Forensic Science International: Genetics Supplement Series
Volume 2, Issue 1 , Pages 74-76, December 2009

Automated extraction of DNA and PCR setup using a Tecan Freedom EVO® liquid handler

Section of Forensic Genetics, Department of Forensic Medicine, Faculty of Health Sciences, University of Copenhagen, Denmark

Received 8 July 2009; accepted 9 July 2009. published online 01 September 2009.

Article Outline

Abstract 

We have implemented and validated automated methods for DNA extraction and PCR setup developed for a Tecan Freedom EVO® liquid handler mounted with a Te-MagS™ magnetic separation device. The DNA was extracted using the Qiagen MagAttract® DNA Mini M48 kit. The DNA was amplified using AmpFℓSTR® Identifiler®, Y-filer® (Applied Biosystems), GenePrint® FFFL and PowerPlex® Y (Promega). The methods were validated for fresh whole blood and blood from deceased according to EN/ISO 17025.

Keywords: Laboratory automation, Nucleic acid extraction, Magnetic beads, Reference samples

 

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1. Introduction 

Routine extraction of DNA from blood may be performed using a variety of different methods. Traditionally, a manual Chelex® based protocol was used [1]. Recently, several small and simple automated extractors such as Qiagen BioRobot® EZ1 Workstation [2], Invitrogen iPrep™ Magnatrix™ from Magnetic Biosolutions [3] and NucliSENS® easyMAG® from bioMérieux became commercially available offering rapid extraction with limited hands-on time [4]. However, the throughput was limited as they only allowed between 6 and 24 samples to be extracted per run. Larger automated extractors such as Qiagen BioRobot® M48 Workstation [5], KingFisher® Flex and Qiagen QIAsymphony SP allow for extraction of up to 96 samples and hence increased throughput. We have developed and validated an automated extraction method relying on the M48 extraction kit (Qiagen) on a Tecan Freedom EVO® 150/8 automated liquid handler capable of extracting DNA from up to 96 samples including controls and subsequent PCR set up in less than 3.5h.

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2. Materials and methods 

2.1. Samples 

Aliquots of blood were taken from the collection tubes and placed in new sample tubes (1.5mL, Eppendorf GmbH, Hamburg, Germany) at the day of arrival. For samples extracted using the manual Chelex® protocol, 3μL was used while 6μL was used for the automated extraction method on the Tecan Freedom EVO.

2.2. Manual chelex® 

The extraction protocol described previously [1], was modified. In brief, 1mL autoclaved MilliQ filtered water was added to each sample tube containing 3μL blood, followed by mixing for 20s on an IKA Vibrax® VXR shaker (IKA® Works, Inc., Wilmington, NC). Following incubation for 30min at room temperature (RT), samples were mixed again and centrifuged at 13,000 Relative Centrifugal Force (RCF) for 5min in a bench top centrifuge (Eppendorf 5417 C). The supernatant was carefully decanted and discarded. To each sample, 200μL of a 5% Chelex® solution pH>10.5 (Chelex® 100 Resin, 100–200 mesh Na-form, BIO-RAD Laboratories, Richmond, CA) was added. The Chelex® particles were prevented from sedimenting by continuous stirring on a magnetic stirrer. Following a brief mixing step, the samples were incubated for 1h at 56°C in a heating block (Techne Dri-Block, DB-3D, Techne, Cambridge, UK). The samples were mixed vigorously for 20s, incubated for 8min at 100°C, vigorously mixed for 20s and centrifuged at 13,000 RCF for 5min. The supernatant contained approximately 0.5ng/μL extracted DNA and was used for the subsequent PCR [6], [7].

2.3. Tecan freedom EVO 

Sample tubes containing 6μL blood located in the bottom of the tube loaded in 16-tube carriers (Tecan) were inserted on the deck of the robot. Extraction was accomplished using components of the MagAttract DNA Blood Midi M48 Kit (Qiagen GmbH, Hilden, Germany). Reagent volumes were adjusted according to the number of samples being processed. Magnetic bead extraction was performed in lid-less 1.5mL tubes (Sarstedt, Numbrecht Germany) held in 6 by 8 tube racks designed to fit into the Te-MagS™ separation device (Tecan). When more than 48 samples were processed, two tube racks were utilized. One was placed onto the Te-MagS™ while the other was kept in a hotel on the instrument until needed. Following magnetic separation and incubation at 56°C for 5min, the supernatant containing the extracted DNA was transferred to the original sample containing tubes.

2.4. PCR 

PCR master mix was prepared according to the manufacturer's instructions with modifications: For the AmpFlSTR® Identifiler® PCR Amplification Kit, the AmpFℓSTR® Yfiler® PCR Amplification Kit (Applied Biosystems (AB), Foster City, CA), the total reaction volume was reduced to 11μL. For the GenePrint® Fluorescent STR Systems FFFL Multiplex PCR amplification kit and the PowerPlex® Y System PCR amplification kit (Promega, Madison, WI), the total reaction volume was reduced to 9 and 10μL, respectively. For samples extracted by the manual Chelex® method, PCR was performed in individual 0.2mL MicroAmp™ PCR tubes (AB). For samples extracted with the automated method, PCR setup was accomplished using a different script on the Tecan Freedom EVO® allowing addition of additional samples extracted previously or by other procedures. PCR conditions were according to the manufacturer's recommendations and amplifications were performed on Gold-plated 96-well GeneAmp® PCR System 9700 (AB).

2.5. Data analysis 

Multicolor fluorescence capillary electrophoresis of amplified STR profiles was performed on ABI 3130xL Genetic Analyzers (AB). Results were analyzed using Genescan Analysis version 3.7 (AB). Allele calls were made using Genotype version 3.7 (AB) macros.

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3. Results and discussion 

The automated methods replaced a manual Chelex®-100 based extraction protocol and subsequent single-tube PCR setup. The protocol was labor intensive, not easily scalable, limited in throughput and had a risk of sample misplacement. The automated methods enabled extraction of DNA from up to 96 samples including controls and addition of PCR master mix within 3.5h leading to a more than 3-fold, easily scalable increase in throughput. Furthermore, the method returned the DNA containing solutions to the original sample tubes reducing the risk of sample misplacement and eliminating the need for labor intensive number controls. However, this approach increases the risk of introducing PCR inhibiters. Therefore, the original sample tubes were washed with MilliQ water during magnetic extraction.

The quality of the extracted DNA was evaluated by comparing positive controls from 76 different Chelex® based extractions with positive controls from 86 different extractions performed on the Tecan Freedom EVO (Fig. 1). The results show no apparent difference between the two extraction methods. Both methods resulted in equally amplifiable DNA and the balance in terms of signal intensity between the various loci was maintained.

  • View full-size image.
  • Fig. 1. 

    Comparison of fluorescence signal intensities from PCR products using automated and manually extracted DNA as template. The error bars represent the standard error of the mean (SEM).

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4. Conclusion 

We have demonstrated that (1) DNA extraction with magnetic beads and (2) PCR setup for accredited, forensic genetic DNA typing can be implemented on an automated liquid handler leading to reduced risk of sample misplacement, reduction of manual work and an easily scalable throughput without compromising the quality of the extracted DNA. The scripts are freely available.

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Role of funding 

None.

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Conflict of interest 

None.

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Acknowledgement 

None.

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References 

  1. Walsh PS, Metzger DA, Higuchi R. Chelex 100 as a medium for simple extraction of DNA for PCR-based typing from forensic material. Biotechniques. 1991;10:506–513
  2. Anslinger K, Bayer B, Rolf B, Keil W, Eisenmenger W. Application of the BioRobot EZ1 in a forensic laboratory. Leg. Med. (Tokyo). 2005;7:164–168
  3. Valgren C, Wester S, Hansson O. A comparison of three automated DNA purification methods in Forensic casework. Forensic Sci. Int.: Genet. Suppl. Ser. 2008;1:76–77
  4. Dundas N, Leos NK, Mitui M, Revell P, Rogers BB. Comparison of automated nucleic acid extraction methods with manual extraction. J. Mol. Diagn. 2008;10:311–316
  5. Nagy M, Otremba P, Kruger C, Bergner-Greiner S, Anders P, Henske B, et al. Optimization and validation of a fully automated silica-coated magnetic beads purification technology in forensics. Forensic Sci. Int. 2005;152:13–22
  6. Hallenberg C, Morling N. Population study and validation of the Y-STR pentaplex for use in forensic case work. Int. Congress Ser. 2003;1239:379–381
  7. Hallenberg C, Nielsen K, Simonsen B, Sanchez J, Morling N. Y-chromosome STR haplotypes in Danes. Forensic Sci. Int. 2005;155:205–210

PII: S1875-1768(09)00002-X

doi:10.1016/j.fsigss.2009.07.002

Forensic Science International: Genetics Supplement Series
Volume 2, Issue 1 , Pages 74-76, December 2009

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