Stanford
Genome Technology Center
Technology
Development Group
| Instruments
Oligonucleotide Synthesizer |
Oligonucleotide
Synthesizer
A facility has been developed at the Center which is capable of synthesizing oligonucleotides that are vital to the DNA sequencing process. The single strand sections are known as primers and are necessary to locate specific sites and regions on a DNA molecule and allows sequencing of precisely known regions of the molecule to take place. Use of Stanford Developed Synthesizer MachinesThe facility currently is comprised of three AMOS (Automated Multiplex Oligonucleotide Synthesizer) machines 1, 2 and other pieces of ancillary equipment. The AMOS machines were developed at Stanford University for the express purpose of producing large-quantity, low-cost oligos for large scale DNA sequencing operations. They allow the low-cost synthesis of a precisely known sequence of nucleotide bases as specified in a computer file provided by a researcher. The unique feature of these machines is that they produce the nucleotide sequences on a high throughput scale. Each machine is capable of synthesizing 96 different oligos simultaneously in the format of an industry standard 96 well micro-titer plate. A typical run takes about 3 1/2 to 4 hours to synthesize a plate. With pre-synthesis and post-synthesis preparations, typically two plates can be produced per machine in a typical 8 hour day. The automated design of the machine also allows the possibility of an additional unattended overnight run where a short final procedure can be completed at the beginning of the next day. Thus between the three machines it is potentially possible to process nine plates which leads to a potential throughput of 864 oligos per day.The length of typical primers is between 20 and 30 bases. This length is determined by the requirements of the researcher or sequencing team. However, the machines are capable of generating oligos up to approximately 90 bases in length. The majority of the production is centered on the shorter lengths. For a typical 30 base oligo and 20 working days per month, the potential production rate of this facility is over 1/2 million bases per month. As the machines have come on line in the facility and as the operation has become more efficient, the production rate has risen from 75,000 bases per month at the end of 1996 to over 200,000 bases per month by August 1997. Computer Controlled Automated SystemA computer file of the required oligos is provided by a researcher to the facility. This file is composed of 96 primer sequences and primer names. This is delivered to the facility either on a floppy disk or over the computer network. This file is read by the AMOS instrument and the process begins.The synthesis process is strictly chemical in nature and is based on a well developed solid-support based synthesis chemistry3,4 . Each well of the 96 well plate is initially seeded with a slurry of microscopic controlled-pore glass beads (cpg) with the first base of the specified sequence already attached. Thus, each well receives one of four types of beads according to a map generated by the machine software. 11 different reagents are employed in the synthesis protocol and these are delivered by valves and jets into wells of the 96-well plate with the appropriate timing, sequence and position control. Machine Design PhilosophyThe philosophy of the machine design is to have the liquid reagent handling system (this includes reagent sources, valves, manifolds and tubing) to be fixed and non-moving. The filter bottom 96-well plate is supported in a moving chamber and is accurately positioned to allow delivery of the reagents into the appropriate wells from fixed position jets. The 96-well plate is contained within a controlled positive pressure atmosphere of inert argon to exclude the harmful effects of oxygen and water vapor.A predefined protocol controls every aspect of the synthesis sequence as the bases are added to the nucleotide chain. The four major steps of the protocol are:
Simplicity of operationThe machine has been designed to be "user friendly". The software has been designed to provide a series of "virtual instrument panels" as part of the graphical user interface. Thus, all operation is controlled by "virtual" push buttons on the computer screen. Performance is monitored during operation by on-screen indicators. Detailed machine operation is controlled by tables of operating parameters which are set during initial calibration and then typically not subsequently changed.The setup and operation of the machine typically requires only the importing of the files containing the prescribed oligonucleotide sequences and restocking of the reagents used in the operation. The tubing and jets which deliver the reagents to the 96-well plate are automatically primed with the reagents under computer control at the beginning of each run. At the end of each run the lines are purged of the synthesizing reagents. By purging with a neutral flushing agent, buildup of deposits or clogging of lines is prevented. Using computer control for these operations saves technician time and also prevents introduction of human error in these operations. The liquids are moved through the system under the pressure of dry argon gas applied to the reagent bottles, thus no pumps are required.. The synthesis reaction takes place in the bottom of the wells of the 96-well plate. The bottom of each well contains a filter which serves the double purpose of a base support for the cpg beads and a barrier which holds the liquids within the well under the normal low argon pressure in the reaction chamber. When it is necessary to discharge liquids from the well, the pressure is raised within the chamber and the liquids are forced through the filter into a waste collection chamber. Figure 1 shows a view of the machine. The controlling computer monitor is seen in the background. The view shows the operator removing a sampling plate which holds a detritylation product. These "trityl" samples are placed in a plate reader which monitors the optical density of the liquid produced by the deblocking step of the synthesis. This gives a measure of the efficiency of the synthesis operation as it proceeds. Figure 2 is a close-up view of the valves, manifolds and tubing which deliver the reagent liquids to the wells of the 96-well plate. The system uses 119 electrically actuated valves to control the flow of liquids and gases in the system. The 96-well plate is moved back an forth beneath the nozzle assembly by an accurate linear drive system. The system knows precisely the location of the wells at any point in time and places the appropriate well beneath a liquid jet as required. The amount of liquid discharged is accurately controlled by the computer by the timing of the opening of the valves. Contact the Stanford Office of Technology Licensing for more information on this instrument. 1 Lashkari, D.A. et al, An automated multiplex oligonucleotide synthesizer: Development of high-throughput, low-cost DNA synthesis, (1995) Proc. Nat. Acad. Sci . USA 92, 7912-7915 2 Lashkari, D. A., Development of an Automated Multiplex Oligonucleotide Synthesizer and Its Application in Genome Analysis, Ph.D. Dissertation, Stanford University, Stanford, CA, June 1996 3 Beaucage, S.L. & Caruthers, M.H. (1981) Tetrahedron Lett. 22, 1859-1862 4 McBride, L.J. & Caruthers, M.H. (1983) Tetrahedron Lett. 24, 245-248 |
