nucleic acid extraction automation overview (early 2011)

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Nucleic Acid Extraction Automation in Molecular Diagnostic part 1 Patrick Merel Biomedical Innovation Platform (PTIB), Pessac, France [email protected] 1 1

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An overview of Nucleic Acid Extraction procedures and automation by Patrick Merel, University Hospital of Bordeaux, France.

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Page 1: Nucleic Acid Extraction Automation Overview (early 2011)

Nucleic Acid Extraction Automationin Molecular Diagnostic

part 1

Patrick MerelBiomedical Innovation Platform (PTIB), Pessac, France

[email protected]

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Page 2: Nucleic Acid Extraction Automation Overview (early 2011)

DNA and RNA from...

Blood, Plasma, Cells … Body Fluids

Nucleic Acid (NA) extraction in molecular diagnostic

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Quality of DNA/RNA is crucial for the PCR, RT/PCR steps

Extraction procedures often require centrifugation or filtration

Which are difficult steps to automate

Or, which require complex instrumentation

However, there are now numerous options for automation of NA extraction steps

Semi-automated protocols (microtiterplate based)

Automated protocols on robotic workstations

Dedicated and automatic workstations

But first, let’s go shopping for the perfect NA extraction technology...

NA Extraction Steps Automation

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Organic extraction is a conventional technique that uses organic solvents to extract contaminants from cell lysates.

The cells are lysed using a detergent, and then mixed with phenol, chloroform, and isoamyl alcohol.

The correct salt concentration and pH must be used during extraction to ensure that contaminants are separated into the organic phase and that DNA remains in the aqueous phase.

DNA is usually recovered from the aqueous phase by alcohol precipitation.

NA organic extraction

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NA Phenol-chloroform extraction

Equal volumes of a phenol:chloroform mixture and the aqueous DNA sample are mixed, forming a biphasic mixture.

The proteins will partition into the organic phase while the DNA (as well as other contaminants such as salts, sugars, etc.) remain in the aqueous phase.

It is then followed by precipitation with ethanol, as ethanol precipitates the nucleic acid.

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Nucleic Acid Extraction by centrifugation

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DNA extraction by Salting-Out

•Salting-out is another conventional technique where proteins and other contaminants are precipitated from the cell lysate using high concentrations of salt such as potassium acetate or ammonium acetate.

•The precipitates are removed by centrifugation, and the DNA is recovered by alcohol precipitation.

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Most nucleic acid purification techniques that use silica in combination with chaotropic reagents such as guanidine hydrochloride are covered by U.S. Patent No. 5,234,809, European Patent No. 0,389,063, and equivalents in other countries. It is commonly known as the "Boom" patents and is owned

by Biomerieux.

DNA Separation by Silica Adsorption

•This method is based on the selective adsorption of nucleic acids to a silica-gel membrane in the presence of high concentrations of chaotropic salts.

•Use of optimized buffers in the lysis procedure ensures that only DNA is adsorbed while cellular proteins, and metabolites remain in solution and are subsequently washed away.

•Ready-to-use DNA is then eluted from the silica-gel membrane using a low-salt buffer.

•No alcohol precipitation is required but filtration or combination of filtration+centrifugation procedures are needed.

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For DNA

or RNA

Silica beads based NA extraction

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Mainly single tube based,but various options for

microtiterplate processing are now available

Centrifugation + filtration NA extraction

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Filtration + centrifugation NA extraction

Filtration only procedures available for single unit processing, and

microtiter plate based products for robotic handling.

Most of the time filtration procedures end with a centrifugation

step.

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Magnetic beads based NA extraction

High quality nucleic acids

Scalable

No centrifugation

No vacuum steps

Rapid purification

Reproducible12

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Dynal Dynabeads Technology

•Dynabeads a re super pa ramagne t i c , monosized polymer particles, providing a consistent, defined surface for the adsorbtion or coupling of various bioreactive molecules.

•The beads are magnetisable and super paramagnetic, meaning they are only magnetic in a magnetic field.•Due to this property, the beads can easily be resuspended when the magnetic field is removed. •The technology behind the beads was licensed to Dyno Industrier in 1980, which later formed Dynal Biotech together with Apothekernes Laboratorium in 1986.

Scanning Electron Microscopic pictures of the monosized Dynabeads® (A) and alternative

magnetisable particles from other suppliers (B-E). Courtesy of Mikal Heldal, University of Bergen,

Norway. 13

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Page 14: Nucleic Acid Extraction Automation Overview (early 2011)

๏Lysis and binding 5+5 min

๏Wash, Rinse 3x1 min

๏Elution 30 s RT

๏or 5 min 65C

๏Total @ RT <15 min

•Chaotropic soln (GTC) + silica magnetic particles

•Chaotropic solution, removes non-bound matter

•EtOH, removes GTC

•Water, removes EtOH

•Water or low salt buffer

Dynal Dynabeads Procedure

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Page 15: Nucleic Acid Extraction Automation Overview (early 2011)

Agencourt SPRI Technology

•The Agencourt SPRI (Solid Phase Reversible Immobilization) technology is a patented purification technology developed at MIT's Whitehead Institute (Hawkins, et. al., Nucleic Acids Res. 1995 (23): 4742-4743).

•Nucleic acids are immobilized onto paramagnetic microparticles (1µM magnetic carboxylate-modified base particles) using specific buffer conditions. Flexibility is added to the system through modification of the binding buffer, which alters the type and size of immobilized nucleic acid.

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Agencourt SPRI Technology

Lysis 30 min RTBind 5 min

Capture 15 minWashing1 2x10 minWashing2 2x6 min

Capture+Elution 2+10 min

Total @ RT 100 min16

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Magnetic Extraction Pros and Cons

Pros•Usually very fast procedures

•Robotic friendly•Rapid growing segment for instrumentation

•Cheaper ?•Potential for specific extraction

Cons•Manipulation of small volume samples

•For small to medium quantity of DNA•Magnetic stand/plate needed•Usually generic extraction

•Not all procedures equal in regards of time requirement

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Page 18: Nucleic Acid Extraction Automation Overview (early 2011)

NA extraction by hybridization

Different companies have developed over the past years, specific hybridization capture procedures:

RNAture (Hitachi Chemical), Xtrana, ProLinx, Gen-Probe, DNA Research Innovation (DRI now part of Invitrogen/Life Technologies) have been the most visible.

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Gen-Probe Target Capture Technology

Target Capture•Maximizes assay sensitivity & specificity. •RNA target is “captured” with magnetic beads.•Eliminates inhibitors in swab and urine samples.

•Allows for simplified specimen processing.•Patented process with specific probes coated magnetic particles.

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How it works ChargeSwitch Technology provides a switchable surface charge that is dependent on the pH of surrounding buffer to purify nucleic acids (Figure 1). In conditions of pH < 6.5, the CST surface has a positive charge that selectively binds the negatively charged nucleic acid backbone. Proteins and other contaminants are removed in an aqueous wash buffer. For elution, the charge on the CST surface is switched off by raising the pH to 8.5. Purified nucleic acid elutes instantly and is ready for downstream use.

Advantages of the ChargeSwitch Technology • Novel, pH dependent switch action • Avoids introduction of ethanol, organic solvents or chaotropic salts • Improved downstream performance for high specification applications • Unmatched high sensitivity for micro samples

Invitrogen ChargeSwitch Technology

ChargeSwitch gDNA50-100 µl

Lysis 1 min RTCapture 1 min

Washing 3x1 minElution 1+4 min

Total @ RT 10 min

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Very recent new approach for NA extraction

Enzyme based NA extraction

DNA extraction using a thermostable proteinase.…hands off, closed tube and all you need is a thermal cycler.

WorkFlow

automated on the epMotion 5075MC

Zygem: PrepGEMand ForensicGEM

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Very recent new approach for NA extraction

Boreal Genomics instruments are based on SCODA: a novel, proprietary electrophoresis technology that selectively concentrates nucleic acids from large sample volumes to a small, concentrated amount of gel or buffer. Advantages:

•Up to 100X more efficient for contaminant rejection•High yield even in very dilute samples•Simple operation•Target length selection during extraction•Protection of high molecular weight targets•Potential for sequence specific extraction including short RNA/DNA fragments

This is accomplished by means of rotating electric fields that act uniquely on long, charged polymers and leave other molecules unaffected.

1x5mL or 4x1 mL input

• Target run times < 30 min

• Digital high voltage control

• Unattended operation

• Lysate to purified sample in one step

• Extraction in 10 – 50 uL of buffer

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NA extraction Automation: why?

Time consuming step in MDxSample throughput

Hand on timeReproducibility

StandardizationCost

Instrumentation availability

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NA extraction automation options

✓Microtiter Plate based Procedures

✓Generic Robotic Instrumentation

✓Dedicated Robotic Instrumentation

✓In Vitro Diagnostic Robotic Instrumentation

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Microtiterplate based NA extraction

•Qiagen and Macherey-Nagel have been pioneers in providing DNA extraction protocols on 96 well-plates

Qiagen, http://www.qiagen.com

DNAeasy, RNAeasy, QIAamp, QIAfilter

Macherey-Nagel, http://www.macherey-nagel.com

NucleoBond, NucleoSpin, NucleoTrap, Multi-96

•Additional companies providing 96 well-plate compatible kits for DNA/RNA extraction:

Promega, BioLine, Stratagene, Millipore, Ambion, Invitrogen…

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High Throughput DNA extraction with Macherey-Nagel

The NucleoSpin Multi96 blood kit from macherey-Nagel allows rapid parallel DNA extraction from blood, serum, plasma, cell cultures, buffy coats.

Features:

silica membrane technology

complete removal of PCR inhibitors

no organic solvents

200 µl whole blood or 107 lympho. or cultured cells can be processed

Simultaneous processing of 192 samples

2 x 96 well plates

or

n x 8 strips on a 96 deep-well block

Processing time >2hrs

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Microtiterplate compatible viral DNA extraction

Bench top centrifuge for Nucleospin multi 96

DNA extraction.

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HighThroughput DNA extraction from centrifugation to filtration

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High Throughput DNA extraction from centrifugation to filtration

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Semi-Automation of the QIAamp 96 Spin Blood Kit on the Biomek 2000

Setup of the Beckman Coulter Biomek 2000

Tablet

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Qiagen QIAamp 96 Spin Blood Kit setup on the Biomek 2000

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Centrifugation of the Filtration Unit + Disposable Deep-Well

Semi-Automation of the QIAamp 96 Spin Blood Kit on the Biomek 2000

Centrifugation of the Filtration Unit + Storage Deep-Well

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NA extraction automation options

✓Microtiter Plate based Procedures

✓Generic Robotic Instrumentation

✓Dedicated Robotic Instrumentation

✓In Vitro Diagnostic Robotic Instrumentation

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Generic Robotic Instrumentation

✓Develop your own procedure

based on manual protocols

some are robotic friendly

✓Follow reagents providers recommendations

✓Follow robotic instrument providers recommendations

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Generic Robotic Workstations with available molecular testing applications.

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Company Instrument IVD-D or CE-IVD compliant; comments

NA extraction applications

available

PCR applications available

Web Address

Agilent Automation Solutions

Biocell, Bravo, BenchCel

No; very high throughput Yes Yes chem.agilent.com/

Aurora Instruments Versa serie, Versa mini no Yes Yes aurora-instr.com/

Beckman Coulter, Inc. Biomek 2000,3000Biomek NX,FX No Yes Yes beckmancoulter.com/Beckman Coulter, Inc. Biomek 2000,3000Biomek NX,FX No Yes Yes beckmancoulter.com/

Caliper LifeSciences

SciClone ALH3000 No Yes Yes

caliperls.com/Caliper LifeSciences

SciClone ALH3000 No Yes Yes

caliperls.com/Caliper LifeSciences Zephyr No Yes Yes caliperls.com/Caliper LifeSciences

Twister II+AB7900 No No Yes

caliperls.com/

Cybio CyBi-Dilus, -RoboSpense No Yes Yes cybio-ag.com/

EppendorfepMotion 5070

No; MC with integrated PCR Yes Yes eppendorf.com/Eppendorf epMotion 5075

LH, VAC, MC, TMX

No; MC with integrated PCR Yes Yes eppendorf.com/

Hamilton STAR, STARlet, Nimbus

Yes; NAT pooling system Yes Yes hamiltoncomp.com/

Labcorp-Protedyne BioCube, Radius No; very high throughput Yes Yes protedyne.com/

Perkin-Elmer Janus CS Autoplex, Forensic No Yes Yes las.perkinelmer.com/

Tecan Freedom Evo Yes Yes Yes tecan.com/

ThermoCRS CRS DNA No; very high throughput Yes Yes thermo.com/

Xiril Xiril 75, 100, 150 No Yes Yes xiril.com/

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ThermoCRS Extraction platforms

High throughput (up to 14, 96-well microplates/hour)Proven reliabilityUses well-established DNA purification protocolsAdaptable to any magnetic bead basedseparation methodUnattended 24 hour operation and remote monitoringPOLARA™ Open Architecture Laboratory Automation Software

The CRS DNA Purification System is an automation tool for beadbased methods using magnetic particles. This includes binding of DNA

to magnetic beads, washing of the bound material, elution from the solidphase and transfer to a final destination plate.

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Agilent Automation Solutions-Bravo and BioCel

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BioCel

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Agilent Automation Solutions: Bravo and BenchCel

Bravo

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A flexible workstation providing high-throughput gDNA isolation

• Up to 10 microplates can be processed in one run without user intervention using the Invitrogen Charge Switch EasyPlex procedure• Microplate processing time is approximately 10 min per microplate (excluding incubation time ~ 30min)

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In May 2007, Protedyne became a wholly owned subsidiary of Laboratory Corporation of America.

Protedyne BioCube Platforms

Protedyne BioCubes

LX, SX, EX

Performance: Approximately 7,000 DNA samples can be prepared per eight-hour shift using a Protedyne 96-well,

vacuum-based plasmid DNA purification procedure.

Also, QiaAmp Genomic DNA on the BioCube: 14 plates per 8

hour shift

Protedyne Radius

Protedynes bench-top robotic system

a protocol for the Invitrogen ChargeSwitch EasyPlex gDNA kit using the gDNA isolation workstation in which up to 10 microplates (96-well) can be processed simultaneously.

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Hamilton Robotics Platforms

STARlet STAR STARplus

MICROLAB 4000 MICROLAB 4200

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Nimbus

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Tecan Extraction Platforms

The MiniPrep , f rom Drug Discovery (mother - daughter plate replication), to Molecular Biology (Sequencing preparation, PCR a m p l i fi c a t i o n a s s a y s ) a n d Diagnostics (Immunophenotyping, ELISA Assays).

The Genesys serie with four or eight tip arm for high throughput. NA extraction automation has been fully automated using the magnetic separator MagS and solid phase extraction through columns. The extraction is usually performed in 96 well cartridge blocks. These are placed on vacuum chambers on the instrument worktable and can be evacuated by software controlled vacuum pump VacS.

The Freedom Evo family from 1m to 2m deck size. A special mention for the IVD-D compliant Freedom Evo Clinical.

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Invitrogen reagents automation and large volumes procedures

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Xiril Robotic Platforms

X75 X100

Xiril Robotic Workstations have a 1-tip, 2-tip or 4-tip liquid handling arm with selectable and exchangeable pump volumes. Each pump is equipped with a tip adaptor that fits disposable tips with or without filters.

For added flexibility and increased sample throughput, the Xiril X100 and X150 can be configured with an additional liquid handling arm for specific Life Science applications. Macherey, Agencourt, Promega application notes available.

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Beckman Coulter Biomek Platforms

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Beckman Coulter NX and Biomek Platforms

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Robotic Workstation Industrials “Partners”

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Beckman Coulter Hamilton Thermo-Jouan- CRS Robotics

PerkinElmer Tecan Self-Contained units

Biomek 2000, Biomek 3000 MicroLab, Starplus, Starlet

CRS DNA Purification System

MultiProbe IIEX, DNAtrack, Janus

Genesis RSP 100, 150, 200Evo

Agilent Biocel, Bravo, Protedyne BioCube

Biomek FX, Biomek NX AutoSprint MiniPrep 60 MolBio

Multimek

Promega, Xtrana, Agencourt, Qiagen

Millipore, Agowa, Roche BilaTec Agencourt, Agowa, Macherey-Nagel,

Promega

Qiagen, Macherey-Nagel, Agowa

Various, Invitrogen

Filtration, Magnetic, hybridation

Filtration, purification Magnetic Filtration, Magnetic Filtration, Magnetic all types

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Robotic friendly NA extraction reagents

๏ Automated Protocols

๏ Automated SV 96 Total RNA Isolation System Protocol

๏ Automated Wizard® Magnetic 96 DNA Plant System Protocol

๏ Automated Wizard® SV 96 Genomic DNA Purification System Protocol

๏ Automated Wizard® SV 96 PCR Clean-Up System Protocol

๏ Automated Wizard® MagneSil™ 384-Well PCR Clean-Up System Protocol

๏ Automated Wizard® MagneSil™ 384-Well Sequencing Reaction Clean-Up System Protocol

๏ Automated Wizard® MagneSil® 96-Well PCR Clean-Up System Protocol

๏ Automated Wizard® Magnetic 96 DNA Plant System: Scaled Up Protocol for Increased DNA Yield

๏ Automated Wizard® MagneSil® Sequencing Reaction Clean-Up System Protocol for 96-Well Plates

๏ Automated MagneSil® ONE, Fixed Yield Plant Genomic DNA Purification Protocol

๏ MagneSil® KF, Genomic System

Example of Promega’s reagents

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Page 48: Nucleic Acid Extraction Automation Overview (early 2011)

NA extraction automation options

✓Microtiter Plate based Procedures

✓Generic Robotic Instrumentation

✓Dedicated Robotic Instrumentation

✓In Vitro Diagnostic Robotic Instrumentation

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Dedicated Workstations For NA extraction 1/4

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Applied BioSystemsApplied BioSystems

AutogenAutogenAutogenAutogen

Beckman Coulter, Inc.Beckman Coulter, Inc.

Bee RoboticsBioer

BiomérieuxBiomérieux

Bioneer

ABI Prism 6100 Filtration 96 96/30min jusqu’à 1m de cellules

ARN, ADN appliedbiosystems.com

ABI Prism 6700 Filtration; archivage, préparation PCR

96 96/75min jusqu’à 1m de cellules

ARN, ADN

appliedbiosystems.com

AutoGenflex 3000 Centrifugation 48 48/4-6hr jusqu’à 5mL ADN, ARN, plasmides, cosmides

autogen.com

AutoGenPrep 2000

Centrifugation

48 48/4-6hr 1mL ADN, tissus, plasmides, cosmides, levures, plantes

autogen.com

AutoGenPrep 245

Centrifugation

24 24/3.5hr 700µL ARN, ADN, tissus, plasmides

autogen.com

AutoGenPrep 965

Centrifugation

4x96 384/4-6hr Jusqu’à 250µL ou 1mL

ADN, plasmides, cosmides

autogen.com

Vidiera NsP Filtration; quantification, normalisation, préparation PCR

96 96/2.5hr Jusqu’à 300µL ADN, ADN et ARN viral

beckman.com

SPRI-TE Magnétique/Magtration; cartouches de réactifs

10 10/30min NS ADN

beckman.com

QuatroProbe Magnétique 48 NS 200µL à 1mL NS beerobotics.com

Gene-Pure NPA-32 Magnétique/Magnetic rod

32 NS 50 à 800µl ADN, ARN bioer.com.cn

Extractor Méthode de Boom 10 120/8hr ADN 100µL, jusqu’à 2mL

ADN, ARN biomerieux.com

EasyMag Magnétique/Méthode de Boom

24 60min Jusqu’à 1mL

biomerieux.com

HT-Prep Pro Filtration 96 60min Jusqu’à 300µL ADN, ARN bioneer.co.kr

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Dedicated Workstations For NA extraction 2/4

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ChemagenChemagen

Corbett LS part of QiagenEppendorf

FujiFilmFujiFilmFujiFilm

GeneSystemsGenomic

HandyLab part of BD

Invitrogen

Magnetic Biosolutions AB

Mole

NordiagNordiag

Promega

Chemagic Magnétique/Magnetic rod

12, 96 150/8h 10mL (45min/12), 4000/8h 1.5mL (15min/96)

10µL à 10mL DNA, RNA, RNAv, mRNA

chemagen.de

Prepito

Magnétique/Magnetic rod

1-12 30min 10µL à 600µL

DNA, RNA, RNAv, mRNA

chemagen.de

QIAxtractor FIltration 8-96 8/45min, 96/96min 180 à 200µL DNA, RNA, RNAv, mRNA

corbettlifescience.com

epMotion 5075 VA, TMX

Filtration 1-96 NS NS DNA, RNA, tissus, plasmides

eppendorf.com

QuickGene-610 Filtration sur Membrane poreuse de 80µM

6 12min 2mL ADN fujifilm.com

QuickGene-810Filtration sur Membrane poreuse de 80µM

8 6min 200µl ADN, ARN, tissus, plasmides

QuickGene-mini80

Filtration sur Membrane poreuse de 80µM

8 6min (manuel) 200µl ADN, ARN, tissus, plasmides, cellules

GeneExtract Filtration 6 Semi-automatique > 1L de liquide ADN genesystems.fr

ExtraGene Centrifugation 48 240/jour 0.5 à 10mL ADN, ARN, Proteines genomics-tools.com

Lynx Magnétique; cartouches de réactifs

24 35min NS ADN, ARN handylab.com

iPrep Magnétique/Magtration; cartouches de réactifs

12 26min 350µL ADN invitrogen.com

Magnatrix 1200 Magnétique/Magnatrix 12-96 20min/12 NS ADN, ARN magbio.com

Magnatrix 8000plus

Magnétique/Magnatrix

8-192 NS NS

GeneMole Magnétique; cartouches de réactifs

16 NS 50µL à 1mL ADN, ARN, cellules, tissus

molegenetics.com

Arrow Magnetic, Bug’s and Beads

1-12 30min 200-500µL DNA, RNA nordiag.com

Bullet 1-96 1.5hr 20 à 200µL DNA, RNA

nordiag.com

Maxwell 16 Magnétique/Magnetic rod; cartouches de réactifs

16 30min 50 à 400µL ADN www.promega.com

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Dedicated Workstations For NA extraction 3/4

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Precision System SciencePrecision System SciencePrecision System SciencePrecision System SciencePrecision System SciencePrecision System Science

QiagenQiagenQiagenQiagenQiagenQiagenQiagen

Mag. Syst. 6Mx Magnétique/Magtration; cartouches de réactifs

6-48 NS 100 à 1000µl ADN, ARN pssbio.com

Mag. Syst. 8Lx Magnétique/Magtration; cartouches de réactifs

1-8 50min 1-10mL ADN

pssbio.com

Magt. Syst.12GC Magnétique/Magtration; cartouches de réactifs

1-12 30min 100 à 200µL ADN, ARN, ADN et ARN viral, tissus, plasmides

pssbio.com

SX-6G Magnétique/Magtration 48 80min 25 à 500µL ADN, ARN

pssbio.com

SX-8G Magnétique/Magtration 96 80min 5 à 40µL ADN, ARN

pssbio.com

SX-96GC Magnétique/Magtration 4x96 NS 10 à 40µL ADN, ARN

pssbio.com

Mag. Syst.Ba-X Magnétique/Magtration 1-3 0.2ml,1ml, 3ml ADN, ARN

BioRobot Universal SystemFiltration + centrifugation + Magnétique; PCR setup

4x96 4x96/2-3hr jusqu’à 200µL ADN, ADN et ARN viral, plasmides

qiagen.com

BioRobot MDX DSP** Filtration + centrifugation + Magnétique ; PCR setup; CE IVD-D

4x96 4x96/2-3hr jusqu’à 200µL ADN, ADN et ARN viral

qiagen.com

EZ1, EZ1 advanced, EZ1 advanced XL

Magnétique/Magtration; cartouches de réactifs; CE-IVD-D

6, 6, 14 20min 200 à 350µL ADN, ADN et ARN viral, tissus

qiagen.com

BioRobot 48/96 Magnétique/Magtration 0,5 48/2hr 100 à 300µL ADN, ADN/ARN, ARN, mARN

qiagen.com

BioSprint 15/96 Magnétique/Magnetic rod

0,15625 20min 10µL à 1mL ADN, ADN/ARN, ARN, mARN

qiagen.com

AutoPure LS Centrifugation 16 96/jour Jusqu’à 10mL ADN

qiagen.com

QIACube Filtration + centrifugation

12 65min jusqu’à 200µL ADN, ADN et ARN viral, plasmid

qiagen.com

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Qiagen QIAsymphonySP, RGQ Magnetic, Magnetic rod 300 LBC/16hrs; 1-96;

1-72 2hrs up to 1ml DNA, RNA qiagen.com/

Dedicated Workstations For NA extraction 4/4

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Roche DiagnosticsRoche DiagnosticsRoche Diagnostics

Thermo LabSystemsThermo LabSystems

MagNAPure 2.0/96 Magnetic, Magtration; PCR prep

32/96 2hr/1hr 20 à 300µL DNA, RNA, DNA/RNA, mRNA

roche-applied-science.com

MagNAPure Compact SystemMagnetic, Magtration; cartridge

8 20min 100µL à 1mL

DNA, RNA, DNA/RNA, mRNA

roche-applied-science.com

AmpliPrep Magnetic,PCR prep, CE IVD-D, continuous flow

1-72 24/1hr 100 à 750µL DNA, RNAv

roche-applied-science.com

KingFisher Magnetic,Magnetic rod 2x12 24/17min, 29min, 41min

20 à 200µL DNA, RNA, mRNA, proteins, cells

thermo.com/

KingFisher ML/96

Magnetic,Magnetic rod

15-96 15/14min, 36min, 43min

10µL à 1mL

DNA, RNA, mRNA, proteins, cells

thermo.com/

QIAEnsembleSP Magnetic, Magnetic rod 3000 LBC 16hrs

LBC, Urine Vials, PreservCyt, surepath Vials, ETU tubes

DNA qiagen.com/

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A closed system for the process of 8 samples / 4 hrs.

A precursor in dedicated workstation

The ABI 340 extractor

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From bench to automation

Phenol Chloroform extraction & Alcohol precipitation made automatic

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Automated Platforms 1

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Centrifugation Based

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AutoGen Centrifugation based Workstations

AutoGen extractors

AutoGenFlex Star

AutoGen 960-965 (4x96 samples in 4hrs)

Gene Prep

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Autogen - 960-965 Extractors

1. Processing Method: • Single phase organic extraction2. Protocols: DNA extraction from whole blood, blood spots, human tissues and cells (including ES cells), plasmids, BACs, microbes and plants. 3. Sample Volume:• Up to 1.8 mls per well4. Maximum Sample Throughput:• 384 (96 deep well plate x 4) per run5. Processing Time:• Four hours for 4 plates

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AutoGen - AutoGenFlex StarAn AutoGen and QIAGEN Collaboration

High precision gripper-type robot hand

Transports sample tubes to and from workstation, dispensing station and centrifuge

Removable sample and DNA storage racks

Heater to allow for efficient digesting of the sample and drying of purified DNA

-4-position swing type buckets-Variable speed and duration settings-HEPA-Filter for biohazard reduction

A u t o G e n F l e x i s c o m p l e t e l y automated and easy to use, with no manual intervention required. Isolate up to 80 x 10ml whole blood samples per day

58Qiagen's proven FlexiGene chemistry

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AutoGen - Gene PREP

•Automates entire extraction process including on board digestion.•Capable of extraction from multiple sample types.•Low cost per prep using AutoGen’s reliable solution phase extraction chemistry.•Higher throughput based on new design which shortens processing time.•Produces high quality and high yields of DNA.•Optional tube stacker allows for unattended processing of up to 192 samples.

48 samples in 2 hours (faster time to result).Direct application of extracted nucleic acid to your next procedure (saves labor and time).

The GENE PREP is the latest fully automated DNA extraction system from AutoGen.

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On-Board Centrifuge and XYZ Robot

Heating ShakerOptional Tube Unit Stacker

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Gentra/Qiagen AUTOPURE LS

Complete automation of process for large volume samples

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Genomic ExtraGene Platform

ExtraGene is a multi-protocol platform based on centrifugation.

ExtraGene process up to 48 samples in 20 ml tubes. The running time depends on the protocol.

Up to two improved centrifuges, with a mechanized hatch on the top

The robotic arm moves a special tool for tubes cap opening, closing, tube handling, nozzles for syringe pumps and precision peristaltic pumps

On the front side, a drawer supports tubes racks, tips racks and liquid and solid waste collector

ExtraGene is contained in a fume-hod (optional)

Generic reagents = Much more extractions with your budget

Full automation = Much more extractions with your work force

Separation by centrifugation = Higher yeld and lower cost (less than 1.5€ / extraction)

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genomics-tools.com

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Automated Platforms 2

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Filtration Based

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Qiagen Extraction Platforms

BioRobot 3000

BioRobot 9604

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Filtration based platforms

The BioRobot 8000

The BRmdx

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Filtration based platform on a centrifuge

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The QIAcube process QIAGEN spin columns, enabling seamless integration of automated, low-throughput sample prep into your laboratory workflow. No change of purification chemistry is required, assuring fast startup and immediate results. All steps in the purification procedure are fully automated – including sample lysis – and up to 12 samples can be processed per run.

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FujiFilm QuickGene 810

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Complete Automation with the QuickGene HT

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Beckman Coulter Inc. Vidiera NsP

Primary tubes on racks

NA extraction by filtration

Qiagen chemistry

Quantification by OD

Normalization

PCR setup

Archieving

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Automated Platforms 3

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Magnetic Beads Based

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Biomerieux Nuclisens MiniMag

12 to 24 sample batch

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“High throughput” nucleic acid automated extraction with minimum hands-on-time and a very fast turnaround time (40 minutes for 24 extractions).

Biomerieux Nuclisens EasyMag

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PSS, Japana precursor in magnetic extraction automation

Magtration Technology

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PSS Workstations-1

PSS SX-6G PSS SX-8G

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PSS Workstations-2

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PSS Workstations-new concept

Pre-packed reagents for magnetic extraction procedures

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A key evolution for NA extraction Instrumentation: predisposable reagents

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PSS Workstations-new concept

Individual barcoding for reaction cartridges

CCD camera procedure monitoring

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PSS Workstations-new concept

Large volume magnetic extraction- PSS-8LX

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0.2 to 3 ml sample volume handling

1 to 10 ml sample volume

50 min run

pre-filled reaction cartridge

The new Magtration System BA-X

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Beckman Coulter Inc. SPRI-TE

Automated processing of 1-10 samples in 30 minutes

✓ High-performance SPRI magnetic bead technology

✓ Pre-packaged, ready-to-use reagents and labware

✓ Simple to operate✓ Method card driven protocols✓ UV decontamination

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Nordiag Magnetic Extraction

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The affordable NorDiag Arrow instrument makes automated sample preparation possible for a l l l a bo ra t o r i e s . The new innovative tip pump assembly will allows for a wide range of sample volumes for extraction. The tip pump assembly is a closed system, reducing the possibility for contamination. Touch screen operation which makes the instrument very user friendly.

The NorDiag Bullet, 8 tip config.

96 samples from primary tube to PCR*/SDA** setup in less than 1.5 hours, using the NorDiag Bullet instrument

The BUGS’n BEADS technology is based on an initial adsorption of bacteria/cells to magnetic particles, followed by a magnetic separation where the sample mater ia l i s removed. This step removes most of the inhibitors that are often found in samples. The next step is lysis of cells and the release of nucleic acids, followed by immobilisation of nucleic acids to magnetic particles.

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Qiagen Magnetic Extraction

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EZ1 Advanced

BioRobot M48 workstation BioSprint 96 Workstation QIAsymphony Workstation

EZ1 Advanced XL

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Roche Magnetic Extraction

COBAS® AmpliPrep-System

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2010 Evolution in Roche Automated Platforms

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The Magna Pure 96 System

Speed - 96 sample extraction in <1hrEasy loadingPrefilled reagent traysFewer disposablesRun surveillanceConnectivity

Time from sample to results: 2 hrs

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Magnetic rods

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QiagenBioSprint

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Promega Maxwell 16 System

Magnetic rods

16 sample batch

16 samples/30 min

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Chemagic by Chemagen

96 rods for <200 µl samples in 96 well plates

4000 samples/day (200µl/6µg)

12 rods for <10 ml samples in 15 or 50 ml tubes

12 samples/40 min (7ml/210µg)

Distributed in the US by Parallabs86

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Chemagic by Chemagen

1 – 12 samples per run๏ integrated buffer dispensing๏ revolutionary resuspension technology๏ standard plastic devices๏ chemagic Kits and protocols for a huge variety of

sample materials๏ quantitative yield, highest purity๏ sample sizes up to 600 µl๏ bar code reading available

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NA extraction automation options

✓Microtiter Plate based Procedures

✓Generic Robotic Instrumentation

✓Dedicated Robotic Instrumentation

✓In Vitro Diagnostic Robotic Instrumentation

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Abbott Molecular solutions

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m2000sp m2000rt

RealTime HIV-1

RealTime HCV

RealTime HCV Genotype II

RealTime HBV

RealTime High Risk HPV

RealTime CT/NG

RealTime CT

RealTime mS9

Parvo B19 PCR Kit

HSV 1/2 PCR Kit

CMV PCR Kit

VZV PCR Kit

EBV PCR Kit

M. tuberculosis

SARS RT-PCR Kit

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Abbott Molecular solutions

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Abbott M24sp (Freedom EVO Clinical 75)

1 to 24 sample batch processingNA extraction, Open ModeNo PCR setupCE-marked

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Biomerieux NucliSens EasyMag Platform

“High throughput” nucleic acid automated extraction with minimum hands-on-time and a very fast turnaround time (40

minutes for 24 extractions).91

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The Biomerieux Nuclisens EasyMag On Site

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Qiagen MDx, CE-IVD Instrument

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Qiagen platforms: New Generation

The QIAsymphony SP

•from 1 to 96 samples in batches of 24

•volumes up to 1 ml

•Magnetic Rod separation

•in-process sample loading

•bar code reading

•expandable

942008 2010 2011?

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Qiagen QIAsymphony RGQ

The QIAsymphony RGQ:

from DNA extraction with the QSsp, to PCR setup with the QAas and PCR with the Rotor-Gene Q (1 to 72 samples per run)

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HIV, HCV, HBV and CMV, EBV, HSV, VZV, BKV1 Artus RealTime PCR kits available for the Rotor-Gene Q. More to come.

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Qiagen platforms: Next Generation

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The QIAensemble

Qiagen

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Roche Cobas AmpliPrep

Cobas Ampliprep + 2xTaqMan 48

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COBAS AmpliPrep COBAS TaqManHamilton Pipettor

Pool Size = 6

Roche WNV TaqScreen platform

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Roche Cobas S201 System

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Roche Cobas p630 System

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The cobas p 630 Instrument provides fully automated pre-analytical solution for primary tube handling.

It can be used in conjunction with the COBAS® AmpliPrep/TaqMan® System and can support up to three COBAS® AmpliPrep Instruments.

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Roche Cobas 4800 System

Streamlined workflow

• Nucleic acid extraction from multiple types of specimens is fully automated, with the fewest manual steps• Swab and urine samples in cobas PCR Media in asingle run • Multiple liquid cytology media types in a single run • No manual preprocessing• Cobas PCR Media tubes and liquid cytology vials can be loaded directly into the system• Load-and-Go reagents are already bar-coded and ready to use, requiring no additional mixing• Bidirectional connectivity to the LIS enables the automated reporting of results

Flexibility to meet different needs• Adapts to the needs and throughput of each lab• Can run up to 376 samples in a day but is still efficient for labs with less intensive testing needs

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Roche Cobas S401 System

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Siemens Medical Solutions Diagnostics

Primary tube sampling

Up to 96 samples per run

Proprietary extraction technology using mag. particles

Siemens

Competition

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From sample Prep Automation to ? with the BD Viper

The BD Viper System is a patented approach to laboratory automation using an industrial class of robotics known as Selective Compliance Assembly Robot Arm (SCARA).

This classification indicates that the robot is multi-jointed (4-axis), similar to the human arm.

The BD Viper Sample Processor automates the sample hand l ing associated with high volume amplified molecular tes t ing of Chlamydia trachomatis and Neisseria gonorrhoeae performed on the BD ProbeTec ET System.

More than 500 samples for CT and GC can be processed per 8-hour shift.

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The BD Viper with XTR Technology

ThroughputUp to 736 CT/CG results in 8.5 hour shift

Time to First Results3 hours 15 minutes

Amplification and Detection TechnologyStrand displacement amplification with real-time amplification and detection

Contamination PreventionClosed solid barrier amplification system

Specimen Collection TypeVaginal & endocervical swabs, urethral swabs, urine, liquid-base cytology (LBC). All collection devices utilize pierceable caps during processing

On-Board Sample Capacity96 samples including controls per run

Patient Sample Identification LocationA membrane switch pad records the location of the tube. Capac i ty to read mult ip le barcode configurations: 2 of 5, code 39, code 128, codabar

Sample Dispensing System6-station pipettor with liquid-level sensing

Sample Volume800 µL

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I wish I could

see the XTR

• On-board sample extraction utilizing proprietary BD Fox™ chemistry

• Walk-away platform with "load and go" processing• Automation using SCARA robotic technology• Multiple sample types – Liquid-based cytology,

male urethral swabs, endocervical swabs, vaginal swabs, and urines

• Zero reagent preparation – Ready-to-use reagents• Automated sample cap piercing• Liquid-level sensing of samples and reagents• Patient sample location identification

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Gen-Probe DTS 800 Systems

DTS® 800 SystemsGenerates up to 800 results in 8 hours

Includes the primary equipment and ancillary items to process APTIMA assays

DTS: Simplified Sample ProcessingThe DTS family of systems uses an exclusive penetrable cap to minimize contamination and results in safer, more convenient sample removal.

The design also reduces the time and effort required to remove the cap and obtain swab samples from the tube.

DTS simplifies sample transport, minimizes handling and greatly reduces laboratory cross-contamination.・In addition, bar code

technology simplifies the preparation of worklists.

Sample Preparation SystemThe Ten-Tube Unit (TTU) design

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The Very 1st Fully Integrated MDx Platform

Complete automation of extraction, amplification and detection

one operator can test 500 samples with the APTIMA Combo 2 assay in approximately 8.5 hours, or 1,000 samples in approximately 13.5 hours. In addition, one operator can run two TIGRIS systems

simultaneously.

APTIMA Combo 2 assay for Chlamydia trachomatis (CT) and Neisseria gonorrhoeae (GC)

Procleix HIV-1/HCV Assay for simultaneously detection of HIV-1 and HCV

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Gen-Probe - TIGRIS

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Gen-Probe® Panther New System

1 the PANTHER system can continue running unattended, processing 500 samples in 12 hours.

2 Primary tube sampling with random access loading for maximum flexibility and productivity.

3 Multiple assays can be run from a single sample, and operators have continuous access to samples, reagents and consumables.

4 A customer-driven design, intuitive software for ease of use, and remote diagnost ics capabilities.

5 Extensive process controls that include positive sample identification, liquid level detection, reagent dispense verification, and radio frequency identification (RFID) tags on fluid containers.

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In Europe, the PANTHER system can initially be used to perform Gen-Probe's nucleic acid tests for Chlamydia and gonorrhea. These include the APTIMA Combo 2(R), APTIMA(R) CT (Chlamydia) and APTIMA GC (gonorrhea) assays.

Random accessOptimized workflow and throughputSTAT test prioritization Multiple tests from a single sample

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Fully Automated MDX Platforms EVolution

IVD industry Challengers:

Cepheid GeneXpert -> Cepheid Infinity

BD MAX System

Icubate, Idaho, Iquum, GeneFluidics

Upcoming or in Dev.

Hitachi High-Tech Open Platform

BCI UniCel DxN

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What Future for NA Extraction in MDx?

✓Automation

✓Magnetic beads = most popular✓Pre-dispensed reagents

✓Size of batch determination not final (6, 8, 24, 32, 48, 72, 96)✓96 sample batch mainly found on “generic” robotics

✓Semi-random and random access platforms start to be popular

✓Evolution through complete integration of NA extraction + PCR + Detection

✓More questions unsolved about sample preservation

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Archiving NA samples

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DNA Storage

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Storage of NA Samples at -80°C

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Freedom EVO / REMP SSS Factory

The Freedom EVO® and the REMP (Small Size Store™ (SSS) together enable the storage, retrieval and reformatting of compounds and DNA samples.

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Biophile ultra-low temperature automated

Biophile (part of TekCel)

a combination of freezer, automation and information technologies.

BIOPHILE, Inc., is the first to market -80C ultra-low temperature automated storage and retrieval systems.

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High Throughput Clinical Sample StorageARUP implemented the original Automated Transport and Sorting System built by MDS AutoLab of Toronto, Ontario on November 17, 1998. This system served ARUP's needs extremely well for more than five years, reaching a level of 18,000-22,000 new patient specimens per day and a total volume of tubes transported and sorted each day of 27,000 to 32,000.

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Sample Storage at room Temperature

DNA storage at room temperature instead of -20 or -80°C

๏Biomatrica, San Diego, CASampleMatrix is a dissolvable compound that stabilizes DNA at room temperature

SampleMatrix forms a protective seal around the sample as it dries, effectively ‘shrink-wrapping’ the DNA.

๏Genevault, Carlsbad, CAFTA paper on plate: GenPlates or GenTegra: Inorganic matrix

Outsourcing sample storage

๏Imagene, Bordeaux/Paris, FranceEncapsulating DNA, RNA or proteins in an aluminium-capsule under zero atmosphere

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Sample Storage at room Temperature

Biomatrica Preserve and store DNA at room temperature

Biomatrica has developed SampleMatrix, to stabilize and protect biological materials at room temperature without degradation. The SampleMatrix technology was designed by combining extremophile biology that enables long-term survival of organisms in extremely dry environments with synthetic chemistry.

Samples are preserved through formation of a protective thermo-stable barrier during a simple air-drying process. Samples are protected from degradation from heat and UV light.

DNAstable enables you to store plasmid and genomic DNA samples at room temperature. When you are ready to use your samples, 100% of the DNA can be recovered in 10 minutes or less and then used directly in downstream applications. By exploiting these unique characteristics, Biomatrica products enable researchers to stabilize biological samples for long periods of time.

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Sample Storage at room Temperature

GenVaultGenPlates’ proprietary format features FTA paper elements built into each wells for transport and long term storage of biosamples at ambient temperature, in the dry-state.

Upon application of 10ul of biosample directly to the 6mm disc of FTA paper, cells lyse and release DNA. The DNA becomes entwined in the fibrous network of the cellulose matrix and is dried completely at ambient temperature.

When DNA is required for downstream analysis, high-quality double-stranded DNA from FTA paper can be reliably recovered using the GenSolve chemistry.

Samples stabilized and dried down with GenTegra and stored for over 12 months at ambient laboratory temperature (21°C to 25°C) perform identically to DNA stored at -20°C in all downstream applications. Long-term accelerated stability studies of DNA at elevated temperature (76°C) indicate that DNA stabilized with GenTegra will retain its molecular integrity for decades at ambient laboratory temperatures.

Ensuring the dryness of DNA samples prior to storage is an important factor in preserving DNA integrity in ambient temperature storage conditions.

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Sample Storage at room Temperature

Imagene Room temperature DNA preservation

This technology relies in a DNAshell capsule which utilizes encapsulation under a controlled anhydrous and anoxic atmosphere of preliminarily desicated and purified DNA, into small sealed, corrosion-proof metallic capsules. This process prevents DNA from being altered by any degradation factors (water, oxygen, light).

Completely anhydrous, anoxic and perfectly airtight, the DNAshell capsules provides decisive advantages compared to freezing, enhancing stability, safety, transport and distribution of DNA. It also lowers operation and maintenance costs. In addition, the laser-marked Data Matrix codes on the capsules enable easy traceability and inventory of each DNA sample.

Ensuring the dryness of DNA samples prior to storage is an important factor in preserving DNA integrity in ambient temperature storage conditions.

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DNA can therefore be preserved at room temperature for unlimited periods.

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GenVault’s Dynamic Archive is an automated system for archiving and retrieving G e n P l a t e s a t r o o m temperature. The system utilizes a robot to access the shuttles that contain trays on which GenPlates are housed. Up to 31,500 geneplates.

GenVault’s DeskTop Archive is the smallest in the series of room-temperature biosample archiving systems available from GenVault. The “desktop” size system allows one to test-drive the concept of b i o s a m p l e s t o r a g e a t roomtemperature at low cost and low risk. Up to 1200 biosamples.

Robotics Archive or Personal Archive

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Biomatrica's dry storage cabinets employ passive control devices to ensure a stable humidity environment and are useful for laboratories where moisture levels or fluctuation is an issue. The cabinets can maintain relative humidity at <40% for the optimal room temperature storage of samples stabilized in Biomatrica’s products. Dry storage cabinets require no energy input and help your lab save money and "go green."

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Sample Management Softwares

Biomatrica

Greiner

Central Labo Europehttp://www.central-labo-europe.com/

http://www.biomatrica.com/

http://www.greinerbioone.com/

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And also: Genvault, GenoLogics...etc.

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Sample Management Software: LabCollector

http://www.labcollector.com/features.php

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Time for Questions

Or Time for Lunch?

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