U.S. patent application number 10/387969 was filed with the patent office on 2003-09-18 for printing molecular library arrays.
This patent application is currently assigned to Affymetrix, Inc.. Invention is credited to Fodor, Stephen P.A., Goldberg, Martin. J., Goss, Virginia, McGall, Glenn, Pease, R. Fabian, Rava, Richard P., Stryer, Lubert, Winkler, James L..
Application Number | 20030175409 10/387969 |
Document ID | / |
Family ID | 23563722 |
Filed Date | 2003-09-18 |
United States Patent
Application |
20030175409 |
Kind Code |
A1 |
Pease, R. Fabian ; et
al. |
September 18, 2003 |
Printing molecular library arrays
Abstract
A method and apparatus for selectively applying a print material
onto a substrate for the synthesis of an array of oligonucleotides
at selected regions of a substrate. The print material includes a
barrier material, a monomer sequence, a nucleoside, a deprotection
agent, a carrier material, among other materials. The method and
apparatus also relies upon standard DMT based chemistry, and a
vapor phase deprotection agent such as solid TCA and the like.
Inventors: |
Pease, R. Fabian; (Stanford,
CA) ; McGall, Glenn; (San Jose, CA) ;
Goldberg, Martin. J.; (Saratoga, CA) ; Rava, Richard
P.; (Redwood City, CA) ; Fodor, Stephen P.A.;
(Palo Alto, CA) ; Goss, Virginia; (Santa Barbara,
CA) ; Stryer, Lubert; (Stanford, CA) ;
Winkler, James L.; (San Diego, CA) |
Correspondence
Address: |
BANNER & WITCOFF, LTD.
28 STATE STREET
28th FLOOR
BOSTON
MA
02109-9601
US
|
Assignee: |
Affymetrix, Inc.
Santa Clara
CA
|
Family ID: |
23563722 |
Appl. No.: |
10/387969 |
Filed: |
March 13, 2003 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
10387969 |
Mar 13, 2003 |
|
|
|
09841405 |
Apr 24, 2001 |
|
|
|
09841405 |
Apr 24, 2001 |
|
|
|
09427850 |
Oct 26, 1999 |
|
|
|
6239273 |
|
|
|
|
09427850 |
Oct 26, 1999 |
|
|
|
09093843 |
May 22, 1998 |
|
|
|
09093843 |
May 22, 1998 |
|
|
|
08635272 |
Apr 19, 1996 |
|
|
|
5831070 |
|
|
|
|
08635272 |
Apr 19, 1996 |
|
|
|
08395604 |
Feb 27, 1995 |
|
|
|
5599695 |
|
|
|
|
Current U.S.
Class: |
427/2.11 ;
536/25.3 |
Current CPC
Class: |
B01J 2219/00382
20130101; B01J 2219/00454 20130101; B01J 2219/00626 20130101; B01J
2219/00378 20130101; B41M 3/00 20130101; C40B 60/14 20130101; B01J
2219/0061 20130101; C07H 21/00 20130101; B01J 2219/00722 20130101;
B01J 2219/00427 20130101; B01J 2219/00621 20130101; B01J 2219/00637
20130101; B01J 2219/00596 20130101; B01J 2219/00585 20130101; G03F
7/00 20130101; B01J 2219/00639 20130101; B01J 2219/0059 20130101;
B01J 2219/00612 20130101; B01J 2219/00529 20130101; B01J 2219/00659
20130101; B01J 2219/00605 20130101; C40B 40/06 20130101; B01J
2219/00608 20130101; C40B 50/14 20130101; B01J 19/0046 20130101;
B01J 2219/00385 20130101; B01J 2219/00527 20130101 |
Class at
Publication: |
427/2.11 ;
536/25.3 |
International
Class: |
B05D 003/00; C07H
021/04 |
Claims
What is claimed is:
1. A method of forming polymers having diverse monomer sequences on
a substrate, said method comprising: providing a substrate
comprising a linker molecule layer thereon, said linker molecule
layer comprising a linker molecule and a protective group; applying
a barrier layer overlying said linker molecule layer, said applying
step forming selected exposed regions of said linker molecule
layer; and exposing said selected exposed regions of said linker
molecule layer to a vapor comprising a deprotecting agent.
2. The method of claim 1 wherein said deprotection agent is an
acidic vapor selected from a group consisting of TCA, DCA, and
HCl.
3. The method of claim 1 wherein said deprotection agent is at a
temperature ranging from about 20.degree. C. to about 50.degree.
C.
4. The method of claim 1 wherein said applying step is selected
from a group consisting of relief press printing, letter press
printing, gravure printing, intaglio printing, stencil printing,
and lithography.
5. The method of claim 1 wherein said barrier layer is selected
from a group consisting of a lacquer, an epoxy, an oil, a
polyester, and a polyurethane.
6. The method of claim 1 wherein said barrier layer is a
liquid.
7. The method of claim 1 wherein said deprotection agent comprises
a carrier gas.
8. The method of claim 1 wherein said deprotection agent comprises
a water vapor.
9. A method of deprotecting selected regions of a substrate, said
method comprising: providing a substrate comprising a layer of
linker molecules thereon, each of said linker molecules having a
protective group; applying a vapor deprotection agent to selected
regions of said linker molecule layer.
10. The method of claim 9 wherein said deprotection agent is
selected from a group consisting of TCA, DCA, and HCl.
11. The method of claim 9 wherein said deprotection agent is at a
temperature ranging from about 20.degree. C. to about 50.degree.
C.
12. The method of claim 9 wherein said applying step occurs through
forced convention.
13. The method of claim 9 wherein said deprotection agent comprises
a carrier gas.
14. The method of claim 9 wherein said deprotection agent comprises
a water vapor.
15. A method of applying a medium in selected regions of a
substrate, said method comprising the steps of: providing a
partially completed substrate comprising an array of monomers, said
partially completed substrate having a top surface; selectively
applying a medium comprising an element selected from a group
consisting of a barrier material, a receptor, a deprotection agent,
a monomer group, a carrier material, and an activator to selected
regions of said substrate top surface.
16. The method of claim 15 wherein said medium is selected from a
group consisting of TCA, DCA, HCL, and any other acidic vapor.
17. The method of claim 15 wherein said medium is at a temperature
ranging from about 20.degree. C. to about 50.degree. C.
18. The method of claim 15 wherein said medium is a deprotection
agent.
19. The method of claim 18 wherein said deprotection agent
comprises a carrier gas.
20. The method of claim 18 wherein said deprotection agent
comprises a water vapor.
21. The method of claim 15 wherein said selectively applying step
is selected from a group consisting of relief press printing,
letter press printing, gravure printing, intaglio printing, and
stencil printing.
22. The method of claim 15 wherein said selectively applying step
occurs through a drop-on-demand printhead.
23. A method of synthesizing an oligonucleotide comprising the
steps of: coupling a first portion of said oligonucleotide to said
substrate, said first portion of said oligonucleotide comprising a
removable protecting group; removing said protecting group with a
vapor phase deprotection agent to expose a functional group on said
first portion of said oligonucleotide; and covalently bonding a
second portion of said oligonucleotide to said first portion of
said oligonucleotide.
24. The method as recited in claim 23 wherein said surface of said
substrate is selectively protected by a mask during said removing
step.
25. The method as recited in claim 24 further comprising repeating
said removing and covalently bonding steps to form an array of
oligonucleotides.
26. The method of claim 24 wherein said vapor phase deprotection
agent is selected from a group consisting of TCA, DCA, and HCl.
27. The method of claim 24 wherein said mask is selected from a
group consisting of an epoxy, a silicone oil, a metal, a silicon
material, a lacquer, a oil, and a polyester.
28. The method of claim 24 wherein said mask is held in place by
electrostatic force.
Description
BACKGROUND OF THE INVENTION
[0001] The present invention relates to the synthesis and placement
of materials at known locations. In particular, one embodiment of
the invention provides a method and associated apparatus for the
selective application of an array of oligonucleotides on a
substrate by way of standard dimethoxytrityl (DMT) based chemistry.
The invention may be applied in the field of preparation of an
oligomer, a peptide, a nucleic acid, an oligosaccharide, a
phospholipid, a polymer, or a drug congener preparation, especially
to create sources of chemical diversity for use in screening for
biological activity.
[0002] Industry utilizes or has proposed various techniques to
synthesize arrays of oligonucleotides. One such technique is the
use of small rubber tubes as reaction chambers to make up a single
dimensional array by the sequential addition of reagents. This
technique has advantages by the use of standard DMT based
chemistry. However, a limitation with resolution often exists with
such technique. Typically the smallest cell size is about 1
millimeter in dimension. This method also does not enable the
synthesis of a sufficiently large number of polymer sequences for
effective economical screening. A further limitation is an
inability to form an array of, for example, oligonucleotides at
selected regions of a substrate.
[0003] Other representative techniques are described in U.S. Pat.
No. 5,143,854 and WO93/09668 which is hereby incorporated by
reference for all purposes. Such techniques are finding wide use
and are considered pioneering in the industry. In some
applications, however, it is desirable to have alternative
techniques and chemistries for synthesis of compound libraries.
[0004] It would be desirable to have a method and apparatus for
making high density arrays of oligonucleotides using DMT-based
chemistry and other suitable oligonucleotide synthesis chemistries,
as is a method and apparatus for conventional phosphoramidite-based
synthesis of a spatially defined array of oligomers (e.g.,
polynucleotides, polypeptides, oligosaccharides, and the like) each
having a substantially predetermined sequence of residues (i.e.,
polymerized monomer units).
SUMMARY OF THE INVENTION
[0005] According to the present invention, a method and apparatus
to form an array of polymers, such as oligonucleotides and related
polymers (e.g., peptide nucleic acids) at selected regions of a
substrate using conventional linkage chemistries (e.g., standard
DMT-based oligonucleotide synthesis chemistry) is provided. The
method and apparatus includes use of selected printing techniques
in distributing materials such as barrier materials, deprotection
agents, base groups, nucleosides, nucleotides, nucleotide analogs,
amino acids, imino acids, carrier materials, and the like to
selected regions of a substrate. Each of the printing techniques
may be used in some embodiments with, for example, standard
DMT-based chemistry for synthesis of oligonucleotides, and in
particular selected deprotecting agents in vapor form.
[0006] In a specific embodiment, the present invention provides a
method of forming polymers having diverse monomer sequences on a
substrate. In an embodiment, the method is used to synthesize
oligonucleotides having predetermined polynucleotide sequence(s) on
a solid substrate, typically in the form of a spatially defined
array, wherein the sequence(s) of an oligonucleotide is
positionally determined. The present method includes steps of
providing a substrate with a linker molecule layer thereon. The
linker molecule layer has a linker molecule and a protective group.
The present method also includes a step of applying a barrier layer
overlying at least a portion of the linker molecule layer. The
barrier layer shields the underlying portion from contact with a
reagent capable of otherwise reacting with the underlying portion
and applied subsequent to application of the barrier layer, thereby
substantially precluding a predetermined chemical reaction from
occurring on areas of the substrate overlaid with the barrier
material. The applying step forms selected exposed regions of the
linker molecule layer. A step of exposing the selected exposed
regions of the linker molecule layer (e.g., regions not overlaid
with the barrier material) to a reagent, typically in vapor phase,
and often comprising a deprotecting agent is also included.
[0007] In an alternative specific embodiment, the present method
includes a method of applying a medium in selected regions of a
substrate. The present method includes steps of providing a
substrate with a top surface, and selectively applying a medium
having an element selected from a group consisting of a barrier
material, a receptor, a deprotection agent, a monomer group, a
carrier material, and an activator to selected regions of the
substrate top surface.
[0008] In an embodiment, the invention provides a method for
synthesizing a spatial array of polymers of diverse monomeric
sequence (e.g., such as a collection of oligonucleotides having
unique sequences), wherein the composition (e.g., nucleotide
sequence) of each polymer is positionally defined by its location
in the spatial array. In general, the method employs a masking step
whereby a spatially distributed barrier material is applied to a
substrate to block at least one step of a monomer addition cycle
from occurring on a portion of the substrate overlaid by the
barrier material. The method comprises applying a barrier material
to a first spatially defined portion of a substrate, said substrate
optionally also comprising a layer of linker molecules and/or
nascent polymers (e.g., nascent oligonucleotides), whereby the
barrier material overlaying said first spatially defined portion of
said substrate shields the underlying portion from contact with a
subsequently applied reagent capable of otherwise reacting with the
underlying portion and necessary for a complete monomer addition
cycle whereby a monomer unit is covalently linked to a nascent
polymer or linker, thereby substantially precluding a chemical
reaction from occurring on said first spatially defined portion
which is overlaid with the barrier material and providing a
remaining unshielded portion of said substrate (i.e., portion(s)
not overlaid with the barrier material) available for contacting
said subsequently applied reagent and undergoing said chemical
reaction necessary for a complete monomer addition cycle (i.e.,
polymer elongation). The subsequently applied reagent is typically
a monomer (e.g., nucleotide, nucleoside, nucleoside derivative,
amino acid, and the like), a deprotecting agent for removing
protecting group(s) which block polymer elongation (e.g., removal
of DMT groups by acid hydrolysis), a coupling agent (e.g.,
phosphoramidites, such as cyanoethyl phosphoramidite nucleosides),
a capping agent (e.g., acetic anhydride and 1-methylimidazole),
and/or an oxidation agent (e.g., iodine; such as in
iodine:water:pyridine:tetrahydrofuran mixture). The method further
provides that, subsequent to the application of the barrier
material, the reagent(s) is/are applied and permitted to chemically
react with the unshielded portion of the substrate for a suitable
time period and under suitable reaction conditions. Following
reaction of the unshielded portion with the reagent(s), monomer
addition is completed and the barrier material is removed (not
necessarily in that order), resulting in a monomer addition to
polymer(s) in the unshielded portion of the substrate and
substantial lack of monomer addition to polymer(s) in the shielded
portion of the substrate, during said monomer addition cycle.
[0009] In an embodiment, the masking step, wherein,a barrier
material is applied to a spatially defined portion of the substrate
and used to shield said spatially defined portion to block a
monomer addition cycle on said spatially defined portion, is
employed repetitively. A first barrier mask is applied to overlay a
first spatially defined portion of a substrate creating: (1) a
first shielded portion overlain by said barrier mask, and (2) a
first unshielded portion comprising the portion of the substrate
not overlain by said barrier mask. The application of the first
barrier mask is followed by completion of a first monomer addition
cycle, whereby a monomer unit is covalently added to the first
unshielded portion to extend or initiate a nascent polymer bound to
said substrate, typically covalently, and whereby said first
monomer addition cycle substantially fails to result in addition of
a monomer unit to nascent polymers in the first shielded portion.
The first barrier mask is removed, concomitant with, prior to, or
subsequent to the completion of said first monomer addition cycle,
and one or more subsequent cycles of applying a subsequent barrier
mask, which may overlay subsequent shielded portions which is/are
spatially distinct from said first shielded portion, and performing
at least one subsequent monomer addition cycle(s) followed after
each cycle by barrier removal, and optionally, reapplication of a
barrier mask and initiation of a further monomer addition cycle
until polymers of a predetermined length (number of incorporated
monomer units) are produced.
[0010] In an aspect of the invention, a repetitive
masking/synthesis process can be comprised of the following
steps:
[0011] (1) application of barrier material to substrate having a
reactive surface capable of covalently bonding to a monomer unit or
reacting with a deprotecting agent or other reagent necessary for
completion of a monomer addition cycle, said reactive surface being
derivatived with a linker and/or a monomer unit or nascent polymer
(e.g., a 3'-linked nucleoside or 3'-linked polynucleotide), wherein
said barrier material covers a portion of said reactive surface
creating a covered portion, said covered portion being a shielded
portion and being substantially incapable of reacting with a
monomer unit or reagent necessary for completion of a monomer
addition cycle, and the remaining portion of the substrate being an
unshielded portion capable of reacting with a monomer unit or
reagent necessary for completion of a monomer addition cycle;
[0012] (2) contacting the substrate with reagents necessary for
completion of a monomer addition cycle, wherein a monomer unit is
covalently attached to the reactive surface of the substrate (e.g.,
a linker, a 3'-linked nucleoside, or 3'-linked nascent
polynucleotide) in an unshielded portion;
[0013] (3) removing the barrier material; and
[0014] (4) repeating steps 1, 2, and 3 from 0 to 5000 cycles,
preferably from 2 to 250 cycle, more usually from 4 to 100 cycles,
and typically from about 7 to 50 cycles, until a predetermined
polymer length is produced on a portion of the substrate. The
pattern of barrier material applied in each cycle may be different
that the prior or subsequent cycle(s), if any, or may be the same.
Often, in step (2), at least one reagent necessary for completion
of a monomer addition cycle is applied in vapor phase.
[0015] In an embodiment of the invention is provided a substrate
having a spatial array of polymers of predetermined length produced
by the method described supra.
[0016] In one aspect of the invention is provided a method for
applying a barrier material or reagent necessary for a monomer
addition cycle to a substrate, said method comprising transferring
the barrier material or reagent as a charged droplet by
electrostatic interaction, such as, for example, in an inkjet or
bubble jet print head or similar device. In an embodiment, the
barrier material or reagent is suitable for use in polynucleotide
(oligonucleotide) synthesis. In an embodiment, the substrate is a
silicon or glass substrate or a charged membrane (e.g., nylon 66 or
nitrocellulose).
[0017] An aspect of the invention provides a method for
synthesizing polynucleotides on a substrate, said method comprising
application of at least one reagent necessary for addition of a
nucleotide to a nascent polynucleotide or linker molecule bound to
a substrate, wherein said application is performed with the reagent
present substantially in vapor phase.
[0018] A further understanding of the nature and advantages of the
present invention may be realized by reference to the latter
portions of the specification and attached drawings.
BRIEF DESCRIPTION OF THE DRAWINGS
[0019] FIGS. 1-3 illustrate simplified cross-sectional views of a
substrate being processed according to the present invention;
[0020] FIGS. 4-13 illustrate selected printing techniques according
to the present invention;
[0021] FIG. 14 illustrates a simplified cross-sectional view of an
apparatus used to achieve local selectivity;
[0022] FIG. 15 illustrates a jig used for contacting a mask to a
substrate without smearing;
[0023] FIG. 16 is a photograph of a fluorescent image of a
fluoreprimed workpiece that was selectively shielded from liquid
deprotection by a lacquer;
[0024] FIG. 17 is a photograph of dots of uncured epoxy and pump
oil overlying a workpiece;
[0025] FIG. 18 illustrates a SEM photograph of a liquid uncured
epoxy pattern on a glass workpiece;
[0026] FIG. 19 illustrates a SEM photograph of a 100 micron
resolution sample with an epoxy barrier pattern;
[0027] FIG. 20 illustrates a SEM photograph of a 75 micron
resolution sample with an epoxy barrier pattern;
[0028] FIG. 21 is a photograph of a fluorescent.pattern from vapor
deprotection through an uncoated silicon stencil mask;
[0029] FIG. 22 is a close-up version of the photograph of FIG.
21;
[0030] FIG. 23 is a photograph of an epoxy paint pattern
transferred from a nickel grid;
[0031] FIGS. 24 and 25 are photographs of fluorescent images
resulting from vapor phase deprotection through an epoxy
pattern;
[0032] FIG. 26 illustrates a 2.times.2 array of oligonucleotides
formed by masking out deprotection agents after A (vertical mask)
and a first T in the synthesis of 3'-CGCATTCCG;
[0033] FIG. 27 is a scanned output of an array after hybridizing
with 10 nM target oligonucleotide 5'-GCGTAGGC-fluorescein for 15
minutes at 15 C;
[0034] FIGS. 28 and 29 are scanned outputs after hybridizing to a
newly-made sample of the same target sequence of FIGS. 26 and
27;
[0035] FIG. 30 is an array of same oligos as in FIGS. 26 and 27
made by displacing the reaction chamber when added bases A and the
first T in the sequence 3'-CGCATTCCG;
[0036] FIGS. 31 and 32 illustrate scanned outputs after hybridizing
with 10 nM 5'-GCGTAGGC-fluorescein.
DESCRIPTION OF THE SPECIFIC EMBODIMENT
[0037] Glossary
[0038] The following terms are intended to have the following
general meanings as they are used herein:
[0039] 1. Ligand: A ligand is a molecule that is recognized by a
particular receptor. Examples of ligand that can be investigated by
this invention include, but are not restricted to, agonists and
antagonists for cell membrane receptors, toxins and venoms, viral
epitopes, hormones (e.g., opiates, steroids, etc.), hormone
receptors, peptides, enzymes, enzyme substrates, cofactors, drugs,
lectins, sugars, oligonucleotides, nucleic acids, oligosaccharides,
proteins, and monoclonal antibodies.
[0040] 2. Monomer: A member of the set of small molecules which are
or can be joined together to form a polymer. The set of monomers
includes but is not restricted to, for example, the set of common
L-amino acids, the set of D-amino acids, the set of synthetic
and/or natural amino acids, the set of nucleotides and the set of
pentoses and hexoses. The particular ordering of monomers within a
polymer is referred to herein as the "sequence" of the polymer. As
used herein, monomers refers to any member of a basis set for
synthesis of a polymer, which include for example and not
limitation, polynucleotides, polypeptides, and small molecules such
as benzodiazepines, .beta.-turn mimetics, and protoprostaglandins,
among others. For example, dimers of the 20 naturally occurring
L-amino acids form a basis set of 400 monomers for synthesis of
polypeptides. Different basis sets of monomers may be used at
successive steps in the synthesis of a polymer. Furthermore, each
of the sets may include protected members which are modified after
synthesis. The invention is described herein primarily with regard
to the preparation of molecules containing sequences of monomers
such as amino acids, but could readily be applied in the
preparation of other polymers. Such polymers include, for example,
both linear and cyclic polymers of nucleic acids, polysaccharides,
phospholipids, and peptides having either .alpha.-, .beta.-, OR
.omega.-amino acids, heteropolymers in which a known drug is
covalently bound to any of the above, polynucleotides,
polyurethanes, polyesters, polycarbonates, polyureas, polyamides,
polyethyleneimines, polyarylene sulfides, polysiloxanes,
polyimides, polyacetates, or other polymers which will be apparent
upon review of this disclosure. Such polymers are "diverse" when
polymers having different monomer sequences are formed at different
predefined regions of a substrate. Methods of cyclization and
polymer reversal of polymers are disclosed in application Ser. No.
07/796,727 filed Nov. 22, 1991 (now U.S. Pat. No. 5,242,974 issued
Sep. 7, 1993, entitled "POLYMER REVERSAL ON SOLID SURFACES,"
incorporated herein by reference for all purposes. One set of
polymers is polynucleotides and peptide nucleic acids.
[0041] 3. Peptide: A polymer in which the monomers are alpha amino
acids and which are joined together through amide bonds,
alternatively referred to as a polypeptide. In the context of this
specification it should be appreciated that the amino acids may be
the L-optical isomer or the D-optical isomer. Peptides are often
two or more amino acid monomers long, and often more than 20 amino
acid monomers long. Standard abbreviations for amino acids are used
(e.g., P for proline). These abbreviations are included in Stryer,
Biochemistry, Third Ed., 1988, which is incorporated herein by
reference for all purposes. Peptide analogs are commonly used in
the pharmaceutical industry as non-peptide drugs with properties
analogous to those of the template peptide. These types of
non-peptide compound are termed "peptide mimetics" or
"peptidomimetics" (Fauchere, J. (1986) Adv. Drug Res. 15: 29; Veber
and Freidinger (1985) TINS p.392; and Evans et al. (1987) J. Med.
Chem 30: 1229, which are incorporated herein by reference) and are
often developed with the aid of computerized molecular modeling.
Peptide mimetics that are structurally similar to therapeutically
useful peptides may be used to produce an equivalent therapeutic or
prophylactic effect. Generally, peptidomimetics have one or more
peptide linkages optionally replaced by a linkage selected from the
group consisting of: --CH.sub.2NH--, --CH.sub.2S--,
--CH.sub.2--CH.sub.2--, --CH.dbd.CH-- (cis and trans),
--COCH.sub.2--, --CH(OH)CH.sub.2--, and --CH.sub.2SO--, by methods
known in the art and further described in the following references:
Spatola, A. F. in "Chemistry and Biochemistry of Amino Acids,
Peptides, and Proteins," B. Weinstein, eds., Marcel Dekker, New
York, p. 267 (1983); Spatola, A. F., Vega Data (March 1983), Vol.
1, Issue 3, "Peptide Backbone Modifications" (general review);
Morley, J. S., Trends Pharm Sci (1980) pp. 463-468 (general
review); Hudson, D. et al., Int J Pept Prot Res (1979) 14:177-185
(--CH.sub.2NH--, CH.sub.2CH.sub.2--); Spatola, A. F. et al., Life
Sci (1986) 38:1243-1249 (--CH.sub.2--S); Hann, M. M., J Chem Soc
Perkin Trans I (1982) 307-314 (--CH--CH--, cis and trans);
Almquist, R. G. et al., J Med Chem (1980) 23:1392-1398
(--COCH.sub.2--); Jennings-White, C. et al., Tetrahedron Lett
(1982) 23:2533 (--COCH.sub.2--); Szelke, M. et al., European Appln.
EP 45665 (1982) CA: 97:39405 (1982) (--CH(OH)CH.sub.2--); Holladay,
M. W. et al., Tetrahedron Lett (1983) 24:4401-4404
(--C(OH)CH.sub.2--); and Hruby, V. J., Life Sci (1982) 31:189-199
(--CH.sub.2--S--); each of which is incorporated herein by
reference. A particularly preferred non-peptide linkage is
--CH.sub.2NH--. Such peptide mimetics may have significant
advantages over polypeptide embodiments, including, for example:
more economical production, greater chemical stability, enhanced
pharmacological properties (half-life, absorption, potency,
efficacy, etc.), altered specificity (e.g., a broad-spectrum of
biological activities), reduced antigenicity, and others.
Systematic substitution of one or more amino acids of a consensus
sequence with a D-amino acid of the same type (e.g., D-lysine in
place of L-lysine) may be used to generate more stable peptides. In
addition, constrained peptides (including cyclized peptides)
comprising a consensus sequence or a substantially identical
consensus sequence variation may be generated by methods known in
the art (Rizo and Gierasch (1992) Ann. Rev. Biochem. 61: 387,
incorporated herein by reference); for example, by adding internal
cysteine residues capable of forming intramolecular disulfide
bridges which cyclize the peptide.
[0042] 4. Receptor: A molecule that has an affinity for a given
ligand. Receptors may be naturally-occurring or manmade molecules.
Also, they can be employed in their unaltered state or as
aggregates with other species. Receptors may be attached,
covalently or noncovalently, to a binding member, either directly
or via a specific binding substance. Examples of receptors which
can be employed by this invention include, but are not restricted
to, antibodies, cell membrane receptors, monoclonal antibodies and
antisera reactive with specific antigenic determinants (such as on
viruses, cells or other materials), drugs, polynucleotides, nucleic
acids, peptides, cofactors, lectins, sugars, polysaccharides,
cells, cellular membranes, and organelles. Receptors are sometimes
referred to in the art as anti-ligands. As the term receptors is
used herein, no difference in meaning is intended. A "Ligand
Receptor Pair" is formed when two macromolecules have combined
through molecular recognition to form a complex. Specific examples
of receptors which can be investigated by this invention include
but are not restricted to:
[0043] a) Microorganism receptors: Determination of ligands which
bind to receptors, such as specific transport proteins or enzymes
essential to survival of microorganisms, is useful in a new class
of antibiotics. Of particular value would be antibiotics against
opportunistic fungi, protozoa, and those bacterial resistant to the
antibiotics in current use.
[0044] b) Enzymes: For instance, the binding site of enzymes such
as the enzymes responsible for cleaving neurotransmitters;
determination of ligands which bind to certain receptors to
modulate the action of the enzymes which cleave the different
neurotransmitters is useful in the development of drugs which can
be used in the treatment of disorders of neurotransmission.
[0045] c) Antibodies: For instance, the invention may be useful in
investigating the ligand-binding site on the antibody molecule
which combines with the epitope of an antigen of interest;
determining a sequence that mimics an antigenic epitope may lead to
the development of vaccines of which the immunogen is based on one
or more of such sequences or led to the development of related
diagnostic agents or compounds useful in therapeutic treatments
such as for auto-immune diseases (e.g., by blocking the binding of
the "self" antibodies).
[0046] d) Nucleic Acids: Sequences of nucleic acids may be
synthesized to establish DNA or RNA binding sequences.
Polynucleotides, which include oligonucleotides, are composed of
nucleotides, typically linked 5' to 3' by a phosphodiester bond or
phosphorothiolate bond or the like. The term "corresponds to" is
used herein to mean that a polynucleotide sequence is homologous
(i.e., is identical, not strictly evolutionarily related) to all or
a portion of a reference polynucleotide sequence, or that a
polypeptide sequence is identical to a reference polypeptide
sequence. In contradistinction, the term "complementary to" is used
herein to mean that the complementary sequence is homologous to all
or a portion of a reference polynucleotide sequence. For
illustration, the nucleotide sequence "TATAC" corresponds to a
reference sequence "TATAC" and is complementary to a reference
sequence "GTATA". Polynucleotides can include nucleotides having a
variety of bases, including but not limited to: adenine, thymine,
cytosine, guanine, uridine, inosine, deazaguanosine,
N.sup.2-dimethylguanosine, 7-methylguanosine, N.sup.6-.DELTA..sup.2
isopentenyl-2-methylthioadenosine, 2'-O-methyladenine,
2'-O-methylthymine, 2'-O-methylcytosine, 2'-O-methylguanine,
pseudouridine, dihydrouridine, 4-thiouridine, and the like.
[0047] e) Catalytic Polypeptides: Polymers, preferably
polypeptides, which are capable of promoting a chemical reaction
involving the conversion of one or more reactants to one or more
products. Such polypeptides generally include a binding site
specific for at least one reactant or reaction intermediate and an
active functionality proximate to the binding site, which
functionality is capable of chemically modifying the bound
reactant. Catalytic polypeptides and others are described in, for
example, PCT Publication No. WO 90/05746, WO 90/05749, and WO
90/05785, which are incorporated herein by reference for all
purposes.
[0048] f) Hormone Receptors: For instance, the receptors for
insulin and growth hormone. Determination of the ligands which bind
with high affinity to a receptor is useful in the development of,
for example, an oral replacement of the daily injections which
diabetics must take to relieve the symptoms of diabetes, and in the
other case, a replacement for the scarce human growth hormone which
can only be obtained from cadavers or by recombinant DNA
technology. Other examples are the vasoconstrictive hormone
receptors; determination of those ligands which bind to a receptor
may lead to the development of drugs to control blood pressure.
[0049] g) Opiate receptors: Determination of ligands which bind to
the opiate receptors in the brain is useful in the development of
less-addictive replacements for morphine and related drugs.
[0050] 5. Substrate: A material having a rigid or semi-rigid
surface. In many embodiments, at least one surface of the substrate
will be substantially flat, although in some embodiments it may be
desirable to physically separate synthesis regions for different
polymers with, for example, wells, raised regions, etched trenches,
or the like. According to other embodiments, small beads may. be
provided on the surface which may be released upon completion of
the synthesis. Often, the substrate is a silicon or glass surface,
or a charged membrane, such as nylon 66 or nitrocellulose.
[0051] 6. Protective Group: A material which is bound to a monomer
unit and which may be selectively removed therefrom to expose an
active site such as, in the specific example of an amino acid, an
amine group. In the specific example of a polynucleotide
synthesized via phosphoramidite chemistry, a protecting group can
be a trityl ether (DMT ether) group linked to a nucleotide via a
5'-hydroxyl position.
[0052] 7. Predefined Region: A predefined region is a localized
area on a substrate which is, was, or is intended to be used for
formation of a selected polymer and is otherwise referred to herein
in the alternative as a "selected" region or simply a "region." The
predefined region may have any convenient shape, e.g., circular,
rectangular, elliptical, wedge-shaped, etc. In some embodiments, a
predefined region and, therefore, the area upon which each distinct
polymer sequence is synthesized is smaller than about 1 cm.sup.2,
more preferably less than 1 mm.sup.2, still more preferably less
than 0.5 mm.sup.2, and in some embodiments about 0.125 to 0.5
mm.sup.2. In most preferred embodiments the regions have an area
less than about 10,000 .mu.m.sup.2 or, more preferably, less than
100 .mu.m.sup.2. Within these regions, the polymer synthesized
therein is preferably synthesized in a substantially pure form. A
shielded portion or unshielded portion can be a predefined
region.
[0053] 8. Substantially Pure: A polymer is considered to be
"substantially pure" within. a predefined region of a substrate
when it exhibits characteristics that distinguish it from other
predefined regions. Typically, purity will be measured in terms of
biological activity or function as a result of uniform sequence.
such characteristics will typically be measured by way of binding
with a selected ligand or receptor. Preferably the region is
sufficiently pure such that the predominant species in the
predefined region is the desired sequence. According to preferred
aspects of the invention, the polymer is 5% pure, more preferably
more than 10% pure, preferably more than 20% pure, and more
preferably more than 80% pure, more preferably more than 90% pure,
more preferably more than 95% pure, where purity for this purpose
refers to the ratio of the number of ligand molecules formed in a
predefined region having a desired sequence to the total number of
molecules formed in the predefined region.
[0054] 9. Monomer Addition Cycle: A monomer addition cycle is a
cycle comprising the chemical reactions necessary to produce
covalent attachment of a monomer to a nascent polymer or linker,
such as to elongate the polymer with the desired chemical bond
(e.g., 5'-3' phosphodiester bond, peptide bond, etc.). For example
and not to limit the invention, the following steps typically
comprise a monomer additon cycle in phosphoramidite-based
oligonucleotide synthesis: (1) deprotection, comprising removal of
the DMT group from a 5'-protected nucleoside (which may be part of
a nascent polynucleotide) wherein the 5'-hydroxyl is blocked by
covalent attachment of DMT, such deprotection is usually done with
a suitable deprotection agent (e.g., a protic acid: trichloroacetic
acid or dichloroacetic acid), and may include physical removal
(e.g., washing, such as with acetonitrile) of the removed
protecting group (e.g., the cleaved dimethyltrityl group), (2)
coupling, comprising reacting a phosphoramidite nucleoside(s),
often activated with tetrazole, with the deprotected nucleoside,
(3) optionally including capping, to truncate unreacted nucleosides
from further participation in subsequent monomer addition cycles,
such as by reaction with acetic anhydride and N-methylimidazole to
acetylate free 5'-hydroxyl groups, and (4) oxidation, such as by
iodine in tetrahydrofuran/water/pyridine, to convert the trivalent
phosphite triester linkage to a pentavalent phosphite triester,
which in turn can be converted to a phosphodiester via reaction
with ammonium hydroxide. Thus, with respect to phosphoramidite
synthesis of polynucleotides, the following reagents are typically
necessary for a complete monomer addition cycle: trichloroacetic
acid or dichloroacetic acid, a phosphoramidite nucleoside, an
oxidizing agent, such as iodine (e.g., iodine/water/THF/pyridine),
and optionally N-methylimidazole for capping.
[0055] 10. Specific hybridization is defined herein as the
formation of hybrids between a probe polynucleotide (e.g., a
polynucleotide of the invention which may include substitutions,
deletion, and/or additions) and a specific target polynucleotide
(e.g., an analyte polynucleotide) wherein the probe preferentially
hybridizes to the specific target polynucleotide and substantially
does not hybridize to polynucleotides consisting of sequences which
are not substantially identical to the target polynucleotide.
However, it will be recognized by those of skill that the minimum
length of a polynucleotide required for specific hybridization to a
target polynucleotide will depend on-several factors: G/C content,
positioning of mismatched bases (if any), degree of uniqueness of
the sequence as compared to the population of target
polynucleotides, and chemical nature of the polynucleotide (e.g.,
methylphosphonate backbone, phosphorothiolate, etc.), among
others.
[0056] General
[0057] The present invention provides for the use of a substrate
with a surface. In preferred embodiments, linker molecules are
provided on a surface of the substrate. The purpose of the linker
molecules, in certain embodiments, is to facilitate receptor
recognition of the synthesized polymers. In preferred embodiments,
the linker molecules each include a protection group. A layer of
barrier material may be applied to the surface of the substrate,
and in particular the linker molecule layer. The barrier material
is selectively applied by way of a variety of printing techniques
to form exposed regions. A step of deprotection by way of
deprotection agents may then be applied to the exposed regions.
Preferably, the deprotection step occurs with use of deprotection
agents in the vapor phase. This sequence of steps may be used for
the selected synthesis of an array of oligonucleotides.
[0058] The present invention also provides for use of selected
printing techniques to apply deprotection agents, barrier
materials, nucleosides, and the like for the synthesis of an array
of oligonucleotides. Preferably, the type of printing technique
should be able to transfer a sufficient volume of print material to
selected regions of the substrate in an easy, accurate, and cost
effective manner. Examples of various printing techniques for the
synthesis of for example an array of oligonucleotides are described
herein. Further examples of these embodiments of the present
invention may be applied to the synthesis of arrays of DNA as
explained by application Ser. No. 07/796,243 in the name of Winkler
et al., and U.S. Pat. No. 5,143,854 in the name of Pirrung et al.,
which are both hereby incorporated by reference for all
purposes.
[0059] Examples of suitable phosphoramidite synthesis methods are
described in the User Manual for Applied Biosystems Model 391, pp.
6-1 to 6-24, available from Applied Biosystems, 850 Lincoln Center
Dr., Foster City, Calif. 94404, and are generally known by those
skilled in the art.
[0060] Chemical synthesis of polypeptides is known in the art and
are described further in Merrifield, J. (1969) J. Am. Chem. Soc.
91: 501; Chaiken I. M. (1981) CRC Crit. Rev. Biochem. 11: 255;
Kaiser et al. (1989) Science 243: 187; Merrifield, B. (1986)
Science 232: 342; Kent, S. B. H. (1988) Ann. Rev. Biochem. 57: 957;
and Offord, R. E. (1980) Semisynthetic Proteins, Wiley Publishing,
which are incorporated herein by reference).
[0061] Once synthesized, polynucleotide arrays of the invention
have many art-recognized uses. For example and not limitation, the
synthesized sequences may be used as hybridization probes or PCR
amplimers to detect the presence of a specific DNA or mRNA, for
example to diagnose a disease characterized by the presence of an
elevated mRNA level in cells, to identify a disease allele, or to
perform tissue typing (i.e., identify tissues characterized by the
expression of a particular mRNA), and the like. The sequences may
also be used for detecting genomic gene sequences in a DNA sample,
such as for forensic DNA analysis (e.g., by RFLP analysis, PCR
product length(s) distribution, etc.) or for diagnosis of diseases
characterized by amplification and/or rearrangements of a
characteristic gene.
EMBODIMENTS OF THE PRESENT INVENTION
[0062] An embodiment of the present invention may be briefly
outlined by way of the following method.
[0063] 1. Provide a substrate.
[0064] 2. Optionally, form a layer of linker molecules on the
substrate.
[0065] 3. Mechanically apply a barrier pattern on the linker
molecules with exposed regions.
[0066] 4. Deprotect the linker molecules in the exposed regions
with standard DMT chemistry.
[0067] 5. Strip barrier pattern.
[0068] 6. Apply remaining synthesis steps.
[0069] This sequence of steps provides for an embodiment with use
of a barrier layer with standard DMT chemistry. This provides for a
desired selectivity, easy in synthesis, low costs, high contrast,
high resolution, among other features. Of course, this sequence of
steps is shown for illustrative purposes only, and should not limit
the scope of the appended claims herein.
[0070] An alternative embodiment of the present invention may be
briefly outlined by way of the following method.
[0071] 1. Provide a substrate.
[0072] 2. Optionally, form a layer of linker molecules on the
substrate.
[0073] 3. Selectively apply a print media by way of a printing
technique (not a photosensitive printing techniques) on the linker
molecules.
[0074] 4. Apply remaining synthesis steps.
[0075] This sequence of steps allows for the selective application
of a print medium onto a substrate by way of the various printing
techniques described herein. These printing techniques simply do
not use any exotic photosensitive type materials, although later
photosensitive steps can be combined with the teachings herein. In
preferred embodiments, deprotection agents may be introduced onto
the substrate in vapor form. Accordingly, the present invention
provides for the selective application of a variety of print media
onto a substrate without necessitating the use of conventional
photosensitive materials.
[0076] FIG. 1 illustrates one embodiment according to the present
method. A substrate 12 is shown in cross-section. The substrate may
be biological, nonbiological, organic, inorganic, or a combination
of any of these, existing as particles, strands, precipitates,
gels, sheets, tubing, spheres, containers, capillaries, pads,
slices, films, plates, slides, and the like. The substrate may have
any convenient shape, such as a disc, square, sphere, circle, etc.
The substrate is preferably flat but may take on a variety of
alternative surface configurations. For example, the substrate may
contain raised or depressed regions on which the synthesis takes
place. The substrate and its surface preferably form a rigid
support on which to carry out the reactions described herein. For
instance, the substrate may be a functionalized glass, Si, Ge,
GaAs, GaP, SiO.sub.2, SiN.sub.4, modified silicon, or any one of a
wide variety of gels or polymers such as (poly)tetrafluoroethylene,
polypropylene, polyethylene, (poly)vinylidenedifluoride,
poly-styrene, polycarbonate, or combinations thereof. Other
substrate materials will be readily apparent to those of skill in
the art upon review of this disclosure. In a preferred embodiment
the substrate is flat glass or single-crystal silicon with surface
relief features of less than 10 microns. In another preferred
embodiment, the substrate is a polypropylene material.
[0077] Surfaces on the solid substrate will usually, though not
always, be composed of the same material as the substrate. Thus,
the surface may be composed of any of a wide variety of materials,
for example, polymers, plastics, resins, polysaccharides, silica or
silica-based materials, carbon, metals, inorganic glasses,
membranes, or any of the above-listed substrate materials. In some
embodiments the surface may provide for the use of caged binding
members which are attached firmly to the surface of the substrate.
Preferably, the surface will contain reactive groups, which could
be carboxyl, amino, hydroxyl, or the like. Most preferably, the
surface will have surface Si-OH functionalities, such as are found
on silica surfaces. For synthesis of polynucleotides by
phosphoramidite chemistry, a linker consisting of
(--COCH2CH2CONHCH2CH2CH2-siloxane bond-glass substrate) may be used
to attach to a DMT-protected nucleoside via formation of a carboxyl
bond to the 3' hydroxyl of the nucleoside.
[0078] The substrate 12 includes a surface 14 with a layer of
linker (or spacer) molecules 16 thereon. The linker molecules are
preferably of sufficient length to permit polymers in a completed
substrate to interact freely with molecules exposed to the
substrate. The linker molecules should be about 4 to about 40 atoms
long to provide sufficient exposure. The linker molecules may be,
for example, aryl acetylene, ethylene glycol oligomers containing
2-10 monomer units, diamines, diacids, amino acids, among others,
and combinations thereof. Alternatively, the linkers may be the
same molecule type as that being synthesized (i.e., nascent
polymers), such as oligonucleotides or oligopeptides.
[0079] In a preferred embodiment, the linker molecules are PEG
linker. Of course, the type of linker molecules used depends upon
the particular application.
[0080] The linker molecules can be attached to the substrate via
carbon-carbon bonds using, for example,
(poly)trifluorochloroethylene surfaces, or preferably, by siloxane
bonds (using, for example, glass or silicon oxide surfaces).
Siloxane bonds with the surface of the substrate may be formed in
one embodiment via reactions of linker molecules bearing
trichlorosilyl groups. The linker molecules may optionally be
attached in an ordered array, i.e., as parts of the head groups. In
alternative embodiments, the linker molecules are absorbed to the
surface of the substrate.
[0081] The linker molecules or substrate itself and monomers used
herein are provided with a functional group to which is bound a
protective group. Preferably, the protective group is on the distal
or terminal end of the linker molecule opposite the substrate. The
protective group may be either a negative protective group (i.e.,
the protective group renders the linker molecules less reactive
with a monomer upon exposure) or a positive protective group (i.e.,
the protective group renders the linker molecules more reactive
with a monomer upon exposure). In the case of negative protective
groups an additional step of reactivation will be required. In some
embodiments, this will be done by heating.
[0082] In a subsequent step, the substrate 12 includes a barrier
pattern 18 with selected exposed regions 20 formed thereon. Each of
the exposed regions corresponds to an "opening" in the barrier
material where it is desirable to remove protecting groups from the
linker molecules. The protecting groups may be removed from the
linker molecules by immersion in the deprotecting solution.
Examples of the deprotecting solution include trichloroacetic acid,
hydrochloric acid, among others.
[0083] The barrier pattern can be made of any suitable material
capable of masking certain regions of the linker molecule layer to
protect such regions from subsequent processing. The barrier
pattern may include, for example, materials such as a lacquer, an
oil, a mask stencil, a silicone mask, an epoxy, a silicone oil, a
polyester, a silicon membrane mask, a liquid capable of providing a
barrier to protecting groups, a solid capable of providing a
barrier to protecting groups, among others, and combinations
thereof. The lacquer may include a lacquer such as Pactra 63-1 and
others, often having characteristics formulated to withstand hot
fuel. An epoxy may include any suitable epoxy type material such as
West 105 and others. Selected oils are a rotary pump oil such as
Mowioc MC110, a silicone oil such as Dow Corning 704, and others.
Polyester type materials may include TAP SB and the like, and
combinations thereof.
[0084] The barrier pattern is applied as a liquid or a vapor by a
variety of techniques. Examples of selected ways to apply the
barrier material include brush, spray techniques, selected printing
techniques, and others.
[0085] Selected printing techniques may be used for the application
of liquid barrier materials. The selected techniques of printing
include a relief or letter press (the oldest form), gravure or
intaglio, stencil printing, lithography, among others. FIGS. 4-17,
which will be described in more detail below, illustrate a variety
of printing techniques used in applying the barrier material. Some
of the techniques as applied in the printing industry were from
Printing Technology, J. Michael Adams, David D. Faux, Lloyd J.
Ricker (3d.Ed., Delmar, 1988), which is hereby incorporated by
reference for all purposes.
[0086] After optionally deprotecting the linker molecule layer, the
barrier material is then stripped by methods of wet chemical strip,
acetone, IPA, and others. The linker molecule layer is then washed
or otherwise contacted with a first monomer layer such as receptor
"A" in FIG. 3. The first monomer reacts with the activated
functional-groups of the linker molecules which have been
deprotected. FIG. 3 illustrates a simplified cross-sectional
diagram of the substrate 12, linker molecule layer 12, and monomer
layer "A." The sequence of steps illustrated by FIGS. 1-3 may be
repeated to achieve the desired sequence of monomers at selected
regions to form an array of oligonucleotides, peptides, other
polymers, and the like.
[0087] FIGS. 4-7 illustrate techniques of printing as relief or
letter press 50, gravure or intaglio 60, stencil printing 70, and
lithography 80, respectively. Relief printing 50 relies upon the
use of raised features 52 to transfer printing medium to a
substrate. As for Gravure or intaglio printing 60, it uses sunken
features 62 to apply the desired shape to a substrate. Stencil
printing 70 which includes screen printing, shadow masking, spray
painting, and others, occurs through mechanical openings 72 of a
stencil 74. Lithography is a form of printing regions 82 of a
surface that are chemically treated to selectively retain print
medium. Each of these techniques may be used for the application of
a barrier material, a carrier material, a deprotecting agent, or a
polymer unit pattern onto a substrate.
[0088] More recent forms of printing include xerography (which
includes laser printing), ink jet printing (or print medium jet
printing), and others. FIGS. 8-13 illustrate the more recent forms
of printing.
[0089] FIG. 8 illustrates a form of xerography printing 90.
Xerography printing is directed to printing by way of an electrical
charge pattern. Steps of xerography printing often include steps of
charging 91, exposure 92, development 93, transfer 94, fixing 95,
and cleaning 96.
[0090] FIGS. 9-10 illustrates two forms of printing known as ink
jet printing or in this case print medium jet printing. In this
type of printing, print medium is forced through an array of
orifices that is scanned across a workpiece, and is therefore
really a form or derivative of stencil printing. FIG. 9 illustrates
a continuous ink jet process 110. The continuous print medium jet
process includes a substrate 111, a catcher assembly 112, a recycle
113, a deflector ribbon 114, a charge plate 115, an orifice plate
116, among other elements. The print medium jet type printer may
deliver a pattern of selected print medium in a single pass. A
resolution of such printing technique can be as low as about 200
microns and less.
[0091] The printhead includes a substrate 121, a heater assembly
122, a barrier 123, print medium 124, a nozzle plate 125, and the
print medium jet 126. The heater assembly 122 may be formed from a
underlayer 127 overlying the substrate, a resistive heater element
128 overlying the underlayer, a conductor 129, and a overlying
layer of passivation 130. The drop-on-demand printhead has the
capability of delivering controlled amounts of fluids such as
barrier medium, carrier material, monomer units, and the like onto
the surface of a workpiece.
[0092] A simplified cross-sectional view of an offset rotary press
140 is illustrated by FIG. 11. The offset rotary press includes a
impression cylinder 141, a blanket cylinder 142, a plate cylinder
143, print medium rollers 144, a solvent roller 145, and a
substrate 146. The image is transferred (or offset) from the plate
cylinder to the blanket cylinder, which reverses the image. The
image is then passed to the press substrate (or sheet) as it moves
between the blanket cylinder and the impression cylinder.
[0093] Another form of offset printing such as gravure offset
printing 150 is illustrated by FIG. 12. Gravure offset printing
includes steps of print medium transfer 151, transfer 152, and
printing 153. A doctor blade 154 may be used to force medium 155
into grooves 158 during the print medium transfer step. The print
medium is then transferred onto a blanket cylinder 156, often
covered with a rubber blanket 157. The blanket cylinder then prints
the print medium onto a substrate. An example of gravure offset
printing is illustrated in Mikami et al., IEEE Transactions on
Electron Devices 41, 306, (March 1994), which is hereby
incorporated by reference for all purposes. Mikami et al. applies
offset printing for the manufacture of arrays of thin film
transistors for flat panel displays. By way of gravure offset
printing, features sizes down to about 30 micrometers have been
made.
[0094] Still a further printing technique 160 is illustrate by FIG.
13. The printing technique includes use of a plate cylinder 162, a
plurality of distribution rollers 164, a plurality of form rollers
166, a print medium fountain roller 168, a ductor roller 169, and
other elements. This type of technique provides for a more uniform
distribution of barrier medium, carrier medium, deprotecting agents
or polymer units (or conventional ink) in selected
applications.
[0095] In preferred embodiments, spatially selectivity and in
particular local selectivity is achieved by way of trapping liquid
under an in-contact stencil mask. It should be noted that local
selectivity refers to the process of forming the liquid barrier at
a selected or predefined region. A liquid may attach to solid
surfaces and pull them together with selected values of surface and
interfacial energies. The pull is characterized as a pressure P in
the following relationship:
P=T/R
[0096] where
[0097] P is the suction pressure between the two surfaces;
[0098] T is the surface tension of the liquid; and
[0099] R is the radius of curvature of the meniscus.
[0100] The relationship assumes a small contact angle, but not so
small as to cause liquid to creep along surfaces of either solid.
An example of this technique is illustrated in D. B. Tuckerman and
R. F. W. Pease, Paper presented at U.S./Japan VLSI Symposium,
Kaanapaali, Hi. 1983 (Tuckerman et al.), which is hereby
incorporated by reference for all purposes. Tuckerman et al.
discloses the use of DC 704 oil to attach integrated circuit chips
in this manner.
[0101] A suitable value for R often requires that the volume of
liquid just fills the gap between the solid surfaces of the barrier
and substrate. Too little liquid and the gap empties to drain
certain regions, thereby creating no deprotection at such regions.
Too much liquid corresponds to a larger R, and the reduction of the
attractive force P. Of course, the selected amount of liquid
depends upon the particular application.
[0102] FIG. 14 illustrates a simplified cross-sectional view of an
apparatus 170 used to achieve local selectivity. The apparatus
includes a glass workpiece 172, a stencil 174, an upper electrode
176, a mask mount (or lower electrode) 178, and other features. The
glass workpiece is positioned between the upper electrode and the
mask mount. A voltage such as one of about 20,000 volts is applied
to the upper electrode, while the mask mount (or lower electrode
includes a potential at about 0 volt. The difference in voltages
provides an electrostatic force for the attachment of the workpiece
to the stencil. An attractive pressure between the stencil and the
workpiece ranges from about 0.5 gm-force/cm.sup.2 to about 50
gm-force/cm.sup.2 and is preferably about 5 gm-force/cm.sup.2.
[0103] The attachment between the stencil and the workpiece allows
for deprotection agents to be introduced onto exposed regions 179
of the workpiece. For example, a deprotection agent 177 such as
vapor phase TCA passes over exposed regions of the workpiece,
thereby causing selective deprotection of such regions.
[0104] In a preferred embodiment, the apparatus includes a jig 180
for aligning a stencil 181 coated with a liquid barrier material
onto the workpiece 183, as illustrated by FIG. 15. The jig
substantially prevents the liquid barrier material from smearing
onto unselected regions of cells on the workpiece. As shown, the
jig includes a workpiece holder 184, a stencil mount 185, sliding
surfaces 186, among other features. The stencil mount includes a
gasket 187, a stencil holder 188, the stencil mask 181, a spacer
189, sliding surfaces 186, and other features.
[0105] To align the coated stencil onto the workpiece, the coated
stencil is firmly placed onto the stencil holder of the stencil
mount. The stencil mount then inserts into a cavity 191 of the
workpiece holder. The coated stencil firmly abuts the surface of
the workpiece. The tolerance between the sliding surfaces is about
1.0 micron to about 10.0 microns, and is preferably at about 2.5
microns. An attractive pressure between the stencil and the
workpiece ranges from about 0.8 gm-force/cm.sup.2 to about 50
gm-force/cm.sup.2 and is preferably about 5 gm-force/cm.sup.2. A
gasket made of a material such as teflon, polyisoprene, polymethyl
methacrylate, and others is located between the spacer and the
stencil mask mount. This gasket absorbs shock between the stencil
and workpiece to seal them together upon an applied pressure.
[0106] A further alternative embodiment provides a high resolution
stencil mask for ion beam proximity printing, ion beam projection
lithography, and the like. The high resolution stencil mask
includes, for example, a silicon membrane mask, an epoxy barrier
mask, and others. An other example of a high resolution stencil
mask may include electroformed nickel, among others. A high
resolution stencil mask includes feature sizes of about 0.5 micron
to about 50 microns, but less than about 100 microns. A high
resolution stencil mask also includes a thickness of about 2 to
about 20 microns, and is preferably about 5 microns and less. An
example of such mask is made by Nanostructures, Inc.
[0107] The high resolution stencil mask attaches to the workpiece
by the aforementioned techniques. For example, the stencil mask
directly attaches to the surface of the workpiece by placement.
Alternatively, the stencil mask may be attached to the workpiece
with an interfacial fluid. Further, the stencil mask attaches to
the workpiece with use of an electrostatic force, among other
forces. The apparatus of FIGS. 14 and 15 may also be used to attach
the high resolution stencil onto the workpiece. Of course, the type
of high resolution stencil mask depends upon the particular
application.
[0108] In an alternative embodiment, the present invention provides
for the use of vapor phase deprotection agents. The vapor phase
deprotection agents may be introduced at low pressure, atmospheric
pressure, among others. The use of such vapor phase deprotection
agents allows for ease in processing the work piece with linker
molecules and barrier material.
[0109] Deprotection may be carried out in a directional stream of
deprotection agent at low pressure. Low pressure deprotection
occurs by first removing the work piece with linker molecules and
barrier material from the synthesizer. The work piece is then
transferred into a vacuum chamber, preferably an extremely low
pressure vacuum chamber. The deprotection agent is then bled into
the vacuum chamber at a selected rate to promote directionality of
the deprotection agent stream. Preferably, the vacuum chamber
includes pressures ranging from about 10.sup.-5 torr to about 1
torr to promote the directionality of the stream. For example, the
deprotection agents may include either a 2% solution of TCA in DCP,
solid TCA, among others, and combinations thereof. By use of the
TCA/DCP solution or solid TCA, the vapor pressure ranges from about
0.1 millitorr to about 1 torr to promote a directional stream of
deprotection agents.
[0110] In a preferred embodiment, the deprotecting steps occur at
atmospheric pressure. At atmospheric pressure, the work piece is
held over a solution of deprotection agents at atmospheric
pressure, and preferably at room temperature. An example of a
deprotection agent includes TCA, DCP, and the like, and
combinations thereof. The TCA may be mixed with DCP to form a 2%
solution. Alternatively, the TCA may be used in pure form at room
temperature, or at a temperature ranging from about 10.degree. C.
to about 50.degree. C., and preferably at about 20.degree. C. The
work piece is held over the TCA type deprotection agents for about
1 minute or less. The TCA can also be blown against the workpiece
by way of forced convection and the like, and even mixed with a
water vapor, a carrier gas, or the like.
[0111] An advantage of the vapor phase deprotection agent, even at
atmospheric pressure, is the lack of mechanical action to disturb
any physical barrier pattern material. A further advantage with the
vapor phase deprotection agent is the ease in use with selected
work pieces.
EXAMPLES
[0112] 1. Use of Lacquer Barrier Material
[0113] An experiment was carried out with a standard 2 inch by 3
inch derivatized glass slide. An ABI Model 392 synthesizer was used
to apply PEG linker-CC-DMT (linker layer-2 polymer units-protecting
group) on one side of the glass slide. The glass slide, also known
as the work piece, was then removed from the reaction chamber of
the synthesizer for the application of a barrier material.
[0114] To find a suitable barrier material, patterns of candidate
materials were applied with a fine paint brush to the linker layer.
The fine paint brush produced features made of the barrier material
down to about 100 microns. The barrier material was allowed to dry
in air, typically at room temperature. The barrier material is
intended to provide an enclosure over selected regions of the
linker layer. The regions outside the selected regions were exposed
for further processing such as a step of deprotection and further
synthesis.
[0115] In this experiment, a lacquer known as Pactra 63-1 was found
to be an effective barrier material. A fine brush applied the
lacquer as dots from about 0.1 mm to about 1 mm in dimension to the
linker layer. The lacquer was dried for several hours at room
temperature, before subsequent processing.
[0116] A step of deprotection followed the application of the
barrier material. In this experiment, the workpiece was immersed
into a deprotecting solution to remove the protecting group from
the linker layer.
[0117] After deprotecting, the barrier material was stripped. In
this experiment, stripping occurred by the use of acetone, gently
wiped across the lacquer. The use of acetone in this technique did
not affect subsequent coupling processes.
[0118] After stripping, the workpiece was reinserted back into the
reaction chamber of the synthesizer. In the reaction chamber, the
workpiece was fluoreprime coupled. After removing the workpiece
from the reaction chamber, a solution of ethylene diamine/ammonia
was used to immerse the workpiece for about 5 to 10 minutes.
[0119] The workpiece was then placed into a confocal scanning
fluorescence microscope for inspection. The deprotected regions
fluoresced strongly, and the successfully shielded regions (or
selected regions) by the barrier material did not fluoresce. This
experiment shows the effectiveness of the lacquer barrier
material.
[0120] To achieve proper control over the experiment, the
experimental and control groups were synthesized and sampled as
shown in Table 1.
1TABLE 1 Sample and Control Groups for Barrier Material Formation
Experiment. # TYPE PROCESS RESULT 1 Control Apply barrier pattern,
strip, No and scan. fluorescence. 2 Control Apply barrier pattern,
strip, Complete fluoreprime, and scan, fluorescence. 3 Sample Apply
barrier pattern, strip, Selective deprotect, fluoreprime, and
scan.
[0121] FIG. 16 is a photograph of fluorescent image of the
fluoreprimed workpiece that was selectively shielded from liquid
deprotection by lacquer. The photograph 200 shows a workpiece 202
with exposed regions 204, and protected regions 205. As noted in
Table 1, the layer of barrier material which includes the lacquer
effectively shielded the protecting groups, from deprotection. The
contrast ratio between the exposed regions and the protected
regions 205 is about 20:1.
[0122] From this experiment, it is concluded that at least one type
of material such as the lacquer can serve as an effective barrier
material for complete deprotection and complete protection. The
lacquer barrier material is easily applied and dries at room
temperature. It is also concluded that at least one type of
material such as acetone effectively strips the lacquer from the
linker layer but does not affect the coupling process.
[0123] 2. Use of Vapor-Phase Deprotection
[0124] Vapor phase deprotection was carried out on a standard
workpiece. The workpiece was a standard 2 inch by 3 inch
derivatized glass slide. An ABI Model 392 synthesizer was used to
apply PEG linker-CC-DMT (linker layer-2 polymer units-protecting
group) on one side of the glass slide. The glass slide also known
as the work piece was fluoreprimed, as before. Experiments were
performed at a variety of selected barrier materials and
deprotection agent pressures as follows.
[0125] A. Low Pressure Deprotection
[0126] To prove the principles of low pressure deprotection, an
experiment was carried out were a deprotection agent is introduced
onto the fluoreprimed workpiece at a vacuum. In this experiment,
the workpiece was removed from the synthesizer, and inserted into a
vacuum chamber. The deprotection agent was bled in through a leak
valve at a selected pressure, as measured by vacuum gauges. The
vacuum gauges read pressures ranging from about 0.1 millitorr to
about 1 torr. At these pressures, the deprotection agent stream
should be substantially directional. Two forms of deprotection
agents were used in this experiment as follows.
2TABLE 2 Low Pressure Deprotection Experiments # PROCESS RESULT 1
Apply 2% solution of TCA in DCP at Deprotection pressures from
about 0.1 millitorr to partially about 1 torr. completed. 2 Apply
TCA from vapor of solid TCA at Deprotection pressures from about
0.1 millitorr to partially about 1 torr. completed.
[0127] From Table 2, it is clear that applying the deprotection
agents at low pressures provided for at least partial deprotection
from the deprotection agents. An advantage with the use of the
deprotection agents at low pressures is the directionality of the
stream of deprotection agents. By controlling the directionality of
the deprotection agents, mechanical masks such as stencil masks may
be used as a barrier for subsequent processing without being in
(intimate) contact with the workpiece.
[0128] B. Atmospheric Pressure Gas-Phase Deprotection
[0129] An experiment was also performed where the deprotection
agents were introduced onto the workpiece at atmospheric pressure.
The workpieces were prepared as in the previous experiment at low
pressure. A lacquer known as Pactra 63-1 was applied as dots from
about 0.1 mm to about 1 mm in dimension to the linker molecule
layer of each of the workpieces. The lacquer was dried for several
hours at room temperature, before additional processing. The
workpieces were then subjected to different deprotecting steps as
shown in Table 3.
3TABLE 3 Atmospheric Pressure Deprotection Experiments # PROCESS
RESULT 1 Hold workpiece over a test tube Complete protection under
containing a solution of about the lacquer dots and 2% TCA/DCP for
about 60 partial deprotection seconds. elsewhere. 2 Hold workpiece
over a vial Complete protection under containing solid TCA at about
the lacquer dots and 20.degree. C. for about 60 seconds. complete
deprotection elsewhere. 3 Hold workpiece over a vial Partial
protection under containing solid TCA heated to the lacquer dots
and about 70.degree. C. for about 60 complete deprotection seconds.
elsewhere. The lacquer dots did not hold up to the hot TCA, which
appeared to condense on the workpiece.
[0130] Each of the samples 1, 2, and 3 was fluoreprimed after
deprotection, and scanned to determine the effectiveness of the
deprotection agents. From the samples as noted in Table 3, the
process of sample 2 has results that appear to be the most
effective. Deprotection at room temperature (about 20.degree. C.)
with use of solid TCA provided complete protection under the
lacquer dots, and complete deprotection elsewhere, clearly
desirable results.
[0131] C. Atmospheric Pressure Deprotection with Liquid Barrier
Materials
[0132] Atmospheric deprotection was also carried out using a
variety of different barrier materials. These barrier materials
include an epoxy such as West 105 (an uncured epoxy resin), a
rotary pump oil such as Mowioc MC 110, a silicone oil such as Dow
Corning 704, and a polyester such as TAP SB. The workpieces were
made as before and the experiments were carried out with the solid
TCA deprotection agent at room temperature. Samples were held over
the solid TCA for a period of about 20 to about 60 seconds. All of
the liquids appeared to act as effective barriers, that is,
protection occurred underlying the liquid regions. The West 105
epoxy appeared to give the best results by having the crispest
edges, and was therefore chosen for later experiments as described
herein.
[0133] FIG. 17 is a photograph of dots of uncured epoxy and pump
oil overlying a workpiece. The photograph 300 shows a workpiece 302
with exposed regions 304, and protected regions 306, 308. As shown,
the protected regions include painted-on patches of uncured epoxy
306 (the patches on the left-side of the dotted line), and patches
of vacuum pump oil 308 (the patches on the right-side of the dotted
line). From the photograph, the patches of uncured epoxy appeared
to have crisper edges than the patches of vacuum pump oil. The
contrast ratio for the uncured epoxy was about 20:1.
[0134] D. Atmospheric Pressure Deprotection with Local
Selectivity
[0135] Experiments were performed with use of the apparatus of FIG.
14 and a liquid interface between a workpiece and a stencil mask.
The stencil mask was electroformed nickel, and coated uniformly
with a layer of liquid barrier material such as an epoxy or a
polyester. In this experiment, the liquid was prepared by
dissolving the West 105 epoxy material in acetone. A solution of
0.1 ml. epoxy in 10 ml. acetone was applied to glass slide via a
Sonotek ultrasonic spray nozzle. The glass slide was then spun at
about 3000 revolutions per minute such that the surface with
solution is normal to the axis of revolution. Of course, the
rotation speed and duration depends upon the desired film thickness
of remaining solution. The glass slide was then placed film side
down onto the stencil mask. An electrode at about 22 kV on a back
surface of the glass slide applies electrostatic force onto the
workpiece, typically at about an attractive pressure of about 200
Pa (or about 2.times.10.sup.-3 atmospheres or 2
grams-force/cm.sup.2). The glass slide is then removed from the
mask which retains a coating of barrier material.
[0136] Alignment of the coated stencil mask with workpiece occurred
with use of the jig apparatus of FIG. 15. The jig apparatus brought
the stencil mask in contact with the workpiece without smearing
more than a small fraction of a cell size, typically about 2.5
microns in this experiment. A mask using a 1,000 mesh/inch (25
micron pitch) grid was used as a stencil mask.
[0137] FIG. 18 illustrates a SEM photograph 400 at 1300 times
magnification of a liquid uncured epoxy pattern on a glass
workpiece (following removal from stencil mask). As shown, the
photograph includes a glass workpiece 402, a grid pattern of
uncured epoxy 403, and exposed regions 404. The grid pattern bars
are about 7 microns across. This photograph demonstrates the
accuracy of the aforementioned apparatus for the placement of an
epoxy barrier material onto a workpiece surface without
smearing.
[0138] FIG. 19 illustrates a SEM photograph 500 of a 100 micron
resolution sample. The photograph 500 shows a workpiece 502 with
exposed regions 504, and protected regions 506. The workpiece was
prepared as before. A stencil mask made of nickel was coated with
West 105 epoxy (an uncured epoxy resin) and electrostatically held
against the workpiece. Each of the spaces between the protected
regions was about 100 microns in length from inner edge to inner
edge. The contrast ratio between the exposed and protected regions
was about 200:1. This photograph clearly shows the effectiveness of
present apparatus and West 105 epoxy. It should be noted that the
arc shaped region 508 was caused by West 105 epoxy applied with a
fine paint brush.
[0139] FIG. 20 illustrates a SEM photograph of a 75 micron
resolution sample. The workpiece was prepared as before. The
photograph 600 shows a workpiece 602 with exposed regions 604, and
protected regions 606. The workpiece was prepared as before. A
stencil mask made of nickel was coated with West 105 epoxy (an
uncured epoxy resin) and electrostatically held against the
workpiece. Each of the spaces between the protected regions is
about 75 microns in length from inner edge to inner edge. The width
of each of the bars is about 25 microns. The contrast ratio between
the exposed and protected regions was less than that of FIG. 19. As
even narrower width bars were attached to the workpiece, the
contrast ratio between the exposed and protected regions decreased.
The photograph 600 shows a contrast ratio of about 4:1 to about
2:1.
[0140] E. Atmospheric Pressure Deprotection Silicon Membrane
Mask
[0141] An experiment was performed with high resolution silicon
stencil masks being fabricated for ion-beam proximity printing and
ion beam projection lithography. A mask available from
Nanostructures, Inc. with a membrane thickness of about 2 to 4
microns was used. The mask was mounted in openings of about 3
millimeter on an aluminum frame. The masks structure with frame was
electrostatically attached to the workpiece without use of an
interfacial fluid (because of risk of rupturing the fragile
membrane). The workpiece was made, as previously noted, in the
manner described above. A step of vapor phase deprotection occurred
on the workpiece with mask structure attached. The workpiece was
fluoreprimed and scanned, as the previous experiments. In this
experiment, the results were encouraging, as illustrated in the
photograph of FIGS. 21 and 22.
[0142] FIG. 21 is the photograph of fluorescent pattern from the
vapor deprotection through the uncoated silicon stencil mask. The
photograph 700 includes a workpiece 702 with exposed regions 704
and protected regions 706. As noted, the protected regions were
protected by way of the silicon mask. The fluorescent contrast
ratios exceeded 10:1 for features of about 50 microns, which are
clearly desirable results. No substantial "undercutting" (reaction
under the stencil) occurred, presumably due to partial separation
caused by particulate contamination.
[0143] Other regions also suffered from undercutting, as can be
seem by the relative sizes of the arrowed features 708 in the FIG.
22 photograph. The undercutting at such regions as defined by the
arrow were believed to be caused by particulate contamination
preventing good contact. Of course, this problem may be cured, at
least in part, by proper process controls, and the like.
[0144] In other experiments, less undercutting occurred for smaller
features such as about 5 microns and less. It is believed the
smaller dimensions due to the smaller features had better contact
between workpiece and mask structure. The smaller features are more
compliant so the electrostatic force between mask and workpiece
surface is more effective at maintaining good contact.
[0145] F. Atmospheric Pressure Deprotection with 105 Epoxy
Barrier
[0146] Experiments were also performed to test the masking strength
of 105 epoxy manufactured by West, against vapor phase
deprotection. The workpiece samples were prepared, as previously
noted, same as the previous experiments up to the deprotecting
step. A film of 105 epoxy was spin coated to a thickness of about
0.8+/-0.3 microns (about 1 milligram over about a 4.times.4
cm.sub.2 surface). Thin fragments of silicon were placed at a
region near the center of the field of view, and small marks were
scored manually nearby, to create an unmasked region. The regions
underlying the silicon film were completely masked, the scored
regions completely unmasked, and the exposed regions of epoxy film
were in question. After placement of such regions, the workpiece
was air dried in a desiccator for about 24 hours, before the vapor
phase deprotection step.
[0147] A vapor phase deprotection step was performed on the sample
workpiece described above. The sample was then scanned. The
fluorescent count rate for each of the regions are listed in Table
4 below.
4TABLE 4 Fluorescent Count Rate for 105 Epoxy Samples # REGION
FLUORESCENT COUNT RATE 1 Region of exposed 105 epoxy on about 390
to about 490 the workpiece. 2 Region of silicon fragments about 330
to about 390 covering the 105 epoxy. 3 Region of scored 105 epoxy
to about 1530 to about 1630 form unprotected area.
[0148] The Table 4 illustrates that thin film epoxy blocks at least
about 75% of the deprotecting action, for example. Further tests
may need to be performed to achieve higher contrasts, between
protected and unprotected regions.
[0149] Another experiment was performed using a fine grid pattern
of 105 epoxy applied to a workpiece. The workpiece was made similar
to the technique as described above up to the deprotecting step. A
fine, clear, epoxy grid pattern of 25 microns half-pitch was
applied to a center region of the workpiece. No epoxy was placed
around such center region. Additional widths (down to about 25
microns) of epoxy were applied with an ultra-fine paint brush.
[0150] After about 24 hours of air drying in a desiccator, the
workpiece under went a step of vapor phase deprotection. A scanned
fluorescent image was uniformly bright except for the center region
masked by the epoxy pattern. In the masked region, the grid pattern
was visible but contrast and brightness were lower. Signal strength
in the nominally clear regions of the grid pattern was
significantly lower than that from regions more than about 100
microns from the epoxy pattern. Accordingly, it appears as if the
West 105 material inhibits the deprotection within regions of about
100 microns of such material.
[0151] Other experiments were performed using finer marks of the
105 epoxy, an epoxy paint (TAP "Copon" Clear), a polyester resin
(TAP plastics), and a Dow Corning 704 silicone oil. At low
magnification the results were as before, that is, the fluorescent
image of the oil marks had poor acuity, but the polyester and epoxy
matter were fine. However, at high magnification (resolution at
about 100 microns and less) the 105 epoxy did have less sharp of an
edge than either the epoxy paid or the polyester marks. In
particular, the epoxy paint pattern showed the 1 micron pixel size
of the scanner, and was therefore determined the preferred barrier
material.
[0152] G. Atmospheric Pressure Deprotection With TAP "Copon" Clear
Paint
[0153] In the experiment with use of epoxy paint, printed patterns
of the TAP "Copon" clear paint were formed by painting on a
solution of 1:4 (paint:spray thinner) on to a 500 mesh/inch nickel
grid (Buckbee Mears). The grid with fresh paint was then
electrostatically attached to the workpiece, transferring the paint
pattern on the surface of the workpiece. The grid was then removed
from the surface of the workpiece, leaving the paint pattern
behind.
[0154] FIG. 23 is a photograph 800 of the paint pattern transferred
from the nickel grid. The photograph 800 includes a workpiece 802
with an epoxy paint pattern 804 and exposed regions 806. The
distance in each of the exposed regions is about 25 microns, and
the pattern includes bars, each having a width of about 20 microns.
This type of printing is an example of high resolution gravure
printing, as described above. The transferred paint pattern acted
as the barrier material during subsequent deprotecting steps. As
shown, the paint patterns were crisp (and fine lined) to create an
effective mask for printing a barrier pattern to obtain a diverse
array of oligonucleotides.
[0155] FIGS. 24 and 25 are photographs of fluorescent images
resulting from vapor phase deprotection through an epoxy pattern,
similar to the one shown by the photograph of FIG. 23. Both of the
photograph show good contrast ratios between the exposed and
protected regions. The photographs do not show any visible
proximity effect and the contrast ratio exceeds 10:1, clearly
desirable results.
[0156] 3. Hybridization Experiments
[0157] To demonstrate the effectiveness of the aforementioned
techniques on the synthesis of oligonucleotides, selected
experiments were performed. 2.times.2 arrays of oligonucleotides
were prepared on substrates 1002 using silicon fragments (pieces of
silicon material), which were electrostatically attached as crude
masks at base #4 (A) and #5 (T). FIG. 26 illustrates a 2.times.2
array 1000 of oligonucleotides formed by masking out the deprotect
agents after A (vertical mask 1009) and the first T in the
synthesis of 3'-CGCATTCCG 1004. A 10 nM target
5'-GCGTAGGC-fluorescein at 15 C was exposed in the flow cell to the
array, which was then scanned with a scanner. The four probes were
3'-CGCATCCG (match) 1005, 3'-CGCTCCG (deletion) 1006, 3'-CGCTTCCG
(substitution) 1007, and 3'-CGCATTCCG (addition) 1004.
[0158] FIG. 27 is a photograph of a representative scanned
fluorescent output, which shows the counts obtained in the four
areas. The matched area is the most strongly fluorescing,
indicating the strongest hybridization to the match and the weakest
to the deletion. The white spots appear most frequently in the
matching region and was a characteristic of all experiments using
this particular target. Another sample was hybridized with a more
freshly prepared target oligo and obtained better results (as in
FIG. 28) that were further enhanced by hybridizing at 10.degree. C.
for 15 minutes and then scanning at 15.degree. C. The counts in the
matching region 1005 were more than doubled with only a modest
increase in the unmatched regions as shown in FIG. 29.
[0159] Because the synthesis of the probes, with the exception of
the addition, involved at least one removal from the synthesizer
for masked deprotection, control experiments were performed forming
the other three probes as uninterrupted sequences on the reverse
side of three separate substrates. These sequences were hybridized
and scanned to check for any significant difference in counts
between those grown uninterruptedly on the reverse side with those
grown on the front using the masking. There were no significant
differences with those grown on the front using the masking. Hence
we conclude that one or two interruptions for masked vapor-phase
deprotection introduces no significant undesired perturbation of
the growth of the probes.
[0160] A further check was made on an array 2000 of four oligos of
FIG. 26) by moving the reaction chamber around the substrate at
bases 4 and 5 as shown by FIG. 31 to compare directly the behavior
of a (simple) array made with vapor-phase deprotection at one or
two bases with the conventional ABI chemistry throughout. The
results are shown in FIGS. 28 and 29. The results were similar to
those obtained with the array made with vapor-phase deprotection
including the enhanced selectivity and signal from the matching
area following hybridization at 10 C for 15 minutes. Thus no
detectable difference between the results obtained with vapor-phase
deprotection with those made conventionally were seen.
[0161] While the above is a full description of the specific
embodiments, various modifications, alternative constructions, and
equivalents may be used. For example, while the description above
is in terms of the synthesis of oligonucleotide arrays, it would
be. possible to implement the present invention with peptides,
small molecules, other polymers, or the like. Alternatively, the
embodiments may also be in context to peptides, other polymers, or
the like.
[0162] Therefore, the above description and illustrations should
not be taken as limiting the scope of the present invention which
is defined by the appended claims.
* * * * *