Tecan Journal Edition 1/2006
More power to your workstation with Tecan’s Freedom EVOware® software page 8
From primary blood to DNA – fully automated DNA extraction page 10
Tecan and REMP – the perfect synergy page 18
Cover shows Eskil Trollhagen, Project Manager Applications International, Tecan AG Switzerland
W E LCO M E
Welcome to the first Tecan Journal of the new year! 2006 is set to be a busy year for Tecan as we continue on our theme of ‘Talk to Tecan’ and learn from you, our customers, how best to provide ground-breaking automation solutions. 2005 was a successful year for Tecan and we hope to continue our period of growth throughout 2006. The acquisition of REMP half way through the year has now been successfully implemented and we look forward to the added expertise and knowledge our REMP colleagues will bring to the entire Tecan product and service offering. Several new and exciting products are planned for launch in 2006, including specific application modules for
platforms, new readers, and new generations of washers and OEM pump and pipetting components. Many of these new developments are in direct response to market trends and changes in regulatory requirements in all the application areas that Tecan serves. Of these, biopharmaceutical and clinical diagnostics remain important application areas, and forensics is a growing and exciting focus for Tecan. There is a real demand for increased sample throughput in this discipline and we are well placed to give customers the reliability they need in such highly innovative and regulated environments.
I hope that we can continue to deepen and prosper our relationship with you as customers and partners. I wish you all a successful and prosperous new year for 2006. Thomas Bachmann Chief Executive Officer (CEO)
Tecan Journal 1/2006
CO N T E N T S
Welcome and Contents Thomas Bachmann, CEO, welcomes you… pages 2-3
More power to your workstation with Freedom EVOware® An overview of the different software packages available, including the new and improved Freedom EVOware v1.2
Watch out this year for new developments from the Tecan pipeline Find out about Tecan’s new Pressure Monitored Pipetting and MCA tools
Freedom EVOware takes control in San Diego Jeff Reid describes how Freedom EVOware helps researchers at UCSD generate a genome-wide RNAi library pages 8-9
Bioser Medikal places eight Freedom EVOlyzers® in Turkey Tecan’s Turkey distributors are kept busy pages 4-5
Training brings the World together Tecan leads a quality system regulation training workshop in China Demonstrating the new InfiniteTM and Freedom EVO® in China Hands-on training demonstrations for Tecan’s distributors pages 6-7
Automated, high throughput preparation of ProteinChip® Arrays for SELDI-TOF MS profiling Automating the search for serum biomarkers from breast cancer patients pages 14-15
Automated genomic DNA extraction from buccal cells with Invitrogen’s ChargeSwitch® Technology Tecan and Invitrogen develop nucleic acid purification kits for automation with the Freedom EVO pages 16-17
From primary blood to DNA – fully automated DNA extraction The Radboud University Medical Center Nijmegen uses Chemagen magnetic beads with a Freedom EVO Workstation to extract DNA from blood samples pages 10-11
Naming the dead – the role of high throughput mitochondrial DNA sequencing The Armed Forces DNA Identification Library in the US processes thousands of samples each year with the Tecan GenesisTM RWS 200 Workstation pages 12-13
Tecan and REMP – the perfect synergy In conversation with Bernhard Iseli, COO of REMP pages 18-19
Events for the first half of 2006 page 20
Tecan Journal 1/2006
LGAT L OEBST A L PNR EOW DU S CTS
Watch out this year for new developments from the Tecan pipeline
Find out about Tecan’s new Pressure Monitored Pipetting and MCA tools
Tecan’s new Pressure Monitored Pipetting (PMP) tool is an independent option for Freedom EVO® platforms that brings a number of benefits to biopharma applications, including DNA extraction and screening assays. With PMP, the pipetting quality can be observed online for both aspirations and dispensations, and faults including tip leakage, bubbles, occluded tips or inappropriate dispensed sample volumes can quickly be detected. The PMP can also detect the liquid level of nonconducting liquids (as organic solvents) with pressure Liquid Level Detection (pLLD). The PMP also allows Liquid Level Detection by simultaneously using the conventional capacitive Liquid Level Detection (cLLD) with pLLD (dual LLD). PMP is an innovative new technology. The PMP algorithm uses known pipetting parameters, such as pipetting speed, volume and tip type, to calculate a model curve. This is then fitted to the measured pressure curve to allow for unknown pipetting parameters, such as sample viscosity or surface tension, prior to failure detection / determination, which is performed by a repeatable and sensitive failure detection algorithm. The PMP integrates easily into the Freedom EVO 100, 150 or 200 equipped with disposable tips, and comes with its own software module that monitors and analyzes pipetting. The software module is fully integrated in Freedom EVOware®. The measured pressure curves can be stored
Close up of a tip Tecan Journal 1/2006
by the software, allowing you to retrieve data when you need to. The sample traceability and sensitive error detection of the PMP bring considerably increased process stability and process security to many liquid handling applications. The PMP has already been verified for serum and aqueous solutions, for the whole volume range from 10 μl up to 1000 μl, and will be developed for other liquid classes. Tecan will release the PMP for research use in biopharma applications by April 2006. A new option for Freedom EVO – a multi-channel arm Showing again the evolutionary concept of the Freedom EVO, Tecan is adding a new multi-channel arm to the multi-functional platform. With up to 44 positions on the worktable and integration flexibility on all sides, including below the worktable, Freedom EVO with its new multi-channel arm is fully compatible with existing modules and options, such as vacuum and magnetic bead separation, incubation and reading capabilities. In addition to the multi-channel arm Freedom EVO can have up to two other arms enabling true parallel processing for higher throughput and productivity. The Freedom EVO with its new multichannel arm can be seen at the LabAutomation exhibition at Tecan booth #305.
A P P L I C AT I O N D I A G N O ST I C S
Bioser Medikal places eight ® Freedom EVOlyzers in Turkey Throughout 2005, the Company has placed no fewer than eight Freedom EVOlyzer 100/4 at five different sites in Turkey, and Bioser’s Freedom EVOlyzer team has implemented over 15 different assays from renowned reagent manufacturers.“We trust the quality and reliability of Tecan’s products and we particularly like the Freedom EVOlyzer because of its high throughput and continuous loading capability,” said Mr Cepni.
Mr Güngur Cepni, CEO of Bioser Medikal
Over the years, Tecan has enjoyed working with numerous distributors and Bioser Medikal, based in Istanbul, Turkey, is a great example of how such a collaboration can help each party to grow and prosper. Since its first contact with Tecan in 1994, Bioser has placed over 60 units throughout Turkey, including Freedom EVOlyzer®, Genesis™ RMP, Minilyser™ and Genesis FE500™ instruments. As well as Tecan, Bioser has also teamed up with other global partners such as Dade Behring and Equipar.
The Freedom EVOlyzer, Tecan's new generation of IVD-D (98/79/EC) compliant ELISA, is available in 100 cm, 150 cm to 200 cm worktable sizes. Equipped with 2, 4 or 8 channels, either steel tips or disposable tips, and an independent robotic manipulator (RoMa) arm for plate transport, the Freedom EVOlyzer can achieve a high degree of
“I strongly believe in a steady increase of automation in Turkish labs and hospitals. The demand for high quality operations and less labor intensive procedures is to be seen everywhere,” explained Mr Güngur Cepni, CEO, Bioser Medikal. “Furthermore, compliance with international quality regulations such as the European IVDD (98/79/EC) is already an important part of our market.”
parallelism which leads to maximum capacity. Continuous loading and parallel sample pipetting also ensure high throughput for the smaller sizes of the instrument. The Freedom EVOlyzer’s integrated features, such as panels, sensors, lights and warning alarms, enable close monitoring of all actions, providing high safety and reliability. “The trust is the key. If you want to grow and earn a defendable place in the market, you have to earn the customers’ trust in your company and in the products you offer.” Mr Cepni concluded: “We have been doing this since day one and that is why we have succeeded.” Minilyser™ is a trademark of REMP AG, Oberdiessback, Switzerland
Izmit (Kocaeli) Eskisehir
Tecan’s Turkey distributors are kept busy
Tecan Journal 1/2006
Q UA L I T Y A S S U R A N C E
Training brings the World together
At Tecan, we pride ourselves on an excellent reputation for training and, in the past year, our expertise in this area has helped to develop and encourage good relations with regulatory authorities and governing bodies all over the World. In May 2005, Tecan was invited by China’s State Food and Drug Administration (SFDA) to carry out quality system regulation (QSR) training for its members. A delegation comprising Mark Wang, Tecan’s Chief Representative in China, Günter Weisshaar, the Vice President of Quality Assurance and Regulatory Affairs, and Christie Rice, Tecan’s Global
The 28 SFDA participants at the Tecan workshop Tecan Journal 1/2006
Compliance Director and former employee of the US FDA, traveled to the SFDA Center for Drug Evaluation in Beijing to conduct the workshop. The course covered medical device regulations based on US guidelines, including complaint handling, adverse event reporting, corrective and preventive action, recalls and market withdrawals, all in accordance with the 21 CFR part 820 FDA regulations. The 28 participants, all employees of the SFDA, are responsible for building up Chinese regulations to match those in the US and Europe, beginning with a Chinese Compulsory Certificate for registration of new products. The information they took from the course will help to establish national guidelines for investigating incidents when something goes wrong, whether that’s
with medical devices or the companies supplying them. The workshop was very well received and was recorded and published on the SFDA homepage. Tecan also received official thanks from the SFDA and has been invited back for a second course to take place in January 2006. This workshop, for a smaller group of SFDA employees, will concentrate more closely on how to investigate complaints, the procedures involved and how to statistically evaluate the whole reporting structure. We are delighted to have been invited back to China and will continue to use our expertise in training, whether that’s in compliance and regulatory issues or in product and software workshops, to build and strengthen relations with customers, business partners and government bodies alike.
Demonstrating the new Infinite™ and Freedom EVO® in China Tecan was proud to introduce Infinite, the new multimodal microplate reader, and the Freedom EVO liquid handling platform to distributors at a recent training meeting in China. The five-day event was organized by the Tecan Group Beijing Representative Office and took place in the China World Hotel (Dec 5 2005) and the Jianguo Hotel Beijing (Dec 6 to 9 2005).
Christie Rice (Director Global Compliance, Tecan US), Mrs Yi Mei (Director of Blood Management Division, Ministry of Health) and Günter Weisshaar (Executive Vice President Global Quality & Regulatory Affairs, Tecan Group Ltd.)
Participants came from Tecan’s distributors for China, including Eastwin, Union Net Credit and Chinawealth, and Tecan’s distributors for Singapore, including In vitro Technologies, Siber Hegner Sdn Bhd, SciMed Asia Pte Ltd., Everlight Biotech Co Ltd., DKSH and Pozitronics. Tecan was represented by delegates from Switzerland, Austria, Singapore, the US and India. Invitrogen was also invited to participate in the training meeting and to explore the possibility of further co-operation with Tecan.
Half of the week’s meeting was devoted to the Infinite, which was introduced by Dieter Popp and Gerlinde ZerzaSchnitzhofer, and included comprehensive, hands-on training for all technical aspects of the plate reader, its software and its applications, as well as sales advice. The product managers Jason Meredith and Christoph Jung then took the floor with the new Freedom EVO, and introduced the audience to the benefits of the new Freedom EVOware® features as well as a variety of biopharma applications appropriate to the platform.
Mr He Ju-Cheng (General Manager of Vastec, the Tecan China distributor in the Hospital Segment), Dr Zhao Rong-Ping (Head of Immunology Division of the Clinical Diagnostic Laboratory at Shanghai Renji Hospital) and Mark Wang (Tecan Beijing Representative Office)
Mark Wang and Christie Rice
Q UA L I T Y A S S U R A N C E
Hands-on training demonstrations for Tecan’s distributors Tecan Journal 1/2006
AU TO M AT E D L I Q U I D H A N D L I N G
More power to your workstation with Freedom EVOware®
Freedom EVOware lets you take control of your lab workflow and protocols, combining both pipetting actions and scheduling in a single package. Available in Standard or Plus versions, Freedom EVOware is designed to grow with the size of your workstation and provide simple instrument control for your complex needs. The system is guarded from unauthorized entry, providing discrete levels of access through its password protection system and is ideal for laboratories working to 21CFR part 11 regulations, since it generates the electronic logs required for audit trails, electronic signatures and multi-level password control. New users will find both Freedom EVOware Standard and Freedom EVOware Plus intuitive and easy to follow. Daily maintenance routines can be controlled from the start-up screens or users can click to run a pre-existing program. Users who choose to create a new protocol enter the heart of the program where the Tecan Journal 1/2006
image of their workstation acts as a virtual reference point for their design. The library of worktable and script templates helps users get started without delay, and a range of built-in wizards and dynamic HTML info-pads guide you through common modifications. More advanced users wishing to design their protocols from scratch will find all the information they need within the Freedom EVOware program. Users choose the function they require, then ‘drag and drop’ the robotic moves into the design of their protocol. Similarly, this time-saving ‘drag and drop’ feature allows you to access labware
definitions and over 75 new or third-party device drivers. A database of liquid classes helps to define parameters so that accurate pipetting and liquid handling is achieved for almost any type of liquid you are dealing with and, in all cases, the online help is ready to point the way. Having designed a new protocol, users are able to view it as a 3D simulation before it is transferred to the Freedom EVO® Workstation, providing the opportunity to correct or fine tune any steps. Maximizing throughput and optimizing your resources is always a priority and this is where Freedom EVOware Plus comes to the fore. Incorporating all the features available in the Standard version, Freedom EVOware Plus also has an integrated scheduling facility designed specifically for high throughput, multi-plate applications. Its scheduling function allows users to create complex workflows, where the processing of a second batch of samples can begin before the first has finished, creating an entirely optimized protocol. Using Freedom EVOware Plus to design and run your protocols means your workstation need never be idle again.
Freedom EVOware takes control in San Diego Tecan’s Freedom EVOware has given researchers at the University of California in San Diego, US, the ability to program their Freedom EVO 200 Workstation to meet all their needs as they generate a genome wide RNAi library. Drosophila is used as a model organism and creation of
AU TO M AT E D L I Q U I D H A N D L I N G
An overview of the different software packages available, including the new and improved Freedom EVOware v1.2
Freedom EVO 96
Freedom EVOsim LiHa RoMa
the library involves amplifying its 20,000 genes using specific primers, prior to making double stranded RNA.
down by a large number of options that you are never going to use. Depending on what you want to achieve, you can really do a lot with Freedom EVOware. It lets you get as complex as you like in terms of workstation protocols and it gives you a greater degree of freedom to create more elegant programs.”
New and improved Freedom EVOware v1.2
The magnitude of the project initially presented some difficulties as Jeff Reid, staff research associate, explained:
“The large number of primer pairs involved in amplifying the genes and the scale of the PCR reaction preparation meant that carrying out the PCR process manually was not feasible. The obvious answer was to automate the procedure using our Freedom EVO Workstation, and so we turned to Freedom EVOware to design the protocol.” “We have found Freedom EVOware to be a good program, which is very userfriendly and reliable,” Jeff continued. “Compared to other, similar programs I have used, the Freedom EVOware desktop interface is definitely a lot cleaner. You can optimize standard protocols by accessing the commands and flight types that you need without being slowed
The laboratory’s workstation has a single, eight-channel liquid handling (LiHa) arm , a robotic manipulator (RoMa) arm and a Te-MO™ 96 multichannel pipettor. The TeMO 96 aliquots primer pairs and transfers them to 96- and 384-well plates containing genomic DNA, ready for the first round of PCR. The products of this first PCR reaction are then transferred, again by the Te-MO 96, to a fresh plate and act as templates for the second amplification step. Special primer sets are introduced during the second round of amplification to minimize crosscontamination of the samples. As the project progresses, the final PCR products will be prepared for the generation of double stranded RNA and the resulting RNAi library will be normalized for use in transfection studies.
What do you want to do?
The project has so far amplified 7,000 genes ready for conversion into RNAi molecules, with the amplification of the remaining genes part of the laboratory’s long-term goal. The team is proposing to use the RNAi library in high throughput light microscopy and positron emission topography studies to see what effects the presence of the RNAi molecules has on specific gene function in Drosophila.
Freedom EVOware version 1.2 is now available in both Standard and Plus forms for all Freedom EVO Workstations, and offers the added advantage of full integration with Tecan’s new i-control software for the series of InfiniteTM microplate readers. The Application Programming Interface (API) enhancements give programmers the freedom to control Freedom EVOware remotely and to install their own error handling routines, as well as extending Freedom EVOware’s device support through the driver toolkit. This latest version provides the simultaneous control of up to three workstation arms, including two LiHas and the enhanced Pick and Place arm. It will also control the new PosIDTM 3 positive identification system and gives improved movement in the Z-axis of the Te-MagS™. Freedom EVOware v1.2 operates on all four Freedom EVO Workstations (75, 100, 150 and 200) and is available for existing customers1. Tecan software engineers and product care teams are on hand to answer questions from customers directed through your local Tecan sales organization, providing technical support as a priority. 1
Existing customers should contact their local Tecan representatives to check for compatibility
Tecan Journal 1/2006
A P P L I C AT I O N B I O P H A R M A
From primary blood to DNA – fully automated DNA extraction The Department of Human Genetics at the Radboud University Nijmegen Medical Center provides care for patients and families with hereditary diseases, and performs ground-breaking research into the relationship between genes and diseases. The main areas of interest for the Center’s DNA laboratory are hereditary cancers, kidney disease, hereditary deafness and blindness, mental retardation and neuromuscular disease, including mitochondrial disorders. The laboratory’s research activities require a reliable, highly reproducible and standardized method for DNA extraction from full blood samples. We are using a Tecan Freedom EVO® 150 Workstation, combined with a Chemagic Magnetic Separation Module I from Chemagen, to automate our DNA extraction method using magnetic beads. Figures 1 and 2 show the experimental set-up for the DNA extraction, which includes a Tecan Freedom EVO 150 Workstation with PosIDTM barcode reader, and a liquid handling (LiHa) arm with eight tips in mixed configuration. A robotic manipulator (RoMa) arm with a centric gripper transports buckets with BD Falcon™ tubes through an opening in the benchtop (under the deck) onto the carrier axis of the Chemagic Magnetic Separation Module I (Chemagen), which is adjacent to the Tecan Workstation. There is also a dispenser for larger volumes on the Workstation. The extraction is performed in 50 ml Falcon™ tubes, which are organized in a 4 x 3 (=12) configuration in the buckets, in order to fit into the Chemagic Magnetic Separation Module I (see Figure 3). The module consists of an electromagnet and a metal 12-rod head. The rods are dipped into the Falcon™ tubes, which contain the sample and a magnetic bead suspension, so that when the electromagnet is switched on, the rods are magnetized and the beads are separated.
DNA extraction from full blood without aliquoting No aliquoting or sample distribution is necessary for this application, so the original primary blood samples are used. Each sample’s barcode is read from the primary tube at the beginning of the process and passed through the whole process to the final 2 ml Eppendorf vials containing the extracted DNA. All of the barcodes and the sample positions are stored and documented within the LIMS. The LiHa arm is used to prepare the tubes, which contain approximately 7 ml blood with lysis buffer, M-PVA Magnetic Beads, washing buffer and elution buffer. The RoMa arm moves the buckets with the samples under deck onto the carrier axis of the separator and the Tecan software starts the Chemagic protocol for the magnetic separation. Centrifugation steps do not need to be carried out with this protocol and sample preparation using Chemagen’s Chemagic kits can be completed very quickly. The high magnetite content of the magnetic beads makes extractions from any sample volume very easy and, consequently, the low levels of nonspecific binding ensure that highly pure nucleic acids are isolated from crude samples and critical sample materials. The isolated DNA can then be used directly in a variety of downstream applications.
Frans Hol, PhD, Clinical Molecular Geneticist, Radboud University Medical Center Nijmegen, Dept. Human Genetics – 120, PO Box 9101, NL-6500 HB Nijmegen, The Netherlands Tecan Journal 1/2006
The final product, the nucleic acid stock solution, is quantified by measuring the OD 280 / 260 with a Tecan GENios™ reader, and normalized in order to get a standard working concentration. The OD 280 value represents the DNA quality and the OD 260 represents the DNA concentration; typical yields from this procedure are 30 μg DNA/ml sample. Downstream processing The nucleic acid stock solution is suitable for use in a number of downstream processes, including Southern blotting, PCR, multiplex ligation-dependent probe amplification (MLPA), sequencing and conformation sensitive capillary electrophoresis. Performance, throughput and validation With the current set-up, the average throughput is 100-150 samples per week and 5,000 samples per year. With each two hour extraction run, 12 samples can be extracted as a batch and the maximum throughput per day is four runs with a total of 48 samples. The instrument set-up and the method have been thoroughly validated by comparison with manual methods and the results have been published. Quality assessment studies have been undertaken by the European Molecular Diagnostics Network in 1-2 cycles per year with results scoring at the end of the study, and this is comparable with the German „Ringversuche” method.
A P P L ICCU ATSTO ION M EBRI OSPUHPAPROMRT A
Figure 1: Fully automated DNA extraction set-up: Tecan Freedom EVO 150 Workstation and Chemagic Magnetic Separation Module I (Chemagen). Tecan Freedom EVO 150 Workstation  with PosID barcode reader , LiHa arm with 8 tips in mixed configuration  and RoMa arm with centric gripper . Buckets are transported through an opening in the benchtop under deck onto the carrier axis  of the Chemagic Magnetic Separation Module I (Chemagen) .
Figure 3: Chemagic Magnetic Separation Module I (Chemagen). Electromagnet  and metal 12-rod head .
Automating DNA extraction procedures has several advantages: • The entire process is fully automated, from the primary blood sample to the final DNA as a nucleic acid stock solution, ready for further experiments
• It is much faster (two hours) compared with the manual method, which needs an overnight incubation • It results in a much better and more reproducible quality of DNA • The DNA has a lower viscosity and so can be pipetted much more easily in downstream processes
Figure 2: Fully automated DNA extraction set-up in detail. PosID barcode reader , LiHa arm , RoMa arm with centric gripper , heat incubator  and buckets  with samples. The Radboud University Medical Center Nijmegen uses Chemagen magnetic beads with a Freedom EVO Workstation to extract DNA from blood samples
• The set-up allows excellent documentation of the process and results, and complete sample identification and tracking • Combining Tecan instruments with Chemagen kits and the Magnetic Separation Module has been well established by Tecan, and both instruments can be controlled using one software package • The system has been running very reliably, for over a year, and this is particularly important since aliquots of original blood samples are not stored, meaning that if a sample is lost then it would be necessary to go back to the patient for a second blood sample Falcon™ is a trademark of Becton, Dickinson and Company.
Tecan Journal 1/2006
A P P L I C AT I O N B I O P H A R M A
Naming the dead â€“ the role of high throughput mitochondrial DNA sequencing Timothy McMahon PhD, Head of Validation Services, Armed Forces DNA Identification Laboratory, Maryland, USA
The delicate process of identifying the remains of American soldiers killed in action, whether from recent conflicts including Iraq or older campaigns such as Korea, Vietnam and World War II, falls to the Armed Forces DNA Identification Laboratory (AFDIL) in Maryland. While the more recent casualties can be identified from fresh bone and tissue samples, the older skeletal remains present more of a problem as their nuclear genomes are often too degraded for standard nuclear
DNA, can be extracted and the Hypervariable regions one and two (HV1 and HV2) amplified using primer sets or mini primer sets, depending on the level of degradation. The results of these amplifications are then compared to reference samples collected from maternal relatives of missing forces personnel and, together with historical records, anthropological and after-action battle reports, are used to propose a formal identification.
The Armed Forces DNA Identification Library in the US processes thousands of samples each year with the Tecan Genesis 200 Workstation
DNA (nucDNA) identification methods. Instead, small sequences of intact mitochondrial DNA (mtDNA), which are present in higher concentrations in degraded remains compared to genomic Tecan Journal 1/2006
AFDIL receives over 800 bone samples from unidentified bodies and between 1,500 and 2,000 family reference samples per year. The bone samples, because of their general condition, require manual
DNA extraction and sequencing but the reference samples are more straightforward to process and are sequenced using seven primers that cover over 1,122 bases of the mtDNA control region that contains HV1 and HV2 as well as mini variable regions 1 and 2. Samples are processed in groups of 93 plus two negative controls and a PCR positive control. Preparing individual sequencing reaction plates for each primer and the actual physical requirement of placing them individually into the thermal cyclers and purifying the samples after sequencing is therefore manually intensive and equates to as much as 12 man-hours. There was obviously a need to find a faster, automated method of preparing the sequencing reactions and a Tecan GenesisTM RWS 200 Workstation was chosen for the task. The Genesis 200 Workstationâ€™s flexible programming and the ability to use a probe detection system instead of tips provided the full automation and versatility that AFDIL required, and other aspects, including sensitivity and low contamination levels, were also critical in the choice of instrument. However, it was ultimately the flexibility in programming and the ability to mount thermal cyclers and a heat sealer on the workstation platform that made the Genesis 200 the perfect choice for this application. The system has been designed to be completely automated, allowing the user to walk away once the instrument is running and return the following morning when sequencing is complete. It is fitted with a liquid handling (LiHa) arm with eight TeflonÂŽ coated fixed tips and a
A P P L I C AT I O N B I O P H A R M A
robotic manipulator (RoMa) arm, and the platform simultaneously houses the four thermal cyclers that perform the sequencing reaction. Purified mtDNA is plated out in seven 96-well plates by the LiHa arm and combined with the individual primer sequencing master mix (primer, sequencing dilution buffer and half reaction BigDye v1.1). The RoMa arm transfers each plate to the sealing unit and then moves the first four plates into the thermal cyclers. Once the sequencing reaction is complete, the RoMa arm removes the plates to the cooled deck and the remaining plates are then sequenced. Cross-contamination of samples is kept to a minimum by regular bleach washes of the fixed LiHa tips. The result of this hands-free process has been a significant drop in the number of samples that have had to be re-sequenced. Previously, on average 4.9 % of the samples would have to be re-sequenced, but using the workstation the failure rate has fallen by a factor of 10, saving time, money and reagents. Similarly, the time taken to process the samples has been cut by almost two thirds, giving technicians more time to concentrate on sequencing the bone samples and for data analysis. The sequence information generated from the reference samples is stored on an internal database ready for comparative searches, but the results from the bone samples have to undergo a stringent in-house review process before the sequence is reported to the Joint Pacific Accounting Command Central Identification Laboratory (JPAC-CIL). JPAC-CIL then submit a comparison request for the sample to AFDIL and we compare it to the internal family reference database to see if a positive ID can be made by mtDNA. For a good sample this can take up to two months although, if the DNA is badly degraded, the process will take longer. In many cases the identity of the remains is already suspected based on other information gathered by JPAC-CIL, and this allows the sample to be screened against a targeted set of reference samples. For some remains, maternal relatives may have to be found in order to generate a
conclusive identification. In all cases, automation of the reference sample sequencing process using the Tecan Genesis 200 Workstation has undoubtedly helped to answer some decade-old questions of identity and bring peace to the families of soldiers lost in conflict. Teflon速 is a registered trademark of E.I. Dupont de Nemours, Wilmington DE, USA
Tecan Genesis 200 Workstation at the AFDIL in Maryland, US
Timothy McMahon, Head of Validation Services, Armed Forces DNA Identification Laboratory, Maryland, USA
Timothy McMahon in the laboratory
Tecan Journal 1/2006
A P P L I C AT I O N B I O P H A R M A
Automated, high throughput preparation of ProteinChip® Arrays for SELDI-TOF MS profiling Laurent Jacotot*, Philippe Vaglio*, Anthony Gonçalves 1,2 4, Yves Toiron 1, Hagay Sobol 3,4, Jean-Paul Borg 1, Xavier Saunier 5, Emmanuel Russo 5 *Modul-Bio S.A.S., 232 Boulevard Sainte Marguerite, 13009 Marseille, France; 1Molecular Pharmacology, 2Medical Oncology, 3Genetic Oncology, Cancer Institute of Marseille, Institut Paoli-Calmettes and 4UMR599 Institut National de la Santé et de la Recherche Médicale (INSERM) 8University of la Méditerranée, UFR of Médecine, Marseille, France; 5 Tecan France S.A.S, 26 avenue Tony Garnier, 69007 Lyon, France.
The Institut Paoli-Calmettes in Marseille, France, has begun a global search for serum biomarkers with diagnostic, prognostic and/or predictive values in clinical samples from breast cancer patients. As part of the regional anticancer center’s clinical proteomics program, the researchers are using Tecan’s Freedom EVO® 150 platform to automate sample preparation for surface enhanced laser desorption/ionization-time of flight (SELDI-TOF) mass spectrometry (MS) to identify markers of interest.
the likely efficacy of specific anti-cancer treatments, as well as help to identify new molecular targets for innovative therapeutic strategies. Traditionally, biomarkers have been investigated using 1-D or 2-D gel electrophoresis to separate proteins, coupled to mass spectrometry for protein identification1. However, these procedures are complicated and require large amounts of material, meaning that large-scale application of these methods has generally been restricted to preclinical and basic science studies.
Comparing protein expression levels in cancer patients’ serum with normal serum samples may lead to the identification of important biomarkers that can predict for malignancy of tumors. These biomarkers can also provide valuable information concerning
SELDI-TOF MS allows relatively high throughput protein analysis of very complex biological samples, yet needs only limited preprocessing. The technology combines chromatographic fractionation of the proteome using specific protein chips and TOF MS analysis
In order to make the SELDI-TOF MS analysis more reproducible on a high throughput basis, researchers at the Institut decided to set up complete automation of the pre-analytical steps, i.e. chromatographic capture of proteins on ProteinChip® Arrays. To achieve this, they approached Modul-Bio S.A.S., a software and robotics company based in Marseille, France, that custom-builds flexible robotics systems and LIMS for biological applications, including DNA sequencing, protein interactions or chemical compound screening processes. Laurent Jacotot, Chief Executive Officer at Modul-Bio, explained, “We custom build to what people need, based on available off-the-shelf systems. For this project, we recommended the Tecan Freedom EVO 150 platform because its programming flexibility meant we could easily add the Ciphergen SELDI-TOF protocol to it. The Institut also wanted to integrate a Teleshake shaker (Variomag) and other equipment with the Tecan’s Gemini™ software system, so we developed an application to fully control the speed, motion and sequence of all the devices and movements.”
Figure 1: Automating the search for serum biomarkers from breast cancer patients
Figure 2: Tecan’s Freedom EVO 150 platform
Tecan Journal 1/2006
that can be applied to various clinical samples, including biological fluids such as serum2. Samples are directly bound to Ciphergen ProteinChip® Arrays and analyzed with SELDI-TOF MS profiling, using the ProteinChip® Biomarker system. Preparation of the ProteinChip® Arrays requires equilibration of the ProteinChips® with specific binding buffer, washing, sample spotting, incubation with shaking, washing, a short drying phase and application of the energy absorbing molecule (matrix) application.
Modul-Bio’s automated preparation of the ProteinChip® Arrays vastly increased the sample throughput for researchers at the Institut Paoli-Calmettes. The system was specially developed to provide extremely
A P P L I C AT I O N B I O P H A R M A
high-precision liquid volume handling, including liquid level sensing, single or multiple dispensing options and flexibility for different liquid classes. Importantly, automated sample processing generates improved sample quality and data reliability, due to the decrease in possible human error, and makes it easier for laboratories to carry out repetition of data sets. Using the Freedom EVO with eight
Figure 3: Modul-Bio’s Swing Java™ interface
pipetting tips and two liquid handling (LiHa) arms, the entire process can be performed within three hours for 96 samples, which equates to 192 or 288 samples each day and the potential to complete 384 samples in a day. Modul-Bio also developed a Swing Java™ interface for this application (Figures 1-3), seamlessly integrated with Gemini, to control the adapted Ciphergen protocols on the Freedom EVO (Figure 4). This interface allows the user to choose the number and volume of serum samples to be bound on Ciphergen ProteinChips® and the type of ProteinChip® used in each column of the ProteinChip® cassette (standard MTP format). The arrays are available with different chromatographic properties, including hydrophobic, hydrophilic, anion exchange, cation exchange and immobilized-metal affinity surfaces.
References 1. Hanash SM. (2000) Biomedical applications of twodimensional electrophoresis using immobilized pH gradients: current status. Electrophoresis 21: 1202-1209. 2. Fung ET, Thulasiraman V, Weinberger SR, Dalmasso EA. (2001) Protein biochips for differential profiling. Curr Opin Biotechnol 12: 65-69. 3. Gonçalves A, Esterni B, Bertucci F, Sauvan R, Chabannon C, Cubizolles M, Bardou VJ, Houvenaegel G, Jacquemier J, Granjeaud S, Meng X-Y, Fung ET, Birnbaum D, Maraninchi D, Viens P, Borg J-P. (2005) Post-operative serum proteomic profiles may predict metastatic relapse in high-risk primary breast cancer patients receiving adjuvant chemotherapy. Oncogene (in press) 4. Petricoin EF, Ardekani AM, Hitt BA, Levine PJ, Fusaro VA, Steinberg SM, Mills GB, Simone C, Fishman DA, Kohn
Depending on the different type of protein chip, specific protocols with specific buffer are then executed by the Tecan system. This set-up has been used at the Institut Paoli-Calmettes to investigate serum samples from two different groups of patients. In the first study, samples from high-risk, early breast cancer patients were analyzed for serum biomarkers that could predict for metastatic relapse or metastasis-free survival. The second study investigated samples from patients certified with and without BRCA1 mutations, which have been implicated in a large number of hereditary breast cancers, in order to generate plasma protein biomarkers that could be associated with the presence of the mutation3.
SELDI-TOF MS techniques make it possible to analyze biomarkers from small sample volumes requiring only limited preprocessing phases, and the availability of tools such as Tecan’s Freedom EVO platform and Ciphergen’s ProteinChip® Arrays and Biomarker System make largescale analyses much easier. Coupled to appropriate bioinformatic tools, SELDI-TOF MS has shown to be a very promising method for probing serum to identify protein patterns and/or biomarkers related to various stages and types of solid tumors4-9, which could serve as early diagnostic markers. JAVA™ is a trademark of Sun Microsystems, Inc. in the United States and other countries ProteinChip® is a registered trademark of Ciphergen Biosystems Inc., Fremont, CA, USA
Figure 4: Tecan’s Freedom EVO 150 platform and Ciphergen ProteinChips® cassette
EC, Liotta LA. (2002) Use of proteomic patterns in serum to identify ovarian cancer. Lancet 359: 572-577. 5. Adam BL, Qu Y, Davis JW, Ward MD, Clements MA, Cazares LH, Semmes OJ, Schellhammer PF, Yasui Y, Feng Z, Wright GL Jr. (2002) Serum protein fingerprinting coupled with a pattern-matching algorithm distinguishes prostate cancer from benign prostate hyperplasia and healthy men. Cancer Res 62: 3609-3614. 6. Petricoin EF III, Ornstein DK, Paweletz CP, Ardekani A, Hackett PS, Hitt BA, Velassco A, Trucco C, Wiegand L, Wood K, Simone CB, Levine PJ, Linehan WM, EmmertBuck MR, Steinberg SM, Kohn EC, Liotta LA. (2002) Serum proteomic patterns for detection of prostate cancer. J Natl Cancer Inst 94: 1576-1578.
using surface-enhanced laser desorption and ionization mass spectrometry. Clin Cancer Res 10: 860-868. 8. Wadsworth JT, Somers KD, Cazares LH, Malik G, Adam BL, Stack BC Jr, Wright GL Jr, Semmes OJ. (2004) Serum protein profiles to identify head and neck cancer. Clin Cancer Res 10: 1625-1632. 9. Zhang Z, Bast RC Jr, Yu Y, Li J, Sokoll LJ, Rai AJ, Rosenzweig JM, Cameron B, Wang YY, Meng X-Y, Berchuck A, van Haaften-Day C, Hacker NF, de Bruijn HWA, van der Zee AGJ, Jacobs IJ, Fung ET, Chan DW. (2004) Three biomarkers identified from serum proteomic analysis for the detection of early stage ovarian cancer. Cancer Res 64: 5882-5890.
7. Koopmann J, Zhang Z, White N, Rosenzweig J, Fedarko N, Jagannath S, Canto MI, Yeo CJ, Chan DW, Goggins M. (2004) Serum diagnosis of pancreatic adenocarcinoma
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A P P L I C AT I O N B I O P H A R M A
Automated genomic DNA extraction from buccal cells with Invitrogen’s ChargeSwitch® Technology Extracting genomic DNA (gDNA) from buccal swabs or pelleted mouthwashes is extremely useful as a quick, non-invasive technique for collection and isolation of DNA. This method is used for DNA extraction in many applications, including genotyping, detection of disease markers and for comparison to crime scene samples. In response to the demand for increased sample throughput in these applications, Tecan and Invitrogen have developed a range of ChargeSwitch® Technology-based nucleic acid purification kits that are fully automated using Tecan robots. The quantity of cells and, therefore, DNA obtained from a buccal swab or pelleted mouthwash can often be very low. As a result, techniques used for purification of DNA from buccal cells must be sensitive and reliable, as well as ensure that the DNA obtained is suitable for relevant downstream applications such as PCR, sequencing and STR analysis. Currently, there are a number of automated techniques for the purification of gDNA from buccal cells, all of which often use harsh ionic chaotropes, such as guanidinium isothiocyanate, hazardous organic reagents such as ethanol, phenol, chloroform or isopropranol (IPA), or expensive and undesirable alcohols. All of these reagents can penetrate during the purification process and this can have a detrimental effect on the efficiency of enzymes such as Taq® polymerase, resulting in reaction failure, as well as potentially causing problems for liquid handling systems, such as salt precipitation in lines. ChargeSwitch® Technology avoids the use of any chaotropes, organic solvents or alcohols and delivers high yield, high purity nucleic acid in a rapid and cost effective manner.
Nucleic acid sample preparation with ChargeSwitch® Technology ChargeSwitch® Technology is a unique chemistry that acts as a pH-dependent ionic switch, which is “switched on” at a lower pH, becoming positively charged and binding DNA. When “switched off” by raising the pH, the charge is neutralized, allowing purified DNA to be released. The ChargeSwitch® protocol is summarized on the opposite page.
Automated purification of gDNA from buccal cells using ChargeSwitch® Technology on Tecan workstations (Figure 1), such as the GenesisTM or Freedom EVO®, is a reliable, walkaway process with many advantages. Automated protocols for isolating gDNA from buccal cells generally require either a centrifugation or vacuum step, which can cause a bottleneck in the process.
During gDNA isolation, variations in template quantity may have an effect on the final STR profile, where too much template may lead to overamplification and saturation, while too little template may result in partial profiles or failures. Through limitation of binding capacity, ChargeSwitch® Technology can provide a normalized yield of purified gDNA in the range of 1-3 ng/ml. At this concentration, the need for quantification is removed and users can readily modify the amount of beads added in order to adjust the normalization. Furthermore, using the ChargeSwitch® Genomic DNA Purification protocol (ChargeSwitch® gDNA Normalized Buccal Cell Kit, Cat. No.
(a) Low throughput
(b) Medium throughput
(c) High throughput
(d) Ultra high throughput
Automating gDNA isolation
Figure 1: Tecan automated platforms
Tecan Journal 1/2006
Using ChargeSwitch® Technology, coated onto the highest quality magnetic beads available, vacuum and centrifugation steps are no longer necessary, making the technology ideally suited to high throughput environments.
A P P L I C AT I O N B I O P H A R M A
ChargeSwitch® protocol Digest
1. Add 1 ml of digestion reagent to a buccal cell swab in a 96-well deep well plate.
5. Separate beads on the magnet and discard supernatant.
2. Incubate at 37-55°C for 20 minutes.
6. Resuspend ChargeSwitch® beads in 1 ml of wash buffer and wait for a pellet to reform over the magnet.
Bind 3. Transfer digest supernatant into a fresh 96-well deep well plate containing purification reagents and ChargeSwitch® beads. 4. Mix and incubate for 5 minutes.
7. Remove supernatant and repeat wash step.
Elute 8. Remove plate from magnet and resuspend ChargeSwitch® beads in 150 μl of elution buffer.
CS11020) can remove the need for quantitation prior to STR analysis, saving time and increasing throughput (Figure 2). When fully automated on Tecan workstations, the ChargeSwitch® protocol offers complete walkaway to minimize risk of cross contamination (Figure 3). ChargeSwitch® avoids the use of enzyme inhibitors, increasing the performance of enzymatic reactions. The high yielding kit can provide larger yields for less sensitive downstream reactions, or for when DNA needs to be archived. For more information about ChargeSwitch® Technology and its use in genomic DNA extraction from buccal cells, please go to www.invitrogen.com/naprep.
Figure 3: Low risk of cross contamination
Single buccal swabs were placed in alternate microtitre plate wells (highlighted in purple) to demonstrate that the adjacent empty wells would not be contaminated by DNA from other samples. DNA was purified using the ChargeSwitch® Genomic DNA Purification protocol (Buccal Cells, High Yield) on the Tecan Genesis robotic workstation. The DNA content of each microtitre plate well was then measured using Quant-it™ PicoGreen® analysis (values are shown in ng/μl). Results indicate that cross contamination of samples does not occur. PicoGreen® is a registered trademark and Quant-it™ is a trademark of Molecular Probes, Inc
Figure 2: High yield data
Yield of gDNA purified from eight samples using the ChargeSwitch® Genomic DNA Purification protocol (ChargeSwitch® gDNA Buccal Cell Kit, Cat No. CS11021) on the Tecan Genesis. The variation in concentration is due to the amount of epithelial cells collected in the sampling process.
REMP Tube Technology™ and Small Size Store™ are trademarks of REMP AG, Oberdiessbach, Switzerland ChargeSwitch® is a registered trademark of Invitrogen Taq® is a registered trademark of Fermentas
ChargeSwitch® protocols are also available for automated plasmid micropreps with no centrifugation or vacuum filtration, automated PCR cleanup with adjustable size exclusion and automated gDNA purification from blood with no centrifugation or vacuum filtration.
Tecan Journal 1/2006
Tecan and REMP – the perfect synergy
In June 2005, the Tecan Group of companies acquired REMP. Bernhard Iseli, COO of REMP and an employee for four years, talks about the change of direction for REMP now that it is part of the Tecan Group. Firstly, would you say that the acquisition of REMP by Tecan came as a shock to REMP’s employees? I wouldn’t say a shock but perhaps a surprise! The management team at REMP was always very clear in communicating its plans to take the Company in this direction and I believe this was well understood by the employees. In reality, our employees were just waiting to see what would happen and from the beginning they saw this as a very positive move that gave them more personal security as part of a larger company group. So surprising, yes, but not a shock!
Tecan Journal 1/2006
And six months on, do REMP employees already feel part of the Tecan Group? Yes they do. Everyone realised there would be changes but we are very comfortable with the whole situation. Tecan has been helpful and supportive from the start and we have been made very welcome – even at Tecan’s 25th birthday party. I’m quite sure that an important factor has been that we are both Swiss companies. There have been relatively few changes – we have the same culture, the same understanding, the same
language and a similar work ethic – and that has been a real plus. At the same time we all recognize the great opportunities that have now opened up for REMP; we have the financial security and capacity to introduce our products to entirely new markets all over the World. How is Tecan helping you to make the most of these opportunities? Well, first of all we immediately acquired over a hundred additional sales people! By January 2006, all Tecan sales personnel will be trained and familiar with REMP products and this has been a
REMP’s headquarters in Oberdiessbach, Switzerland
huge step for us. For this to happen effectively we have developed a clear structure to the product portfolio. How have your existing customers reacted to the acquisition? Mainly positively. They too are pleased that there is a strong partner behind REMP and they see advantages to pooling together products and particularly services. Service has traditionally been a very important aspect of REMP’s offering, with over 90% of our systems on yearly or three-yearly maintenance contracts. In this field we see the biggest potential to increase our services as a group. And from which new markets do you expect to attract new customers?
complement each other. For example, REMP’s revolutionary large-scale automated storage and retrieval systems integrate seamlessly with Tecan’s stateof-the-art automation platforms, and Tecan has a whole portfolio of DNA extraction, normalization and other genomics tools that will fit perfectly with bio-fluid storage and consumables. And what about future product development? Do you expect to draw some aspects of Tecan and REMP products together? Not yet. We are still following a clear transition plan led by the integration team whereby we are systematically pulling together all aspects of the business; production, sales, marketing etc.
At the end of 2005, we perfected the product portfolio and decided which products we want to bring to which markets. I am sure that product development will follow soon and I personally would say that the first step, the easiest step, would be to work at a software level. Once that step has been taken in principle, further development will almost certainly be driven by a need by a customer to fully integrate REMP and Tecan products with each other; a need, for example, to have a seamless system that takes samples from a store to a Freedom EVO® platform. From that process we will be able to make sense of how best to bring the technologies together for the market as a whole.
Well, our main market has always been the pharmaceutical/drug discovery market, specifically large store solutions for compound management. Of course, we want to grow in our existing markets and the new small size store product has opened up new avenues for this. However, at the same time, we are currently developing a biosample store in collaboration with a customer and this -80˚ technology is set to become a major focus for next year. There is a great deal of interest in this project in the life science market and, although it is an entirely new field for us, it will bring us even closer to Tecan’s areas of interest. There is already an overlap of Tecan and REMP markets though, isn’t there? Yes, certainly there are many areas of overlap which will make the transition of the two companies a fairly natural progression. Together, they comprehensively cover applications in the drug discovery, biotech, diagnostic and general laboratory arenas and the product portfolios of both companies
Central to our product range are the sample stores. REMP offers the ideal storage solution, whether for -80°C, -20°C or room temperature, for hundreds or millions of samples, vials or tube tacks, fully automated or manual.
Tecan Journal 1/2006
Meet Tecan at these events during the first half of 2006 Europe 3ème Assises de Genetique Humaine et Medicale
January 26-28 2006
RNAi 2006 – Advances in RNA interference research
March 22-23 2006
Genomes to Systems Conference
March 22-24 2006
March 28-31 2006
April 25-28 2006
38th European Human Genetics Conference
May 06-09 2006
May 8-11 2006
June 28-30 2006
European Academy of Forensic Science
June 14-16 2006
March 25-28 2006
Pharmaceutical Society of Japan
March 28 2006
Protein Science Society of Japan
April 24-26 2006
International Bio Expo
May 17-19 2006
International Congress of Biochemistry and Molecular Biology
June 18-23 2006
Palm Springs, CA
January 21-25 2006
MD & M West Tradeshow
January 31-Feb 02 2006
AAFS 58th Annual Meeting
February 20-25 2006
March 12-17 2006
April 01-05 2006
May 22-24 2006
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 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, email@example.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. Gemini™, Genesis™, Genesis FE500™, GENios™, Infinite™, PosID™, Te-MagS™ and Te-MO™ 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. © 2006 Tecan Trading AG, Switzerland, all rights reserved.
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Tecan Journal 1/2006
REMP (USA) Inc. +1 508 429 2200
REMP Deutschland GmbH +49 6126 5831 0
REMP Nippon AG +81 3 3539 1771