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DNA profiles obtained from urine in snow
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
DNA profiles obtained from urine in snow Charlotte Dufvaa,⁎, Emilia Karlssona, Christina Forsberga, Ricky Ansella,b a b
National Forensic Centre, Swedish Police Authority, Linköping, Sweden Department of Physics, Chemistry and Biology (IFM), Linköping University, Linköping, Sweden
A R T I C LE I N FO
A B S T R A C T
Keywords: Crime scene DNA evidence Snow Urine
Urine can be a potential important source of evidence when occurring at crime scenes. In case of outdoor scenes including snow a yellow colour could indicate the existence of human urine. In an effort to be able to investigate urine as a crime scene sample we have evaluated protocols for analysing DNA from urine in snow. Two different tests were performed with a smaller and a larger volume of urine dispensed on snow. The tubes were put into a freezer to mimic winter conditions. A Urine Preservative was added to some of the samples. DNA profiles were compared between samples extracted using a Urine DNA Isolation Kit and samples extracted with a Chelex-based method. In addition, a test was performed with the aim to mimic a potential crime scene. With a smaller volume of urine the best quality DNA profiles were obtained using the Urine DNA Isolation Kit without the Urine Preservative. When a larger volume of urine was handled and in the crime scene setup, there were no clear difference between the two extraction methods. Instead, the variation observed was between individuals.
1. Introduction Urine can potentially be important evidence when present at crime scenes. Wintertime, in case of outdoor scenes, yellow snow could indicate the presence of human urine. In urine however, cell destruction and DNA degrading may occur, making it a challenging material [1]. In this study we present an evaluation of two different protocols for analysing DNA from urine in snow. 2. Materials and methods Two tests with male urine dispensed on snow in 50 mL tubes were performed; a smaller volume from three individuals and a larger volume from four other individuals. The tubes were put into a freezer to mimic winter conditions. A Urine Preservative (Norgen Biotek Corporation) was added to some of the samples one day before the extractions were performed when the samples were thawing. The samples were thereafter stored at room temperature. Samples without preservative were stored in a refrigerator after thawing. Comparison was also made with urine stored at room temperature. Quantification (Quantifiler HP DNA Kit, Thermo Fisher Scientific) and typing results (PowerPlex ESX 16 Fast System, Promega Corporation) from samples with or without preservative in combination with two different extraction methods, a Urine DNA Isolation Kit (Slurry Format, Norgen Biotek Corporation) and a Chelex-based method were compared. DNA
⁎
extraction with the Urine DNA Isolation Kit was performed according to the manufactures recommendations [2]. The Chelex-based extraction method started with centrifugation of the 50 mL tubes for 4 min at 12,300×g (RCF). After discarding the supernatant the extract was transferred to a 1.5 mL tube and centrifuged for 3 min at 11,000×g (RCF). After discarding the supernatant 170 μL lysis buffer (5% Chelex (w/w), (Bio-Rad Laboratories), 0.1 mg/mL Proteinase K (Sigma-Aldrich) and 0.2% Tween 20 (w/w) (Sigma-Aldrich)) was added [3]. Finally, the samples were heated twice (56 °C for 45–75 min, and 100 °C for 20–40 min) and vortexed in between. In addition, another test was performed with three men urinating in snow to mimic a crime scene scenario. After 4 h the snow was collected. The procedure was repeated and the first samples were extracted with the urine kit and the later samples were extracted with the Chelexbased method. The preservative was used only in combination with the urine kit. 3. Results and discussion When results for samples containing the smaller volumes of urine (3 and 6 mL) were compared the urine kit gave better results for “Male 1”, Table 1. The Chelex method in combination with the preservative gave blank DNA profiles although a concentration was obtained for all three men. Thus, when using the Chelex method, it seems beneficial to store the samples in refrigerator.
Corresponding author. E-mail address: [email protected] (C. Dufva).
https://doi.org/10.1016/j.fsigss.2019.10.084 Received 5 September 2019; Accepted 7 October 2019 1875-1768/ © 2019 Elsevier B.V. All rights reserved.
Please cite this article as: Charlotte Dufva, et al., Forensic Science International: Genetics Supplement Series, https://doi.org/10.1016/j.fsigss.2019.10.084
Forensic Science International: Genetics Supplement Series xxx (xxxx) xxx–xxx
C. Dufva, et al.
Table 1 Mean value for quantification (ng/μL) and typing results (number of markers with alleles > 50 rfu) for 3 and 6 mL urine in snow from three men analysed with two different extraction methods with or without added preservative, n = 3. The storage after thawing was in room temperature (RT) or in refrigerator (approximately 6 °C). Male (volume urine)
Extraction method
Preservative
Storage after thawing
Mean concentration (ng/μL)
Mean value STR marker (0–16)
1 (3 mL)
Chelex
Yes No No Yes No No Yes No No Yes No No Yes No No Yes No No
RT RT 6 °C RT RT 6 °C RT RT 6 °C RT RT 6 °C RT RT 6 °C RT RT 6 °C
0.0014 ± 0.0006 0.0005 ± 0.0002 0.0006 ± 0.0002 0.0023 ± 0.0004 0.0031 ± 0.0002 0.011 ± 0.0008 0.16 ± 0.08 0.03 ± 0.01 0.015 ± 0.007 0.07 ± 0.01 0.08 ± 0.03 0.11 ± 0.01 0.0088 ± 0.0006 0.0020 ± 0.0002 0.0083 ± 0.0035 0.0016 ± 0.0004 0.0034 ± 0.001 0.019 ± 0.009
0±0 7 ± 0.6 12 ± 2 15 ± 1 9 ± 1.5 16 ± 0 0±0 16 ± 0 13 ± 3.6 16 ± 0 16 ± 0 16 ± 0 0±0 15 ± 1.5 16 ± 0 16 ± 0 16 ± 0 16 ± 0
Urine kit
2 (6 mL)
Chelex
Urine kit
3 (6 mL)
Chelex
Urine kit
individuals. All samples generated complete DNA profiles. “Male 3” in the mimicked “crime scene” seemed to be an outlier as the urine contained significantly higher DNA concentrations (Fig. 1B). In addition, following these tests true casework samples have been analysed with success. Two samples of yellow snow collected at different locations at one crime scene were put into tubes and sent on ice to the laboratory for analysis. By using the Chelex method DNA concentrations of 0.4 ng/μL and 0.06 ng/μL were obtained generating single source DNA profiles from two individuals. 4. Conclusions With a smaller volume of urine the highest quality DNA profiles were obtained using the Urine DNA Isolation Kit without the Urine Preservative. When a larger volume of urine was handled as well as in the crime scene setup, there were no clear difference between the two extraction methods. Instead, the greatest variation observed was between individuals. When the protocols are compared there is an obvious difference in hands-on time. Ten samples with the Urine Kit protocol takes approximately 2.5 h compared to 30 min for the Chelex method. In addition, the Urine Kit is much more expensive approximately 9.3 EUR per sample without and 18.7 EUR with preservative. In contrast, the cost for Chelex lysis buffer is approximately 0.2EUR per sample. Based on differences in hands-on time and cost, given the results, the Chelexbased extraction method is the preferable choice. References Fig. 1. DNA-quantification results for: (A) samples containing 25 mL urine in snow from four men analysed with two different extraction methods with or without added preservative (B) samples from a mimicked crime scene setup from three men analysed with two different extraction methods.
[1] S.H. Zhang, S.M. Zhao, Z.M. Zhao, C.T. Li, Genotyping of urinary samples stored with EDTA for forensic applications, Genet. Mol. Res. 11 (2012) 3007–3012. [2] Urine DNA Isolation Kit (Slurry Format) Product Insert, Product # 48800. Norgen Biotek Corporation. [3] P.S. Walsh, D.A. Metzger, R. Higuchi, Chelex® 100 as a medium for simple extraction of DNA for PCR-based typing from forensic material, BioTechniques 10 (1991) 506–513.
When a larger volume of urine (25 mL) was handled as well as in the crime scene setup, there were no clear difference between the two extraction methods, Fig. 1. Instead, the variation observed was between
2
Contents lists available at ScienceDirect
Forensic Science International: Genetics Supplement Series journal homepage: www.elsevier.com/locate/fsigss
DNA profiles obtained from urine in snow Charlotte Dufvaa,⁎, Emilia Karlssona, Christina Forsberga, Ricky Ansella,b a b
National Forensic Centre, Swedish Police Authority, Linköping, Sweden Department of Physics, Chemistry and Biology (IFM), Linköping University, Linköping, Sweden
A R T I C LE I N FO
A B S T R A C T
Keywords: Crime scene DNA evidence Snow Urine
Urine can be a potential important source of evidence when occurring at crime scenes. In case of outdoor scenes including snow a yellow colour could indicate the existence of human urine. In an effort to be able to investigate urine as a crime scene sample we have evaluated protocols for analysing DNA from urine in snow. Two different tests were performed with a smaller and a larger volume of urine dispensed on snow. The tubes were put into a freezer to mimic winter conditions. A Urine Preservative was added to some of the samples. DNA profiles were compared between samples extracted using a Urine DNA Isolation Kit and samples extracted with a Chelex-based method. In addition, a test was performed with the aim to mimic a potential crime scene. With a smaller volume of urine the best quality DNA profiles were obtained using the Urine DNA Isolation Kit without the Urine Preservative. When a larger volume of urine was handled and in the crime scene setup, there were no clear difference between the two extraction methods. Instead, the variation observed was between individuals.
1. Introduction Urine can potentially be important evidence when present at crime scenes. Wintertime, in case of outdoor scenes, yellow snow could indicate the presence of human urine. In urine however, cell destruction and DNA degrading may occur, making it a challenging material [1]. In this study we present an evaluation of two different protocols for analysing DNA from urine in snow. 2. Materials and methods Two tests with male urine dispensed on snow in 50 mL tubes were performed; a smaller volume from three individuals and a larger volume from four other individuals. The tubes were put into a freezer to mimic winter conditions. A Urine Preservative (Norgen Biotek Corporation) was added to some of the samples one day before the extractions were performed when the samples were thawing. The samples were thereafter stored at room temperature. Samples without preservative were stored in a refrigerator after thawing. Comparison was also made with urine stored at room temperature. Quantification (Quantifiler HP DNA Kit, Thermo Fisher Scientific) and typing results (PowerPlex ESX 16 Fast System, Promega Corporation) from samples with or without preservative in combination with two different extraction methods, a Urine DNA Isolation Kit (Slurry Format, Norgen Biotek Corporation) and a Chelex-based method were compared. DNA
⁎
extraction with the Urine DNA Isolation Kit was performed according to the manufactures recommendations [2]. The Chelex-based extraction method started with centrifugation of the 50 mL tubes for 4 min at 12,300×g (RCF). After discarding the supernatant the extract was transferred to a 1.5 mL tube and centrifuged for 3 min at 11,000×g (RCF). After discarding the supernatant 170 μL lysis buffer (5% Chelex (w/w), (Bio-Rad Laboratories), 0.1 mg/mL Proteinase K (Sigma-Aldrich) and 0.2% Tween 20 (w/w) (Sigma-Aldrich)) was added [3]. Finally, the samples were heated twice (56 °C for 45–75 min, and 100 °C for 20–40 min) and vortexed in between. In addition, another test was performed with three men urinating in snow to mimic a crime scene scenario. After 4 h the snow was collected. The procedure was repeated and the first samples were extracted with the urine kit and the later samples were extracted with the Chelexbased method. The preservative was used only in combination with the urine kit. 3. Results and discussion When results for samples containing the smaller volumes of urine (3 and 6 mL) were compared the urine kit gave better results for “Male 1”, Table 1. The Chelex method in combination with the preservative gave blank DNA profiles although a concentration was obtained for all three men. Thus, when using the Chelex method, it seems beneficial to store the samples in refrigerator.
Corresponding author. E-mail address: [email protected] (C. Dufva).
https://doi.org/10.1016/j.fsigss.2019.10.084 Received 5 September 2019; Accepted 7 October 2019 1875-1768/ © 2019 Elsevier B.V. All rights reserved.
Please cite this article as: Charlotte Dufva, et al., Forensic Science International: Genetics Supplement Series, https://doi.org/10.1016/j.fsigss.2019.10.084
Forensic Science International: Genetics Supplement Series xxx (xxxx) xxx–xxx
C. Dufva, et al.
Table 1 Mean value for quantification (ng/μL) and typing results (number of markers with alleles > 50 rfu) for 3 and 6 mL urine in snow from three men analysed with two different extraction methods with or without added preservative, n = 3. The storage after thawing was in room temperature (RT) or in refrigerator (approximately 6 °C). Male (volume urine)
Extraction method
Preservative
Storage after thawing
Mean concentration (ng/μL)
Mean value STR marker (0–16)
1 (3 mL)
Chelex
Yes No No Yes No No Yes No No Yes No No Yes No No Yes No No
RT RT 6 °C RT RT 6 °C RT RT 6 °C RT RT 6 °C RT RT 6 °C RT RT 6 °C
0.0014 ± 0.0006 0.0005 ± 0.0002 0.0006 ± 0.0002 0.0023 ± 0.0004 0.0031 ± 0.0002 0.011 ± 0.0008 0.16 ± 0.08 0.03 ± 0.01 0.015 ± 0.007 0.07 ± 0.01 0.08 ± 0.03 0.11 ± 0.01 0.0088 ± 0.0006 0.0020 ± 0.0002 0.0083 ± 0.0035 0.0016 ± 0.0004 0.0034 ± 0.001 0.019 ± 0.009
0±0 7 ± 0.6 12 ± 2 15 ± 1 9 ± 1.5 16 ± 0 0±0 16 ± 0 13 ± 3.6 16 ± 0 16 ± 0 16 ± 0 0±0 15 ± 1.5 16 ± 0 16 ± 0 16 ± 0 16 ± 0
Urine kit
2 (6 mL)
Chelex
Urine kit
3 (6 mL)
Chelex
Urine kit
individuals. All samples generated complete DNA profiles. “Male 3” in the mimicked “crime scene” seemed to be an outlier as the urine contained significantly higher DNA concentrations (Fig. 1B). In addition, following these tests true casework samples have been analysed with success. Two samples of yellow snow collected at different locations at one crime scene were put into tubes and sent on ice to the laboratory for analysis. By using the Chelex method DNA concentrations of 0.4 ng/μL and 0.06 ng/μL were obtained generating single source DNA profiles from two individuals. 4. Conclusions With a smaller volume of urine the highest quality DNA profiles were obtained using the Urine DNA Isolation Kit without the Urine Preservative. When a larger volume of urine was handled as well as in the crime scene setup, there were no clear difference between the two extraction methods. Instead, the greatest variation observed was between individuals. When the protocols are compared there is an obvious difference in hands-on time. Ten samples with the Urine Kit protocol takes approximately 2.5 h compared to 30 min for the Chelex method. In addition, the Urine Kit is much more expensive approximately 9.3 EUR per sample without and 18.7 EUR with preservative. In contrast, the cost for Chelex lysis buffer is approximately 0.2EUR per sample. Based on differences in hands-on time and cost, given the results, the Chelexbased extraction method is the preferable choice. References Fig. 1. DNA-quantification results for: (A) samples containing 25 mL urine in snow from four men analysed with two different extraction methods with or without added preservative (B) samples from a mimicked crime scene setup from three men analysed with two different extraction methods.
[1] S.H. Zhang, S.M. Zhao, Z.M. Zhao, C.T. Li, Genotyping of urinary samples stored with EDTA for forensic applications, Genet. Mol. Res. 11 (2012) 3007–3012. [2] Urine DNA Isolation Kit (Slurry Format) Product Insert, Product # 48800. Norgen Biotek Corporation. [3] P.S. Walsh, D.A. Metzger, R. Higuchi, Chelex® 100 as a medium for simple extraction of DNA for PCR-based typing from forensic material, BioTechniques 10 (1991) 506–513.
When a larger volume of urine (25 mL) was handled as well as in the crime scene setup, there were no clear difference between the two extraction methods, Fig. 1. Instead, the variation observed was between
2