Tecan Journal Edition 03/2006

The world’s biggest, singleautomated forensicanalysis systempage 14-19

Redefining the geneticbasis of learning disabilitypage 12-13

TILLING in Dresden page 22-23

Cover shows Caroline Huber, Product Manager ClinicalDiagnostic and Friedrich Jost, Head SoftwareDevelopment Clinical Diagnostic

Tecan Journal

ISSN 1660-5276

Edition 3/2006

2 W E LCO M E

Tecan Journal 3/2006

Delighting our customersis at the heart of Tecan’s activities

Our customers’ needs are the drivingforce for everything we do at Tecan - from our dedication to quality and thedevelopment of new and innovativesolutions for our customers’ challenges,to fielding the highly skilled service forcethat our customers rely on throughoutan instrument’s lifetime - all of us atTecan strive to not only meet ourcustomers’ expectations, but to exceedthem, day in, day out.

Of course, we cannot do this alone, andwe rely heavily on you, our customers,to help us understand your needs andexpectations, and guide us in reachingour goals. This partnership, in many ways,is reflected in this, the third edition of theTecan Journal for 2006, which is also ourlargest issue ever. We are proud to bringyou reports from the recent customer-focused events in China and Japan, andpresent a number of innovationsdeveloped in association with customersto meet specific application needs,including nanoscale automated liquidhandling and a platform for automatedion channel screening.

Throughout more than 25 years inbusiness, we have been proud to be thepartner our customers trust and it was a special privilege to develop the newDNA sample processing system with the South African Police Service, highlightedin the following pages. Installation wascompleted earlier this year and we aredelighted to be able to share with yousome details of the system which, atnearly 40 meters long, is the world’slargest single automated forensicanalysis system.

Finally, to help us better understand you,our customers, and enable us to focus ourefforts more effectively, we will shortly becontacting some customers with a survey.Should any of you be contacted for thiseffort, I would personally very muchappreciate you taking a brief momentto let us know exactly where we stand.

We hope you enjoy this issue of the TecanJournal and look forward to continuingworking with you to meet your needs.

Thomas BachmannChief Executive Officer (CEO)

Tecan strives to not only meetour customers’expectations, but toexceed them, day in,day out.

Delighting our customersThomas Bachmann, CEO, thanks you,our customerspage 2

Latest products:Tecan launches Infinite™ F500 The latest addition to Tecan’s high enddetection instruments

The new Flask Flipper™ module,Te-PoolSafe™ option and new RoMa arm for Freedom EVO® 75Increasing the functionality of Tecan’s smallest robotic workstationpages 4-5

The International Union of Biochemistryand Molecular Biology (IUBMB) 2006Tecan shows new products in Japan page 6

Meeting the directors of the blood banksin South West ChinaA great opportunity for Tecan to makecontact with blood banks in the regionpage 7

The Genesis FE500™Taking control of sample preparation page 8

Automated processing of whole bloodsamples for monitoring ofimmunosuppressants by LC-MS/MSTecan’s Freedom EVO can be used for thequantification of tacrolimus by LC-MS/MSpages 9-11

Redefining the genetic basis of learning disabilityDominic McMullan, Principal ClinicalScientist, reportspages 12-13

The world’s biggest, single automatedforensic analysis system is hungry for samplespages 14-19

Automating metabolic inhibition assays –on a nanoscaleIntegrating a nanopipettor with a Tecanworkstation and Safire2™ microplatereader for miniaturized assayspages 20-21

TILLING in DresdenLarge-scale detection of single nucleotidepolymorphismspages 22-23

High performance ion channel screeningnpi electronic has revolutionizedautomated drug screeningpages 24-25

Managing lead optimization compoundswith REMP technologyUsing REMP’s Small-Size Store™ to managethousands of drug discovery samplespages 26-27

Events for 2006Meet Tecan at these events during thenext six months page 28


3CO N T E N T S

44 L AT E ST P R O D U C T S

Tecan’s latest products

This new multifunctional microplatereader provides outstanding sensitivityand measurement speeds, and is able tomeasure a 1536-well plate in under 30seconds. The F500 is the optimaldetection platform for assay developmentand screening laboratories, for labora-tories in the cosmetics industry and,thanks to its modular pricing, for smallerresearch laboratories.

The Infinite F500 has a range of measure-ment modes, including fluorescenceintensity top reading, absorbance, andtime-resolved fluorescence. Fluorescenceintensity bottom reading, fluorescencepolarization, homogenous time-resolvedfluorescence, and luminescencemeasurement modes are available asoptions. The new instrument offers anumber of improvements over existingmicroplate readers, such as integratedinjectors that can operate at variablevolumes for three different syringe sizes,very fast switching of filter modules and

Tecan launches the new InfiniteTM F500 high-end microplate readerThe Infinite 500 series was launched in August 2006 and is the latest addition to Tecan’s portfolio of high-end detection instruments, combining the features andmodularity of Tecan’s highly successful GENios Pro™ and GENios Ultra™ systems.

Making of Infinite F500. Members of the development team (from left to right):Christian Oberdanner (Application Specialist Infinite F500), Daniela Inführ, Martin Überegger,Stefan Rainer (Manufacturing Infinite F500)

The new Infinite F500 from Tecan

automated z-focusing in all top readingmodes. The easy-to-use and flexiblemicroplate reader uses Tecan’s i-control™and Magellan™ software packages and iscompatible with Tecan’s Freedom EVO®series of liquid handling workstations,allowing complete automation of assayand analysis.

Important announcement: Launchtutorial for the Infinite F500 at SBS

A special tutorial will take place at SBS on 19 September 2006 at 2:00 pm inroom 619 entitled “Infinite F500 - a newmultifunctional microplate detectionsystem from Tecan for biomolecularscreening assays”. If you would like toattend this tutorial then please contactMonica Jenkins([email protected])for further details.


5L AT E ST P R O D U C T S


New RoMa arm forFreedom EVO® 75Tecan has released the new RoMa 75robotic manipulator arm for theFreedom EVO 75 liquid handlingworkstation. This option fits theFreedom EVO 75’s smaller dimensions,without compromising on the highquality performance and mechanicsthat you expect from Tecan’s modules.

The new arm increases thefunctionality of Tecan’s smallestrobotic workstation by movingmicroplates and disposable tip racks to and from other modules such ashotels, vacuum and magnetic beadseparation modules and shakers.It can also load microplates intorobotic incubators, plate washers anddetection instruments such as Tecan’sSunrise™ reader, thereby enabling fully automated plate processing.The RoMa 75 is controlled by theFreedom EVOware software package(version 1.21 and above). Most installedFreedom EVO 75 instruments can easilybe upgraded with this new option.

5L AT E ST P R O D U C T S

The flask handling module assists the cellharvesting process by shaking, knocking cells off the surface and holding flasks in the uprightposition for liquid addition or removal

Tecan has collaborated with the Instituteof Chemistry and Biotechnology of theZurich University of Applied Sciences in Winterthur (Division of Cell Biology,Prof U. Graf-Hausner) to establish anumber of cell detachment methods,demonstrating that the Freedom EVO’snew flask handling module can assist inprocessing a range of diverse cell lines.Studies on various cell types have shownthat when cells were grown to nearconfluency in Corning RoboFlask® vessels,and harvested either manually or by theFreedom EVO equipped with the FlaskFlipper, no statistical differences in thecell counts and cell viability wereobserved (data available from Tecan).

Accurate samplepooling with the newTe-PoolSafe™Tecan’s new PoolSafe Option (Te-PoolSafe)is a fast and sensitive liquid arrival checkfor blood pooling processes in thepreparation of nucleic acid testing (NAT).The Te-PoolSafe measures, checks anddocuments every liquid arrival of eachsample dispensed into the pool tube,bringing enhanced safety and security to all pooling and testing applications.

The Te-PoolSafe runs on Logic™ andFreedom EVOware® software and caneasily be integrated with all existingFreedom EVO and Genesis™ liquid handlingplatforms. The module’s extremely highsensitivity, combined with its ability tomake very rapid measurements, makes itan important addition to every blood bankand NAT laboratory.

Not available for sale in the United States ofAmerica and Canada

Manual cell passaging is a thing of the past!Manually harvesting adherent cells fromtissue culture flasks is one of the mostcommon and tedious processes in the cell culture lab – but Tecan is about tochange that! The new Flask Flipper™module is available for Freedom EVO®liquid handling workstations of any sizeto harvest, passage, split and seedadherent cells in microplate-sized,automation-friendly cell culture flasks.Such flasks have recently been introducedby recognized consumable manufacturers,including the RoboFlask® vessel byCorning Life Sciences. The Flask Flipperassists the cell harvesting process byshaking, knocking cells off the surfaceand holding flasks in the upright positionfor liquid addition or removal.

6 G LO B A L N E W S


The 20th IUBMB International Congressof Biochemistry and Molecular Biology,covering all topics within the fields ofmolecular biology and biochemistry,was held from June 18-23 at the KyotoInternational Conference Hall,Takaragaike Prince Hotel, in Japan.Run once every three years in variouslocations around the world, it has been39 years since the congress last visitedJapan. At the opening ceremony, the8,000 participants from 71 countrieswere graced by the presence of HisImperial Highness the Crown Prince.

As a joint event with the symposia of the Japanese Biochemical Society and the Molecular Biology Society of Japan,normally held in autumn, the programwith the theme “Life - MolecularIntegration & Biological Diversity” wasaimed at giving young researchers, whocarry the future of Japan’s life science,the opportunity to directly experiencesome of the world’s top class research.Prominent researchers presented 11plenary lectures, symposia across 89themes, and 6,000 posters.

In the Scientific Exhibition, with 271participating companies, Tecan Japandisplayed the Freedom EVO® with the

new 96-channel pipetting head(MultiChannel Arm, MCA), Cellerity™’sFlask Flipper™ module, and the Infinite™200 microplate reader. Nicolas Sandoz,Product Manager of the MCA, and RolandDurner, Tecan’s Global Product Manager,attended the event to promote theCellerity. After visiting Tecan usersthroughout Japan during the week priorto the exhibition, Nicolas Sandoz andRoland Durner offered further expertiseat the Tecan exhibition booth,demonstrating products seen in Japan forthe first time. REMP’s Small-Size Store™(SSS) and devices related to the REMPTube Technology™ were also exhibited.

Many existing users of Tecan’s automatedsystems visited our IUBMB exhibitionstand to find out more about the MCA,a new option on the Freedom EVO.The Infinite microplate reader, which hasbeen very popular since its launch last year,has maintained a high level of interest, andthe exhibition offered many opportunitiesto obtain feedback from experiencedusers of the instrument. The knockingsound from Cellerity’s Flask Flippermodule dislodging adherent cells fromthe inner surface of flasks attracted muchattention, prompting many people tolisten to the descriptions from Tecan staff.

The International Union of Biochemistry and Molecular Biology (IUBMB) 2006Tecan shows new products in Japan

Meeting the directors of theblood banks in South West China

On April 11-16 this year, Tecan was invitedto participate in the Director Forum ofSouth West China Blood Centers.The forum, organized and hosted bythe Yunnan Blood Center at Kunming,was a major event for the blood centersof South West China, covering the fiveprovinces of Yunnan, Sichuan, Chongqing,Guizhou and Guangxi. Around 50attendees were present, including the 15 directors and presidents of the blood centers from all five provinces.The Director of the Yunnan Blood Centerat Kunming, Mr Yang Tong-Han, wasinstrumental in planning and organizingthe event.

The blood banks in the Yunnan Provincehave been loyal Tecan customers for over seven years, since they obtainedmany Tecan instruments funded by agovernment loan. All of the 16 cities andautonomous regions in Yunnan are usingTecan’s Genesis RSP™ workstation,Columbus™ washer and Sunrise™ readers.The forum was a great opportunity forTecan to make contact with the otherblood banks in the region.

Günter Weisshaar, Executive Vice-President Global Quality and RegulatoryCompliance, together with Tecan China’sWilliam Shi, Field Application Engineer,and Mark Wang, Tecan China’s ChiefRepresentative, attended the forum.They were also joined by Mr Edgar Doerig,

Head of the Commercial and EconomicSection of the Swiss Embassy in China.

On behalf of Tecan – the sole instrumentsupplier invited for this event – GünterWeisshaar and Mark Wang made a jointpresentation about quality and safety inblood banks, and William then introducedthe attendees to Tecan’s new products andservice. Mr Doerig, of the Swiss Embassy,made the closing remarks in the session,assuring the representatives of the bloodbanks of Tecan’s total compliance with allregulatory affairs of Europe and the world.Having visited the company headquartershimself and spoken to various Tecanmanagers, Mr Doerig expressed hisconfidence in the high quality productsand good service provided by Tecan.

The forum was also an ideal opportunityfor Tecan China to introduce Chinawealth,Tecan’s new distributor in the blood centersector of China. Chinawealth’s Mr ZhangMing, Product Manager, and Ms WangHuan, South West China Sales Manager,were present, and Tecan China andChinawealth jointly contributed the cost of coach travel for the event.

The timing of the forum was perfect as, inmid-April, the event coincided with the lunar new year of the Dai Nationality,who live mainly in Xishuangbanna andDehong of the Yunnan province. Forumattendees later participated in the

promotion of voluntary blood donation at the traditional Water SplashingFestival at Jinghong in Xishuangbanna.

The event helped to raise awareness of Tecan’s products and organizationamongst the users, and gained valuablefeedback, which will allow Tecan toprovide an improved service structurecombined with better channels ofcommunication between the users and Tecan China.


7G LO B A L N E W S

(from left to right): Yang Tong-Han,Director of Yunnan Blood Center Kunming;Günter Weisshaar, Tecan’s Executive Vice-President Global Quality andRegulatory Compliance; Edgar Doerig,Head of the Commercial and EconomicSection of the Swiss Embassy in China;Mark Wang, Tecan China’s ChiefRepresentative

Mark Wang, Tecan China’s Chief Representative

8 P L AT F O R M S

Tecan Journal 3/2006 Maria Torstensson-Jungestål and the three Genesis FE500s at Uppsala

The department of clinical chemistry at Uppsala University Hospital in Sweden is one of several high throughput laboratories to rely on Tecan’s Genesis FE500™ front-end,pre-analytical system for dealing with large numbers of routine clinical samples.Like any large teaching hospital, Uppsala receives more than 4000 blood samples a day for clinical chemistry tests. The department began with just one Genesis FE500 but the workload has steadily increased over the last five years and there are now three instruments, all configured to prepare samples for as many as 650 clinical chemistry parameters, including allergens for allergy profiles.

The Genesis FE500 combines pre-analytical functions including pre-sorting, centrifugation, volume check,decapping, secondary tube labeling,aliquoting and destination sorting intoassorted sample and analyzer racks, alltogether on a small footprint instrument.Before the Genesis FE500 systems werein place in Uppsala, several staffmembers were dedicated entirely tosample preparation and the wholeprocedure was completely open toinaccuracy and human error.

As Åke Jansson, laboratory engineer inthe department, explained: “The problemof human error is just taken out of the

equation now and the increase in ourworkload is interesting. We now have the capacity to handle huge numbers of samples and have become a centralinput for the entire hospital. With thethroughput we have today, it would be impossible to handle so many tubesmanually, with aliquoting, secondarytube labeling, and everything else that is involved.”

“We looked around at other front-endinstruments available before we boughteach Genesis FE500 system and the Tecansystem’s flexibility was always theoutstanding feature for us. The GenesisFE500 gives us total flexibility by

transferring samples into tubes, includingdirectly into the sample racks for ourmain clinical chemistry analyzers, twoARCHITECT ci8200® systems from AbbottDiagnostics and one Modular E170 fromRoche AG. At the outset it was very easyto configure once we were fully aware of our laboratory’s patterns of work andorganization and, once set, the GenesisFE500 simply gets on with everything,virtually unattended.”

ARCHITECT ci8200 is a registered trademark of Abbott Laboratories

taking control of sample preparationThe Genesis FE500™


9A P P L I C AT I O N D I AG N O ST I C

Automated processing of whole blood samples for monitoring ofimmunosuppressants by LC-MS/MS

After transplantation of solid organs, lifelong treatment withimmunosuppressive drugs is mandatory to avoid rejections.Therapeutic drug monitoring of the main immunosuppressant drugs,cyclosporin A, tacrolimus, sirolimus and everolimus, is essential to allowtailoring of the dosage according to individual patients’ whole blood drug concentrations.

Immunoassays are still the predominantanalytical technique used for therapeuticdrug monitoring of these drugs, but thetests are expensive and their analyticalquality, in terms of specificity andreproducibility, is limited.

More recently, LC-MS/MS has beenintroduced in many laboratories as analternative technology forimmunosuppressant monitoring.However, its use in the clinical laboratoryhas been restricted for several reasons,including high instrument costs, the needfor development of instrument-specificanalytical protocols and the need forskilled technicians. Compared with gaschromatography (GC)-MS, therequirements for sample clean-up withLC-MS/MS are only limited, but proteinremoval is mandatory and solid phaseextraction (SPE) or solvent extraction may be necessary for robust and highly

precise quantitative methods. While SPEcan easily be automated by columnswitching and applying permanentlyused extraction columns, manual samplehandling for protein precipitation causesa substantial workload for larger scale LC-MS/MS immunosuppressantmonitoring. Automation of this first stepin sample preparation represents aparticular challenge when using wholeblood, because sedimentation of bloodcells can be observed within a fewminutes, but complete resuspensionmust be achieved immediately beforequantitative sample pipetting.

At the Hospital of the University of Munich,our laboratory was analyzing about 70samples per day for sirolimus andcyclosporin A using one LC-MS/MS system.All sample preparation and instrument

handling for the daily series was performedby one technician, including an on-lineSPE method following manual proteinprecipitation. We decided to also switch ourtacrolimus monitoring from immunoassaytechnology to LC-MS/MS, after implementa-tion of a further LC-MS/MS system.However, since this would requireprocessing approximately 80 additionalsamples per day and since the availabilityof further technicians is critical, we decidedto develop an automated liquid handlingsystem for sample preparation.

Our method allows, for the first time,direct automated processing of largeseries of whole blood samples forimmunosuppressant monitoring by LC-MS/MS; the entire analytical systemproved highly precise and convenient.

Michael Vogeser (left),Institute of ClinicalChemistry, and Ute Spohrer,Hospital Pharmacy, Hospitalof the University of Munich,Munich, Germany

Liquid chromatography with tandem mass spectrometry (LC-MS/MS) is an efficient and powerfultechnology for the routine determination of immunosuppressants in whole blood but, until now,its application has been limited by time-consuming manual sample preparation. Using a TecanFreedom EVO® liquid handling workstation, we have developed an automated sample preparationprotocol for the quantification of tacrolimus in whole blood by LC-MS/MS.

Close up of pipetting needles

10

The system comprises sample dispensingand protein precipitation with theFreedom EVO® 100/4, automated SPE ofdeproteinized samples by on-line SPEwith column switching, and LC-MS/MSanalyte detection with a Waters Alliance2795 HPLC separation module coupled toa Waters Micromass® Quattro Ultima PtTM

MS/MS system. Barcode reading andresuspension of the samples, transfer ofwhole blood aliquots into a deep-wellplate, addition of internal standardsolution, mixing and protein precipitationby addition of an organic solvent are allperformed by the Freedom EVO.The workstation is equipped with a liquidlevel detection system, a clot detectionsystem, a washing station, a chilledreagent carrier for up to six troughs, aplate carrier on a horizontal shaker, anadditional static plate carrier, a barcodereader, sample trays for blood collectiontubes, a liquid handling (LiHa) arm and arobotic manipulator (RoMa) arm. Aftercentrifugation of the plate with a Rotanta460 centrifuge from Hettich (Tuttlingen,Germany), the deproteinizedsupernatants are submitted to on-lineSPE using column switching prior to LC-MS/MS analysis. Throughout theentire process, the only manual actionsrequired are decapping of the tubes andtransferring the deep-well plate from therobotic system to a centrifuge and finallyto the HPLC autosampler (table 1).

Whole blood pools were used to assessthe reproducibility of the entire analyticalsystem for measuring tacrolimusconcentrations. A total coefficient ofvariation of 1.7% was found for the entireautomated analytical process (n = 40).Close agreement between tacrolimusresults obtained after either manual orautomated sample preparation wasobserved. We were able to achievecompletely automated resuspension of

the samples before quantitative pipettingby repeated aspiration and dispensing ofthe sample material with disposable 1 mlpipetting tips. Careful optimization of theliquid handling steps finally resulted in ahighly precise analytical solution,outperforming manual samplepreparation: the intra-assay coefficientsof variation (CVs) for tacrolimus results infive series of samples ranged from 2.4%to 5.3% when the samples were preparedmanually, compared with just 1.0% to1.3% when samples were prepared usingthe automated protocol. The validation of our innovative analytical system wasperformed with tacrolimus as the targetanalyte, but it can reasonably beassumed that similar data will beobtained for the simultaneous analysis ofsirolimus, everolimus and cyclosporin A.

Full automation of immunosuppressantquantification by LC-MS/MS offersimportant improvements with respect toboth the routine laboratory workflow and

the analytical quality. In addition toimproved reproducibility, our automationprotocol avoids the risk of labeling errors,as barcodes can be read from the primarypatients’ samples and the information isdirectly transferred into the LC-MS/MSsample list. Importantly, automation also minimizes the hands-on time ofspecialized technicians and reduces direct handling of infectious material.

MicroMass is a registered trademark and QuattroUltima Pt is a trademark of Waters Corporation


A P P L I C AT I O N D I AG N O ST I C

Full automation of immunosuppressant quantification by LC-MS/MS offers important improvements with respect to boththe routine laboratory workflow and the analytical quality.

Configuration of the Freedom EVO workstation used, including sample trays, washing station, chilledreagent trough holder, tip holder, tip dropping station and horizontal shaker with deep-well plate

11A P P L I C AT I O N D I AG N O ST I C


Table 1:Protocol of the LC-MS/MS front endautomated pipetting system forsample preparation.

Manual step/user interaction:

1. Barcode-labeled whole blood patient samples are placed into 16-position sample trays. Samples are resuspended by gently rotating the traysoverhead. Tubes are decapped manually and the trays positioned into thepipetting system.

2. The safety door is closed and the preparation process is started by giving thenumber of samples loaded to the system software.

Automated pipetting system’s steps methodology example:

3. Sample barcodes are read tray by tray (the trays are moved along a barcode reader).

4. Internal standard solution is pipetted from the respective reagent troughinto the wells of the 96 position 2 ml deep-well plate using separate tips for each well.

5. Each set of four whole blood samples is resuspended simultaneously usingdisposable tips. Firstly, sample is aspirated at the bottom position of thesample tube and dispensed on the upper liquid level with level tracking.Subsequently, sample is aspirated at the upper liquid level and dispensed at the bottom position. These respective two mixing steps are replicated.Aspiration is done slowly, dispense is done rapidly.

6. After changing the tips, sample is pipetted from 50% of the upper liquidlevel into the wells of the deep-well plate in a sequence corresponding to thesample sequence in the trays. After completion of pipetting of the wholeseries, the deep-well plate is shaken.

7. A sample data file is written in Microsoft Excel format, giving the deep-well position and the corresponding barcode-read tube identification for each sample.

8. Using new tips for each well, the precipitation solution is pipetted intosample wells.

9. The deep-well plate is shaken for two minutes.

10. In the second deep-well plate positioned on the instrument, system liquid is dispensed into a number of wells corresponding to the number of samplesprocessed (‘tare-plate’ for balancing the subsequent centrifugation).

Manual step/user interaction:

11. The sample plate is sealed with an adhesive film; sample plate and tareplate are placed manually into the centrifuge and centrifuged for 10 min.

12. The Excel sample list is transferred from the PC of the pipetting system into a template sample list of the MS/MS-system (using a USB memory stick).

12 M I C R OA R R AY


About one in 200 babies worldwide isborn with a chromosomal abnormalityand many of these children have mentalretardation and learning disabilities.Chromosomal imbalances, such asdeletions, microdeletions, translocationsor inversions, underlie some of thesedisorders and the genetic diagnosis ofthese patients has conventionally reliedon cytogenetic chromosomal analysistechniques, such as karyotyping,fluorescence in situ hybridization (FISH) or conventional comparative genomichybridization (CGH). However, the limitedresolution of these approaches meansthat subtle chromosomal rearrangementsinvolving fewer than 5-10 mega base pairs(Mb) cannot be detected, and manyaffected children go undiagnosed.

Over the past few years, scientists havestarted to develop microarray-basedtechnologies, such as array-based CGH(aCGH), to investigate genetic changes in samples from patients with learningdisabilities, since these techniques havethe potential to detect abnormalitieswith much greater resolution thanstandard analysis methods. ConventionalCGH experiments compare genomic DNAfrom patient samples with referenceDNA. The two sets of DNA are labeledwith different fluorochromes andcompetitively hybridized to normalhuman metaphase chromosomes so thatcopy number changes can be easilyidentified. By applying CGH to micro-arrays, the test and reference materialsare hybridized to thousands of definedDNA probes concurrently, so that a highresolution screen of all chromosomes canbe completed in a single, cost effectiveexperiment.

At the West Midlands Regional GeneticsLaboratory, we are using a Tecan HS4800™ Pro hybridization station toautomate aCGH for screening of patientswith learning disability anddysmorphology. The laboratory is thelargest NHS diagnostics genetic unit inthe UK, processing over 27,000 sampleseach year from the local population aswell as from medical institutions in theUK and Europe, covering a full spectrumof diagnoses for genetic diseases.

There are some classic chromosomalabnormalities which we know how todetect, and we largely use FISH for those.But we are particularly interested in thevery large proportion of mentalretardation patients who do not have agenetic diagnosis for their disease, andstarted using aCGH, which is becoming a frontline approach now.

The patients have been referred to thelaboratory following assessment byclinical geneticists and are typicallymoderately affected children withdocumented developmental delays.The extent to which a person is affectedby a genetic copy number imbalancedepends substantially but not wholly onthe exact genetic material lost or gained.Being able to pinpoint the exact geneticchanges in a patient, and knowing thegenes’ functions, might allow doctors to give parents a better prediction of achild’s future development. Ultimatelythis may also impact on treatment.

The application of microarrays in clinicaldiagnostics is fundamentally reliant onaccurate results delivered automaticallywithin a comprehensive quality controlframework. Through their collaboration,

Redefining the genetic basisof learning disabilityDominic McMullan, Principal Clinical Scientist, Microarray Diagnostics,West Midlands Regional Genetics Laboratory, Birmingham, UK

Tecan and BlueGnome have optimizedthe Tecan HS Pro series of automatedhybridization stations and BlueGnomeprotocols for automated processing ofthe CytoChip™ diagnostic bacterialartificial chromosome (BAC) microarrays,which we are using for our screening.

We had some initial aCGH pilot studiesusing manual methods, which sometimesproduced uneven hybridization, and westraightaway saw the need forautomation, largely because of theincreased reliability and reproducibility itoffers. Also, because we are relying oncommercially available BAC arrays, wecannot afford to have any failings withthem - we want to maximize our successrate as far as we can, which is why weturned to the HS 4800 Pro, chosen forseveral reasons. One thing that really

Dom McMullan loads slides into the Tecan HS 4800 Pro

13M I C R OA R R AY


should be detectable, given high qualityraw data. The CytoChip™ is unusual inthat it is tiled with increased BAC densityacross areas of certain well-characterizedcytogenetic syndromes relating torecurrent chromosomal abnormalities.Our laboratory was involved in thisdesign feature, which enables pick-up ofpotentially missed changes as well asdetection of novel changes. In otherwords some patients might not have aclassic phenotype, but may have a classicabnormality. For the patients that we arelooking at, we should have up to a 20%pick-up rate using this technique,according to previously published studies;i.e. up to 20% of those patients shouldhave something that we could not detectwith traditional methods. We have foundthat we are now picking up genomicchanges that previously would not bedetectable; we are almost starting again,in terms of redefining new syndromes,by increasing the resolution.

CytoChip is a trademark of BlueGnome.At present, the HS 4800 Pro is intended to beused for general purpose in Europe and forresearch use only in the USA.

helped us to make the decision is theautomated slide drying, which no otherinstrument on the market has. Afterinjecting our probe, the operator walksaway, leaving the machine to hybridize,wash and dry the slides, ready forscanning, so the whole process becomesless dependent on core lab hours andoperator-related inconsistency. The recentdevelopment of segmented chambersallows us to perform two separatehybridizations on one slide without anycrossover. This is very important for aCGH, because we can test patient- andreference-labeled DNA in opposingfluorochromes on two different arrays,so removing any potential dye effects orbias. The HS Pro is easy to use and itsinherent agitation makes the hybridiza-tion very even across the slide, increasingthe signal-to-noise ratio. The agitationsetting allows more viscous hybridizationbuffers to be used than previously, whichis key, because aCGH buffers containdextran sulfate and so are more viscous.

The CytoChip™ BAC microarrays arespotted with 3,200 BAC clones to give aspatial resolution of approximately 1 Mb;any abnormalities detected by the arrayswe then directly confirm with FISH.Its resolution means that, effectively,there is a BAC for every 1 Mb of thegenome. On current evidence, themajority of pathogenic imbalances

Metaphase FISH image of chromosome 15qdeletion

Plot of aCGH showing chromosome 15qdeletion

BlueGnome CytoChipTM: typical result of adye swap experiment showing a deletionof 7 clones on the p arm of chromosome 6

The HS 4800 Pro

The BlueGnome CytoChip™

14 F O R E N S I C S


It’s 37 meters long but only 4 meterswide; it’s controlled by 27 PCs andincludes eight Tecan Freedom EVO®workstations. It took the project team 23.7man years to complete, during whichtime they traveled over one millionkilometers and spent 4.5 years away fromhome. This is no ordinary laboratoryinstrument, but the new, fully automatedgenetic sample processing system (GSPS),a DNA analysis system that was speciallycommissioned for the Biology Unit of theSouth African Police Service’s (SAPS)Forensic Science Laboratory (FSL).

The SAPS receives crime samples fromacross a country that is twice the size of Texas – with only two laboratories inwhich to analyze them. This has led,unsurprisingly, to a backlog of caseswaiting to be dealt with and the urgentneed for an automated system to processcurrent and past samples. Regular readersof the Tecan Journal may remember theannouncement in 2004 of the award of anEUR 8.35 million tender to Tecan toprovide, install and service this forensicDNA typing system. After years ofplanning, the project was finallycompleted in February 2006 and thesystem began the rigorous validationsrequired before sample processing begins.The GSPS will be able to accept in excessof 800 samples for DNA extraction andfurther downstream processing in a day,significantly improving the speed at whichDNA analysis results can be obtained.

The world’s biggest,single automated forensic analysis system is hungry for samples

Johann van Niekerk, a GSPS Operatorattached to the Technical/SpecialistSupport section of the SAPS FSL BiologyUnit, explained the mammoth scale ofthis project: “We’re moving into a newforensic laboratory in a refurbishedbuilding that was previously the AndrewMcColm Hospital, in Pretoria. This hugearea has been fitted out and transformedto create a laboratory space with, forexample, evidence recovery and manualDNA processing laboratories, administrativeoffices and support space. It also housesthe automated GSPS, which covers threedifferent laboratory spaces over a 40 mlong by 6 m wide footprint.”

Christo Weitz, Project Manager for theGSPS, commented: “With regard to ourrequirements for a robotic sampleprocessing system, we didn’t have to

compromise at all on any of thespecifications, because when we lookedat Tecan’s proposal for the GSPS, it wasabundantly clear that they understoodexactly what it was that we wanted andthey provided us with exactly thatsolution.”

The Tecan SAPS team, headed by BeatGlauser and largely based in Munich, hasbeen working on this project since early2004 and this massive platform washardly a straightforward installation.The 37 m process line inhabits threerestricted access spaces that operate in acleanroom-like manner, each with antechambers and its own separate,climate-controlled environment, toprevent chain of evidence breaches andcross-over contamination – the GSPS-related hardware alone took four weeks

Robot fetches a rack with samples for further processing

One-to-one teaching of the client by a Tecan engineer

15F O R E N S I C S

them into a number of individualprocesses that are often carried out bydifferent operators. This is not only muchmore labor-intensive and timeconsuming, but results in significantdwell times between the differentprocess steps and the resulting variabilitycan severely compromise the data,making them less comparable.

241,791 lines of software code

Tecan provided all the GSPS software,which works on three different levels forcomplete and seamless integration of all the components within workcells,between workcells and across the entiresystem. Tecan’s flexible assay composerand task scheduler (FACTS™) softwareincludes supervisory software for thesystem and control software for individual instruments in addition to theinstruments’ individual software from

to install. However, the biggest challengefor Tecan was the inclusion of a numberof third party instruments, such as DNAanalyzers from Applied Biosystems, intothe GSPS; all of these required full LIMSand control software integration,achieved by the Tecan team throughrethinking, redesign and consultation.The final set-up is documented in 162controlled Tecan documents and,incredibly, 149 third party manuals!

Fitting the pieces together

The complete GSPS is run by 27 PCs andconsists of four 6-axis articulated roboticarms on servo-driven tracks, runningthrough three separate areas that arejoined by inter-laboratory airlocks.The system uses eight Freedom EVOautomated liquid handling workstations,of which four perform cell isolation andDNA extraction steps, two perform PCRnormalization and set-up, and twoperform fragment separation set-up.In addition, there are three thermalcyclers, three Applied Biosystems geneticanalyzers (for fragment separation) andtwo sequence detection systems(for DNA quantification through to real-time PCR) integrated into thestructure. Automated sample storageand retrieval systems also operate withinthe production line, as well as all thenecessary equipment for cell isolation(such as centrifuges), laboratory platesupport (such as Te-Stacks™, shakers andheaters), laboratory plate sealers and foilpiercing instruments, ‘sandwich’ assemblysystems for genetic analyzers, and variousother laboratory essentials, all of whichwere supplied and incorporated into thesystem by Tecan and its localsubcontractor, Vertex Automation.

The GSPS is made up of robotic workcellsthat are fully interlinked throughintegrated ‘islands’ of automation. Fullyintegrating all the processes into a singlesystem is vital not only to the set-up andthroughput, but also to the quality ofdata achieved. Performing the differentanalysis procedures manually separates

their respective vendors; it communicatesbetween the instruments and co-ordinatesall of their activities. A system processsupervisor (SPS) software application wasalso developed and this serves as theLIMS of the facility, interfacing withindividual instruments’ supplied LIMS aswell as pulling all the different instrumentstogether. The SPS also interfaces withSTRLAB, a client-supplied LIMS, whichmanages sample submissions and resultanalysis processes.

“The software is really what makes thiswhole thing tick, and it’s all Tecansoftware,” explained Arnold Greyling,Technical/Support Manager of the SAPSFSL Biology Unit. “We’re using FreedomEVOware® software for the liquidhandling systems, but then each of ourlabs or subportions of the labs is run byits FACTS software and then, to completethe integration between the differentlabs and the FACTS installation, Tecancreated a top level SPS application.”

The system’s dynamic task scheduling isessential for eliminating dwell time andreducing the casework turnaround time,by ensuring that programmed tasks arecarried out at the optimum rate andactively rescheduling according toavailable system resources. Different


A delicate task: The fragment analyzer is loaded by the robot after base plate and top clamp are added

After inversion the separated samples aretransferred to the purification step

16 F O R E N S I C S


parallel processes can be simultaneouslyperformed and continuous loading of plates, use of alternative devices or running emergency samples is stillpossible, without interrupting the process.

The in-built autonomous managementprocess is capable of running for 24 hoursa day, every day of the year, so staff canbe more usefully employed in caseworkmanagement (front end processes) oranalyzing and verifying results and data(back end processes), depending on theirexpertise. This means that automationwill not mean loss of jobs for staff at theFSL – if anything, the laboratory mayneed to employ more people, as DirectorPierre Joubert, head of the SAPS FSLBiology Unit, explained: “The kind of staffthat were previously responsible for themain day-to-day activities, such as DNAextraction or PCR set-up, can beredeployed to perform evidence recoveryand sample submission to the GSPS - thesharp end of the system. We actuallyneed more people just to feed thesystem! The beauty of it is that many of our staff will now be given moreinteresting things to do, it’s about lettinghumans do what they do best and lettingmachines do what they do best.”

to minimize cross-contamination: therobotic sample indexing lab, the DNAextraction and PCR set-up lab, and theDNA amplification and fragmentseparation lab. It is vital that everysample transfer step is carried out in a safe, accurate and precise manner inorder to minimize cross-contaminations.

Area 1: Robotic sample indexing

Samples are initially transferred intobarcoded, robotic sample indexing tubesand then sorted according to type(e.g. semen, or blood and similar bodilyfluids) by a selective compliancearticulated robot arm (SCARA). Precisesample sorting is a critical first step andit must ensure that samples fromrecovered evidence are securely andaccurately transferred to predefined welllocations in laboratory sample plates fordownstream automated processing,while maintaining full sample traceability.

Area 2: DNA extraction and PCR set-up

Samples are transported to the secondlaboratory area, where cellular materialfrom the samples is resuspended into asecond plate and lyzed for DNA isolation.The samples from two 48-well plates arethen transferred into a 96-well plate forDNA extraction. These steps are designedto achieve reliable, automated DNApurification from up to 96 forensicsamples. The technologies used provideuniform DNA recovery and purity acrossthe 96-well plate, with no detectable

Helping to reduce crime

A smaller scale automated sampleprocessing system has also been builtin South Africa, which will deal with DNAtyping of reference blood samples only,while this new GSPS is expected to processthe majority of the country’s crimesamples. It will bring a number ofimportant advantages to the SAPS, inaddition to massively increasing the FSL’ssample throughput and improving thequality of the analyses. The improvedcasework turnaround times will help toproduce investigative leads from cold hitsas well as minimizing a criminal’s potentialwindow of activity, allowing speedierapprehension of criminals, exoneration ofthe innocent, prevention of crime and, inthe long term, considerable cost saving.

In one end...The processing line as a whole consists of four primary processes: robotic sampleindexing; cell isolation, differential lysisand DNA extraction; PCR normalizationand set-up; DNA amplification andfragment separation. The system runsacross three defined laboratory areas for the different steps, in order toaccommodate sample traceability and

The inverter wheel is loaded with lyzed samples

Robot loads a plate into the air lock to send it tothe next laboratory


17F O R E N S I C S

cross-contamination between samples,allowing effective removal of PCRinhibitors. During these processes, therobots need to be able to repeatedlydeliver samples to a range of high densitymicroplates; provide full flexibility forperforming complete processes includingplate replication, serial plate dilutions andin-plate serial dilutions; and provide fastand automated interchange betweenfixed tips and disposable tips accordingto the process required. Real-time PCRset-up is performed through anautomated pipetting system, whichallows reproducible delivery of templateDNA, DNA primers and other components(buffers, nucleotides) that are typical of a DNA amplification reaction mix.DNA solutions are automaticallynormalized to a constant concentrationbased on information obtained throughreal-time PCR.

Area 3: DNA amplification and fragmentseparation

The plates undergo DNA amplificationusing thermal cyclers and fragment

separation processes. For real-time PCR,the quantitative relationship betweenthe amount of starting target sampleand the amount of PCR product at anygiven cycle number is determined bydetecting specific nucleic acid amplifica-tion products as they accumulate in realtime. For the GSPS, this process has to berobotic-amenable and capable of worklistimport and data export capabilities.Fragment separation is carried out usingcapillary electrophoresis, with anautomated pipetting system that allowsreproducible delivery of amplified DNA,other components (buffers, internal lanestandards, formamide) and sizestandards, in a predetermined pattern.Fragment separation plate assemblyrequires an instrument to assembleelectrophoresis ‘sandwich’ platescontaining amplified DNA into a carrierplate. Fragment separation data are thentransferred for analysis and verified,and/or transferred for repeat or newanalyses. A number of secondaryprocesses are also involved, including a sample storage, retrieval and delivery

system; short-term sample archivemanagement; robotic sample conveyance;inter-laboratory pass-through chambers;lab plate cover management and anemergency shut-down and recoveryprocedure.

…and out the otherThe DNA profile generated by the systemis then analyzed by humans and used forpolice work and court cases. Once theresults are approved, the so-called expertwitnesses - the same people that initiallyprepared the samples - will present thedata in court.

Lysis buffer is added to the samples

The RSI workstation is busy sorting the samples

18

F O R E N S I C S


South African Police Service’s project number crunching8.35 million: number of Euros provided by the EU to fund the project, to reduce crime in South Africa

~10 million: number of Euros spent by the SAPS to refurbish the GSPS building and set up the biology unit

1.1 million: number of Euros VAT it cost to pay for the 9.6 tons GSPS at customs

1 million: number of kilometers the Tecan SAPS team traveled during their 65 trips between South Africa andGermany (equates to 27 times around the world)

950: number of nights the Tecan SAPS team spent in South Africa

~700: number of long nights the Tecan SAPS design team spent setting up the system in Munich

151,192: total lines of code used to program the entire GSPS

65,120: number of lines of comments in the GSPS software

25,479: number of empty lines in the GSPS software

828: number of DNA samples that the GSPS will process each day

200: maximum number of DNA samples that can currently be processed daily, using manual methods

149: number of third party instrument manuals for the GSPS

51: number of crates needed to pack up the GSPS in Munich to fly to South Africa

23.7: number of man years that were worked by the Tecan SAPS team, from date of the contract signature (3 March 2004) until the system was passed to the SAPS for operational qualification (3 February 2006)

9.2: number of man years that were needed to write the system’s control software

80: number of laboratory technicians that will prepare, register and load the samples into the GSPS

12: number of results analysts that will be studying the data

5: number of laboratory staff that will restock the system each day (three hours per day)

1: number of laboratory staff that will be required to run the entire system

0: number of hairs the project manager has left on his head after completing the project!

SAPS Team Event

To celebrate the SAPS installation havingsuccessfully reached the OperationalQualification milestone, where the systemis handed over to the client, the core teamof people working on the South Africanproject met in a restaurant in Mollis, inthe valley of Glarus, Switzerland, early oneFriday afternoon in June.

All the team had been told was that theyshould arrive on time and bring sunglasses,sunscreen and a camera. After welcomingeveryone we drove on for another fiveminutes and parked our cars in front of the little airport where a helicopter wasstanding in front of a hangar.

Everyone’s eyes widened. Is it reallywaiting for us? YES! After all thoseaeroplane trips between Germany andSouth Africa, this was somethingcompletely different. We were taken byhelicopter in two groups to the PlanuraHütte, in the middle of an alpinesurrounding, where the host of the hutwas already expecting us.

After enjoying food, drinks and the viewit was time to return: there was a quickannouncement on the radio and, shortlyafterwards, the silence was broken by theflap-flap-flap of the helicopter. Feelinglike true VIPs we flew back to the base.


19F O R E N S I C S

From there we took our cars to the hotelin Wädenswil, where we checked in andproceeded to Wädi-Bräu, the localmicrobrewery. The brew master agreed to broaden our (already profound)knowledge on liquid handling and, inbetween the explanations, we returnedto the restaurant for our next course.A bit late (and with a beer or two) wethen returned happily to the hotel.

What a day!

A job well done!

20 A P P L I C AT I O N B I O P H A R M A


Automating metabolicinhibition assays - on a nanoscale

Brett Litten is a senior DMPK scientist andautomation specialist at AstraZeneca’sAlderley Park site in Cheshire, UK, wherehe is jointly responsible for newtechnology and the design and deliveryof various automated in vitro assays andin vivo related processes within thedepartment. The company has significantlyinvested in Tecan’s technologies over theyears and one of Brett’s latest projectshas been to integrate a Nanodrop™ IIeight-channel nanopipettor fromInnovadyne Technologies with a TecanGenesis RWS™ 200 Workstation andTecan Safire2™ microplate reader.

This configuration is being used toincrease throughput in the cytochromeP450 inhibition assay. In this assay, theactivity of five major human cytochromeP450 enzymes (CYPs) is determined usinga number of substrates, which aremetabolized to fluorescent products.Inhibition of the CYPs results in adecrease in the amount of fluorescencemetabolite formed and, by using a rangeof test drug entities, the potency ofinhibition (IC50) can be assessed. Brettintegrated the Nanodrop™ with the Tecanworkstation in order to miniaturize thisassay for use with 1536-well plates, as heexplained: “We required a higher platedensity to help reduce assay costs andincrease throughput. The Nanodrop™allows us to use a 1536-well plate suchthat a greater number of drug entities atdifferent concentrations can be testedagainst the major P450 enzymes perassay. Furthermore, using 1536-well platesleads to a reduction in the number oftimes the assay is run per week for ourcurrent research purposes and, in turn,frees up more resources. The process isfully automated, including fluorescenceanalysis with the Safire2 plate reader, soour researchers can get on with othertasks during the assay.”

Innovadyne’s Nanodrop™ II is a smallscale, high accuracy, high precision nano-dispenser, which is able to efficientlyhandle very small volumes of liquid, as lowas 100 nl. The instrument will also happilydispense microliter-range volumes, so ithas quite wide-ranging uses.

“The Nanodrop™’s attraction is that itextends the current usable range of ourTecan workstations, which are generallyspeaking of a larger volume criterion.Due to the Nanodrop™’s small size, it iseasy to install into an existing robotplatform,” said Brett. “It is very simple tointegrate, and uses a combination oftraditionally robust syringe aspirationcoupled to Innovadyne’s micro-solenoiddispense valve assembly for deliveringaccurate quantities of liquid.”

In order to run the assay, the 1536-wellplates are loaded on to the Tecan work-station with the manually preparedreagents and enzyme solutions.The workstation’s Gemini™ software is then used to execute the assay,controlling the various sequences for theNanodrop™’s processes, including spikingthe wells with 100 nl of test compound.Once the Nanodrop™ has prepared theplates, the Tecan robotic manipulator(RoMa) arm transfers them to the Tecanincubator, also housed on the system,

AstraZeneca is one of the world’s leading pharmaceutical companies focused onproducing effective medicines that make a real difference in important areas ofhealthcare. In today’s environment, the constant streamlining of laboratoryprocesses in order to increase efficiency and save valuable resources is an importantaspect for such companies, and taking advantage of the latest advances inautomation technologies can help to get ahead.

The Nanodrop™ II


21

for necessary preincubations andincubations. The RoMa arm can thenremove the plates after the requiredreaction time and place them on thespecified site on the Nanodrop™ for thenext steps before finally transferring theplate to the Safire2 for detection.

The Safire2 is a double monochromatorplate reader that is capable of reading96- to 384- and 1536-well plates influorescence, fluorescence polarization,absorbance and luminescence modes.The metabolic inhibition assay usesfluorescence substrates that aremetabolized by the enzymes and producea fluorescent signal, which can bequantified to indicate the potency ofthe test compound to inhibit theenzyme-substrate metabolic reaction.

“We chose the Safire2 because we neededa detection system that could read 1536-well plates in a very short space oftime, so we could minimize evaporationand keep the automated process as fastas possible,” Brett explained. “We haveactually integrated it so it is physicallyconnected to our Tecan workstation andis directly accessed by the RoMa arm.The Safire2 is the fastest unit I’ve everused for reading plates, capable ofreading 1536-well plates in just37 seconds! We read our 1536-well plates using several fixed wavelengthmethods to accommodate the differentexcitation-emission wavelengthsrequired for our assay.”

Once the plate is read, the rawfluorescence data are stored to a text file

and automatically transferred to anetwork server from where analysts canprocess the data to generate inhibitionIC50 values.

Since Brett’s original bespoke integrationof the instruments, Tecan and Innovadynehave been working together to produceoff-the-shelf systems where theNanodrop™ is fully integrated withTecan’s Freedom EVO® liquid handlingworkstations. These systems will besuitable for diverse assay processesrequiring smaller volumes – for furtherinformation please contact MatthewWebb, Tecan UK Limited.

Nanodrop™ is a trademark of InnovadyneTechnologies, Inc.

“The Safire2 is the fastest unit I’ve ever used for reading plates, capable of reading 1536-well platesin just 37 seconds!”

A P P L I C AT I O N B I O P H A R M A

Brett Litten and the Genesis 200 workstation The Nanodrop™ II and the Safire2 on board theGenesis 200

22 A P P L I C AT I O N B I O P H A R M A


TILLING in Dresden:large-scale detection of singlenucleotide polymorphisms

TILLING (Targeting Induced Local LesionIN Genomes), a powerful reverse geneticstool, was developed to identify singlenucleotide polymorphisms (SNP) inmutagenized individuals by PCRamplification of a gene of interest fromeach individual, followed by SNPdetection1,2. However, because theidentification of nonsense mutationsleading to loss of gene function is arelatively rare event, it is necessary toscreen thousands of mutagenizedindividuals to find a presumptive nullmutation in a gene of interest. TILLING is mainly based on PCR followed byenzymatic digests or direct sequencing of PCR-fragments, and is thereforeperfectly suited to automation. At theMPI CBG, we chose the Tecan GenesisRWS™ 200 workstation to automate allpipetting steps.

Our current set-up consists of a liquidhandling arm with eight Teflon®-coatedtips (for 384-well plates), water-cooledtroughs for all reagents and cooledcarriers that represent the pipettingplatform, a robotic manipulator (RoMa)arm and hotels for PCR-plate storage.The RoMa arm transfers PCR platesbetween an integrated thermocycler withfour 384-well blocks, a cooled pipettingplatform and a cooled incubator. Pads in

the motorized lids of the PCR machinesseal microplates and prevent crosscontamination of samples.

Mutagenesis and library generation

The basis of a successful TILLING projectis the generation of libraries of heavilymutagenized individuals. Alkylatingmutagens such as ethylnitrosourea (ENU)or ethane methyl sulfonate (EMS) caneasily be applied to zebrafish, fruit fliesand nematode worms, inducing pointmutations that are randomly distributedthroughout the genome of treatedindividuals. By crossing these mutagenizedanimals to untreated individuals theinduced SNPs are propagated to the nextgeneration, producing large numbers ofindividuals carrying new mutations.A classical TILLING library consists ofseveral thousand live mutagenizedanimals (zebrafish and Drosophila) orrecoverable frozen individuals (C. elegans),and their corresponding genomic DNA.All genomic DNA preparations werecarried out in deep-well 96-well plates,diluted and dispensed as 5 μl aliquotsinto 384-well plates (DNA concentrationof about 0.2-1 ng/μl). The plates werestored frozen and could be used directlyfor PCR. Simple washing steps of theTeflon® coated tips with system waterprevents cross contamination whenpipetting different templates.

Screening procedures

For zebrafish projects, we screen using anested PCR approach originally describedby Wienholds et al.2 In this approach, thenormalization step is at the PCR level and not at the level of the genomic DNA,which was a factor at the start of theproject when photometric plate readerswere not available at the MPI in Dresden.Again, all pipetting steps are performedautomatically. The inner primer pairs inthe nested step contain universal tails,which serve as templates either foruniversal primers that are labeled with fluorescent dyes or, alternatively,as direct templates for universalsequencing primers.

The first PCR is set up to a total volumeof 10 μl in 384-well plates containing thegenomic template DNA. For the secondround of PCR, appropriate amounts of the products from the first PCR aretransferred to fresh 384-well plates thatalready contain the mastermix for thesecond PCR. For SNP detection by directsequencing we check the quality of thesecond PCR products by agarose gelelectrophoresis beforehand. Primers and dNTPs were removed anddephosphorylated by Exonuclease I /shrimp alkaline phosphatase incubation.Again, the Genesis 200 performs allpipetting steps, and the microplates are

(from left) Sylke Winkler,Claudia Seidel and Nicola Gscheidel

Sylke Winkler, Claudia Seidel, Stefan Meissner,Nicola Gscheidel and Michael BrandBiotechnology Centre and Department of Genetics, DresdenUniversity of Technology (TU Dresden) and Max Planck Institute of Molecular Cell Biology and Genetics (MPI-CBG), Dresden

23A P P L I C AT I O N B I O P H A R M A

manually loaded onto the eight 384-wellPCR blocks as they become available.Our current capacity, including qualitychecks by agarose gel electrophoresis,is 3,072 samples in 1.5 working days.

For the Drosophila and C. elegans projects,we follow the TILLING protocol asdescribed by Colbert et al.3 After the firstPCR is performed and diluted in the sameway, the second PCR is run with tailedinner primers and fluorescently labeledprimers (LI-COR™ IRDye700® andIRDye800®), resulting in labeled PCRproducts which can be pooled up tofourfold, denatured and re-annealed toinduce heteroduplex formation betweenmutated and wild type PCR products.Heteroduplexes are cleaved by theendonuclease Cel-1, and the resultingfluorescently labeled cleavage fragmentsare purified and separated by highresolution SDS-PAGE on slab gelsequencers. Using Gemini™ software, afully automated protocol was set up forthis TILLING assay, which includes manydifferent pipetting steps. The RoMa armtransfers PCR-plates between thethermocycler blocks and the pipettingplatform, and all mixes are madeavailable in cooled troughs. Four 384-wellplates (1,536 samples) could be handledwithin 11 hours, plus an additional two to three hours purification time.

The detection of presumptive mutationsrequires automation of a screening set-upto allow the handling of huge numbersof samples. For example, with heavilymutagenized zebrafish about four to fivemillion base pairs have to be screened toisolate a relatively rare nonsensemutation (stop codons account for 5% ofall SNPs). We are currently able to handlemore than 9,000 samples per weekwhich, with a modest detection rate of500 base pairs per screening round,equates to 4.5 million base pairs ofgenomic information per week. Therefore,our set-up allows the identification ofone nonsense mutation plus manyadditional missense mutations eachweek, and demonstrates the suitability ofan automated TILLING approach toroutinely generate mutants for functionalgenomic studies.

Teflon is a registered trademark of E.I.Dupont.IRDye is a registered trademark of LI-COR, Inc.

Literature

1. McCallum, C.M., Comai, L., Greene, E.A.,Henikoff, S.: Targeted screening for inducedmutations. Nature Biotechnology 18:455 (2000).

2. Wienholds, E., Schulte-Merker, S.,Walderich, B., Plasterk, R.H.A.: Target-selected inactivation of the zebrafish rag1gene. Science 297: 99 (2002).

3. Colbert, T., Till, B.J., Tompa, R., Reynolds, S.,Steine, M.N., Yeung, A.T., McCallum, C.M.,Comai, L., Henikoff, S.: High-throughputscreening for induced point mutations.Plant Physiology 126: 480 (2001).

4. Winkler, S., Schwabedissen, A., Backasch,D., Bökel, C., Seidel, C., Bönisch, S., Fürthauer,M., Kuhrs, A., Cobreros, L., Brand, M.,Gonzalez-Gaitan, M.: Target-selectedmutant screen by TILLING in Drosophila. Genome Research15: 718 (2005).

For review: Stemple, D.L.: TILLING – a high-throughput harvest form functionalgenomics. Nature Reviews 5: 1 (2004).

The Dresden TILLING project was initiated as a collaborative effort for zebrafish Danio rerio(Michael Brand, TU Dresden and MPI-CBG;Carl-Philipp Heisenberg, MPI-CBG), for thenematode worm Caenorhabditis elegans(Anthony Hyman, MPI-CBG) and for the fruitfly Drosophila melanogaster 4 (MarcosGonzález-Gaitán, MPI-CBG). Worktable and PCRintegration were designed with support fromHannes Grabner and Jan Wagner from theTechnology Development Studio (TDS) of theMPI-CBG. The Dresden TILLING project isfinanced by the Sixth Framework Programme of the European Commission ‘Zebrafish Modelsfor Human Development and Disease’(ZF-MODELS), the Dresden University ofTechnology, and the Max Planck Institute of Molecular Cell Biology and Genetics.


Close up of the eightchannel liquid handlingarm pipetting into a 384-well plate

Sylke Winkler and theGenesis 200

The ScreeningTool was developedthrough a collaboration between npielectronic and Prof Steffen Hering fromthe Department of Pharmacology andToxicology at the University of Vienna,who originally invented the miniaturerecording chamber (patent pending).It was designed to automate thestandard, two-electrode, voltage clampscreening procedure in a 15 μl bath,covered by a glass plate with a funnel forprecise, automated drug application bythe liquid handling system. This set-upallows very fast solution changes andrequires only minimal amounts of samplefor the procedure. The design depends on

one of npi electronic’s TURBO TECamplifiers, which are specially developedfor recording of bioelectrical signals fromXenopus oocytes, and Tecan’s MSP9250liquid handling system.

“The Tecan liquid handling system is idealfor the ScreeningTool because it has allthe necessary features. In particular, thetime it takes to apply the drug and thetime it takes in between applications isvery short,” explained Mr Hans ReinerPolder, managing director of npi electronic.“Automating the drug application helpsto get more reproducible data as thereliability of the instrument, based on thefeatures of the Tecan liquid handling

system, is excellent. The uptake of thedrug into the pipette and the wash-inand wash-out procedures are also fullyautomated, further increasing thereliability. This completely avoids cross-contamination between individualapplications and the quality of theexperiment is improved considerably. Thesystem can be easily programmed by thecustomer, using scripting language thatwas developed in cooperation withTecan.”

Using the Tecan liquid handling systemmeans that only the pipette tip headneeds to be filled with solution (~100 μl),so the ScreeningTool requires justfractions of the amount of sample thatother instruments need to perform acomplete experiment. All alternativeinstruments are based on tubing, whichhas to be filled with solution, resulting ina lot of sample wastage and considerabletime delays. Another important feature isthat this system can be programmed toensure that cells are always kept at thesame ionic milieu in between applications,which increases the reliability of theexperiments as well as maintaining thecell’s condition. Instead of using only afew solutions for one cell, more than ahundred solutions can be used on one single cell, allowing many moreexperiments to be performed in a shorterperiod of time.

24 CO M P O N E N T S


npi electronic has revolutionized automated drug screening with the launch of theScreeningTool, an automated system for screening of voltage- or ligand-gated ionchannels in Xenopus oocytes, with millisecond resolution and high speed voltageclamping. The innovative instrument depends on a Tecan OEM liquid handlingsystem for automated drug delivery, integrated with a miniature chamber designand unique amplifier to provide unprecedented speed, reproducibility and efficiencyfor high quality drug screening applications.

High performanceion channel screening

hERG channel inhibition by 10 μM Amiodarone (black filled circles) at 1 Hz


25CO M P O N E N T S

“Combining the Tecan MSP9250 with Prof Hering’s special chamber makes thisinstrument unique and very specializedfor its applications; there is no competinginstrument that could fulfil all theseparameters at the same time,” said Hans Reiner.

Voltage clamping is performed usingnpi’s TURBO TEC series of amplifiers,as Hans Reiner explained, “The npiamplifiers are based on a unique,sophisticated design that we developedyears ago and their high speed ofresponse makes them superior to similarsystems. The amplifiers also have a veryhigh degree of precision, so they are idealfor Prof Hering’s chamber design.”

The ScreeningTool’s flexible fluidapplication and high performancevoltage clamp make it highly suitable fordrug screening on ligand and voltagegated ion channels. The system isspecifically designed for Xenopus oocytes,which are widely used for drug screeningand for pharmacological safety tests.Xenopus oocytes are often used toanalyze the interaction between certain

drugs and ion channels or receptors thatcould be expressed in the oocytes, such as investigations of the hERG channel - a special potassium channel derived fromthe heart. Some drugs affect this channeland alter the heart beat, causingarrhythmia, and such cases would notnormally be suitable for furtherdevelopment.

This is the first time that npi electronichas used a Tecan OEM component for oneof their products and Hans Reiner is veryimpressed with the collaboration service,“We had a lot of support from Tecan andestablished a good relationship, whichhelped us to produce a professionalversion of the software that is suitablefor use in the pharmaceutical industry,within a very short time period.”

“In the future we plan to develop otherversions of the ScreeningTool designedfor different cell types and are veryinterested to continue the collaborationwith Tecan. We had a very goodexperience with this product and I havealready recommended Tecan to all ourpartner companies.”

“We had a very goodexperience with this product and I have alreadyrecommended Tecanto all our partnercompanies.”

Automated, high speed ion channel screening with millisecond resolution The Screening Tool is automated using Tecan’s MSP9250

Managing lead optimizationcompounds with REMP technology

“Our automation laboratory servesprincipally the lead optimization stage ofdrug discovery, so we receive severalhundred samples per week frommedicinal chemistry labs,” said Dr ClaudeDufresne, Senior Investigator, who hasbeen working at the Merck site since1988 and has been developingautomation methods and systems formost of the past ten years. “From thosesamples, we prepare serial dilution platesfor distribution to various biological assaylaboratories and for our own uses.We also store the samples in order tosupply biology labs with samples forretesting and secondary assays; that’swhy we needed the SSS.”

The samples are usually chemicalcompounds that have been synthesizedas part of particular research programsand are based on lead compounds thatmay have come out of a high throughputscreening campaign. The samples mightbe part of a chemical series based onthese structures and evolved throughtraditional medicinal chemistryprocesses.

“We receive compounds from most of the research programs at our site, so thesamples relate to a range of differentdrug discovery programs,” Dr Dufresneexplained. “Some of our principalresearch interests include diabetes,obesity and atherosclerosis, for example.

26 S A M P L E M A N AG E M E N T


The central automation facility was createdin 2005 to provide, in part, an integratedcompound management service for theMerck Rahway Site. A robust and efficientsample storage and retrieval system is avital part of the service.

The Rahway Facility for Automation and ScreeningTechnologies, which is based in New Jersey, USA,and is part of Merck Research Laboratories, is using aREMP Small-Size Store™ (SSS) to manage its thousands of samples needed for drug discovery investigations.

Dr Claude Dufresne

We collaborate with scientists in specifictherapeutic areas not only by providingthe sample management service, but alsoby assisting with assay development,optimization and implementation onautomated systems. In this function, wealso carry out some of the primary assaysin support of medicinal chemistry.”

The automation laboratory manages itscompound storage entirely throughREMP technologies, including the SSSwith its in-built Tube Punching Technology,the REMP 96 Tube Technology™ con-sumables, the 2D Code Scanner, and theeight position manual capper/decapper(REMP MCD8™), which the scientists havealso automated using their ownengineering and software.

“Initially it was the 96 Tube Technologythat really attracted us, and then addingthe SSS to make use of this technologywas a real bonus,” Dr Dufresne said.“The REMP mini-tube and Tube PunchingTechnology is really a breakthrough forsample storage. Traditionally, compoundswould be stored in deep-well plates,meaning that if you needed to cherry picksamples, then you would need to useliquid handling to aspirate samples fromindividual wells. By using the SSS with itsin-built Tube Punching Technology wedon’t need this step, we can just punchout the tubes that we want straight intoa delivery rack, meaning that in one

system we can physically retrieveindividual samples, as opposed toretrieving whole plates of samples.”

Using the SSS means that cherry pickingoccurs within the environmentallycontrolled storage system, so there are no freeze/thaw cycles on samples, andstorage temperatures ranging from -20˚C, +4˚C or even more ambienttemperatures, such as the 18°C used atRahway, can be used.

“When samples are stored in vials, inboxes or racks, and placed in fridges orfreezers, the management of that samplecollection is a significant time drain,”continued Dr Dufresne. “It can easily take hours a day for someone to gothrough the activities of maintaining andretrieving individual samples, just for thedaily work - but this all goes away withautomated storage!”

REMP Tube Technology Consumablesconsist of individually sealed or cappedtubes secured within their own TubeRack. The tube transfer technologygreatly improves the reliability of tubehandling and the unique punchingtechnique greatly reduces handling errorrates, because the transfer is performedby a single axis movement.This technique is faster and more reliablecompared to conventional pick and placemethods, and also eliminates potentialcross-contamination, exposure to air ordilution effects, which are often seenwith samples undergoing traditionalliquid transfer. The tubes virtuallyeliminate the risk of sample degradationand can be either individually sealed, forsingle use, or capped in an automated ormanual fashion for multiaccess.

The scalability of the SSS means that upto two extension units can be added tothe SSS, tripling the storage capacity andthis is also important for the Rahwayautomation laboratory, as Dr Dufresneexplained: “Another big attraction of theREMP technology for us was size; weneeded a really small store, because wedid not have much spare lab space at thetime, and our needs in supporting thelead optimization could be met with justone or two of these units. Our currentlevel of storage is only a few thousandsamples, although it is now growingquite rapidly as we are beginning to storemultiple concentrations and multiplecopies of each sample. We use these unitsfor short term storage of samplesinvolved in active projects as, at the endof a project, all of the samples relating tothat project will go to the main samplerepository where millions of compoundsare stored. Since it is very difficult topredict usage levels down the road as wecontinue to transform our researchoperations, it is comforting to know thatdoubling or tripling our local storagecapacity would be an easy upgrade.”

The above text does not imply endorsementof Merck & Co.


27S A M P L E M A N AG E M E N T

REMP Small-Size Store

REMP Tube Technology

28 E V E N T S 2 0 0 6

Tecan Journal 3/2006 www.tecan.com

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Meet Tecan at these events before the end of the year

Tecan Journal, Customer Magazine of Tecan Trading AG., ISSN 1660-5276Design: OTM/London www.otmcreate.comPhotography: Marc Wetli/Zürich www.wetli.com, Günter Bolzern/Zürich www.bolzern.net,Susanne Völlm/Zürich www.susannevoellm.ch, Beat Glauser/www.beatglauser.comEditor: kdm/UK www.kdm-communications.comPrint: DAZ Druckerei Albisrieden AG/Zurich www.daz.chAddress: Tecan Switzerland AG, Marketing Communications, Seestrasse 103, CH-8708Männedorf, Switzerland, [email protected],www.tecan.comTecan Group Ltd. makes every effort to include accurate and up-to-date information within thispublication, however, it is possible that omissions or errors might have occurred. Tecan Group Ltd.cannot, therefore, make any representations or warranties, expressed or implied, as to the accuracyor completeness of the information provided in this publication. Changes in this publicationcan be made at any time without notice. All mentioned trademarks are protected by law.

For technical details and detailed procedures of the specifications provided in this documentplease contact your Tecan representative.This brochure may contain reference to applications and products which are not available in allmarkets. Please check with your local sales representative.Cellerity, Columbus, FACTS, FE500, Flask Flipper, Gemini, Genesis, Genesis RSP, Genesis RWS, GENios,HS 4800, i-control, Infinite, Logic, Magellan, Safire2, Sunrise,Te-Stacks and Te-PoolSafe are trademarksand Freedom EVO, Freedom EVOlyzer and Freedom EVOware are registered trademarks of Tecan Group Ltd., Männedorf, Switzerland.Tecan is in major countries a registered trademark of Tecan Group Ltd., Männedorf, Switzerland.MCD8, Small-Size Store and Tube Technology are trademarks of REMP AG, Oberdiessbach,Switzerland.© 2006 Tecan Trading AG, Switzerland, all rights reserved.

Headquarters:Tecan Group Ltd., Seestrasse 103, CH-8708 Männedorf, SwitzerlandT +41 44 922 88 88 F +41 44 922 88 89 [email protected] www.tecan.com

China

CCLab 2006 (with VASTEC) Qingdao Sept 15-19 2006

Drug Discovery and Development Shanghai Nov 20-21 2006

Europe

50° Siga Annual Congress Ischia Sept 10-14 2006

IFR 128 Journees Screening Strasbourg Sept 11-18 2006

Conferences on arrayCGH and Molecular Cytogenetics Leuven Sept 13-16 2006

4th International Forum Life Science Automation Rostock Sept 14-15 2006

Biospain Biotec Madrid Sept 18-20 2006

DGTI and ISCT Europe Frankfurt Sept 19-22 2006

Biotech Forum and Scanlab Copenhagen Sept 26-28 2006

The Lausanne Genomics Days Lausanne Oct 5-6 2006

1st Fraunhofer Life Science Symposium Leipzig Oct 22-24 2006

Het Instrument Utrecht Oct 30- Nov 3 2006

Medica Düsseldorf Nov 15-18 2006

Japan

Genomics Drug Discovery Key Technology 2006 Kyoto Sept 19 2006

Japanese Association for Food Immunology Tokyo Oct 23-24 2006

Japanese Society for the Study of Xenobiotics Tokyo Nov 29-Dec 1 2006

USA

Society for Biomolecular Sciences (SBS) Seattle, WA Sept 17-21 2006

17th International Symposium of Human Identification (ISHI) Nashville, TN Oct 9-12 2006

American Society of Human Genetics (ASHG) New Orleans, LA Oct 9-13 2006

Neuroscience Atlanta, GA Oct 14-18 2006

American Association of Blood Banks (AABB) Miami Beach, FL Oct 21-24 2006

American Society of Cell Biology (ASCB) San Diego, CA Dec 9-13 2006

Tecan Journal Edition 03/2006

Documents

Transcript of Tecan Journal Edition 03/2006