Tecan Journal Edition 03/2006

ISSN 1660-5276

Tecan Journal Edition 3/2006

The world’s biggest, single automated forensic analysis system page 14-19

Redefining the genetic basis of learning disability page 12-13 Cover shows Caroline Huber, Product Manager Clinical Diagnostic and Friedrich Jost, Head Software Development Clinical Diagnostic

TILLING in Dresden page 22-23

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W E LCO M E

Delighting our customers is at the heart of Tecan’s activities

Tecan strives to not only meet our customers’ expectations, but to exceed them, day in, day out.

Our customers’ needs are the driving force for everything we do at Tecan from our dedication to quality and the development of new and innovative solutions for our customers’ challenges, to fielding the highly skilled service force that our customers rely on throughout an instrument’s lifetime - all of us at Tecan strive to not only meet our customers’ expectations, but to exceed them, day in, day out.

Of course, we cannot do this alone, and we rely heavily on you, our customers, to help us understand your needs and expectations, and guide us in reaching our goals. This partnership, in many ways, is reflected in this, the third edition of the Tecan Journal for 2006, which is also our largest issue ever. We are proud to bring you reports from the recent customerfocused events in China and Japan, and present a number of innovations developed in association with customers to meet specific application needs, including nanoscale automated liquid handling and a platform for automated ion channel screening.

Throughout more than 25 years in business, we have been proud to be the partner our customers trust and it was a special privilege to develop the new DNA sample processing system with the South African Police Service, highlighted in the following pages. Installation was completed earlier this year and we are delighted to be able to share with you some details of the system which, at nearly 40 meters long, is the world’s largest single automated forensic analysis system.

Finally, to help us better understand you, our customers, and enable us to focus our efforts more effectively, we will shortly be contacting some customers with a survey.

Should any of you be contacted for this effort, I would personally very much appreciate you taking a brief moment to let us know exactly where we stand.

We hope you enjoy this issue of the Tecan Journal and look forward to continuing working with you to meet your needs.

Thomas Bachmann Chief Executive Officer (CEO)

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CO N T E N T S

Delighting our customers Thomas Bachmann, CEO, thanks you, our customers page 2 Latest products: Tecan launches Infinite™ F500 The latest addition to Tecan’s high end detection instruments

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

Meeting the directors of the blood banks in South West China A great opportunity for Tecan to make contact with blood banks in the region page 7

Automating metabolic inhibition assays – on a nanoscale Integrating a nanopipettor with a Tecan workstation and Safire2™ microplate reader for miniaturized assays pages 20-21

The Genesis FE500™ Taking control of sample preparation page 8

Automated processing of whole blood samples for monitoring of immunosuppressants by LC-MS/MS Tecan’s Freedom EVO can be used for the quantification of tacrolimus by LC-MS/MS pages 9-11

Redefining the genetic basis of learning disability Dominic McMullan, Principal Clinical Scientist, reports pages 12-13

TILLING in Dresden Large-scale detection of single nucleotide polymorphisms pages 22-23

High performance ion channel screening npi electronic has revolutionized automated drug screening pages 24-25

Managing lead optimization compounds with REMP technology Using REMP’s Small-Size Store™ to manage thousands of drug discovery samples pages 26-27

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

The world’s biggest, single automated forensic analysis system is hungry for samples pages 14-19

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

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

Tecan’s latest products Tecan launches the new InfiniteTM F500 high-end microplate reader The 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 and modularity of Tecan’s highly successful GENios Pro™ and GENios Ultra™ systems.

This new multifunctional microplate reader provides outstanding sensitivity and measurement speeds, and is able to measure a 1536-well plate in under 30 seconds. The F500 is the optimal detection platform for assay development and screening laboratories, for laboratories in the cosmetics industry and, thanks to its modular pricing, for smaller research laboratories.

The new Infinite F500 from Tecan

The Infinite F500 has a range of measurement modes, including fluorescence intensity top reading, absorbance, and time-resolved fluorescence. Fluorescence intensity bottom reading, fluorescence polarization, homogenous time-resolved fluorescence, and luminescence measurement modes are available as options. The new instrument offers a number of improvements over existing microplate readers, such as integrated injectors that can operate at variable volumes for three different syringe sizes, very fast switching of filter modules and

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)

Tecan Journal 3/2006

automated z-focusing in all top reading modes. The easy-to-use and flexible microplate reader uses Tecan’s i-control™ and Magellan™ software packages and is compatible with Tecan’s Freedom EVO® series of liquid handling workstations, allowing complete automation of assay and analysis.

Important announcement: Launch tutorial for the Infinite F500 at SBS A special tutorial will take place at SBS on 19 September 2006 at 2:00 pm in room 619 entitled “Infinite F500 - a new multifunctional microplate detection system from Tecan for biomolecular screening assays”. If you would like to attend this tutorial then please contact Monica Jenkins (monica.jenkins@tecan.com) for further details.

L AT E ST P R O D U C T S Tecan has collaborated with the Institute of Chemistry and Biotechnology of the Zurich University of Applied Sciences in Winterthur (Division of Cell Biology, Prof U. Graf-Hausner) to establish a number of cell detachment methods, demonstrating that the Freedom EVO’s new flask handling module can assist in processing a range of diverse cell lines.

Studies on various cell types have shown that when cells were grown to near confluency in Corning RoboFlask® vessels, and harvested either manually or by the Freedom EVO equipped with the Flask Flipper, no statistical differences in the cell counts and cell viability were observed (data available from Tecan).

Accurate sample pooling with the new Te-PoolSafe™

The flask handling module assists the cell harvesting process by shaking, knocking cells off the surface and holding flasks in the upright position for liquid addition or removal

Manual cell passaging is a thing of the past!

Manually harvesting adherent cells from tissue culture flasks is one of the most common and tedious processes in the cell culture lab – but Tecan is about to change that! The new Flask Flipper™ module is available for Freedom EVO® liquid handling workstations of any size to harvest, passage, split and seed adherent cells in microplate-sized, automation-friendly cell culture flasks.

Such flasks have recently been introduced by recognized consumable manufacturers, including the RoboFlask® vessel by Corning Life Sciences. The Flask Flipper assists the cell harvesting process by shaking, knocking cells off the surface and holding flasks in the upright position for liquid addition or removal.

Tecan’s new PoolSafe Option (Te-PoolSafe) is a fast and sensitive liquid arrival check for blood pooling processes in the preparation of nucleic acid testing (NAT).

The Te-PoolSafe measures, checks and documents every liquid arrival of each sample dispensed into the pool tube, bringing enhanced safety and security to all pooling and testing applications.

The Te-PoolSafe runs on Logic™ and Freedom EVOware® software and can easily be integrated with all existing Freedom EVO and Genesis™ liquid handling platforms. The module’s extremely high sensitivity, combined with its ability to make very rapid measurements, makes it an important addition to every blood bank and NAT laboratory.

Not available for sale in the United States of America and Canada

New RoMa arm for Freedom EVO® 75 Tecan has released the new RoMa 75 robotic manipulator arm for the Freedom EVO 75 liquid handling workstation. This option fits the Freedom EVO 75’s smaller dimensions, without compromising on the high quality performance and mechanics that you expect from Tecan’s modules.

The new arm increases the functionality of Tecan’s smallest robotic workstation by moving microplates and disposable tip racks to and from other modules such as hotels, vacuum and magnetic bead separation modules and shakers.

It can also load microplates into robotic incubators, plate washers and detection instruments such as Tecan’s Sunrise™ reader, thereby enabling fully automated plate processing.

The RoMa 75 is controlled by the Freedom EVOware software package (version 1.21 and above). Most installed Freedom EVO 75 instruments can easily be upgraded with this new option.

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GLOBAL N EWS

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

The 20th IUBMB International Congress of Biochemistry and Molecular Biology, covering all topics within the fields of molecular biology and biochemistry, was held from June 18-23 at the Kyoto International Conference Hall, Takaragaike Prince Hotel, in Japan.

Run once every three years in various locations around the world, it has been 39 years since the congress last visited Japan. At the opening ceremony, the 8,000 participants from 71 countries were graced by the presence of His Imperial 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 program with the theme “Life - Molecular Integration & Biological Diversity” was aimed at giving young researchers, who carry the future of Japan’s life science, the opportunity to directly experience some of the world’s top class research.

Prominent researchers presented 11 plenary lectures, symposia across 89 themes, and 6,000 posters.

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

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new 96-channel pipetting head (MultiChannel Arm, MCA), Cellerity™’s Flask Flipper™ module, and the Infinite™ 200 microplate reader. Nicolas Sandoz, Product Manager of the MCA, and Roland Durner, Tecan’s Global Product Manager, attended the event to promote the Cellerity. After visiting Tecan users throughout Japan during the week prior to the exhibition, Nicolas Sandoz and Roland Durner offered further expertise at the Tecan exhibition booth, demonstrating products seen in Japan for the first time. REMP’s Small-Size Store™ (SSS) and devices related to the REMP Tube Technology™ were also exhibited.

Many existing users of Tecan’s automated systems visited our IUBMB exhibition stand to find out more about the MCA, a new option on the Freedom EVO. The Infinite microplate reader, which has been very popular since its launch last year, has maintained a high level of interest, and the exhibition offered many opportunities to obtain feedback from experienced users of the instrument. The knocking sound from Cellerity’s Flask Flipper module dislodging adherent cells from the inner surface of flasks attracted much attention, prompting many people to listen to the descriptions from Tecan staff.

GLOBAL N EWS

Meeting the directors of the blood banks in South West China (from left to right): Yang Tong-Han, Director of Yunnan Blood Center Kunming; Günter Weisshaar, Tecan’s Executive Vice-President Global Quality and Regulatory Compliance; Edgar Doerig, Head of the Commercial and Economic Section of the Swiss Embassy in China; Mark Wang, Tecan China’s Chief Representative

On April 11-16 this year, Tecan was invited to participate in the Director Forum of South West China Blood Centers.

The forum, organized and hosted by the Yunnan Blood Center at Kunming, was a major event for the blood centers of South West China, covering the five provinces of Yunnan, Sichuan, Chongqing, Guizhou and Guangxi. Around 50 attendees were present, including the 15 directors and presidents of the blood centers from all five provinces.

The Director of the Yunnan Blood Center at Kunming, Mr Yang Tong-Han, was instrumental in planning and organizing the event.

The blood banks in the Yunnan Province have been loyal Tecan customers for over seven years, since they obtained many Tecan instruments funded by a government loan. All of the 16 cities and autonomous regions in Yunnan are using Tecan’s Genesis RSP™ workstation, Columbus™ washer and Sunrise™ readers.

The forum was a great opportunity for Tecan to make contact with the other blood banks in the region.

Günter Weisshaar, Executive VicePresident Global Quality and Regulatory Compliance, together with Tecan China’s William Shi, Field Application Engineer, and Mark Wang, Tecan China’s Chief Representative, attended the forum.

They were also joined by Mr Edgar Doerig,

Head of the Commercial and Economic Section of the Swiss Embassy in China.

On behalf of Tecan – the sole instrument supplier invited for this event – Günter Weisshaar and Mark Wang made a joint presentation about quality and safety in blood banks, and William then introduced the attendees to Tecan’s new products and service. Mr Doerig, of the Swiss Embassy, made the closing remarks in the session, assuring the representatives of the blood banks of Tecan’s total compliance with all regulatory affairs of Europe and the world.

Having visited the company headquarters himself and spoken to various Tecan managers, Mr Doerig expressed his confidence in the high quality products and good service provided by Tecan.

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

The event helped to raise awareness of Tecan’s products and organization amongst the users, and gained valuable feedback, which will allow Tecan to provide an improved service structure combined with better channels of communication between the users and Tecan China.

The forum was also an ideal opportunity for Tecan China to introduce Chinawealth, Tecan’s new distributor in the blood center sector of China. Chinawealth’s Mr Zhang Ming, Product Manager, and Ms Wang Huan, South West China Sales Manager, Mark Wang, Tecan China’s Chief Representative were present, and Tecan China and Chinawealth jointly contributed the cost of coach travel for the event.

The timing of the forum was perfect as, in mid-April, the event coincided with the lunar new year of the Dai Nationality, who live mainly in Xishuangbanna and Dehong of the Yunnan province. Forum attendees later participated in the

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P L AT F O R M S

The Genesis FE500™ taking control of sample preparation 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 into assorted sample and analyzer racks, all together on a small footprint instrument.

Before the Genesis FE500 systems were in place in Uppsala, several staff members were dedicated entirely to sample preparation and the whole procedure was completely open to inaccuracy and human error.

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

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equation now and the increase in our workload is interesting. We now have the capacity to handle huge numbers of samples and have become a central input for the entire hospital. With the throughput we have today, it would be impossible to handle so many tubes manually, with aliquoting, secondary tube labeling, and everything else that is involved.”

“We looked around at other front-end instruments available before we bought each Genesis FE500 system and the Tecan system’s flexibility was always the outstanding feature for us. The Genesis FE500 gives us total flexibility by

transferring samples into tubes, including directly into the sample racks for our main clinical chemistry analyzers, two ARCHITECT ci8200® systems from Abbott Diagnostics and one Modular E170 from Roche AG. At the outset it was very easy to configure once we were fully aware of our laboratory’s patterns of work and organization and, once set, the Genesis FE500 simply gets on with everything, virtually unattended.”

ARCHITECT ci8200 is a registered trademark of Abbott Laboratories

Maria Torstensson-Jungestål and the three Genesis FE500s at Uppsala

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

Automated processing of whole blood samples for monitoring of immunosuppressants by LC-MS/MS Liquid chromatography with tandem mass spectrometry (LC-MS/MS) is an efficient and powerful technology 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 Tecan Freedom EVOÂŽ liquid handling workstation, we have developed an automated sample preparation protocol for the quantification of tacrolimus in whole blood by LC-MS/MS. Michael Vogeser (left), Institute of Clinical Chemistry, and Ute Spohrer, Hospital Pharmacy, Hospital of the University of Munich, Munich, Germany

After transplantation of solid organs, lifelong treatment with immunosuppressive drugs is mandatory to avoid rejections.

Therapeutic drug monitoring of the main immunosuppressant drugs, cyclosporin A, tacrolimus, sirolimus and everolimus, is essential to allow tailoring of the dosage according to individual patients’ whole blood drug concentrations.

Immunoassays are still the predominant analytical technique used for therapeutic drug monitoring of these drugs, but the tests are expensive and their analytical quality, in terms of specificity and reproducibility, is limited.

More recently, LC-MS/MS has been introduced in many laboratories as an alternative technology for immunosuppressant monitoring.

However, its use in the clinical laboratory has been restricted for several reasons, including high instrument costs, the need for development of instrument-specific analytical protocols and the need for skilled technicians. Compared with gas chromatography (GC)-MS, the requirements for sample clean-up with LC-MS/MS are only limited, but protein removal is mandatory and solid phase extraction (SPE) or solvent extraction may be necessary for robust and highly

Close up of pipetting needles

precise quantitative methods. While SPE can easily be automated by column switching and applying permanently used extraction columns, manual sample handling for protein precipitation causes a substantial workload for larger scale LC-MS/MS immunosuppressant monitoring. Automation of this first step in sample preparation represents a particular challenge when using whole blood, because sedimentation of blood cells can be observed within a few minutes, but complete resuspension must be achieved immediately before quantitative sample pipetting.

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

All sample preparation and instrument

handling for the daily series was performed by one technician, including an on-line SPE method following manual protein precipitation. We decided to also switch our tacrolimus monitoring from immunoassay technology to LC-MS/MS, after implementation of a further LC-MS/MS system.

However, since this would require processing approximately 80 additional samples per day and since the availability of further technicians is critical, we decided to develop an automated liquid handling system for sample preparation.

Our method allows, for the first time, direct automated processing of large series of whole blood samples for immunosuppressant monitoring by LC-MS/MS; the entire analytical system proved highly precise and convenient.

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

The system comprises sample dispensing and protein precipitation with the Freedom EVOÂŽ 100/4, automated SPE of deproteinized samples by on-line SPE with column switching, and LC-MS/MS analyte detection with a Waters Alliance 2795 HPLC separation module coupled to a Waters MicromassÂŽ Quattro Ultima PtTM MS/MS system. Barcode reading and resuspension of the samples, transfer of whole blood aliquots into a deep-well plate, addition of internal standard solution, mixing and protein precipitation by addition of an organic solvent are all performed by the Freedom EVO. The workstation is equipped with a liquid level detection system, a clot detection system, a washing station, a chilled reagent carrier for up to six troughs, a plate carrier on a horizontal shaker, an additional static plate carrier, a barcode reader, sample trays for blood collection tubes, a liquid handling (LiHa) arm and a robotic manipulator (RoMa) arm. After centrifugation of the plate with a Rotanta 460 centrifuge from Hettich (Tuttlingen, Germany), the deproteinized supernatants are submitted to on-line SPE using column switching prior to LC-MS/MS analysis. Throughout the entire process, the only manual actions required are decapping of the tubes and transferring the deep-well plate from the robotic system to a centrifuge and finally to the HPLC autosampler (table 1).

Whole blood pools were used to assess the reproducibility of the entire analytical system for measuring tacrolimus concentrations. A total coefficient of variation of 1.7% was found for the entire automated analytical process (n = 40).

Close agreement between tacrolimus results obtained after either manual or automated sample preparation was observed. We were able to achieve completely automated resuspension of

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

the samples before quantitative pipetting by repeated aspiration and dispensing of the sample material with disposable 1 ml pipetting tips. Careful optimization of the liquid handling steps finally resulted in a highly precise analytical solution, outperforming manual sample preparation: the intra-assay coefficients of variation (CVs) for tacrolimus results in five series of samples ranged from 2.4% to 5.3% when the samples were prepared manually, compared with just 1.0% to 1.3% when samples were prepared using the automated protocol. The validation of our innovative analytical system was performed with tacrolimus as the target analyte, but it can reasonably be assumed that similar data will be obtained for the simultaneous analysis of sirolimus, everolimus and cyclosporin A.

the analytical quality. In addition to improved reproducibility, our automation protocol avoids the risk of labeling errors, as barcodes can be read from the primary patients’ samples and the information is directly transferred into the LC-MS/MS sample list. Importantly, automation also minimizes the hands-on time of specialized technicians and reduces direct handling of infectious material.

MicroMass is a registered trademark and Quattro Ultima Pt is a trademark of Waters Corporation

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

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

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

Table 1: Protocol of the LC-MS/MS front end automated pipetting system for sample 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 trays overhead. Tubes are decapped manually and the trays positioned into the pipetting system.

2. The safety door is closed and the preparation process is started by giving the number 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 trough into 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 using disposable tips. Firstly, sample is aspirated at the bottom position of the sample 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 liquid level into the wells of the deep-well plate in a sequence corresponding to the sample sequence in the trays. After completion of pipetting of the whole series, 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 into sample 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 samples processed (‘tare-plate’ for balancing the subsequent centrifugation).

Manual step/user interaction:

11. The sample plate is sealed with an adhesive film; sample plate and tare plate 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).

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M I C R OA R R AY

Redefining the genetic basis of learning disability Dominic McMullan, Principal Clinical Scientist, Microarray Diagnostics, West Midlands Regional Genetics Laboratory, Birmingham, UK

About one in 200 babies worldwide is born with a chromosomal abnormality and many of these children have mental retardation and learning disabilities.

Chromosomal imbalances, such as deletions, microdeletions, translocations or inversions, underlie some of these disorders and the genetic diagnosis of these patients has conventionally relied on cytogenetic chromosomal analysis techniques, such as karyotyping, fluorescence in situ hybridization (FISH) or conventional comparative genomic hybridization (CGH). However, the limited resolution of these approaches means that subtle chromosomal rearrangements involving fewer than 5-10 mega base pairs (Mb) cannot be detected, and many affected children go undiagnosed.

Over the past few years, scientists have started to develop microarray-based technologies, such as array-based CGH (aCGH), to investigate genetic changes in samples from patients with learning disabilities, since these techniques have the potential to detect abnormalities with much greater resolution than standard analysis methods. Conventional CGH experiments compare genomic DNA from patient samples with reference DNA. The two sets of DNA are labeled with different fluorochromes and competitively hybridized to normal human metaphase chromosomes so that copy number changes can be easily identified. By applying CGH to microarrays, the test and reference materials are hybridized to thousands of defined DNA probes concurrently, so that a high resolution screen of all chromosomes can be completed in a single, cost effective experiment.

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At the West Midlands Regional Genetics Laboratory, we are using a Tecan HS 4800™ Pro hybridization station to automate aCGH for screening of patients with learning disability and dysmorphology. The laboratory is the largest NHS diagnostics genetic unit in the UK, processing over 27,000 samples each year from the local population as well as from medical institutions in the UK and Europe, covering a full spectrum of diagnoses for genetic diseases.

There are some classic chromosomal abnormalities which we know how to detect, and we largely use FISH for those.

But we are particularly interested in the very large proportion of mental retardation patients who do not have a genetic diagnosis for their disease, and started using aCGH, which is becoming a frontline approach now.

The patients have been referred to the laboratory following assessment by clinical geneticists and are typically moderately affected children with documented developmental delays.

The extent to which a person is affected by a genetic copy number imbalance depends substantially but not wholly on the exact genetic material lost or gained.

Being able to pinpoint the exact genetic changes in a patient, and knowing the genes’ functions, might allow doctors to give parents a better prediction of a child’s future development. Ultimately this may also impact on treatment.

The application of microarrays in clinical diagnostics is fundamentally reliant on accurate results delivered automatically within a comprehensive quality control framework. Through their collaboration,

Dom McMullan loads slides into the Tecan HS 4800 Pro

Tecan and BlueGnome have optimized the Tecan HS Pro series of automated hybridization stations and BlueGnome protocols for automated processing of the CytoChip™ diagnostic bacterial artificial chromosome (BAC) microarrays, which we are using for our screening.

We had some initial aCGH pilot studies using manual methods, which sometimes produced uneven hybridization, and we straightaway saw the need for automation, largely because of the increased reliability and reproducibility it offers. Also, because we are relying on commercially available BAC arrays, we cannot afford to have any failings with them - we want to maximize our success rate as far as we can, which is why we turned to the HS 4800 Pro, chosen for several reasons. One thing that really

M I C R OA R R AY

helped us to make the decision is the automated slide drying, which no other instrument on the market has. After injecting our probe, the operator walks away, leaving the machine to hybridize, wash and dry the slides, ready for scanning, so the whole process becomes less dependent on core lab hours and operator-related inconsistency. The recent development of segmented chambers allows us to perform two separate hybridizations on one slide without any crossover. This is very important for aCGH, because we can test patient- and reference-labeled DNA in opposing fluorochromes on two different arrays, so removing any potential dye effects or bias. The HS Pro is easy to use and its inherent agitation makes the hybridization very even across the slide, increasing the signal-to-noise ratio. The agitation setting allows more viscous hybridization buffers to be used than previously, which is key, because aCGH buffers contain dextran sulfate and so are more viscous.

The CytoChip™ BAC microarrays are spotted with 3,200 BAC clones to give a spatial resolution of approximately 1 Mb; any abnormalities detected by the arrays we then directly confirm with FISH. Its resolution means that, effectively, there is a BAC for every 1 Mb of the genome. On current evidence, the majority of pathogenic imbalances

should be detectable, given high quality raw data. The CytoChip™ is unusual in that it is tiled with increased BAC density across areas of certain well-characterized cytogenetic syndromes relating to recurrent chromosomal abnormalities.

Our laboratory was involved in this design feature, which enables pick-up of potentially missed changes as well as detection of novel changes. In other words some patients might not have a classic phenotype, but may have a classic abnormality. For the patients that we are looking 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 should have something that we could not detect with traditional methods. We have found that we are now picking up genomic changes that previously would not be detectable; we are almost starting again, in terms of redefining new syndromes, by increasing the resolution.

Metaphase FISH image of chromosome 15q deletion

CytoChip is a trademark of BlueGnome.

At present, the HS 4800 Pro is intended to be used for general purpose in Europe and for research use only in the USA. Plot of aCGH showing chromosome 15q deletion

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

The HS 4800 Pro

The BlueGnome CytoChip™

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FORENSICS

The world’s biggest, single automated forensic analysis system is hungry for samples It’s 37 meters long but only 4 meters wide; it’s controlled by 27 PCs and includes eight Tecan Freedom EVO® workstations. It took the project team 23.7 man years to complete, during which time they traveled over one million kilometers and spent 4.5 years away from home. This is no ordinary laboratory instrument, but the new, fully automated genetic sample processing system (GSPS), a DNA analysis system that was specially commissioned for the Biology Unit of the South African Police Service’s (SAPS) Forensic Science Laboratory (FSL).

The SAPS receives crime samples from across a country that is twice the size of Texas – with only two laboratories in which to analyze them. This has led, unsurprisingly, to a backlog of cases waiting to be dealt with and the urgent need for an automated system to process current and past samples. Regular readers of the Tecan Journal may remember the announcement in 2004 of the award of an EUR 8.35 million tender to Tecan to provide, install and service this forensic DNA typing system. After years of planning, the project was finally completed in February 2006 and the system began the rigorous validations required before sample processing begins.

The GSPS will be able to accept in excess of 800 samples for DNA extraction and further downstream processing in a day, significantly improving the speed at which DNA analysis results can be obtained.

Johann van Niekerk, a GSPS Operator attached to the Technical/Specialist Support section of the SAPS FSL Biology Unit, explained the mammoth scale of this project: “We’re moving into a new forensic laboratory in a refurbished building that was previously the Andrew McColm Hospital, in Pretoria. This huge area has been fitted out and transformed to create a laboratory space with, for example, evidence recovery and manual DNA processing laboratories, administrative offices and support space. It also houses the automated GSPS, which covers three different laboratory spaces over a 40 m long by 6 m wide footprint.”

Christo Weitz, Project Manager for the GSPS, commented: “With regard to our requirements for a robotic sample processing system, we didn’t have to

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

compromise at all on any of the specifications, because when we looked at Tecan’s proposal for the GSPS, it was abundantly clear that they understood exactly what it was that we wanted and they provided us with exactly that solution.”

The Tecan SAPS team, headed by Beat Glauser and largely based in Munich, has been working on this project since early 2004 and this massive platform was hardly a straightforward installation.

The 37 m process line inhabits three restricted access spaces that operate in a cleanroom-like manner, each with ante chambers and its own separate, climate-controlled environment, to prevent chain of evidence breaches and cross-over contamination – the GSPSrelated hardware alone took four weeks

Robot fetches a rack with samples for further processing

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FORENSICS

to install. However, the biggest challenge for Tecan was the inclusion of a number of third party instruments, such as DNA analyzers from Applied Biosystems, into the GSPS; all of these required full LIMS and control software integration, achieved by the Tecan team through rethinking, redesign and consultation.

The final set-up is documented in 162 controlled Tecan documents and, incredibly, 149 third party manuals!

Fitting the pieces together The complete GSPS is run by 27 PCs and consists of four 6-axis articulated robotic arms on servo-driven tracks, running through three separate areas that are joined by inter-laboratory airlocks.

The system uses eight Freedom EVO automated liquid handling workstations, of which four perform cell isolation and DNA extraction steps, two perform PCR normalization and set-up, and two perform fragment separation set-up.

In addition, there are three thermal cyclers, three Applied Biosystems genetic analyzers (for fragment separation) and two sequence detection systems (for DNA quantification through to real-time PCR) integrated into the structure. Automated sample storage and retrieval systems also operate within the production line, as well as all the necessary equipment for cell isolation (such as centrifuges), laboratory plate support (such as Te-Stacks™, shakers and heaters), laboratory plate sealers and foil piercing instruments, ‘sandwich’ assembly systems for genetic analyzers, and various other laboratory essentials, all of which were supplied and incorporated into the system by Tecan and its local subcontractor, Vertex Automation.

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

them into a number of individual processes that are often carried out by different operators. This is not only much more labor-intensive and time consuming, but results in significant dwell times between the different process steps and the resulting variability can 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 for complete and seamless integration of all the components within workcells, between workcells and across the entire system. Tecan’s flexible assay composer and task scheduler (FACTS™) software includes supervisory software for the system and control software for individual instruments in addition to the instruments’ individual software from

The GSPS is made up of robotic workcells that are fully interlinked through integrated ‘islands’ of automation. Fully integrating all the processes into a single system is vital not only to the set-up and throughput, but also to the quality of data achieved. Performing the different analysis procedures manually separates

their respective vendors; it communicates between the instruments and co-ordinates all of their activities. A system process supervisor (SPS) software application was also developed and this serves as the LIMS of the facility, interfacing with individual instruments’ supplied LIMS as well as pulling all the different instruments together. The SPS also interfaces with STRLAB, a client-supplied LIMS, which manages sample submissions and result analysis processes.

“The software is really what makes this whole thing tick, and it’s all Tecan software,” explained Arnold Greyling, Technical/Support Manager of the SAPS FSL Biology Unit. “We’re using Freedom EVOware® software for the liquid handling systems, but then each of our labs or subportions of the labs is run by its FACTS software and then, to complete the integration between the different labs and the FACTS installation, Tecan created a top level SPS application.”

The system’s dynamic task scheduling is essential for eliminating dwell time and reducing the casework turnaround time, by ensuring that programmed tasks are carried out at the optimum rate and actively rescheduling according to available system resources. Different

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parallel processes can be simultaneously performed and continuous loading of plates, use of alternative devices or running emergency samples is still possible, without interrupting the process.

The in-built autonomous management process is capable of running for 24 hours a day, every day of the year, so staff can be more usefully employed in casework management (front end processes) or analyzing and verifying results and data (back end processes), depending on their expertise. This means that automation will not mean loss of jobs for staff at the FSL – if anything, the laboratory may need to employ more people, as Director Pierre Joubert, head of the SAPS FSL Biology Unit, explained: “The kind of staff that were previously responsible for the main day-to-day activities, such as DNA extraction or PCR set-up, can be redeployed to perform evidence recovery and sample submission to the GSPS - the sharp end of the system. We actually need more people just to feed the system! The beauty of it is that many of our staff will now be given more interesting things to do, it’s about letting humans do what they do best and letting machines do what they do best.”

Helping to reduce crime A smaller scale automated sample processing system has also been built in South Africa, which will deal with DNA typing of reference blood samples only, while this new GSPS is expected to process the majority of the country’s crime samples. It will bring a number of important advantages to the SAPS, in addition to massively increasing the FSL’s sample throughput and improving the quality of the analyses. The improved casework turnaround times will help to produce investigative leads from cold hits as well as minimizing a criminal’s potential window of activity, allowing speedier apprehension of criminals, exoneration of the innocent, prevention of crime and, in the long term, considerable cost saving.

In one end...

The processing line as a whole consists of four primary processes: robotic sample indexing; cell isolation, differential lysis and DNA extraction; PCR normalization and set-up; DNA amplification and fragment separation. The system runs across three defined laboratory areas for the different steps, in order to accommodate sample traceability and

The inverter wheel is loaded with lyzed samples

to minimize cross-contamination: the robotic sample indexing lab, the DNA extraction and PCR set-up lab, and the DNA amplification and fragment separation lab. It is vital that every sample transfer step is carried out in a safe, accurate and precise manner in order to minimize cross-contaminations.

Area 1: Robotic sample indexing Samples are initially transferred into barcoded, robotic sample indexing tubes and then sorted according to type (e.g. semen, or blood and similar bodily fluids) by a selective compliance articulated robot arm (SCARA). Precise sample sorting is a critical first step and it must ensure that samples from recovered evidence are securely and accurately transferred to predefined well locations in laboratory sample plates for downstream automated processing, while maintaining full sample traceability.

Area 2: DNA extraction and PCR set-up Samples are transported to the second laboratory area, where cellular material from the samples is resuspended into a second plate and lyzed for DNA isolation.

The samples from two 48-well plates are then transferred into a 96-well plate for DNA extraction. These steps are designed to achieve reliable, automated DNA purification from up to 96 forensic samples. The technologies used provide uniform DNA recovery and purity across the 96-well plate, with no detectable Robot loads a plate into the air lock to send it to the next laboratory

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Lysis buffer is added to the samples

The RSI workstation is busy sorting the samples

cross-contamination between samples, allowing effective removal of PCR inhibitors. During these processes, the robots need to be able to repeatedly deliver samples to a range of high density microplates; provide full flexibility for performing complete processes including plate replication, serial plate dilutions and in-plate serial dilutions; and provide fast and automated interchange between fixed tips and disposable tips according to the process required. Real-time PCR set-up is performed through an automated pipetting system, which allows reproducible delivery of template DNA, DNA primers and other components (buffers, nucleotides) that are typical of a DNA amplification reaction mix.

DNA solutions are automatically normalized to a constant concentration based on information obtained through real-time PCR. Area 3: DNA amplification and fragment separation The plates undergo DNA amplification using thermal cyclers and fragment

separation processes. For real-time PCR, the quantitative relationship between the amount of starting target sample and the amount of PCR product at any given cycle number is determined by detecting specific nucleic acid amplification products as they accumulate in real time. For the GSPS, this process has to be robotic-amenable and capable of worklist import and data export capabilities.

Fragment separation is carried out using capillary electrophoresis, with an automated pipetting system that allows reproducible delivery of amplified DNA, other components (buffers, internal lane standards, formamide) and size standards, in a predetermined pattern.

Fragment separation plate assembly requires an instrument to assemble electrophoresis ‘sandwich’ plates containing amplified DNA into a carrier plate. Fragment separation data are then transferred for analysis and verified, and/or transferred for repeat or new analyses. A number of secondary processes are also involved, including a sample storage, retrieval and delivery

system; short-term sample archive management; robotic sample conveyance; inter-laboratory pass-through chambers; lab plate cover management and an emergency shut-down and recovery procedure.

‌and out the other The DNA profile generated by the system is then analyzed by humans and used for police work and court cases. Once the results are approved, the so-called expert witnesses - the same people that initially prepared the samples - will present the data in court.

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South African Police Service’s project number crunching 8.35 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 and Germany (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!

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SAPS Team Event

A job well done!

To celebrate the SAPS installation having successfully reached the Operational Qualification milestone, where the system is handed over to the client, the core team of people working on the South African project met in a restaurant in Mollis, in the valley of Glarus, Switzerland, early one Friday afternoon in June.

All the team had been told was that they should arrive on time and bring sunglasses, sunscreen and a camera. After welcoming everyone we drove on for another five minutes and parked our cars in front of the little airport where a helicopter was standing in front of a hangar.

Everyone’s eyes widened. Is it really waiting for us? YES! After all those aeroplane trips between Germany and South Africa, this was something completely different. We were taken by helicopter in two groups to the Planura Hütte, in the middle of an alpine surrounding, where the host of the hut was already expecting us.

After enjoying food, drinks and the view it was time to return: there was a quick announcement on the radio and, shortly afterwards, the silence was broken by the flap-flap-flap of the helicopter. Feeling like true VIPs we flew back to the base.

From there we took our cars to the hotel in Wädenswil, where we checked in and proceeded to Wädi-Bräu, the local microbrewery. The brew master agreed to broaden our (already profound) knowledge on liquid handling and, in between the explanations, we returned to the restaurant for our next course.

A bit late (and with a beer or two) we then returned happily to the hotel.

What a day!

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

Automating metabolic inhibition assays on a nanoscale AstraZeneca is one of the world’s leading pharmaceutical companies focused on producing effective medicines that make a real difference in important areas of healthcare. In today’s environment, the constant streamlining of laboratory processes in order to increase efficiency and save valuable resources is an important aspect for such companies, and taking advantage of the latest advances in automation technologies can help to get ahead.

Brett Litten is a senior DMPK scientist and automation specialist at AstraZeneca’s Alderley Park site in Cheshire, UK, where he is jointly responsible for new technology and the design and delivery of various automated in vitro assays and in vivo related processes within the department. The company has significantly invested in Tecan’s technologies over the years and one of Brett’s latest projects has been to integrate a Nanodrop™ II eight-channel nanopipettor from Innovadyne Technologies with a Tecan Genesis RWS™ 200 Workstation and Tecan Safire2™ microplate reader.

The Nanodrop™ II Tecan Journal 3/2006

This configuration is being used to increase throughput in the cytochrome P450 inhibition assay. In this assay, the activity of five major human cytochrome P450 enzymes (CYPs) is determined using a number of substrates, which are metabolized to fluorescent products.

Inhibition of the CYPs results in a decrease in the amount of fluorescence metabolite formed and, by using a range of test drug entities, the potency of inhibition (IC50) can be assessed. Brett integrated the Nanodrop™ with the Tecan workstation in order to miniaturize this assay for use with 1536-well plates, as he explained: “We required a higher plate density to help reduce assay costs and increase throughput. The Nanodrop™ allows us to use a 1536-well plate such that a greater number of drug entities at different concentrations can be tested against the major P450 enzymes per assay. Furthermore, using 1536-well plates leads to a reduction in the number of times the assay is run per week for our current research purposes and, in turn, frees up more resources. The process is fully automated, including fluorescence analysis with the Safire2 plate reader, so our researchers can get on with other tasks during the assay.”

Innovadyne’s Nanodrop™ II is a small scale, high accuracy, high precision nano-dispenser, which is able to efficiently handle very small volumes of liquid, as low as 100 nl. The instrument will also happily dispense microliter-range volumes, so it has quite wide-ranging uses.

“The Nanodrop™’s attraction is that it extends the current usable range of our Tecan workstations, which are generally speaking of a larger volume criterion.

Due to the Nanodrop™’s small size, it is easy to install into an existing robot platform,” said Brett. “It is very simple to integrate, and uses a combination of traditionally robust syringe aspiration coupled to Innovadyne’s micro-solenoid dispense valve assembly for delivering accurate quantities of liquid.”

In order to run the assay, the 1536-well plates are loaded on to the Tecan workstation with the manually prepared reagents and enzyme solutions.

The workstation’s Gemini™ software is then used to execute the assay, controlling the various sequences for the Nanodrop™’s processes, including spiking the wells with 100 nl of test compound.

Once the Nanodrop™ has prepared the plates, the Tecan robotic manipulator (RoMa) arm transfers them to the Tecan incubator, also housed on the system,

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Brett Litten and the Genesis 200 workstation

for necessary preincubations and incubations. The RoMa arm can then remove the plates after the required reaction time and place them on the specified site on the Nanodrop™ for the next steps before finally transferring the plate to the Safire2 for detection.

The Safire2 is a double monochromator plate reader that is capable of reading 96- to 384- and 1536-well plates in fluorescence, fluorescence polarization, absorbance and luminescence modes.

The metabolic inhibition assay uses fluorescence substrates that are metabolized by the enzymes and produce a fluorescent signal, which can be quantified to indicate the potency of the test compound to inhibit the enzyme-substrate metabolic reaction.

The Nanodrop™ II and the Safire2 on board the Genesis 200

“We chose the Safire2 because we needed a detection system that could read 1536-well plates in a very short space of time, so we could minimize evaporation and keep the automated process as fast as possible,” Brett explained. “We have actually integrated it so it is physically connected to our Tecan workstation and is directly accessed by the RoMa arm.

The Safire2 is the fastest unit I’ve ever used for reading plates, capable of reading 1536-well plates in just 37 seconds! We read our 1536-well plates using several fixed wavelength methods to accommodate the different excitation-emission wavelengths required for our assay.”

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

and automatically transferred to a network server from where analysts can process the data to generate inhibition IC50 values.

Since Brett’s original bespoke integration of the instruments, Tecan and Innovadyne have been working together to produce off-the-shelf systems where the Nanodrop™ is fully integrated with Tecan’s Freedom EVO® liquid handling workstations. These systems will be suitable for diverse assay processes requiring smaller volumes – for further information please contact Matthew Webb, Tecan UK Limited.

Nanodrop™ is a trademark of Innovadyne Technologies, Inc.

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

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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 single nucleotide polymorphisms Sylke Winkler, Claudia Seidel, Stefan Meissner, Nicola Gscheidel and Michael Brand Biotechnology Centre and Department of Genetics, Dresden University of Technology (TU Dresden) and Max Planck Institute of Molecular Cell Biology and Genetics (MPI-CBG), Dresden

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

TILLING (Targeting Induced Local Lesion IN Genomes), a powerful reverse genetics tool, was developed to identify single nucleotide polymorphisms (SNP) in mutagenized individuals by PCR amplification of a gene of interest from each individual, followed by SNP detection1,2. However, because the identification of nonsense mutations leading to loss of gene function is a relatively rare event, it is necessary to screen thousands of mutagenized individuals to find a presumptive null mutation in a gene of interest. TILLING is mainly based on PCR followed by enzymatic digests or direct sequencing of PCR-fragments, and is therefore perfectly suited to automation. At the MPI CBG, we chose the Tecan Genesis RWS™ 200 workstation to automate all pipetting steps.

Our current set-up consists of a liquid handling arm with eight Teflon®-coated tips (for 384-well plates), water-cooled troughs for all reagents and cooled carriers that represent the pipetting platform, a robotic manipulator (RoMa) arm and hotels for PCR-plate storage.

The RoMa arm transfers PCR plates between an integrated thermocycler with four 384-well blocks, a cooled pipetting platform and a cooled incubator. Pads in

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the motorized lids of the PCR machines seal microplates and prevent cross contamination of samples.

Mutagenesis and library generation The basis of a successful TILLING project is the generation of libraries of heavily mutagenized individuals. Alkylating mutagens such as ethylnitrosourea (ENU) or ethane methyl sulfonate (EMS) can easily be applied to zebrafish, fruit flies and nematode worms, inducing point mutations that are randomly distributed throughout the genome of treated individuals. By crossing these mutagenized animals to untreated individuals the induced SNPs are propagated to the next generation, producing large numbers of individuals carrying new mutations.

A classical TILLING library consists of several thousand live mutagenized animals (zebrafish and Drosophila) or recoverable frozen individuals (C. elegans), and their corresponding genomic DNA. All genomic DNA preparations were carried out in deep-well 96-well plates, diluted and dispensed as 5 μl aliquots into 384-well plates (DNA concentration of about 0.2-1 ng/μl). The plates were stored frozen and could be used directly for PCR. Simple washing steps of the Teflon® coated tips with system water prevents cross contamination when pipetting different templates.

Screening procedures For zebrafish projects, we screen using a nested PCR approach originally described

by Wienholds et al.2 In this approach, the normalization step is at the PCR level and not at the level of the genomic DNA, which was a factor at the start of the project when photometric plate readers were not available at the MPI in Dresden.

Again, all pipetting steps are performed automatically. The inner primer pairs in the nested step contain universal tails, which serve as templates either for universal primers that are labeled with fluorescent dyes or, alternatively, as direct templates for universal sequencing primers.

The first PCR is set up to a total volume of 10 μl in 384-well plates containing the genomic template DNA. For the second round of PCR, appropriate amounts of the products from the first PCR are transferred to fresh 384-well plates that already contain the mastermix for the second PCR. For SNP detection by direct sequencing we check the quality of the second PCR products by agarose gel electrophoresis beforehand. Primers and dNTPs were removed and dephosphorylated by Exonuclease I / shrimp alkaline phosphatase incubation.

Again, the Genesis 200 performs all pipetting steps, and the microplates are

A P P L I C AT I O N B I O P H A R M A manually loaded onto the eight 384-well PCR blocks as they become available.

Our current capacity, including quality checks 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 as described by Colbert et al.3 After the first PCR is performed and diluted in the same way, the second PCR is run with tailed inner primers and fluorescently labeled primers (LI-COR™ IRDye700® and IRDye800®), resulting in labeled PCR products which can be pooled up to fourfold, denatured and re-annealed to induce heteroduplex formation between mutated and wild type PCR products.

Heteroduplexes are cleaved by the endonuclease Cel-1, and the resulting fluorescently labeled cleavage fragments are purified and separated by high resolution SDS-PAGE on slab gel sequencers. Using Gemini™ software, a fully automated protocol was set up for this TILLING assay, which includes many different pipetting steps. The RoMa arm transfers PCR-plates between the thermocycler blocks and the pipetting platform, and all mixes are made available in cooled troughs. Four 384-well plates (1,536 samples) could be handled within 11 hours, plus an additional two to three hours purification time.

The detection of presumptive mutations requires automation of a screening set-up to allow the handling of huge numbers of samples. For example, with heavily mutagenized zebrafish about four to five million base pairs have to be screened to isolate a relatively rare nonsense mutation (stop codons account for 5% of all SNPs). We are currently able to handle more than 9,000 samples per week which, with a modest detection rate of 500 base pairs per screening round, equates to 4.5 million base pairs of genomic information per week. Therefore, our set-up allows the identification of one nonsense mutation plus many additional missense mutations each week, and demonstrates the suitability of an automated TILLING approach to routinely generate mutants for functional genomic studies.

Teflon is a registered trademark of E.I.Dupont.

IRDye is a registered trademark of LI-COR, Inc.

Close up of the eight channel liquid handling arm pipetting into a 384-well plate

Sylke Winkler and the Genesis 200

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

2. Wienholds, E., Schulte-Merker, S., Walderich, B., Plasterk, R.H.A.: Targetselected inactivation of the zebrafish rag1 gene. 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-throughput screening 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-selected mutant screen

by TILLING in Drosophila. Genome Research 15: 718 (2005).

For review: Stemple, D.L.: TILLING – a high-throughput harvest form functional genomics. 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 the nematode worm Caenorhabditis elegans (Anthony Hyman, MPI-CBG) and for the fruit fly Drosophila melanogaster 4 (Marcos González-Gaitán, MPI-CBG). Worktable and PCR integration were designed with support from Hannes Grabner and Jan Wagner from the Technology Development Studio (TDS) of the MPI-CBG. The Dresden TILLING project is financed by the Sixth Framework Programme of the European Commission ‘Zebrafish Models for Human Development and Disease’ (ZF-MODELS), the Dresden University of Technology, and the Max Planck Institute of Molecular Cell Biology and Genetics.

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CO M P O N E N T S

High performance ion channel screening npi electronic has revolutionized automated drug screening with the launch of the ScreeningTool, an automated system for screening of voltage- or ligand-gated ion channels in Xenopus oocytes, with millisecond resolution and high speed voltage clamping. The innovative instrument depends on a Tecan OEM liquid handling system for automated drug delivery, integrated with a miniature chamber design and unique amplifier to provide unprecedented speed, reproducibility and efficiency for high quality drug screening applications.

The ScreeningTool was developed through a collaboration between npi electronic and Prof Steffen Hering from the Department of Pharmacology and Toxicology at the University of Vienna, who originally invented the miniature recording chamber (patent pending).

It was designed to automate the standard, two-electrode, voltage clamp screening procedure in a 15 μl bath, covered by a glass plate with a funnel for precise, automated drug application by the liquid handling system. This set-up allows very fast solution changes and requires only minimal amounts of sample for the procedure. The design depends on

one of npi electronic’s TURBO TEC amplifiers, which are specially developed for recording of bioelectrical signals from Xenopus oocytes, and Tecan’s MSP9250 liquid handling system.

“The Tecan liquid handling system is ideal for the ScreeningTool because it has all the necessary features. In particular, the time it takes to apply the drug and the time it takes in between applications is very short,” explained Mr Hans Reiner Polder, managing director of npi electronic.

“Automating the drug application helps to get more reproducible data as the reliability of the instrument, based on the features of the Tecan liquid handling

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

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system, is excellent. The uptake of the drug into the pipette and the wash-in and wash-out procedures are also fully automated, further increasing the reliability. This completely avoids crosscontamination between individual applications and the quality of the experiment is improved considerably. The system can be easily programmed by the customer, using scripting language that was developed in cooperation with Tecan.”

Using the Tecan liquid handling system means that only the pipette tip head needs to be filled with solution (~100 μl), so the ScreeningTool requires just fractions of the amount of sample that other instruments need to perform a complete experiment. All alternative instruments are based on tubing, which has to be filled with solution, resulting in a lot of sample wastage and considerable time delays. Another important feature is that this system can be programmed to ensure that cells are always kept at the same ionic milieu in between applications, which increases the reliability of the experiments as well as maintaining the cell’s condition. Instead of using only a few solutions for one cell, more than a hundred solutions can be used on one single cell, allowing many more experiments to be performed in a shorter period of time.

CO M P O N E N T S

Automated, high speed ion channel screening with millisecond resolution

“Combining the Tecan MSP9250 with Prof Hering’s special chamber makes this instrument unique and very specialized for its applications; there is no competing instrument that could fulfil all these parameters at the same time,” said Hans Reiner.

Voltage clamping is performed using npi’s TURBO TEC series of amplifiers, as Hans Reiner explained, “The npi amplifiers are based on a unique, sophisticated design that we developed years ago and their high speed of response makes them superior to similar systems. The amplifiers also have a very high degree of precision, so they are ideal for Prof Hering’s chamber design.”

The ScreeningTool’s flexible fluid application and high performance voltage clamp make it highly suitable for drug screening on ligand and voltage gated ion channels. The system is specifically designed for Xenopus oocytes, which are widely used for drug screening and for pharmacological safety tests.

Xenopus oocytes are often used to analyze the interaction between certain

drugs and ion channels or receptors that could be expressed in the oocytes, such as investigations of the hERG channel a special potassium channel derived from the heart. Some drugs affect this channel and alter the heart beat, causing arrhythmia, and such cases would not normally be suitable for further development.

This is the first time that npi electronic has used a Tecan OEM component for one of their products and Hans Reiner is very impressed with the collaboration service, “We had a lot of support from Tecan and established a good relationship, which helped us to produce a professional version of the software that is suitable for use in the pharmaceutical industry, within a very short time period.”

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The Screening Tool is automated using Tecan’s MSP9250

“We had a very good experience with this product and I have already recommended Tecan to all our partner companies.”

“In the future we plan to develop other versions of the ScreeningTool designed for different cell types and are very interested to continue the collaboration with Tecan. We had a very good experience with this product and I have already recommended Tecan to all our partner companies.”

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SAMPLE MANAGEMENT

Managing lead optimization compounds with REMP technology The Rahway Facility for Automation and Screening Technologies, which is based in New Jersey, USA, and is part of Merck Research Laboratories, is using a REMP Small-Size Store™ (SSS) to manage its thousands of samples needed for drug discovery investigations.

Dr Claude Dufresne

“Our automation laboratory serves principally the lead optimization stage of drug discovery, so we receive several hundred samples per week from medicinal chemistry labs,” said Dr Claude Dufresne, Senior Investigator, who has been working at the Merck site since 1988 and has been developing automation methods and systems for most of the past ten years. “From those samples, we prepare serial dilution plates for distribution to various biological assay laboratories and for our own uses.

We also store the samples in order to supply biology labs with samples for retesting and secondary assays; that’s why we needed the SSS.”

The central automation facility was created in 2005 to provide, in part, an integrated compound management service for the Merck Rahway Site. A robust and efficient sample storage and retrieval system is a vital part of the service.

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The samples are usually chemical compounds that have been synthesized as part of particular research programs and are based on lead compounds that may have come out of a high throughput screening campaign. The samples might be part of a chemical series based on these structures and evolved through traditional medicinal chemistry processes.

“We receive compounds from most of the research programs at our site, so the samples relate to a range of different drug discovery programs,” Dr Dufresne explained. “Some of our principal research interests include diabetes, obesity and atherosclerosis, for example.

SAMPLE MANAGEMENT

system we can physically retrieve individual samples, as opposed to retrieving whole plates of samples.”

Using the SSS means that cherry picking occurs within the environmentally controlled storage system, so there are no freeze/thaw cycles on samples, and storage temperatures ranging from -20˚C, +4˚C or even more ambient temperatures, such as the 18°C used at Rahway, can be used.

REMP Small-Size Store

We collaborate with scientists in specific therapeutic areas not only by providing the sample management service, but also by assisting with assay development, optimization and implementation on automated systems. In this function, we also carry out some of the primary assays in support of medicinal chemistry.”

The automation laboratory manages its compound storage entirely through REMP technologies, including the SSS with its in-built Tube Punching Technology, the REMP 96 Tube Technology™ consumables, the 2D Code Scanner, and the eight position manual capper/decapper (REMP MCD8™), which the scientists have also automated using their own engineering and software.

“Initially it was the 96 Tube Technology that really attracted us, and then adding the SSS to make use of this technology was a real bonus,” Dr Dufresne said.

“The REMP mini-tube and Tube Punching Technology is really a breakthrough for sample storage. Traditionally, compounds would be stored in deep-well plates, meaning that if you needed to cherry pick samples, then you would need to use liquid handling to aspirate samples from individual wells. By using the SSS with its in-built Tube Punching Technology we don’t need this step, we can just punch out the tubes that we want straight into a delivery rack, meaning that in one

“When samples are stored in vials, in boxes or racks, and placed in fridges or freezers, the management of that sample collection is a significant time drain,” continued Dr Dufresne. “It can easily take hours a day for someone to go through the activities of maintaining and retrieving individual samples, just for the daily work - but this all goes away with automated storage!”

REMP Tube Technology Consumables consist of individually sealed or capped tubes secured within their own Tube Rack. The tube transfer technology greatly improves the reliability of tube handling and the unique punching technique greatly reduces handling error rates, because the transfer is performed by a single axis movement.

This technique is faster and more reliable compared to conventional pick and place methods, and also eliminates potential cross-contamination, exposure to air or dilution effects, which are often seen with samples undergoing traditional liquid transfer. The tubes virtually eliminate the risk of sample degradation and can be either individually sealed, for single use, or capped in an automated or manual fashion for multiaccess.

The scalability of the SSS means that up to two extension units can be added to the SSS, tripling the storage capacity and this is also important for the Rahway automation laboratory, as Dr Dufresne explained: “Another big attraction of the REMP technology for us was size; we needed a really small store, because we did not have much spare lab space at the time, and our needs in supporting the lead optimization could be met with just one or two of these units. Our current level of storage is only a few thousand samples, although it is now growing quite rapidly as we are beginning to store multiple concentrations and multiple copies of each sample. We use these units for short term storage of samples involved in active projects as, at the end of a project, all of the samples relating to that project will go to the main sample repository where millions of compounds are stored. Since it is very difficult to predict usage levels down the road as we continue to transform our research operations, it is comforting to know that doubling or tripling our local storage capacity would be an easy upgrade.”

The above text does not imply endorsement of Merck & Co.

REMP Tube Technology Tecan Journal 3/2006

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28

EVENTS 2006

Meet Tecan at these events before the end of the year China CCLab 2006 (with VASTEC)

Qingdao

Sept 15-19 2006

Drug Discovery and Development

Shanghai

Nov 20-21 2006

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

Kyoto

Sept 19 2006

Europe

Japan Genomics Drug Discovery Key Technology 2006 Japanese Association for Food Immunology

Tokyo

Oct 23-24 2006

Japanese Society for the Study of Xenobiotics

Tokyo

Nov 29-Dec 1 2006

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

USA

Tecan Journal, Customer Magazine of Tecan Trading AG., ISSN 1660-5276 Design: OTM/London www.otmcreate.com Photography: Marc Wetli/Zürich www.wetli.com, Günter Bolzern/Zürich www.bolzern.net, Susanne Völlm/Zürich www.susannevoellm.ch, Beat Glauser/www.beatglauser.com Editor: kdm/UK www.kdm-communications.com Print: DAZ Druckerei Albisrieden AG/Zurich www.daz.ch Address: Tecan Switzerland AG, Marketing Communications, Seestrasse 103, CH-8708 Männedorf, Switzerland, journal@tecan.com,www.tecan.com Tecan Group Ltd. makes every effort to include accurate and up-to-date information within this publication, 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 accuracy or completeness of the information provided in this publication. Changes in this publication can 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 document please contact your Tecan representative.

This brochure may contain reference to applications and products which are not available in all markets. 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 trademarks and 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.

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Tecan Journal 3/2006

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