U.S. patent application number 11/022377 was filed with the patent office on 2005-12-22 for apparatus and method for polymer synthesis using arrays.
Invention is credited to Brennan, Thomas M..
Application Number | 20050281719 11/022377 |
Document ID | / |
Family ID | 22500483 |
Filed Date | 2005-12-22 |
United States Patent
Application |
20050281719 |
Kind Code |
A1 |
Brennan, Thomas M. |
December 22, 2005 |
Apparatus and method for polymer synthesis using arrays
Abstract
A polymer synthesis apparatus (20) for building a polymer chain
including a head assembly (21) having an array of nozzles (22) with
each nozzle coupled to a reservoir (23) of liquid reagent (24), and
a base assembly (25) having an array of reaction wells (26). A
transport mechanism (27) aligns the reaction wells (26) and
selected nozzles (22) for deposition of the liquid reagent (24)
into selected reaction wells (26). A sliding seal (30) is
positioned between the head assembly (21) and the base assembly
(25) to form a common chamber (31) enclosing both the reaction well
(26) and the nozzles (22) therein. A gas inlet (70) into the common
chamber (31), upstream from the nozzles (22), and a gas outlet (71)
out of the common chamber (31), downstream from the nozzles (22),
sweeps the common chamber (31) of toxic fumes emitted by the
reagents. Each reaction well (26) includes an orifice (74)
extending into the well (26) which is of a size and dimension to
form a capillary liquid seal to retain the reagent solution (76) in
the well (26) for polymer chain growth therein. A pressure
regulating device (82) is provided for controlling a pressure
differential, between a first gas pressure exerted on the reaction
well (26) and a second gas pressure exerted on an exit (80) of the
orifice, such that upon the pressure differential exceeding a
predetermined amount, the reagent solution (76) is expelled from
the well (26) through the orifice (74). A method of synthesis of a
polymer chain in a synthesis apparatus (20) is also included.
Inventors: |
Brennan, Thomas M.; (San
Francisco, CA) |
Correspondence
Address: |
DORSEY & WHITNEY LLP
555 CALIFORNIA STREET, SUITE 1000
SUITE 1000
SAN FRANCISCO
CA
94104
US
|
Family ID: |
22500483 |
Appl. No.: |
11/022377 |
Filed: |
December 21, 2004 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
11022377 |
Dec 21, 2004 |
|
|
|
10171497 |
Jun 12, 2002 |
|
|
|
10171497 |
Jun 12, 2002 |
|
|
|
09689272 |
Oct 10, 2000 |
|
|
|
09689272 |
Oct 10, 2000 |
|
|
|
09162348 |
Sep 28, 1998 |
|
|
|
09162348 |
Sep 28, 1998 |
|
|
|
08453972 |
May 30, 1995 |
|
|
|
5814700 |
|
|
|
|
09162348 |
Sep 28, 1998 |
|
|
|
08452967 |
May 30, 1995 |
|
|
|
5837858 |
|
|
|
|
08453972 |
|
|
|
|
08142593 |
Oct 22, 1993 |
|
|
|
5472672 |
|
|
|
|
08452967 |
|
|
|
|
08142593 |
Oct 22, 1993 |
|
|
|
5472672 |
|
|
|
|
Current U.S.
Class: |
422/130 |
Current CPC
Class: |
B01J 2219/005 20130101;
B01J 2219/00686 20130101; B01J 2219/00328 20130101; C40B 50/14
20130101; C40B 60/14 20130101; C07H 21/00 20130101; B01J 2219/00364
20130101; C40B 40/06 20130101; B01J 2219/00423 20130101; B01J
2219/00585 20130101; B01J 2219/00722 20130101; B01J 2219/00454
20130101; B01J 2219/00418 20130101; B01J 2219/0059 20130101; B01J
2219/0072 20130101; C07K 1/045 20130101; C07B 2200/11 20130101;
C07K 1/04 20130101; B01J 2219/0031 20130101; B01J 2219/00313
20130101; B01J 19/0046 20130101; B01J 2219/00695 20130101; B01J
2219/00315 20130101; B01J 2219/00689 20130101; B01J 2219/00369
20130101; B01J 2219/00459 20130101; B01J 2219/00725 20130101; G01N
35/1072 20130101; B01J 2219/00596 20130101; B01J 2219/00286
20130101; C40B 40/10 20130101; B01J 2219/00416 20130101; B01J
2219/00414 20130101 |
Class at
Publication: |
422/130 |
International
Class: |
B01J 019/00 |
Claims
1. A polymer synthesis apparatus for building a polymer chain by
sequentially adding polymer units to a solid support in a liquid
reagent comprising: a base assembly including a reaction well, and
having at least one orifice extending into said well; at least one
solid support disposed in said well for growing and immobilizing a
polymer chain thereon; reagent solution in said well in contact
with said solid support and at least one polymer unit of said
polymer chain affixed to said solid support; retaining means
positioned in said well, and formed and dimensioned to
substantially prevent passage of said solid support through said
orifice; said orifice having an entrance into said well and an
exit, and being of a size and dimension to form a capillary liquid
seal to retain said reagent solution in said well to enable polymer
chain growth therein when a pressure differential between a first
gas pressure exerted on said reaction well and a second gas
pressure exerted on said orifice exit is less than a predetermined
amount; and a pressure regulating device for controlling said
pressure differential such that upon said pressure differential
exceeding said predetermined amount, said reagent solution being
expelled from said well through said orifice.
2. The polymer synthesis apparatus as defined in claim 1 wherein,
said retaining means comprises a filter member disposed in said
well between said one orifice and said support structure.
3. The polymer synthesis apparatus as defined in claim 2 wherein,
said filter member is composed of a glass fiber frit.
4. The polymer synthesis apparatus as defined in claim 2 wherein,
said filter member and said orifice cooperate in a manner forming
said capillary liquid seal.
5. The polymer synthesis apparatus as defined in claim 1 further
including: a top chamber mechanism cooperating with a top surface
of said base assembly to form a top chamber enclosing said reaction
well therein, said regulating device communicating with said top
chamber to regulate said pressure differential.
6. The polymer synthesis apparatus as defined in claim 1 wherein,
said solid support is provided by controlled pore glass (CPG).
7. The polymer synthesis apparatus as defined in claim 1 wherein,
said polymer chain is an oligonucleotide.
8. The polymer synthesis apparatus as defined in claim 1 further
including: a bottom chamber mechanism cooperating with a bottom
surface of said base assembly to form a bottom chamber enclosing
said orifice therein, said regulating device communicating with
said bottom chamber to regulate said pressure differential.
9. A polymer synthesis apparatus for building a polymer chain by
sequentially adding polymer units in a reagent solution, said
apparatus comprising: a head assembly having a plurality of nozzles
mounted thereto in generally spaced-apart relation, each nozzle
being coupled to a reservoir of liquid reagent for controlled
delivery therethrough; a base assembly having at least one reaction
well; a transport mechanism coupled to at least one of said head
assembly and said base assembly to produce relative movement
therebetween to position said reaction well and a selected one
nozzle in alignment for deposition of a liquid reagent into said
reaction well for synthesis of a polymer chain; and a sliding seal
positioned between said head assembly and said base assembly forms
a common chamber enclosing both said reaction well and said nozzles
therein.
10. The polymer synthesis apparatus as defined in claim 9 wherein,
each said nozzle terminates proximate a substantially planar bottom
surface of said head assembly, and said base assembly includes a
substantially planar top surface in opposed relation to said head
bottom surface.
11. The polymer synthesis apparatus as defined in claim 10 wherein,
said sliding seal is provided by a balloon seal having one end
affixed to one of said head bottom surface and said base top
surface, and an opposite end in sliding contact with the other of
said base top surface and said head bottom surface.
12. The polymer synthesis apparatus as defined in claim 11 wherein,
said opposite end tapers radially inwardly toward an interior
portion of said chamber in a manner such that upon increase in
pressure in said chamber, the seal integrity between said opposite
end and the other of said base top surface and said head bottom
surface increases.
13. The polymer synthesis apparatus as defined in claim 11 wherein,
said seal opposite end includes a substantially stick-free coating
or layer in sliding contact with the other of said base top surface
and said head bottom surface.
14. The polymer synthesis apparatus as defined in claim 11 wherein,
said coating or layer comprises TEFLON.RTM..
15. The polymer synthesis apparatus as defined in claim 11 wherein,
the other of said base top surface and said head bottom surface in
sliding contact with said seal opposite end includes a
substantially stick-free coating or layer therebetween.
16. The polymer synthesis apparatus as defined in claim 15 wherein,
said coating or layer comprises TEFLON.RTM..
17. The polymer synthesis apparatus as defined in claim 12 wherein,
said seal one end is mounted to said base bottom surface, and said
seal opposite end is in sliding contact with said base top
surface.
18. The polymer synthesis apparatus as defined in claim 9 wherein,
said transport mechanism includes a stepped motor operably coupled
to one of the head assembly and the base assembly for incremental
movement thereof.
19. The polymer synthesis apparatus as defined in claim 9 wherein,
said transport mechanism is operably coupled to said base assembly
for movement thereof.
20-71. (canceled)
72. A method of synthesis for building a polymer chain by
sequentially adding polymer units to at least one solid support for
growing a polymer chain thereon in a liquid reagent, said method
comprising the steps of: depositing a liquid reagent in a reaction
well in contact with at least one solid support and at least one
polymer unit of said polymer chain affixed to said solid support to
sequentially add a polymer unit to said polymer chain, said well
having at least one drain orifice extending out of said well, and
being of a size and dimension to form a capillary liquid seal to
retain said reagent solution in said well to enable polymer chain
growth on said solid support; creating a gas pressure differential
within said reaction well such that said pressure differential
exerted on an exit of said drain orifice exceeds a predetermined
amount necessary to overcome said capillary liquid seal and expel
said reagent solution from said well through said drain orifice;
and controlling said pressure differential through a pressure
regulating device for one of raising said pressure differential
above said predetermined amount and lowering said pressure
differential below said predetermined amount, wherein said pressure
differential is created by forming a vacuum.
Description
TECHNICAL FIELD
[0001] The present invention relates, generally, to polymer
synthesis apparatus, and, more particularly, relates to polymer
synthesis apparatus using arrays.
BACKGROUND ART
[0002] In the recent past, oligonucleotides have played an
increasing and more pivotal role in diagnostic medicine, forensic
medicine and molecular biology research. One primary function of
these polymers, in particular, is their use in gene probe assays
for the detection of specific nucleic acid sequences.
[0003] Gene probe assays are used for a variety of purposes,
including: genetic counseling; tissue typing; and molecular biology
research. For example, an individual may be tested to see if he or
she carries the gene for Huntington's disease or cystic fibrosis.
Another use of gene probe assays include determining compatibility
prior to tissue transplantation, and for matching up tissue or
blood samples for forensic medicine. Finally, in molecular biology
research, these assays are extensively employed to explore
homologies among genes from different species, and to clone genes
for which only a partial nucleic acid or amino acid sequence is
known, such as in the polymerase chain reaction (pcr).
[0004] These types of assays typically test for the presence of a
specific nucleic acid sequence, usually a DNA sequence, although
RNA sequences may be employed as well. As is well known in the
field, this is accomplished utilizing oligonucleotides synthesized
to have specific, predetermined sequences. The sequence of an
oligonucleotide probe may be based on known amino acid sequences,
known DNA sequences, or may be a "guess" probe, based on homology
to known or putative DNA or amino acid sequences. Since DNA is a
"degenerate" code, and several DNA sequences will result in a
single amino acid sequence, gene probe assays based on an amino
acid sequence frequently utilize pools of related oligonucleotides;
each having a different specific sequence of nucleotides. Thus it
is often necessary to create a large number of related but distinct
oligonucleotides to clone a single gene.
[0005] In addition to the actual sequence, there are a number of
parameters which may be altered in the synthesis of
oligonucleotides. It is also frequently necessary to synthesize
these polymers in a variety of lengths, since generally, the longer
the oligonucleotide probe, the more specific the gene probe assay
will be; specificity may or may not be desired in any particular
application. Oligonucleotides may be made of deoxyribonucleotides,
or ribonucleotides, or mixtures. Alternatively, oligonucleotides
with modified or non-standard bases or non-radioactive labels
incorporated may be desirable. Similarly, oligonucleotides with
altered ribose-phosphate backbones may be created for some
applications.
[0006] There are several other uses of oligonucleotides besides the
gene probe assay use. For example, the formation of a single
stranded oligonucleotide with a specific sequence may be used in
the formation of extremely stable triplex DNA structures. Other
uses include direct construction of synthetic genes and plasmid
vectotrts by ligating or joining together the component
5'-phosphate oligonucleotides.
[0007] Accordingly, as the use of synthetic oligonucleotides has
increased, so has its demand. In turn, this has spawned development
of new synthesis apparatus and methodologies for basic procedures
for custom sequence defined oligonucleotides. These apparatus and
methods, however, are generally very expensive to employ and not
readily available for mass production thereof. Typically, the
present generation automated DNA sequential synthesizers place a
derivatized solid support, such as controlled pore glass (CPG),
into an individual reaction chamber of a column to provide a stable
anchor on which to initiate solid phase synthesis. Using a series
of complex valving, and pumps coupled to the column, the
appropriate selected reagents are sequentially filtrated through
the chamber in a predetermined manner. Contact of the reagent with
the polymer units pre-affixed to the CPG, which is retained and
supported in the chamber by a sample support porous frit, causes a
reaction resulting in sequenced growth thereon.
[0008] While each column of this assembly is effective to rapidly
mass produce a homogenous population of sequence defined
oligonucleotides, the current assemblies only offer four (4) column
capabilities. Increased column capacity is limited due to physical
limitations of the valving configuration. Hence, only four
independent synthesis cycles can be performed simultaneously.
Further, since the synthesis apparatus is not generally amenable to
integrated automation with other robotic lab instrumentation, an
operator must intervene to load and remove each individual
synthesis column manually. Such handling increases human error.
[0009] A more important limitation is that all the reagents are
funneled through a common manifold passage. Only one reagent or
combination thereof, thus, can be simultaneously deposited in
selected columns. For example, the reagent "tetrazole" cannot be
deposited in column one while a particular amidite reagent is
simultaneously being deposited in column four. In addition, for
each independent synthesis or reaction, the common manifold passage
and associated valving must be flushed with a cleansing reagent so
that residual amidite or deblocking reagents will not be
undesirably deposited in a column. This approach wastes time, as
well as increasing operator costs.
[0010] Synthesis of arrays of bound oligonucleotides or peptides is
also generally known in the art. In one approach to parallel
synthesis, known as the Tea-bag?? method or disk design, an array
of individual packets or disks of solid support beads are
physically sorted into four (4) amidite subsets for treatment with
the selected amidite. After each packet of beads has been treated
with the common reagent, the packets must again be manually
resorted into the four subsets for the subsequent synthesis cycle.
Such sorting and resorting becomes too burdensome and labor
intensive for the preparation of large arrays of
oligonucleotides.
[0011] Another approach using arrays is the pin dipping method for
parallel oligonucleotide synthesis. Geysen, J. Org. Chem. 56, 6659
(1991). In this method, small amounts of solid support are fused to
arrays of solenoid controlled polypropylene pins, which are
subsequently dipped into trays of the appropriate reagents. The
density of arrays, however, is limited, and the dipping procedure
employed is cumbersome in practice.
[0012] Disclosed at the Southern, Genome Mapping Sequence
Conference, May 1991, Cold Spring Harbour, N.Y., is still another
scheme for oligonucleotide array synthesis in which selected areas
on a glass plate are physically masked and the desired chemical
reaction is carried out on the unmasked portion of the plate. The
problem with this method is that it is necessary to remove the old
mask and apply a new one after each interaction. Fodor et al.,
Science 251, 767 (1991) describes another method for synthesizing
very dense 50 micron arrays of peptides (and potentially
oligonucleotides) using mask-directed photochemical deprotection
and synthetic intermediates. This method is limited by the slow
rate of photochemical deprotection and by the susceptibility to
side reactions (e.g., thymidine dimer formation) in oligonucleotide
synthesis. Khrapko et al., FEBS Letters 256, 118 (1989) suggest
simplified synthesis and immobilization of multiple
oligonucleotides by direct synthesis on a two-dimensional support,
using a printer-like device capable of sampling each of the four
nucleotides into given dots on the matrix. However, no particulars
about how to make or use such a device are provided.
[0013] In summary, the related art generally contains numerous
ideas and information related to the synthesis of arrays of
oligonucleotides or peptides for the determination of nucleotide
sequences or the amino acid sequences of specific binding peptides.
However, existing or suggested methods are limited, and do not
conveniently and reliably mass produce very large arrays necessary
for effective large-scale sequencing.
DISCLOSURE OF INVENTION
[0014] Accordingly, it is an object of the present invention to
provide a polymer synthesis apparatus and method for building
sequence defined polymer chains.
[0015] Another object of the present invention is to provide a
polymer synthesis apparatus and method for preparing large quantity
arrays of oligonucleotides or peptides in a reproducible and rapid
manner.
[0016] Yet another object of the present invention is to provide a
polymer synthesis apparatus and method which reduces reagent waste
during the preparation of large quantity arrays of
oligonucleotides.
[0017] Still another object of the present invention is to provide
a polymer synthesis apparatus and method for preparing large
quantity arrays of oligonucleotide at reduced costs.
[0018] It is a further object of the present invention to provide a
polymer array synthesis apparatus and method which is durable,
compact, easy to maintain, has a minimum number of components, is
easy to use by unskilled personnel, and is economical to
manufacture.
[0019] In accordance with the foregoing objects, one embodiment of
the present invention provides a polymer synthesis apparatus for
building a polymer chain by sequentially adding polymer units in a
reagent solution. The synthesis apparatus comprises a head assembly
having a plurality of nozzles mounted thereto in generally
spaced-apart relation. Each nozzle is coupled to a reservoir of
liquid reagent for controlled delivery therethrough. Further, a
base assembly is included having at least one reaction well, and a
transport mechanism coupled to at least one of the head assembly
and the base assembly to produce relative movement therebetween.
This positions the reaction well and a selected one nozzle in
alignment for deposition of a liquid reagent into the reaction well
for synthesis of a polymer chain. A sliding seal is positioned
between the head assembly and the base assembly to form a common
chamber enclosing both the reaction well and the nozzles
therein.
[0020] The synthesis apparatus may further include an inlet into
the common chamber positioned upstream from the nozzles, and an
outlet out of the common chamber positioned downstream from the
nozzles. A pressurized gas source is coupled to the inlet for
continuously streaming a gas through the common chamber from the
chamber upstream to the chamber downstream and out of the outlet to
sweep the common chamber of toxic fumes emitted by the reagents, as
well as keep air and moisture from seeping in.
[0021] In another aspect of the present invention, a polymer
synthesis apparatus is provided comprising a base assembly
including a reaction well, and having at least one orifice
extending into the well. At least one solid support is disposed in
the well for growing and immobilizing a polymer chain thereon.
Reagent solution in the well is in contact with the solid support
and at least one polymer unit of the polymer chain affixed to the
solid support. Further, a retaining device is included positioned
in the well, and is formed and dimensioned to substantially prevent
passage of the solid support through the orifice. The orifice has
an entrance into the well and an exit out of the well, and is of a
size and dimension to form a capillary liquid seal to retain the
reagent solution in the well to enable polymer chain growth
therein. To retain the solution in the well, a pressure
differential between a first gas pressure exerted on the reaction
well and a second gas pressure exerted on the orifice exit must be
less than a predetermined amount. Finally, a pressure regulating
device is provided for controlling the pressure differential such
that upon the pressure differential exceeding the predetermined
amount, the reagent solution is expelled from the well through the
orifice.
[0022] The present invention also includes a method of synthesis of
a polymer chain in a synthesis apparatus comprising the steps of:
A) aligning the reaction well and a selected one nozzle through the
transport mechanism coupled to at least one of the head assembly
and the base assembly to produce relative movement therebetween;
and B) depositing a liquid reagent into the well from the reagent
reservoir through the one nozzle to enable synthesis of a polymer
chain. Finally, C) sweeping toxic fumes, emitted by the reagents,
from the common chamber through passage of a gas from a pressurized
gas source, coupled to an inlet into the common chamber and
positioned upstream from the nozzles, and out of the chamber
through an outlet out from the common chamber and positioned
downstream from the nozzles.
[0023] Another method of polymer synthesis is provided for building
a polymer chain by sequentially adding polymer units to at least
one solid support for growing and immobilizing a polymer chain
thereon in a liquid reagent. The method comprises the steps of A)
depositing a liquid reagent in the reaction well, having a properly
sized orifice, in contact with at least one solid support and at
least one polymer unit of the polymer chain affixed to the solid
support, and forming a capillary liquid seal to retain the reagent
solution in the well to enable polymer chain growth on the solid
support. The next step includes B) applying a first gas pressure to
the reaction well such that a pressure differential between the
first gas pressure and a second gas pressure exerted on an exit of
the orifice exceeds a predetermined amount necessary to overcome
the capillary liquid seal and expel the reagent solution from the
well through the orifice.
BRIEF DESCRIPTION OF THE DRAWING
[0024] The assembly of the present invention has other objects and
features of advantage which will be more readily apparent from the
following description of the Best Mode of Carrying Out the
Invention and the appended claims, when taken in conjunction with
the accompanying drawing, in which:
[0025] FIG. 1 is an exploded top perspective view of the polymer
synthesis array apparatus constructed in accordance with the
present invention.
[0026] FIG. 2 is bottom perspective view of a head assembly of the
polymer synthesis array apparatus of FIG. 1 illustrating the
recessed nozzle ends.
[0027] FIG. 3 is a side elevation view, in cross-section of the
polymer synthesis array apparatus of FIG. 1 and showing the
sweeping action of the flow of inert gas through the common
chamber.
[0028] FIG. 4 is a front elevation view, in cross-section of the
polymer synthesis array apparatus of FIG. 1 and illustrating the
head assembly pivotally mounted to a frame assembly.
[0029] FIG. 5 is an enlarged side elevation view, in cross-section,
of the polymer synthesis array apparatus taken substantially along
the line 5-5 of FIG. 3 and showing the capillary liquid seal formed
between the liquid reagent solution and the corresponding frit and
orifice.
[0030] FIG. 6 is an enlarged side elevation view, in cross-section,
of the polymer synthesis array apparatus taken substantially along
the line 6-6 of FIG. 3 and illustrating the balloon seal
gasket.
[0031] FIG. 7 is an enlarged, schematic, top perspective view of a
delivery assembly mounted to the head assembly of the polymer
synthesis array apparatus.
[0032] FIGS. 8A and 8B depict the results of capillary
electrophoresis runs of oligonucleotides Nos. 14 and 15,
respectively, from Example 1.
BEST MODE OF CARRYING OUT THE INVENTION
[0033] While the present invention will be described with reference
to a few specific embodiments, the description is illustrative of
the invention and is not to be construed as limiting the invention.
Various modifications to the present invention can be made to the
preferred embodiments by those skilled in the art without departing
from the true spirit and scope of the invention as defined by the
appended claims. It will be noted here that for a better
understanding, like components are designated by like reference
numerals throughout the various figures.
[0034] Attention is now directed to FIGS. 1 and 2 where a polymer
synthesis apparatus, generally designated 20, is shown for building
a polymer chain by sequentially adding polymer units to a solid
support in a liquid reagent. Incidently, while apparatus 20 is
particularly suitable for building sequence defined
oligonucleotides, the present invention may be employed for
synthesis of any polymer chain. Hence, the term "polymer unit" will
be defined as a moiety that is bound to other moieties of the same
or a different kind to form a polymer chain, such as
olignucleotides and peptide chains.
[0035] In one embodiment, the synthesis apparatus 20, briefly,
comprises a head assembly, generally designated 21, having a
plurality of nozzles 22 (FIG. 2) mounted thereto in generally
spaced-apart relation. Each nozzle 22 is coupled to a reservoir 23
(FIG. 7) of liquid reagent 24 for controlled delivery therethrough.
Further, a base assembly, generally designated 25, is included
having at least one reaction well 26, and a transport mechanism,
generally designated 27 (FIG. 3), coupled to at least one of head
assembly 21 and base assembly 25 to produce relative movement
therebetween. This positions a selected reaction well 26 and a
selected nozzle 22 in alignment for deposition of a selected liquid
reagent 24 into the reaction well for synthesis of a polymer chain.
A sliding seal, generally designated 30, is positioned between the
head assembly and the base assembly to form a common chamber 31
(FIG. 3) which encloses both the reaction well and the nozzles
therein.
[0036] In the preferred embodiment, an array of wells 26 (FIG. 1)
is provided formed in a microtiter plate 32 which is carried by a
sliding plate 33 of base assembly 25. The synthesis apparatus is
particularly configured to employ a 96-well microtiter plate (not
all wells shown for ease of illustration), aligned in 12 equally
spaced-apart rows 34, each extending transverse to a longitudinal
axis 35 of elongated base assembly 25, by 8 equally spaced-apart
columns 36 wide. Microtiter plate 32 is preferably fabricated from
a chemically inert material such as polypropylene. It of course
will be appreciated that any number of wells, or arrangement of
rows and columns, could be employed without departing from the true
spirit and nature of the present invention.
[0037] FIGS. 1 and 2 further illustrate that nozzles 22, mounted to
mounting blocks 37 of head assembly 21, are further aligned in an
array of nozzle rows 40 and columns 41 similar to the array of
wells 26. In the preferred form, the number of nozzle columns 41,
each extending parallel to a longitudinal axis 42 of head assembly
21, is equivalent to the number of well columns 36. Accordingly,
each nozzle 22 in a particular bank or row 40 of nozzles
corresponds to and is aligned with a respective column 36 of wells
26. The nozzles in any one row 40 and column 41 are also equally
spaced-apart by the same distance as the spacing between the well
rows 34 and columns 36 which permit simultaneous alignment between
one or more well rows 34 with selected nozzle rows 40 during a
single cycle. That is, the array of wells can be aligned with the
array of nozzles along a plurality of positions for simultaneous
deposition.
[0038] This array technique for sequence defined oligonucleotide
synthesis first evolved from the high density oligonucleotide array
chip assembly for hybridization sequencing disclosed in U.S. patent
application Ser. No. 07/754,614, filed Sep. 4, 1991, now pending.
Hence, this array technique, alone, is not claimed as a novel
feature of the present invention. That assembly, however, is
neither suitable nor appropriate for the large-scale production
capabilities of the present invention as will be described
henceforth.
[0039] To simplify organization and manufacture of polymer chains,
a delivery assembly 43 (FIG. 7) of the synthesis apparatus, for
controlling delivery of the liquid reagents through the array of
nozzles, communicably couples all nozzles 22 in a particular bank
or row 40 to a common independent liquid reagent reservoir 23,
while each bank or row 40 of nozzles is coupled to a different
liquid reagent applied in a particular polymer synthesis. For
instance, the first row 40 of nozzles 22 may only dispense the
activator tetrazole, while the second row of nozzles dispenses the
amidite thymidine. In oligonucleotide synthesis, this order of
liquid reagent distribution may continue down the line for the
amidites adenosine, cytosine, and guanine, the auxiliary base AnyN,
the solvent wash/reaction solvent acetonitrile, the Cap acetic
anhydride, the Cap N-methylimidazole, iodine and the deblockers
dichloracetic acid or trichloracetic acid; all of which are
reagents used for the synthesis of defined sequence
oligonucleotides. Incidently, only five rows of nozzles are shown
for the ease of illustration. Further, it will be understood that
any nozzle contained in the array may be communicably coupled to
any reagent reservoir.
[0040] The delivery assembly 43 of the present invention
communicably couples each bank of nozzles to a common reagent
reservoir 23 through independent dispensing tubes 44. Each tube
includes a passageway 45 (FIG. 6) and has one end coupled to the
respective nozzle and an opposite end terminating in the liquid
reagent 24 contained in the reservoir. These tubes are press-fit
into apertures 46 extending through mounting blocks 37 and a head
plate 47 of head assembly 21. The flexible and semi-resilient
nature of each tube 44, preferably TEFLON.RTM. which are more
resistant to deterioration upon contact with the reagents, provides
an adequate seal between the tubing exterior and the respective
aperture.
[0041] A distal end of each tube 44, as shown in FIGS. 2 and 6,
forms nozzle 22 which extends into, but not past, transversely
positioned slots 50 which are formed in a bottom surface 51 of head
plate 47. Accordingly, each independent nozzle 22 is recessed so as
not to interfere with a top surface 52 of base sliding plate 33
during relative sliding movement therebetween. Further, the
independent extension of each nozzle into slots 50 promotes removal
or discarding of residual liquid reagent accumulated at the end of
the nozzle after delivery of reagent therefrom.
[0042] There are two important concerns in liquid reagent delivery
through nozzles 22: 1) how to eject a droplet cleanly so that a
drop is not left hanging on the end of the nozzle; and 2) how to
keep the contents of the reaction chamber from splashing when the
stream of reagent is delivered into the well. Further, the ejection
velocity of the reagent from the nozzle must be sufficient to
induce mixing between the first and second delivered reagent in the
reaction chamber. Very small droplets can be ejected cleanly at
high ejection velocities, but do not have sufficient kinetic energy
to overcome the surface tension of the liquid already in the well
to cause mixing. In contrast, larger droplets also eject cleanly at
high ejection velocities, but tend to splash the contents into
adjacent wells. At lower ejection velocities, the reagents tend to
leave the last drop hanging from the nozzle tip, which is also a
function of the cross-sectional area of the tip. Moreover, the flow
rate of liquids through small capillary tubing varies directly with
the delivery pressure and inversely with the length of the tube and
inversely with the diameter. All these variables must be taken into
consideration when developing delivery pressure and nozzle
configurations, as well as the materials of construction, so that
the reagents can be expelled cleanly without leaving a residual
drop of liquid reagent hanging from the nozzle tip. Hence,
depending on the liquid reagent, it may be more beneficial to
dispense it in a continuous stream, a series of pulses or in
droplet form.
[0043] Each reagent reservoir 23, as shown in FIG. 7, includes a
pressure tube 53 coupled to a compressor device (not shown) which
pressurizes reservoir air space 54 to drive the stored liquid
reagent from the reservoir and through respective dispensing tubes
44. Delivery of reagents through dispensing tubes 44 is controlled
by an array of independent valve assemblies 55 each mounted in-line
therewith. These valve assemblies are preferably provided by
solenoid driven micro shutoff valves, each capable of opening and
closing in less than 5 milliseconds to deliver accurate volumes of
liquid reagent.
[0044] To assure a constant delivery pressure across each
dispensing tube 44, and hence, a constant delivery rate of liquid
reagent through any nozzle in a bank or row 40 of nozzles 22, each
dispensing tube will independently terminate in the liquid reagent
contained in the reagent reservoir. Thus, independent of the number
of nozzles set to deliver, this configuration will not suffer
uneven rate delivery caused by varying line pressure.
[0045] In accordance with the present invention, base assembly 25
and head assembly 21 cooperate with transport mechanism 27 for
relative movement between the base assembly and the head assembly
to align the array of wells with the array of nozzles at a
plurality of positions. Preferably, the transport mechanism moves
the base assembly along (phantom lines in FIG. 3) its longitudinal
axis 35 such that the wells of each row 34 remain aligned with the
nozzle columns 41. Base assembly 25 is, thus, slidably supported by
frame assembly 57 (FIGS. 3 and 4) for reciprocating movement in the
direction of arrows 60. Sliding plate 33, carrying microtiter plate
32, is slidably received in a track mechanism 61 of frame assembly
57 for aligned movement. Hence, by controlling the delivery of
reagent through selected nozzles (via valve assemblies 55), and
through manipulation of the transport mechanism, a plurality of
homogenous populations of sequence defined polymers can be
simultaneously synthesized in selected wells in a rapid and
reproducible manner.
[0046] Transport mechanism 27 includes a stepped motor assembly 62,
schematically represented in FIG. 3, which is operably coupled to
sliding plate 33. Hence, base assembly 25 cooperates with track
mechanism 61 and the stepped motor for linear incremental movement
to align the array of wells with the array of nozzles at a
plurality of positions. It will be understood, however, that the
transport mechanism can be provided by any motor/track
configuration which moves the base assembly relative the head
assembly.
[0047] Before polymer synthesis begins or after the synthesis
process has ended, the array of wells may be positioned outside of
common chamber 31 and exposed to the open environment for access by
moving the wells 26 (via movement of base assembly 25) to the
extreme right or left of sliding seal 30. Hence, the well array may
be cleaned or loaded with solid support material, to be discussed.
Further, the wells and nozzles, once inside the common chamber, may
be accessed through a hinge assembly 63, as shown in FIG. 4, which
pivotally mounts head assembly 21 to frame assembly 57.
[0048] Referring now to FIGS. 1, 3 and 5, the sliding seal of the
present invention will be described. In oligo-nucleotide synthesis
by phosphoramidite coupling, there are two major chemical
engineering requirements because the coupling reactions are rapid
and irreversible: water and oxygen are preferably both be excluded
from the common reaction chamber during synthesis. Phosphoramidites
are sensitive to hydrolysis by tracing of water, and to oxidation
by contact with air. Sliding seal 30, therefore, is disposed
between the bottom surface 51 of head assembly 21 and the top
surface 52 of base assembly 25 to environmentally contain both the
reaction wells and the nozzles in a common chamber 31. Further, as
will be described in greater detail below, by streaming an inert
gas through common chamber 31 to sweep the air and water traces
from the chamber, hydrolysis and oxidation can be minimized, if not
eliminated.
[0049] Sliding seal 30 must be formed to maintain environmental
containment while permitting the base assembly to slide relative
the head assembly. In the preferred form, sliding seal 30 is
provided by an elastic, rectangular-shaped, hydrofoil or balloon
seal gasket having an upper end mounted to head bottom surface 51,
and an opposite or lower end 64 in sliding contact with base top
surface 52. This special gasket, preferably composed of rubber or
the like, increases seal integrity between gasket lower end 64 and
base top surface 52 as the pressure in common chamber increases.
FIG. 5 illustrates that gasket lower end 64 peripherally tapers
inwardly toward an interior of common chamber 31. Upon increase in
chamber pressure, the walls of gasket seal 30 expand outwardly
whereby the surface area contact between the gasket lower end and
the base top surface increases for better sealing engagement
therebetween.
[0050] To facilitate sliding contact, while maintaining
environmental containment, gasket seal 30 includes a stick-free
coating or layer 65 (FIG. 5), preferably TEFLON.RTM., between the
gasket lower end and the base top surface. This layer further
serves the purpose of protecting the sealing gasket from surface
absorption of residual liquid reagents which tend to deteriorate
the gasket upon contact, due in part to the elastic nature thereof.
As shown in FIG. 5, top surface 52 of the base assembly may also
include a coating or layer 66 of TEFLON.RTM. to promote sliding
contact and for protection of the top surface from residual
reagent.
[0051] It will be understood that the seal between gasket lower end
64 and base top surface 52 need not be hermetic. The primary
function of the seal gasket is to exclude oxygen from the reaction
chamber. Thus, it is important to normally maintain a minimum
positive pressure inside common chamber 31 at all times during
synthesis which is slightly greater than atmospheric pressure so
that the flow of gas, should a leak occur, would be outward. This
minimum positive pressure differential is generally about {fraction
(1/100)} psi to about {fraction (1/10)} psi.
[0052] As previously indicated and as viewed in FIGS. 3, 5 and 6,
it is desirable to flush the air and water traces from the reaction
head space of the chamber with an inert gas, preferably argon, to
minimize hydrolysis and oxidation of the amidites during synthesis.
It is further desirable to continuously stream the inert gas
through the head space to protect sensitive amidites from the
capping and deblocker reagents, such as aqueous iodine vapor or
trichloroacetic acid, which will react with the amidites This is
accomplished by introducing a flow of inert gas (represented by
arrows 67) through common chamber 31 from a gas inlet 70,
positioned upstream from the array of nozzles 22, which exits the
chamber through a gas outlet 71 positioned downstream from the
nozzles. Gas inlet 70 is coupled to a gas source (not shown)
through inlet tube 72 (FIG. 1) which further provides the positive
pressure inside common chamber 31 necessary to exclude the oxygen
from the environment. Since the head space of the common chamber is
relatively small (exaggerated in the FIGURES for illustration), a
sufficient flow of gas past the nozzles can be fashioned to sweep
or flush the chamber without expending large volumes of gas.
[0053] The gas inlet is preferably provided by an elongated inlet
slot 70 (FIG. 2) extending into head plate 47 and aligned
transverse to the longitudinal axis 42 of head plate 47. This shape
and orientation induces a substantially laminar flow of inert gas
from upstream inlet 70 to downstream outlet 71 which minimizes
cross flow of gas across the nozzles and sweeps the dead zones of
stagnant reagent vapors from the chamber. It will be noted that the
gas inlet may also be provided by a series of apertures extending
transversely across head plate 47, and that gas outlet 71 may
further be provided by an elongated slot or series of
apertures.
[0054] Because of the gas flow through the chamber, the acid and
moisture sensitive phophoramides are positioned upstream from the
deblocker and capping reagents, forming a diffusion gas barrier,
which maximizes the sweeping effect. Accordingly, in FIG. 3, the
phosphoramides would be dispensed from nozzles 22 closer to gas
inlet 70, while the cappers, washers and deblockers would be
dispensed from the nozzles situated closer to gas outlet 71,
downstream from the phosphoramide dispensing nozzles.
[0055] In another aspect of the present invention, as best viewed
in FIGS. 5 and 6, polymer synthesis apparatus 20 is provided with
reaction wells 26 having at least one orifice, generally designated
74, extending into the well. At least one solid support 75 is
disposed in the well for growing and immobilizing a polymer chain
thereon. Reagent solution 76 in well 26 is in contact with solid
support 75 and at least one polymer unit of the polymer chain
affixed to the solid support. Orifice 74 has an entrance 77 into
well 26 from the common chamber side and an exit 80 out of the well
into a lower catch basin 81 below. Importantly, the orifice is of a
size and dimension to form a capillary liquid seal with reagent
solution 76 contained therein to retain the reagent solution in the
well enabling polymer chain growth therein. To further retain
solution 76 in wells 26, a pressure differential between a common
chamber gas pressure exerted on the reagent solution in reaction
wells 26 and a second gas pressure exerted on orifice exits 80
(illustrated by arrows 79 in FIG. 6) must be less than a
predetermined amount. Finally, a pressure regulating device 82 is
provided for controlling the pressure differential such that upon
the pressure differential exceeding the predetermined amount, the
reagent solution 76 is expelled from well 26 through orifice 74
(FIG. 5).
[0056] Briefly, after proper alignment between selected wells 26'
with selected nozzles 22' (FIG. 6), using the array technique and
novel apparatus above-mentioned, the liquid reagents can be
deposited into selected wells 26'. The deposited reagent solutions
collects across the correctly dimensioned orifice 74, in
combination with a relatively small pressure differential (not
greater than the predetermined amount), to form a meniscus across
orifice 74 and creating a capillary liquid seal to retain the
solution in the well without draining through the orifice. This
seal effectively separates the common reaction chamber from the
environment of lower catch basin 81 below. After a sufficient
amount of time has passed to complete the synthesis reaction
(generally about one minute), the reagent solution is purged from
well 26 through orifice 74 and into lower catch basin 81 by
increasing the gas pressure differential above the predetermined
amount which overcomes the capillary forces in the orifice (FIG.
5). Subsequently, the purged reagent solutions may be drawn out of
the catch basin through a drain outlet 83. This process is repeated
for each synthesis cycle (i.e., deblocking, washing, coupling,
capping and oxidizing steps) until the desired defined synthesis
polymer is fabricated.
[0057] A retaining device, generally designated 84, is included
positioned in the bottom of well 26 between orifice 74 and the
solid support 75 which is formed and dimensioned to substantially
prevent passage of the solid support through the orifice. Retaining
device 84 is preferably provided by a polyethylene or glass fiber
frit which acts as a filter membrane permitting the reagent
solution to flow therethrough while retaining the solid support and
polymer chain grown thereon in the well. Hence, the porosity of the
frit is also a factor in the formation of the capillary liquid seal
and in the determination of the pressure differential necessary to
purge the reaction well.
[0058] To regulate and control pressure differential between common
chamber 31 and lower catch basin 81, as mentioned, pressure
regulating device 82 is provided operably coupled therebetween. In
the preferred embodiment, pressure regulating device 82 is
integrated with the gas flow assembly employed to flush the head
space in common chamber 31 of reagent toxins. Upon the inert gas
freely sweeping the chamber from gas inlet 70 to gas outlet 71, the
minimum pressure differential is generally retained between about
{fraction (1/100)} psi to about {fraction (1/10)} psi. This
pressure differential which is sufficiently positive to prevent
seepage of environmental air into the common chamber, while being
insufficient to overcome the capillary forces of the capillary
liquid seal in each well. By preventing or restricting the outflow
of inert gas through gas outlet 71, the pressure inside chamber 31
can be increased, thereby increasing the pressure differential to
purge the wells (FIG. 5) if the catch basin pressure is not
increased at the same or greater rate.
[0059] The pressure regulating device 82, hence, includes a chamber
valve 85 (FIG. 1) coupled to gas outlet 71 for controlling the
outflow of inert gas sweeping common chamber 31. Accordingly, by
sufficiently closing or restriction flow through chamber valve 85,
the pressure differential can be raised above the predetermined
amount so that the wells can be purged of reagent solution
simultaneously. Similarly, by sufficiently opening chamber valve
85, the pressure differential may be lowered below the
predetermined amount when it is desired to retain the deposited
liquid solution in the selected wells.
[0060] The liquid reaction solution will not leak out of or be
purged from well orifice 74 until there is a sufficient head of
liquid in the well or a sufficient gas pressure differential
between the common chamber and the lower catch basin to overcome
the capillary forces in the orifice. The rate of gravity-driven and
pressure-driven leakage from the orifice is primarily governed by
the viscosity of the solvent, the porosity of the frit, the size of
the orifice, and the gas pressure differential. For instance, a 10
.mu.UHMW polyethylene frit and a 0.015 in.sup.2 orifice will
support at least 0.79 in liquid head of acetonitrile (having a
viscosity of about 0.345 centipose (7.2.times.10.sup.-5
(lbf.multidot.s)/ft.sup.2 at an operating temperature of about
68.degree. F.)) before beginning to overcome the capillary forces
in the orifice. On the other hand, by increasing the pressure
differential above the predetermined amount (generally about 1 psi)
to about 5 psi, purging of the well will occur rapidly. In
practice, it is necessary to maintain a pressure differential
between about 2.5 psi to about 5 psi to sufficiently purge the
reaction wells simultaneously of reagent solution. As the
individual wells begin to empty, the flow rate of inert gas through
the empty wells of the microtiter plate substantially increases
which decreases the pressure in common chamber 31. Accordingly,
this decrease in interior pressure further decreases the purging or
draining rate of the reagent solution through the orifices, an
effect magnified by retaining filter membrane 84.
[0061] It will be appreciated that the pressure differential may
also be created by forming a vacuum in lower catch basin 81 to
purge the reaction wells. FIG. 1 illustrates that an access opening
86 into lower catch basin 81 may be sealed by a cover 87, and drain
outlet 83 may be coupled to a vacuum pump which creates a vacuum in
the basin. The pressure differential may also be created from a
combination of positive pressure in common chamber 31 and a vacuum
in catch basin 81. Further, since the reagent solution is allowed
to collect in the reaction well for reaction thereof rather than
continuously streaming through the chamber, as is employed by some
other prior art assemblies, reagent consumption is substantially
minimized thereby saving costs. Labor costs are also reduced by
minimizing each cycle time.
[0062] To coordinate all the simultaneous functions, a control
mechanism 90 (FIG. 7) is operably coupled between transport
mechanism 27, valve assemblies 55 and pressure regulating device
82. A sequence file can be input which contains an ordered list of
the well position, scale, final deblocking instruction and the ATGC
and N (odd Base) sequence for each oligonucleotide. This file
cooperates with a command file used to indicate the actual number
and order of Deblock/Wash/Couple/Cap/Oxidize steps and the length
of time for Wait and pressure and/or vacuum Drain steps which
define the complete coupling cycle.
[0063] Oligonucleotides are typically synthesized on solid supports
of controlled pored glass (CPG) having the first nucleotide
previously linked to the CPG through the 3'-succinate linkage.
Hence, upon preparation for polymer synthesis, each well is
individually loaded with the correct CPG derivative. While one
could begin a full plate of synthesis using only one CPG
derivative, for example T (i.e., dT-Icaa-CPG), it is more desirable
to perform array synthesis where any base can be in the first
position. However, individually weighing and transferring 0.5 mg
quantities of the appropriate dry CPG derivative to each well can
be tedious and time consuming.
[0064] In accordance with the present invention, a balanced density
slurry technique is employed to deposit the correct amount of CPG
into a reaction well. By suspending the CPG in a suspension
solution, a desired amount of CPG can be accurately deposited in a
well by pipetting, either automatically or manually, a
corresponding volume of suspension solution therein. For example, a
non-settling 10%-1% weight/volume suspension of CPG can be prepared
in a 2.5:1 volume/volume dibromomethane-dichlorometha- ne solution.
Subsequently, the CPG can be washed and purged of suspension
solution before synthesis using the technique mentioned above.
[0065] In another aspect of the present invention, a method of
synthesis of a polymer chain is provided comprising the steps of:
A) aligning reaction well 26 and a selected one nozzle 22 of
synthesis apparatus 20 through transport mechanism 27 coupled to at
least one of head assembly 21 and base assembly 25 to produce
relative movement therebetween; and B) depositing a liquid reagent
24 into well 26 from reagent reservoir 23 through the one nozzle to
enable synthesis of a polymer chain. Finally, C) sweeping toxic
fumes, emitted by the reagents, from common chamber 31 through
passage of a gas from a pressurized gas source, coupled to an inlet
70 into common chamber 31 and positioned upstream from the nozzles,
and out of the chamber through an outlet 71 out from the common
chamber and positioned downstream from the nozzles.
[0066] Another method of polymer synthesis is provided for building
a polymer chain by sequentially adding polymer units to at least
one solid support for growing and immobilizing a polymer chain
thereon in a liquid reagent. The method comprises the steps of A)
depositing liquid reagent 24 in reaction well 26, having a properly
sized orifice 74, in contact with at least one solid support 75 and
at least one polymer unit of the polymer chain affixed to solid
support 75, and forming a capillary liquid seal to retain the
reagent solution in well 26 to enable polymer chain growth on solid
support 75. The next step includes B) applying a first gas pressure
to reaction chamber 31 such that a pressure differential between
the first gas pressure and a second gas pressure exerted on an exit
80 of orifice 74 exceeds a predetermined amount necessary to
overcome the capillary liquid seal and expel the reagent solution
from well 26 through orifice 74.
[0067] By repeating the steps of the two above-mentioned methods,
one continuous chain of polymer units can be formed.
[0068] The following example serves to more fully describe the
manner of using the above-described invention, as well as to set
forth the best mode contemplated for carrying out various aspects
of the invention. It is to be understood that this example in no
way serves to limit the true scope of the invention, but rather are
presented for illustrative purposes. It is to be understood that
any method of oligonucleotide synthesis may be utilized in the
present invention.
EXAMPLE 1
Synthesis of an Array of 15 Oligonucleotides
[0069] The general synthesis procedure follows published procedures
for the phosphoramidite and hydrogen phosphonate methods of
oligonucleotide synthesis; for example, the methods outlined in
Oligonucleotides and Analogues: A Practical Approach, F. Eckstein,
Ed. IRL Press, Oxford University; Oligonucleotide Synthesis: A
Practical Approach, Gait, Ed., IRL Press, Washington D.C.; and U.S.
Pat. Nos. 4,458,066, 4,500,707 and 5,047,524, all hereby
incorporated by reference. It is to be understood that other
methods of oligonucleotide synthesis may be used in the present
invention.
[0070] In general, the basic steps of the synthesis reaction are as
follows, with appropriate acetonitrile washing steps:
[0071] a) the first nucleoside, which has been protected at the 5'
position, is derivatized to a solid support, usually controlled
pore glass (CPG), or is obtained prederivatized;
[0072] b) the sugar group of the first nucleoside is deprotected or
detritlyated, using tricholoracetic-methylene chloride acid, which
results in a colored product which may be monitored for reaction
progress;
[0073] c) the second nucleotide, which has the phosphorus, sugar
and base groups protected, is added to the growing chain, usually
in the presence of a tetrazole catalyst;
[0074] d) unreacted first nucleoside is capped to avoid
perpetuating errors, using acetic anhydride and
N-methylimidazole;
[0075] e) the phosphite triester is oxidized to form the more
stable phosphate triester, usually using iodine reagents;
[0076] f) the process is repeated as needed depending on the
desired length of the oligonucleotide; and
[0077] g) cleavage from the solid support is done, usually using
aqueous ammonia at elevated temperatures over a period of
hours.
[0078] Accordingly, a sequence file was generated which indicated
the 3'-5' sequence and well number for each oligonucleotide to be
synthesized, the scale of the reaction for each nucleotide, and
whether a final detritylation was to be performed on the product at
the end of the reaction. The software was designed to support
simultaneous independent synthesis of oligonucleotides of different
lengths and scale. The sequence of the 15 different
oligonucleotides is shown in Table 1 below.
1TABLE 1 Number Sequence 1 AAG TCT TGG ACT TAG AAG CC 2 GGG TCA CTT
CTT TGT TTT CG 3 CCT TTA CTT TTC GGT CAA GG 4 TGA CTG GTA GGT CCA
CTG CA 5 ACC TCA CTT CGG TAA CTT AAA 6 CCA AAT TTT GAG GTA ACC ACT
T 7 GTG CTA TCC GGG ACA CCA 8 GGA AGA TCC ACG AGT CGT TT 9 CGT CCA
CCT CTT CTT TGA AGA 10 TGG TAG TAA TCC TAC CGT CT 11 GCG AAG GTC
TAC TCT CGT C 12 GTT CAG AGT AAG GAG TCG TGG 13 GTG GAA CCA TAG ACA
ACC ACA TAT 14 TTG TTC ATC TTT GTG TAG GGT AC 15 AGA AAA GTC ACG
GTC ACA CT
[0079] A command file was generated which contained the sequence of
reaction steps performed during a coupling cycle. The basic
commands were WASH (volume), DEBLOCK (volume), COUPLE, CAP,
OXIDIZE, WAIT (seconds) and DRAIN (pressure/vacuum seconds), as
shown in Table 2 below:
2TABLE 2 Step Number Step Volume(.lambda.) Time(sec) 1 Deblock 125
2 Wait 5 3 Drain 15 4 Wash 200 5 Drain 15 6 Wash 300 7 Drain 20 8
Wash 200 9 Drain 20 10 Couple 11 Couple 12 Wait 150 13 Drain 10 14
Wash 200 15 Drain 15 16 Cap 17 Wait 40 18 Drain 10 19 Wash 200 20
Drain 15 21 Oxidize 40 22 Wait 10 23 Drain 300 24 Wash 20 25 Drain
200 26 Wash 20 27 Drain
[0080] A configuration file was generated which contained the
operating characteristics of the machine, the liquid flow rates,
the concentration and molar equivalents used for each of the
reagents, as shown in Table 3 below.
3TABLE 3 reagent concentration flow rate molar excess acetonitrile
220.lambda./sec trichloroacetic- 125.lambda./sec methylene chloride
acid tetrazole 0.45 M 210.lambda./sec 250 acetic anhydride
210.lambda./sec 500 N-methylimidazole 210.lambda./sec 500 iodine
210.lambda. sec 150 phosphoramidite 0.1 M 210.lambda. sec
[0081] 96 well plates were used which have a conical lower section
which has been recessed to receive a pressed-fit 0.196 inch filter
disk. Below the filter was the flow restriction and capillary
liquid seal orifice, 0.020 inch diameter.times.0.350 inch length.
The outside diameter of the capillary seal at the exit tip was
0.035 inch. When fitted with Whatman GF/A filters, the gas flow
rate through a completely dry plate at an upper chamber pressure of
1 psi was 10 liters per minute. At 0.02 psi upper chamber pressure,
the capillary seal at the bottom of the synthesis well supported an
acetonitrile liquid head of approximately 1 cm above the filter
before slow leakage commenced. The plates were fabricated of molded
polypropylene.
[0082] To each well was added the correct amount of CPG with
derivatized first base, either A, T, C, or G. For example, 0.5 mg
CPG was used in a 20 nanomole scale reaction. The CPG beads were
added as a slurry in dibromomethane-chloroform pipetted into each
well. This step was performed in ordinary lab atmosphere prior to
loading the plate into the machine. After loading the plate into
the machine, a WASH acetonitrile cycle was done to rinse the
slurry, and to verify that all wells were draining properly.
[0083] The nozzle valve reservoirs were flushed with argon and then
filled with fresh reagents from the supply bottles by pressurized
transfer to avoid air and water contamination. The valve reservoirs
were then pressurized to the configuration pressure (4 psi). The
nozzle tubing was then primed by dispensing a small amount of
liquid with the sliding plate in the purge position for each
reagent.
[0084] During reagent addition and reaction wait periods, the
laminar gas sweep flow across the reaction chamber was regulated to
0.4 liter per minutes with an internal pressure of approximately
0.02 psi. During spent reagent removal and washing cycles, the
chamber pressure was increased to a maximum of 5 psi. At 2.5 psi,
the synthesis wells drained at a rate of approximately 150
.lambda./sec.
[0085] The machine was then switched into AUTOmatic mode in order
to commence actual synthesis. The machine paused during the first,
second and last cycles in order to allow collection of the DEBLOCK
trityl products for calorimetric analysis. This was accomplished by
sliding a second 96-well microtiter plate into the lower
vacuum/drain chamber, immediately beneath the capillary exit tips
of the synthesis plate. The optical density of detritylation
solution at 490 nanometers was read using a plate reader. The value
for the first cycle confirmed the amount of CPG loaded into a well,
and comparison of the second cycle with the last cycle permitted
calculation of the synthesis coupling efficiency. The final cycle
value also indicated the amount of oligonucleotide which will be
obtained after sidechain deprotection by ammonia cleavage.
[0086] After the machine completed the synthesis in all the wells,
the synthesis plate was removed from the machine. The synthesis
plate was stacked on top of a second 96 well deprotection plate
(Beckman deep-well titer plate 267001). The oligonucleotides were
cleaved from the support by pipetting 200 microliters of
concentrated NH.sub.4OH into each well followed by incubation at
25.degree. C. for 15 minutes. The ammonium cleavage solutions were
eluted through the filter and capillary exit into the wells of the
deprotection plate by pressure, vacuum or preferably brief
centrifugation of the stack. This cleavage step was repeated twice
with fresh aliquots of ammonia.
[0087] If the phosphoramidites used for synthesis had benzoyl and
isobutyryl protecting groups, the final deprotection was completed
by heating the crude ammonia cleavage product. The wells of the
deprotection titer plate were sealed with a dimpled silicon rubber
cap (Beckman 267002), and the caps held in place using a
spring-loaded plate press. The sealed apparatus was heated in an
air oven or water bath at 55.degree. C. for 8-15 hours, and then
cooled in an ice bath. The deprotection plate was removed from the
press and centrifuged briefly prior to removing the cap.
[0088] The ammonia solution was then evaporated to dryness using a
Savant 210 Speed Vac equipped with a microplate rotor. The
oligonucleotide pellets thus obtained were generally sufficiently
pure for use directly in most sequencing primer and PCR
applications. The amount of oligonucleotide predicted by the last
trityl assay was verified by optical density at 260 nanometers, and
the homogeneity of the product assayed by HPLC or capillary gel
electrophoresis. Capillary electrophoresis analysis was performed
on an Applied Biosystems 270A instrument according to the
manufacturer's protocois for oligonucleotides 14 and 15 from the
above run are shown in FIGS. 8A and 8B, respectively. Typical
yields for a 20 nanomolar scale synthesis of a 20-mer were 2.5 OD
(85 micrograms).
* * * * *