy and Molina, in their review, discussed the presence of trace amounts of metals in the buffer as a potential cause of degradation [12]. Such traces of metals can generate a Fenton reaction, leading to DNA degradation. Because it seems that DNA is better preserved when stored at higher concentrations [19], increasing the DNA concentration prior to matrix storage should also be tested. Interlaboratory experiments involving the storage of DNA using this matrix could also be of great value, as they would permit the comparison of storage protocols, as well as the investigation of the potential origins of DNA degradation (e.g., purity of DNA). These new data, combined with our previous results [18] and results from other groups [15,19], suggest that Qiasafe is useful for the conservation of DNA at room temperature for medium to high DNA quantities. In regard to low DNA quantities, as suggested above, some optimization will be required.
Nevertheless, this technology could be used for the safe and low-cost transportation of DNA samples. Acknowledgments This study was supported by the Federal Ministry of Justice of Belgium. The authors would like to thank Dr. S. Desmyter from the National Institute of Criminalistics and Criminology for his help with this work. Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:10.1016/j.fsigen.2013.11.009. References [1] D.L. Davis, E.P. O’Brien, C.M. Bentzley, Analysis of the degradation of oligonucleotide strands during the freezing/thawing processes using MALDI-MS, Anal. Chem. 72 (2000) 5092–5096. [2] S.B. Lee, C.A. Crouse, M.C. Kline, Optimizing storage and handling of DNA extracts, Forensic Sci. Rev. 2 (2010) 131–144. [3] V.N. Lyscov, Y. Moshkovsky, DNA cryolysis, Biochim. Biophys. Acta 190 (1969) 101–110. [4] K. Shikama, Effect of freezing and thawing on the stability of double helix of DNA, Nature 207 (1965) 529–530. [5] J. Bonnet, M. Colotte, D. Coudy, V. Couallier, J. Portier, B. Morin, S. Tuffet, Chain and conformation stability of solid-state DNA: implications for room temperature storage, Nucleic Acids Res. 38 (2010) 1531–1546. [6] M. Colotte, D. Coudy, S. Tuffet, J. Bonnet, Adverse effect of air exposure on the stability of DNA stored at room temperature, Biopreserv. Biobank. 9 (2011) 47–50. [7] S. Arsenault, K. McWeeny, D. Bilodeau, D. Gaudet, Establishment of a sustainable biobank, Genet. Eng. Biotechnol. News 28 (2008). [8] T. Lindahl, Instability and decay of the primary structure of DNA, Nature 362 (1993) 709–715. [9] S. Smith, P.A. Morin, Optimal storage conditions for highly dilute DNA samples: a role for trehalose as a preserving agent, J. Forensic Sci. 50 (2005) 1101–1108. [10] M. Sakurai, T. Furuki, K. Akao, D. Tanaka, Y. Nakahara, T. Kikawada, M. Watanabe, T. Okuda, Vitrification is essential for anhydrobiosis in an African chironomid, Polypedilum vanderplanki, Proc. Natl. Acad. Sci. U. S. A. 105 (2008) 5093–5098. [11] D.A. Wharton, C.J. Marshall, How do terrestrial Antarctic organisms survive in their harsh environment? J. Biol. 8 (2009) 29. [12] T.J. Anchordoquy, M.C. Molina, Preservation of DNA, Cell Preserv. Technol. 5 (2007) 180–188. [13] M. Uritani, M. Takai, K. Yoshinaga, Protective effect of disaccharides on restriction endonucleases during drying under vacuum, J. Biochem. 117 (1995) 774–779. [14] J.H. Crowe, Trehalose as a ‘‘chemical chaperone’’: fact and fantasy, Adv. Exp. Med. Biol. 594 (2007) 143–158. [15] S.B. Lee, K.C. Clabaugh, B. Silva, K.O. Odigie, M.D. Coble, O. Loreille, M. Scheible, R.M. Fourney, J. Stevens, G.R. Carmody, T.J. Parsons, A. Pozder, A.J. Eisenberg, B. Budowle, T. Ahmad, R.W. Miller, C.A. Crouse, Assessing a novel room temperature DNA storage medium for forensic biological samples, Forensic Sci. Int. Genet. 6 (2012) 31–40. [16] V.I. Bruskov, L.V. Malakhova, Z.K. Masalimov, A.V. Chernikov, Heat-induced formation of reactive oxygen species and 8-oxoguanine, a biomarker of damage to DNA, Nucleic Acids Res. 30 (2002) 1354–1363. [17] E. Wan, M. Akana, J. Pons, J. Chen, S. Musone, P.Y. Kwok, W. Liao, Green technologies for room temperature nucleic acid storage, Curr. Issues Mol. Biol. 12 (2010) 135–142. [18] C. Frippiat, S. Zorbo, D. Leonard, A. Marcotte, M. Chaput, C. Aelbrecht, F. Noel, Evaluation of novel forensic DNA storage methodologies, Forensic Sci. Int. Genet. 5 (2011) 386–392. [19] S.E. Howlett, H.S. Castillo, L.J. Gioeni, J.M. Robertson, J. Donfack, Evaluation of DNAstable for DNA storage at ambient temperature, Forensic Sci. Int. Genet. (2013), http://dx.doi.org/10.1016/j.fsigen.2013.09.003. [20] M. Hogan, GenVault’s ambient temperature technologies for biospecimen managament, P3G Meeting, 2009 http://p3g.org/sites/default/files/site/default/files/ Hogan_Michael_GenVault_Luxembourg_092009.pdf (accessed 23.09.13).
