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
<11 min, allowing a plate of 96 20-mers to be synthesized in
<5 hr. Oligonucleotide synthesis quality is comparable to
commercial machines, with average coupling efficiencies routinely >98% across the entire 96-well plate. No significant
well-to-well variations in synthesis quality have been observed
in >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
The publication costs of this article were defrayed in part by page charge
payment. This article must therefore be hereby marked "advertisement" in
accordance with 18 U.S.C. §1734 solely to indicate this fact.
Abbreviations: AMOS, automated multiplex oligonucleotide synthesizer; CPG, controlled pore glass.
*To whom reprint requests should be addressed.
7912
Applied Biological Sciences: Lashkari et aL
Proc. Natl. Acad. Sci. USA 92 (1995)
Valve Banks
N Amidite
G Amidite
C Amidite
A Amidite
T Amidite
Activator
,
Top Plate
Outlet Argon Manifold
rvw
w"
7913
Inlet Argon Manifold
,e
z1"
i
Perimeter IInflating
Seal
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
I
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x
Area of Seal Detail
x
Solenoid
Valve Bank
I I
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-X
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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-
/-
Inflating Seal
Gas Pressure Enhances Seal
UUUAUUU4UR
I~~~~
Z'
.
~~~~~
/ Reaction Plate
Waste Collection Chamber
FIG. 2. Cross section of a valve bank, nozzle setup, top plate, and
slider with reaction plate holder. Tubing from reagent bottles leads
into valves having individual nozzles leading through the top plate.
Spacing of the tubing matches spacing of the wells in the synthesis plate
and reagents are dripped into appropriate wells during synthesis. Spent
reagents are purged through each well into a collection chamber
leading to a waste bottle.
op PateI
Thin Teflon heet
'Sliding platet
FIG. 3. Close-up view of perimeter inflatable seal. A thin Teflon
strip (0.005 inch) is attached around the perimeter of the top plate. It
is gently pressed against the lower sliding plate by a backing elastic
sheet and interior gas pressure.
7914
Proc. Natl. Acad. Sci. USA 92 (1995)
Applied Biological Sciences: Lashkari et al.
Polypropylene well
CPG support
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 <4.5 hr not including deprotection and lyophilization times. The synthesizer is currently generating large
numbers of high-quality oligonucleotides with >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 <2% for full plate syntheses; the
major cause of failures has been the dislodging of well filters
during synthesis, resulting in loss of CPG material. No failures
have resulted from clogged valves, plugged drain holes, valve
misfirings, or improper seal functions. In general, with fresh
reagents synthesis quality is as high as'any commercial instrument.
There are no substantial well-to-well variations in synthesis
quality. Spectral analyses of trityl material from 38 synthesis
(-3600 oligonucleotides) were compared with respect to
standard deviation (SD) across an entire plate, along a single
row (representing the same reagent valves) and across different rows. Each value for SD was normalized to the data's
average, allowing comparison between different syntheses.
Data taken after cycle 4 and cycle 10 for all 38 plates have been
averaged and are shown in Table 1. As would be expected,
there is less variation among oligonucleotides of the same row
since wells of a single row have the same reagent delivery
components. The average SD of about 20% of mean value is
reasonable given cumulative effects of variations in tubing,
valves, CPG retention, trityl material collected, and initial
CPG loading. Cross-contamination between wells does not
occur as evidenced by the absence of trityl compounds in blank
wells next to wells containing oligonucleotides (D.A.L., un-
publishe4 data).
Applied Biological Sciences: Lashkari et aL
Proc. Natl. Acad. Sci. USA 92 (1995)
7915
Table 2. Reagent costs for oligonucleotide synthesis on
the AMOS
Cost,
Amount/
base
Reagent
dollars/unit
Dollars/base
Amidites
0.0025 g
$35/g
$0.09
Activator
0.0010 g
(tetrazole)
$0.02
$15/g
16 ,ul
Capping solutions
$0.075/ml
$0.001
Oxidizer
25 ,ul
$0.075/ml
$0.002
Deblocking solution 0.3 ml
$0.04/ml
$0.012
Acetonitrile
1.3 ml
$0.008/ml
$0.01
Total
$0.135
Costs are based on a 20-nmol synthesis scale. In addition to the
reagent costs, one-time costs of $0.035 for CPG and $0.10 per
oligonucleotide for the plate should also be factored into the total cost
of the oligonucleotide.
12
10
8
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.
.
2
.
.
.
.
3
4
5
6
7
Time, min
FIG. 5. HPLC chromatogram of a 24-mer synthesized on the
AMOS. The unpurified oligonucleotide was deprotected as described
and resuspended in distilled H20 and analyzed on an NPN-18C (50 x
4.6 mm i.d.) column (Sarasep, Santa Clara, CA). Buffer A: 100 mM
triethanolamine, pH 7.0. Buffer B: 100 mM triethanolamine, pH
7.0/25% acetonitrile. Gradient: 0-11.5 min, 24-38% buffer B. mAU,
milliabsorbance units.
A principal benefit of this instrument is significant cost
savings due to reduced reagent consumption and labor costs.
The instrument incorporates smaller synthesis scales, eliminates the rinsing of lines, and makes it possible to achieve at
least a 10-fold reduction in the cost of oligonucleotides as
compared to existing instruments. In addition, with the high
capacity of the instrument, one full-time user can synthesize up
to 480 oligonucleotides per machine per week. This is equal to
an -10-fold increase in the number of oligonucleotides synthesized by an individual. Savings based on reagent usage and
labor are substantial and enable the researcher to generate
large numbers of oligonucleotides at a fraction of the cost of
commercial instruments (Table 2). This translates into a 10fold reduction in costs of synthesis per oligonucleotide.
Oligonucleotides are essential components of many applications in molecular biology. The synthesis chemistry is robust
and commercial oligonucleotide synthesizers have taken advantage of the chemistry to provide oligonucleotides of high
quality and purity. The devices, however, generate products in
Table 1. Synthesis variation based on trityl analysis
Sample SD as % of
mean
Cycle 4
Cycle 10
Entire plate
19
23
Per row (i.e., same valves)
14
18
Per column (i.e., across valves)
16
20
Values are based on trityl data of >3600 oligonucleotides. The least
variation is seen between wells serviced by the same reagent components, as would be expected. Larger variations at cycle 10 relative to
cycle 4 are due to cumulative effects of minor differences in tubing,
valves, and reaction wells.
individual tubes in relatively large quantities at prohibitive
synthesis costs. The 96-well AMOS was developed to address
these issues. It provides high-quality oligonucleotides in a
standardized format and its high capacity and efficient reagent
use reduce the overall cost of synthesis dramatically.
The instrument alters the underlying method of oligonucleotide synthesis. Rather than sequentially deliver reagents to a
stationary column via a single common line, the AMOS moves
the entire reaction chamber to the reagents. This fundamental
change allows highly parallel synthesis of oligonucleotides in an
efficient manner with no sacrifice of product quality. The
design is in no way limited to the 96-well format-the synthesis
method can be incorporated into devices that synthesize many
more oligonucleotides in almost any desired configuration.
With the development of the AMOS, large numbers of
oligonucleotides are now readily available for mapping and
sequencing projects. More importantly, many other applications can now be envisioned. Large-scale gene synthesis has
become possible using oligonucleotides as short as 45 bases in
length. This enables efficient construction of genes and systematic mutational analysis. Other applications include genome-wide mutagenesis strategies for Saccharomyces cerevisiae (8) and PCR-based mapping methods requiring large
numbers of oligonucleotides. Low-cost, high-volume oligonucleotide synthesis will have an important impact on traditional
molecular biology strategies and allow many techniques once
thought to be too costly to now become practical.
We thank A. Smith, W. C. Reynolds, D. Botstein, H. Heyneker, D.
Preuss, V. Smith, and D. Shoemaker for helpful discussions and comments; and J. Ohms and P. Oefner for aid in CE and HPLC diagnostics.
DAL was supported by National Institutes of Health Predoctoral
Training Program in Genetics Grant NIHGM07790. S.P.H.-S. was supported by Grant NIHHGO0205. R.M.N. was supported by Grant
NIHNS26237. This research was supported by Grant NIHHGO0205 (to
R.W.D.). T.B. was supported by Grant DOEDEFG0393ER6165.
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