U.S. patent application number 11/138840 was filed with the patent office on 2005-11-24 for photolithographic method and system for efficient mask usage in manufacturing dna arrays.
This patent application is currently assigned to Affymetrix, INC. Invention is credited to Hubbell, Earl A., Mittmann, Michael P..
Application Number | 20050260507 11/138840 |
Document ID | / |
Family ID | 25242680 |
Filed Date | 2005-11-24 |
United States Patent
Application |
20050260507 |
Kind Code |
A1 |
Mittmann, Michael P. ; et
al. |
November 24, 2005 |
Photolithographic method and system for efficient mask usage in
manufacturing DNA arrays
Abstract
Systems, methods, and products are described for synthesizing
probe arrays of polymers. A mask is used that includes reticle
areas, each of which includes a number of reticles associated with
a same synthesis area on a substrate. A method includes (a)
aligning the mask with respect to the substrate so that a first
reticle of a first reticle area is aligned with a first synthesis
area and so that a second reticle of the first reticle area is
aligned with a first discard area on the substrate; (b) coupling
monomers on the first synthesis area at locations determined by the
first reticle; (c) re-aligning the mask with respect to the
substrate so that the second reticle is aligned with the first
synthesis area; and (d) coupling monomers on the first synthesis
area at locations determined by the second reticle. The monomers
may be, for example, nucleotides, amino acids or saccharides.
Inventors: |
Mittmann, Michael P.; (Palo
Alto, CA) ; Hubbell, Earl A.; (Los Angeles,
CA) |
Correspondence
Address: |
AFFYMETRIX, INC
ATTN: CHIEF IP COUNSEL, LEGAL DEPT.
3380 CENTRAL EXPRESSWAY
SANTA CLARA
CA
95051
US
|
Assignee: |
Affymetrix, INC
Santa Clara
CA
|
Family ID: |
25242680 |
Appl. No.: |
11/138840 |
Filed: |
May 26, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11138840 |
May 26, 2005 |
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09824931 |
Apr 3, 2001 |
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6949638 |
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60265103 |
Jan 29, 2001 |
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Current U.S.
Class: |
430/5 |
Current CPC
Class: |
B01J 2219/00596
20130101; G16B 25/30 20190201; B01J 2219/00527 20130101; B01J
2219/00695 20130101; G16B 25/00 20190201; B01J 2219/00711 20130101;
B01J 2219/00659 20130101; B01J 2219/00605 20130101; B01J 2219/0059
20130101; B82Y 30/00 20130101; B01J 2219/00722 20130101; B01J
2219/00585 20130101; B01J 2219/00608 20130101; B01J 2219/00689
20130101; C40B 60/14 20130101; G01N 33/54366 20130101; B01J
2219/00432 20130101; C40B 40/06 20130101; B01J 19/0046
20130101 |
Class at
Publication: |
430/005 |
International
Class: |
G03F 001/00 |
Claims
What is claimed is:
1-28. (canceled)
29. A method for synthesizing probe arrays of polymers on a
substrate using a mask having a plurality of reticle areas, wherein
each reticle area comprises a plurality of reticles, each of which
is associated with a same synthesis area on the substrate, the
method comprising the steps of: (a) aligning the mask with respect
to the substrate so that a first reticle of a first reticle area is
aligned with a first synthesis area associated with the plurality
of reticles of the first reticle area, and so that a second reticle
of the first reticle area is aligned with a first discard area on
the substrate; (b) coupling monomers on the first synthesis area at
locations determined by the first reticle; (c) re-aligning the mask
with respect to the substrate so that the second reticle is aligned
with the first synthesis area; and (d) coupling monomers on the
first synthesis area at locations determined by the second
reticle.
30. The method of claim 29, wherein: when the first reticle is
aligned with the first synthesis area, every reticle of the first
reticle area other than the first reticle is aligned with a discard
area on the substrate.
31. The method of claim 29, further comprising the step of: dicing
the substrate at least partially within the first discard area.
32. The method of claim 31, wherein: the dicing physically
separates a probe array, including the first synthesis area on the
substrate, from at least one other synthesis area on the
substrate.
33. The method of claim 31, wherein: the plurality of reticles in
each reticle area are arranged in a same pattern.
34. The method of claim 33, wherein: the pattern comprises rows and
columns of reticles.
35. The method of claim 34, wherein: the dicing of the substrate is
done in a straight line lying entirely within one or more discard
areas including the first discard area.
36. The method of claim 29, wherein: the monomers are selected from
the group consisting of nucleotides, amino acids or
saccharides.
37. The method of claim 29, wherein: step (b) further comprises
coupling a first monomer and step (d) further comprises coupling
the first monomer or a second monomer.
38. The method of claim 29, wherein: steps (b) and (d) each further
comprise directing light through the aligned reticles to de-protect
the locations for coupling.
39. A system for synthesizing probe arrays of polymers on a
substrate, comprising: (a) a mask having a plurality of reticle
areas, wherein each reticle area comprises a plurality of reticles,
each of which is associated with a same synthesis area on the
substrate; (b) an aligner constructed and arranged to (i) align the
mask with respect to the substrate so that a first reticle of a
first reticle area is aligned with a first synthesis area
associated with the plurality of reticles of the first reticle
area, and so that a second reticle of the first reticle area is
aligned with a first discard area on the substrate, and (ii)
re-align the mask with respect to the substrate so that the second
reticle is aligned with the first synthesis area; and (c) a
synthesizer constructed and arranged to (i) couple monomers on the
first synthesis area at locations determined by the first reticle
when the first reticle is aligned with the first synthesis area,
and (ii) couple monomers on the first synthesis area at locations
determined by the second reticle when the second reticle is aligned
with the first synthesis area.
40. The system of claim 39, wherein: the monomers are selected from
the group consisting of nucleotides, amino acids or
saccharides.
41. The system of claim 39, wherein: the synthesizer further is
constructed and arranged to direct light through the aligned
reticles to de-protect the locations for coupling.
42-48. (canceled)
49. A computer program product for synthesizing polymers on a
substrate using a mask having a plurality of reticle areas, wherein
each reticle area comprises a plurality of reticles, each of which
is associated with a same synthesis area on the substrate, the
product comprising a computer usable medium storing control logic
that, when executed on a computer system, performs a method
comprising the steps of: (a) aligning the mask with respect to the
substrate so that a first reticle of a first reticle area is
aligned with a first synthesis area associated with the plurality
of reticles of the first reticle area, and so that a second reticle
of the first reticle area is aligned with a first discard area on
the substrate; (b) coupling monomers on the first synthesis area at
locations determined by the first reticle; (c) re-aligning the mask
with respect to the substrate so that the second reticle is aligned
with the first synthesis area; and (d) coupling monomers on the
first synthesis area at locations determined by the second
reticle.
50. The computer program product of claim 49, wherein the method
further comprises the step of: (e) dicing the substrate at least
partially within the first discard area.
51. The computer program product of claim 49, wherein: the monomers
are selected from the group consisting of nucleotides, amino acids
or saccharides.
Description
RELATED APPLICATIONS
[0001] The present application claims priority from U.S.
Provisional Patent Application Ser. No. 60/265,103, entitled "RAPID
FLEXIBLE CONTENT ARRAY AND ONLINE ORDERING SYSTEM," filed Jan. 29,
2001, hereby incorporated herein by reference in its entirety for
all purposes.
FIELD OF THE INVENTION
[0002] The present invention is related to systems, methods, and
products providing lithographic masks used to form high-density
probes of biological materials on a substrate.
BACKGROUND
[0003] U.S. Pat. No. 5,424,186 to Fodor, et al., describes a
technique for, among other things, forming and using high density
arrays of probes comprising molecules such as oligonucleotide, RNA,
peptides, polysaccharides, and other materials. Arrays of
oligonucleotides or peptides, for example, are formed on the
surface by sequentially removing a photo-removable group from a
surface, coupling a monomer to the exposed region of the surface,
and repeating the process. Nucleic acid probe arrays synthesized in
this manner, such as Affymetrix.RTM. GeneChip.RTM. probe arrays
from Affymetrix, Inc. of Santa Clara, Calif., have been used to
generate unprecedented amounts of information about biological
systems. Analysis of these data may lead to the development of new
drugs and new diagnostic tools.
[0004] A typical step in the process of synthesizing these probe
arrays is to design a mask that will define the locations on a
substrate that are exposed to light. Some systems and methods
useful in the design and/or use of such masks are described in the
following U.S. Pat. No. 5,571,639 to Hubbell, et al.; U.S. Pat. No.
5,593,839 to Hubbell, et al.; U.S. Pat. No. 5,856,101 to Hubbell,
et al.; U.S. Pat. No. 6,153,743 to Hubbell, et al.; and U.S. Pat.
No. 6,188,783 to Balaban, et al., each of which is hereby
incorporated herein by reference for all purposes. Notwithstanding
the advances described in these patents, it is desirable to
identify additional techniques for designing and using masks in the
manufacture of probe arrays.
SUMMARY OF THE INVENTION
[0005] Systems, methods, computer program products, masks, and
probe arrays produced thereby, are described with reference to
illustrative, non-limiting, embodiments. For example, while certain
systems, methods, computer software products, masks, and probe
arrays are described with respect to the manufacture and/or use of
Affymetrix.RTM. GeneChip.RTM. probe arrays, these descriptions are
merely illustrative. Other implementations are possible, for
example, with respect to other types of probe arrays. Moreover,
possible implementations are not limited to probe arrays. That is,
the synthesized polymers need not be used as probes but may be
employed with respect to any of a variety of conventional
combinatorial chemistry purposes and uses.
[0006] In accordance with some embodiments, a method is described
for synthesizing polymers on a substrate using one or more masks.
In some implementations, the polymers synthesized on the substrate
comprise probes in a probe array. The masks each include reticle
areas made up of reticles. A reticle is made up of areas
transparent to the photolithographic radiation (hereafter, simply
referred to for convenience as "light"), and also is made up of
occluded areas through which the light does not pass. The
transparent areas are sometimes referred to as "flashes." Each of
the reticles in a particular reticle area is associated with the
same synthesis area on the substrate. In those implementations in
which the combinatorial chemistry is directed to producing probe
arrays, the term "synthesis area" is used herein to refer to an
area of the substrate on which probes are synthesized that are
intended to be included in the synthesized probe arrays. In these
implementations, a probe of a probe array generally may be a
polymer having a sequence such that the probe is capable of
hybridizing with potential targets, or having a sequence serving as
a control to assess the hybridization process. In contrast, the
term "discard area" is used herein to refer to an area on the
substrate through which dicing lines may be cut to physically
separate the substrate into two or more probe arrays. A part or all
of a discard area may contain synthesized polymers that are not
intended for use as probes (or, in implementations other than probe
arrays, are not intended for the purpose of the combinatorial
chemistry). That is, in the illustrated implementations, although
polymers may be synthesized in discard areas, these polymers may be
discarded or otherwise ignored or destroyed rather than used as
probes in a synthesized probe array. The polymers in discard areas
may have sequences suitable for hybridization or control, but need
not. In some implementations, the term "discard area" may be used
more broadly to also include areas not used for cutting but used
for joining the substrate to packaging, or for other purposes not
including the purpose of providing a synthesis area.
[0007] The method includes the following steps: (a) for each
reticle area, sequentially aligning two or more of the reticles of
that reticle area with the associated synthesis area; and (b) for
each sequential alignment, coupling monomers on the substrate at
locations determined by the aligned reticles. The monomers of this
method, and of other embodiments and implementations described
herein, may include nucleotides, amino acids, or saccharides, for
example.
[0008] In accordance with this method, the reticle areas are
substantially contiguously arranged on the mask, and the plurality
of reticles within each of the reticle areas are substantially
contiguously arranged within the reticle area. The term
"substantially contiguously" is used in this context to mean that
reticles in a reticle area may abut each other, and that reticle
areas may abut each other. That is, there generally need not be any
spaces between the reticles or between the reticle areas. However,
the term "substantially contiguously" is used broadly herein to
include implementations, such as that illustrated in the detailed
description below, in which narrow boundary areas are provided
between reticles and/or between reticle areas. As described in
greater detail below, these narrow boundary areas may be provided
for a variety of practical reasons related to making masks,
scanning labeled probe-target pairs, and other reasons. Notably,
however, they are not provided for the reason of reserving, by
themselves, a space on the substrate for dicing.
[0009] In some implementations of the method, each reticle area may
be made up of reticles arranged in a particular pattern that is the
same for all reticle areas. For example, the pattern may be an
array of rows and columns in which the rows have a height H (which
is the height of the reticles, plus a boundary area, if any) and
the columns may have a width W (which is the width of the reticles,
plus a boundary area, if any). In some aspects of these
implementations, the sequential alignment of step (a) may include
translating the mask with respect to the substrate by a sequence of
steps. The translation distance at each step is determined by the
height H or the width W. For example, a step may consist of a
translation W to the right or, as another example, a translation W
to the right and a translation H down. The translation between the
mask and substrate is relative, and may thus be accomplished by
moving the mask and keeping the substrate immobile, by moving the
substrate and keeping the mask immobile, or by moving both to
varying degrees.
[0010] In some implementations of the method, step (b) may include
coupling a same monomer for each of the aligned reticles. Step (b)
may also include directing light through the aligned reticles to
de-protect the locations for coupling.
[0011] Other embodiments described herein are directed to systems
for synthesizing polymers on a substrate. In one implementation,
the system includes a mask having a plurality of reticle areas,
wherein each reticle area includes two or more reticles, each of
which is associated with a same synthesis area on the substrate.
The system also has an aligner that, for each reticle area,
sequentially aligns two or more of the reticles of that reticle
area with the associated synthesis area. Another element of the
system is a synthesizer that, for each sequential alignment, causes
monomers to be coupled on the substrate at locations determined by
the aligned reticles. In these implementations, the reticle areas
are substantially contiguously arranged on the mask, and the
reticles within each of the reticle areas are substantially
contiguously arranged within the reticle area.
[0012] Further embodiments are directed to a mask for synthesizing
polymers on a substrate. The mask has substantially contiguously
arranged reticle areas and, within each reticle area, substantially
contiguous reticles. Each of the reticles in a same reticle area is
associated with a same synthesis area on the substrate and is
constructed and arranged for synthesizing polymers by enabling the
coupling of monomers on the same synthesis area at locations
determined by the reticles.
[0013] Yet other embodiments are directed to a method for
manufacturing a mask for synthesizing polymers on a substrate. The
method includes the step of identifying two or more reticle areas
substantially contiguously arranged on the mask. Another step is to
construct and arrange two or more substantially contiguous reticles
within each reticle area, each of which is associated with a same
synthesis area on the substrate. The reticles further are
constructed and arranged for synthesizing polymers by enabling the
coupling of monomers on the same synthesis area at locations
determined by the reticles of a particular reticle area.
[0014] Also described herein is a probe array including polymers
synthesized on a substrate by a method that includes the steps of:
(a) providing at least one mask having two or more reticle areas,
wherein each reticle area comprises two or more reticles, each of
which is associated with a same synthesis area on the substrate;
(b) for each reticle area, sequentially aligning two or more of the
plurality of reticles of that reticle area with the associated
synthesis area; and (c) for each sequential alignment, coupling
monomers on the substrate at locations determined by the aligned
reticles. The two or more reticle areas are substantially
contiguously arranged on the mask, and the two or more reticles
within each of the reticle areas are substantially contiguously
arranged within the reticle area.
[0015] In other embodiments, a computer program product is
described for synthesizing polymers on a substrate using a mask
having a plurality of reticle areas, wherein each reticle area
comprises two or more reticles, each of which is associated with a
same synthesis area on the substrate. The product includes a
computer usable medium storing control logic that, when executed on
a computer system, performs a method including the steps of: (a)
for each reticle area, sequentially aligning two or more of the
reticles of that reticle area with the associated synthesis area;
and (b) for each sequential alignment, coupling monomers on the
substrate at locations determined by the aligned reticles. The
reticle areas are substantially contiguously arranged on the mask,
and the reticles within each of the reticle areas are substantially
contiguously arranged within the reticle area.
[0016] Also described herein is a method for synthesizing probe
arrays of polymers on a substrate using a mask having two or more
reticle areas, wherein each reticle area includes two or more
reticles, each of which is associated with a same synthesis area on
the substrate. The method includes the following steps: (a)
aligning the mask with respect to the substrate so that a first
reticle of a first reticle area is aligned with a first synthesis
area associated with the two or more reticles of the first reticle
area, and so that a second reticle of the first reticle area is
aligned with a first discard area on the substrate; (b) coupling
monomers on the first synthesis area at locations determined by the
first reticle; (c) re-aligning the mask with respect to the
substrate so that the second reticle is aligned with the first
synthesis area; and (d) coupling monomers on the first synthesis
area at locations determined by the second reticle.
[0017] In general, the preceding steps may be repeated so that each
of the reticles of a reticle area is aligned during a synthesis
step with the synthesis area associated with that reticle area.
When a reticle is aligned with the synthesis area, the other
reticles of the same reticle area typically are not aligned with
that, or another, synthesis area. Rather, they typically are
aligned with a discard area. A further step in some implementations
of the method is to dice the substrate. The dicing is done at least
partially within the first discard area. Typically, the dicing
physically separates a probe array, including the first synthesis
area on the substrate, from at least one other synthesis area on
the substrate.
[0018] A further embodiment described herein consists of a system
for synthesizing probe arrays of polymers on a substrate. The
system includes a mask having two or more reticle areas, wherein
each reticle area comprises a plurality of reticles, each of which
is associated with a same synthesis area on the substrate. Also
included in the system is an aligner that (i) aligns the mask with
respect to the substrate so that a first reticle of a first reticle
area is aligned with a first synthesis area associated with the
plurality of reticles of the first reticle area, and so that a
second reticle of the first reticle area is aligned with a first
discard area on the substrate, and (ii) re-aligns the mask with
respect to the substrate so that the second reticle is aligned with
the first synthesis area. Another element of the system is a
synthesizer that (i) couples monomers on the first synthesis area
at locations determined by the first reticle when the first reticle
is aligned with the first synthesis area, and (ii) couples monomers
on the first synthesis area at locations determined by the second
reticle when the second reticle is aligned with the first synthesis
area. Typically, the synthesizer further is constructed and
arranged to direct light through the aligned reticles to de-protect
the locations for coupling.
[0019] Yet another embodiment consists of a probe array comprising
polymers synthesized on a substrate by a method that includes the
following steps: (a) providing at least one mask having a plurality
of reticle areas, wherein each reticle area comprises a plurality
of reticles, each of which is associated with a same synthesis area
on the substrate; (b) aligning the mask with respect to the
substrate so that a first reticle of a first reticle area is
aligned with a first synthesis area associated with the plurality
of reticles of the first reticle area, and so that a second reticle
of the first reticle area is aligned with a first discard area on
the substrate; (c) coupling monomers on the first synthesis area at
locations determined by the first reticle; (d) re-aligning the mask
with respect to the substrate so that the second reticle is aligned
with the first synthesis area; and (e) coupling monomers on the
first synthesis area at locations determined by the second reticle.
A further embodiment is a computer program product for synthesizing
polymers on a substrate using a mask having a plurality of reticle
areas. Each reticle area includes a plurality of reticles, each of
which is associated with a same synthesis area on the substrate.
The product includes a computer usable medium storing control logic
that, when executed on a computer system, performs a method
including: (a) aligning the mask with respect to the substrate so
that a first reticle of a first reticle area is aligned with a
first synthesis area associated with the plurality of reticles of
the first reticle area, and so that a second reticle of the first
reticle area is aligned with a first discard area on the substrate;
(b) coupling monomers on the first synthesis area at locations
determined by the first reticle; (c) re-aligning the mask with
respect to the substrate so that the second reticle is aligned with
the first synthesis area; and (d) coupling monomers on the first
synthesis area at locations determined by the second reticle.
[0020] The preceding embodiments and implementations are not
necessarily inclusive or exclusive of each other and may be
combined in any manner that is non-conflicting and otherwise
possible, whether they be presented in association with a same, or
a different, embodiment or implementation. The description of one
embodiment or implementation is not intended to be limiting with
respect to others. Also, any one or more function, step, operation,
or technique described elsewhere in this specification may, in
alternative embodiments or implementations, be combined with any
one or more function, step, operation, or technique described in
the summary. Thus, the above embodiments and implementations are
illustrative rather than limiting.
BRIEF DESCRIPTION OF THE DRAWINGS
[0021] The above and other embodiments and implementations will be
more clearly appreciated from the following detailed description
when taken in conjunction with the accompanying drawings. In the
drawings, like reference numerals indicate like structures or
method steps and the leftmost digit of a reference numeral
indicates the number of the figure in which the referenced element
first appears (for example, the element 120 appears first in FIG.
1).
[0022] FIG. 1 is a functional block diagram of one embodiment of a
system for designing and manufacturing masks;
[0023] FIGS. 2A and 2B are simplified graphical representations of
a representative reticle and a mask in accordance with a
conventional arrangement of reticles on a mask;
[0024] FIGS. 2C through 2F are simplified graphical representations
of a conventional method for synthesizing probes on a substrate,
and then dicing the substrate to provide probe arrays, using one or
more masks of the conventional type shown in FIG. 2B;
[0025] FIGS. 3A through 3F are simplified graphical representations
of a method for synthesizing probes on a substrate, and then dicing
the substrate to provide probe arrays, using one or more masks,
each of which has reticles used in separate synthesis steps;
[0026] FIGS. 4A through 4E are simplified graphical representations
of another method for synthesizing probes on a substrate, and then
dicing the substrate to provide probe arrays, using one or more
masks, each of which has reticles used in separate synthesis
steps;
[0027] FIG. 5C is a simplified graphical representations of one
embodiment of a mask having substantially contiguous reticle areas
(a representative of which is shown in greater detail in FIG. 5B)
made up of substantially contiguous reticles (a representative of
which is shown in greater detail in FIG. 5A);
[0028] FIGS. 6A through 6C are simplified graphical representations
of another embodiment of a mask having substantially contiguous
reticles and reticle areas;
[0029] FIGS. 6D through 6H are simplified graphical representations
of one embodiment of a method for synthesizing probes on a
substrate, and then dicing the substrate to provide probe arrays,
using one or more masks such as those of FIGS. 6A through 6C;
[0030] FIGS. 6I and 6J are simplified graphical representations
showing aspects of the substrate of FIGS. 6E through 6H in greater
detail;
[0031] FIG. 6K is a simplified graphical representation of yet
another embodiment of a mask having substantially contiguous
reticles and reticle areas, and FIGS. 6L and 6M are simplified
graphical representations of another implementation of the method
for synthesizing probes on a substrate, and then dicing the
substrate to provide probe arrays, using the mask of FIG. 6K;
[0032] FIG. 7 is a flow diagram of one embodiment of a method for
synthesizing probes on a substrate, and then dicing the substrate
to provide probe arrays, using one or more masks having
substantially contiguous reticles and reticle areas; and
[0033] FIG. 8 is a functional block diagram of one embodiment of a
probe array synthesis system suitable for making probe arrays using
masks having substantially contiguous reticles and reticle
areas.
DETAILED DESCRIPTION
[0034] Detailed descriptions are now provided with respect to
systems, methods, software program products, masks produced
thereby, probe arrays produced thereby, and combinations
thereof.
Synthesized Probe Arrays
[0035] Various techniques and technologies may be used for
synthesizing dense arrays of biological materials on or in a
substrate or support. For example, Affymetrix.RTM. GeneChip.RTM.
arrays are synthesized in accordance with techniques sometimes
referred to as VLSIPS.TM. (Very Large Scale Immobilized Polymer
Synthesis) technologies. Some aspects of VLSIPS.TM. technologies
are described in the following U.S. Pat. No. 5,424,186 to Fodor, et
al.; U.S. Pat. No. 5,143,854 to Pirrung, et al.; U.S. Pat. No.
5,445,934 to Fodor, et al.; U.S. Pat. No. 5,744,305 to Fodor, et
al.; U.S. Pat. No. 5,831,070 to Pease, et al.; U.S. Pat. No.
5,837,832 to Chee, et al.; U.S. Pat. No. 6,022,963 to McGall, et
al.; and U.S. Pat. No. 6,083,697 to Beecher, et al. Each of these
patents is hereby incorporated by reference in its entirety. The
probes of these arrays in some implementations consist of
oligonucleotides, which are synthesized by methods that include the
steps of activating regions of a substrate and then contacting the
substrate with a selected monomer solution. The regions are
activated with a light source shown through a mask in a manner
similar to photolithography techniques used in the fabrication of
integrated circuits. Other regions of the substrate remain inactive
because the mask blocks them from illumination. By repeatedly
activating different sets of regions and contacting different
monomer solutions with the substrate, a diverse array of polymers
is produced on the substrate. Various other steps, such as washing
unreacted monomer solution from the substrate, are employed in
various implementations of these methods.
[0036] Additional techniques for synthesizing and using
high-density probe arrays are described in U.S. Pat. No. 5,384,261
to Winkler, et al. These techniques include systems for
mechanically protecting portions of a substrate and selectively
de-protecting and coupling materials to the substrate using
light-directed methods. Still further techniques for probe array
synthesis are provided in U.S. Pat. No. 6,121,048 to Zaffaroni, et
al. The '261 and '048 patents also are incorporated herein by
reference for all purposes.
[0037] The probes of these synthesized probe arrays typically are
used in conjunction with tagged biological samples such as cells,
proteins, genes or EST's, other DNA sequences, or other biological
elements. These samples, referred to herein as "targets," are
processed so that, typically, they are spatially associated with
certain probes in the probe array. For example, one or more
chemically tagged biological samples, i.e., the targets, are
distributed over the probe array. Some targets hybridize with at
least partially complementary probes and remain at the probe
locations, while non-hybridized targets are washed away. These
hybridized targets, with their "tags" or "labels," are thus
spatially associated with the targets' complementary probes. The
hybridized probe and target may sometimes be referred to as a
"probe-target pair." Detection of these pairs can serve a variety
of purposes, such as to determine whether a target nucleic acid has
a nucleotide sequence identical to or different from a specific
reference sequence. See, for example, U.S. Pat. No. 5,837,832,
referred to and incorporated above. Other uses include gene
expression monitoring and evaluation (see, e.g., U.S. Pat. No.
5,800,992 to Fodor, et al.; U.S. Pat. No. 6,040,138 to Lockhart, et
al.; and International App. No. PCT/US98/15151, published as
WO99/05323, to Balaban, et al.), genotyping (U.S. Pat. No.
5,856,092 to Dale, et al.), or other detection of nucleic acids.
The '992, '138, and '092 patents, and publication WO99/05323, are
incorporated by reference herein in their entireties for all
purposes.
[0038] Probes typically are able to detect the expression of
corresponding genes or EST's by detecting the presence or abundance
of mRNA transcripts present in the target. This detection may, in
turn, be accomplished by detecting labeled cRNA that is derived
from cDNA derived from the mRNA in the target. In general, a group
of probes, sometimes referred to as a probe set, contains
sub-sequences in unique regions of the transcripts and does not
correspond to a full gene sequence. Further details regarding the
design and use of probes are provided in U.S. Pat. No. 6,188,783,
incorporated above, and in PCT Application Ser. No. PCT/US
01/02316, filed Jan. 24, 2001, and hereby incorporated herein in
its entirety for all purposes.
[0039] Labeled targets in hybridized probe arrays may be detected
using various commercial devices, sometimes referred to as
"scanners." Scanners image the targets by detecting fluorescent or
other emissions from the labels, or by detecting transmitted,
reflected, or scattered radiation. A typical scheme employs optical
and other elements to provide excitation light and to selectively
collect the emissions. Also generally included are various
light-detector systems employing photodiodes, charge-coupled
devices, photomultiplier tubes, or similar devices to register the
collected emissions. For example, a scanning system for use with a
fluorescent label is described in U.S. Pat. No. 5,143,854,
incorporated by reference above. Other scanners or scanning systems
are described in U.S. Pat. Nos. 5,578,832; 5,631,734; 5,834,758;
5,936,324; 5,981,956; 6,025,601; 6,141,096; 6,185,030; and
6,201,639, and in PCT Application PCT/US99/06097 (published as
WO99/47964), each of which is hereby incorporated by reference in
its entirety for all purposes.
Mask Design System 101 and Mask Manufacturing System 150
[0040] FIG. 1 includes a functional block diagram of an
illustrative mask design system 101 for designing the masks used to
produce synthesized probe arrays. In particular, system 101
provides data, represented as mask specification files 112, that
may be used by mask manufacturing system 150 to produce masks 155.
Probe information 107 is provided to system 101. Information 107
specifies the desired sequences of polymers constituting probes
derived and presented in accordance with known techniques and
systems such as described, for example, in U.S. Pat. No. 5,571,639,
incorporated by reference above. Desired mask characteristics 106
also are provided to system 101. Characteristics 106 may include,
for example, specification of substantially contiguous reticles in
substantially contiguous reticle areas, as described in greater
detail below with respect to the illustrative masks of FIGS. 5A-C,
6A-C, 6K, and with respects to aspects of the method steps
represented in FIG. 7. Illustrative mask design application 199 is
any of a variety of conventional software applications, such as a
computer-aided design application, that may be used to generate
mask specification files 112 based, at least in part, on desired
mask characteristics 106 and probe information 107. Aspects of
computer-aided design systems are described in U.S. Pat. Nos.
5,593,839 and 5,856,101, both of which have been incorporated
herein. Application 199 typically is loaded via an input device 102
(such as a floppy disk or CD-ROM reader) into a memory device
(e.g., RAM or hard drive) of computer 100 for execution, as
represented by mask design application executables 199A. Computer
100 may be any type of computer platform such as a workstation, a
personal computer, a server, or any other present or future
computer. Computer 100 typically includes known components such as
a processor 105, an operating system 110, a system memory 120,
memory storage devices 125, and input-output controllers 130, all
of which typically communicate in accordance with known techniques
such as via system bus 104.
[0041] As will be appreciated by those of ordinary skill in the
relevant art, mask specification files 112 include data specifying
a layout of reticles on one or more masks consistent with desired
mask characteristics 106 and probe information 107. As also will be
appreciated by those of ordinary skill in the relevant art, probe
specification files 114 include information specifying the order in
which monomers may be applied in a probe array synthesizer that
synthesizes probe arrays using masks 155. Although the word "files"
is used for convenience of illustration with respect to files 112
and 114, the word is used broadly to include any type of data
structure or technique for storing or transmitting data in any form
or format.
[0042] Mask manufacturing system 150 may be any of a variety of
conventional systems for producing masks, or a mask-producing
system of a type that may be developed in the future. Masks 155
produced by system 150 typically comprise lithographic members such
as chrome on glass, as one illustrative and non-limiting example.
Reticles on the mask selectively direct light to a substrate during
an exposure in accordance with known techniques. As noted above
with respect to an illustrative example, the light may activate
linker molecules at sites on the substrate determined by openings
in the reticles through which the light passes. Mask manufacturing
system 150 is capable of producing masks 155 of conventional design
or of an improved design in accordance with aspects of the present
invention, depending on whether desired mask characteristics 106
are conventional or otherwise.
Probe Arrays Synthesized Using Masks of FIGS. 2A-B
[0043] FIGS. 2A and 2B are simplified graphical representations of
a mask having a conventional arrangement of reticles for
synthesizing probe arrays. FIGS. 2C through 2F are simplified
graphical representations of a conventional method for synthesizing
probe arrays using one or more masks of the conventional type shown
in FIGS. 2A and 2B.
[0044] FIG. 2B shows mask 155A having illustrative dimensions of 45
millimeters by 45 millimeters. Mask 155A includes reticles 210A
through 210Y, generally and collectively referred to hereafter as
reticles 210. FIG. 2A shows representative reticle 210A having
illustrative dimensions of 6 millimeters by 6 millimeters. Each of
reticles 210 includes open or occluded areas for respectively
specifying the coupling, or not, of a monomer in this example. As
will be appreciated by those of ordinary skill in the relevant art,
the selective coupling of monomers as specified by a reticle may
take place on millions of locations on a substrate. For example, a
reticle may specify selective coupling locations for thousands of
probe sets corresponding, for example, to thousands of genes or
EST's of potential interest. Moreover, each of the probe sets may
include tens of probes, each of which may include many thousands of
polymers having a same sequence.
[0045] As indicated in FIGS. 2A and 2B, adjacent ones of reticles
210 are surrounded by a dicing offset 212 that, in this example, is
3 millimeters. As will be described in greater detail below, dicing
offset 212 is provided on the reticle so that corresponding areas
of the substrate will not contain probes. The substrate is diced,
i.e., cut, through the areas thus provided. It will be appreciated
by those of ordinary skill in the relevant art that mask 155A is a
simplified representation because, among other things, it does not
show alignment features typically provided to facilitate the
alignment of masks and substrates during probe synthesis. For
example, a distinctive feature, such as a checkerboard pattern (not
shown), may be provided in some parts of the mask occupied by
dicing offsets 212 and/or by one or more of reticles 210. Alignment
features may thus displace reticles in some instances so that, for
example, mask 155A includes 23 reticles rather than the 25 reticles
shown in this example.
[0046] In accordance with conventional techniques well known to
those of ordinary skill in the relevant art, a number of masks
having reticles arranged in the fashion shown in FIG. 2B may be
used to synthesize probe arrays. Mask 155A-1 of FIG. 2C is one such
mask having reticles designed for a first light-exposure step. Mask
155A-1 includes reticles 210A-1 to 210Y-1 (generally and
collectively referred to as reticles 210-1), and dicing offsets
212. In the first light-exposure step, mask 155A-1 is aligned over
wafer 240 as represented in FIG. 2D. Wafer 240 is a substrate
having dimensions commensurate with the areas on which probes are
to be synthesized, as determined by reticles 210, including
reticles 210-1. For convenience wafer 240 is shown as having the
same dimensions as mask 155A, but it need not be so. Light is
directed through the reticles in order to selectively enable
coupling of a monomer to areas on wafer 240. Each of reticles 210-1
determines the locations of monomer coupling in an area that will
correspond to a probe array after dicing of the wafer. For example,
reticle 210A-1 determines monomer coupling in a first
light-exposure step in the synthesis of an area of wafer 240 that
will become probe array 230A. Array 230A after the first
light-exposure step is represented in FIG. 2D as area 230A-1 of
wafer 240. For convenience, this and other areas may hereafter be
referred to generally and collectively as probe arrays 230-1.
[0047] A second light-exposure step is represented in FIGS. 2G and
2H. As will be understood by those of ordinary skill in the
relevant art, various other synthesis steps (not shown) typically
are undertaken between the first and second light-exposure steps,
such as washing a monomer and other reagents over wafer 240 so as
to couple monomers at locations determined by reticles 210-1 and to
add photo-chemically removable protecting groups so that the next
cycle of synthesis can be initiated. Mask 155A-2 is used in this
second light-exposure step, and reticles 210-2 of this second step
are aligned over the same areas as were corresponding reticles
210-1 of step one in this example. A monomer is washed over wafer
240, thereby coupling them to locations now having zero, one, or
two monomers, depending on whether the corresponding location in
masks 155A-1 and corresponding location in masks 155A-2 were both
occluded, not occluded in mask 155A-1 and occluded in mask 155A-2,
or not occluded in both masks, respectively. Thus, wafer 240
includes, after this second cycle, probe array areas 230-1,2 (shown
for convenience simply as "1, 2" in FIG. 2H) having probes at
locations determined by reticles 210-1 of mask 155A-1 and reticles
210-2 (shown for convenience simply as "2" in FIG. 2G) of mask
155A-2.
[0048] These synthesis cycles may continue using dozens of masks
155A, each of which has reticles 210 arranged in a same
configuration and aligned over the same areas of wafer 240. The
last of these dozens of masks is represented in FIG. 2E as mask
155A-N. FIG. 2F shows wafer 240 after the last of the synthesis
cycles has been completed, at which time wafer 240 consists of
probe array areas 230A-230Y, each having probes determined by
reticles in corresponding positions of masks 155A-1 through 155A-N.
For example, reticle 210A-1 through reticle 210A-N has sequentially
been aligned with probe array area 230A(1-N) over the N cycles. In
accordance with conventional techniques, wafer 240 then is diced
through dicing offsets 212 as indicated by horizontal dicing lines
232A-232D (hereafter, "dicing lines 232") and vertical dicing lines
234A-234D (hereafter, "dicing lines 234"). The result is the
physical separation from wafer 240 of 25 synthesized probe arrays
230.
[0049] Notably, the dicing operation in accordance with this
conventional approach typically requires that a significant portion
of masks 155A be set aside for dicing offsets 212. The example of
3-millimeter offset areas separating each probe array area is
typical in order to accommodate the thickness of the cut made by
conventional techniques. Also, the number of masks needed in
accordance with the conventional approach of FIGS. 2A-2F is equal
to the number of synthesis cycles, i.e., the number of times that
each probe array area is exposed to light. For example, in some
Affymetrix.RTM. GeneChip.RTM. arrays, 25-mer oligonucleotide probes
are synthesized in 75 synthesis cycles; that is, 75 masks are used.
The mask set required for synthesis of these arrays thus is
relatively expensive, and the synthesis process is relatively
lengthy.
Probe Arrays Synthesized Using Masks of FIGS. 3A-D and 4A-D
[0050] FIG. 3A is a simplified graphical representation of another
mask design that provides the advantage of generally requiring a
smaller mask set, although the disadvantage noted above with
respect to the dedication of wafer area to dicing offsets is not
alleviated. Illustrative mask 155B of FIG. 3A is made up of 16
reticles consisting of four reticles in each of four groups.
Reticles 310A-1 through 310D-1 are aligned during a first
light-exposure step over probe array areas of wafer 340 shown in
FIG. 3C as areas 330A-1 through 330D-1. (As in the previous
examples, alignment features and the details of the reticles are
not shown, but their implementation in accordance with various
techniques will readily be appreciated by those of ordinary skill
in the relevant art.) Rather than employing a second mask for the
second light-exposure step, however, mask 155B is rotated and/or
translated so that the second set of four reticles, 310A-2 through
310D-2, are aligned over the same probe array areas of wafer 340.
This alignment is shown in FIGS. 3C and 3D wherein reticles 310A-2
through 310D-2 (shown simply as reticles "2" for convenience) are
aligned over wafer 340 having monomers synthesized in the first
cycle. The result, after the second synthesis cycle is completed,
is represented in FIG. 3E where wafer 340 is shown with reticle
areas 330A-330D having monomers coupled to them in accordance with
light-exposure steps 1 and 2. In the illustrated example, four such
light-exposure steps may be undertaken using the same mask 155B.
Additional masks having the same arrangement of reticles may then
be used to complete the N cycles of N light-exposure steps,
resulting in wafer 340 as shown in FIG. 3F. Four probe arrays may
then be diced from wafer 340 by cutting along dicing lines 332 and
334.
[0051] Thus, while the number of masks used to synthesize probe
arrays 330A through 330D is one-fourth the number used to
synthesize probe arrays 230A through 230Y of the previous example,
the number of probe arrays from the mask set is reduced from 25 to
four. As is now evident, both the number of probe arrays
synthesized with a mask set and the number of masks in a mask set
can be altered by changing the size and/or geometry of the mask and
reticles. As one of many possible examples, mask 155B could have
been made with four groups of nine reticles (i.e., 36 reticles per
mask for the synthesis of nine probe arrays using four
light-exposure steps per mask) by increasing the size of the mask
and/or reducing the size of the reticles. Similarly, the mask of
this alternative example could consist of nine groups of four
reticles (i.e., 36 reticles per mask for the synthesis of four
probe arrays using nine light-exposure steps per mask).
[0052] Many arrangements of reticles are possible that allow
multiple synthesis cycles to be accomplished using each mask. One
alternative arrangement is illustrated in FIGS. 4A-E. FIG. 4A is a
simplified graphical representation of a mask 155C that has six
groups of two reticles each (i.e., 12 reticles per mask for the
synthesis of two probe arrays using six light-exposure steps per
mask). Reticles of the first group, i.e., reticles 410A-1 and
410B-1, are aligned over probe array areas 430A and 430B in a first
light-exposure step, as shown in FIG. 4B. As shown in FIG. 4C,
reticles 410A-2 and 410B-2 (shown for convenience simply as "2")
are aligned over the same probe array area of wafer 440 in a second
light-exposure step. This process is repeated for the third group,
as shown in FIG. 4D, and for the remaining three of the six groups
of illustrative mask 155C. If more than six synthesis cycles are
used, the process is similarly repeated for additional groups in
additional masks in which reticles are arranged in the
configuration of mask 155C. The result is shown in FIG. 4E in which
dicing line 432 is cut to separate wafer 440 into probe array 430A
and probe array 430B, each synthesized using N light-exposures. As
will now be evident, any number of groups, having any number of
reticles each, could similarly be constructed. As one of many
possible examples, mask 155C could have been arranged to have 21
groups of two reticles each (i.e., 42 reticles per mask for the
synthesis of two probe arrays using 21 light-exposure steps per
mask, not shown). In all of these arrangements, a dicing offset of
an illustrative 3 millimeters is employed between reticles of the
same group (i.e., between reticles exposed during the same
light-exposure step).
[0053] Thus, in all of the preceding variations noted with respect
to FIGS. 3A-F and 4A-E, the disadvantage remains that reticles are
separated by dicing offsets 312 and 412. As noted, these dicing
offsets are reserved for the purpose of allowing dicing of probe
arrays through locations on wafers 340 and 440, respectively, as
determined by the separation of reticles in the same group (i.e.,
reticles exposed during the same light-exposure step). The
inclusion of dicing offsets 312 and 412 reduce the area of masks
155B and 155C, respectively, that can be dedicated to reticles.
Probe Arrays Synthesized Using Masks of FIGS. 5A-C, 6I, 6K, and
6L
[0054] A number of advantages, as compared for instance to the
previous mask examples, can be attained by employing a mask design
such as that now described in relation to FIGS. 5A-C, 6I, 6K, and
6L. For convenience, and for reasons that will be described below,
this advantageous design may sometimes be referred to hereafter as
the "interleaved" design, and masks having this design are
sometimes referred to as "interleaved" masks. Significantly, the
interleaved design need not reserve areas of the mask so that those
reserved areas will provide, by themselves, an area on a wafer
through which dicing cuts may be made. Rather, as shown in FIG. 5C,
reticles in many implementations of interleaved masks may be spaced
as closely together as desired without regard to reserving space on
the substrate for dicing. In some implementations in which reticles
are less wide than the width needed to make a dicing cut, a small
boundary area may be provided so that cuts may be made through the
boundary area together with discard areas. Notably, the small
boundary areas of these implementations are supplemented by the
discard areas to provide for dicing, as contrasted with the dicing
offsets of previous examples that themselves were sufficiently wide
to provide an area for a dicing cut.
[0055] Interleaved mask 155D of FIG. 5C is assumed for illustrative
and comparative purposes to be 45 millimeters by 45 millimeters,
the same dimensions illustratively assumed for conventional mask
155A of FIG. 2B. Whereas the reticles of conventional mask 155A are
separated from each other by 3 millimeter dicing offsets, the
reticles of interleaved mask 155D are substantially contiguous on
the mask. As shown in FIG. 5A, the reticles of interleaved mask
155D are smaller than those of conventional mask 155A; e.g., they
are illustratively assumed to be 4.0 millimeters by 4.0
millimeters. One hundred reticles (generally and collectively
referred to as reticles 510) may thus be placed on mask 155D (as
compared to 25 larger reticles, with dicing offsets on mask 155A).
In the illustrated implementation, reticles 510 are separated from
each other by a small boundary that is 0.5 millimeters wide. In
other implementations, this boundary could be arbitrarily small,
including implementations in which there are no boundaries between
reticles; i.e., they abut each other.
[0056] There are a variety of practical reasons, not related to
providing discard areas on a wafer, for optionally providing
boundaries of non-zero width. One reason for providing
non-zero-width boundaries is related to the process of scanning
probe arrays. As shown in FIG. 5B, reticles on an interleaved mask
may be described as being arranged in groups referred to herein as
"interleaved reticle areas." For example, in FIG. 5B, reticles
510A-1, 510A-2, 510A-3, and 510A-4 constitute an interleaved
reticle area 510A. Each of the reticles in interleaved reticle area
510A is associated with a same synthesis area on a wafer, referred
to for illustration of this example as the first synthesis area.
Reticle 510A-1 is aligned with the first synthesis area during a
first light-exposure step, reticle 510A-2 is aligned with the same
first synthesis area during a second light-exposure step, and so
on. As described in greater detail below in relation to FIGS. 6D
through 6H, light shown through reticles 510A-2, 510A-3, and 510A-4
(i.e., the "out-of-step" reticles for the first cycle) during the
first light-exposure step typically will result in the coupling of
a monomer used during the first synthesis cycle on areas of the
substrate surrounding the first synthesis area. Similarly, light
shown through reticles 510A-1, 510A-3, and 510A-4 (the
"out-of-step" reticles for the second cycle) during the second
light-exposure step typically will result in the coupling of a
monomer used during the second synthesis cycle on areas of the
substrate surrounding the first synthesis area, and so on for the
third and fourth cycles. However, as described in greater detail
below, the resulting polymers that may be coupled to the substrate
surrounding the first synthesis area by out-of-step reticles
generally are not related to any intended probe sequence and, in
any event, typically are not used, or intended to be used, as
probes. Thus, the area surrounding the first synthesis area that
may contain polymers synthesized by alignment of out-of-step
reticles may be referred to for convenience as "discard areas."
Even though polymers in discard areas located along the boundaries
with the first synthesis area are not synthesized for the purpose
of hybridizing with potential targets, hybridize may occur.
[0057] When irradiated by a scanner, any labeled targets that have
hybridized with polymers in discard areas around the first
synthesis area will provide detectable excitation radiation. This
excitation radiation may interfere with the identification of
intended probes within the first synthesis area. For example, a
human operator or a software algorithm attempting to identify
corners of the probe array created from the first synthesized area
may be confused by emissions detected from labeled targets that
happened to hybridize with the polymer in discard areas near the
corners. Identification of corners of a probe array is a typical
method for imposing an "alignment grid" to help in identifying
probes from scanned images, as described in greater detail in U.S.
Pat. No. 6,090,555 to Fiekowsky, et al., which is hereby
incorporated by reference herein in its entirety for all purposes.
By providing a small boundary between reticles, a small separation
is provided between a synthesis area and its surrounding discard
areas, and thus the likelihood of confusion may be reduced.
However, providing a non-zero boundary for this reason is optional
and need not be implemented in various implementations.
[0058] Another reason for optionally providing boundaries of
non-zero width is that the design of the machinery used to handle,
align, and/or synthesize masks and/or wafers may make it convenient
to employ masks and/or wafers of particular outside dimensions. For
instance, a mask or wafer having the 45 millimeter by 45 millimeter
dimensions of the present example may be more convenient than one
of 40 millimeters on a side. Thus, whereas 100 reticles of 4
millimeters by 4 millimeters each could fit on a square mask of 40
millimeters on a side and such an implementation is optional, this
arrangement of abutting reticles would merely leave empty space on
the edges of a square mask or wafer having 45 millimeters on a
side. Thus, a separation of 0.5 millimeters may be provided between
reticles of this size on a square mask of 45 millimeters on a side
without affecting the efficiency of mask usage. It may be
advantageous in some cases to increase the size of each reticle
from 4.0 to 4.5 millimeters on a side, thus allowing the synthesis
of additional probes using abutting reticles. However, it also is
possible that the smaller reticle size is sufficient for synthesis
of the desired number of probes, and there thus may be no reason to
increase the reticle size. Also, the equipment used for packaging,
handling, or processing (e.g., hybridizing, washing, scanning)
probe arrays may make it convenient to have probe arrays of a
particular size. Thus, while larger reticles and thus larger probe
arrays might be possible for masks or wafers of a particular size,
there may be no reason to use the larger reticles.
[0059] Yet another practical reason that boundaries of non-zero
width may be employed is if the width of a discard area is less
than the minimum width needed for dicing, and each synthesis area
is separated from its neighboring synthesis areas by no more than
one discard area. For example, if each reticle is a square having
sides of 2.53 millimeters, and the interleaved reticle areas have
four reticles that abut each other, then the discard areas around
each synthesis area on a wafer will be 2.53 millimeters. For
implementations in which the area reserved for the dicing cut must
be no less than 3 millimeters wide, then an addition of 0.5
millimeters increases the effective size of the discard area to
provide the extra margin for dicing.
[0060] The use of interleaved masks to synthesize polymers on a
substrate is now further described in relation to FIGS. 6A through
6M. FIG. 6A is a graphical representation of an interleaved mask
155E-1 similar to mask 155D of FIG. 5C except that fewer reticles
are included for ease of illustration. Mask 155E-1 is one of a set
of masks 155E that may be used in accordance with this example.
Mask 155E-1 has 36 reticles grouped together in interleaved reticle
areas having four reticles each. It will be understood that these
are merely illustrative numbers both with respect to the number of
reticles in the mask and the number of reticles in an interleaved
reticle area. The four reticles in interleaved reticle area 610A
are labeled 610A-1 through 610A-4, the four reticles in interleaved
reticle area 610B are labeled 610B-1 through 610B-4, and so on for
interleaved reticle areas 610C through 610K (all of which are
generally and collectively referred to as interleaved reticle areas
610). Reticles 610A-1, 610B-1, and so on through 610K-1, are
aligned with a synthesis area during a first light-exposure step.
Similarly, reticles 610A-2, 610B-2, and so on through 610K-2, are
aligned with a synthesis area during a second light-exposure step,
and so on for all four steps for which mask 155E-1 is used. For
ease of illustration, this nomenclature is shown in simplified form
in FIG. 6B and subsequent figures, wherein only the step numbers
are shown and the interleaved reticle areas are not explicitly
labeled. FIG. 6C shows a second mask of the set, interleaved mask
155E-2, having the same configuration as mask 155E-1 but used for
synthesis of polymers during light exposure steps 5 through 8.
[0061] FIG. 6D shows interleaved mask 155E-1 aligned with wafer 600
for a first synthesis cycle. Wafer 600 is shown as being slightly
smaller than mask 155E-1, but this difference is provided for ease
of illustration only. Wafer 600 may also be the same size, or
larger than, masks 155E. In FIG. 6D, first-step reticles in each
interleaved reticle area (i.e., reticles 610A-1, 610B-1, and so on
through 610K-1) of mask 155E-1 are aligned with synthesis areas.
Synthesis areas are highlighted for illustrative purposes by dark
lines. During the first synthesis cycle, light is shown through the
first-step reticles to de-protect linker molecules on wafer 600 as
determined by the non-occluded portions of each first-step reticle.
Light may (but need not) also be shown during this first
light-exposure step through all the other reticles of mask 155E-1,
but any polymers thus synthesized are in discard areas of wafer
600.
[0062] FIG. 6E shows interleaved mask 155E-1 aligned with wafer 600
for a second synthesis cycle. Either wafer 600, mask 155E-1, or
both may be moved to accomplish this alignment, and/or optical
elements may be used to accomplish the alignment without
necessarily moving either wafer 600 or mask 155E-1. In FIG. 6E,
second-step reticles in each interleaved reticle area (i.e.,
reticles 610A-2, 610B-2, and so on through 610K-2) of mask 155E-1
are aligned with synthesis areas. Reticle 610A-2 is aligned with
the same synthesis area with which reticle 610A-1 was aligned
during the first synthesis cycle, reticle 610B-2 is aligned with
the same synthesis area with which reticle 610B-1 was aligned
during the first synthesis cycle, and so on for each interleaved
reticle area. That is, each reticle of a same interleaved reticle
area is aligned with a particular synthesis area common to the
reticles of that interleaved reticle area during the cycle in which
that reticle is used for synthesis of probes. Typically,
out-of-step reticles (i.e., those not used during a particular
cycle for synthesis of probes and thus not aligned with a synthesis
area) are aligned with discard areas.
[0063] FIGS. 6F and 6G show interleaved mask 155E-1 aligned with
wafer 600 for third and fourth synthesis cycles, respectively. In
similar fashion, mask 155E-2, and other masks that may be included
in mask set 155E, are sequentially aligned and then re-aligned over
synthesis areas.
[0064] FIG. 6H shows wafer 600 after N synthesis cycles, where each
mask of mask set 155E had been used for four cycles and then
replaced with the next in the set. As shown in FIG. 6H, wafer 600
includes synthesis areas 630A through 630I (generally and
collectively referred to as synthesis areas 630). Dicing lines 612
and 614 may be cut so as to physically separate wafer 600 into nine
substrates suitable for packaging or otherwise to be used as probe
arrays. It will be understood that the number, orientation, and
placement of dicing lines 612 and 614 in FIG. 6H are illustrative
only. For example, if it is desired to dice wafer 600 so that the
area of the separated substrates are as close as possible to that
of the synthesis area, then the dicing lines could be shifted
closer to the synthesis areas and additional dicing lines added as
necessary.
[0065] FIGS. 6I and 6J provide further detail regarding the dicing
of wafer 600 after the N light-exposure steps have been completed.
FIG. 6I shows the upper left portion of wafer 600 consisting of
synthesis area 630A after N light-exposure steps, surrounded by
discard areas 632A, 634A, and 636A. This region of a wafer
consisting of a synthesis area surrounded by discard areas may
sometimes be referred to herein as a single-chip interleaved area,
such as area 640 of the example of FIG. 6I. The term "single-chip"
is used to indicate that dicing lines may be cut, in any manner,
through the discard areas surrounding the synthesis area in order
to provide a separate probe array (e.g., a separate GeneChip.RTM.
probe array). As can be seen from FIG. 6D, reticles 610A-2, 610A-3,
and 610A-4 are aligned with discard areas 632A, 634A, and 636A,
respectively, during light-exposure step one. FIG. 6E shows that,
during light-exposure step two, reticle 610A-3 is aligned with
discard area 636A, reticle 610B-1 is aligned with discard area
632A, and reticle 610B-4 is aligned with discard area 634A. FIGS.
6F and 6G similarly show the alignment of reticles with both
synthesis areas and discard areas during light-exposure steps three
and four, respectively. FIG. 6J shows how dicing lines 612 and 614
may pass through discard areas so that synthesis areas 630 may be
diced to provide physically separate probe arrays.
[0066] FIGS. 6K through 6M show an implementation of the
interleaved mask design using another arrangement of reticles in
interleaved reticle areas. In this example, each interleaved
reticle area, such as area 645A of FIG. 6K, includes six reticles
arranged in two rows and three columns. Thus, for example, reticles
647A-1 through 647A-6 may be sequentially aligned over synthesis
area 650A of single-chip interleaved area 660 of wafer 670 (shown
in FIGS. 6L and 6M) during light-exposure steps one through six,
respectively. In the manner described above, discard areas 652A,
654A, 656A, 658A, and 659A are formed around synthesis area 650A
during these, and other, light exposure steps. The resulting
synthesis areas 650A through 650I are shown after N light-exposure
steps in FIG. 6M, surrounded by discard areas through which cuts
may be made, for example, along dicing lines 662 and 664.
[0067] It will be understood that the examples of FIGS. 5A through
5C, and 6A through 6M, are illustrative only, and that many
combinations of numbers of rows and columns of reticles in
interleaved reticle areas may be used in alternative
implementations. Moreover, patterns other than rows and columns may
also be employed to constitute interleaved reticle areas. Also, it
is not required that reticles be square, as shown for convenience
in these figures. They may be any other shape.
[0068] As will now be appreciated, a significant advantage of using
interleaved masks is that the number of masks in a mask set
generally may be substantially reduced as compared to designs in
which portions of the mask are dedicated to dicing offsets. At the
same time, the productivity of interleaved masks generally need not
be compromised in terms of the number of probe arrays synthesized.
These advantages are demonstrated, for example, by comparing
conventional mask 155A of FIG. 2B with interleaved mask 155D of
FIG. 5C. The size of each mask has illustratively been assumed to
be the same: 45 millimeters on a side. As noted, details such as
the use of alignment features are not considered with respect to
either mask for sake of clarity and ease of illustration. The
number of probe arrays synthesized from a single mask set is also
the same for each of the masks: 25 synthesis areas yielding 25
probe arrays. It is now assumed that the probe arrays are
synthesized using N light-exposure steps. Employing the
conventional design of mask 155A, N masks are therefore used.
Employing the interleaved design of mask 155D, N/4 masks (or N/4
rounded down to an integer, plus 1, if N is not evenly divisible by
4) are used. As noted, the savings in terms both of time and
expense typically are therefore significant. Moreover, these
savings can be driven further by employing interleaved designs in
which additional steps are implemented by reticles of the same
mask, as in mask 155F of FIG. 6K in which six light-exposure steps
are implemented using each mask.
[0069] As is evident, increasing the number of light-exposure steps
implemented on each mask, assuming the mask size remains the same,
generally is accomplished by reducing the size of the reticles.
Smaller reticles generally results in smaller synthesis areas,
which generally means, assuming a constant probe density, that
fewer probes may be synthesized on each synthesis area and thus
fewer probes are included in the resulting probe arrays. However,
this effect is ameliorated by at least two considerations. First,
the mask area is efficiently used in accordance with the
interleaved design because little or no space need be provided
between reticles. Thus, more information can generally be carried
by an interleaved mask than by conventional masks in which dicing
offsets, generally carrying no information, are included.
[0070] Second, there are important applications in which the
savings in time and money achieved by using interleaved masks far
outweighs any reduction in the number of probes in the resulting
probe arrays. For example, there is considerable demand among users
for custom-made arrays that can be provided relatively quickly and
inexpensively. A user of custom-made arrays may not require that
tens of thousands of genes or EST's be probed in a single array.
Rather, the user may require probe arrays representing far fewer
sequences, e.g., 100 to 1,000 genes or EST's. Moreover, the user
may not need large numbers of the customized arrays. For example, a
dozen arrays may be sufficient. For user demand of this type, the
previous example of a reticle of 2.53 millimeters on a side
generally provides sufficient probe density using conventional
probe synthesis technology while producing sufficient number of
probe arrays using far fewer masks as compared to conventional mask
designs. For example, if the boundary area between reticles has a
width of 0.47 millimeters (so that the boundary area plus discard
area is sufficient for a 3 millimeter dicing cut), then the
illustrative square mask of 45 millimeters on a side can
accommodate 15.times.15=225 reticles. If these 225 reticles are
applied to 15 light-exposure steps, then a mask set generally
produces 15 square synthesis areas of 2.53 millimeters on a side
and 15 probe arrays of this size. It is illustratively assumed that
75 light-exposure steps are used for complete synthesis of probes
(e.g., 25-mer oligonucleotides). Thus, only five interleaved masks
(75 divided by 15) are required in the illustrative mask set. Using
the conventional approach represented by mask 155A, 75 masks would
be needed. The fifteen-fold reduction in the size of the mask set
achieved using the interleaved masks provides significant savings
in time and cost while satisfying user demand with respect to probe
density and number of probe arrays supplied.
[0071] FIG. 7 is a simplified flow chart showing illustrative steps
that may be employed to implement the use of interleaved masks as
described above. It will be understood that many variations to
these steps are possible, and that the steps may be alternatively
characterized. For example, step 702 is to determine a group of
non-contiguous synthesis areas on a substrate, each separated from
each of its nearest neighbors by a discard area. Aspects of this
step could alternatively be characterized as providing a mask
having substantially contiguous interleaved reticle areas, each
consisting of substantially contiguous reticles that may
sequentially be aligned over a same synthesis area on a substrate.
Step 704 is to select an initial group of reticles on a selected
mask. For example, all first-step reticles in each of the reticle
areas of a first mask could be selected. Step 705 is to align each
of the selected group of reticles with one of the determined group
of synthesis areas. Thus, in this example, the first-step reticles
are each aligned with a synthesis area. Step 710 is to couple
monomers onto the synthesis areas at locations determined by the
aligned reticles. For example, monomers are coupled to locations on
the synthesis areas determined by non-occluded portions of the
first-step reticles, i.e., those portions through which light shown
during the first light-exposure step so as to de-protect molecules
on the substrate. As indicated by decision elements 720 and 730,
and steps 732 and 722, this process is repeated for all of the
reticles in the interleaved reticle areas for each of the masks in
the mask set (i.e., for step 2 through step N reticles). Then, as
indicated in step 740, the substrate may be diced through areas
including discard areas to provide physically separated substrates
suitable for further processing and packaging as probe arrays.
[0072] The foregoing steps, and others that may be used in numerous
variations of the interleaved mask design, may be implemented, for
example, in the form of computer instructions and data such as
represented by desired mask characteristics 106 of FIG. 1. As
described above, mask design system 101 employs mask design
application 199 to operate on characteristics 106 and probe
information 107 to provide mask specification files 112 and probe
specification files 114. Mask manufacturing system 150 uses mask
specification files 112 to produce masks 155. FIG. 8 is a
simplified functional block diagram of a probe array synthesis
system 800 suitable for producing synthesized probe arrays 810
based on files 112 and 114, masks 155, and various other materials
(e.g., reagents 804, substrates, packaging, and other materials
806) well known to those of ordinary skill in the relevant
arts.
[0073] System 800 includes a computer or controller 810, which may
be any type of general purpose computer, as described above with
respect to computer 100, or a dedicated processor or controller.
System 800 also includes aligner 820 that, typically under the
control of computer or controller 810, performs the sequential
alignments of masks and substrates as specified, for example, by
aspects of mask specification files 112. Thus, alignment steps of
FIG. 7, or other steps for implementing an interleaved mask design,
are provided to system 800 as data and/or instructions in files 112
and implemented by masks 155 produced in accordance with mask
characteristics 106 embodying interleaved mask designs. A further
element of system 800 is synthesizer 830 that also typically
operates under the control of computer or controller 810. In
accordance with techniques well known to those of ordinary skill in
the art, synthesizer applies reagents 804 and materials 806 to form
polymers on a substrate, and to dice the substrate, all as
described above.
[0074] Having described various embodiments and implementations, it
should be apparent to those skilled in the relevant art that the
foregoing is illustrative only and not limiting, having been
presented by way of example only. Many other schemes for
distributing functions among the various functional elements of the
illustrated embodiment are possible. The functions of any element
may be carried out in various ways in alternative embodiments. For
example, some or all of the functions described as being carried
out by computer or controller 810 could be carried out by aligner
820 and/or synthesizer 830.
[0075] Also, the functions of several elements may, in alternative
embodiments, be carried out by fewer, or a single, element.
Similarly, in some embodiments, any functional element may perform
fewer, or different, operations than those described with respect
to the illustrated embodiment. Also, functional elements shown as
distinct for purposes of illustration may be incorporated within
other functional elements in a particular implementation. Also, the
sequencing of functions or portions of functions generally may be
altered. Certain functional elements, files, data structures, and
so on, may be described in the illustrated embodiments as located
in system memory of a particular computer. In other embodiments,
however, they may be located on, or distributed across, computer
systems or other platforms that are co-located and/or remote from
each other. For example, any one or more of data files or data
structures described as co-located on and "local" to a server or
other computer may be located in a computer system or systems
remote from the server or other computer. In addition, it will be
understood by those skilled in the relevant art that control and
data flows between and among functional elements and various data
structures may vary in many ways from the control and data flows
described above or in documents incorporated by reference herein.
More particularly, intermediary functional elements may direct
control or data flows, and the functions of various elements may be
combined, divided, or otherwise rearranged to allow parallel
processing or for other reasons. Also, intermediate data structures
or files may be used and various described data structures or files
may be combined or otherwise arranged. Numerous other embodiments,
and modifications thereof, are contemplated as falling within the
scope of the present invention as defined by appended claims and
equivalents thereto.
* * * * *