An automated multiplex oligonucleotide synthesizer: development of ...

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Development of high-throughput, low-cost DNA synthesis .... Custom software then sends a stream of binary control bits to a set of serial to parallel converters, ...
Proc. Natl. Acad. Sci. USA Vol. 92, pp. 7912-7915, August 1995 Applied Biological Sciences

An automated multiplex oligonucleotide synthesizer: Development of high-throughput, low-cost DNA synthesis (oligonucleotides/phosphoramidite chemistry/sequencing/automation)

DEVAL A. LASHKARI*, ScoTr P. HUNICKE-SMITH, RIcHARD M. NORGREN, RONALD W. DAVIS, AND THOMAS BRENNAN Stanford DNA Sequence and Technology Center, Departments of Genetics and Biochemistry, Stanford University School of Medicine, Stanford, CA 94305

Contributed by Ronald W Davis, April 25, 1995

ABSTRACT

These aspects of oligonucleotide synthesis-output format, throughput, and cost of synthesis-have been addressed in the development of an automated multiplex oligonucleotide synthesizer (AMOS). This instrument can rapidly synthesize up to 96 different oligonucleotides in a standard 96-well format with low reagent consumption. Routine phosphoramidite chemistry (5, 6) is performed on a controlled pore glass (CPG) matrix in a modified 96-well tray enclosed in an argon flow chamber. Oligonucleotides can be synthesized on a variety of scales and can be of different lengths within a given synthesis. Additionally, the instrument is capable of adding modified bases containing, for example, biotin, phosphate, or fluorescent labels to the oligonucleotides.

An automated oligonucleotide synthesizer has been developed that can simultaneously and rapidly synthesize up to 96 different oligonucleotides in a 96-well microtiter format using phosphoramidite synthesis chemistry. A modified 96-well plate is positioned under reagent valve banks, and appropriate reagents are delivered into individual wells containing the growing oligonucleotide chain, which is bound to a solid support. Each well has a filter bottom that enables the removal of spent reagents while retaining the solid support matrix. A seal design is employed to control synthesis environment and the entire instrument is automated via computer control. Synthesis cycle times for 96 couplings are 6000 oligonucleotides synthesized to date. The reduced reagent usage and increased capacity allow the overall synthesis cost to drop by at least a factor of 10. With the development of this instrument, it is now practical and cost-effective to synthesize thousands to tens of thousands of oligonucleotides.

MATERIALS AND METHODS

Many biological problems are difficult to address due to the limited availability of experimental components or the prohibitive costs of certain reagents. This is especially true with respect to oligonucleotides. Projects requiring hundreds of oligonucleotides have not been practical or easily feasible. Individual research groups could make use of large numbers of oligonucleotides if they were readily available for a multitude of DNA constructions. For example, whole genes could be quickly synthesized containing many sequence variations. Additionally, large numbers of oligonucleotides are not obtainable in convenient formats and their costs have been high. These reagents are critical to primer-directed sequencing strategies (1) and various methods for mapping genomes (2-4). Not only are oligonucleotides expensive but they are also typically synthesized in a low-throughput manner prohibitive of large-scale use. Current machines produce oligonucleotides in small numbers as individual samples and it is cumbersome and potentially error-prone to generate and use large numbers of these individual products. The genome sequencing and mapping projects must face these limitations as they scale up their efforts; for maximum efficiency and accuracy, most large-scale projects would prefer reagents and samples in a universal, multiple sample setup, such as the 96-well tray. This format interfaces well with other instrumentation such as robotic workstations and automated sequencers.

The AMOS consists of reagent bottles connected by Teflon tubing to valves that deliver reagents into wells of a reaction plate, which is held by a slider and positioned using a drive motor (Fig. 1). The individual components are detailed below. Reagent Sources and Tubing. All synthesis reagents (Glen Research, Sterling, VA) are maintained under argon pressure at 4 psi (1 psi = 6.89 kPa). Bottles have eight Teflon lines leading to the individual valves responsible for reagent delivery control. Reagent concentrations are as listed by the manufacturer. All reagents are delivered through Teflon tubing of 0.05 inch i.d. and 1/16 inch o.d. (1 inch = 2.54 cm) (Cole Parmer, Niles, IL). Line volumes are 100 ,ul per line from bottle to valve to delivery nozzle. Argon supply lines are also of similar material. Valves. Teflon tubing from the reagent bottles leads to miniature solenoid valves (part no. LFVX0500450A, Lee, Westbrook, CT) arranged in banks of eight, one bank per reagent (Fig. 2). Valves were chosen on the basis of a low internal volume of 10 ,ll and material compatibility with reagents. Although multiple valves can be actuated simultaneously, each valve is individually controlled. Individual control is essential for making oligonucleotides of different length and sequence. Additionally, volumes as low as 10 ,ul can be delivered accurately, minimizing reagent consumption. Stationary and Sliding Plates and Seal. Tubing exits the valves and is press fit into a top stationary plate made entirely of aluminum, fixing the tubing position and providing an airtight seal{Fig. 2). The ends of the tubing are blunt-cut to act as nozzles for reagent delivery and are laterally spaced to match the spacing of wells in a 96-well plate. The nozzle ends are situated 5 mm above the reaction plate to prevent spraying of the liquid stream to adjacent wells. A thin Teflon membrane is suspended around the perimeter of the top plate. It is gently pushed against the lower sliding plate by resilient elastic backing. The seal thus excludes

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Abbreviations: AMOS, automated multiplex oligonucleotide synthesizer; CPG, controlled pore glass. *To whom reprint requests should be addressed.

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Axis of motion FIG. 1. Side-view schematic of the AMOS. The top plate has valve banks with reagent delivery nozzles leading into the space between the top plate and slider. Argon enters upstream of amidite valve banks and exits downstream of the deblocking solution valve bank, sweeping through the inner chamber. Argon is maintained internally by the perimeter inflating seal. Slider and reaction plate position is controlled by the lead screw and drive motor.

ambient air during synthesis and forms a still tighter seal as internal pressure is increased (Fig. 3). Below the top stationary plate is a sliding plate also made of aluminum but coated with a Teflon sheet; this sheet passivates the surface and decreases sliding friction against the gas seal. The sliding plate holds and positions the 96-well reaction plate. Electronics and Computer Control. The user supplies two separate text files for synthesis control; the first specifies the synthesis protocol per coupling step and the second contains 96 sequences and final conditions for each oligonucleotide. Custom software then sends a stream of binary control bits to a set of serial to parallel converters, one per reagent. These converters control the firing of the eight individual valves for each reagent. In coordination with valve firings, the software controls the sliding plate's position with a linear motion controlling device (part no. RD355A/D2303, Industrial Devices, Novato, CA) while monitoring actuator position via a shaft-mounted optical encoder. The encoder allows positioning to within ±0.005 inch. During synthesis, the software requires no manual intervention but allows the user to specify trityl collection steps as well as failed wells in which synthesis should not be continued. Reaction Plate and Solid Support Matrix. The reaction plate is a modified deep well plate with each tapered well having a capacity of 700 ,tl (Fig. 4). The bottoms of the wells are drilled with 1/64-inch holes and fitted with a glass fiber filter (GF/A filters, Whatman). The combination of filter and

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small drain hole serves to support the CPG during synthesis and to hold reagents in the well by surface tension until overcome by controlled gas pressure applied from above the plate. The phosphoramidite synthesis chemistry is performed on a CPG matrix derivitized with the 3' base (Glen Research, Sterling, VA) in 500 A and 1000 A pore size. The CPG is dispensed into each well by creating a slurry of CPG in chloroform/dibromomethane, 1.5:2.0 (vol/vol), solution. Typical synthesis scales are 20 nmol and the amounts of CPG per well are calculated based on manufacturer's loading of 3' base to CPG. Synthesis. The reagent delivery valves and plate positioning are coordinated by a computer. Control software developed in-house accepts text files containing oligonucleotide sequences, synthesis protocols, and setup configuration data. Synthesis protocols are based on standard phosphoramidite chemistry (5, 6). The slider bar, controlled by the linear motor arm and encoder, positions the reaction plate underneath the appropriate bank of eight reagent valves and the valves are electronically activated as needed to deliver reagents simultaneously to an entire row of eight wells in the plate (Fig. 2). Reagents are delivered directly onto the solid support, which is sufficiently agitated by the delivery process to guarantee proper mixing. The synthesis protocol is a modification of typical procedures. To ensure that the coupling, detritylation, and oxidation steps are as complete as possible, reagents remain in the wells between 5 and 30 sec before expulsion by argon pressure. Acetonitrile is used to rinse wells in-between reagent deliveries when appropriate. Additionally, argon is constantly flowing across the inner chamber to prevent con-

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Polypropylene well

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Support filter Drain hole FIG. 4. Cross section of reaction well. Wells of a polypropylene deep-well 96-well plate are tapered and have a 1/64-inch drain hole. A porous filter is press fit into each well; CPG is maintained above the filter in the area where reagents are delivered.

tamination of amidite and activator nozzles with vapors from the deblocking and oxidizer solutions. For postsynthesis deprotection, the reaction plate is removed from the instrument and a standard 96-well deep-well (Beckman) collection plate is placed underneath the reaction plate. Two hundred microliters of concentrated ammonium hydroxide is added to each well for 20 min. The plate is then centrifuged at 1000 rpm for 1 min in an RC-3B centrifuge with an HL-2B rotor (Sorvall, Newtown, CIT). The ammonia containing the eluted oligonucleotide is now in the secondary collection plate. After repeating the process two more times, the second plate is sealed with a cover and placed in a 58°C water bath for 10 hr. After deprotection, the plate is put in a concentrator from Savant (part no. SC210A) for lyophilization; lyophilized final products are thus delivered in the standard 96-well format. Synthesis Quality Analysis. Quantitative trityl analysis is performed on each individual sample at regular intervals during synthesis. A flat-bottom 96-well plate (Polyfiltronics, Rockland, MA) is positioned within the waste collection chamber underneath the reaction plate. Eluant containing the trityl material is collected into the corresponding wells and quantitated at A = 470 nm on a V-Max plate reader from Molecular Devices. In the event of a failure in a particular well, the software controlling the synthesizer can be instructed to abort synthesis in that well while continiuing synthesis at other positions within the plate.

RESULTS AND DISCUSSION The design of the instrument revolves around creating a suitable environment in which to perform the phosphoramidite synthesis chemistry. The chemistry is critically sensitive to water and air contamination, so complete control of the reaction environment is essential. Additionally, a method for removing spent reagents from wells is required. To address these issues, a constant directional flow of argon gas is maintained in conjunction with a seal design (Fig. 3) that serves three purposes: (i) exclusion of ambient water and air, (ii) prevention of diffusion from acidic and water-based reagents from their respective nozzles to the sensitive amidites, and (iii) thorough drainage of the reaction wells with positive pressure created by temporary closure of the argon outlet. In this way, free movement of the slider against the top plate is possible while maintaining a proper seal. The inflatable seal allows argon to be maintained within the ,pace between' the top plate and the bottom slider. Argon enters the chamber at one end sieeps through' the space, and exits the opposite end. Since the seal must aso permit the movement of the bottom slider during synthesis, it "is attached

to the top stationary plate and rests against the bottom slider. To aid in expulsion of spent reagents from the reaction plate, the argon outlet valve is closed, increasing the pressure within the chamber. As pressure increases, the Teflon seal presses against the bottom slider with more force, creating an even tighter seal. The relative positions of the valve'blocks and delivery nozzles also play a role in synthesis quality. The amidites and activator nozzles are upstream of the all other reagents with respect to the direction of flow of argon (Fig. 1). This prevents their exposure to vapors from the deblocking acid and water containing oxidizer that are downstream of all reagents and closest to the argon outlet. The AMOS has made oligonucleotide synthesis more efficient. The individual columns used for synthesis on commercial devices have been replaced by a single 96-well plate. This avoids individual sample handling and provides a more standardized output format compatible with other instruments. Sample plates can be handled by robotic devices and errors in sample manipulation can be minimized. Reagent consumption is also more efficient. One major cost factor in commercial instruments is the reagent usage due to repeated purging and priming of a single reagent delivery line to the stationary synthesis column. The AMOS circumvents the need for washing and priming of lines by providing each reagent with its own dedicated line and moving the entire reaction plate to the reagent. The throughput of the instrument is high-with the existing protocol, the cycle time is 11 min for a maximum of 96 couplings. Thus, an entire plate of 96 different 20-mers can be synthesized in 6000 oligonucleotides synthesized to date. The lengths range from 6-mers to 98-mers. The oligonucleotides have been successfully used in applications that include the polymerase chain reaction

(PCR), site-directed mutagenesis, primer-directed sequencing, cloning, multiple ligations, protein binding studies, and gene mapping (7). Synthesis quality has not been sacrificed for the increased throughput. Coupling efficiencies based on trityl analysis throughout syntheses of entire plates have consistently averaged >98%. High-quality oligonucleotides are routinely obtained with little detectable well-to-well variation and the oligonucleotides require no purification before use (Fig. 5). Failure rates have averaged