The concept for a semi-automated processing system for DNA analysis of crime scene samples was developed at the Landeskriminalamt Baden-Württemberg (LKA BW) and comprises the extraction of genomic DNA from human cells by ChargeSwitch® magnetic bead technology (CST), quantification of purified DNA by real-time PCR, amplification of short tandem repeats (STRs) by PCR and DNA fragment length analysis of STRs by capillary electrophoresis. Three liquid handling workstations from Tecan, a real-time PCR device and a 16-channel capillary electrophoresis (CE) system, both from Applied Biosystems (AB), are linked via laboratory data network. Transmission and management of sample and analysis data is enabled by a Laboratory Information and Management System (LIMS). Suitability for a wide range of stain types, early exclusion of DNA-free samples, barcode sample identification and prevention of cross-contaminations guarantee efficiency and high quality standards.
As DNA profiling has become one of the most efficient and required tools in criminal investigations, forensic laboratories are challenged with increasing workloads. To step up sample throughput and analysis efficiency, we developed a concept for a semi-automated processing system with a medium throughput of 20,000 samples annually, covering the whole range of biological sample types (e.g. saliva, blood, semen, epithelial cells, buccal swabs). Highest analysis sensitivity and minimum DNA contamination risk should be guaranteed in consideration of economical aspects.
2. Material and methods
Operational steps and hardware components of the DNA analysis automation concept are listed in Table 1. A workflow scheme is shown in Fig. 1. Further details are described elsewhere [
- Haak B.
- et al.
Evaluation of a semi-automated, magnetic bead-based DNA-extraction method for genetic fingerprinting of forensic casework samples.
Forensic Sci. Int. 2008; 1: 35-36
- Schwenzer R.
- et al.
Implementation of a robotized real-time-PCR setup for the use of the AB Quantifiler™ Human DNA Quantification Kit.
Forensic Sci. Int. 2008; 1: 68-70
Table 1Operational steps and hardware components of the DNA analysis automation concept
|Operational steps||Hardware components||Applications|
|DNA extraction||Tecan Freedom EVO® 150 liquid handling workstation I||ChargeSwitch® Technology (Invitrogen)|
|Real-time PCR setup||Tecan Freedom EVO® 150 liquid handling workstation II||Quantifiler™ Human DNA Quantification Kit (AB)|
|Real-time PCR||AB 7500 Real-Time PCR System||SDS v1.2.3 (AB)|
|Normalization/negative selection||Special created Software tool (Tecan, ERL)|
|STR PCR setup||Tecan Freedom EVO® 150 liquid handling workstation II||Kits of various manufacturers|
|STR PCR||AB 9700 GeneAmp PCR System|
|Amplification product pipeting/preparation for CE/rearrangement||Tecan MiniPrep™ 75 liquid handling workstation III||Special created Software tool (Tecan, ERL)|
|Fragment analysis||AB Prism 3130xl Genetic Analyzer||Data Collection Software v3.0 (AB)|
|Data analysis||Genemapper® ID S v3.2 (AB)|
Sample identification by integrated barcode reading is performed with reference to work lists provided by the SQL server-based LIMS. Without ionic chaotropes (potential PCR inhibitors) and offering long-term stability of eluted DNA (double-stranded conformation), CST magnetic bead extraction of DNA is promising, especially for efficient STR analysis. The full range of different crime scene samples is covered by our CST protocol. The single tube format used for DNA extraction allows to work with samples of relatively large sizes and to handle the purified DNA individually further on. Besides the magnetic mixing function of the TeMagS module specially programmed travel pathways of the robots’ liquid channel arms represent an essential measure to avoid cross-contaminations. Quantification data is used to normalize template DNA for STR PCR. Unpromising concentrations of human DNA below an empirically determined cutoff value are routinely not submitted to STR PCR amplification. The sample preparation for CE comprises a rearrangement of the plate positions of individual amplification products to avoid CE runs without PCR products. The complete analysis process from cell lysates to STR results is accomplished within 15 h (Table 2).
Table 2Time flow of the semi-automated process for DNA profiling of forensic casework samples
|Time flow (bulk of 88 samples)|
|Manual separation of lysates from stain carriers||50 min|
|CST™ extraction + transfer to plate (Tecan I)||2 h|
|Real-time PCR setup (Tecan II)||30 min|
|Real-time PCR (AB 7500)||1 h 45 min|
|Evaluation of Real-time PCR data||20 min|
|STR PCR setup (Tecan II)||45 min|
|STR PCR (AB 9700)||3 h 30 min|
|CE sample preparation (Tecan III)||1 h|
|CE (AB 3130xl)||4 h|
|Total||14 h 40 min|
To cope with the increasing demand for forensic DNA analysis capacity we established a semi-automated system for DNA profiling that was adapted to our specific demands and is now performing routinely the efficient, contamination free and high quality DNA analysis of diverse biological samples for routine casework at the LKA BW. To avoid errors by human failure, barcode sample identification and convenient transmission and management of sample and analysis data via LIMS were established. Our measures, especially the early pick out of DNA-free samples, were found to minimize significantly analysis time and costs. As a result personal capacity was set free for other time-consuming and labour-intense tasks like the microscopic preparation of perpetrators’ skin particles from relevant casework objects. Next, analysis data should be incorporated automatically via LIMS into reports and expertises.
Conflict of interest
Support by all co-workers of the LKA BW for their contributions and especially the expert support of Carsten Aprill (Field Automation Specialist from Tecan Germany, Crailsheim) and Ernst Radda (ERL, Vienna; Tecan) is greatly acknowledged.
- Evaluation of a semi-automated, magnetic bead-based DNA-extraction method for genetic fingerprinting of forensic casework samples.Forensic Sci. Int. 2008; 1: 35-36
- Implementation of a robotized real-time-PCR setup for the use of the AB Quantifiler™ Human DNA Quantification Kit.Forensic Sci. Int. 2008; 1: 68-70
Accepted: November 7, 2007
Received in revised form: October 19, 2007
Received: August 24, 2007
© 2008 Elsevier Ireland Ltd. Published by Elsevier Inc. All rights reserved.