U.S. patent application number 11/015257 was filed with the patent office on 2006-07-20 for substrate preparation process.
This patent application is currently assigned to Affymetrix, INC.. Invention is credited to Martin Diggelman, Martin Goldberg, Earl Hubbell, Glenn McGall, MacDonald Morris, Nam Quoc Ngo, Richard P. Rava, Jennifer Tan, Mel Yamamoto.
Application Number | 20060160099 11/015257 |
Document ID | / |
Family ID | 31949801 |
Filed Date | 2006-07-20 |
United States Patent
Application |
20060160099 |
Kind Code |
A1 |
Goldberg; Martin ; et
al. |
July 20, 2006 |
Substrate preparation process
Abstract
The present invention provides novel processes for the large
scale preparation of arrays of polymer sequences wherein each array
includes a plurality of different, positionally distinct polymer
sequences having known monomer sequences. The methods of the
invention combine high throughput process steps with high
resolution photolithographic techniques in the manufacture of
polymer arrays.
Inventors: |
Goldberg; Martin; (Saratoga,
CA) ; Diggelman; Martin; (Nierdorf, CH) ;
Hubbell; Earl; (Mountain View, CA) ; McGall;
Glenn; (Palo Alto, CA) ; Ngo; Nam Quoc;
(Campbell, CA) ; Morris; MacDonald; (Felton,
CA) ; Yamamoto; Mel; (Fremont, CA) ; Tan;
Jennifer; (Newark, CA) ; Rava; Richard P.;
(Redwood City, CA) |
Correspondence
Address: |
AFFYMETRIX, INC;ATTN: CHIEF IP COUNSEL, LEGAL DEPT.
3420 CENTRAL EXPRESSWAY
SANTA CLARA
CA
95051
US
|
Assignee: |
Affymetrix, INC.
Santa Clara
CA
|
Family ID: |
31949801 |
Appl. No.: |
11/015257 |
Filed: |
December 16, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10722032 |
Nov 25, 2003 |
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11015257 |
Dec 16, 2004 |
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09716507 |
Nov 20, 2000 |
6706875 |
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10722032 |
Nov 25, 2003 |
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09244568 |
Feb 4, 1999 |
6307042 |
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09716507 |
Nov 20, 2000 |
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08634053 |
Apr 17, 1996 |
5959098 |
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09244568 |
Feb 4, 1999 |
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Current U.S.
Class: |
435/6.19 ;
427/2.11; 435/287.2 |
Current CPC
Class: |
B01J 2219/00725
20130101; B01J 2219/00585 20130101; B01J 2219/00529 20130101; C40B
40/06 20130101; C40B 60/14 20130101; C07H 21/00 20130101; B01J
2219/00605 20130101; B01J 2219/00596 20130101; B01J 2219/00432
20130101; B01J 2219/00641 20130101; B01J 2219/0059 20130101; B82Y
30/00 20130101; C07K 1/047 20130101; B01J 2219/00711 20130101; B01J
2219/00527 20130101; B01J 2219/00626 20130101; B01J 2219/00608
20130101; B01J 2219/00617 20130101; B01J 2219/00722 20130101; C07B
2200/11 20130101; B01J 2219/00637 20130101; C40B 40/10 20130101;
B01J 19/0046 20130101; B01J 2219/00659 20130101; B01J 2219/00612
20130101; B01J 2219/00689 20130101 |
Class at
Publication: |
435/006 ;
435/287.2; 427/002.11 |
International
Class: |
C12Q 1/68 20060101
C12Q001/68; C12M 1/34 20060101 C12M001/34; B05D 3/02 20060101
B05D003/02 |
Claims
1. A method of forming an array of polymers on a surface of a
substrate, comprising: providing a substrate having a first surface
coated with functional groups protected with a photolabile
protecting group, and a second surface having a layer disposed
thereon, said layer including one or more of an index matching
compound, a light absorbing compound and an antireflective
compound; and sequentially activating and coupling monomers in
different selected regions of said substrate to form a plurality of
different polymer sequences in different known locations on said
surface of said substrate, wherein said activating step comprises
directing an activation radiation at said first surface of said
substrate.
2-49. (canceled)
50. A method for synthesizing a plurality of biopolymers on the
surface of a support, said method comprising: (a) placing said
support into a reaction chamber and applying to said surface said
biopolymers or precursors of said biopolymers, (b) removing said
support from said reaction chamber and placing said support into a
flow chamber, (c) introducing a liquid reagent for conducting said
synthesis into said flow chamber, (d) removing said liquid reagent
from said flow chamber wherein the pressure in said chamber is
maintained substantially atmospheric during said removing. (e)
removing said support from said flow chamber and (f) repeating
steps (a)-(e) to form said plurality of biopolymers on the surface
of said support.
51. A method according to claim 50 wherein liquid reagent is
removed from said flow chamber under vacuum.
52. A method according to claim 50 wherein liquid reagent is
removed from said flow chamber by simultaneously venting and
applying a vacuum to said flow chamber.
53. A method according to claim 52 wherein said venting and said
applying a vacuum are carried out at opposite ends of said flow
chamber.
54. A method according to claim 50 wherein said method further
comprises holding said liquid reagent in said flow chamber for a
predetermined period of time.
55. A method according to claim 50 wherein said support is
glass.
56. A method according to claim 50 further comprising introducing a
pressurized inert gas into said flow chamber after step (c) and
simultaneously evacuating said flow chamber.
57. A method according to claim 50 wherein said biopolymers are
polynucleotides.
58. A method according to claim 50 wherein said liquid reagent for
conducting said synthesis comprises an oxidizing agent or an agent
for removing a protecting group.
59. A method according to claim 50 wherein said biopolymers are
synthesized on said surface in multiple arrays and said support is
subsequently diced into individual arrays of biopolymers on a
support.
60. A method according to claim 59 further comprising exposing the
array to a sample and reading the array.
61. A method according to claim 60 comprising forwarding data
representing a result obtained from a reading of the array.
62. A method according to claim 61 wherein the data is transmitted
to a remote location.
63. A method according to claim 62 comprising receiving data
representing a result of an interrogation obtained by the reading
of the array.
64. A method for synthesizing an array of biopolymers on the
surface of a support wherein said synthesis comprises a plurality
of monomer additions, said method comprising after each of said
monomer additions: (a) placing said support into a flow chamber,
(c) introducing a liquid reagent for conducting said synthesis into
said flow chamber, (d) removing said reagent from said flow chamber
by simultaneously venting said chamber and applying a vacuum to the
interior of said chamber, (e) removing said support from said flow
chamber and (f) repeating steps (a)-(e) to form said plurality of
biopolymers on the surface of said support.
65. A method according to claim 64 wherein said venting and said
applying a vacuum are carried out at opposite ends of said flow
chamber.
66. A method according to claim 64 wherein said method further
comprises holding said liquid reagent in said flow chamber for a
predetermined period of time.
67. A method according to claim 64 wherein said support is
glass.
68. A method according to claim 64 further comprising introducing a
pressurized inert gas into said flow chamber after step (d) and
simultaneously evacuating said flow chamber.
69. A method according to claim 64 wherein said biopolymers are
polynucleotides.
70. A method according to claim 64 wherein said liquid reagent for
conducting said synthesis is an oxidizing agent or an agent for
removing a protecting group.
71. A method according to claim 64 wherein said biopolymers are
synthesized on said surface in multiple arrays and said support is
subsequently diced into individual arrays of biopolymers on a
support.
72. A flow cell assembly for conducting at least one reaction in
the synthesis of an array of biopolymers on the surface of a
support, said flow cell comprising: (a) a flow cell chamber, (b) a
manifold in fluid communication with said chamber, said manifold
comprising at least a wash reagent inlet, an inlet for a reagent
for conducting a step of said synthesis, and a vent, and (c) a
vacuum source in fluid communication with said flow cell
chamber.
73. A flow cell assembly according to claim 72 further comprising a
fluid level sensor and a controller for controlling said inlets,
said vent and said vacuum source.
74. A flow cell assembly according to claim 72 further comprising a
gas inlet.
75. An apparatus for synthesizing an array of biopolymers on the
surface of a support, said apparatus comprising: (a) one or more
flow cell assemblies of claim 72, (b) one or more fluid dispensing
stations in fluid communication with one or more of said plurality
of flow cell assemblies, (c) a station for monomer addition to said
surface of said support, and (d) a mechanism for moving a support
to and from said station for monomer addition and a flow cell and
from one flow cell to another flow cell.
76. An apparatus according to claim 75 further comprising a
controller for controlling the movement of said mechanism.
77. An apparatus according to claim 75 wherein said mechanism is a
robotic arm.
78. A method comprising using an array, prepared by an apparatus
according to claim 75, by exposing the array to a sample and
reading the array.
79. A method according to claim 78 comprising forwarding data
representing a result obtained from a reading of the array.
80. A method according to claim 79 wherein the data is transmitted
to a remote location.
81. A method according to claim 80 comprising receiving data
representing a result of an interrogation obtained by the reading
of the array.
Description
BACKGROUND OF THE INVENTION
[0001] Methods for synthesizing a variety of different types of
polymers are well known in the art. For example, the "Merrifield"
method, described in Atherton et al., "Solid Phase Peptide
Synthesis," IRL Press, 1989, which is incorporated herein by
reference for all purposes, has been used to synthesize peptides on
a solid support. In the Merrifield method, an amino acid is
covalently bonded to a support made of an insoluble polymer or
other material. Another amino acid with an alpha protecting group
is reacted with the covalently bonded amino acid to form a
dipeptide. After washing, the protecting group is removed and a
third amino acid with an alpha protecting group is added to the
dipeptide. This process is continued until a peptide of a desired
length and sequence is obtained.
[0002] Methods have also been developed for producing large arrays
of polymer sequences on solid substrates. These large "arrays" of
polymer sequences have wide ranging applications and are of
substantial importance to the pharmaceutical, biotechnology and
medical industries. For example, the arrays may be used in
screening large numbers of molecules for biological activity, i.e.,
receptor binding capability. Alternatively, arrays of
oligonucleotide probes can be used to identify mutations in known
sequences, as well as in methods for de novo sequencing of target
nucleic acids.
[0003] Of particular note, is the pioneering work described in U.S.
Pat. No. 5,143,854 (Pirrung et al.) and PCT Application No.
92/10092 disclose improved methods of molecular synthesis using
light directed techniques. According to these methods, light is
directed to selected regions of a substrate to remove protecting
groups from the selected regions of the substrate. Thereafter,
selected molecules are coupled to the substrate, followed by
additional irradiation and coupling steps. By activating selected
regions of the substrate and coupling selected monomers in precise
order, one can synthesize an array of molecules having any number
of different sequences, where each different sequence is in a
distinct, known location on the surface of the substrate.
[0004] These arrays clearly embody the next step in solid phase
synthesis of polymeric molecules generally, and polypeptides and
oligonucleotides, specifically. Accordingly, it would be desirable
to provide methods for preparation of these arrays, which methods
have high throughput, high product quality, enhanced
miniaturization and lower costs. The present invention meets these
and other needs.
SUMMARY OF THE INVENTION
[0005] The present invention generally provides novel processes for
the efficient, large scale preparation of arrays of polymer
sequences wherein each array includes a plurality of different,
positionally distinct polymer sequences having known monomer
sequences. In one embodiment, the methods of the present invention
provide for the cleaning and stripping of substrate wafers to
remove oil and dirt from the surface, followed by the
derivatization of the wafers to provide photoprotected functional
groups on the surface. Polymer sequences are then synthesized on
the surface of the substrate wafers by selectively exposing a
plurality of selected regions on the surface to an activation
radiation to remove the photolabile protecting groups from the
functional groups and contacting the surface with a monomer
containing solution to couple monomers to the surface in the
selected regions. The exposure and contacting steps are repeated
until a plurality of polymer arrays are formed on the surface of
the substrate wafer. Each polymer array includes a plurality of
different polymer sequences coupled to the surface of the substrate
wafer in a different known location. The wafers are then separated
into a plurality of individual substrate segments, each segment
having at least one polymer array formed thereon, and packaged in a
cartridge whereby the surface of said substrate segment having the
polymer array formed thereon is in fluid contact with the
cavity.
[0006] In another embodiment, the present invention provides
methods of forming polymer arrays by providing a substrate having a
first surface coated with functional groups protected with a
photolabile protecting group, and a second surface having a layer
that includes one or more of an index matching compound, a light
absorbing compound and an antireflective compound. The method then
provides for the sequential activation and coupling of monomers in
different selected regions of the first surface of the substrate to
form a plurality of different polymer sequences in different known
locations on the surface of the substrate, by directing an
activation radiation at the first surface of the substrate.
[0007] In yet another embodiment, the present invention provides a
method of forming a plurality of polymer arrays using a batch
process. In particular, this method comprises the steps of
activating a plurality of substrate wafers by exposing selected
regions on each of a plurality of substrate wafers then contacting
them with a monomer containing solution in a batch.
[0008] In a further embodiment, the present invention provides a
method of synthesizing polymers on substrates by first derivatizing
the substrate with an aminoalkyltrialkoxysilane.
[0009] In an additional embodiment, the present invention provides
a method for forming an array of polymers on a substrate using
light-directed synthesis wherein the exposing step comprises
directing an activation radiation at selected regions on the
surface of said substrate by shining the activation radiation
through a photolithographic mask having transparent regions and
opaque regions where the transparent regions are smaller than the
selected regions. As a result, the activation radiation shone
through the transparent regions in the mask is diffracted to expose
the selected regions.
[0010] The present invention also provides methods of forming
arrays of polymer sequences having enhanced synthesis efficiencies
through the incorporation of monomers which have lipophilic
chemical groups coupled thereto.
[0011] The present invention also provides methods of forming
polymer arrays using the above-described methods, but wherein the
deprotection and coupling steps in adjacent selected regions of the
substrate surface are aligned to minimize differences in synthesis
steps between adjacent regions.
[0012] In still another embodiment, the present invention provides
polymer arrays and methods of forming them on a tubular substrate
by the sequential activation of and coupling of monomers to
selected segments of the tubular substrate surface.
[0013] In an additional embodiment, the present invention provides
methods of photoprotecting functional groups that are coupled to
solid supports by exposing the functional group to a
photoprotecting group transfer agent having the formula: ##STR1##
wherein R.sub.1 is a photolabile protecting group and X is a
leaving group.
BRIEF DESCRIPTION OF THE DRAWINGS
[0014] FIG. 1 schematically illustrates light directed
oligonucleotide synthesis using photolithographic methods.
[0015] FIGS. 2A-C are flow diagrams illustrating the overall
process of substrate preparation. FIG. 2A is a flow dagram
illustrating the overall process. FIGS. 2B and 2C are flow diagrams
of the synthesis steps for individual and batch processes,
respectively.
[0016] FIGS. 3A and 3B show schematic illustrations of alternate
reactor systems for carrying out the combined photolysis/chemistry
steps of used in the methods of the present invention.
[0017] FIGS. 4A and 4B schematically illustrate different isolated
views of a flow cell incorporated into the reactor systems of FIGS.
3A and 3B. FIG. 4C shows a schematic illustration of an integrated
reactor system including computer control and substrate translation
elements.
[0018] FIG. 5A shows the alkylation of the exocyclic amine
functional group of deoxyguanosine with dimethoxytritylchloride
(DMT-Cl) and subsequent coupling of a MenPOC protecting group to
the 3' hydroxyl group of a nucleoside phosphoramidite. FIG. 5B
shows the synthetic route for production of Fmoc-phosphoramidites.
FIG. 5C shows a synthetic route for introduction of a lipophilic
substituent to the photoprotecting group MeNPOC.
[0019] FIG. 6A shows a schematic representation of a device
including a six vessel reaction chamber bank, for carrying out
multiple parallel monomer addition steps separate from the
photolysis step in light directed synthesis of oligonucleotide
arrays. FIG. 6B shows a detailed view of a single reaction
chamber.
[0020] FIG. 7 illustrates a substrate wafer fabricated with a
plurality of probe arrays which wafer also includes alignment
marks.
[0021] FIG. 8 illustrates one embodiment of an array cartridge into
which an array substrate is placed for use.
[0022] FIGS. 9A and 9B show the coupling of fluorescent nucleotides
to a substrate surface using photolithographic methods in 50 and
100 .mu.m features, using back-side and front-side exposure,
respectively. FIGS. 9C and 9D show a plot of fluorescence intensity
as a function of substrate position at the border between two
features for back-side and front-side exposure as indicated. FIG.
9C illustrates the contrast difference from a top view of the plots
while FIG. 9D shows a side view.
[0023] FIG. 10 is a bar chart showing a comparison of silanation
methods using 5 different silanes to derivatize the surface of
glass substrates (3-acetoxypropyltrimethoxysilane ("OAc");
3-glycidoxypropyltrimethoxysilane ("Epoxy");
4-(hydroxybutyramido)propyltriethoxysilane ("Mono"); 3
-aminopropyltriethoxysilane ("APS"); and 3-N,N-bis(2-hydroxyethyl)
aminopropyl; triethoxysilane ("bis")). Shown are the surface
density of reactive groups as shown by fluorescence staining
(black) and fluorescence intensity of a standard hybridization
experiment following synthesis of oligonucleotides on the surface
of substrates derivatized using these silanes (grey).
[0024] FIG. 11 shows the reprotection of deprotected hydroxyl
groups on a glass substrate with MeNPOC-tetrazolide as a function
of time of exposure to the MeNPOC-tetrazolide and addition of
catalyst.
DESCRIPTION OF THE PREFERRED EMBODIMENT
I. Definitions
[0025] Probe: A probe, as defined herein, is a surface-immobilized
molecule that is recognized by a particular target. These may also
be referred to as ligands. Examples of probes encompassed by the
scope of this invention include, but are not limited to, agonists
and antagonists of cell surface receptors, toxins and venoms, viral
epitopes, hormone receptors, peptides, peptidomimetics, enzymes,
enzyme substrates, cofactors, drugs, lectins, sugars,
oligonucleotides, nucleic acids, oligosaccharides, proteins or
monoclonal antibodies, natural or modified, e.g., reshaped,
chimeric, etc.
[0026] Array: An array is a preselected collection of different
polymer sequences or probes which are associated with a surface of
a substrate. An array may include polymers of a given length having
all possible monomer sequences made up of a specific basis set of
monomers, or a specific subset of such an array. For example, an
array of all possible oligonucleotides of length 8 includes 65,536
different sequences. However, as noted above, an oligonucleotide
array also may include only a subset of the complete set of probes.
Similarly, a given array may exist on more than one separate
substrate, e.g., where the number of sequences necessitates a
larger surface area in order to include all of the desired polymer
sequences.
[0027] Functional group: A functional group is a reactive chemical
moiety present on a given monomer, polymer or substrate surface.
Examples of functional groups include, e.g., the 3' and 5' hydroxyl
groups of nucleotides and nucleosides, as well as the reactive
groups on the nucleobases of the nucleic acid monomers, e.g., the
exocyclic amine group of guanosine, as well as amino and carboxyl
groups on amino acid monomers.
[0028] Monomer/Building block: A monomer or building block is a
member of the set of smaller molecules which can be joined together
to form a larger molecule or 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 natural or synthetic
amino acids, the set of nucleotides (both ribonucleotides and
deoxyribonucleotides, natural and unnatural) and the set of
pentoses and hexoses. As used herein, monomer refers to any member
of a basis set for synthesis of a larger molecule. A selected set
of monomers forms a basis set of monomers. For example, the basis
set of nucleotides includes A, T (or U), G and C. In another
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 in any of the successive steps
in the synthesis of a polymer. Furthermore, each of the sets may
include protected members which are modified after synthesis.
[0029] Feature: A feature is defined as a selected region on a
surface of a substrate in which a given polymer sequence is
contained. Thus, where an array contains, e.g., 100,000 different
positionally distinct polymer sequences on a single substrate,
there will be 100,000 features.
[0030] Edge: An edge is defined as a boundary between two features
on a surface of a substrate. The sharpness of this edge, in terms
of reduced bleed over from one feature to another, is termed the
"contrast" between the two features.
[0031] Protecting group: A protecting group is a material which is
chemically bound to a reactive functional group on a monomer unit
or polymer and which protective group may be removed upon selective
exposure to an activator such as a chemical activator, or another
activator, such as electromagnetic radiation or light, especially
ultraviolet and visible light. Protecting groups that are removable
upon exposure to electromagnetic radiation, and in particular
light, are termed "photolabile protecting groups."
II. Abbreviations
[0032] ACN Acetonitrile
[0033] Bz Benzoyl
[0034] CE .beta.-cyanoethyl
[0035] CEP Cyanoethylphosphoramidite
[0036] DCM dichloromethane
[0037] DIEA Diiminoethylamine
[0038] dG Deoxyguanosine
[0039] DMAP Dimethylaminopyridine
[0040] DMC N,N-dimethylcarbamoyl
[0041] DMF Dimethylformamide
[0042] DMT 4,4'-Dimethoxytrityl
[0043] DPC Diphenylcarbamoyl
[0044] HOAT 1-Hydroxy-7-azabenzotriazole
[0045] HOBT 1-hydroxybenzotriazole
[0046] Ibu Isobutyryl
[0047] MeNP .alpha.-methyl-o-nitropiperonyl
[0048] MeNPOC .alpha.-methyl-o-nitropiperonyloxycarbonyl
[0049] MeNV .alpha.-methyl-o-nitroveratryl
[0050] MeNVOC .alpha.-methyl-o-nitroveratryloxycarbonyl
[0051] MMT 4-Methoxytrityl
[0052] NMI n-methylimidazole
[0053] NMP n-methylpyrollidinone
[0054] NP o-Nitropiperonyl
[0055] NPE 2-(p-nitrophenyl)ethyl
[0056] NPSE 2-(p-nitrophenylsulfonyl)ethyl
[0057] NV o-Nitroveratryl
[0058] NPOC o-Nitropiperonyloxycarbonyl
[0059] NVOC o-Nitroveratryloxycarbonyl
[0060] PAC Phenoxyacetyl
[0061] PYMOC 1-Pyrenylmethyloxycarbonyl
[0062] SSPE Saline, Sodium Phosphate, EDTA Buffer
[0063] TEA Triethylamine
[0064] THF Tetrahydrofuran
III. Process Overview
[0065] The present invention generally provides processes and
devices for reproducibly and efficiently preparing arrays of
polymer sequences on solid substrates. The overall process is
illustrated in FIG. 2A. Generally, the process 1 begins with a
series of substrate preparation steps 10 which may include such
individual processing steps as stripping cleaning and
derivatization of the substrate surface to provide uniform reactive
surfaces for synthesis. The polymer sequences are then synthesized
on the substrate surface in the synthesis step 20. Following
polymer synthesis, the substrates are then separated into
individual arrays 40, and assembled in cartridges that are suitable
for ultimate use 60. In alternate embodiments, the present
invention also provides for the synthesis of the polymer sequences
on the substrate surface using either an individual or batch
process mode. A comparison of these two synthesis modes is shown in
FIG. 2B. In the individual processing mode, the activation and
monomer addition steps are combined in a single unit operation 22.
For example, a single substrate wafer is placed in a reactor system
where it is first subjected to an activation step to activate
selected regions of the substrate. The substrate is then contacted
with a first monomer which is coupled to the activated region.
Activation and coupling steps are repeated until the desired array
of polymer sequences is created. The arrays of polymer sequences
are then subjected to a final deprotection step 30.
[0066] In the batch processing mode, a number of substrate wafers
are subjected to an activating step 24. The activated substrate
wafers are then pooled 26 and subjected to a monomer addition step
28. Each substrate wafer is then subjected individually to
additional activation steps followed by pooling and monomer
addition. This is repeated until a desired array of polymer
sequences is formed on the substrate wafers in a series of
individual arrays. These arrays of polymer sequences on the
substrate wafers are then subjected to a final deprotection step
30.
IV. Substrate Preparation
[0067] The term "substrate" refers to a material having a rigid or
semi-rigid surface. In many embodiments, at least one surface of
the substrate will be substantially flat or planar, 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. Preferred substrates
generally comprise planar crystalline substrates such as silica
based substrates (e.g. glass, quartz, or the like), or crystalline
substrates used in, e.g., the semiconductor and microprocessor
industries, such as silicon, gallium arsenide and the like. These
substrates are generally resistant to the variety of synthesis and
analysis conditions to which they may be subjected. Particularly
preferred substrates will be transparent to allow the
photolithographic exposure of the substrate from either
direction.
[0068] Silica aerogels may also be used as substrates. Aerogel
substrates may be used as free standing substrates or as a surface
coating for another rigid substrate support. Aerogel substrates
provide the advantage of large surface area for polymer synthesis,
e.g., 400 to 1000 m.sup.2/gm, or a total useful surface area of 100
to 1000 cm.sup.2 for a 1 cm.sup.2 piece of aerogel substrate. Such
aerogel substrates may generally be prepared by methods known in
the art, e.g., the base catalyzed polymerization of (MeO).sub.4Si
or (Eto).sub.4Si in ethanol/water solution at room temperature.
Porosity may be adjusted by altering reaction coondition by methods
known in the art.
[0069] Individual planar substrates generally exist as wafers which
can have varied dimensions. The term "wafer" generally refers to a
substantially flat sample of substrate from which a plurality of
individual arrays or chips may be fabricated. The term "array" or
"chip" is used to refer to the final product of the individual
array of polymer sequences, having a plurality of different
positionally distinct polymer sequences coupled to the surface of
the substrate. The size of a substrate wafer is generally defined
by the number and nature of arrays that will be produced from the
wafer. For example, more complex arrays, e.g., arrays having all
possible polymer sequences produced from a basis set of monomers
and having a given length, will generally utilize larger areas and
thus employ larger substrates, whereas simpler arrays may employ
smaller surface areas, and thus, less substrate.
[0070] Typically, the substrate wafer will range in size of from
about 1''.times.1'' to about 12''.times.12'', and will have a
thickness of from about 0.5 mm to about 5 mm. Individual substrate
segments which include the individual arrays, or in some cases a
desired collection of arrays, are typically much smaller than the
wafers, measuring from about 0.2 cm.times.0.2 cm to about 5
cm.times.5 cm. In particularly preferred aspects, the substrate
wafer is about 5''.times.5'' whereas the substrate segment is
approximately 1.28 cm.times.1.28 cm. Although a wafer can be used
to fabricate a single large substrate segment, typically, a large
number of substrate segments will be prepared from a single wafer.
For example, a wafer that is 5''.times.5'' can be used to fabricate
upwards of 49 separate 1.28 cm.times.1.28 cm substrate segments.
The number of segments prepared from a single wafer will generally
vary depending upon the complexity of the array, and the desired
feature size.
[0071] Although primarily described in terms of flat or planar
substrates, the present invention may also be practiced with
substrates having substantially different conformations. For
example, the substrate may exist as particles, strands,
precipitates, gels, sheets, tubing, spheres, containers,
capillaries, pads, slices, films, plates, slides, etc. In a
preferred alternate embodiment, the substrate is a glass tube or
microcapillary. The capillary substrate provides advantages of
higher surface area to volume ratios, reducing the amount of
reagents necessary for synthesis. Similarly, the higher surface to
volume ratio of these capillary substrates imparts more efficient
thermal transfer properties. Additionally, preparation of the
polymer arrays may be simplified through the use of these capillary
based substrates. For example, minimizing differences between the
regions on the array, or "cells", and their "neighboring cells" is
simplified in that there are only two neighboring cells for any
given cell (see discussion below for edge minimization in chip
design). Spatial separation of two neighboring cells on an array
merely involves the incorporation of a single blank cell, as
opposed to full blank lanes as generally used in a flat substrate
conformation. This substantially conserves the surface area
available for polymer synthesis. Manufacturing design may also be
simplified by the linear nature of the substrate. In particular,
the linear substrate may be moved down a single mask in a direction
perpendicular to the length of the capillary. As it is moved, the
capillary will encounter linear reticles (translucent regions of
the mask), one at a time, thereby exposing selected regions within
the capillary or capillary. This can allow bundling of parallel
capillaries during synthesis wherein the capillaries are exposed to
thicker linear reticles, simultaneously, for a batch processing
mode, or individual capillaries may be placed on a mask having all
of the linear reticles lined up so that the capillary can be
stepped down the mask in one direction. Subsequent capillaries may
be stepped down the mask at least one step behind the previous
capillary. This employs an assembly line structure to the substrate
preparation process.
[0072] As an example, a standard optimization chip for detecting 36
simultaneous mutations using a flat geometry chip and an
optimization tiling strategy, is 44.times.45 features (1980 probes
and blanks), with 36 blocks of 40 probes each (1440 probes), plus
15 blanks per block (540 blank probes). A capillary format,
however, can incorporate the same number of test probes in a
smaller space. Specifically, in a capillary substrate, 36 strings
of 40 probes will have only one blank space separating each probe
group (35 blank probes), for a total of 1475 features.
[0073] Finally, linear capillary based substrates can provide the
advantage of reduced volume over flat geometries. In particular,
typical capillary substrates have a volume in the 1-10 .mu.l range,
whereas typical flow cells for synthesizing or screening flat
geometry chips have volumes in the range of 100 .mu.l.
[0074] A. Stripping and Rinsing
[0075] In order to ensure maximum efficiency and accuracy in
synthesizing polymer arrays, it is generally desirable to provide a
clean substrate surface upon which the various reactions are to
take place. Accordingly, in some processing embodiments of the
present invention, the substrate is stripped to remove any residual
dirt, oils or other fluorescent materials which may interfere with
the synthesis reactions, or subsequent analytical use of the
array.
[0076] The process of stripping the substrate typically involves
applying, immersing or otherwise contacting the substrate with a
stripping solution. Stripping solutions may be selected from a
number of commercially available, or readily prepared chemical
solutions used for the removal of dirt and oils, which solutions
are well known in the art. Particularly preferred stripping
solutions are composed of a mixture of concentrated H.sub.2SO.sub.4
and H.sub.2O.sub.2. Such solutions are generally available from
commercial sources, e.g., Nanostrip.TM. from Cyantek Corp. After
stripping, the substrate is rinsed with water and in preferred
aspects, is then contacted with a solution of NaOH, which results
in regeneration of an even layer of hydroxyl functional groups on
the surface of the substrate. In this case, the substrate is again
rinsed with water, followed by a rinse with HCl to neutralize any
remaining base, followed again by a water rinse. The various
stripping and rinsing steps may generally be carried out using a
spin-rinse-drying apparatus of the type generally used in the
semiconductor manufacturing industry.
[0077] Gas phase cleaning and preparation methods may also be
applied to the substrate wafers using, e.g., H.sub.2O or O.sub.2
plasma or reactive ion etching (RIE) techniques that are well known
in the art.
[0078] B. Derivatization
[0079] Following cleaning and stripping of the substrate surface,
the surface is derivatized to provide sites or functional groups on
the substrate surface for synthesizing the various polymer
sequences on that surface. In particular, derivatization provides
reactive functional groups, e.g., hydroxyl, carboxyl, amino groups
or the like, to which the first monomers in the polymer sequence
may be attached. In preferred aspects, the substrate surface is
derivatized using silane in either water or ethanol. Preferred
silanes include mono- and dihydroxyalkylsilanes, which provide a
hydroxyl functional group on the surface of the substrate. Also
preferred are aminoalkyltrialkoxysilanes which can be used to
provide the initial surface modification with a reactive amine
functional group. Particularly preferred are
3-aminopropyltriethoxysilane and 3-aminopropyltrimethoxysilane
("APS"). Derivatization of the substrate using these latter amino
silanes provides a linkage that is stable under synthesis
conditions and final deprotection conditions (for oligonucleotide
synthesis, this linkage is typically a phosphoramidate linkage, as
compared to the phosphodiester linkage where hydroxyalkylsilanes
are used). Additionally, this amino silane derivatization provides
several advantages over derivatization with hydroxyalkylsilanes.
For example, the aminoalkyltrialkoxysilanes are inexpensive and can
be obtained commercially in high purity from a variety of sources,
the resulting primary and secondary amine functional groups are
more reactive nucleophiles than hydroxyl groups, the
aminoalkyltrialkoxysilanes are less prone to polymerization during
storage, and they are sufficiently volatile to allow application in
a gas phase in a controlled vapor deposition process (See
below).
[0080] Additionally, silanes can be prepared having protected or
"masked" hydroxyl groups and which possess significant volatility.
As such, these silanes can be readily purified by, e.g.,
distillation, and can be readily employed in gas-phase deposition
methods of silanating substrate surfaces. After coating these
silanes onto the surface of the substrate, the hydroxyl groups may
be deprotected with a brief chemical treatment, e.g., dilute acid
or base, which will not attack the substrate-silane bond, so that
the substrate can then be used for polymer synthesis. Examples of
such silanes include acetoxyalkylsilanes, such as
acetoxyethyltrichlorosilane, acetoxypropyltrimethoxysilane, which
may bedeprotected after application using, e.g., vapor phase
ammonia and methylamine or liquid phase aqueous or ethanolic
ammonia and alkylamines. Epoxyalkylsilanes may also be used, such
as glycidoxypropyltrimethoxysilane which may be deprotected using,
e.g., vapor phase HCl, trifluoroacetic acid or the like, or liquid
phase dilute HCl.
[0081] The physical operation of silanation of the substrate
generally involves dipping or otherwise immersing the substrate in
the silane solution. Following immersion, the substrate is
generally spun as described for the substrate stripping process,
i.e., laterally, to provide a uniform distribution of the silane
solution across the surface of the substrate. This ensures a more
even distribution of reactive functional groups on the surface of
the substrate. Following application of the silane layer, the
silanated substrate may be baked to polymerize the silanes on the
surface of the substrate and improve the reaction between the
silane reagent and the substrate surface. Baking typically takes
place at temperatures in the range of from 90.degree. C. to
120.degree. C. with 110.degree. C. being most preferred, for a time
period of from about 1 minute to about 10 minutes, with 5 minutes
being preferred.
[0082] In alternative aspects, as noted above, the silane solution
may be contacted with the surface of the substrate using controlled
vapor deposition methods or spray methods. These methods involve
the volatilization or atomization of the silane solution into a gas
phase or spray, followed by deposition of the gas phase or spray
upon the surface of the substrate, usually by ambient exposure of
the surface of the substrate to the gas phase or spray. Vapor
deposition typically results in a more even application of the
derivatization solution than simply immersing the substrate into
the solution.
[0083] The efficacy of the derivatization process, e.g., the
density and uniformity of functional groups on the substrate
surface, may generally be assessed by adding a fluorophore which
binds the reactive groups, e.g., a fluorescent phosphoramidite such
as Fluoreprime.TM. from Pharmacia, Corp., Fluoredite.TM. from
Millipore, Corp. or FAM.TM. from ABI, and looking at the relative
fluorescence across the surface of the substrate.
V. Synthesis
[0084] General methods for the solid phase synthesis of a variety
of polymer types have been previously described. Methods of
synthesizing arrays of large numbers of polymer sequences,
including oligonucleotides and peptides, on a single substrate have
also been described. See U.S. Pat. Nos. 5,143,854 and 5,384,261 and
Published PCT Application No WO 92/10092, each of which is
incorporated herein by reference in its entirety for all
purposes.
[0085] As described previously, the synthesis of oligonucleotides
on the surface of a substrate may be carried out using light
directed methods as described in., e.g., U.S. Pat. Nos. 5,143,854
and 5,384,261 and Published PCT Application No WO 92/10092, or
mechanical synthesis methods as described in U.S. Pat. No.
5,384,261 and Published PCT Application No. 93/09668, each of which
is incorporated herein by reference. Preferably, synthesis is
carried out using light-directed synthesis methods. In particular,
these light-directed or photolithographic synthesis methods involve
a photolysis step and a chemistry step. The substrate surface,
prepared as described herein comprises functional groups on its
surface. These functional groups are protected by photolabile
protecting groups ("photoprotected"), also as described herein.
During the photolysis step, portions of the surface of the
substrate are exposed to light or other activators to activate the
functional groups within those portions, i.e., to remove
photoprotecting groups. The substrate is then subjected to a
chemistry step in which chemical monomers that are photoprotected
at at least one functional group are then contacted with the
surface of the substrate. These monomers bind to the activated
portion of the substrate through an unprotected functional
group.
[0086] Subsequent activation and coupling steps couple monomers to
other preselected regions, which may overlap with all or part of
the first region. The activation and coupling sequence at each
region on the substrate determines the sequence of the polymer
synthesized thereon. In particular, light is shown through the
photolithographic masks which are designed and selected to expose
and thereby activate a first particular preselected portion of the
substrate. Monomers are then coupled to all or part of this portion
of the substrate. The masks used and monomers coupled in each step
can be selected to produce arrays of polymers having a range of
desired sequences, each sequence being coupled to a distinct
spatial location on the substrate which location also dictates the
polymer's sequence. The photolysis steps and chemistry steps are
repeated until the desired sequences have been synthesized upon the
surface of the substrate.
[0087] Basic strategy for light directed synthesis of
oligonucleotides on a VLSIPS.TM. Array is outlined in FIG. 1. The
surface of a substrate or solid support, modified with
photosensitive protecting groups (X) is illuminated through a
photolithographic mask, yielding reactive hydroxyl groups in the
illuminated regions. A selected nucleotide, typically in the form
of a 3'-O-phosphoramidite-activated deoxynucleoside (protected at
the 5' hydroxyl with a photosensitive protecting group), is then
presented to the surface and coupling occurs at the sites that were
exposed to light. Following capping and oxidation, the substrate is
rinsed and the surface is illuminated through a second mask, to
expose additional hydroxyl groups for coupling. A second selected
nucleotide (e.g., 5'-protected, 3'-O-phosphoramidite-activated
deoxynucleoside) is presented to the surface. The selective
deprotection and coupling cycles are repeated until the desired set
of products is obtained. Pease et al., Proc. Natl. Acad. Sci.
(1994) 91:5022-5026. Since photolithography is used, the process
can be readily miniaturized to generate high density arrays of
oligonucleotide probes. Furthermore, the sequence of the
oligonucleotides at each site is known. Such photolithographic
methods are also described in U.S. Pat. No. 5,143,854, U.S. Pat.
No. 5,489,678 and Published PCT Application No. WO 94/10128 each of
which is incorporated herein by reference in its entirety for all
purposes. In the large scale processes of the present invention, it
is typically preferred to utilize photolithographic synthesis
methods.
[0088] Using the above described methods, arrays may be prepared
having all polymer sequences of a given length which are composed
of a basis set of monomers. Such an array of oligonucleotides, made
up of the basis set of four nucleotides, for example, would contain
up to 4.sup.n oligonucleotides on its surface, where n is the
desired length of the oligonucleotide probe. For an array of 8mer
or 10mer oligonucleotides, such arrays could have upwards of about
65,536 and 1,048,576 different oligonucleotides respectively.
Generally, where it is desired to produce arrays having all
possible polymers of length n, a simple binary masking strategy can
be used, as described in U.S. Pat. No. 5,143,854.
[0089] Alternate masking strategies can produce arrays of probes
which contain a subset of polymer sequences, i.e., polymers having
a given subsequence of monomers, but are systematically substituted
at each position with each member of the basis set of monomers. In
the context of oligonucleotide probes, these alternate synthesis
strategies may be used to lay down or "tile" a range of probes that
are complementary to, and span the length of a given known nucleic
acid segment. The tiling strategy will also include substitution of
one or more individual positions within the sequence of each of the
probe groups with each member of the basis set of nucleotides.
These positions are termed "interogation positions." By reading the
hybridization pattern of the target nucleic acid, one can determine
if and where any mutations lie in the sequence, and also determine
what the specific mutation is by identifying which base is
contained within the interogation position. Tiling methods and
strategies are discussed in substantial detail in U.S. patent
application Ser. No. 08/143,312 filed Oct. 26, 1993, and
incorporated herein by reference in its entirety for all
purposes.
[0090] Tiled arrays may be used for a variety of applications, such
as identifying mutations within a known oligonucleotide sequence or
"target". Specifically, the probes on the array will have a
subsequence which is complementary to a known nucleic acid
sequence, but wherein at least one position in that sequence has
been systematically substituted with the other three
nucleotides.
[0091] Use of photolabile protecting groups during polymer
synthesis has been previously reported, as described above.
Preferred photolabile protecting groups generally have the
following characteristics: they prevent selected reagents from
modifying the group to which they are attached; they are stable to
synthesis reaction conditions (that is, they remain attached to the
molecule); they are removable under conditions that minimize
potential adverse effects upon the structure to which they are
attached; and, once removed, they do not react appreciably with the
surface or surface bound oligomer. In some embodiments, liberated
byproducts of the photolysis reaction can be rendered unreactive
toward the growing oligomer by adding a reagent that specifically
reacts with the byproduct.
[0092] The removal rate of the photolabile protecting groups
generally depends upon the wavelength and intensity of the incident
radiation, as well as the physical and chemical properties of the
protecting group itself. Preferred protecting groups are removed at
a faster rate and with a lower intensity of radiation. Generally,
photoprotecting groups that undergo photolysis at wavelengths in
the range from 300 nm to approximately 450 nm are preferred.
[0093] Generally, photolabile or photosensitive protecting groups
include ortho-nitrobenzyl and ortho-nitrobenzyloxycarbonyl
protecting groups. The use of these protecting groups has been
proposed for use in photolithography for electronic device
fabrication (see, e.g., Reichmanis et al., J. Polymer Sci. Polymer
Chem. Ed. (1985) 23:1-8, incorporated herein by reference for all
purposes).
[0094] Examples of additional photosensitive protecting groups
which may be used in the light directed synthesis methods herein
described, include, e.g., 1-pyrenylmethyloxycarbonyl,
.alpha.,.alpha.-dimethyl-3,5-dimethoxybenzyloxycarbonyl,
4-methoxyphenacyloxycarbonyl, 3'-methoxybenzoinyloxycarbonyl,
3',5'-dimethoxybenzoinyl-oxycarbonyl
2',3'-dimethoxybenzoinyl-oxycarbonyl,
2',3'-(methylenedioxy)benzoinyloxycarbonyl,
N-(5-bromo-7-nitroindolinyl)carbonyl
3,5-dimethoxybenzyloxycarbonyl, and
.alpha.-(2-methyleneanthraquinone)oxycarbonyl.
[0095] Particularly preferred photolabile protecting groups for
protection of either the 3' or 5'-hydroxyl groups of nucleotides or
nucleic acid polymers include the o-nitrobenzyl protecting groups
described in Published PCT Application No. WO 92/10092. These
photolabile protecting groups include, e.g.,
nitroveratryloxycarbonyl(NVOC), nitropiperonyl oxycarbonyl(NPOC),
.alpha.-methyl-nitroveratryloxycarbonyl (MeNVOC),
.alpha.-methyl-nitropiperonyloxycarbonyl(MeNPOC),
1-pyrenylmethyloxycarbonyl (PYMOC), and the benzylic forms of each
of these (i.e., NV, NP, MeNV, MeNP and PYM, respectively), with
MeNPOC being most preferred.
[0096] Protection strategies may be optimized for different
phosphoramidite nucleosides to enhance synthesis efficiency.
Examples of such optimized synthesis methods are reported in, e.g.,
U.S. patent application Ser. No. 08/445,332 filed May 19, 1995.
Generally, these optimization methods involve selection of
particular protecting groups for protection of the O.sup.6 group of
guanosine, which can markedly improve coupling efficiencies in the
synthesis of guanosine containing oligonucleotides. Similarly,
selection of the appropriate protecting group for protection of the
N.sup.2 group of guanosine can also result in such an improvement,
in absence of protection of the O.sup.6 group. For example,
suitable protecting groups for protection of the N.sup.2 group,
where the O.sup.6 group is also protected, include, e.g., mono- or
diacyl protecting groups, triarylmethyl protecting groups, e.g.,
DMT and MMT, and amidine type protecting groups, e.g.,
N,N-dialkylformamidines. Particularly preferred protecting groups
for the N.sub.2 group include, e.g., DMT, DMF, PAC, Bz and Ibu.
[0097] Protection of the O.sup.6 group will generally be carried
out using carbamate protecting groups such as --C(O)NX.sub.2, where
X is alkyl, or aryl; or the protecting group --CH.sub.2CH.sub.2Y,
where Y is an electron withdrawing group such as cyano,
p-nitrophenyl, or alkyl- or aryl-sulfonyl; and aryl protecting
groups. In a particularly preferred embodiment, the O.sup.6 group
is protected using a diphenylcarbamoyl protecting group (DPC).
[0098] Alternatively, improved coupling efficiencies may be
achieved by selection of an appropriate protecting group for only
the N.sup.2 group. For example, where the N.sup.2-PAC protecting
group is substituted with an Ibu protecting group, a substantial
improvement in coupling efficiency is seen, even without protection
of the O.sup.6 group.
[0099] A variety of modifications can be made to the
above-described synthesis methods. For example, in some
embodiments, it may be desirable to directly transfer or add
photolabile protecting groups to functional groups, e.g., NH.sub.2,
OH, SH or the like, on a solid support. For these methods,
conventional peptide or oligonucleotide monomers or building blocks
having chemically removable protecting groups are used instead of
monomers having photoprotected functional groups. In each cycle of
the synthesis procedure, the monomer is coupled to reactive sites
on the substrate, e.g., sites deprotected in a prior photolysis
step. The protecting group is then removed using conventional
chemical techniques and replaced with a photolabile protecting
group prior to the next photolysis step.
[0100] A number of reagents will effect this replacement reaction.
Generally, these reagents will have the following generic
structure: ##STR2## where R.sub.1 is a photocleavable protecting
group and X is a leaving group, i.e., from the parent acid HX. The
stronger acids typically correspond to better leaving groups and
thus, more reactive acylating agents.
[0101] Examples of suitable leaving groups include a number of
derivatives having a range of properties, e.g., relative
reactivity, solubility, etc. These groups generally include simple
inorganic ions, i.e., halides, N.sub.3.sup.-, and the like, as well
as compounds having the following structures: ##STR3## where
R.sub.2 is alkyl, substituted alkyl or aryl, R.sub.3 is hydrogen,
alkyl, thioalkyl, aryl; R.sub.4 is an electron withdrawing group
such as NO.sub.2, SO.sub.2--R.sub.2, or CN; R.sub.5 is a sterically
hindered alkyl or aryl group such as adamantyl, t-butyl and the
like; and R.sub.6 is alkyl or aryl substituted with electronegative
substituents. Examples of these latter leaving groups include:
##STR4##
[0102] Conditions for carrying out this transfer are similar to
those used for coupling reaction in solid phase peptide synthesis,
or for the capping reaction in solid phase oligonucleotide
synthesis. The solid phase amine, hydroxyl or thiol groups are
exposed to a solution of the protecting group coupled to the
leaving group, e.g., MeNPOC--X in a non-nucleophilic organic
solvent, e.g., DMF, NMP, DCM, THF, ACN, and the like, in the
presence of a base catalysts, such as pyridine, 2,6-lutidine, TEA,
DIEA and the like. In cases where acylation of surface groups is
less efficient under these conditions, nucleophilic catalysts such
as DMAP, NMI, HOBT, HOAT and the like, may also be included to
accelerate the reaction through the in situ generation of more
reactive acylating agents. This would typically be the case where a
derivative is preferred for its longer term stability in solution,
but is not sufficiently reactive without the addition of one or
more of the catalysts mentioned above. On automated synthesizers,
it is generally preferable to choose a reagent which can be stored
for longer terms as a stable solution and then activated with the
catalysts only when needed, i.e., in the reactor system flow cell,
or just prior to the addition of the reagent to the flow cell.
[0103] In addition to the protection of amine groups and hydroxyl
groups in peptide and oligonucleotide synthesis, the reagents and
methods described herein may be used to transfer photolabile
protecting groups directly to any nucleophilic group, either
tethered to a solid support or in solution.
[0104] A. Individual Processing
[0105] 1. Flow Cell/Reactor System
[0106] In one embodiment, the substrate preparation process of the
present invention combines the photolysis and chemistry steps in a
single unit operation. In this embodiment, the substrate wafer is
mounted in a flow cell during both the photolysis and chemistry or
monomer addition steps. In particular, the substrate is mounted in
a reactor system that allows for the photolytic exposure of the
synthesis surface of the substrate to activate the functional
groups thereon. Solutions containing chemical monomers are then
introduced into the reactor system and contacted with the synthesis
surface, where the monomers can bind with the active functional
groups on the substrate surface. The monomer containing solution is
then removed from the reactor system, and another photolysis step
is performed, exposing and activating different selected regions of
the substrate surface. This process is repeated until the desired
polymer arrays are created.
[0107] Reactor systems and flow cells that are particularly suited
for the combined photolysis/chemistry process include those
described in, e.g., U.S. Pat. No. 5,424,186, which is incorporated
herein by reference in its entirety for all purposes.
[0108] A schematic illustration of a device for carrying out the
combined photolysis/chemistry steps of the individual process, is
shown in FIGS. 3A and 3B. These figures show a cross-sectional view
of alternate embodiments of the reactor system 100. Referring first
to FIG. 3B, the device includes a flow cell which is made up of a
body 102 having a cavity 104 disposed in one surface. The cavity
generally includes fluid inlets 108 and outlets 110 for flowing
fluid into and through the cavity. The cavity may optionally
include ridges 106 on the back surface of the cavity to aid in
mixing the fluids as they are pumped into and through the cavity.
The substrate 112 is mounted over the cavity whereby the front
surface of the substrate wafer 114 (the surface upon which the
arrays are to be synthesized) is in fluid communication with the
cavity. The device also includes a fluid delivery system in fluid
connection with the fluid inlet 108 for delivering selected fluids
into the cavity to contact the first surface of the substrate. The
fluid delivery system typically delivers selected fluids, e.g.,
monomer containing solutions, index matching fluids, wash
solutions, etc., from one or more reagent reservoirs 118, into the
cavity via the fluid inlet 108. The delivery system typically
includes a pump 116 and one or more valves to select from the
various reagent reservoirs.
[0109] For carrying out the photolysis reactions, the device 100
also typically includes a light source 124, as described above. The
light source is shown through a photolithographic mask 128 and is
directed at the substrate 112. Directing the light source at the
substrate may generally be carried out using, e.g., mirrors 122
and/or lenses 120 and 126. Alternatively, as shown in FIG. 3B, the
mask 128 may be placed directly over the substrate 112, i.e.
immediately adjacent to the substrate, thereby obviating the need
for intervening lenses.
[0110] FIGS. 4A and 4B show different views of schematic
illustrations of one embodiment of the flow cell portion of the
device, e.g., the body substrate combination. As shown in FIGS. 4A
and 4B, a panel 320 is mounted to the body 102 to form the bottom
surface of the cavity 104. Silicone cement or other adhesive may be
used to mount the panel and seal the bottom of the cavity. In
particularly preferred aspects, panel 320 will be a light
absorptive material, such as yellow glass, RG1000 nm long pass
filter, or other material which absorbs light at the operating
wavelengths, for eliminating or minimizing reflection of impinging
light. As a result, the burden of filtering stray light at the
incident wavelength during synthesis is significantly lessened. The
glass panel also provides a durable surface for forming the cavity
since it is relatively immune to corrosion in the high salt
environments or other conditions common in DNA synthesis reactions
or other chemical reactions.
[0111] The substrate wafer 112 is mated to a surface 300. The first
surface 114 of wafer comprises the photolabile protecting groups
coupled to functional groups coupled to the substrate surface, as
described above. In some embodiments, vacuum pressure may be used
to mate the wafer to the surface 300. In such embodiments, a groove
304, which may be about 2 mm deep and 2 mm wide, is formed on
surface 300. The groove communicates with an opening 303 that is
connected to a vacuum source, e.g., a pump. The vacuum source
creates a vacuum in the groove and causes the substrate wafer to
adhere to surface 300.
[0112] A groove 310 may be formed on surface 300 for seating a
gasket 311 therein. The gasket ensures that the cavity is sealed
when the wafer is mated to the flow cell. Alignment pins 315 may be
optionally provided on surface 300 to properly align the substrate
wafer on the flow cell.
[0113] Inlet port 307 and outlet port 306 are provided for
introducing fluids into and flowing fluids out of the cavity. The
flow cell provides an opening 301 in which a flow tube 340 is
passed through for coupling to inlet port 307. Likewise, a flow
tube 341 is passed through opening 302 for coupling with outlet
port 306. Fittings 345 are employed to maintain the flow tubes in
position. Openings 301 and 302 advantageously position the flow
tubes so that the flow cell can easily and conveniently be mounted
on the synthesis system.
[0114] A pump, which is connected to one of the flow tubes,
circulates a selected fluid into the cavity and out through the
outlet port for recirculation or disposal. The selected fluids may
include, e.g., monomer containing solutions, index matching fluids,
wash solutions or the like. Although described in terms of a pump,
a variety of pressurized delivery systems may be used to deliver
fluids to the cavity. Examples of these alternate systems utilize
argon gas to circulate the selected fluid into and through the
cavity. Simultaneously, the flow of argon gas may be regulated to
create bubbles for agitating the fluid as it is circulated through
the system. Agitation is used to mix the fluid contents in order to
improve the uniformity and/or yield of the reactions.
[0115] As shown, inlet and outlet ports 306 and 307, respectively,
are located at opposite ends of the panel. This configuration
improves fluid circulation and regulation of bubble formation in
the cavity. In one embodiment, the outlet and inlet are located at
the top and bottom ends of the cavity, respectively, when the flow
cell is mounted vertically on the synthesizer. Locating the outlet
and inlet at the highest and lowest positions in the cavity,
respectively, facilitates the removal of bubbles from the
cavity.
[0116] In some embodiments, the flow cell may be configured with a
temperature control system to permit the synthesis reactions to be
conducted under optimal temperature conditions. Examples of
temperature control systems include refrigerated or heated baths,
refrigerated air circulating devices, resistance heaters,
thermoelectric peltier devices and the like.
[0117] In some instances, it may be desirable to maintain the
volume of the flow cell cavity as small as possible so as to more
accurately control reaction parameters, such as temperature or
concentration of chemicals. In addition to the benefits of improved
control, smaller cavity volumes may reduce waste, as a smaller
volume requires a smaller amount of material to carry out the
reaction.
[0118] For particularly small cavity volumes, a difficulty may
arise where bubbles in the reaction fluids can become trapped in
the cavity, which may result in incomplete exposure of the
substrate surface to the reaction fluid. In particular, when a
fluid fills into a very shallow channel or slit, it will tend to
fill the shallowest areas first, due to relatively strong capillary
forces in those areas. If the channel is too shallow, inconsistency
and non-flatness of the substrate which results in uneven capillary
forces, will lead to an uneven fluid front during filling. As the
liquid front loses its even shape, liquid may surround air or gas
pockets to produce trapped bubbles. Accordingly, where particularly
small cavity volumes are desired, a flow cell may be employed
wherein the top and bottom surfaces of the flow cell are
nonparallel, being narrower at the inlet of the flow cell, and
growing wider toward the outlet. Uniform filling of the flow cell
ensures that the fluid front maintains a straight shape, thereby
minimizing the potential of having bubbles trapped between the
surfaces.
[0119] A schematic illustration of one embodiment of an integrated
reactor system is shown in FIG. 3C. The device includes an
automated peptide synthesizer 401. The automated peptide
synthesizer is a device which flows selected reagents through a
flow cell 402 under the direction of a computer 404. In a preferred
embodiment the synthesizer is an ABI Peptide Synthesizer, model no.
431A. The computer may be selected from a wide variety of computers
or discrete logic including for, example, an IBM PC-AT or similar
computer linked with appropriate internal control systems in the
peptide synthesizer. The PC is provided with signals from the ABI
computer indicative of, for example, the beginning of a photolysis
cycle. One can also modify the synthesizer with a board that links
the contacts of relays in the computer in parallel with the
switches to the keyboard of the control panel of the synthesizer to
eliminate some of the keystrokes that would otherwise be required
to operate the synthesizer.
[0120] Substrate 406 is mounted on the flow cell, forming a cavity
between the substrate and the flow cell. Selected reagents flow
through this cavity from the peptide synthesizer at selected times,
forming an array of peptides on the face of the substrate in the
cavity. Mounted above the substrate, and preferably in contact with
the substrate is a mask 408. Mask 408 is transparent in selected
regions to a selected wavelength of light and is opaque in other
regions to the selected wavelength of light. The mask is
illuminated with a light source 410 such as a UV light source. In
one specific embodiment the light source 410 is a model no. 82420
made by Oriel. The mask is held and translated by an x-y
translation stage 412. Translation stages may be obtained
commercially from, e.g., Newport Corp. The computer coordinates the
action of the peptide synthesizer, translation stage, and light
source. Of course, the invention may be used in some embodiments
with translation of the substrate instead of the mask.
[0121] 2. Photolysis Step
[0122] As described above, photolithographic methods are used to
activate selected regions on the surface of the substrate.
Specifically, functional groups on the surface of the substrate or
present on growing polymers on the surface of the substrate, are
protected with photolabile protecting groups. Activation of
selected regions of the substrate is carried out by exposing
selected regions of the substrate surface to activation radiation,
e.g., light within the effective wavelength range, as described
previously. Selective exposure is typically carried out by shining
a light source through a photolithographic mask. Alternate methods
of exposing selected regions may also be used, e.g., fiberoptic
faceplates, etc. For the individual process methods, e.g., the
integrated photolysis/chemistry process, the substrate is mounted
in the reactor system or flow cell such that the synthesis surface
of the substrate is facing the cavity and away from the light
source. As the light source is shown on the surface opposite that
upon which the photoprotective groups are provided, this method of
exposure is termed "back-side" photolysis.
[0123] Because the individual feature sizes on the surface of the
substrate prepared according to the processes described herein can
typically range as low as 1-10 .mu.m on a side, the effects of
reflected or refracted light at the surface of the substrate can
have significant effects upon the ability to expose and activate
features of this size. One method of reducing the occurrence of
reflected light is to incorporate a light absorptive material as
the back surface of the flow cell, as described above. Refraction
of the light as it enters the flow cell, i.e., crosses the
substrate/flow cell interface, through the back surface of the
substrate can also result in a loss in feature resolution at the
synthesis surface of the substrate resulting from refraction and
reflection. To alleviate this problem, during the photolysis step,
it is generally desirable to fill the flow cell with an index
matching fluid ("IMF") to match the refractive index of the
substrate, thereby reducing refraction of the incident light and
the associated losses in feature resolution. The index matching
fluid will typically have a refractive index that is close to that
of the substrate. Typically, the refractive index of the IMF will
be within about 10% that of the substrate, and preferably within
about 5% of the refractive index of the substrate. Refraction of
the light entering the flow cell, as it contacts the interface
between the substrate and the IMF is thereby reduced. Where
synthesis is being carried out on, e.g., a silica substrate, a
particularly preferred IMF is dioxane which has a refractive index
roughly equivalent to the silica substrate.
[0124] The light source used for photolysis is selected to provide
a wavelength of light that is photolytic to the particular
protecting groups used, but which will not damage the forming
polymer sequences. Typically, a light source which produces light
in the UV range of the spectrum will be used. For example, in
oligonucleotide synthesis, the light source typically provides
light having a wavelength above 340 nm, to effect photolysis of the
photolabile protecting groups without damaging the forming
oligonucleotides. This light source is generally provided by a
Hg-Arc lamp employing a 340 nm cut-off filter (i.e., passing light
having a wavelength greater than 340-350 nm). Typical photolysis
exposures are carried out at from about 6 to about 10 times the
exposed half-life of the protecting group used, with from 8-10
times the half-life being preferred. For example, MeNPOC, a
preferred photolabile protecting group, has an exposed half-life of
approximately 6 seconds, which translates to an exposure time of
approximately 36 to 60 seconds.
[0125] Photolithographic masks used during the photolysis step
typically include transparent regions and opaque regions, for
exposing only selected portions of the substrate during a given
photolysis step. Typically, the masks are fabricated from glass
that has been coated with a light-reflective or absorptive
material, e.g., a chrome layer. The light-reflective or absorptive
layer is etched to provide the transparent regions of the mask.
These transparent regions correspond to the regions to be exposed
on the surface of the substrate when light is shown through the
mask.
[0126] In general, it is desirable to produce arrays with smaller
feature sizes, allowing the incorporation of larger amounts of
information in a smaller substrate area, allowing interogation of
larger samples, more definitive results from an interogation and
greater possibility of miniaturization. Alternatively, by reducing
feature size, one can obtain a larger number of arrays, each having
a given number of features, from a single substrate wafer. The
result is substantially higher product yields for a given process.
This technique, generally referred to as "die shrinking" is
commonly used in the semiconductor industry to enhance product
outputs or to reduce chip sizes following a over-sized test run of
a manufacturing process.
[0127] In seeking to reduce feature size, it is important to
maximize the contrast between the regions of the substrate exposed
to light during a given photolysis step, and those regions which
remain dark or are not exposed. By "contrast" is meant the
sharpness of the line separating an exposed region and an unexposed
region. For example, the gradient of activated to nonactivated
groups running from an activated or exposed region to a nonexposed
region is a measure of the contrast. Where the gradient is steep,
the contrast is high, while a gradual gradient indicates low or
poor contrast.
[0128] One cause of reduced contrast is "bleed-over" from exposed
regions to non-exposed regions during a particular photolysis step.
In certain embodiments, contrast between features is enhanced
through the front side exposure of the substrate. Front side
exposure reduces effects of diffraction or divergence by allowing
the mask to be placed closer to the synthesis surface.
Additionally, and perhaps more importantly, refractive effects from
the light passing through the substrate surface prior to exposure
of the synthesis surface are also reduced or eliminated by
front-side exposure. This is discussed in greater detail below.
[0129] Contrast between features may also be enhanced using a
number of other methods. For example, the level of contrast
degradation between two regions generally increases as a function
of the number of differential exposures or photolysis steps between
the two regions, i.e., incidences where one region is exposed while
the other is not. The greater the number of these incidences, the
greater the opportunity for bleed over from one region to the other
during each step and the lower the level of contrast between the
two regions regions. Translated into sequence information, it
follows that greater numbers of differences between polymers
synthesized in adjacent regions on a substrate can result in
reduced contrast between the regions. Namely, the greater the
number of differences in two polymer sequences, the greater the
number of incidences of a region bearing the first polymer being
exposed while the other was not. These effects are termed "edge"
effects as they generally occur at the outer edges of the
feature.
[0130] It is thus desirable to minimize these edge effects to
enhance contrast in synthesis. Accordingly, in one aspect, the
present invention provides a method of enhancing contrast by
reducing the number of differential synthesis/photolysis steps
between adjacent polymer sequence containing regions throughout an
array.
[0131] One method of edge minimization is to divide the polymers to
be sequenced into blocks of related polymers, leaving blank lanes
between the blocks to prevent bleed-over into other blocks. While
this method is effective in reducing edge effects, it requires the
creation of a specific algorithm for each new tiling strategy. That
is, the layout of each block in terms of probe location will depend
upon the tiled sequence. In one aspect, the present invention
provides methods for aligning polymer synthesis steps on an array
whereby the number of differential synthesis steps is reduced,
and/or the syntheses in adjacent regions of the array are optimized
for similarity.
[0132] The following example illustrates a typical synthesis
strategy. Assuming a simple array where a single possible mutation
is being explored at the third position in the sequence TGTATCA. An
array of complementary probes might be as follows: TABLE-US-00001
#1 ACATAGT #2 ACTTAGT #3 ACGTAGT #4 ACCTAGT
where position 3 has been substituted with each of the four
nucleotides. In synthesizing this array, monomer addition is
typically cycled through the four nucleosides in a given preset
order, e.g., 1-A, 2-C, 3-G, 4-T. Thus, for the array shown above,
the first "A" in each of the sequences would be coupled in the
first cycle. The second "C" would be coupled in the second monomer
addition cycle. Each of the substituted positions would then be
coupled in their respective cycle, e.g., the "A" in probe #1 would
be coupled in the fifth cycle, while the "T", "G", and "C" would be
coupled in the sixth, seventh, and eighth cycles, respectively.
[0133] Up to this point, each probe has been exposed to a minimal
number of differential exposures, as described above. However, the
monomer addition steps following the substituted monomer give rise
to some difficulties in this regard. For example, it would be
possible to couple the "T" in the fourth position in probe #1 at
the sixth cycle while the "T" in the remaining probes would have to
be added at the tenth cycle, because they could not be added before
the preceding monomer in the sequence. The remaining synthesis
steps for probe 1 would then be out of sequence with those of the
remaining probes, resulting in an increased number of differential
sequence steps between probe 1 and the remaining probes. By
aligning the addition of the "T" monomer in probe #1 with that of
the remaining probes, the number of differential synthesis steps is
minimized. Specifically, by waiting until the tenth cycle to add
the "T" in probe #1, the number of differential exposures between
the probes is minimized to only that number necessary to
incorporate the various mutations or substitutions.
[0134] The methods described herein utilize a generalized synthesis
method for aligning synthesis steps to accomplish the
above-described goal. These generalized methods can be followed
regardless of the particular tiling strategy used or targeted
sequence.
[0135] In particular, the methods described herein, identify each
probe by a generic structure which is effectively independent of
the actual targeted sequence. This generic description of a probe
sequence is termed an "image", a collection of polymer sequences is
termed a "picture", and a local translation, e.g., in a larger
targeted sequence, is termed a "frame". The entire picture and
frame structure is termed a "collage".
[0136] Each position in the probe is designated by the position
number in the frame, or targeted sequence segment, followed by a
number that indicates the rotation from the wild type monomer, with
the wild type monomer being "0". By rotation is meant the number of
cycles required to go from the wild type monomer to the substituted
monomer in the addition cycle (note that a "0" and a "4" are the
same monomer in terms of nucleotides). For example, if a given wild
type sequence has an "A" in a given position, a substitution to a
"G" would be identified by a rotation of "3", assuming a monomer
addition or synthesis cycle of A, C, T, G.
[0137] In terms of the above example, probe #1, being the same as
the wild type target as also described above, would be identified
as: TABLE-US-00002 #1 <1, 0> <2, 0> <3, 0> <4,
0> <5, 0> <6, 0> <7, 0>
[0138] where each position is not rotated from the wild type, or is
"unmodified." The remaining sequences would be identified as:
TABLE-US-00003 #2 <1, 0> <2, 0> <3, 1> <4,
0> <5, 0> <6, 0> <7, 0> #3 <1, 0> <2,
0> <3, 2> <4, 0> <5, 0> <6, 0> <7,
0> #4 <1, 0> <2, 0> <3, 3> <4, 0> <5,
0> <6, 0> <7, 0>
indicating a rotation in the third position for each of the
nucleoside monomers.
[0139] Sequence positions which are in the same layer are aligned
to be added in the same synthesis cycle. The "depth" of the
sequence or the "layer" in which a given monomer is found, are
determined by counting each occurrence where an unmodified base
follows a modified base. Each sequence has a depth of at least one.
For example, the sequence "X" indicated by the
<1,1><2,0><3,0> has a depth of 2, where
<2,0> and <3,0> are in the second layer. Similarly, the
sequence "Y" identified as <1,0><2,1><3,0> has a
depth of two where <1,0> is in the first layer and
<3,0> is in the second layer. Aligning these two sequences,
it can be seen that the monomer <3,0> in sequences X and Y
may be aligned as it exists in the same layer.
[0140] In contrast, the sequence
"Z"<1,0><2,0><3,1> has a depth of one with
<1,0><2,0> in the first layer. Thus, the position
<2,0> in the sequence X would not be aligned with the same
position in sequence Z as they exist in different layers.
[0141] A specific example of the collage method is illustrated
using the following sequence/tiling strategy. A targeted sequence
is complementary to the sequence CTTA. Thus, written in the
above-described generic style, the wild type sequence would be
designated <1,0><2,0><3,0><4,0>. Assuming a
simplified tiling strategy where each position was to be
substituted with a monomer rotated one from the wild type, the
array would have the generic description: TABLE-US-00004 #1 <1,
1> <2, 0> <3, 0> <4, 0> #2 <1, 0> <2,
1> <3, 0> <4, 0> #3 <1, 0> <2, 0> <3,
1> <4, 0> #4 <1, 0> <2, 0> <3, 0> <4,
1>
[0142] which would correspond to the sequences: TABLE-US-00005 #1 G
T T A #2 C A T A #3 C T A A #4 C T T C
[0143] The assignment of bases of each layer to a particulr cycle
is termed a "frame." For example, the frame for the above synthesis
would be as follows: TABLE-US-00006 Layer 1 Layer 2 <1, 0> =
2 <---- <2, 0> = 4 <2, 0> = 8 <3, 0> = 8
<3, 0> = 12 <4, 0> = 13
[0144] Once monomers in the same layer are aligned, the synthesis
is carried out with the following aligned cycle assignments:
TABLE-US-00007 Cycle A C G T A C G T A C G T A C G T Layer 2 #1 3 8
12 13 #2 2 5 12 13 #3 2 4 5 13 #4 2 4 8 10 Layer 1
[0145] The bases in the first layer are assigned the cycles closest
to the start of the synthesis. The modified bases (between the
layers) are assigned the next available cycles. The second layer is
assigned a set of cycles as close as possible to the start of
synthesis consistent with the bases already assigned (i.e., without
altering the base ordering of any of the probes). Subsequent layers
are assigned in a similar manner. This method allows maximum
alignment of synthesis cycles throughout the frame being
synthesized, while minimizing the total length of synthesis (e.g.,
number of steps).
[0146] Another method of minimizing bleed-over in the photolysis
steps is to reduce the size of the transmissive or translucent
portion of the mask, thus preventing unintentional exposure of
adjoining regions caused by diffraction of the light shown through
the mask. In particular, typical photolysis steps can have a
duration of up to 8 to 10 times the half-life of the
photodeprotection reaction. Thus, photoprotection can be up to 50%
complete where the light intensity is only 12% of optimal levels,
i.e., the level required for complete or near complete
photodeprotection. Typically, such intensity levels may be reached
well outside the feature boundary as defined by the transmissive
portion of the mask.
[0147] Reducing the size of the transmissive portion of the mask
allows diffraction, scattering and divergence at the edges of each
feature without that diffraction interfering with neighboring
features. Thus, the region of incomplete photolysis can be centered
on the desired boundary between features. As a result, the total
area of the chip that is compromised in a multi-step synthesis is
minimized because bleed-over effects from each region are centered
in the boundary rather than well into the neighboring feature.
Accordingly, in one aspect of the present invention provides a
method of minimizing bleed-over in adjoining cells by reducing the
size of the transmissive portion of the mask, such that the zone of
divergent light shown through the mask is centered on the desired
feature border. As an example, a mask exposing a rectangular
feature can be reduced by, e.g., 20 .mu.m in each dimension, thus
allowing greater homogeneity at the edges of 100 .mu.m features. In
preferred aspects, the translucent region of the mask will be from
about 2% to about 25% smaller in each dimension of the size of the
region which is to be exposed. In more preferred aspects, the
translucent portion of the mask will be from about 10% to about 25%
smaller in each dimension.
[0148] 3. Chemistry Step
[0149] Following each photolysis step, a monomer building block is
introduced or contacted with the synthesis surface of the
substrate. Typically, the added monomer includes a single active
functional group, for example, in the case of oligonucleotide
synthesis, a 3'-hydroxyl group. The remaining functional group that
is involved in linking the monomer within the polymer sequence,
e.g., the 5'-hydroxyl group of a nucleotide, is generally
photoprotected. The monomers then bind to the reactive moieties on
the surface of the substrate, activated during the preceding
photolysis step, or at the termini of linker molecules or polymers
being synthesized on the substrate.
[0150] Typically, the chemistry step involves solid phase polymer
synthesis methods that are well known in the art. For example,
detailed descriptions of the procedures for solid phase synthesis
of oligonucleotides by phosphoramidite, phosphite-triester,
phosphotriester, and H-phosphonate chemistries are widely
available. See, for example, Itakura, U.S. Pat. No. 4,401,796;
Caruthers et al., U.S. Pat. Nos. 4,458,066 and 4,500,707; Beaucage
et al., Tetrahedron Lett., 22:1859-1862 (1981); Matteucci et al.,
J. Amer. Chem. Soc., 103:3185-3191 (1981); Caruthers et al.,
Genetic Engineering, 4:1-17 (1982); Jones, chapter 2, Atkinson et
al., chapter 3, and Sproat et al., chapter 4, in Gait, ed.
Oligonucleotide Synthesis: A Practical Approach, IRL Press,
Washington D.C. (1984); Froehler et al., Tetrahedron Lett.,
27:469-472 (1986); Froehler et al., Nucleic Acids Res.,
14:5399-5407 (1986); Sinha et al. Tetrahedron Lett., 24:5843-5846
(1983); and Sinha et al., Nucl. Acids Res., 12:4539-4557
(1984).
[0151] In operation, during the chemistry/monomer addition step,
the IMF is removed from the flow cell through an outlet port. The
flow cell is then rinsed, e.g., with water and/or acetonitrile.
Following rinsing, a solution containing an appropriately protected
monomer to be coupled in the particular synthesis step is added.
For example, where the synthesis is of oligonucleotide probe
arrays, being synthesized in the 3' to 5' direction, a solution
containing a 3'-O-activated phosphoramidite nucleoside,
photoprotected at the 5' hydroxyl is introduced into the flow cell
for coupling to the photoactivated regions of the substrate.
Typically, the phosphoramidite nucleoside is present in the monomer
solution at a concentration of from 1 mM to about 100 mM, with 10
mM nucleoside concentrations being preferred. Typically, the
coupling reaction takes from 30 seconds to 5 minutes and preferably
takes about 1.5 minutes.
[0152] Following coupling, the monomer solution is removed from the
flow cell, the substrate is again rinsed, and the IMF is
reintroduced into the flow cell for another photolysis step. The
photolysis and chemistry steps are repeated until the substrate has
the desired arrays of polymers synthesized on its surface.
[0153] For each photolysis/chemistry cycle, it will generally be
desirable to maximize coupling efficiencies in order to maximize
probe densities on the arrays. Coupling efficiencies may be
improved through a number of methods. For example, coupling
efficiency may be increased by increasing the lipophilicity of the
building blocks used in synthesis. Without being bound to any
theory of operation, it is believed that such lipophilic building
blocks have enhanced interaction at the surface of the preferred
crystalline substrates. The lipophilicity of the building blocks
may generally be enhanced using a number of strategies. In
oligonucleotide synthesis, for example, the lipophilicity of the
nucleic acid monomers may be increased in a number of ways. For
example, one can increase the lipophilicity of the nucleoside
itself, the phosphoramidite group, or the protecting group used in
synthesis.
[0154] Modification of the nucleoside to increase its lipophilicity
generally involves specific modification of the nucleobases. For
example, deoxyguanosine (dG) may be alkylatea on the exocyclic
amino group (N2) with DMT-Cl, after in situ protection of both
hydroxyl groups as trimethylsilylethers (See, FIG. 5A). Liberation
of the free DMT protected nucleoside is achieved by base catalyzed
methanolosis of the di-TMS ether. Following standard procedures,
two further steps are used resulting in the formation of
5'-MeNPOC-dG-phosphoramidites. The DMT group is used because the
normally used 5'-DMT-phosphoramidites show high coupling
efficiencies on silica substrate surfaces and because of the ease
of synthesis for the overall compound. The use of acid labile
protecting groups on the exocyclic amino groups of dG allows
continued protection of the group throughout light-directed
synthesis. Similar protection can be used for other nucleosides,
e.g., deoxycytosine (dC). Protection strategies for nucleobase
functional groups, including the exocyclic groups are discussed in
U.S. patent application Ser. No. 08/445,332 filed May 19, 1995,
previously incorporated herein by reference.
[0155] A more lipophilic phosphoramidite group may also be used to
enhance synthesis efficiencies. Typical phosphoramidite synthesis
utilizes a cyanoethyl-phosphoramidite. However, lipophilicity may
be increased through the use of, e.g., an Fmoc-phosphoramidite
group. Synthesis of Fmoc-phosphoramidites is shown in FIG. 5B.
Typically, a phosphorus-trichloride is reacted with four
equivalents of diisopropylamine, which leads to the formation of
the corresponding monochloro-bisamino derivative. This compound
reacts with the Fmoc-alcohol to generate the appropriate
phosphatidylating agent.
[0156] As with the phosphoramidite group, the photolabile
protecting groups may also be made more lipophilic. For example, a
lipophilic substituent, e.g., benzyl, naphthyl, and the like, may
be introduced as an alkylhalide, through .alpha.-akylation of a
nitroketone, as shown in FIG. 5C. Following well known synthesis
techniques, one generates the chloroformate needed to introduce the
photoactive lipophilic group to the 5' position of a
deoxyribonucleoside.
[0157] B. Batch Processing
[0158] In a second embodiment of the substrate preparation process,
each of the photolysis and chemistry steps involved in the
synthesis operation are provided as separate unit operations. This
method provides advantages of efficiency and higher feature
resolution over the single unit operation process. In particular,
the separation of the photolysis and chemistry steps allows
photolysis to be carried out outside of the confines of the flow
cell. This permits application of the light directly to the
synthesis surface, i.e., without first passing through the
substrate. This "front-side" exposure allows for greater definition
at the edges of the exposed regions (also termed "features") by
eliminating the refractive influence of the substrate and allowing
placement of the mask closer to the synthesis surface. A comparison
illustrating the improved resolution of front-side synthesis is
shown in FIGS. 8A-8D.
[0159] In addition to the benefits of front side exposure, the
batch method provides advantages in the surface area of a substrate
wafer that may be used in synthesizing arrays. In particular, by
combining photolysis/chemistry aspects in the individual process
methods, the operation of mounting the substrate wafer on the flow
cell can result in less than the entire surface of the substrate
wafer being used for synthesis. In particular, where the substrate
wafer is used to form one wall of the flow cell, as is typically
the case in these combined methods, engineering constraints
involved in mounting of the flow cell can result in a reduction in
the available substrate surface area. This is particularly the case
where a vacuum chuck system is used to mount the substrate on the
flow cell, where the vacuum chuck system requires a certain amount
of surface area to hold the substrate on the flow cell with
sufficient force.
[0160] In batch mode operation, the chemistry step is generally
carried out by immersing the entire substrate wafer in the monomer
solution, thus allowing synthesis over most if not all of the
substrate wafer's synthesis surface. This results in a higher chip
yield per substrate wafer than in the individual processing
methods. Additionally, as the chemistry steps are generally the
time limiting steps in the synthesis process, monomer addition by
immersion permits monomer addition to multiple substrates at a
given time, while more substrates are undergoing the photolysis
steps.
[0161] For example, where synthesis is performed in the individual
processing operation, as described above, the engineering
constraints in vacuum mounting a substrate to a flow cell can
result in a significant decrease in the size of a synthesis area on
the substrate wafer. For example, in one process, a substrate wafer
having dimensions of 5.times.5'' has only 2.5.times.2.5'' available
as a synthesis surface, which when separated into chips of typical
dimensions (e.g., 1.28 cm.times.1.28 cm) typically results in 16
potential chips per wafer. The same sized wafer, when subjected to
the batch mode synthesis can have a synthesis area of about
4.3.times.4.3'', which can produce approximately 49 chips per
wafer.
[0162] In general, a number of substrate wafers is subjected to the
photolysis step. Following photolysis, the number of wafers is
placed in a rack or "boat" for transport to the station which
performs the chemistry steps, whereupon one or more chemistry steps
are performed on the wafers, simultaneously. The wafers are then
returned to the boat and transported back to the station for
further photolysis. Typically, the boat is a rack that is capable
of carrying several wafers at a time and is also compatible with
automated systems, e.g., robotics, so that the wafers may be loaded
into the boat, transported and placed into the chemistry station,
and following monomer addition returned to the boat and the
photolysis station, all through the use of automated systems.
[0163] Initial substrate preparation is the same for batch
processing as described in the individual processing methods,
above. However, beyond this initial substrate preparation, the two
process take divergent paths. In batch mode processing, the
photolysis and chemistry steps are performed separately. As is
described in greater detail below, the photolysis step is generally
performed outside of the flow cell. This can cause some
difficulties, as there is no provision of an IMF behind the
substrate to prevent the potentially deleterious effects of
refraction and reflection of the photolytic light source. In some
embodiments, however, the same goal is accomplished by applying a
coating layer to the back-side of the substrate, i.e., to the
non-synthesis surface of the substrate. The coating layer is
typically applied after the substrate preparation process, but
prior to derivatization. This coating is typically selected to
perform one or more of the following functions: (1) match the
refractive index of the substrate to prevent refraction of light
passing through the substrate which may interfere with the
photolysis; and (2) absorb light at the wavelength of light used
during photolysis, to prevent back reflection which may also
interfere with photolysis.
[0164] Typically, suitable coating materials may be selected from a
number of suitable materials which have a refractive index
approximately equal to that of the substrate and/or absorb light at
the appropriate wavelength. In particular, index matching coatings
are typically selected to have a refractive index that is within at
about 10% that of the substrate, and preferably within about 5%.
Similarly, light absorbing coatings are typically selected whereby
light at the photolytic wavelength is absorbed, which in preferred
aspects is light in the ultraviolet range, e.g., between 280 nm and
400 nm. Light absorbing coatings and index matching coatings may be
combined to provide combined protection against refraction and
reflection, or a single coating material may be selected which
possesses both of the desired properties.
[0165] Preferred polymers will typically be selected to be
compatible with the various reaction conditions which would be
encountered during the synthesis process, e.g., insoluble in and
non-reactive with synthesis reagents, and resistant to the
mechanical forces involved in handling and manipulating the
substrate, throughout the synthesis process. Additionally,
preferred coating materials are easily removable upon completion of
the synthesis process, e.g., in the final deprotection step or in a
final coating removal step.
[0166] Examples of suitable coating materials include
anti-reflective coatings that are well known in the art and
generally commercially available, e.g., magnesium fluoride
compounds, which are light-absorbing in the desired wavelength
range, polymethylmethacrylate coatings (PMMA), which have a
refractive index comparable to glass substrates, and polyimide
coatings which are both light-absorbing in the desired wavelength
range, and have a refractive index close to that of a glass
substrate. Polyimide coatings are most preferred.
[0167] Application of the coating materials may be carried out by a
variety of methods, including, e.g., vapor deposition, spray
application, and the like. In preferred aspects, the coating
solution will be applied to the substrate using a spin-coating
method. Typically, this involves spinning the substrate during
deposition of the coating solution on the substrate surface that is
to be coated. The spinning substrate results in spreading of the
coating solution radially outward on the surface of the
substrate.
[0168] Application of the coating material using the spin-coating
process usually employs a two-speed spinning of the substrate. The
application of the coating material to the surface of the substrate
and initial spreading of the coating solution are usually carried
out at low rotational speeds and for relatively short duration. For
example, to apply 1 ml of a 12% solids w/v polymer coating solution
to a 4.3''.times.4.3'' substrate, initial spreading is carried out
at 500 r.p.m. for 10 seconds. Elimination of excess polymer
solution and evening of the polymer layer are carried out at higher
rotational speeds and for substantially longer durations. For
example in the application described above, the second spinning
step is carried out at approximately 3000 r.p.m. for 30 seconds. It
will be understood by those of skill in the art, that the above
described parameters for spin-coating can be varied within the
scope of the present invention. For example, where higher
concentration (w/v) polymer solutions are used, it may be desirable
to increase one or both rotational speeds, as well as the time at a
given speed. Similarly, where the polymer concentration in the
polymer solution is reduced, lower speeds and shorter spin times
may be used.
[0169] Following application, the polymer coating is then cured on
the surface of the substrate. Curing is typically carried out by
heating the coated substrate. In preferred processes, the curing
process involves a two-step heating process. The first step
involves a "soft-bake" heating of the coated substrate to initially
cure the polymer coating. This soft-bake step typically takes place
at relatively low temperatures for relatively short periods, i.e.,
85.degree. C. for 5 minutes. The second step of the curing process
is a final curing of the polymer coating which is typically carried
out at higher temperatures for longer periods, i.e.,
220-360.degree. C., for approximately 60 minutes. In preferred
aspects, a polymer coating applied to the back side of the
substrate will be from about 1 to about 50 .mu.m thick, and more
preferably, from about 5 to about 20 .mu.m thick, with polymer
coating of about 10 .mu.m thick being most preferred.
[0170] The back-side coated substrate is then subjected to
derivitization, rinsing and baking, according to the above
described methods.
[0171] As described previously, the steps of photolysis and monomer
addition in the batch mode aspects of the present invention are
performed in separate unit operations. Separation of photolysis and
chemistry steps allows a more simplified design for a photolyzing
apparatus. Specifically, the apparatus need not employ a flow cell.
Additionally, the apparatus does not need to employ a particular
orientation to allow better filling of the flow cell. Accordingly,
the apparatus will typically incorporate one or more mounting
frames to immobilize the substrate and mask during photolysis, as
well as a light source. The device may also include focusing
optics, mirrors and the like for directing the light source through
the mask and at the synthesis surface of the substrate. As
described above, the substrate is also placed in the device such
that the light from the light source impacts the synthesis surface
of the substrate before passing through the substrate. As noted
above, this is termed "front-side" exposure.
[0172] Typically a photolysis step requires far less time than a
typical chemistry step, e.g., 60 seconds as compared to 10 minutes.
Thus, in the individual processing mode where the photolysis and
chemistry steps are combined, the photolysis machinery sits idle
for long periods of time during the chemistry step. Batch mode
operation, on the other hand, allows numerous substrates to be
photolyzed while others are undergoing a particular chemistry step.
For example, a number of substrate wafers may be exposed for a
given photolysis step. Following photolysis, the several substrate
wafers may be transferred to a number of reaction chambers for the
monomer addition step. While monomer addition is being carried out,
additional substrate wafers may be undergoing photolysis.
[0173] FIG. 6A schematically illustrates a bank of reaction
chambers for carrying simultaneous monomer addition steps on a
number of separate substrates in parallel. As shown, the bank of
reaction chambers is configured to simultaneously perform identical
synthesis steps in each of the several reaction chambers. Each
reaction chamber 602 is equipped with a fluid inlet 604 and outlet
606 for flowing various fluids into and through the reaction
chamber. The fluid inlet of each chamber is generally fluidly
connected to a manifold 608 which connects all of the reaction
chambers, in parallel, to a single valve assembly 610. Typically,
rotator valves are preferred for this aspect of the apparatus. The
valve assembly allows the manifold to be fluidly connected to one
of a plurality of reagent vessels 612-622. Also included is a pump
624 for delivering the various reagents to the reaction chamber.
Although primarily described as performing the same synthesis steps
in parallel, the bank of reaction chambers could also be readily
modified to carry out to perform multiple independent chemistry
steps. The outlet ports 606 from the reaction chambers 602 are
typically fluidly connected to a waste vessel (not shown).
[0174] FIG. 6B shows a schematic representation of a single
reaction chamber for performing the chemistry steps of the batch
process, e.g., monomer addition. As shown, the reaction chamber
employs a "clam-shell" design wherein the substrate is enclosed in
the reaction chamber 602 when the door 652 is closed against the
body 654 of the apparatus. More particularly, the substrate wafer
660 is mounted on the chamber door and held in place, e.g., by a
vacuum chuck shown as vacuum groove 670. When the door 652 is
closed, the substrate wafer 668 is placed into the reactor cavity
656 on the body of the device. The reactor cavity is surrounded by
a gasket 658, which provides the seal for the reaction chamber when
the door is closed. Upon closing the door, the substrate wafer is
pressed against the gasket and the pressure of this contact seals
the reaction chamber. The reaction chamber includes a fluid inlet
604 and a fluid outlet 606, for flowing monomer solutions into and
out of the reaction chamber.
[0175] The apparatus may also include latches 666, for locking the
reaction chamber in a sealed state. Once sealed, reagents are
delivered into the reaction chamber through fluid inlet 662 and out
of the reaction chamber through fluid outlet 664. The reaction
chamber also typically includes a temperature control element for
maintaining the reaction chamber at the optimal synthesis
temperature. As shown, the reaction chamber includes automatic
alignment pins 672, e.g., solenoid or servo operated, for aligning
a substrate wafer on the vacuum groove 670.
[0176] Following a monomer addition step, the substrate wafers are
each subjected to a further photolysis step. The process may
generally be timed whereby during a particular chemistry step, a
new series of wafers is being subjected to a photolysis step. This
dramatically increases the throughput of the process.
[0177] Following overall synthesis of the desired polymers on the
substrate wafers, permanent protecting groups, e.g., those which
were not removed during each synthesis step, typically remain on
nucleobases and the phosphate backbone of synthetic
oligonucleotides. Removal of these protecting groups is usually
accomplished with a concentrated solution of aqueous ammonium
hydroxide. While this method is effective for the removal of the
protecting groups, these conditions can also cleave the synthetic
oligomers from the support (usually porous silica particles) by
hydrolyzing an ester linkage between the oligo and a functionalized
silane derivative that is bonded to the support. In VLSIPS
oligonucleotide arrays, it is desirable to preserve the linkage
connecting the oligonucleotides to the glass after the final
deprotection step. For this reason, synthesis is carried out
directly on glass which is derivatized with a
hydroxyalkyl-trialkoxysilane (e.g.,
bis(hydroxyethyl)aminopropylsilane). However, these supports are
not completely stable to the alkaline hydrolysis conditions used
for deprotection. Depending upon the duration, substrates left in
aqueous ammonia for protracted periods can suffer a loss of probes
due to hydroxide ion attack on the silane bonded phase.
[0178] Accordingly, in preferred embodiments, final deprotection of
the polymer sequences is carried out using anhydrous organic
amines. In particular, primary and secondary alkylamines are used
to effect final deprotection. The alkylamines may be used undiluted
or in a solution of an organic solvent, e.g. ethanol, acetonitrile,
or the like. Typically, the solution of alkyl amine will be at
least about 50% alkylamine (v/v). A variety of primary and
secondary amines are suitable for use in deprotection, including
ammonia, simple low molecular weight (C.sub.1-4)alkylamines, and
substituted alkylamines, such as ethanolamine and ethylenediamine.
More volatile amines are preferred where removal of the
deprotection agent is to be carried out by evaporation, whereas the
less volatile amines are preferred in instances where it is
desirable to maintain containment of the deprotection agent and
where the solutions are to be used in repeated deprotections.
Solutions of ethanolamine or ethylenediamine in ethanol have been
used in deprotecting synthetic oligonucleotides in solution. See,
Barnett, et al., Tet. Lett. (1981) 22:991-994, Polushin, et al,
(1991) N.A.R. Symp. Ser. No. 24:49-50 and Hogrefe, et al. N.A.R.
(1993) 21:2031-2038.
[0179] Depending upon the protecting groups to be removed, the time
required for complete deprotection in these solutions ranges from
several minutes for "fast" base-protecting groups, e.g. PAC or
DMF-protected A, C or G and Ibu-protected C, to several hours for
the standard protecting groups, e.g. benzoyl-protected A, C or G
and Ibu-protected G,. By comparison, even the fast protecting
groups require 4-8 hours for complete removal in aqueous ammonia.
During this time, a significant percentage (e.g., 20-80%) of probes
are cleaved from a glass substrate through hydrolytic cleavage of
the silane layer, whereas after 48 hours of exposure to 50%
ethanolic ethylenediamine solution, 95% of the probes remain on the
substrate.
VI. Assembly of Probe Array Cartridges
[0180] Following synthesis, final deprotection and other finishing
steps, e.g. polymer coat removal where necessary, the substrate
wafer is assembled for use as individual substrate segments.
Assembly typically employs the steps of separating the substrate
wafer into individual substrate segments, and inserting or
attaching these individual segments to a housing which includes a
reaction chamber in fluid communication with the front surface of
the substrate segment, e.g., the surface having the polymers
synthesized thereon.
[0181] Methods of separating and packaging substrate wafers are
described in substantial detail in Published PCT Application No.
95/33846, which is hereby incorporated herein by reference in its
entirety for all purposes.
[0182] Typically, the arrays are synthesized on the substrate wafer
in a grid pattern, with each array being separated from each other
array by a blank region where no compounds have been synthesized.
These separating regions are termed "streets". The wafer typically
includes a number of alignment marks located in these streets.
These marks serve a number of purposes, including aligning the
masks during synthesis of the arrays as described above, separation
of the wafer into individual chips and placement of each chip into
its respective housing for subsequent use, which are both described
in greater detail below. An illustration of a wafer including these
alignment marks is shown in FIG. 7. As shown, substrate wafer 700
includes individual arrays 710 separated by streets 720 and
includes alignment marks 730.
[0183] Generally, the substrate wafer can be separated into a
number of individual substrates using scribe and break methods that
are well known in the semiconductor manufacturing industry. For
example, well known scribe and break devices may be used for
carrying out the separation steps, e.g., a fully programmable
computer controlled scribe and break devices, such as a DX-III
Scriber-Breaker manufactured by Dynatex International.TM., or the
LCD-1 scriber/dicer manufactured by Loomis Industries. The steps
typically involve scribing along the desired separation points,
e.g., between the individual synthesized arrays on the substrate
wafer surface, followed by application of a breaking force along
the scribe line. For example, typical scribe and break devices
break the wafer by striking the bottom surface of the wafer along
the scribe lines with an impulse bar, or utilizing a three point
beam substrate bending operation. The shock from the impulse bar
fractures the wafer along the scribe line. Because the majority of
force applied by the impulse bar is dissipated along the scribe
line, the device is able to provide high breaking forces without
exerting significant force on the substrate itself, allowing
separation of the wafer without damaging the individual chips.
[0184] In alternative methods, the wafer may be separated into
individual segments by, e.g., sawing methods, such as those
described in U.S. Pat. No. 4,016,855.
[0185] Once the wafer is separated into individual segments, these
segments may be assembled in a housing that is suited for the
particular analysis for which the array will be used. Examples of
methods and devices for assembling the substrate segments or arrays
in cartridges are described in, e.g., U.S. patent application Ser.
No. 08/485,452, previously incorporated by reference. Typically,
the housing includes a body having a cavity disposed within it. The
substrate segment is mounted over the cavity on the body such that
the front side of the segment, e.g., the side upon which the
polymers have been synthesized, is in fluid communication with the
cavity. The bottom of the cavity may optionally include a light
absorptive material, such as a glass filter or carbon dye, to
prevent impinging light from being scattered or reflected during
imaging by detection systems. This feature improves the
signal-to-noise ratio of such systems by significantly reducing the
potential imaging of undesired reflected light.
[0186] The cartridge also typically includes fluid inlets and fluid
outlets for flowing fluids into and through the cavity. A septum,
plug, or other seal may be employed across the inlets and/or
outlets to seal the fluids in the cavity. The cartridge also
typically includes alignment structures, e.g., alignment pins,
bores, and/or an asymmetrical shape to ensure correct insertion
and/or alignment of the cartridge in the assembly devices,
hybridization stations, and reader devices.
[0187] An illustration of one embodiment of the array cartridge is
shown in FIG. 8. FIG. 8 shows a top view 802, end view 804, side
view 806 and bottom view 808 of the array cartridge 800. The body
of the array cartridge may generally be fabricated from one or more
parts or casings 810-814 that are made using a number of
manufacturing techniques. In preferred aspects, the cartridge is
fabricated from two or more injection molded plastic parts.
Injection molding enables the parts to be formed inexpensively.
Also, assembling the cartridge from two parts simplifies the
construction of various features, such as the internal channels for
introducing fluids into the cavity. As a result, the cartridges may
be manufactured at a relatively low cost.
[0188] The top and bottom views of the cartridge include alignment
structures, such as alignment holes 816 and 818. As shown, these
alignment holes are disposed through the body of the cartridge,
however, those of ordinary skill will appreciate that other
alignment structures, e.g., alignment pins, etc., would be equally
useful. As shown in the bottom view 808, alignment holes 816 and
818 also include an annular bevelled region to assist in insertion
of complementary alignment pins on the hybridization station.
[0189] Referring to the top view 802 of the cartridge 800, cavity
820 includes a flat bottom peripheral portion 822, a bevelled
portion 824 extending from the flat bottom peripheral portion, and
a flat upper portion 826 surrounding the bevelled portion. The
array includes an outer periphery which rests against the flat
bottom peripheral portion 822. The bevelled portion aligns the chip
onto the flat bottom peripheral portion 822. As shown, the top
casing 814 extends outside the middle and bottom casings, 812 and
810, respectively, to provide a nonflush edge 828. The alignment
structures 816 and 818, as well as the non flush edge 828, ensure
proper orientation of the cartridge in the hybridization station,
as well as other devices used in producing and reading polymer
arrays. Surrounding mounting structures 816 and 818 are annular
recesses 817 and 819, respectively, which aid in guiding the
cartridge onto complementary mounting structures on the various
devices.
[0190] As shown in the bottom view 808, the cartridge includes
inlet and outlet ports 830 and 834, which include a bevelled
annular region 832 and 836 surrounding these ports, respectively,
to assist with fluid flow therethrough. Typically, the inlet and
outlet ports will include septa disposed across the ports (not
shown). Bottom casing 810 also includes a cavity 838, located
adjacent the array, which cavity may be adapted for receiving a
temperature monitoring and/or controlling device. As shown the
cavity 838 has an annular recessed region 839 surrounding it, to
ensure that the temperature controller may be inserted with maximum
ease.
[0191] The array cavity 820 is preferably located at a center of
the bottom casing, but may also be at other locations. The cavity
may be round, square, rectangular, or any other shape, and
orientation. The cavity is preferably smaller than the surface area
of the chip to be placed thereon, and has a volume sufficient to
perform hybridization and the like. In one embodiment, the cavity
includes dimensions such as a length of about 0.6 inch, a width of
about 0.6 inch and a depth of about 0.07 inch.
[0192] In a preferred embodiment, the bottom casing with selected
cavity dimensions may be removed from the middle and top casings,
and replaced with another bottom casing with different cavity
dimensions. This allows a user to attach a chip having a different
size or shape by changing the bottom casing, thereby providing ease
in using different chip sizes, shapes, and the like. Of course, the
size, shape, and orientation of the cavity will depend upon the
particular application. The body of the cartridge may generally be
fabricated from one or more parts made using a number of
manufacturing techniques. In preferred aspects, the cartridge is
fabricated from two or more injection molded plastic parts.
Injection molding enables the casings to be formed inexpensively.
Also, assembling the cartridge from two parts simplifies the
construction of various features, such as the internal channels for
introducing fluids into the cavity. As a result, the cartridges may
be manufactured at a relatively low cost.
[0193] The substrate segment may be attached to the body of the
cartridge using a variety of methods. In preferred aspects, the
substrate is attached using an adhesive. Preferred adhesives are
resistant to degradation under conditions to which the cartridge
will be subjected. In particularly preferred aspects, an
ultraviolet cured adhesive attaches the substrate segment to the
cartridge. Devices and methods for attaching the substrate segment
are described in Published PCT Application No. 95/33846, previously
incorporated by reference. Particularly preferred adhesives are
commercially available from a variety of commercial sources,
including Loctite Corp. and Dymax Corp.
[0194] A variety of modifications can be incorporated in the
assembly methods and devices that are generally described herein,
and these too are outlined in greater detail in published PCT
Application No. 95/33846.
[0195] Upon completion, the cartridged substrate will have a
variety of uses. For example, the cartridge can be used in a
variety of sequencing by hybridization ("SBH") methods, sequence
checking methods, diagnostic methods and the like. Arrays which are
particularly suited for sequence checking and SBH methods are
described in, e.g. U.S. patent application Ser. No. 08/505,919,
filed Jul. 24, 1995, Ser. No. 08/441,887, filed May 16, 1995, Ser.
No. 07/972,007, filed Nov. 5, 1992, each of which is incorporated
herein by reference in its entirety for all purposes.
[0196] Typically, in carrying out these methods, the cartridged
substrate is mounted on a hybridization station where it is
connected to a fluid delivery system. The fluid delivery system is
connected to the cartridge by inserting needles into the inlet and
outlet ports through the septa disposed therein. In this manner,
various fluids are introduced into the cavity for contacting the
probes synthesized on the front side of the substrate segment,
during the hybridization process.
[0197] Usually, hybridization is performed by first exposing the
sample with a prehybridization solution. Next, the sample is
incubated under binding conditions for a suitable binding period
with a sample solution that is to be analyzed. The sample solution
generally contains a target molecule, e.g., a target nucleic acid,
the presence or sequence of which is of interest to the
investigator. Binding conditions will vary depending on the
application and are selected in accordance with the general binding
methods known including those referred to in: Maniatis et al.,
Molecular Cloning: A Laboratory Manual (1989), 2nd Ed., Cold Spring
Harbor, N.Y. and Berger and Kimmel, Methods in Enzymology, Volume
152, Guide to Molecular Cloning Techniques (1987), Academic Press,
Inc., San Diego, Calif.; Young and Davis (1983) Proc. Natl. Acad.
Sci. (U.S.A.) 80: 1194, which are incorporated herein by reference.
In some embodiments, the solution may contain about 1 molar of salt
and about 1 to 50 nanomolar of targets. Optionally, the fluid
delivery system includes an agitator to improve mixing in the
cavity, which shortens the incubation period. Finally, the sample
is washed with a buffer, which may be 6.times.SSPE buffer, to
remove the unbound targets. In some embodiments, the cavity is
filled with the buffer after washing the sample.
[0198] Following hybridization and appropriate rinsing/washing, the
cartridged substrate may be aligned on a detection or imaging
system, such as those disclosed in U.S. Pat. No. 5,143,854 (Pirrung
et al.) or U.S. patent application Ser. No. 08/195,889, filed Feb.
10, 1994, Ser. No. 08/465,782, filed Jun. 6, 1995, Ser. No.
08/456,598, filed Jun. 1, 1995, incorporated herein by reference
for all purposes. Such detection systems may take advantage of the
cartridge's asymmetry (i.e., non-flush edge) by employing a holder
to match the shape of the cartridge specifically. Thus, the
cartridge is assured of being properly oriented and aligned for
scanning. The imaging systems are capable of qualitatively
analyzing the reaction between the probes and targets. Based on
this analysis, sequence information of the targets is
extracted.
VII. EXAMPLES
Example-1
Comparison of Front-Side and Back-Side Photolysis
[0199] Two substrate wafers were stripped, silanated and
photoprotected. The substrates were photolyzed through a mask
having rectangular features of 50 and 100 .mu.m on the short side,
for 13 half lives of the photoprotecting group used. The first
substrate was photolyzed from the back-side of the wafer, i.e., the
synthesis surface was facing away from the photolyzing light
source. The second substrate was photolyzed from the front-side,
i.e., the synthesis surface was facing the light source and mask.
Both substrates were then subjected to identical coupling reactions
where a fluorescent 5' protected phosphoramidite was coupled to the
surface of the substrate.
[0200] FIGS. 8A and 8B illustrate the contrast difference between
back-side exposure synthesis and front-side exposure synthesis,
respectively. FIG. 9A shows a fluorescent scan of a substrate
having fluorescent groups coupled directly to the surface of the
substrate using photolithographic techniques, with a mask having 50
.mu.m and 100 .mu.m feature sizes where the activating light was
shown through the back-side of the substrate. FIG. 9B shows the
same synthesis where the activation light was directed at the front
side of the substrate. The definition of the individual features is
greatly enhanced using this front-side photolysis.
[0201] FIGS. 9C and 9D provide a graphic illustration of the
differences in contrast among features prepared using back-side vs.
front-side methods. Specifically, the front-side exposure provides
a much sharper contrast and greater feature definition. This
greater definition permits a much smaller feature size by reducing
bleed-over effects during exposure. While front-side exposure
results in subjecting the synthesis surface to ambient conditions
during photolysis, this has not been found to have any deleterious
effects on the synthesis.
Example-2
Final Deprotection with Ethanolamine and Ethylenediamine
[0202] 1-8mer oligonucleotide probes were synthesized on glass
substrates derivatized with bis(2-hydroxyethyl)
aminopropyltriethoxysilane, according to standard protocols. In
each case, a hexaethyleneglycol-based spacer phosphoramidite was
coupled to the surface before the oligonucleotide sequence, and a
fluorescein-based "tag" phosphoramidite was coupled to the 5' end
of the oligonucleotides, usually in a checkerboard pattern. This
allowed monitoring the loss of probes from the substrates, by
ascertaining a decrease in the surface fluorescence. The substrates
were immersed in either concentrated aqueous ammonia or 50%
ethanolic ethanolamine, or 50% ethanolic ethylenediamine in sealed
containers. At specific times, the substrates were removed, washed
with water, and the surface fluorescence was image was obtained,
against a pH 7.2 phosphate buffer. After each scan, the substrates
were washed again, dried in an inert atmosphere (N.sub.2), and
returned to the deprotection solution. The surface fluorescence of
the substrate immersed in the aqueous ammonia deprotection solution
decayed with a half-time of 8-10 hours. After two days in the
ethanolic amine solutions, only a 5% decay in surface fluorescence
was observed.
Example-3
Comparison of Silanation Methods and Reagents
[0203] For comparison, glass substrates were derivatized with a
number of silanes using solution-phase deposition methods. Mean
functional surface densities were compared by fluorescent staining.
Performance with regard to oligonucleotide synthesis was compared
by synthesizing a 10mer probe sequence on the substrates,
deprotecting, and hybridizing them to a standard fluorescein
labelled oligonucleotide target. Standard oligonucleotide synthesis
cycles (couple-cap-oxidize) were used in all cases, but were
modified slightly to allow for reagent delivery to flowcells for
planar substrates.
[0204] The following silanes, obtained from Huls America were
tested:
[0205] 3-acetoxypropyltrimethoxysilane ("OAc");
[0206] 3-glycidoxypropyltrimethoxysilane ("Epoxy");
[0207] 4-(hydroxybutyramido)propyltriethoxysilane ("Mono");
[0208] 3-aminopropyltriethoxysilane ("APS"); and
[0209] 3-N,N-bis(2-hydroxyethyl)aminopropyl
[0210] triethoxysilane ("bis")
[0211] Precleaned substrates were immersed in a 1% solution of the
silane in 5% water, 95% ethanol, for 5 minutes with gentle
agitation. The substrates were then thoroughly rinsed with alcohol,
dried under N.sub.2, and cured at 100.degree. C. for 15 minutes.
Prior to use, the acetoxypropyl-silanated substrates were soaked in
50% ethanolic ethanolamine for 2 hours, then rinsed and dried.
Similarly, the glycidoxypropyl-silanated substrates were soaked in
0.1 M aqueous HCl for 2 hours, rinsed then dried. All other
substrates were ised withoit further treatment.
[0212] The functional group density was then measured by
fluorescent staining. Specifically,
MeNPOC-hexaethyleneglycol-cyanoethl phosphoramidite was coupled to
the substrate and unreacvted sites were then capped with
(MeO).sub.2PNiPr.sub.2. A portion of the surface was illuminated
through a photolithographic mask for 300 seconds at 365 nm (15
mW/cm.sup.2) to remove the MeNPOC protecting groups. The free
hydroxyls were then labeled with a fluorescein phosphoramidite
(Fluoreprime.TM., Pharmacia Biotech). The substrate was then
deprotected n 50% ethanolic ethylenediamine and surface
fluorescence was measured with a scanning laser confocal
microscope.
[0213] A 10mer oligonucleotide probe sequence (5'-TACCGTTCAG-3')
was synthesized on a selected region of each substrate using
light-directed synthesis. After deprotection in 50% ethanolic
ethylenediamine, the substrte was incubated in a solution of a
complementary fluorescein-labeled oligonucleotide target (10 nM
oligonucleotide in 5.times.SSPE buffer for 6 hours. After briefly
washing the substrate once with 5.times.SSPE, total
surface-hybridized target oligonucleotide was quantitated with a
scanning laser confocal microscope. Staining and hybridization data
are summarized in FIG. 10 which illustrates effective silanation of
glass substrates using each of the above-described silane
reagents.
Example-4
Direct Transfer of Protecting Groups to Hydroxylated Substrates
[0214] Synthesis of MeNPOC-tetrazolide was carried out as follows:
Tetrazole (7.0 g); 100 mmole) was combined with 17.5 ml of DIEA (13
g, 100 mmole) in 100 ml of THF, and a solution of 30 g (110 mmole)
MeNPOC-chloride (See, Pease, et al, supra) in 100 ml THF was added
dropwise over 20 minutes while stirring under argon at 4.degree. C.
Stirring was continued for an additional hour at room temperature.
200 ml of hexane was then added. The precipitate was collected by
filtration, redissolved in 200 ml DCM and washed 3 times with 0.05
M aqueous HCl to remove DIEA.HCL. The organic layer was dried with
NaSO.sub.4 and evaporated to obtain 24.5 g (80%) of the pure
product, which was identified by .sup.1H-NMR, IR and mass
spectrometry.
[0215] MeNPOC-transfer to a hydroxylated substrate with
MeNPOC-tetrazolide was carried out as follows: Using methods
described in the art, e.g., Pease et al., supra, hydroxylated glass
substrates were prepared by silanating the glass with
bis-(hydroxyethyl)aminopropyltriethoxysilane, and then adding a
linker phosphoramidite
(MeNPOC-hexaethyleneglycolcyanoethyl-phosphoramidite) to the
substrates using a standard couple-cap-oxidize cycle. The
substrates were then exposed to light (365 nm at 25 mW/cm.sup.2 for
240 seconds) to remove the MeNPOC protecting groups from the
linker. The free hydroxylated linker substrates were exposed to
freshly mixed solutions of MeNPOC-tetrazolide (0.2M) in ACN
containing 10% v/v 2,6-lutidine .+-.5% w/v NMI or DMAP activator.
After varying periods of time, the MeNPOC-tetrazolide solutions
were removed and N,N-diisopropyl-dimethylphosphoramidite was added
using the standard couple-cap-oxidize cycle in order to cap any
unreacted hydroxyl groups. To assess the extent of MeNPOC transfer,
the substrate was photolysed again, and the reexposed hydroxyls
were reacted with a fluorescent phosphoramidite (Fluoreprime.TM.,
Pharmacia Biotech), added with the same couple-cap-oxidize
protocol. The substrates were finally deprotected with 50%
ethanolic ethanolamine and the mean surface fluorescence was
measured with a laser scanning confocal microscope. FIG. 11 shows
the extent of reprotection with MeNPOC tetrazolide as a function of
time and catalyst.
[0216] While the foregoing invention has been described in some
detail for purposes of clarity and understanding, it will be clear
to one skilled in the art from a reading of this disclosure that
various changes in form and detail can be made without departing
from the true scope of the invention. All publications and patent
documents cited in this application are incorporated by reference
in their entirety for all purposes to the same extent as if each
individual publication or patent document were so individually
denoted.
* * * * *