PLATETRAK APPLICATION NOTE AN004-LH

Drug Discovery Research Clinical Screening

Liquid Handling

PlateTrak™ Application Note

Automation of DNA Purification Using the PlateTrak Automated Microplate Processing System Advances in throughput, reproducibility, and reagent conservation

Michelle Stevens PerkinElmer Life Sciences, Inc.

Kevin McKernan Center for Genome Research, Whitehead Institute

www..com/lifesciences Collaboration between PerkinElmer and the Center for Genome Research at the Whitehead Institute PerkinElmer Life Sciences, Inc., in collaboration with the scientists from the Center for Genome Research at the Whitehead Institute, designed a genomic pipeline of PlateTrak™ systems for the automation of the SPRI™ protocol. SPRI is based on DNA binding to the surface of carboxyl coated magnetic particles for DNA and plasmid purification. Today, the Center is routinely doing 100,000 lanes per day or approximately 2,400,000 per month, making this the fastest microplate-based DNA purification system in use on the .

With the recent increase in genomic research, laboratories are pressed for increased throughput and reliability along with the need to reduce cost. The enormous scale of the Human Genome Project has created a demand for automated systems aimed at high throughput and reproducibility. PerkinElmer Life Sciences, Inc. has completed a collaboration with the Center for Genome Research, at the Whitehead Institute for Biomedical Research, Cambridge, MA to develop expanded capabilities on the PlateTrak conveyor based microplate processing instruments to automate the DNA purification process. The Whitehead Institute is one of the largest contributors to the Human Genome Project. This collaboration led to the development of many exciting new microplate processing solutions on the PlateTrak system that greatly enhance the capabilities for the automation of genomic research. In addition, the ability to reduce the scale of sequencing reactions through advanced pipetting systems produced significant cost savings. The standard procedures developed for isolating DNA typically employ centrifugation and solvent extraction methods that are very difficult to automate. To overcome this, the scientists at the Whitehead Institute developed a new procedure using SPRI (Solid Phase Reversible immobilization) chemistry (Hawkins et al). The SPRI proce- dure is based on DNA binding to the surface of coated paramagnetic particles. These beads display magnetic properties when placed in a magnetic field, but retain no residual magnetism when removed from the magnetic field. This enables rapid and reproducible automation of buffer exchanges and extensive washes required for DNA preparations. The procedure has been developed and optimized for single stranded DNA isolation, such as M13 phage utilizing iron oxide magnetic particles and double stranded plasmid DNA utilizing carboxyl coated magnetic particles. The SPRI procedure allowed the development of an automated procedure in a microplate format with a throughput of five hundred 384-well plates, or 200,000 separate DNA preparations per day. This is the highest throughput achieved in any lab today for genomics. The procedure is rapid, low cost, and provides high quality DNA sequencing results.

2 PerkinElmer paired newly engineered technologies Plate Sealer: Thermal microplate sealer. with standard ones to develop several automated Lift and Transfer Station: Mechanism that enables systems. The following is a list, with a brief microplates to be lifted off and returned to conveyer description, of the separate modules utilized in after aspirating or dispensing. An example would be these systems: compression or expansion of plate libraries. Plate Piercing Module: Module that pierces sealed microplates at an adjustable height for 96- or 384-well plates. Stacker Module: Stacker Module with the capacity to hold a maximum of 50 SBS standard microplates. The cassette can accommodate a variety of plate types including standard plates, deepwell plates, PerkinElmer Tip Racks, and microplates with carriers. Dispense Module: A 96- or 384-tip dispense head that moves only in the Z direction. Reagents or Tip Wash are kept below the conveyer so they can be accessed when a plate is not in position. The heads have a wide volume range depending on tip size. 96-channel head, P235 Disposable Tip (5 µL to 235 µL) 96-channel head, P50 Disposable Tip (0.5 µL to 50 µL) 20 inch MultiPosition Dispense Module with a P250 96-channel head, P20 Disposable Tip Dispense Head on the PlateTrak shown with 250 mL (0.5 µL to 20 µL) bottle mixer cartridge assembly, 96/384 tipwash 384-channel head, P30 Disposable Tip cartridge assembly and a 1 L bottle reservoir cartridge. (0.5 µL to 30 µL) Connecting Diving Boards: Multifunctional diving MultiPosition Dispense Module: A 96- or 384-channel boards used for accessing the conveyer by another dispensing head capable of movements in the X, Y, robotic system. These may be connected to join two and Z directions. The dispense head moves across a separate PlateTrak systems. multiposition grid deck, extending the accessible Wash Module: A Wash Module with a 96 probe mani- stations on the conveyor path. There are 3 model fold with fused aspirate and dispense probes. There is sizes available for varying capacity for plates, reagents, a 96-well magnetic plate lift positioned beneath the tip boxes or plug and play cartridge accessories. deck for magnetic bead washing. 10 inch MPD – 3 position deck, 1 plate stop, Recirculating Chillers: Chilling mechanisms with 2 cartridge slots single or quad positions that keep reagents chilled to 20 inch MPD – 9 position deck, 2 plate stops, 4° C. Quad chillers are ideally suited for the setup of 6 cartridge slots dye primers and terminators for sequencing reactions. 30 inch MPD – 15 position deck, 3 plate Plate to Waste Station: Shuttle mechanism at the end stops, 10 cartridge slots of the conveyor to allow plates to be lifted from the conveyor and discarded to a waste station. Gantry Gripper: Pick and place robotic arm capable of moving plates from and to the conveyor for additional Mixing Bottle and Reservoir: Adjustable speed control processing behind the conveyor. The 30 inch module reagent bottle and reservoir cartridge for keeping mag- has been integrated with 4 position orbital shakers, netic beads in suspension. 8 position magnetic plate deck, and an 8 position Magnetic Plate Shelf: Programmable sliding magnetic magnetic plate shelf. shelf to increase deck capacity for plate handling for SideTrak®: Pick and place robotic arm for the integration separation steps. of peripheral microplate devices to the PlateTrak system. The SideTrak arm moves plates from diving board to external ancillary equipment. Peripheral devices include thermal heat sealers, readers, microplate handlers, microplate hotels and carousels.

3 Transfer Purification Purification Sequencing Pooling Deck Deck #1 Deck #2 Deck Deck

Clones Lysis of SPRI M13 Reformat Sequencing Samples off grown in M13 and procedure: purification purified DNA to reaction for analysis deepwells resuspension Separation of and separation 384-well plates clean-up on the PE and cells of plasmid plasmid DNA of plasmid if needed and 3700 DNA pelleted cell pellets from E. Coli DNA from add the sequencers DNA RNA sequence mixtures for thermocycling

These procedures are currently run in a 96-well These procedures are currently run in format. There will be a conversion to a 384-well a 384-well format. format in the near future.

The instruments for the Whitehead Institute can be In operation, the modules are optimally accessed in broken down into five phases: parallel and sequentially — all are working at the Phase Description same time on different plates and the plates do not move backward. The PlateTrak systems are utilizing 1 Resuspension and Lysis procedure an assembly-line approach to plate processing. 2 & 3 DNA purification, which is handled in Keeping the line moving in one direction offers two steps; optimal throughput. However, since the conveyor 4 Sequencing set up and if needed moves in both the forward and reverse directions, 5 Reaction Pooling or Cleanup modules can be utilized in any order. Following are detailed drawings of each instrument in the system, along with a quick summary of the process during each phase.

30 inch MultiPosition Gripper Tool integrated with an Close-up of orbital shaker. orbital shaker and an 8 position magnetic plate deck.

4 PlateTrak System Layout for Transfer System.

Transfer Deck Process Steps for Transfer Deck Purpose: The purpose of this instrument is two fold: Modules Description 1) M13 Lysis 1-3 Stacker to hold the deepwell plates for processing Single M13 clones grown in deepwell plates that 4 96-Channel Dispenser adds SDS to Microtiter plates to lyse the phage have been centrifuged or filtered to pellet host cells 5-7 30 inch MultiPosition Gripper Tool moves microplates to are placed into the system. These supernatants are shakers and the magnetic plate deck to improve lysis pipetted to new shallow well microplates and SDS 8 Stacker for 96-well Microtiter plates is added to lyse the recombinant M13 phage. Once 9 96-Channel Dispenser aspirates the supernatant from the the M13 plates are lysed, they move directly to deepwell plate and dispenses it to the Microtiter plates the Purification #2 instrument. Since M13 is a 10 Stacker for 96-well Microtiter plates filamentagous virus, it is genetically programmed 11-12 2 position Diving Board for future robotic integration to expel itself from the host bacterial E. Coli cell. 13 Moves deepwell plates to trash after supernatant has Simple centrifugation separates the E. Coli cells been removed and its DNA from the growth media containing the recombinant viral DNA. 2) Plasmid Cell Pellet Resuspension Growth plates are again centrifuged and cells pelleted. The supernatant is discarded, and the plates are placed onto this device. This robot then adds resuspension buffer to the cell pellets and vortexes the plates to resuspend the cell pellets. Once the cells are resuspended, they are transferred from the growth plates to Microtiter® plates that are delivered to the downstream Purification #1 robot. Output: One Microtiter plate for each deepwell plate. Each Microtiter Plate contains M13 Phage or E. Coli cells containing Plasmids which have been resuspened into solution. The M13 plates move directly to the Purification #2 instrument. The plasmid plates move directly to the Purification #1 instrument. This PlateTrak instrument is not currently connected to any other PlateTrak system. Throughput: One 96-well plate per minute.

5 PlateTrak System Layout for DNA Purification System #1.

DNA Purification Deck #1 the source plates. This is now analogous to the M13 preparations after resuspension and lysis. Purpose: The purification decks are where the SPRI There is no E. Coli DNA to compete for magnetic protocol is performed. The purification is based on beads in the second half of the purification protocol size selection of the DNA. Once the host cell has on Purification Deck #2. These output plates move become lysed, the host E. Coli DNA is in solution with onto the Purification #2 instrument. the plasmid DNA. Both molecules share identical bio- chemical properties but can be separated based on This PlateTrak system is connected to DNA size. The E. Coli DNA is 4.2 Million base pairs (Mb), Purification Deck #2 by a conveyor to conveyor link. and most plasmids are usually about 3-10 thousand Throughput: One 96-well plate every 100 seconds. base pairs (Kb). The function of the Purification #1 deck is to perform a differential PEG precipitation with paramagnetic particles. The protocol is designed Process Steps for DNA Purification to selectively precipitate large DNA fragments of the Deck #1 E. Coli cells and proteins to paramagnetic micros- Modules Description pheres without precipitating the plasmid DNA. 1 Stacker for Microtiter plates (source plates) The beads are separated leaving plasmids in solution. 2-3 Dispenses NaOH (caustic) and SDS (detergent) into source This “plasmid enriched supernatant” is then collected plates for bacterial cell lysis and deposited into a new Microtiter plate for further 4-6 MultiPosition Gripper Tool moves plates to orbital shakers purification on the Purification #2 system. The to improve lysis magnetic beads containing the E. Coli DNA are 7 Open Module discarded at a “Plate to Waste” station. 8-9 96-channel dispenser adds PEG (binding) and magnetic General Protocol: Initially, the magnetic beads are bead solution added to lysed phage or lysed E. Coli cells containing 10-12 MultiPosition Gripper Tool moves plates to the orbital shaker and magnetic plate deck for incubation plasmids in the shallow well Microtiter plates togeth- er with a binding buffer of PEG and salt. The PEG and 13 Stacker for Microtiter plates (destination plates) salt concentrations have been optimized for both the 14-15 Reads barcodes and aspirates supernatant from source plates and dispenses it to destination plates single stranded M13 and double stranded DNA binding protocols. 16 Stacker for destination plates 17 For future robotic integration Input Source: 96-well plates of E. Coli bacteria con- 18 Moves source plates to trash and destination plates to taining plasmids. attached PlateTrak Output Product: 96-well Microtiter plates containing plasmid enriched supernatant. The bacterial DNA is bound to the magnetic beads and is discarded with

6 MultiPosition (MPD) module with 96-tip dispense and 96 probe plate washer with magnetic plate lift. bottle mixer to keep beads in suspension.

Magnetic Plate Shelf extended behind the PlateTrak to expand capacity for an additional 8 magnetic plates.

7 PlateTrak System Layout for DNA Purification System #2.

DNA Purification Deck #2 Process Steps for DNA Purification Purpose: The Purification Deck #2 performs the entire #2 Deck purification process for the M13 DNA and the second Modules Description half of the plasmid DNA preparation, which is the 1 Stacker for Microtiter plates (source plates) separation of the plasmid DNA from the RNA in solu- 2-3 Adds PEG and magnetic bead solution tion, utilizing the magnetic beads. This system is also 4-6 MultiPosition Gripper Tool moves plates to orbital shakers utilized for the purification of PCR products with the and magnetic plate deck for incubation same procedures. 7 Wash Module with magnetic plate lift • 1X 70% EtOH wash with final aspirate to remove EtOH Input Source: 96-well plates containing lysed M13 8 Wash Module with magnetic plate lift phage DNA or plasmid DNA in solution. Since this • 2X 70% EtOH wash with final aspirate to remove EtOH system is connected to DNA Purification Deck #1, the 9 Wash Module with magnetic plate lift plasmid DNA plates can be automatically transferred • 1X 70% EtOH wash with final aspirate to remove EtOH to this system. 10 Stacker for Microtiter plates (destination plates) Output Product: Dry Microtiter plates with DNA 11-12 Receives plates from DNA Purification Deck #1 bound to magnetic beads. 13 2 position diving board - for future robotic integration The plates are manually moved to the Sequencing Deck (#5). Throughput: One 96-well plate every minute.

Plate Lift and Transfer mechanism incorporated into Close-up of the plate lift mechanism. a 20 inch MultiPosition Dispense Module to securely transport a 384-well master plate behind the conveyor.

8 PlateTrak System Layout for Sequencing System.

Sequencing Deck plateProcess sealer at theSteps end of for the Sequencingrun for sealing prior Deck to Purpose: The sequencing system can set up sequenc- Modules Description ing reactions utilizing both Dye Primer and Dye 1 Stacker for microtiter plates (source plates) Terminator chemistries in the forward and reverse 2 96-Channel Dispense Module for reconstitution of the DNA directions. The magnetic beads are separated and the and separation from the beads supernatants re-moved for testing and sequencing. 3 Open Module The magnetic beads are re-suspended in a water 4 Stacker for 384-well plates (thermocycled plates) elution buffer with a three minute incubation at room (96-well plates can be used) temperature. The three minute incubation can be 5 Stacker for 384-well plates (96-well plates can be used) avoided with a simple 20 second shake utilizing an 6-7 Lift & Transfer Station and a Magnetic Plate Lift with a in-line shaker mechanism on the conveyor. For dye 96-Channel Dispenser – the Lift & Transfer will move a plate primers, the system will add the sequence mix-tures off the deck. The Magnetic Plate Lift will hold the beads down so the 96-Channel Dispenser can transfer the (Dye Primer and – A, T, C, and G) which are chilled to supernatant to the plate on the Lift and Transfer Module. 4° C in a color-coded four quad chilling station. These If a 384-well plate is used, then four 96-well plates are plates are then reformatted into 384-well plates, and compressed. If not, then the DNA is simply transferred to the forward or reverse mixtures are added to the plate. the destination plate. For dye terminator chemistries, the dye terminator 8 Open Module solution chilled to 4° C is added to the DNA. Purified 9-10 96-Channel Dispenser with 4 color-coded anodized aluminum DNA products from two 96-well plates are added to a chilling stations for addition of the sequencing reagents (A, C, T, and G.) This Module adds the forward primers. 384-well plate with two copies positioned in diagonal 11 Open Module quadrants of the 384-well plate. The forward reaction 12-13 96-Channel Dispenser with 4 color-coded anodized mixture is added to one copy and the reverse mixture aluminum chilling stations for sequencing reagents is added to the second. This ensures that the data will (A, C, T, and G). This Module adds the reverse primers. be accurate even if the plate becomes flipped in orien- 14 Stacker tation throughout the process. The DNA will be ready 15-16 2 position diving board for future robotic integration for the thermocycling operation and subsequent iden- 17-18 SideTrak pick and place robotic arm integrated with a thermal tification on the PerkinElmer 3700 series sequencer. plate sealer and a PlateStak for storage of sealed plates. Input Source: Purified DNA in 96-well Microtiter plates. If appropriate, 384-well plates will be added thermocycling. The plates are loaded to a PlateStak™ for the reformatting. instrument on the SideTrak following sealing. This Output Product: Sealed plates ready for the thermocycler. maintains unidirectional processing for optimum These plates are put on a thermocycler and then moved throughput. to the PE 3700 sequencer. This instrument is integrated Throughput: One 384-well plate every two minutes. to a SideTrak robotic arm, which moves plates into a

9 PlateTrak System Layout for Pooling System.

Process Steps for Pool Deck Modules Description 1 Stacker for 384-well plates (or 96-well plates as purification system) 2 Stacker for 96-well plates 3 Pierces seal on 384-well plates 4 96-Channel Dispenser aspirates from 384-well plates and dispenses to 96-well plates 5-6 96-Channel Dispenser adds PEG (binding) and magnetic bead solution 7-9 MultiPosition Gripper Tool moves plates to orbital shakers and magnetic platedeck for incubation 10 Wash Module with Magnetic Plate Lift 1X wash 70% EtOH wash with final aspirate to remove EtOH 11 Wash Module with MagneticPlate Lift 2X wash 70% EtOH wash with final aspirate to remove EtOH 12 Wash Module with Magnetic Plate Lift 1X wash 70% EtOH wash with final aspirate to remove EtOH Close-up of 20 inch MPD deck grid shown with 13 Stacker for empty 384-well plates (or 96-well plates if used recirculating chiller, magnetic stirrer cartridge, as purification system) and multiple auto tip rack loaders. 14 Stacker for 96-well plates 15-16 2 position Diving Board for future robotic integration 17 Moves plates to trash and can connect to another system

Pooling Deck Purpose: The function of Pooling Decks is two fold. Input Source: The source can be 384-well plates or First a Pooling Deck can take a 384-well cycle plate 96-well Microtiter plates. and compress or “pool” to a single 96-well purification Output Products: There can be two products: 96-well plate for bead based cleanup. This is required when plates ready after bead prep clean-up or dry Microtiter utilizing the dye primer chemistries for sequencing. plates with DNA bound to magnetic beads. This can also function for the second half of the purification steps, and is upgradable to a 384-well This system is not currently connected to another system. bead prep clean-up protocol. Throughput: One 384-well plate every minute.

10 The SPRI technology coupled with the PlateTrak systems developed for the Whitehead Institute have greatly enhanced processing capabilities. One of the primary advantages of this procedure for DNA purification is the reagent savings, in particular Taq FS polymerase, through the ability of the PlateTrak instruments to reproducibly set up small volume reactions. For the sequencing reactions, only 1 mL Taq additions and 3 µL DNA additions are necessary. The yield for the M13 purification is 4-5 µg DNA, and the yield for the plasmid purification is 2-4 µg DNA. There is a total of 2-4 µg in 40 µL from the resuspension. Only 3 µL of this is utilized for the sequencing. Also, 85% of the DNA preparations work with sequencing reads of greater than two-hundred 99% accurate (phred 20) bases. Utilizing 1/16th of the recommended amount of BigDye reagent from PerkinElmer, we attain five- hundred-fifty 99% accurate (phred 20) bases. In addition to generating DNA suitable for sequencing, there are also great results with PCR product purification as assessed by concordance levels with microarrays. In regards to purity, the DNA is suitable for mammalian cell transfections, suggesting very low endotoxin levels. This SPRI-based procedure is rapid, economical (pennies per well), and an excellent means of obtaining DNA sequencing results. It allows the Whitehead Institute to be the largest contributor toward the Human Genome Project by obtaining the highest throughput in any lab with this application. The SPRI protocols can be performed for the purification of plasmid DNA, M13 DNA, and PCR products, as well as, the clean-up of fluorescent sanger sequencing reactions. The combination of PerkinElmer’s automation equip- ment and SPRI chemistry delivers a fully automated purification platform that provides a high yield of high quality DNA suitable for a variety of applications.

11 Worldwide Headquarters: PerkinElmer Life Sciences, Inc., 549 Albany Street, , MA 02118-2512 USA (800) 551-2121 European Headquarters: PerkinElmer Life Sciences, Inc., Imperiastraat 8, BE-1930 Zaventem Belgium Technical Support: in Europe: [email protected] in US and Rest of World: [email protected] Belgium: Tel: 0800 94 540 • : Tel: 0800 90 77 62 • Netherlands: Tel: 0800 02 23 042 • : Tel: 0800 1 81 00 32 • United Kingdom: Tel: 0800 89 60 46 Switzerland: Tel: 0800 55 50 27 • Italy: Tel: 800 79 03 10 • : Tel: 020 79 07 35 • Norway: Tel: 800 11 947 • Denmark: Tel: 80 88 3477 • Spain: Tel: 900 973 255

SPRI is a trademark of Agencourt Bioscience. SPRI is manufactured by Agencourt Bioscience Corp. on behalf of PerkinElmer Life Sciences, Inc. Microtiter is a registered trademark of Dynex Technologies, Inc. All other trademarks or registered trademarks are the property of PerkinElmer Life Sciences, Inc. P10635 © 2002 PerkinElmer Life Sciences, Inc. www.perkinelmer.com/lifesciences