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AS
Forensic Science International: Genetics Supplement Series 4 (2013) e352–e353
Contents lists available at ScienceDirect
Forensic Science International: Genetics Supplement Series journal homepage: www.elsevier.com/locate/FSIGSS
Processing challenging forensic casework samples with new protocols for the QIAsymphony SP/AS M. Scherer *, B. Alsdorf, M. Breitbach, K. Matthaei, A. Schnur, N. Scholle, L. Bochmann, A. Prochnow, H. Engel QIAGEN GmbH, Hilden, Germany
A R T I C L E I N F O
A B S T R A C T
Article history: Received 2 September 2013 Received in revised form 7 October 2013 Accepted 7 October 2013
Novel extraction protocols for the QIAsymphony SP improve DNA yields from forensic casework samples. The Investigator Advanced protocol has a longer heating step that enhances DNA binding to magnetic particles, while the High Efficiency protocol reduces the loss of purified DNA from low elution volumes. In addition, the latest version of the QIAsymphony AS software enables STR PCR setup normalization based on imported quantification data. ß 2013 Elsevier Ireland Ltd. All rights reserved.
Keywords: Forensic casework DNA extraction STR PCR setup Low-volume elution Oil overlay Quantification data QIAsymphony
1. Introduction
2. Materials and methods
High-throughput instruments can support rapid DNA extraction and precise PCR setup in a whole range of research fields. However, dedicated protocols are crucial for samples for special applications, such as forensic casework. The QIAsymphony is a modular platform for fully automated DNA extraction and PCR plate setup. Two novel extraction protocols optimize its performance with forensic samples. The Investigator Advanced (ADV) protocol has a longer heating step that enhances DNA binding to magnetic particles. It functions with a range of lysate volumes and increases the DNA yield at in all cases. The High Efficiency (HE) protocol improves low-volume elution efficiency by applying an oil overlay to eliminate any dead volumes that would typically reduce the overall yield. The platform also has new software functionality for STR PCR setup normalization based on the results of quantification, allowing automatic adaptation of template amounts per reaction to a user-defined target. Minimum DNA template amounts can also be set, automatically excluding samples from downstream STR analysis if no reportable result is expected. This economical approach requires a sensitive quantification method to minimize the risk of false negatives.
2.1. Instruments
* Corresponding author. E-mail address: [email protected] (M. Scherer). 1875-1768/$ – see front matter ß 2013 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.fsigss.2013.10.179
The QIAsymphony SP automates magnetic bead-based DNA extraction and can directly transfer samples to the QIAsymphony AS, which performs automated reaction setup in a variety of formats. The Rotor-Gene Q is a unique rotary thermal cycler for PCR and real-time PCR. The Investigator Advanced (ADV) protocol incubates the sample, binding buffer, and magnetic beads on the lysis station before mixing on the extractor. It is suitable for 200, 500, and 1000 ml lysate. The High Efficiency (HE) protocol applies an oil overlay to volumes of <100 ml. This prevents any eluate being carried by the magnetic beads upon separation. Software version 4.0 allows the upload of quantification data to the QIAsymphony AS, enabling normalization of STR PCR setup, with the possibility to set the template input to a user-defined value. 2.2. Comparison of new and standard extraction protocols To assess the increase in yield with the ADV protocol, a series of saliva or blood dilutions in 4 replications were processed using the Casework 200 and Casework 200 ADV protocols and eluted in 100 ml buffer ATE. To assess the decrease in purified DNA loss with the HE protocol, 1 ml blood samples were processed using the Casework 500 ADV and Casework 500 ADV/HE protocols in elution
M. Scherer et al. / Forensic Science International: Genetics Supplement Series 4 (2013) e352–e353
e353
Fig. 1. Comparison of the ADV and standard protocols for DNA extraction from forensic casework samples. DNA yield from a series of saliva (A) and blood (B) dilutions.
Fig. 2. Comparison of DNA yields from low elution volumes with and without the HE protocol (oil overlay). The HE protocol resulted in a higher DNA yield with both 30 and 50 ml elution volumes.
Fig. 3. Results from normalized STR setup, showing (A) average peak heights across all 16 ESSplex markers and (B) examples of profiles. 4000 RFU is marked in the example profiles as a reference point.
volumes of 30 and 50 ml. DNA quantification was performed using the QIAGEN Investigator Quantiplex Kit on the Rotor-Gene Q. 2.3. Assessment of normalized STR setup A DNA dilution series from 33 pg/ml to 10 ng/ml was created. Samples were loaded onto the QIAsymphony AS (4 replicates each) and an Investigator ESSplex Plus reaction setup with a target input of 500 pg/reaction was performed. PCR was done on an Applied Biosystems GeneAmp 9700 instrument with Gold-plated 96-well Silver Block and capillary electrophoresis was done with a 3500 Genetic Analyzer.
3.2. Normalized STR setup Fig. 3 shows the comparison of average peak heights across all 16 ESSplex markers and gives two examples of profiles generated from the dilution series. 4. Conclusion The new protocols for the QIAsymphony SP and the latest software version for the QIAsymphony AS are highly suited for processing the types of samples encountered in forensic casework.
3. Results
Role of funding
3.1. Performance of ADV and HE extraction protocols
This research was funded by QIAGEN GmbH and performed on QIAGEN premises in Hilden, Germany.
The ADV protocol gave a higher genomic DNA yield than the standard protocol at all dilutions (Fig. 1). The HE protocol decreased the loss of purified DNA in low-elution volumes when compared to the protocol without the oil overlay (Fig. 2).
Conflict of interest None.
Contents lists available at ScienceDirect
Forensic Science International: Genetics Supplement Series journal homepage: www.elsevier.com/locate/FSIGSS
Processing challenging forensic casework samples with new protocols for the QIAsymphony SP/AS M. Scherer *, B. Alsdorf, M. Breitbach, K. Matthaei, A. Schnur, N. Scholle, L. Bochmann, A. Prochnow, H. Engel QIAGEN GmbH, Hilden, Germany
A R T I C L E I N F O
A B S T R A C T
Article history: Received 2 September 2013 Received in revised form 7 October 2013 Accepted 7 October 2013
Novel extraction protocols for the QIAsymphony SP improve DNA yields from forensic casework samples. The Investigator Advanced protocol has a longer heating step that enhances DNA binding to magnetic particles, while the High Efficiency protocol reduces the loss of purified DNA from low elution volumes. In addition, the latest version of the QIAsymphony AS software enables STR PCR setup normalization based on imported quantification data. ß 2013 Elsevier Ireland Ltd. All rights reserved.
Keywords: Forensic casework DNA extraction STR PCR setup Low-volume elution Oil overlay Quantification data QIAsymphony
1. Introduction
2. Materials and methods
High-throughput instruments can support rapid DNA extraction and precise PCR setup in a whole range of research fields. However, dedicated protocols are crucial for samples for special applications, such as forensic casework. The QIAsymphony is a modular platform for fully automated DNA extraction and PCR plate setup. Two novel extraction protocols optimize its performance with forensic samples. The Investigator Advanced (ADV) protocol has a longer heating step that enhances DNA binding to magnetic particles. It functions with a range of lysate volumes and increases the DNA yield at in all cases. The High Efficiency (HE) protocol improves low-volume elution efficiency by applying an oil overlay to eliminate any dead volumes that would typically reduce the overall yield. The platform also has new software functionality for STR PCR setup normalization based on the results of quantification, allowing automatic adaptation of template amounts per reaction to a user-defined target. Minimum DNA template amounts can also be set, automatically excluding samples from downstream STR analysis if no reportable result is expected. This economical approach requires a sensitive quantification method to minimize the risk of false negatives.
2.1. Instruments
* Corresponding author. E-mail address: [email protected] (M. Scherer). 1875-1768/$ – see front matter ß 2013 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.fsigss.2013.10.179
The QIAsymphony SP automates magnetic bead-based DNA extraction and can directly transfer samples to the QIAsymphony AS, which performs automated reaction setup in a variety of formats. The Rotor-Gene Q is a unique rotary thermal cycler for PCR and real-time PCR. The Investigator Advanced (ADV) protocol incubates the sample, binding buffer, and magnetic beads on the lysis station before mixing on the extractor. It is suitable for 200, 500, and 1000 ml lysate. The High Efficiency (HE) protocol applies an oil overlay to volumes of <100 ml. This prevents any eluate being carried by the magnetic beads upon separation. Software version 4.0 allows the upload of quantification data to the QIAsymphony AS, enabling normalization of STR PCR setup, with the possibility to set the template input to a user-defined value. 2.2. Comparison of new and standard extraction protocols To assess the increase in yield with the ADV protocol, a series of saliva or blood dilutions in 4 replications were processed using the Casework 200 and Casework 200 ADV protocols and eluted in 100 ml buffer ATE. To assess the decrease in purified DNA loss with the HE protocol, 1 ml blood samples were processed using the Casework 500 ADV and Casework 500 ADV/HE protocols in elution
M. Scherer et al. / Forensic Science International: Genetics Supplement Series 4 (2013) e352–e353
e353
Fig. 1. Comparison of the ADV and standard protocols for DNA extraction from forensic casework samples. DNA yield from a series of saliva (A) and blood (B) dilutions.
Fig. 2. Comparison of DNA yields from low elution volumes with and without the HE protocol (oil overlay). The HE protocol resulted in a higher DNA yield with both 30 and 50 ml elution volumes.
Fig. 3. Results from normalized STR setup, showing (A) average peak heights across all 16 ESSplex markers and (B) examples of profiles. 4000 RFU is marked in the example profiles as a reference point.
volumes of 30 and 50 ml. DNA quantification was performed using the QIAGEN Investigator Quantiplex Kit on the Rotor-Gene Q. 2.3. Assessment of normalized STR setup A DNA dilution series from 33 pg/ml to 10 ng/ml was created. Samples were loaded onto the QIAsymphony AS (4 replicates each) and an Investigator ESSplex Plus reaction setup with a target input of 500 pg/reaction was performed. PCR was done on an Applied Biosystems GeneAmp 9700 instrument with Gold-plated 96-well Silver Block and capillary electrophoresis was done with a 3500 Genetic Analyzer.
3.2. Normalized STR setup Fig. 3 shows the comparison of average peak heights across all 16 ESSplex markers and gives two examples of profiles generated from the dilution series. 4. Conclusion The new protocols for the QIAsymphony SP and the latest software version for the QIAsymphony AS are highly suited for processing the types of samples encountered in forensic casework.
3. Results
Role of funding
3.1. Performance of ADV and HE extraction protocols
This research was funded by QIAGEN GmbH and performed on QIAGEN premises in Hilden, Germany.
The ADV protocol gave a higher genomic DNA yield than the standard protocol at all dilutions (Fig. 1). The HE protocol decreased the loss of purified DNA in low-elution volumes when compared to the protocol without the oil overlay (Fig. 2).
Conflict of interest None.