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PlateTrak™ Application Note

Automation of DNA PurificationUsing the PlateTrak AutomatedMicroplate Processing System

Advances in throughput, reproducibility,and reagent conservation

Michelle StevensPerkinElmer Life Sciences, Inc.

Kevin McKernan Center for Genome Research, Whitehead Institute

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With the recent increase in genomic research, laboratories are pressed for increased throughput and reliabilityalong with the need to reduce cost. The enormous scale of the Human Genome Project has created a demand forautomated systems aimed at high throughput and reproducibility. PerkinElmer Life Sciences, Inc. has completed acollaboration 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 automatethe DNA purification process. The Whitehead Institute is one of the largest contributors to the Human GenomeProject. This collaboration led to the development of many exciting new microplate processing solutions on thePlateTrak system that greatly enhance the capabilities for the automation of genomic research. In addition, theability to reduce the scale of sequencing reactions through advanced pipetting systems produced significant costsavings.

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 developeda 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 magneticproperties when placed in a magnetic field, but retain no residual magnetism when removed from the magneticfield. This enables rapid and reproducible automation of buffer exchanges and extensive washes required forDNA preparations. The procedure has been developed and optimized for single stranded DNA isolation, such asM13 phage utilizing iron oxide magnetic particles and double stranded plasmid DNA utilizing carboxyl coatedmagnetic particles. The SPRI procedure allowed the development of an automated procedure in a microplateformat with a throughput of five hundred 384-well plates, or 200,000 separate DNA preparations per day. This isthe highest throughput achieved in any lab today for genomics. The procedure is rapid, low cost, and provideshigh quality DNA sequencing results.

Collaboration between PerkinElmer and the Center for GenomeResearch at the Whitehead InstitutePerkinElmer 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 andplasmid purification. Today, the Center is routinely doing 100,000 lanes per day or approximately 2,400,000 permonth, making this the fastest microplate-based DNA purification system in use on the human genome project.

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PerkinElmer paired newly engineered technologieswith standard ones to develop several automatedsystems. The following is a list, with a brief description, of the separate modules utilized inthese systems:

MultiPosition Dispense Module: A 96- or 384-channel dispensing head capable of movements in the X, Y,and Z directions. The dispense head moves across amultiposition grid deck, extending the accessible stations on the conveyor path. There are 3 model sizes available for varying capacity for plates, reagents,tip boxes or plug and play cartridge accessories.

10 inch MPD – 3 position deck, 1 plate stop, 2 cartridge slots

20 inch MPD – 9 position deck, 2 plate stops, 6 cartridge slots

30 inch MPD – 15 position deck, 3 plate stops, 10 cartridge slots

Gantry Gripper: Pick and place robotic arm capable ofmoving plates from and to the conveyor for additionalprocessing behind the conveyor. The 30 inch modulehas been integrated with 4 position orbital shakers, 8 position magnetic plate deck, and an 8 positionmagnetic plate shelf.

SideTrak®: Pick and place robotic arm for the integrationof peripheral microplate devices to the PlateTrak system.The SideTrak arm moves plates from diving board to external ancillary equipment. Peripheral devicesinclude thermal heat sealers, readers, microplate handlers, microplate hotels and carousels.

Plate Sealer: Thermal microplate sealer.

Lift and Transfer Station: Mechanism that enablesmicroplates to be lifted off and returned to conveyerafter aspirating or dispensing. An example would becompression or expansion of plate libraries.

Plate Piercing Module: Module that pierces sealedmicroplates at an adjustable height for 96- or 384-wellplates.

Stacker Module: Stacker Module with the capacity tohold a maximum of 50 SBS standard microplates. Thecassette can accommodate a variety of plate types includingstandard plates, deepwell plates, PerkinElmer Tip Racks,and microplates with carriers.

Dispense Module: A 96- or 384-tip dispense head thatmoves only in the Z direction. Reagents or Tip Washare kept below the conveyer so they can be accessedwhen a plate is not in position. The heads have a widevolume 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)

96-channel head, P20 Disposable Tip(0.5 µL to 20 µL)

384-channel head, P30 Disposable Tip(0.5 µL to 30 µL)

Connecting Diving Boards: Multifunctional divingboards used for accessing the conveyer by anotherrobotic system. These may be connected to join twoseparate PlateTrak systems.

Wash Module: A Wash Module with a 96 probe mani-fold with fused aspirate and dispense probes. There isa 96-well magnetic plate lift positioned beneath thedeck for magnetic bead washing.

Recirculating Chillers: Chilling mechanisms withsingle or quad positions that keep reagents chilled to4° C. Quad chillers are ideally suited for the setup ofdye primers and terminators for sequencing reactions.

Plate to Waste Station: Shuttle mechanism at the endof the conveyor to allow plates to be lifted from theconveyor and discarded to a waste station.

Mixing Bottle and Reservoir: Adjustable speed controlreagent bottle and reservoir cartridge for keeping mag-netic beads in suspension.

Magnetic Plate Shelf: Programmable sliding magneticshelf to increase deck capacity for plate handling forseparation steps.

20 inch MultiPosition Dispense Module with a P250Dispense Head on the PlateTrak shown with 250 mLbottle mixer cartridge assembly, 96/384 tipwash cartridge assembly and a 1 L bottle reservoir cartridge.

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The instruments for the Whitehead Institute can bebroken down into five phases:

Phase Description

1 Resuspension and Lysis procedure2 & 3 DNA purification, which is handled in

two steps;4 Sequencing set up and if needed5 Reaction Pooling or Cleanup

Following are detailed drawings of each instrument inthe system, along with a quick summary of theprocess during each phase.

In operation, the modules are optimally accessed inparallel and sequentially — all are working at thesame time on different plates and the plates do notmove backward. The PlateTrak systems are utilizingan assembly-line approach to plate processing.Keeping the line moving in one direction offersoptimal throughput. However, since the conveyormoves in both the forward and reverse directions,modules can be utilized in any order.

These procedures are currently run in a 96-wellformat. There will be a conversion to a 384-well

format in the near future.

These procedures are currently run in a 384-well format.

Clonesgrown indeepwellsand cellspelleted

Lysis of M13 andresuspensionof plasmidcell pellets

SPRI procedure:Separation ofplasmid DNAfrom E. ColiDNA

M13 purificationand separationof plasmidDNA fromRNA

Sequencingreactionclean-up

Samples offfor analysison the PE3700 DNAsequencers

Reformat purified DNA to384-well platesif needed andadd the

sequence mixtures for

thermocycling

TransferDeck

PurificationDeck #1

PurificationDeck #2

SequencingDeck

PoolingDeck

30 inch MultiPosition Gripper Tool integrated with anorbital shaker and an 8 position magnetic plate deck.

Close-up of orbital shaker.

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Transfer DeckPurpose: The purpose of this instrument is two fold:

1) M13 LysisSingle M13 clones grown in deepwell plates that have been centrifuged or filtered to pellet host cells are placed into the system. These supernatants are pipetted to new shallow well microplates and SDS is added to lyse the recombinant M13 phage. Once the M13 plates are lysed, they move directly to the Purification #2 instrument. Since M13 is a filamentagous virus, it is genetically programmed to expel itself from the host bacterial E. Coli cell. Simple centrifugation separates the E. Coli cells and its DNA from the growth media containing the recombinant viral DNA.

2) Plasmid Cell Pellet ResuspensionGrowth 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. Colicells containing Plasmids which have been resuspenedinto solution. The M13 plates move directly to thePurification #2 instrument. The plasmid plates movedirectly to the Purification #1 instrument.

This PlateTrak instrument is not currently connected toany other PlateTrak system.

Throughput: One 96-well plate per minute.

PlateTrak System Layout for Transfer System.

Modules Description1-3 Stacker to hold the deepwell plates for processing

4 96-Channel Dispenser adds SDS to Microtiter plates to lyse the phage

5-7 30 inch MultiPosition Gripper Tool moves microplates toshakers and the magnetic plate deck to improve lysis

8 Stacker for 96-well Microtiter plates

9 96-Channel Dispenser aspirates the supernatant from thedeepwell plate and dispenses it to the Microtiter plates

10 Stacker for 96-well Microtiter plates

11-12 2 position Diving Board for future robotic integration

13 Moves deepwell plates to trash after supernatant has been removed

Process Steps for Transfer Deck

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DNA Purification Deck #1Purpose: The purification decks are where the SPRIprotocol is performed. The purification is based onsize selection of the DNA. Once the host cell hasbecome lysed, the host E. Coli DNA is in solution withthe plasmid DNA. Both molecules share identical bio-chemical properties but can be separated based onsize. The E. Coli DNA is 4.2 Million base pairs (Mb),and most plasmids are usually about 3-10 thousandbase pairs (Kb). The function of the Purification #1deck is to perform a differential PEG precipitationwith paramagnetic particles. The protocol is designedto selectively precipitate large DNA fragments of theE. Coli cells and proteins to paramagnetic micros-pheres without precipitating the plasmid DNA. The beads are separated leaving plasmids in solution.This “plasmid enriched supernatant” is then collectedand deposited into a new Microtiter plate for furtherpurification on the Purification #2 system. The magnetic beads containing the E. Coli DNA are discarded at a “Plate to Waste” station.

General Protocol: Initially, the magnetic beads areadded to lysed phage or lysed E. Coli cells containingplasmids in the shallow well Microtiter plates togeth-er with a binding buffer of PEG and salt. The PEG andsalt concentrations have been optimized for both thesingle stranded M13 and double stranded DNAbinding protocols.

Input Source: 96-well plates of E. Coli bacteria con-taining plasmids.

Output Product: 96-well Microtiter plates containingplasmid enriched supernatant. The bacterial DNA isbound to the magnetic beads and is discarded with

the source plates. This is now analogous to the M13preparations after resuspension and lysis. There is no E. Coli DNA to compete for magneticbeads in the second half of the purification protocolon Purification Deck #2. These output plates move onto the Purification #2 instrument.

This PlateTrak system is connected to DNAPurification Deck #2 by a conveyor to conveyor link.

Throughput: One 96-well plate every 100 seconds.

Modules Description1 Stacker for Microtiter plates (source plates)

2-3 Dispenses NaOH (caustic) and SDS (detergent) into source plates for bacterial cell lysis

4-6 MultiPosition Gripper Tool moves plates to orbital shakers to improve lysis

7 Open Module

8-9 96-channel dispenser adds PEG (binding) and magnetic bead solution

10-12 MultiPosition Gripper Tool moves plates to the orbital shaker and magnetic plate deck for incubation

13 Stacker for Microtiter plates (destination plates)

14-15 Reads barcodes and aspirates supernatant from source plates and dispenses it to destination plates

16 Stacker for destination plates

17 For future robotic integration

18 Moves source plates to trash and destination plates toattached PlateTrak

Process Steps for DNA Purification Deck #1

PlateTrak System Layout for DNA Purification System #1.

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MultiPosition (MPD) module with 96-tip dispense andbottle mixer to keep beads in suspension.

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

96 probe plate washer with magnetic plate lift.

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DNA Purification Deck #2Purpose: The Purification Deck #2 performs the entirepurification process for the M13 DNA and the secondhalf of the plasmid DNA preparation, which is theseparation of the plasmid DNA from the RNA in solu-tion, utilizing the magnetic beads. This system is alsoutilized for the purification of PCR products with thesame procedures.

Input Source: 96-well plates containing lysed M13phage DNA or plasmid DNA in solution. Since thissystem is connected to DNA Purification Deck #1, theplasmid DNA plates can be automatically transferredto this system.

Output Product: Dry Microtiter plates with DNAbound to magnetic beads.

The plates are manually moved to the SequencingDeck (#5).

Throughput: One 96-well plate every minute.

Modules Description1 Stacker for Microtiter plates (source plates)

2-3 Adds PEG and magnetic bead solution

4-6 MultiPosition Gripper Tool moves plates to orbital shakers and magnetic plate deck for incubation

7 Wash Module with magnetic plate lift• 1X 70% EtOH wash with final aspirate to remove EtOH

8 Wash Module with magnetic plate lift• 2X 70% EtOH wash with final aspirate to remove EtOH

9 Wash Module with magnetic plate lift• 1X 70% EtOH wash with final aspirate to remove EtOH

10 Stacker for Microtiter plates (destination plates)

11-12 Receives plates from DNA Purification Deck #1

13 2 position diving board - for future robotic integration

PlateTrak System Layout for DNA Purification System #2.

Process Steps for DNA Purification #2 Deck

Plate Lift and Transfer mechanism incorporated into a 20 inch MultiPosition Dispense Module to securelytransport a 384-well master plate behind the conveyor.

Close-up of the plate lift mechanism.

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Sequencing DeckPurpose: The sequencing system can set up sequenc-ing reactions utilizing both Dye Primer and DyeTerminator chemistries in the forward and reversedirections. The magnetic beads are separated and thesupernatants re-moved for testing and sequencing.The magnetic beads are re-suspended in a waterelution buffer with a three minute incubation at roomtemperature. The three minute incubation can beavoided with a simple 20 second shake utilizing anin-line shaker mechanism on the conveyor. For dyeprimers, the system will add the sequence mix-tures(Dye Primer and – A, T, C, and G) which are chilled to4° C in a color-coded four quad chilling station. Theseplates are then reformatted into 384-well plates, andthe forward or reverse mixtures are added to the plate.For dye terminator chemistries, the dye terminatorsolution chilled to 4° C is added to the DNA. PurifiedDNA products from two 96-well plates are added to a384-well plate with two copies positioned in diagonalquadrants of the 384-well plate. The forward reactionmixture is added to one copy and the reverse mixtureis added to the second. This ensures that the data willbe accurate even if the plate becomes flipped in orien-tation throughout the process. The DNA will be readyfor the thermocycling operation and subsequent iden-tification on the PerkinElmer 3700 series sequencer.

Input Source: Purified DNA in 96-well Microtiterplates. If appropriate, 384-well plates will be addedfor the reformatting.

Output Product: Sealed plates ready for the thermocycler.These plates are put on a thermocycler and then movedto the PE 3700 sequencer. This instrument is integratedto a SideTrak robotic arm, which moves plates into a

plate sealer at the end of the run for sealing prior to

thermocycling. The plates are loaded to a PlateStak™

instrument on the SideTrak following sealing. Thismaintains unidirectional processing for optimumthroughput.

Throughput: One 384-well plate every two minutes.

Modules Description1 Stacker for microtiter plates (source plates)

2 96-Channel Dispense Module for reconstitution of the DNA and separation from the beads

3 Open Module

4 Stacker for 384-well plates (thermocycled plates) (96-well plates can be used)

5 Stacker for 384-well plates (96-well plates can be used)

6-7 Lift & Transfer Station and a Magnetic Plate Lift with a 96-Channel Dispenser – the Lift & Transfer will move a plateoff the deck. The Magnetic Plate Lift will hold the beads down so the 96-Channel Dispenser can transfer the supernatant to the plate on the Lift and Transfer Module.If a 384-well plate is used, then four 96-well plates are compressed. If not, then the DNA is simply transferred to the destination plate.

8 Open Module

9-10 96-Channel Dispenser with 4 color-coded anodized aluminum chilling stations for addition of the sequencing reagents (A, C, T, and G.) This Module adds the forward primers.

11 Open Module

12-13 96-Channel Dispenser with 4 color-coded anodized aluminum chilling stations for sequencing reagents (A, C, T, and G). This Module adds the reverse primers.

14 Stacker

15-16 2 position diving board for future robotic integration

17-18 SideTrak pick and place robotic arm integrated with a thermal plate sealer and a PlateStak for storage of sealed plates.

PlateTrak System Layout for Sequencing System.

Process Steps for Sequencing Deck

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Pooling DeckPurpose: The function of Pooling Decks is two fold.First a Pooling Deck can take a 384-well cycle plateand compress or “pool” to a single 96-well purificationplate for bead based cleanup. This is required whenutilizing the dye primer chemistries for sequencing.This can also function for the second half of thepurification steps, and is upgradable to a 384-wellbead prep clean-up protocol.

Input Source: The source can be 384-well plates or96-well Microtiter plates.

Output Products: There can be two products: 96-wellplates ready after bead prep clean-up or dry Microtiterplates with DNA bound to magnetic beads.

This system is not currently connected to another system.

Throughput: One 384-well plate every minute.

Modules Description1 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 Lift1X wash 70% EtOH wash with final aspirate to remove EtOH

11 Wash Module with MagneticPlate Lift2X wash 70% EtOH wash with final aspirate to remove EtOH

12 Wash Module with Magnetic Plate Lift1X wash 70% EtOH wash with final aspirate to remove EtOH

13 Stacker for empty 384-well plates (or 96-well plates if usedas purification system)

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

PlateTrak System Layout for Pooling System.

Process Steps for Pool Deck

Close-up of 20 inch MPD deck grid shown with recirculating chiller, magnetic stirrer cartridge, and multiple auto tip rack loaders.

The SPRI technology coupled with the PlateTrak systems developed for the Whitehead Institute have greatlyenhanced processing capabilities. One of the primary advantages of this procedure for DNA purification is thereagent savings, in particular Taq FS polymerase, through the ability of the PlateTrak instruments to reproduciblyset up small volume reactions. For the sequencing reactions, only 1 mL Taq additions and 3 µL DNA additionsare necessary. The yield for the M13 purification is 4-5 µg DNA, and the yield for the plasmid purification is 2-4 µgDNA. 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 (phred20) 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 alsogreat results with PCR product purification as assessed by concordance levels with microarrays. In regards topurity, 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 DNAsequencing results. It allows the Whitehead Institute to be the largest contributor toward the Human GenomeProject 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 wellas, 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 highquality DNA suitable for a variety of applications.

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