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The problem of DNA contamination in forensic case work—How to get rid of unwanted DNA?
Forensic Science International: Genetics Supplement Series 2 (2009) 185–186
Contents lists available at ScienceDirect
Forensic Science International: Genetics Supplement Series journal homepage: www.elsevier.com/locate/FSIGSS
Research article
The problem of DNA contamination in forensic case work—How to get rid of unwanted DNA? A. Preuße-Prange a,*, R. Renneberg b, T. Schwark a, M. Poetsch c, E. Simeoni a, N. von Wurmb-Schwark a a
Institute of Legal Medicine, University Hospital Schleswig-Holstein, Campus Kiel, Germany Institute of Experimental Medicine, Christian-Albrechts-University Kiel & University Hospital Schleswig-Holstein, Campus Kiel and Graduate School Human Development in Landscapes, Christian-Albrechts-University Kiel, Germany c Institute of Legal Medicine, University Hospital of Essen, Germany b
A R T I C L E I N F O
A B S T R A C T
Article history: Received 24 August 2009 Accepted 27 August 2009
The PCR technique has become a powerful and very sensitive tool in a broad field of research, that is, molecular biology, medical diagnostics, population genetics, ancient DNA analysis and forensic casework. However, the high sensitivity down to single molecules can easily cause false-positive PCR results due to different types of contamination. In this study, artificial DNA contaminations (saliva and pure DNA) were treated with UV irradiation and other decontamination procedures. A satisfactory DNA removal could not be achieved, emphasizing the necessity of contamination avoidance. ß 2009 Elsevier Ireland Ltd. All rights reserved.
Keywords: Ancient DNA Contamination UV irradiation
1. Introduction The polymerase chain reaction (PCR) technique is a widely used method for the replication of specific DNA segments and has become a powerful and very sensitive tool in a broad field of research, e.g. molecular biology, medical diagnostics, population genetics, ancient DNA analysis and forensic casework [1,2]. One important consequence resulting from the improved sensitivity in DNA amplification is the necessity to avoid any contaminations caused by foreign unwanted DNA molecules [3]. To prevent DNA contamination several criteria and recommendations have to be followed and fulfilled. A commonly used method for decontamination is UV irradiation. In many protocols UV treatment is mentioned for decontamination and there are several gadgetries such as commercially available DNA workbenches or UV aircleaners. We tested the efficiency of different (chemical and physical) procedures for DNA removal with focus on the commonly recommended UV irradiation. 2. Materials and methods 2.1. Sample preparation Employees of the Institute of Legal Medicine, University Hospital Kiel, Germany, voluntarily donated saliva and blood * Corresponding author at: Institute of Legal Medicine, University Hospital Schleswig-Holstein, Campus Kiel, Arnold-Heller-Str. 12, 24105 Kiel, Germany. Tel.: +49 431 597 3603; fax: +49 431 597 3612. E-mail address: [email protected] (A. Preuße-Prange). 1875-1768/$ – see front matter ß 2009 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.fsigss.2009.08.164
samples for positive and contamination controls. DNA was extracted from blood using the Nucleo Spin Blood Quick Pure kit (Macherey-Nagel, Germany) according to the manufacturer’s instructions. Quantification was done using a self-designed real time PCR. Additionally pure saliva was used which contained 0.2 ng/ml DNA also determined by real time PCR. 2.2. Experimental set-up 1 ml of saliva (0.2 ng/ml DNA) and pure DNA containing solution (96.6 ng/ml DNA) were applied to glass slides and exposed to two UV wavelengths – 254 nm (DNA-Workstation I, Kisker-Biotech) and 312 nm (Fluorescent table, Renner GmbH) – for different periods of time (5 min, 10 min, 15 min, 20 min, 30 min, 1 h, 6 h, 12 h and 24 h) and at two distances (8 cm and 48 cm) from the UV source. After treatment, saliva and pure DNA samples were directly applied to a self-designed mini-STR multiplex PCR [4] with 1 ml DNA from a second volunteer (control-DNA) by re-suspending the samples from the glass slide with the PCR-reaction mix. Additionally, different chemical reagents (DNA-Exitus, AppliChem GmbH; DNA-Away, Carl Roth GmbH + Co. KG; DanKlorix (bleach), Colgate Palmolive GmbH and Ethanol, Bu¨sing & Fasch GmbH & Co. KG) were tested for decontamination. The prepared slides were sprayed with the mentioned chemicals and samples were directly subjected to the PCR after different treatments: (A) Incubation of chemicals for 15 min, then re-suspending the samples from the glass slides with PCR-reaction mix. (B) Incubation for 15 min, then wiping the slides with a paper towel, second treatment with the chemicals for 15 min and application to
186
A. Preuße-Prange et al. / Forensic Science International: Genetics Supplement Series 2 (2009) 185–186
Fig. 1. Treatment with several chemicals. Schematic presentation after PCR and capillary electrophoresis on an ABIPrism 310. The spotted bars show the results after treatment with the tested chemicals once for 15 min and the dark bars the results after incubation twice for 15 min. The results for DNA and saliva are shown in relative fluorescence units.
PCR as described. Negative-controls (swabs from untreated slides) and PCR-negative-controls were performed. 2.3. DNA analysis and detection Fragment analysis was performed on an ABIPrism 310 with 1 ml PCR product each and samples were analyzed using the GeneScanTM Software 3.1.2.; 0.2 ml of GeneScanTM 500 RoxTM Size Standard was added to each sample for size determination (all APPLIED BIOSYSTEMS). 3. Results and discussion Exposure of the artificially contaminated slides at a distance of 8 cm from the UV source led to a reduction of the contamination by 83.4% (DNA) and 56.2% (saliva), with no relation to exposure time. A distance of 48 cm reduced the contamination by 70.7% (DNA) and 53.5% (saliva) on average, showing that the distance between the samples and the light source has an effect on the reduction of the amount of contamination. Furthermore, our findings indicate that isolated DNA is more susceptible to UV irradiation than saliva. Using a wavelength of 254 nm the reduction of unwanted DNA was higher than by using 312 nm (data not shown). These results show that the shorter wavelength eliminates the contamination more efficiently than the longer one. However DNA contamination could not be eliminated completely. The experiments with different chemicals indicate
that in most cases isolated pure DNA is more susceptible to the tested chemicals than saliva (Fig. 1). Even though the results are ambivalent, our study demonstrates that the procedures of DNA decontamination with UV irradiation are rather inefficient. UV irradiation can only reduce the contamination but does not eliminate it completely, demonstrating the importance of contamination avoidance prior to genetic analysis. Role of funding University Research Funding. Conflict of interest statement None. References [1] C. Niederhauser, C. Ho¨felein, B. Wegmu¨ller, J. Lu¨thy, U. Candrian, Reliability of PCR decontamination systems, Genome Res. (1994). [2] J. Tamariz, K. Voynarovska, M. Prinz, T. Caragine, The application of ultraviolet irradiation to exogenous sources of DNA in plasticware and water for the amplification of low copy number DNA, J. Forensic Sci. (2006). [3] K.-H. Esser, W.H. Marx, T. Lisowsky, DNA decontamination: novel DNA-ExitusPlusTM incomparison with conventional reagents, BioTechniques (2006). [4] N. von Wurmb-Schwark, A. Preusse-Prange, A. Heinrich, E. Simeoni, T. Bosch, T. Schwark, A new multiplex-PCR comprising autosomal and y-specific STRs and mitochondrial DNA to analyze highly degraded material, Forensic Sci. Int. Genet. (2009).
Contents lists available at ScienceDirect
Forensic Science International: Genetics Supplement Series journal homepage: www.elsevier.com/locate/FSIGSS
Research article
The problem of DNA contamination in forensic case work—How to get rid of unwanted DNA? A. Preuße-Prange a,*, R. Renneberg b, T. Schwark a, M. Poetsch c, E. Simeoni a, N. von Wurmb-Schwark a a
Institute of Legal Medicine, University Hospital Schleswig-Holstein, Campus Kiel, Germany Institute of Experimental Medicine, Christian-Albrechts-University Kiel & University Hospital Schleswig-Holstein, Campus Kiel and Graduate School Human Development in Landscapes, Christian-Albrechts-University Kiel, Germany c Institute of Legal Medicine, University Hospital of Essen, Germany b
A R T I C L E I N F O
A B S T R A C T
Article history: Received 24 August 2009 Accepted 27 August 2009
The PCR technique has become a powerful and very sensitive tool in a broad field of research, that is, molecular biology, medical diagnostics, population genetics, ancient DNA analysis and forensic casework. However, the high sensitivity down to single molecules can easily cause false-positive PCR results due to different types of contamination. In this study, artificial DNA contaminations (saliva and pure DNA) were treated with UV irradiation and other decontamination procedures. A satisfactory DNA removal could not be achieved, emphasizing the necessity of contamination avoidance. ß 2009 Elsevier Ireland Ltd. All rights reserved.
Keywords: Ancient DNA Contamination UV irradiation
1. Introduction The polymerase chain reaction (PCR) technique is a widely used method for the replication of specific DNA segments and has become a powerful and very sensitive tool in a broad field of research, e.g. molecular biology, medical diagnostics, population genetics, ancient DNA analysis and forensic casework [1,2]. One important consequence resulting from the improved sensitivity in DNA amplification is the necessity to avoid any contaminations caused by foreign unwanted DNA molecules [3]. To prevent DNA contamination several criteria and recommendations have to be followed and fulfilled. A commonly used method for decontamination is UV irradiation. In many protocols UV treatment is mentioned for decontamination and there are several gadgetries such as commercially available DNA workbenches or UV aircleaners. We tested the efficiency of different (chemical and physical) procedures for DNA removal with focus on the commonly recommended UV irradiation. 2. Materials and methods 2.1. Sample preparation Employees of the Institute of Legal Medicine, University Hospital Kiel, Germany, voluntarily donated saliva and blood * Corresponding author at: Institute of Legal Medicine, University Hospital Schleswig-Holstein, Campus Kiel, Arnold-Heller-Str. 12, 24105 Kiel, Germany. Tel.: +49 431 597 3603; fax: +49 431 597 3612. E-mail address: [email protected] (A. Preuße-Prange). 1875-1768/$ – see front matter ß 2009 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.fsigss.2009.08.164
samples for positive and contamination controls. DNA was extracted from blood using the Nucleo Spin Blood Quick Pure kit (Macherey-Nagel, Germany) according to the manufacturer’s instructions. Quantification was done using a self-designed real time PCR. Additionally pure saliva was used which contained 0.2 ng/ml DNA also determined by real time PCR. 2.2. Experimental set-up 1 ml of saliva (0.2 ng/ml DNA) and pure DNA containing solution (96.6 ng/ml DNA) were applied to glass slides and exposed to two UV wavelengths – 254 nm (DNA-Workstation I, Kisker-Biotech) and 312 nm (Fluorescent table, Renner GmbH) – for different periods of time (5 min, 10 min, 15 min, 20 min, 30 min, 1 h, 6 h, 12 h and 24 h) and at two distances (8 cm and 48 cm) from the UV source. After treatment, saliva and pure DNA samples were directly applied to a self-designed mini-STR multiplex PCR [4] with 1 ml DNA from a second volunteer (control-DNA) by re-suspending the samples from the glass slide with the PCR-reaction mix. Additionally, different chemical reagents (DNA-Exitus, AppliChem GmbH; DNA-Away, Carl Roth GmbH + Co. KG; DanKlorix (bleach), Colgate Palmolive GmbH and Ethanol, Bu¨sing & Fasch GmbH & Co. KG) were tested for decontamination. The prepared slides were sprayed with the mentioned chemicals and samples were directly subjected to the PCR after different treatments: (A) Incubation of chemicals for 15 min, then re-suspending the samples from the glass slides with PCR-reaction mix. (B) Incubation for 15 min, then wiping the slides with a paper towel, second treatment with the chemicals for 15 min and application to
186
A. Preuße-Prange et al. / Forensic Science International: Genetics Supplement Series 2 (2009) 185–186
Fig. 1. Treatment with several chemicals. Schematic presentation after PCR and capillary electrophoresis on an ABIPrism 310. The spotted bars show the results after treatment with the tested chemicals once for 15 min and the dark bars the results after incubation twice for 15 min. The results for DNA and saliva are shown in relative fluorescence units.
PCR as described. Negative-controls (swabs from untreated slides) and PCR-negative-controls were performed. 2.3. DNA analysis and detection Fragment analysis was performed on an ABIPrism 310 with 1 ml PCR product each and samples were analyzed using the GeneScanTM Software 3.1.2.; 0.2 ml of GeneScanTM 500 RoxTM Size Standard was added to each sample for size determination (all APPLIED BIOSYSTEMS). 3. Results and discussion Exposure of the artificially contaminated slides at a distance of 8 cm from the UV source led to a reduction of the contamination by 83.4% (DNA) and 56.2% (saliva), with no relation to exposure time. A distance of 48 cm reduced the contamination by 70.7% (DNA) and 53.5% (saliva) on average, showing that the distance between the samples and the light source has an effect on the reduction of the amount of contamination. Furthermore, our findings indicate that isolated DNA is more susceptible to UV irradiation than saliva. Using a wavelength of 254 nm the reduction of unwanted DNA was higher than by using 312 nm (data not shown). These results show that the shorter wavelength eliminates the contamination more efficiently than the longer one. However DNA contamination could not be eliminated completely. The experiments with different chemicals indicate
that in most cases isolated pure DNA is more susceptible to the tested chemicals than saliva (Fig. 1). Even though the results are ambivalent, our study demonstrates that the procedures of DNA decontamination with UV irradiation are rather inefficient. UV irradiation can only reduce the contamination but does not eliminate it completely, demonstrating the importance of contamination avoidance prior to genetic analysis. Role of funding University Research Funding. Conflict of interest statement None. References [1] C. Niederhauser, C. Ho¨felein, B. Wegmu¨ller, J. Lu¨thy, U. Candrian, Reliability of PCR decontamination systems, Genome Res. (1994). [2] J. Tamariz, K. Voynarovska, M. Prinz, T. Caragine, The application of ultraviolet irradiation to exogenous sources of DNA in plasticware and water for the amplification of low copy number DNA, J. Forensic Sci. (2006). [3] K.-H. Esser, W.H. Marx, T. Lisowsky, DNA decontamination: novel DNA-ExitusPlusTM incomparison with conventional reagents, BioTechniques (2006). [4] N. von Wurmb-Schwark, A. Preusse-Prange, A. Heinrich, E. Simeoni, T. Bosch, T. Schwark, A new multiplex-PCR comprising autosomal and y-specific STRs and mitochondrial DNA to analyze highly degraded material, Forensic Sci. Int. Genet. (2009).