Just another DNA extraction system?

Liquid handling marries automated Centrifugation

Dr. Jürgen ZimmermannGenomic Core Facilities EMBL, Heidelberg

2004/03

DNA Extraction from primary sources may be complex

Laboratory NeedsRobust 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% CVSpeed: ~192samples/2h (Offline Lysis)

Chemical Concepts

Beads (Magnetic / Silica)Anion Exchange ChromatographyOrganic Extractions & PrecipitationsContaminant Binding in Filter materialsSilica Filters

NucleoSpin 96 Tissue HC MACHEREY-NAGEL, DürenNucleoSpin 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

Shaking

Centrifugation

Gripper

FeaturesComplete Integration(Including sample digestion)Independent operation of centrifuge & liquid handling systemModular ConceptSample throughput 192/hRCF up to 6.300gOpen to any chemistryBasket Concept

Software

Process Window Deck Layout

Output Input

Incubators

Buffers

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 ExperimentsSPEBAC DNA purificationProtein purificationChemical 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. /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 & YieldWheat leafMaize leaf

Soybean leaf Sunflower leaf

DNA extracts from plants using NucleoSpin 96 Plant HCSample: 50 mg plant leaf - Elution volume: 150 µl CE- 1 % TAE agarose gel10µl (wheat, maize) / 15µl (soybean, sunflower) /Hind III Marker

Plant Tissues: Digestibility

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: /HindIII, M2:1 kb ladder

1 2

wheat, leaf

M A B A B

maize, leaf sunflower, leaf

M A B A B

soybean, leaf

1 2

Plant Tissues: PCRmaize 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 developedDNA quality compatible for RT-PCR, restriction digests, southern & cloningFully Automated system for automated centrifugation, liquid handling, incubation and shakingLiquid handling system and centrifuge can be used independentlyIntegrated multi-utility instrument

Achieved Lab Objectives

Reduced hands on time (load deck)

No human supervision necessary

Increased reproducibility

Modularity

Multipurpose Solution

Acknowledgements

MACHEREY-NAGEL 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