Patrick Merel Biomedical Innovation Platform (PTIB), Pessac, France pmerel@mac.

com

Nucleic Acid Extraction Automation in Molecular Diagnostic part 1

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Nucleic Acid (NA) extraction in molecular diagnostic

DNA and RNA from... Blood, Plasma, Cells Body Fluids

2 2

NA Extraction Steps Automation

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 rst, lets go shopping for the perfect NA extraction technology...

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NA organic extraction

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.

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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

6 6

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.

7 7

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.

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.
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Silica beads based NA extraction

For DNA

or RNA
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Centrifugation + ltration NA extraction

Mainly single tube based, but various options for microtiterplate processing are now available

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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 ltration 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 purication Reproducible
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Dynal Dynabeads Technology

D y n a b e a d s
are super paramagnetic, monosized polymer particles, providing a consistent, dened 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 eld.

Due

to this property, the beads can easily be resuspended when the magnetic eld 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.

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

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

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Dynal Dynabeads Procedure

Chaotropic soln (GTC) + silica magnetic particles Chaotropic solution, removes non-bound matter EtOH, removes GTC Water, removes EtOH Water or low salt buffer

Lysis and binding 5+5 min Wash, Rinse 3x1 min Elution 30 s RT or 5 min 65C
Total @ RT <15 min
14 14

Agencourt SPRI Technology

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

Nucleic

acids are immobilized onto paramagnetic microparticles (1M magnetic carboxylate-modied base particles) using specic buffer conditions. Flexibility is added to the system through modication of the binding buffer, which alters the type and size of immobilized nucleic acid.

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Agencourt SPRI Technology

Lysis 30 min RT Bind 5 min Capture 15 min Washing1 2x10 min Washing2 2x6 min Capture+Elution 2+10 min Total @ RT 100 min
16 16

Magnetic Extraction Pros and Cons

Pros

Usually very fast procedures Robotic friendly Rapid growing segment for instrumentation Cheaper ? Potential for specic 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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NA extraction by hybridization

Different companies have developed over the past years, specic 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 & specicity. RNA target is captured with magnetic
beads. Eliminates inhibitors in swab and urine samples.

Allows for simplied specimen processing. Patented process with specic probes
coated magnetic particles.

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Invitrogen ChargeSwitch Technology

ChargeSwitch gDNA 50-100 l Lysis 1 min RT Capture 1 min Washing 3x1 min Elution 1+4 min Total @ RT 10 min
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. Puried 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 specication applications Unmatched high sensitivity for micro samples

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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.

automated on the epMotion 5075MC

WorkFlow

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 MDx Sample throughput Hand on time Reproducibility Standardization Cost

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, QIAlter 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 l 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 ltration

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High Throughput DNA extraction from centrifugation to ltration

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

Centrifugation of the Filtration Unit + Disposable Deep-Well

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.

Company Instrument IVD-D or CE-IVD compliant; comments No; very high throughput no No No No No No No; MC with integrated PCR Yes; NAT pooling system No; very high throughput No Yes No; very high throughput No NA extraction applications available Yes Yes Yes Yes Yes No Yes Yes Yes Yes Yes Yes Yes Yes PCR applications available Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes cybio-ag.com/ eppendorf.com/ hamiltoncomp.com/ protedyne.com/ las.perkinelmer.com/ tecan.com/ thermo.com/ xiril.com/
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Web Address

Agilent Automation Solutions Aurora Instruments Beckman Coulter, Inc.

Biocell, Bravo, BenchCel Versa serie, Versa mini Biomek 2000,3000 Biomek NX,FX SciClone ALH3000

chem.agilent.com/ aurora-instr.com/ beckmancoulter.com/

Caliper LifeSciences

Zephyr Twister II+AB7900 CyBi-Dilus, RoboSpense epMotion 5070 epMotion 5075 LH, VAC, MC, TMX STAR, STARlet, Nimbus BioCube, Radius Janus CS Autoplex, Forensic Freedom Evo CRS DNA Xiril 75, 100, 150

caliperls.com/

Cybio Eppendorf Hamilton Labcorp-Protedyne Perkin-Elmer Tecan ThermoCRS Xiril

ThermoCRS Extraction platforms

High throughput (up to 14, 96-well microplates/hour) Proven reliability Uses well-established DNA purication protocols Adaptable to any magnetic bead based separation method Unattended 24 hour operation and remote monitoring POLARA Open Architecture Laboratory Automation Software

The CRS DNA Purication System is an automation tool for bead based methods using magnetic particles. This includes binding of DNA to magnetic beads, washing of the bound material, elution from the solid phase and transfer to a nal destination plate.
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Agilent Automation Solutions-Bravo and BioCel

BioCel

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Agilent Automation Solutions: Bravo and BenchCel

A exible 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)

Bravo

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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 purication 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 (96well) can be processed simultaneously.

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

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Hamilton Robotics Platforms

STARlet

STAR

STARplus

Nimbus

MICROLAB 4000 MICROLAB 4200

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Tecan Extraction Platforms

T h e M i n i P re p , f ro m D r u g Discovery (mother - daughter plate replication), to Molecular Biology (Sequencing preparation, PCR amplication assays) and 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

42 42

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 ts disposable tips with or without lters. For added exibility and increased sample throughput, the Xiril X100 and X150 can be congured with an additional liquid handling arm for specic Life Science applications. Macherey, Agencourt, Promega application notes available.
43 43

Beckman Coulter Biomek Platforms

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Beckman Coulter NX and Biomek Platforms

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Robotic Workstation Industrials Partners

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, 200 Evo

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

Automated SV 96 Total RNA Isolation System Protocol

Automated Wizard Magnetic 96 DNA Plant System Protocol

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

Automated Wizard SV 96 Genomic DNA Purication System Protocol

Automated MagneSil ONE, Fixed Yield Plant Genomic DNA Purication Protocol

Automated Wizard SV 96 PCR Clean-Up System Protocol

MagneSil KF, Genomic System

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

Example of Promegas reagents

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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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Dedicated Workstations For NA extraction 1/4

Applied BioSystems ABI Prism 6100 ABI Prism 6700 Autogen AutoGenflex 3000 AutoGenPrep 2000 Filtration Filtration; archivage, prparation PCR Centrifugation 96 96 48 48 96/30min 96/75min 48/4-6hr 48/4-6hr jusqu 1m de cellules jusqu 1m de cellules jusqu 5mL 1mL ARN, ADN ARN, ADN ADN, ARN, plasmides, cosmides ADN, tissus, plasmides, cosmides, levures, plantes ARN, ADN, tissus, plasmides ADN, plasmides, cosmides ADN, ADN et ARN viral ADN NS ADN, ARN
beerobotics.com bioer.com.cn biomerieux.com autogen.com appliedbiosystems.com

AutoGenPrep 245 AutoGenPrep 965 Beckman Coulter, Inc. Vidiera NsP Filtration; quantification, normalisation, prparation PCR Magntique/ Magtration; cartouches de ractifs Magntique Magntique/Magnetic rod Mthode de Boom Magntique/Mthode de Boom Filtration

24 4x96 96

24/3.5hr 384/4-6hr 96/2.5hr

700L Jusqu 250L ou 1mL Jusqu 300L

beckman.com

SPRI-TE Bee Robotics Bioer Biomrieux QuatroProbe Gene-Pure NPA-32 Extractor EasyMag Bioneer HT-Prep Pro

10 48 32 10 24 96

10/30min NS NS 120/8hr 60min 60min

NS 200L 1mL 50 800l

ADN 100L, jusqu ADN, ARN 2mL Jusqu 1mL Jusqu 300L ADN, ARN

bioneer.co.kr

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Dedicated Workstations For NA extraction 2/4

Chemagen Chemagic Magntique/Magnetic rod 12, 96 Prepito QIAxtractor epMotion 5075 VA, TMX QuickGene-610 QuickGene-810 QuickGene-mini80 GeneSystems Genomic GeneExtract ExtraGene Filtration Centrifugation 1-12 8-96 1-96 150/8h 10mL (45min/12), 4000/8h 1.5mL (15min/96) 30min 8/45min, 96/96min NS 12min 6min 6min (manuel) Semi-automatique 240/jour 35min 26min 20min/12 NS NS 30min 1.5hr 30min 10L 10mL DNA, RNA, RNAv, mRNA
chemagen.de

Corbett LS part of Qiagen Eppendorf FujiFilm

FIltration Filtration

10L 600L 180 200L NS 2mL 200l 200l > 1L de liquide 0.5 10mL NS 350L NS NS 50L 1mL 200-500L 20 200L 50 400L

Filtration sur 6 Membrane poreuse de 8 80M 8 6 48

DNA, RNA, RNAv, mRNA DNA, RNA, tissus, plasmides ADN ADN, ARN, tissus, plasmides ADN, ARN, tissus, plasmides, cellules ADN ADN, ARN, Proteines ADN, ARN ADN ADN, ARN

corbettlifescience.com eppendorf.com fujifilm.com

genesystems.fr genomics-tools.com handylab.com invitrogen.com

HandyLab part of BD Lynx Invitrogen Magnetic Biosolutions AB Mole Nordiag iPrep Magnatrix 1200 Magnatrix 8000plus GeneMole Arrow Bullet Promega Maxwell 16

Magntique; 24 cartouches de ractifs Magntique/ 12 Magtration; cartouches de ractifs Magntique/Magnatrix 12-96 8-192 Magntique; 16 cartouches de ractifs Magnetic, Bugs and 1-12 Beads 1-96 Magntique/Magnetic rod; cartouches de ractifs 16

magbio.com

ADN, ARN, cellules, tissus DNA, RNA DNA, RNA ADN

molegenetics.com nordiag.com

www.promega.com

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Dedicated Workstations For NA extraction 3/4

Precision System Science Mag. Syst. 6Mx Mag. Syst. 8Lx Magt. Syst.12GC SX-6G SX-8G SX-96GC Mag. Syst.Ba-X Qiagen Magntique/ Magtration; cartouches de ractifs Magntique/ Magtration; cartouches de ractifs Magntique/ Magtration; cartouches de ractifs Magntique/Magtration 6-48 1-8 1-12 48 NS 50min 30min 80min 80min NS 100 1000l 1-10mL 100 200L 25 500L 5 40L 10 40L 0.2ml,1ml, 3ml 4x96/2-3hr jusqu 200L ADN, ARN ADN ADN, ARN, ADN et ARN viral, tissus, plasmides ADN, ARN ADN, ARN ADN, ARN ADN, ARN ADN, ADN et ARN viral, plasmides ADN, ADN et ARN viral ADN, ADN et ARN viral, tissus ADN, ADN/ARN, ARN, mARN ADN, ADN/ARN, ARN, mARN ADN ADN, ADN et ARN viral, plasmid
qiagen.com pssbio.com

Magntique/Magtration 96 Magntique/Magtration 4x96 Magntique/Magtration 1-3

BioRobot Universal System Filtration + 4x96 centrifugation + Magntique; PCR setup BioRobot MDX DSP** Filtration + 4x96 centrifugation + Magntique ; PCR setup; CE IVD-D EZ1, EZ1 advanced, Magntique/ 6, 6, 14 EZ1 advanced XL Magtration; cartouches de ractifs; CE-IVD-D BioRobot 48/96 BioSprint 15/96 AutoPure LS QIACube Magntique/Magtration 0,5 Magntique/Magnetic rod Centrifugation Filtration + centrifugation 0,15625 16 12

4x96/2-3hr

jusqu 200L

20min

200 350L

48/2hr 20min 96/jour 65min

100 300L 10L 1mL Jusqu 10mL jusqu 200L

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Dedicated Workstations For NA extraction 4/4

QIAsymphony SP, RGQ QIAEnsemble SP Roche Diagnostics MagNAPure 2.0/96 300 LBC/16hrs; 1-96; 2hrs 1-72 16hrs 2hr/1hr 20min 24/1hr 24/17min, 29min, 41min 15/14min, 36min, 43min

Qiagen

Magnetic, Magnetic rod

up to 1ml

DNA, RNA

qiagen.com/

Magnetic, Magnetic rod 3000 LBC

LBC, Urine Vials, PreservCyt, surepath DNA Vials, ETU tubes 20 300L 100L 1mL 100 750L 20 200L 10L 1mL DNA, RNAv DNA, RNA, mRNA, proteins, cells DNA, RNA, DNA/ RNA, mRNA

qiagen.com/ roche-applied-science.com

Magnetic, Magtration; 32/96 PCR prep MagNAPure Compact System Magtration; 8 Magnetic, cartridge AmpliPrep Magnetic,PCR prep, CE 1-72 IVD-D, continuous flow Magnetic,Magnetic rod 2x12 15-96

Thermo LabSystems KingFisher KingFisher ML/96

thermo.com/

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A precursor in dedicated workstation

The ABI 340 extractor

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

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From bench to automation

Phenol Chloroform extraction & Alcohol precipitation made automatic

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Automated Platforms 1

Centrifugation Based

55 55

AutoGen Centrifugation based Workstations

AutoGen extractors

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

AutoGenFlex Star

Gene Prep

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Autogen - 960-965 Extractors

1. Processing Method: Single phase organic extraction 2. 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 well 4. Maximum Sample Throughput: 384 (96 deep well plate x 4) per run 5. Processing Time: Four hours for 4 plates

57 57

AutoGen - AutoGenFlex Star An AutoGen and QIAGEN Collaboration

Removable sample and DNA storage racks Heater to allow for efcient digesting of the sample and drying of puried DNA -4-position swing type buckets -Variable speed and duration settings -HEPA-Filter for biohazard reduction

High precision gripper-type robot hand Transports sample tubes to and from workstation, dispensing station and centrifuge

AutoGenFlex is completely automated and easy to use, with no manual inter vention required. Isolate up to 80 x 10ml whole blood samples per day
Qiagen's proven FlexiGene chemistry
58 58

AutoGen - Gene PREP

On-Board Centrifuge and XYZ Robot

Heating Shaker Optional Tube Unit Stacker

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

Automates entire extraction process including on board digestion. Capable of extraction from multiple sample types. Low cost per prep using AutoGens reliable solution phase extraction

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

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.
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Gentra/Qiagen AUTOPURE LS

Complete automation of process for large volume samples

60 60

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)

genomics-tools.com
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Automated Platforms 2

Filtration Based

62 62

Qiagen Extraction Platforms

BioRobot 3000

BioRobot 9604

63 63

Filtration based platforms

The BioRobot 8000

The BRmdx

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Filtration based platform on a centrifuge

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.

65 65

FujiFilm QuickGene 810

66 66

Complete Automation with the QuickGene HT

67 67

Beckman Coulter Inc.Vidiera NsP

Primary tubes on racks NA extraction by ltration Qiagen chemistry Quantication by OD Normalization PCR setup Archieving

68 68

Automated Platforms 3

Magnetic Beads Based

69 69

Biomerieux Nuclisens MiniMag

12 to 24 sample batch

70 70

Biomerieux Nuclisens EasyMag

High throughput nucleic acid automated extraction with minimum handson-time and a very fast turnaround time (40 minutes for 24 extractions).
71 71

PSS,

Japan

a precursor in magnetic extraction automation

Magtration Technology
72 72

PSS Workstations-1

PSS SX-6G

PSS SX-8G

73 73

PSS Workstations-2

74 74

PSS Workstations-new concept

Pre-packed reagents for magnetic extraction procedures

75 75

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

77 77

PSS Workstations-new concept

Large volume magnetic extraction- PSS-8LX The new Magtration System BA-X

0.2 to 3 ml sample volume handling

1 to 10 ml sample volume 50 min run pre-lled reaction cartridge

78 78

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

79 79

Nordiag Magnetic Extraction

The affordable NorDiag Arrow instrument makes automated sample preparation possible for all laboratories. 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 BUGSn BEADS technology is based on an initial adsorption of bacteria/cells to magnetic particles, followed by a magnetic separation where the sample material is 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.

80 80

Qiagen Magnetic Extraction

EZ1 Advanced

EZ1 Advanced XL

BioRobot M48 workstation

BioSprint 96 Workstation

QIAsymphony Workstation

81 81

Roche Magnetic Extraction

COBAS AmpliPrep-System

82

2010 Evolution in Roche Automated Platforms

The Magna Pure 96 System Time from sample to results: 2 hrs Speed - 96 sample extraction in <1hr Easy loading Prefilled reagent trays Fewer disposables Run surveillance Connectivity
83 83

Magnetic rods
Qiagen BioSprint

84 84

Promega Maxwell 16 System

Magnetic rods 16 sample batch 16 samples/30 min

85 85

Chemagic by Chemagen

96 rods for <200 l samples in 96 well plates 4000 samples/day (200l/6g) 12 rods for <10 ml samples in 15 or 50 ml tubes 12 samples/40 min (7ml/210g)

Distributed in the US by Parallabs

86 86

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 l bar code reading available

87 87

NA extraction automation options

Microtiter Plate based Procedures Generic Robotic Instrumentation Dedicated Robotic Instrumentation In Vitro Diagnostic Robotic Instrumentation
88 88

Abbott Molecular solutions

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. tuberculosi s SARS RTPCR Kit
89 89

Abbott Molecular solutions

Abbott M24sp (Freedom EVO Clinical 75)

1 to 24 sample batch processing NA extraction, Open Mode No PCR setup CE-marked

90 90

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).
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The Biomerieux Nuclisens EasyMag On Site

92 92

Qiagen MDx, CE-IVD Instrument

93 93

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

2008

2010

2011?

94 94

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)

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

Qiagen

The QIAensemble

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Roche Cobas AmpliPrep

Cobas Ampliprep + 2xTaqMan 48

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Roche WNV TaqScreen platform

Hamilton Pipettor COBAS AmpliPrep COBAS TaqMan

Pool Size = 6
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Roche Cobas S201 System

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Roche Cobas p630 System

The cobas p 630 Instrument provides fully automated preanalytical 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

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 efcient for labs with less intensive testing needs

Streamlined workow 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 a single 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

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Roche Cobas S401 System

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Siemens Medical Solutions Diagnostics

Siemens

Competition

Primary tube sampling

Up to 96 samples per run Proprietary extraction technology using mag. particles
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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 classication indicates that the robot is multi-jointed (4-axis), similar to the human arm.

The BD Viper Sample Processor automates the sample handling associated with high volume amplied molecular testing 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

Throughput Up to 736 CT/CG results in 8.5 hour shift

I wish I could see the XTR

Time to First Results 3 hours 15 minutes Amplification and Detection Technology Strand displacement amplification with real-time amplification and detection Contamination Prevention Closed solid barrier amplification system Specimen Collection Type Vaginal & endocervical swabs, urethral swabs, urine, liquid-base cytology (LBC). All collection devices utilize pierceable caps during processing On-Board Sample Capacity 96 samples including controls per run Patient Sample Identification Location A membrane switch pad records the location of the tube. Capacity to read multiple barcode configurations: 2 of 5, code 39, code 128, codabar Sample Dispensing System 6-station pipettor with liquid-level sensing

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

Sample Volume 800 L

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Gen-Probe DTS 800 Systems

DTS: Simplied Sample Processing The 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 simplies sample transport, minimizes handling and greatly reduces laboratory cross-contamination.In addition, bar code technology simplies the preparation of worklists. Sample Preparation System The Ten-Tube Unit (TTU) design DTS 800 Systems Generates up to 800 results in 8 hours Includes the primary equipment and ancillary items to process APTIMA assays

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The Very 1st Fully Integrated MDx Platform

Complete automation of extraction, amplication 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 exibility 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 diagnostics capabilities. 5 Extensive process controls that include positive sample identication, liquid level detection, reagent dispense verication, and radio frequency identication (RFID) tags on uid containers.
Random access Optimized workow and throughput STAT test prioritization Multiple tests from a single sample

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.

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Fully Automated MDX Platforms EVolution

IVD industry Challengers:
Cepheid GeneXpert -> Cepheid Innity 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 nal (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 -80C

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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 rst to market -80C ultra-low temperature automated storage and retrieval systems. 116
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High Throughput Clinical Sample Storage

ARUP 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 ve 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 -80C

Biomatrica, San Diego, CA

SampleMatrix 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, CA
FTA paper on plate: GenPlates or GenTegra: Inorganic matrix Outsourcing sample storage

Imagene, Bordeaux/Paris, France

Encapsulating 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

GenVault
GenPlates 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 brous 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 (21C to 25C) perform identically to DNA stored at -20C in all downstream applications. Long-term accelerated stability studies of DNA at elevated temperature (76C) 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 puried 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.

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

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Robotics Archive or Personal Archive

GenVaults DeskTop Archive is the smallest in the series of roomtemperature biosample archiving systems available from GenVault. The desktop size system allows one to test-drive the concept of biosample storage at roomtemperature at low cost and low risk. Up to 1200 biosamples.

GenVaults Dynamic Archive is an automated system for archiving and retrieving GenPlates at room temperature. The system utilizes a robot to access the shuttles that contain trays on which GenPlates are housed. Up to 31,500 geneplates.

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 Biomatricas products. Dry storage cabinets require no energy input and help your lab save money and "go green."

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Sample Management Softwares

http://www.greinerbioone.com/

Greiner

Central Labo Europe

http://www.central-labo-europe.com/

And also: Genvault, GenoLogics...etc.

http://www.biomatrica.com/
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Biomatrica

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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