- Email: [email protected]
Adaptation and evaluation of the PrepFiler™ DNA extraction technology in an automated forensic DNA analysis process with emphasis on DNA yield, inhibitor removal and contamination security
Forensic Science International: Genetics Supplement Series 2 (2009) 62–63
Contents lists available at ScienceDirect
Forensic Science International: Genetics Supplement Series journal homepage: www.elsevier.com/locate/FSIGSS
Research article
Adaptation and evaluation of the PrepFilerTM DNA extraction technology in an automated forensic DNA analysis process with emphasis on DNA yield, inhibitor removal and contamination security Peter Zimmermann *, Kai Vollack, Barbara Haak, Michelle Bretthauer, Andrea Jelinski, Marga Kugler, Jessica Loidl, Werner Pflug Landeskriminalamt Baden-Wu¨rttemberg, Taubenheimstraße 85, 70372 Stuttgart, Germany
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 26 August 2009
Within the initial step of the forensic DNA analysis process, the DNA extraction efficiency and especially the removal of potential PCR inhibitors is crucial for subsequent steps, e.g. quantification by real-time PCR and amplification of short tandem repeats (STRs). The protocol of the PrepFilerTM Forensic DNA Extraction Kit was optimized for the application on a Tecan liquid handling workstation Freedom EVO1 150. This modified application of the PrepFilerTM technology was compared with respect to DNA yield, sensitivity and the ability to remove potential PCR inhibitors to an established routine method working on the same liquid handling workstation based on ChargeSwitch1 Technology (CST) from Invitrogen. ß 2009 Elsevier Ireland Ltd. All rights reserved.
Keywords: DNA extraction PCR inhibition Automation Liquid handling workstation
1. Introduction In 2007 an automated processing system for the forensic DNA analysis of about 20,000 various crime scene samples of routine casework annually was established at the Landeskriminalamt (Office of Criminal Investigation) Baden-Wu¨rttemberg [1] using a one-for-all protocol for magnetic bead-based DNA extraction with ChargeSwitch1 technology (CST) [2]. On the basis of this routine application running on a liquid handling workstation the PrepFilerTM Forensic DNA Extraction Kit [3] was installed by adapting the existing automation system. 2. Materials and methods For the comparative analysis of both extraction methods blood dilutions and different forensic samples containing difficult stains (e.g. samples contaminated with soil) were prepared in parallel. Cell lysis was performed in 500 ml volumes for 1 h (fresh blood dilutions) or over night (different trace samples), respectively. Both extraction methods were installed on a Freedom EVO1 150 liquid handling workstation equipped with an 8-channel liquid handling arm, two 48 well Te-MagSTM magnetic bead separation modules and a PosIDTM barcode sample identification device (Tecan). As an extra safety precaution to avoid cross-contaminations between * Corresponding author at: Landeskriminalamt Baden-Wu¨rttemberg, Kriminaltechnisches Institut, Taubenheimstraße 85, 70372 Stuttgart, Germany. Tel.: +49 71154011782; fax: +49 71154012735. E-mail address: [email protected] (P. Zimmermann). 1875-1768/$ – see front matter ß 2009 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.fsigss.2009.08.162
samples liquid handling arms do not travel across open wells or tubes other than their targets. Furthermore the possibility of aerosol formation was avoided by using single tubes and two TeMagSTM modules for sample mixing and separation [2] instead of a 96-deep well plate combined with a shaker for mixing. DNA quantification was determined by real-time PCR using the AB QuantifilerTM Human DNA Quantification Kit [4]. 3. Results The adapted PrepFiler protocol was applied for the DNA extraction from different forensic samples and compared to the routine CST protocol already optimized for the whole range of biological sample types [2]. DNA extracted from different blood dilutions (non diluted to 1:729 diluted) showed significantly higher DNA yields using the modified PrepFiler technology versus the ChargeSwitch technology in the range of higher concentrated samples (non diluted to 1:9 diluted) whereas higher diluted samples (1:27 to 1:729 diluted) led to comparable results with both methods. Both methods led to comparable STR profiles after fragment length analyses with an AB Prism 3130xl Genetic Analyzer. DNA extractions from different blood dilutions each containing a constant aliquot of a soil suspension showed higher calculated DNA concentrations in the subsequent real-time PCR with the PrepFiler extracted samples in comparison to CST extracts (Fig. 1A). Evidently PCR inhibition by the added soil contributed to the significant differences in the real-time PCR quantification data as indicated by the CT values of the internal positive control (IPC): whereas the CT values of the IPC were constantly around 25 using the PrepFiler method the CT values of the
P. Zimmermann et al. / Forensic Science International: Genetics Supplement Series 2 (2009) 62–63
63
Fig. 1. Comparison of DNA extraction by PrepFiler and CST. (A) Quantification data of blood dilutions containing soil, (B) CT values of the internal positive controls (IPC) from (A), (C) quantification data of different forensic sample types and (D) CT values of the IPC from C.
IPC varied from 31 to 38 after extraction with the CST (Fig. 1B). The deduced PCR inhibition thereby indicates that the calculated DNA concentration of the DNA from CST is too low as confirmed by comparable STR profiles. Difficult sample types like faeces and blood on leather surface showed a strong PCR inhibition using the CST technology for DNA extraction (CT values above 40 for the IPC, Fig. 1D) and no measurable DNA quantification data from real-time PCR (Fig. 1C). In contrast, the real-time PCR data from PrepFiler extracted DNA showed no inhibition (Fig. 1D). As expected by the observed real-time PCR inhibition from CST extracted DNA, the subsequent STR-PCR with this DNA was also inhibited. After fragment length analyses no signals were detected in the CST extracted faeces samples, whereas full database compatible DNA profiles were obtained with the PrepFiler extracted DNA. The samples from blood isolated on leather material showed less harsh inhibitory effects, but nevertheless DNA isolated by the PrepFiler method led to significantly better DNA profiles. Skin particles transmitted by direct skin contact from persons to objects represent another difficult sample type due to minor DNA amounts in the picogram range that can be expected from such preparations. Preliminary results from DNA extractions of skin particles isolated from different sources like foils, a knife and a woollen hat constantly led to higher DNA quantification data with the CST technology in comparison to the modified PrepFiler method (Fig. 1C). Surprisingly, all in all equal volumes of DNA extracts from both methods produced comparable STR-PCR profiles by fragment length analyses, though different quantification data was gained by real-time PCR. 4. Discussion The PrepFilerTM DNA extraction technology was successfully adapted to our automated forensic DNA analysis process using
a Freedom EVO1 150 liquid handling workstation. Besides high DNA yield and the efficient removal of potential PCR inhibitors, an emphasis was set on extra safety precautions to avoid cross-contaminations between samples. Pathways of the liquid handling arm were programmed not to travel across open wells and magnetic mixing was chosen as an economical solution to avoid potential formation of aerosols. The PrepFilerTM DNA extraction method was shown to be superior in removing PCR inhibitors from samples like faeces, samples from leather or samples contaminated with soil. Our experiments showed that PrepFiler extracted DNA of skin particle samples delivered lower real-time PCR quantification values than CST isolated DNA, but led to comparable DNA profiles, indicating high quality DNA for PCR. However the lower quantification data from skin particle samples indicated that further investigations are necessary to optimize the lysis conditions for routine use. Conflict of interest None. References [1] K. Vollack, et al., Implementation of a semi-automated processing system for DNA profiling of forensic casework samples, Forensic Sci. Int. 1 (2008) 83–85. [2] B. Haak, et al., Evaluation of a semi-automated, magnetic bead-based DNA-extraction method for genetic fingerprinting of forensic casework samples, Forensic Sci. Int. 1 (2008) 35–36. [3] PrepFilerTM Forensic DNA Extraction Kit and Automated Forensic DNA Extraction Kit, Applied Biosystems, 2008. [4] R. Schwenzer, et al., Implementation of a robotized real-time-PCR setup for the use of the AB QuantifilerTM Human DNA Quantification Kit, Forensic Sci. Int. 1 (2008) 68–70.
Contents lists available at ScienceDirect
Forensic Science International: Genetics Supplement Series journal homepage: www.elsevier.com/locate/FSIGSS
Research article
Adaptation and evaluation of the PrepFilerTM DNA extraction technology in an automated forensic DNA analysis process with emphasis on DNA yield, inhibitor removal and contamination security Peter Zimmermann *, Kai Vollack, Barbara Haak, Michelle Bretthauer, Andrea Jelinski, Marga Kugler, Jessica Loidl, Werner Pflug Landeskriminalamt Baden-Wu¨rttemberg, Taubenheimstraße 85, 70372 Stuttgart, Germany
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 26 August 2009
Within the initial step of the forensic DNA analysis process, the DNA extraction efficiency and especially the removal of potential PCR inhibitors is crucial for subsequent steps, e.g. quantification by real-time PCR and amplification of short tandem repeats (STRs). The protocol of the PrepFilerTM Forensic DNA Extraction Kit was optimized for the application on a Tecan liquid handling workstation Freedom EVO1 150. This modified application of the PrepFilerTM technology was compared with respect to DNA yield, sensitivity and the ability to remove potential PCR inhibitors to an established routine method working on the same liquid handling workstation based on ChargeSwitch1 Technology (CST) from Invitrogen. ß 2009 Elsevier Ireland Ltd. All rights reserved.
Keywords: DNA extraction PCR inhibition Automation Liquid handling workstation
1. Introduction In 2007 an automated processing system for the forensic DNA analysis of about 20,000 various crime scene samples of routine casework annually was established at the Landeskriminalamt (Office of Criminal Investigation) Baden-Wu¨rttemberg [1] using a one-for-all protocol for magnetic bead-based DNA extraction with ChargeSwitch1 technology (CST) [2]. On the basis of this routine application running on a liquid handling workstation the PrepFilerTM Forensic DNA Extraction Kit [3] was installed by adapting the existing automation system. 2. Materials and methods For the comparative analysis of both extraction methods blood dilutions and different forensic samples containing difficult stains (e.g. samples contaminated with soil) were prepared in parallel. Cell lysis was performed in 500 ml volumes for 1 h (fresh blood dilutions) or over night (different trace samples), respectively. Both extraction methods were installed on a Freedom EVO1 150 liquid handling workstation equipped with an 8-channel liquid handling arm, two 48 well Te-MagSTM magnetic bead separation modules and a PosIDTM barcode sample identification device (Tecan). As an extra safety precaution to avoid cross-contaminations between * Corresponding author at: Landeskriminalamt Baden-Wu¨rttemberg, Kriminaltechnisches Institut, Taubenheimstraße 85, 70372 Stuttgart, Germany. Tel.: +49 71154011782; fax: +49 71154012735. E-mail address: [email protected] (P. Zimmermann). 1875-1768/$ – see front matter ß 2009 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.fsigss.2009.08.162
samples liquid handling arms do not travel across open wells or tubes other than their targets. Furthermore the possibility of aerosol formation was avoided by using single tubes and two TeMagSTM modules for sample mixing and separation [2] instead of a 96-deep well plate combined with a shaker for mixing. DNA quantification was determined by real-time PCR using the AB QuantifilerTM Human DNA Quantification Kit [4]. 3. Results The adapted PrepFiler protocol was applied for the DNA extraction from different forensic samples and compared to the routine CST protocol already optimized for the whole range of biological sample types [2]. DNA extracted from different blood dilutions (non diluted to 1:729 diluted) showed significantly higher DNA yields using the modified PrepFiler technology versus the ChargeSwitch technology in the range of higher concentrated samples (non diluted to 1:9 diluted) whereas higher diluted samples (1:27 to 1:729 diluted) led to comparable results with both methods. Both methods led to comparable STR profiles after fragment length analyses with an AB Prism 3130xl Genetic Analyzer. DNA extractions from different blood dilutions each containing a constant aliquot of a soil suspension showed higher calculated DNA concentrations in the subsequent real-time PCR with the PrepFiler extracted samples in comparison to CST extracts (Fig. 1A). Evidently PCR inhibition by the added soil contributed to the significant differences in the real-time PCR quantification data as indicated by the CT values of the internal positive control (IPC): whereas the CT values of the IPC were constantly around 25 using the PrepFiler method the CT values of the
P. Zimmermann et al. / Forensic Science International: Genetics Supplement Series 2 (2009) 62–63
63
Fig. 1. Comparison of DNA extraction by PrepFiler and CST. (A) Quantification data of blood dilutions containing soil, (B) CT values of the internal positive controls (IPC) from (A), (C) quantification data of different forensic sample types and (D) CT values of the IPC from C.
IPC varied from 31 to 38 after extraction with the CST (Fig. 1B). The deduced PCR inhibition thereby indicates that the calculated DNA concentration of the DNA from CST is too low as confirmed by comparable STR profiles. Difficult sample types like faeces and blood on leather surface showed a strong PCR inhibition using the CST technology for DNA extraction (CT values above 40 for the IPC, Fig. 1D) and no measurable DNA quantification data from real-time PCR (Fig. 1C). In contrast, the real-time PCR data from PrepFiler extracted DNA showed no inhibition (Fig. 1D). As expected by the observed real-time PCR inhibition from CST extracted DNA, the subsequent STR-PCR with this DNA was also inhibited. After fragment length analyses no signals were detected in the CST extracted faeces samples, whereas full database compatible DNA profiles were obtained with the PrepFiler extracted DNA. The samples from blood isolated on leather material showed less harsh inhibitory effects, but nevertheless DNA isolated by the PrepFiler method led to significantly better DNA profiles. Skin particles transmitted by direct skin contact from persons to objects represent another difficult sample type due to minor DNA amounts in the picogram range that can be expected from such preparations. Preliminary results from DNA extractions of skin particles isolated from different sources like foils, a knife and a woollen hat constantly led to higher DNA quantification data with the CST technology in comparison to the modified PrepFiler method (Fig. 1C). Surprisingly, all in all equal volumes of DNA extracts from both methods produced comparable STR-PCR profiles by fragment length analyses, though different quantification data was gained by real-time PCR. 4. Discussion The PrepFilerTM DNA extraction technology was successfully adapted to our automated forensic DNA analysis process using
a Freedom EVO1 150 liquid handling workstation. Besides high DNA yield and the efficient removal of potential PCR inhibitors, an emphasis was set on extra safety precautions to avoid cross-contaminations between samples. Pathways of the liquid handling arm were programmed not to travel across open wells and magnetic mixing was chosen as an economical solution to avoid potential formation of aerosols. The PrepFilerTM DNA extraction method was shown to be superior in removing PCR inhibitors from samples like faeces, samples from leather or samples contaminated with soil. Our experiments showed that PrepFiler extracted DNA of skin particle samples delivered lower real-time PCR quantification values than CST isolated DNA, but led to comparable DNA profiles, indicating high quality DNA for PCR. However the lower quantification data from skin particle samples indicated that further investigations are necessary to optimize the lysis conditions for routine use. Conflict of interest None. References [1] K. Vollack, et al., Implementation of a semi-automated processing system for DNA profiling of forensic casework samples, Forensic Sci. Int. 1 (2008) 83–85. [2] B. Haak, et al., Evaluation of a semi-automated, magnetic bead-based DNA-extraction method for genetic fingerprinting of forensic casework samples, Forensic Sci. Int. 1 (2008) 35–36. [3] PrepFilerTM Forensic DNA Extraction Kit and Automated Forensic DNA Extraction Kit, Applied Biosystems, 2008. [4] R. Schwenzer, et al., Implementation of a robotized real-time-PCR setup for the use of the AB QuantifilerTM Human DNA Quantification Kit, Forensic Sci. Int. 1 (2008) 68–70.