Just another DNA extraction system? Liquid handling marries automated Centrifugation Dr. Jürgen Zimmermann Genomic Core Facilities EMBL, Heidelberg 2004/03

DNA Extraction from primary sources may be complex

Laboratory Needs Robust process, which can cope with a broad range of even viscous samples conditions Consistency in DNA yield & DNA quality DNA should be used PCR, restrictions, cloning Flexible hardware should allow downstream applications or foreign processes Fully automated process Expandable Scalability

Laboratory Specs DNA yield: up to 20µg–40µg (animal/plant) DNA size: >30kBp DNA Purity: 1.8-2.0 (OD260/280) PCR, Digests Reproducibility: ~5% CV Speed: ~192samples/2h (Offline Lysis)

Chemical Concepts Beads (Magnetic / Silica) Anion Exchange Chromatography Organic Extractions & Precipitations Contaminant Binding in Filter materials Silica Filters NucleoSpin 96 Tissue HC MACHEREY-NAGEL, Düren NucleoSpin 96 Plant HC MACHEREY-NAGEL, Düren

Automation Concepts Vacuum Chambers Magnetic Bead Separators Semi-automated Approaches Dedicated instruments Liquid handling System & Automated Centrifuge Development based on MPII, PerkinElmer

Current Status: Automated DNA extraction from complex samples Vacuum filtration with limited robustness (clogging, cross-contamination) Magnetic beads with limited performance (time consuming, liquid handling in viscous solution, carryover) Contaminant binding with limited specificity & capacity (time consuming, centrifugation speed limited) Dedicated instruments with limited applicability & flexibility

The Instrument Online Incubation Centrifugation Shaking Gripper

Features Complete Integration (Including sample digestion) Independent operation of centrifuge & liquid handling system Modular Concept Sample throughput 192/h RCF up to 6.300g Open to any chemistry Basket Concept

Software Output Input Incubators Buffers Process Window Deck Layout

Applications DNA extraction from animal tissues (mouse tails, mouse embryos, fish embryos, worms) [available] DNA extraction from plant tissues (various plants and organs) [available] Precipitation & Solubilisation Experiments SPE BAC DNA purification Protein purification Chemical Synthesis

Process for Animal Tissues I

Process for Animal Tissues II

Mouse Tails: DNA Yield & Size DNA extracts from 4 mm mouse tail tips using NucleoSpin 96 Tissue HC, 0.8% agarose gel. 1/15 of the eluate is loaded. l/Hind III marker

Mouse Tails: Quality & Reproducibility Genomic DNA from mouse tail clippings is prepared using NucleoSpin® 96 Tissue HC on Spinner. 2µl of each 1:10 dilution of 16 DNA eluates are used as template in a 20µl amplification reaction (40 cycles) in a LightCycler™ analysis (SYBR® Green). The PCR product generated is 212 bp for the Mus musculus cytoplasmic aconitase exon (acoI). A single amplification product is obtained (see melting curve). The obtained Crossing points for all 16 samples show a mean value of 26.02+/- 0.29 (CV 1.12%).

Cross Contamination Test Mouse tail clipping samples and water are arrayed in adjacent wells of a 96-well plate. Genomic DNA is isolated using NucleoSpin 96 Tissue HC on Spinner. 2µl of each eluate is used for PCR (35 cycles) of Mus musculus cytoplasmic aconitase exon (acoI) (212 Bp). PCR product from 10µl of each PCR assay are separated on a 1 % agarose gel. No PCR product is detected in water samples demonstrating the absence of cross-contamination.

Plant Tissues: DNA Size & Yield Wheat leaf Maize leaf Soybean leaf Sunflower leaf DNA extracts from plants using NucleoSpin 96 Plant HC Sample: 50 mg plant leaf - Elution volume: 150 µl CE- 1 % TAE agarose gel 10µl (wheat, maize) / 15µl (soybean, sunflower) l/Hind III Marker

Plant Tissues: Digestibility 1 2 wheat, leaf M A B maize, leaf sunflower, leaf soybean, leaf DNA isolated from different plant species (digested, undigested) Sample amount: 50mg. Elution volume: 150µl CE. 10µl (wheat / maize) or 15µl (soybean / sunflower) of the eluate were run after digestion with EcoRI (A) or undigested (B) on a 1 % TAE agarose gel. M1: l/HindIII, M2:1 kb ladder

Plant Tissues: PCR maize seed, leaf wheat seed, leaf sunflower seed, leaf soybean seed, leaf Amplification of a 750bp fragment from the nad5 gene

Achieved Technical Objectives Robust application for DNA extraction from various complex sources were developed DNA quality compatible for RT-PCR, restriction digests, southern & cloning Fully Automated system for automated centrifugation, liquid handling, incubation and shaking Liquid handling system and centrifuge can be used independently Integrated multi-utility instrument

Achieved Lab Objectives Reduced hands on time (load deck) No human supervision necessary Increased reproducibility Modularity Multipurpose Solution

Acknowledgements MACHEREY-NAGEL Inheco Hettich EMBLEM PerkinElmer EMBL Thomas Zinn Markus Meusel Ulrich Schübel Inheco Bernhard Loibl Hettich Klaus Günter-Eberle EMBLEM Martin Raditsch Gabor Lamm PerkinElmer Ralf Griebel Marek Turewiz Paul Lomax Martin Long EMBL David Ibberson Leo Burger Wolfgang Dilling Vladimir Benes Christian Boulin