2011 course on Molecular Diagnostic Automation - Part 1 - DNA Extraction
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2011 course on Molecular Diagnostic Automation - Part 1 - DNA Extraction



2011 course on Molecular Diagnostic Automation - Part 1 - Nucleic Acid Extraction.

2011 course on Molecular Diagnostic Automation - Part 1 - Nucleic Acid Extraction.
This is from early 2011. Prices and Specifications of instruments may have changed.
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2011 course on Molecular Diagnostic Automation - Part 1 - DNA Extraction 2011 course on Molecular Diagnostic Automation - Part 1 - DNA Extraction Presentation Transcript

  • Patrick Merel Biomedical Innovation Platform (PTIB), Pessac, France pmerel@mac.comNucleic Acid Extraction Automation in Molecular Diagnostic part 1 1 1
  • Nucleic Acid (NA) extraction in molecular diagnosticDNA and RNA from...Blood, Plasma, Cells … Body Fluids 2 2
  • NA Extraction Steps AutomationQuality of DNA/RNA is crucial for the PCR, RT/PCR stepsExtraction procedures often require centrifugation or filtrationWhich are difficult steps to automateOr, which require complex instrumentationHowever, there are now numerous options for automation of NA extraction stepsSemi-automated protocols (microtiterplate based)Automated protocols on robotic workstationsDedicated and automatic workstationsBut first, let’s go shopping for the perfect NA extraction technology... 3 3
  • NA organic extractionOrganic extraction is a conventional technique that usesorganic solvents to extract contaminants from cell lysates.The cells are lysed using a detergent, and then mixed withphenol, chloroform, and isoamyl alcohol.The correct salt concentration and pH must be used duringextraction to ensure that contaminants are separated into theorganic phase and that DNA remains in the aqueous phase.DNA is usually recovered from the aqueous phase by alcoholprecipitation. 4 4
  • NA Phenol-chloroform extractionEqual volumes of a phenol:chloroform mixture and the aqueous DNAsample are mixed, forming a biphasic mixture.The proteins will partition into the organic phase while the DNA (as well asother contaminants such as salts, sugars, etc.) remain in the aqueousphase.It is then followed by precipitation with ethanol, as ethanol precipitates thenucleic acid. 5 5
  • Nucleic Acid Extraction by centrifugation 6 6
  • DNA extraction by Salting-Out•Salting-out is another conventional technique where proteinsand other contaminants are precipitated from the cell lysate usinghigh concentrations of salt such as potassium acetate orammonium acetate.•The precipitates are removed by centrifugation, and the DNA isrecovered by alcohol precipitation. 7 7
  • DNA Separation by Silica Adsorption•This method is based on the selective adsorption of nucleic acids to a silica-gelmembrane in the presence of high concentrations of chaotropic salts.•Use of optimized buffers in the lysis procedure ensures that only DNA isadsorbed while cellular proteins, and metabolites remain in solution and aresubsequently washed away.•Ready-to-use DNA is then eluted from the silica-gel membrane using a low-saltbuffer.•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. 8 8
  • Silica beads based NA extraction For DNA or RNA 9 9
  • Centrifugation + filtration NA extraction Mainly single tube based, but various options for microtiterplate processing are now available 10 10
  • 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. 11 11
  • Magnetic beads based NA extraction High quality nucleic acids Scalable No centrifugation No vacuum steps Rapid purification Reproducible 12 12
  • Dynal Dynabeads Technology•D y n a b e a d s a r e s u p e r p a r a m a g n e t i c ,monosized polymer particles, providing aconsistent, defined surface for the adsorbtion orcoupling of various bioreactive molecules.•The beads are magnetisable and superparamagnetic, meaning they are only magneticin a magnetic field.•Due to this property, the beads can easily beresuspended when the magnetic field isremoved.•The technology behind the beads was licensedto Dyno Industrier in 1980, which later formedDynal Biotech together with ApothekernesLaboratorium 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 13
  • 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 ReversibleImmobilization) technology is a patented purificationtechnology developed at MITs Whitehead Institute(Hawkins, et. al., Nucleic Acids Res. 1995 (23):4742-4743).•Nucleic acids are immobilized onto paramagneticmicroparticles (1µM magnetic carboxylate-modified baseparticles) using specific buffer conditions. Flexibility isadded to the system through modification of the bindingbuffer, which alters the type and size of immobilizednucleic acid. 15 15
  • 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 ConsPros•Usually very fast procedures•Robotic friendly•Rapid growing segment for instrumentation•Cheaper ?•Potential for specific extractionCons•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 17 17
  • NA extraction by hybridizationDifferent companies have developed over the past years, specific hybridization captureprocedures:RNAture (Hitachi Chemical), Xtrana, ProLinx, Gen-Probe, DNA Research Innovation(DRI now part of Invitrogen/Life Technologies) have been the most visible. 18 18
  • Gen-Probe Target Capture TechnologyTarget Capture •Maximizes assay sensitivity & specificity. •Allows for simplified specimen processing. •RNA target is “captured” with magnetic •Patented process with specific probes beads. coated magnetic particles. •Eliminates inhibitors in swab and urine samples. 19 19
  • Invitrogen ChargeSwitch Technology ChargeSwitch gDNA 50-100 µl Lysis 1 min RT Capture 1 min Washing 3x1 min Elution 1+4 min Total @ RT 10 minHow it worksChargeSwitch Technology provides a switchable surface charge that is dependent on the pH ofsurrounding buffer to purify nucleic acids (Figure 1). In conditions of pH < 6.5, the CST surface has apositive charge that selectively binds the negatively charged nucleic acid backbone. Proteins and othercontaminants are removed in an aqueous wash buffer. For elution, the charge on the CST surface isswitched off by raising the pH to 8.5. Purified nucleic acid elutes instantly and is ready fordownstream 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 20 20
  • Very recent new approach for NA extraction Enzyme based NA extraction automated on the epMotion 5075MCDNA extraction using a thermostable proteinase.…hands off, closed tube and all you need is a thermal cycler.WorkFlow Zygem: PrepGEMand ForensicGEM 21
  • Very recent new approach for NA extraction Boreal Genomics instruments are based on SCODA: a •Up to 100X more efficient for contaminant rejection novel, proprietary electrophoresis technology that •High yield even in very dilute samples selectively concentrates nucleic acids from large sample •Simple operation volumes to a small, concentrated amount of gel or buffer. •Target length selection during extraction Advantages: •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 22
  • NA extraction Automation: why? Time consuming step in MDx Sample throughput Hand on time Reproducibility Standardization Cost Instrumentation availability 23 23
  • NA extraction automation options✓Microtiter Plate based Procedures✓Generic Robotic Instrumentation✓Dedicated Robotic Instrumentation✓In Vitro Diagnostic Robotic Instrumentation 24 24
  • Microtiterplate based NA extraction•Qiagen and Macherey-Nagel have been pioneers in providing DNAextraction protocols on 96 well-platesQiagen, http://www.qiagen.com DNAeasy, RNAeasy, QIAamp, QIAfilterMacherey-Nagel, http://www.macherey-nagel.com NucleoBond, NucleoSpin, NucleoTrap, Multi-96•Additional companies providing 96 well-plate compatible kits for DNA/RNAextraction:Promega, BioLine, Stratagene, Millipore, Ambion, Invitrogen… 25 25
  • 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 26 26
  • Microtiterplate compatible viral DNA extraction Bench top centrifuge for Nucleospin multi 96 DNA extraction. 27 27
  • HighThroughput DNA extraction from centrifugation to filtration 28 28
  • High Throughput DNA extraction from centrifugation to filtration 29 29
  • Semi-Automation of the QIAamp 96 Spin Blood Kit on the Biomek 2000 Setup of the Beckman Coulter Biomek 2000 Tablet 30 30
  • Qiagen QIAamp 96 Spin Blood Kit setup on the Biomek 2000 31 31
  • Semi-Automation of the QIAamp 96 Spin Blood Kit on the Biomek 2000Centrifugation of the Centrifugation of the Filtration Unit + Filtration Unit + Disposable Deep-Well Storage Deep-Well 32 32
  • NA extraction automation options✓Microtiter Plate based Procedures✓Generic Robotic Instrumentation✓Dedicated Robotic Instrumentation✓In Vitro Diagnostic Robotic Instrumentation 33 33
  • Generic Robotic Instrumentation✓Develop your own procedure based on manual protocols some are robotic friendly✓Follow reagents providers recommendations✓Follow robotic instrument providers recommendations 34 34
  • Generic Robotic Workstationswith available molecular testing applications. Company Instrument IVD-D or CE-IVD NA extraction PCR applications Web Address compliant; comments applications available available Agilent Automation Biocell, Bravo, No; very high Yes Yes chem.agilent.com/ Solutions BenchCel throughput Versa serie, Versa Aurora Instruments no Yes Yes aurora-instr.com/ mini Biomek 2000,3000 Beckman Coulter, Inc. No Yes Yes beckmancoulter.com/ Biomek NX,FX SciClone ALH3000 No Yes Yes Caliper LifeSciences caliperls.com/ Zephyr No Yes Yes Twister II+AB7900 No No Yes CyBi-Dilus, - Cybio No Yes Yes cybio-ag.com/ RoboSpense epMotion 5070 No; MC with Eppendorf epMotion 5075 Yes Yes eppendorf.com/ integrated PCR LH, VAC, MC, TMX STAR, STARlet, Yes; NAT pooling Hamilton Yes Yes hamiltoncomp.com/ Nimbus system No; very high Labcorp-Protedyne BioCube, Radius Yes Yes protedyne.com/ throughput Janus CS Autoplex, Perkin-Elmer No Yes Yes las.perkinelmer.com/ Forensic Tecan Freedom Evo Yes Yes Yes tecan.com/ No; very high ThermoCRS CRS DNA Yes Yes thermo.com/ throughput Xiril Xiril 75, 100, 150 No Yes Yes xiril.com/ 35 35
  • ThermoCRS Extraction platforms High throughput (up to 14, 96-well microplates/hour) Proven reliability Uses well-established DNA purification 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 Purification System is an automation tool for bead based methods using magnetic particles. This includes binding of DNAto magnetic beads, washing of the bound material, elution from the solid phase and transfer to a final destination plate. 36 36
  • Agilent Automation Solutions-Bravo and BioCel BioCel 37 37
  • Agilent Automation Solutions: Bravo and BenchCel 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)Bravo 38 38
  • 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 a protocol for the Invitrogen ChargeSwitch system EasyPlex gDNA kit using the gDNA isolation workstation in which up to 10 microplates (96- well) can be processed simultaneously.In May 2007, Protedyne became a wholly owned subsidiary of Laboratory Corporation of America. 39 39
  • Hamilton Robotics Platforms STARlet STAR STARplusNimbus MICROLAB 4000 MICROLAB 4200 40 40
  • Tecan Extraction PlatformsT h e M i n i P re p , f ro m D r u g The Genesys serie with four or eight tip arm forDiscovery (mother - daughter plate The Freedom Evo family from high throughput. NA extraction automation hasreplication), to Molecular Biology 1m to 2m deck size. A special been fully automated using the magnetic separator(Sequencing preparation, PCR mention for the IVD-D compliant MagS and solid phase extraction through columns.amplification assays) and Freedom Evo Clinical. The extraction is usually performed in 96 wellDiagnostics (Immunophenotyping, cartridge blocks. These are placed on vacuumELISA Assays). chambers on the instrument worktable and can be evacuated by software controlled vacuum pump VacS. 41 41
  • Invitrogen reagents automation and large volumes procedures 42 42
  • Xiril Robotic Platforms X75 X100Xiril Robotic Workstations have a 1-tip, 2-tip or 4-tip liquid handling arm with selectableand exchangeable pump volumes. Each pump is equipped with a tip adaptor that fitsdisposable tips with or without filters.For added flexibility and increased sample throughput, the Xiril X100 and X150 can beconfigured with an additional liquid handling arm for specific Life Science applications.Macherey, Agencourt, Promega application notes available. 43 43
  • Beckman Coulter Biomek Platforms 44 44
  • Beckman Coulter NX and Biomek Platforms 45 45
  • Robotic Workstation Industrials “Partners” Beckman Coulter Hamilton Thermo-Jouan- CRS PerkinElmer Tecan Self-Contained units RoboticsBiomek 2000, Biomek 3000 MicroLab, Starplus, CRS DNA Purification MultiProbe IIEX, Genesis RSP 100, 150, Agilent Biocel, Bravo, Starlet System DNAtrack, Janus 200 Protedyne BioCube Evo Biomek FX, Biomek NX AutoSprint MiniPrep 60 MolBio Multimek Promega, Xtrana, Millipore, Agowa, Roche BilaTec Agencourt, Agowa, Qiagen, Macherey-Nagel, Various, Invitrogen Agencourt, Qiagen Macherey-Nagel, Agowa Promega Filtration, Magnetic, Filtration, purification Magnetic Filtration, Magnetic Filtration, Magnetic all types hybridation 46 46
  • 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® MagneSil® Sequencing Reaction Clean-Up System Protocol for 96- Well Plates๏ Automated Wizard® Magnetic 96 DNA Plant System Protocol ๏ Automated MagneSil® ONE, Fixed Yield Plant Genomic DNA Purification Protocol๏ Automated Wizard® SV 96 Genomic DNA Purification System Protocol ๏ MagneSil® KF, Genomic System๏ 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 Example of Promega’s reagents Clean-Up System Protocol 47 47
  • NA extraction automation options✓Microtiter Plate based Procedures✓Generic Robotic Instrumentation✓Dedicated Robotic Instrumentation✓In Vitro Diagnostic Robotic Instrumentation 48 48
  • Dedicated Workstations For NA extraction 1/4Applied BioSystems ABI Prism 6100 Filtration 96 96/30min jusqu’à 1m de ARN, ADN appliedbiosystems.com cellules ABI Prism 6700 Filtration; archivage, 96 96/75min jusqu’à 1m de ARN, ADN préparation PCR cellulesAutogen AutoGenflex 3000 Centrifugation 48 48/4-6hr jusqu’à 5mL ADN, ARN, autogen.com plasmides, cosmides AutoGenPrep 2000 48 48/4-6hr 1mL ADN, tissus, plasmides, cosmides, levures, plantes AutoGenPrep 245 24 24/3.5hr 700µL ARN, ADN, tissus, plasmides AutoGenPrep 965 4x96 384/4-6hr Jusqu’à 250µL ou ADN, plasmides, 1mL cosmidesBeckman Coulter, Vidiera NsP Filtration; 96 96/2.5hr Jusqu’à 300µL ADN, ADN et ARN beckman.comInc. quantification, viral normalisation, préparation PCR SPRI-TE Magnétique/ 10 10/30min NS ADN Magtration; cartouches de réactifsBee Robotics QuatroProbe Magnétique 48 NS 200µL à 1mL NS beerobotics.comBioer Gene-Pure NPA-32 Magnétique/Magnetic 32 NS 50 à 800µl ADN, ARN bioer.com.cn rodBiomérieux Extractor Méthode de Boom 10 120/8hr ADN 100µL, jusqu’à ADN, ARN biomerieux.com 2mL EasyMag Magnétique/Méthode 24 60min Jusqu’à 1mL de BoomBioneer HT-Prep Pro Filtration 96 60min Jusqu’à 300µL ADN, ARN bioneer.co.kr 49 49
  • Dedicated Workstations For NA extraction 2/4Chemagen Chemagic Magnétique/Magnetic 12, 96 150/8h 10mL 10µL à 10mL DNA, RNA, RNAv, chemagen.de rod (45min/12), mRNA 4000/8h 1.5mL (15min/96) Prepito 1-12 30min 10µL à 600µLCorbett LS part of QIAxtractor FIltration 8-96 8/45min, 96/96min 180 à 200µL DNA, RNA, RNAv, corbettlifescience.comQiagen mRNAEppendorf epMotion 5075 VA, Filtration 1-96 NS NS DNA, RNA, tissus, eppendorf.com TMX plasmidesFujiFilm QuickGene-610 Filtration sur 6 12min 2mL ADN fujifilm.com QuickGene-810 Membrane poreuse de 8 6min 200µl ADN, ARN, tissus, 80µM plasmides QuickGene-mini80 8 6min (manuel) 200µl ADN, ARN, tissus, plasmides, cellulesGeneSystems GeneExtract Filtration 6 Semi-automatique > 1L de liquide ADN genesystems.frGenomic ExtraGene Centrifugation 48 240/jour 0.5 à 10mL ADN, ARN, Proteines genomics-tools.comHandyLab part of BD Lynx Magnétique; 24 35min NS ADN, ARN handylab.com cartouches de réactifsInvitrogen iPrep Magnétique/ 12 26min 350µL ADN invitrogen.com Magtration; cartouches de réactifsMagnetic Magnatrix 1200 Magnétique/Magnatrix 12-96 20min/12 NS ADN, ARN magbio.comBiosolutions AB Magnatrix 8000plus 8-192 NS NSMole GeneMole Magnétique; 16 NS 50µL à 1mL ADN, ARN, cellules, molegenetics.com cartouches de réactifs tissusNordiag Arrow Magnetic, Bug’s and 1-12 30min 200-500µL DNA, RNA nordiag.com Beads Bullet 1-96 1.5hr 20 à 200µL DNA, RNAPromega Maxwell 16 Magnétique/Magnetic 16 30min 50 à 400µL ADN www.promega.com rod; cartouches de réactifs 50 50
  • Dedicated Workstations For NA extraction 3/4Precision System Mag. Syst. 6Mx Magnétique/ 6-48 NS 100 à 1000µl ADN, ARN pssbio.comScience Magtration; cartouches de réactifs Mag. Syst. 8Lx Magnétique/ 1-8 50min 1-10mL ADN Magtration; cartouches de réactifs Magt. Syst.12GC Magnétique/ 1-12 30min 100 à 200µL ADN, ARN, ADN et Magtration; cartouches ARN viral, tissus, de réactifs plasmides SX-6G Magnétique/Magtration 48 80min 25 à 500µL ADN, ARN SX-8G Magnétique/Magtration 96 80min 5 à 40µL ADN, ARN SX-96GC Magnétique/Magtration 4x96 NS 10 à 40µL ADN, ARN Mag. Syst.Ba-X Magnétique/Magtration 1-3 0.2ml,1ml, 3ml ADN, ARNQiagen BioRobot Universal System Filtration + 4x96 4x96/2-3hr jusqu’à 200µL ADN, ADN et ARN qiagen.com centrifugation + viral, plasmides Magnétique; PCR setup BioRobot MDX DSP** Filtration + 4x96 4x96/2-3hr jusqu’à 200µL ADN, ADN et ARN centrifugation + viral Magnétique ; PCR setup; CE IVD-D EZ1, EZ1 advanced, Magnétique/ 6, 6, 14 20min 200 à 350µL ADN, ADN et ARN EZ1 advanced XL Magtration; cartouches viral, tissus de réactifs; CE-IVD-D BioRobot 48/96 Magnétique/Magtration 0,5 48/2hr 100 à 300µL ADN, ADN/ARN, ARN, mARN BioSprint 15/96 Magnétique/Magnetic 0,15625 20min 10µL à 1mL ADN, ADN/ARN, rod ARN, mARN AutoPure LS Centrifugation 16 96/jour Jusqu’à 10mL ADN QIACube Filtration + 12 65min jusqu’à 200µL ADN, ADN et ARN centrifugation viral, plasmid 51 51
  • Dedicated Workstations For NA extraction 4/4 QIAsymphony 300 LBC/16hrs; 1-96;Qiagen Magnetic, Magnetic rod 2hrs up to 1ml DNA, RNA qiagen.com/ SP, RGQ 1-72 LBC, Urine Vials, QIAEnsemble Magnetic, Magnetic rod 3000 LBC 16hrs PreservCyt, surepath DNA qiagen.com/ SP Vials, ETU tubesRoche Diagnostics MagNAPure 2.0/96 Magnetic, Magtration; 32/96 2hr/1hr 20 à 300µL DNA, RNA, DNA/ roche-applied-science.com PCR prep RNA, mRNA MagNAPure Compact System Magtration; 8 Magnetic, 20min 100µL à 1mL cartridge AmpliPrep Magnetic,PCR prep, CE 1-72 24/1hr 100 à 750µL DNA, RNAv IVD-D, continuous flowThermo LabSystems KingFisher Magnetic,Magnetic rod 2x12 24/17min, 29min, 20 à 200µL DNA, RNA, mRNA, thermo.com/ 41min proteins, cells KingFisher ML/96 15-96 15/14min, 36min, 10µL à 1mL 43min 52 52
  • A precursor in dedicated workstationThe ABI 340 extractor A closed system for the process of 8 samples / 4 hrs. 53 53
  • From bench to automationPhenol Chloroform extraction & Alcohol precipitation made automatic 54 54
  • Automated Platforms 1Centrifugation Based 55 55
  • AutoGen Centrifugation based Workstations AutoGen extractors AutoGen 960-965 (4x96 samples in 4hrs) AutoGenFlex Star Gene Prep 56 56
  • Autogen - 960-965 Extractors1. Processing Method: • Single phase organic extraction2. Protocols: DNA extraction from whole blood, blood spots, humantissues and cells (including ES cells), plasmids, BACs, microbes andplants. 3. Sample Volume:• Up to 1.8 mls per well4. Maximum Sample Throughput:• 384 (96 deep well plate x 4) perrun5. 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 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 High precision gripper-type robot hand Transports sample tubes to and from workstation, dispensing station and centrifugeAutoGenFlex is completelyautomated and easy to use, with nomanual inter vention required.Isolate up to 80 x 10ml whole bloodsamples per day Qiagens proven FlexiGene chemistry 58 58
  • AutoGen - Gene PREP On-Board Centrifuge and XYZ Robot Heating Shaker Optional Tube Unit StackerThe GENE PREP is the latest fullyautomated DNA extraction systemfrom AutoGen. •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.48 samples in 2 hours (faster time to result).Direct application of extracted nucleic acid to your •Higher throughput based on new design which shortens processing time.next procedure (saves labor and time). •Produces high quality and high yields of DNA. •Optional tube stacker allows for unattended processing of up to 192 samples. 59 59
  • Gentra/Qiagen AUTOPURE LSComplete automation of process for large volume samples 60 60
  • Genomic ExtraGene Platform ExtraGene is a multi-protocol platform based oncentrifugation. ExtraGene process up to 48 samples in 20 ml tubes.The running time depends on the protocol. Up to two improved centrifuges, with amechanized hatch on the top The robotic arm moves a special tool fortubes cap opening, closing, tube handling,nozzles for syringe pumps and precisionperistaltic pumps On the front side, a drawer supports tubesracks, tips racks and liquid and solid wastecollector ExtraGene is contained in a fume-hod(optional) Generic reagents = Much more extractionswith your budget Full automation = Much more extractionswith your work force Separation by centrifugation = Higher yeldand lower cost (less than 1.5€ / extraction) genomics-tools.com 61 61
  • Automated Platforms 2Filtration Based 62 62
  • Qiagen Extraction PlatformsBioRobot 3000 BioRobot 9604 63 63
  • Filtration based platforms The BioRobot 8000 The BRmdx 64 64
  • 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 NsPPrimary tubes on racksNA extraction by filtrationQiagen chemistryQuantification by ODNormalizationPCR setupArchieving 68 68
  • Automated Platforms 3Magnetic 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 hands-on-time and a very fast turnaround time (40 minutes for 24 extractions). 71 71
  • PSS, Japan a precursor in magnetic extraction automationMagtration Technology 72 72
  • PSS Workstations-1 PSS SX-6G PSS SX-8G 73 73
  • PSS Workstations-2 74 74
  • PSS Workstations-new conceptPre-packed reagents for magnetic extraction procedures 75 75
  • A key evolution for NA extractionInstrumentation: predisposable reagents 76 76
  • PSS Workstations-new conceptIndividual barcoding for reaction cartridges CCD camera procedure monitoring 77 77
  • PSS Workstations-new conceptLarge 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-filled 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 The BUGS’n BEADS technology is 1.5 hours, using the NorDiag based on an initial adsorption of Bullet instrument 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 XLBioRobot 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 QiagenBioSprint 84 84
  • Promega Maxwell 16 System Magnetic rods 16 sample batch16 samples/30 min 85 85
  • 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 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 µl๏ 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 RealTime RealTime VZV PCR HIV-1 HBV mS9 Kit RealTime RealTime Parvo B19 EBV PCR HCV High Risk PCR Kit Kit HPV RealTime HSV 1/2 M. HCV RealTime PCR Kit tuberculosi Genotype CT/NG s II CMV PCR RealTime Kit SARS RT- CT PCR Kit 89 89
  • Abbott Molecular solutions Abbott M24sp (Freedom EVO Clinical 75)1 to 24 sample batch processingNA extraction, Open ModeNo PCR setupCE-marked 90 90
  • Biomerieux NucliSens EasyMag Platform “High throughput” nucleic acid automated extraction withminimum hands-on-time and a very fast turnaround time (40 minutes for 24 extractions). 91 91
  • 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. 95 95
  • Qiagen platforms: Next Generation The QIAensemble 96 96
  • Roche Cobas AmpliPrep Cobas Ampliprep + 2xTaqMan 48 97 97
  • Roche WNV TaqScreen platform COBAS AmpliPrep COBAS TaqManHamilton Pipettor Pool Size = 6 98 98
  • Roche Cobas S201 System 99 99
  • Roche Cobas p630 System 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. 100 100
  • 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 efficient for labs with less intensive testing needsStreamlined 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 intothe system• Load-and-Go reagents are already bar-coded and ready to use, requiring noadditional mixing• Bidirectional connectivity to the LIS enables the automated reporting of results 101
  • Roche Cobas S401 System 102 102
  • Siemens Medical Solutions Diagnostics Siemens CompetitionPrimary tube samplingUp to 96 samples per runProprietary extractiontechnology using mag. particles 103 103
  • 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 handling associated with high volume amplified 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. 104 104
  • The BD Viper with XTR Technology Throughput Up to 736 CT/CG results in 8.5 hour shift Time to First Results I wish 3 hours 15 minutes I could Amplification and Detection Technology Strand displacement amplification with real-time see the XTR 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 Sample Volume• On-board sample extraction utilizing proprietary • Zero reagent preparation – Ready-to-use reagents 800 µL BD Fox™ chemistry • Automated sample cap piercing• Walk-away platform with "load and go" processing • Liquid-level sensing of samples and reagents• Automation using SCARA robotic technology • Patient sample location identification• Multiple sample types – Liquid-based cytology, male urethral swabs, endocervical swabs, vaginal swabs, and urines 105 105
  • Gen-Probe DTS 800 SystemsDTS: Simplified Sample ProcessingThe DTS family of systems uses an exclusive penetrable cap tominimize contamination and results in safer, more convenientsample removal.The design also reduces the time and effort required to removethe cap and obtain swab samples from the tube.DTS simplifies sample transport, minimizes handling and greatlyreduces laboratory cross-contamination.・In addition, bar code DTS® 800 Systemstechnology simplifies the preparation of worklists. Generates up to 800 results in 8 hours Includes the primary equipment and ancillarySample Preparation System items to process APTIMA assaysThe Ten-Tube Unit (TTU) design 106 106
  • 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 107 107
  • Gen-Probe - TIGRIS 108 108
  • Gen-Probe® Panther New System1 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 diagnostics 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. Random access Optimized workflow and throughput STAT test prioritization Multiple tests from a single sampleIn Europe, the PANTHER system can initially be used to perform Gen-Probes nucleicacid tests for Chlamydia and gonorrhea. These include the APTIMA Combo 2(R),APTIMA(R) CT (Chlamydia) and APTIMA GC (gonorrhea) assays. 109 109
  • Fully Automated MDX Platforms EVolutionIVD industry Challengers: Cepheid GeneXpert -> Cepheid Infinity BD MAX System Icubate, Idaho, Iquum, GeneFluidicsUpcoming or in Dev. Hitachi High-Tech Open Platform BCI UniCel DxN 110 110
  • 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 111 111
  • Archiving NA samples 112 112
  • DNA Storage 113 113
  • Storage of NA Samples at -80°C 114 114
  • 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. 115
  • 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. 116 116
  • High Throughput Clinical Sample StorageARUP implemented the original Automated Transport andSorting System built by MDS AutoLab of Toronto, Ontario onNovember 17, 1998. This system served ARUPs needsextremely well for more than five years, reaching a level of18,000-22,000 new patient specimens per day and a totalvolume of tubes transported and sorted each day of 27,000to 32,000. 117 117
  • Sample Storage at room TemperatureDNA storage at room temperature instead of -20 or -80°C๏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, CAFTA paper on plate: GenPlates or GenTegra: Inorganic matrixOutsourcing sample storage๏Imagene, Bordeaux/Paris, FranceEncapsulating DNA, RNA or proteins in an aluminium-capsule under zeroatmosphere 118 118
  • Sample Storage at room TemperatureBiomatricaPreserve and store DNA at roomtemperatureBiomatrica has developed SampleMatrix, to stabilize andprotect biological materials at room temperature withoutdegradation. The SampleMatrix technology was designed bycombining extremophile biology that enables long-termsurvival of organisms in extremely dry environments withsynthetic chemistry.Samples are preserved through formation of a protectivethermo-stable barrier during a simple air-drying process.Samples are protected from degradation from heat and UVlight.DNAstable enables you to store plasmid and genomic DNAsamples at room temperature. When you are ready to useyour samples, 100% of the DNA can be recovered in 10minutes or less and then used directly in downstreamapplications. By exploiting these unique characteristics,Biomatrica products enable researchers to stabilizebiological samples for long periods of time. 119 119
  • Sample Storage at room TemperatureGenVaultGenPlates’ proprietary format features FTA paper elementsbuilt into each wells for transport and long term storage ofbiosamples at ambient temperature, in the dry-state.Upon application of 10ul of biosample directly to the 6mmdisc of FTA paper, cells lyse and release DNA. The DNAbecomes entwined in the fibrous network of the cellulosematrix and is dried completely at ambient temperature.When DNA is required for downstream analysis, high-qualitydouble-stranded DNA from FTA paper can be reliablyrecovered using the GenSolve chemistry.Samples stabilized and dried down with GenTegra andstored for over 12 months at ambient laboratory temperature(21°C to 25°C) perform identically to DNA stored at -20°C inall downstream applications. Long-term accelerated stabilitystudies of DNA at elevated temperature (76°C) indicate thatDNA stabilized with GenTegra will retain its molecularintegrity for decades at ambient laboratory temperatures.Ensuring the dryness of DNA samples prior to storage is animportant factor in preserving DNA integrity in ambienttemperature storage conditions. 120 120
  • Sample Storage at room TemperatureImageneRoom temperature DNA preservationThis technology relies in a DNAshell capsule which utilizesencapsulation under a controlled anhydrous and anoxicatmosphere of preliminarily desicated and purified DNA, intosmall sealed, corrosion-proof metallic capsules. This processprevents DNA from being altered by any degradation factors(water, oxygen, light).Completely anhydrous, anoxic and perfectly airtight, theDNAshell capsules provides decisive advantages comparedto freezing, enhancing stability, safety, transport anddistribution of DNA. It also lowers operation andmaintenance costs. In addition, the laser-marked Data Matrixcodes on the capsules enable easy traceability and inventoryof each DNA sample.Ensuring the dryness of DNA samples prior to storage is animportant factor in preserving DNA integrity in ambienttemperature storage conditions.DNA can therefore be preserved at roomtemperature for unlimited periods. 121 121
  • Robotics Archive or Personal Archive 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 biosample storage at roomtemperature at low cost and low risk. Up to 1200 biosamples.GenVault’s Dynamic Archive Biomatricas dry storage cabinetsis an automated system for employ passive control devices toarchiving and retrieving ensure a stable humidity environmentGenPlates at room and are useful for laboratories wheretemperature. The system moisture levels or fluctuation is anutilizes a robot to access the issue. The cabinets can maintainshuttles that contain trays on relative humidity at <40% for thewhich GenPlates are housed. optimal room temperature storage ofUp to 31,500 geneplates. samples stabilized in Biomatrica’s products. Dry storage cabinets require no energy input and help your lab save money and "go green." 122 122
  • Sample Management Softwares Greiner http://www.greinerbioone.com/Central Labo Europe http://www.central-labo-europe.com/And also: Genvault, GenoLogics...etc. Biomatrica http://www.biomatrica.com/ 123 123
  • Sample Management Software: LabCollector http://www.labcollector.com/features.php 124 124
  • Time for QuestionsOr Time for Lunch? 125 125