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

2. DNA Extraction from primary sources may be complex

3. 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

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

5. 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

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

7. 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

8. The Instrument
Online Incubation
Centrifugation
Shaking
Gripper

9. 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

10. Software
Output
Input
Incubators
Buffers
Process Window
Deck Layout

11. 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

12. Process for Animal Tissues I

13. Process for Animal Tissues II

14. 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

15. 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 / (CV 1.12%).

16. 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.

17. 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

18. Plant Tissues: Digestibility1 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

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

20. 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

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

22. 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