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
The 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
A 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 Philosophy
The 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:
steps are cycled for each subsequent addition of base to grow the oligonucleotide
chain. Other intermediate steps of the protocol include washing and draining
and are employed so as to insure as optimal a synthesis as possible.
A deblocking step which makes
a site chemically active on the existing base and ready to accept the next
nucleotide. This detritylation step also cuts off a chemical group which
can be collected and measured as a means of monitoring the efficiency of
the synthesis operation.
A coupling step where the appropriate
base is attached to the chain using a phosphoramidite chemical group mixed
with a catalyst activator.
An acetylation step where a
chemical cap is placed on active sites which have not received an amidite
group. This terminates incomplete chains and prevents deletions in the
An oxidation step which forms
an oxygen double bond, converting the newly formed phosphite bond into
a more stable phosphate bond.
Simplicity of operation
The 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
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.
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.
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
Contact the Stanford
Office of Technology Licensing for more information on this
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
D. A., Development of an Automated Multiplex Oligonucleotide Synthesizer
and Its Application in Genome Analysis, Ph.D. Dissertation, Stanford University,
Stanford, CA, June 1996
S.L. & Caruthers, M.H. (1981) Tetrahedron Lett. 22, 1859-1862
L.J. & Caruthers, M.H. (1983) Tetrahedron Lett. 24, 245-248