Oligo Factory

Automated Nucleic Acid Synthesizers Most of today's biological and biomedical laboratories simply could not function without ready access to user-specified oligonucleotides. Many researchers rely on core services or external vendors for their custom nucleotide needs. Other laboratories or departments choose to invest in oligonucleotide synthesizers for their exclusive use. Read on for a review of the basics of solid-phase oligonucleotide synthesis and a summary of some of the commercially available synthesizers. The Basics Phosphoramidite chemistry is the most commonly used method for synthesizing oligonucleotides. Strands are formed by the sequential addition of activated monomers to an immobilized nucleotide chain. The product remains attached to an insoluble support until the final release step. The solid-phase approach is ideal for the automated synthesis of oligonucleotides. All reactions occur in a single vessel, and excess soluble reagents can be added to drive each reaction to completion. At the end of each step, soluble reagents and by-products are readily washed away. DNA/RNA synthesizers are, in essence, complex fluid-handling devices. They automate the production of oligonucleotide chains by controlling the flow of various reagents and nucleotides into a reaction vessel. Unmodified DNA/RNA monomers contain multiple chemical groups that can react with the growing chain. The reactive groups in these monomers must be blocked with protecting chemical groups to decrease the likelihood of unwanted side reactions. Therefore, part of the synthetic process involves removal of these protecting groups at the appropriate times. Most protecting groups remain attached to the growing oligonucleotide chain throughout synthesis and are not removed until the chain is complete. Others are needed to ensure the proper positioning of the next monomer and are removed after each chain elongation cycle. In the phosphoramidite method, a series of deprotection, coupling, capping, and oxidation steps is repeated until the nucleotide chain of interest is formed. The strand is formed 3' to 5', the direction opposite that of nucleic acid synthesis within cells. The first step requires a column that has a protected form of the terminal (3') monomer chemically bound to its matrix. The nascent oligonucleotide chain will stay attached to this support as each activated monomer, one by one, is linked to its 3' neighbor. The column is contained in the reaction vessel, where it reacts with reagents released into the chamber at specific times throughout the cycle. In DNA synthesis, the protected monomers are deoxyribonucleoside 3-phosphoramidites containing dimethoxytrityl (DMT) blocking groups on the 5'-oxygen atoms. These monomers are activated by treatment with a weak acid prior to chain elongation. Deprotection, also known as detritylation, is the first step of each chain elongation cycle. An acid wash removes the DMT blocking group from the terminal monomer. After deprotection, the terminal monomer has a ready-to-react hydroxyl group (OH) at its 5' end. The second step is coupling. Because phosphoramidites react with water, coupling is carried out under anhydrous conditions. In this step, the next activated monomer in the sequence is added to the vessel. An intermediate phosphite triester linkage is formed between the 3' end of the incoming monomer and the 5'-OH of the terminal monomer. The coupling reactions generally go to >98 percent completion in a fairly short time. However, at every coupling step, a small percentage of deprotected terminal monomers will inevitably remain unreacted. The third step is designed to simplify the final purification procedure by preventing most of the unreacted monomers from participating in further coupling reactions. In this step, known as acetylation or capping, the 5'-OH ends of unreacted terminal monomers are blocked via acetylation. The phosphite triester formed in the coupling step is unstable and is susceptible to cleavage by acids and bases. In the fourth step, the phosphite triester (in which P is trivalent) is oxidized to the more stable phosphotriester linkage (in which P is pentavalent). This four-step cycle is repeated for each added monomer. The completed oligonucleotide is then cleaved from the support by treatment with concentrated ammonium hydroxide. A subsequent heat treatment removes the remaining protecting groups. The final product is generally purified by HPLC or PAGE. In some cases, crude oligonucleotides can be passed over a desalting column, then used without further purification. To facilitate the production of modified oligonucleotides, some synthesizers include software for running hydrogen-phosphonate synthesis chemistry. The hydrogen-phosphonate technique is similar to the phosophoramidite method. Slightly different monomers are used, and a different acid is employed for deprotection. Because H-phosphonate diesters are fairly stable, oxidation is required only once--after construction of the chain. This single oxidation step creates an opportunity to modify the entire phosphate backbone. For example, in thiola-tion, an oxidizing agent is used to modify the phosphate backbone with sulfur. The resultant phosphorothioate DNA has been particularly useful in antisense experiments. However, because hydrogen-phosphonate chemistry is significantly less efficient than the phosphoramidite method, it is not routinely used for oligonucleotide synthesis. Overview of the phosphoramidite solid-phase oligonucleotide synthesis cycle Taking Off with OligoPilot(tm)II Amersham Pharmacia Biotech's (Piscataway, N.J.) OligoPilot II is designed for low-cost, large-scale synthesis of DNA. This instrument has a number of key features, including a broad synthesis scale of 10 µmol to 4 mmol, relatively simple scale-up procedures, and low reagent consumption. OligoPilot II employs flow-through instead of conventional batch reactor technology. The flow-through reactor allows the synthesis of oligonucleotides with as little as 1.5 molar equivalents of phosphoramidite monomer. Coupling efficiency is high at >98 percent. Users of the flow-through-based instrument will enjoy the fact that they will accumulate only about one-fourth the waste volume normally expected with most batch reactors. Flow-through operation is also quick, allowing synthesis of 3 mmol of a 25-mer oligonucleotide in less than 35 minutes. The major advantages of flow-through reactor technology result from the fact that it enables the maintenance of a constant phosphoramidite concentration throughout each reaction. Briefly, the coupling mixture is added to the solid support as a "reagent zone" that is pushed through the column. As a result, the coupling mixture is not diluted as the reaction proceeds. In effect, reagents are continuously replaced as they are consumed. The concentration of the phosphoramidite reagent will remain constant and at optimal concentration throughout the complete reaction, even with very small molar excesses. The reaction rate will remain constant throughout the reagent zone. In contrast, during synthesis in a batch reactor, the phosphoramidite concentration decreases throughout the reaction and the overall reaction rate is reduced. OligoPilot II is a pump-driven oligonucleotide synthesizer. The two piston pumps (for wash and detritylation steps) can be used at flow rates of up to 100 ml/min. A low dead-volume pump for introducing phosphoramidites and ancillary reagents can be used at up to 50 ml/min. Five fixed-volume flow-through columns (1.2 ml to 48 ml) are available. An adjustable column capable of holding a volume of 30-150 ml is also provided. Online trityl monitoring provides a continuous display of the coupling efficiency. In the event of a poor coupling, the monitor can stop the synthesis before additional reagents are wasted. An eight-port valve enables the separation of halogenated and nonhalogenated hydrocarbons. This reduces waste-handling costs and makes it possible to separate and reuse other solvents. OligoPilot II features Windows NT 4.0-based UNICORN(tm) version 3.10 software to control, monitor, and evaluate synthesis procedures. Preprogrammed modular method templates for each of the separate steps in synthesis (deprotection, coupling, oxidation/thiolation, wash/purge) are supplied. These modules can be used to create customized synthesis procedures, or new methods may be created from scratch. A sequence editor enables one to simply enter the sequence of any desired oligonucleotide up to 250 bases long. Thiolation can be specified, if desired, as well. The UNICORN software predicts an optimal synthesis routine from the preprogrammed templates based on the sequence entered. The software also enables user-defined alarm and warning limits for monitor signals. UNICORN includes a comprehensive security system and an audit trail for system operation. A synthesis documentation system is included as well. In Step with the ABI 3948 & Expedite(tm) 8909 PE Biosystems' (Foster City, Calif.) ABI 3948 automates the process of sequence entry, oligonucleotide synthesis, and postsynthesis tasks, including cleavage from the support, deprotection, purification, quantification, sample collection, and data management. This system produces up to 48 high-purity primer-length oligonucleotides in 24 hours of unattended operation. Test oligonucleotides analyzed by MicroGel capillary electrophoresis, anion-exchange HPLC, PAGE, and enzymatic digestion/HPLC routinely demonstrate >90 percent product purity. The ABI 3948 is a floor model, self-contained synthesizer under Macintosh computer control. Synthesis, cleavage, and purification all occur on polystyrene-based OneStep(r) columns. The columns are color coded to identify the immobilized monomer: A (green), G (yellow), C (red), and T (blue). The columns contain a mixture of immobilized terminal monomer, as well as underivatized polystyrene for the 5' DMT-selective purification of the completed chain. The ABI 3948 has a rotating turntable, which allows the sequential processing of samples in sets of three OneStep columns. The turntable has 16 rows, each with three column ports, and thus holds up to 48 columns. The unit contains three independently controlled liquid delivery systems. Reagents for synthesis, cleavage, and purification are delivered in parallel through three clamping-jaw, fluid-delivery heads. The cycle time required for addition of each monomer is 3.5 minutes. At the beginning of a synthesis run, the turntable rotates the first set of three columns to the synthesis fluid head. The head clamps down on the columns, and the first round of detritylation, coupling, capping, and oxidation reactions occurs to initiate synthesis. After the first set of three oligonucleotides has completed synthesis, the turntable rotates one position. The columns bearing the now-complete oligonucleotide chains are forwarded to the cleavage fluid-delivery head. The second set of three OneStep columns is concomitantly delivered to the synthesis head. While synthesis begins for the second set, ammonium hydroxide is delivered to the cleavage head. The cleavage step takes 45 minutes. The cleaved oligonucleotides are sent through Teflon tubing that is wrapped in coils around a heated cylinder. Temperature is elevated to 65°C for one hour to effect deprotection, and then the turntable rotates again. The third set of columns is now brought to the synthesis head, and the second set is forwarded to the cleavage head. The temperature is lowered to ambient in the deprotection coils, and the first set of crude, deprotected oligonucleotides passes through the original OneStep columns, where it is purified. The dual role of the OneStep column allows for simple and economical oligonucleotide synthesis and purification. Briefly, underivatized polystyrene in the OneStep column acts as a hydrophobic affinity medium that allows purification of the desired oligonucleotide chain. The 5' DMT groups on completed chains selectively bind to the polystyrene, and impurities are washed away with water. The 5' DMT is removed by an acid treatment, followed by a water wash and elution of the purified oligonucleotide in 1 ml of 20 percent acetonitrile. PE Biosystems reports that oligonucleotide aliquots in 20 percent acetonitrile have shown full activity in DNA sequencing and PCR applications, and that these preparations perform equally as well as HPLC-purified oligonucleotide controls in certain applications. The purified oligonucleotides pass through a UV flowcell for quantification by absorbance at 254 nm, and the concentrations of each sample are recorded. A robotic dispenser sends each sample to its designated spot in a 48-position collection rack with microtiter plate dimensions, which enables easy integration with other automated sampling devices. The purified oligonucleotides are ready for immediate use. Typical yield is approximately two optical density units (ODU), which corresponds to about 10 nmol (60 µg) of a 20-mer. The ABI 3948 conserves reagents through use of fluid sensors positioned at critical points in the liquid delivery lines. For example, fluid sensors placed downstream of the OneStep columns detect the liquid/gas interface in the delivery lines, and feedback control shuts off flow when the minimum sufficient amount of reagent has been delivered and precisely positioned. In contrast, synthesizers that rely on timed deliveries often require excess reagent to compensate for flow-rate variations. According to PE Biosystems, the fluid sensors and the low dead volume of the OneStep columns allow a reagent cost reduction of up to 50 percent compared with other DNA synthesizers. Halogenated and nonhalogenated organic wastes are segregated into 4 l and 10 l containers that are enclosed in a secondary containment tray in the unit's lower cabinet. The Expedite 8909 allows synthesis scale ranges from 50 nmol to 15 µmol for DNA oligonucleotides. This system allows preparation of phosphorothioates and enables preparation of peptide nucleic acid (PNA) molecules, which contain neutral "peptide-like" backbones with nucleobases. The 8909 comes with sequence and protocol editors, as well as software for creating customized reports. Each sequence run is documented, and data can be transferred from individual instruments to a central database. The Multiple Oligonucleotide Synthesis System (MOSS) can expand the capabilities of the Expedite system to allow the unattended synthesis of 16 oligonucleotides. The MOSS unit is placed on top of the 8909 system and requires no additional bench space. Trityl monitoring is available for all column positions. ABI 3948 comes with a Macintosh computer, providing dedicated application controls and monitoring of all synthesis functions, and the 8909 is controlled by a user-supplied PC. Sequences in batch format can be entered from a variety of text applications. An integrated database and a customizable order-entry template allow logging, tracking, and record keeping, and each run is automatically documented. The graphical user interface features "user-friendly" niceties such as point-and-click commands and real-time status screens. In addition to the optimized synthesis protocols, procedures and cycles can be edited for flexible programming. Password protection is provided for security purposes. PE Biosystems' ABI 3948 Something for Everyone--Other Notables Labtronix Inc. (Monroe, Ore.) offers a DNA synthesizer with a 40-column carousel. The unit is controlled by a Pentium 200 MHz computer, which comes with a color monitor and multiserial interface board. The system uses a Windows 98 platform. The synthesis program is configured to suit the user, and can be customized as needed. Labtronix president Corwin Willard explains that a major advantage to using a Labtronix synthesizer is that it "customize[s] each unit (hardware and software) to the customer's specific production needs." Labtronix has manufactured equipment for unique applications such as antisense technology. With each sale, Labtronix includes up to 50 hours' engineering time for postsale support, including software modification. The company also provides installation and training sessions during a two-day site visit. The Polygen synthesizer from Distribio (France) is a rather compact unit. Ten DNA syntheses can be carried out in one run of less than three hours. Company representatives estimate reagent costs at around six cents per base. The mechanics of this synthesizer involve the use of four sliding blocks arranged to enable mutual displacement. The sliders contain a series of holes, and they move together to define the inlets and outlets needed during the synthesis. The Polygen synthesizer does not contain valves. Distribio claims that its slider-based instrument is more reliable than conventional synthesizers because valve-specific problems (such as clogging and leaking seals) are eliminated. The controlling software is available in English, German, or French. Intelligent Bio-Instruments (IBI) offers the 96-channel Parallel DNA Synthesizer for high-throughput oligo production. This machine is particularly attractive for users performing sequencing reactions or producing microarray slides requiring small amounts of many different oligos simultaneously. Use of a mechanical transport system circumvents the problems typically associated with flow-through fluidics; the support is simply dipped into the reagents during each reaction. IBI estimates that by reducing reagent waste (and therefore cost) in this fashion, oligos can be produced for around nine cents per base. If none of the commercially available instruments suits your needs, you may want to contact Engineering by Design (San Jose, Calif.). This company develops engineering solutions for an astonishingly diverse array of applications, and one of its areas of expertise is DNA synthesizers. "With nearly 15 years of success and a history of innovation, we have positioned EBD as an industry resource for solving all equipment and project needs," comments Dale Henson, company president and registered professional engineer. Deborah Wilkinson can be contacted at dwf@sciwriters.com.

Oligo Factory

The Basics

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