U.S. patent application number 11/232584 was filed with the patent office on 2006-05-04 for lithographic mask design and synthesis of diverse probes on a substrate.
This patent application is currently assigned to Affymetrix, INC.. Invention is credited to Earl A. Hubbell, Michael P. Mittmann, Lubert Stryer.
Application Number | 20060094040 11/232584 |
Document ID | / |
Family ID | 25080891 |
Filed Date | 2006-05-04 |
United States Patent
Application |
20060094040 |
Kind Code |
A1 |
Hubbell; Earl A. ; et
al. |
May 4, 2006 |
Lithographic mask design and synthesis of diverse probes on a
substrate
Abstract
Systems and methods of synthesizing probes on a substrate are
provided. One or more shift reticles are utilized to uniformly add
monomers to the substrate at specified locations. The shift
reticles are shifted relative to the substrate between monomer
addition steps. Additionally, characteristics of the desired probes
may be specified at synthesis time.
Inventors: |
Hubbell; Earl A.; (Mountain
View, CA) ; Stryer; Lubert; (Stanford, CA) ;
Mittmann; Michael P.; (Palo Alto, 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: |
25080891 |
Appl. No.: |
11/232584 |
Filed: |
September 21, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09567548 |
May 5, 2000 |
|
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11232584 |
Sep 21, 2005 |
|
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09059779 |
Apr 13, 1998 |
6153743 |
|
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09567548 |
May 5, 2000 |
|
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08767892 |
Dec 17, 1996 |
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09059779 |
Apr 13, 1998 |
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Current U.S.
Class: |
435/6.11 ;
435/287.2 |
Current CPC
Class: |
C07K 1/045 20130101;
B01J 2219/00675 20130101; C40B 50/14 20130101; B01J 2219/00695
20130101; B01J 2219/00585 20130101; B01J 2219/00529 20130101; B01J
2219/00689 20130101; B01J 2219/00722 20130101; C40B 40/10 20130101;
G03F 7/70466 20130101; G03F 7/00 20130101; B01J 2219/00731
20130101; B01J 2219/0059 20130101; B01J 19/0046 20130101; B01J
2219/00432 20130101; B01J 2219/00711 20130101; B82Y 30/00 20130101;
C40B 40/06 20130101; B01J 2219/00596 20130101; C40B 40/12 20130101;
B01J 2219/00608 20130101; B01J 2219/00659 20130101; C40B 40/08
20130101; C40B 60/14 20130101; B01J 2219/00725 20130101; B01J
2219/00527 20130101; C07K 1/047 20130101 |
Class at
Publication: |
435/006 ;
435/287.2 |
International
Class: |
C40B 40/08 20060101
C40B040/08; C12Q 1/68 20060101 C12Q001/68; C12M 1/34 20060101
C12M001/34 |
Claims
1. A method of synthesizing rows of probes including an
interrogation position on a substrate, comprising the steps of
coupling non-interrogation position monomers on the substrate in a
first region having a first width, and coupling rows of
interrogation position monomers on the substrate in a second region
having a second width, the second region being within the first
region and the second width being less than the first width.
2. The method of claim 1, wherein each row of probes includes a
different monomer at the interrogation position.
3. The method of claim 2, wherein the interrogation position
monomers include A, C, G, and T(U).
4. The method of claim 1, the probes that are within the first
region and not the second region do not include interrogation
position monomers.
5. The method of claim 1, wherein coupling the non-interrogation
position monomers on the substrate, comprises coupling monomers on
the substrate at locations specified by at least one shift reticle,
shifting the at least one shift reticle relative to the substrate,
and after shifting the at least one shift reticle, coupling
monomers on the substrate at locations specified by the at least
one shift reticle.
6. The method of claim 5, wherein the probes synthesized on the
substrate include multiple monomers that were coupled to the
substrate utilizing the at least one shift reticle.
7. The method of claim 1, wherein coupling the interrogation
position monomers on the substrate, comprises coupling monomers on
the substrate at locations specified by an interrogation position
reticle, shifting the interrogation position relative to the
substrate, and after shifting the interrogation position reticle,
coupling monomers on the substrate at locations specified by the
interrogation position reticle.
8. The method of claim 7, wherein the interrogation position
reticle is shifted in a direction perpendicular to a direction that
the at least one shift reticle is shifted.
9. A substrate, comprising multiple rows of probes including an
interrogation position monomer coupled to the substrate, and at
least one row of probes that do not include an interrogation
position monomer coupled to the substrate between two rows of
probes the do include an interrogation position monomer.
10. The substrate of claim 9, wherein the multiple rows of probes
including an interrogation position monomer include four rows of
probes wherein each rows of probes includes a different monomer at
the interrogation position.
11. The substrate of claim 10, wherein the interrogation position
monomers include A, C, G, and T(U).
12. The substrate of claim 8, further comprising the step of
shifting the shift reticle relative to the substrate in order to
couple a second layer of different types of monomers on the
substrate at the specified locations.
13. A method of synthesizing probes on a substrate, comprising
coupling monomers on the substrate at locations specified by a
plurality of shift reticles, each shift reticle being for coupling
a specific monomer on the substrate and including a monomer
addition region for coupling an interrogation position monomer,
shifting the plurality of shift reticles relative to the substrate
in a first direction, and after shifting the plurality of shift
reticles, coupling monomers on the substrate at locations specified
by the plurality of shift reticles so that probes are synthesized
on the substrate including different interrogation positions.
14. The method of claim 13, wherein each shift reticle includes
rectangular monomer addition regions that are longer in a second
direction that is perpendicular to the first direction.
15. The method of claim 14, wherein each shift reticle includes an
interrogation position monomer addition region that are at a
different location in the second direction.
16. The method of claim 13, wherein the plurality of shift reticles
include at least one region in a same location that does not couple
monomers on the substrate so that probes of varying lengths may be
synthesized at the same time.
Description
[0001] This is a continuation of application Ser. No. 09/059,779,
filed Apr. 13, 1998, which is a continuation-in-part of application
Ser. No. 08/767,892, filed Dec. 17, 1996, which are both hereby
incorporated by reference for all purposes.
COPYRIGHT NOTICE
[0002] A portion of the disclosure of this patent document contains
material that is subject to copyright protection. The copyright
owner has no objection to the xerographic reproduction by anyone of
the patent document or the patent disclosure in exactly the form it
appears in the Patent and Trademark Office patent file or records,
but otherwise reserves all copyright rights whatsoever
MICROFICHE APPENDIX
[0003] A Microfiche Appendix (1 microfiche, 72 frames) of a
computer program listing of an embodiment of the invention is
included herewith
BACKGROUND OF THE INVENTION
[0004] The present invention is related to computer systems for
generating masks. More particularly, the invention provides systems
and methods for generating and utilizing masks to from probes on a
substrate
[0005] U.S. Pat. No. 5,424,186 describes a pioneering technique
for, among other things, forming and using high density arrays of
molecules such as oligonucleotide, RNA, peptides, polysaccharides,
and other materials. This patent is hereby incorporated by
reference for all purposes. Arrays of oligonucleotides or peptides,
for example, are formed on the surface by sequentially removing a
photoremovable group from a surface, coupling a monomer to the
exposed region of the surface, and repeating the process. These
techniques have been used to form extremely dense arrays of
oligonucleotides, peptides, and other materials. Such arrays are
useful in, for example, drug development, oligonucleotide
sequencing, oligonucleotide sequence checking, and a variety of
other applications. The synthesis technology associated with this
invention has come to be known as "VLSIPS" or "Very Large Scale
Immobilized Polymer Synthesis" technology
[0006] Additional techniques for forming and using such arrays are
described in U.S. Pat. No. 5,384,261, which is also incorporated by
reference for all purposes. Such techniques include systems for
mechanically protecting portions of a substrate (or chip), and
selectively deprotecting/coupling materials to the substrate. These
techniques are now known as "VLSIPS II". Still further techniques
for array synthesis are provided in U.S. application Ser. No.
08/327,512, also incorporated herein by reference for all
purposes
[0007] Dense arrays fabricated according to these techniques are
used, for example, to screen the array of probes to determine which
probe(s) are complementary to a target of interest. According to
one specific aspect of the inventions described above, the array is
exposed to a labeled target. The target may be labeled with a wide
variety of materials, but an exemplary label is a fluorescein
label. The array is then scanned with a confocal microscope based
detection system, or other related system, to identify where the
target has bound to the array. Other labels include, but are not
limited to, radioactive labels, large molecule labels, and
others.
[0008] While meeting with dramatic success, such methods meet with
limitations in some circumstances. For example, during the design
of the layout of molecules in an array according to the above
techniques, it is necessary to design a "mask" that will define the
locations on a substrate that are exposed to light. While such
masks are easily fabricated, they tend to be costly. The design of
such masks is described in U.S. Pat. No. 5,571,639, incorporated
herein by reference for all purposes.
[0009] Often it is desirable to have a specific layout of molecules
in an array for a particular application. For example, PCT
WO95/11995, which is incorporated by reference for all purposes,
describes the synthesis of particular arrays for use in HIV
diagnostics, the diagnosis of genes relevant to certain cancers,
evaluation of the mitochondrial oligonucleotide, and other
applications. In many of these applications there is demand for a
large volume of identical chips, such as in HIV diagnostics. In
many situations, the manufacture of a particular probe array will
require a mask (or mask set) with as many as one hundred reticles
or more. The cost of masks in these situations, while high on a per
mask basis, becomes quite small when viewed in light of the number
of identical arrays that may be synthesized with a particular
mask.
[0010] However, in many other applications, such as particular
research applications, it is desirable to synthesize a relatively
small number of arrays with a particular layout of probes, perhaps
as few as a single array. While this is certainly possible and has
found wide utility in the art, it is costly to fabricate a single
mask (or mask set) for the manufacture of only a few probe arrays.
Accordingly, the "per chip" cost of masks in these situations can
be quite high (on the order of thousands of dollars).
[0011] Accordingly, it is desirable to identify more efficient
techniques for designing and using lithographic masks in the
manufacture of probe arrays and, in particular, reduce the number
of reticles required for a low volume design.
SUMMARY OF THE INVENTION
[0012] The present invention provides techniques for more
economically synthesizing arrays of probes on a substrate. One or
more "shift" reticles are utilized to synthesize many different
probe sets on a substrate. A shift reticle is a reticle that is
shifted (one position or more) after a monomer addition step and
then reused which reduces the number of reticles (or masks)
required. Additionally, the shift masks uniformly add monomers to
the substrate at certain probe locations during synthesis.
Embodiments of the invention allow the length of the probes and
interrogation position to be specified at synthesis time thereby
providing greater flexibility in chip synthesis.
[0013] In one embodiment of the invention, a method of synthesizing
probes on a substrate, comprises the steps of coupling monomers on
the substrate at locations specified by at least one shift reticle,
shifting the at least one shift reticle relative to the substrate,
and after shifting the at least one shift reticle, coupling
monomers on the substrate at locations specified by the at least
one shift reticle, wherein probes including monomers are
synthesized on the substrate
[0014] In another embodiment of the invention, a method of
synthesizing probes on a substrate, comprises the steps of
providing at least one reticle, the at least one reticle for
uniformly adding monomers to the substrate at specified locations,
receiving input as to a characteristic of the probes desired, and
utilizing the at least one reticle to synthesize the desired probes
on the substrate. The characteristic may be the length of the
desired probes, the interrogation position, or the monomer addition
order for synthesizing the desired probes.
[0015] In another embodiment of the invention, a method of
synthesizing probes on a substrate comprises the steps of providing
a set of reticles having monomer addition regions, each reticle for
coupling a different type of monomer on the substrate, utilizing
each reticle of the set to couple a first layer of monomers on the
substrate, the first layer of monomers including different types of
monomers, and shifting each reticle of the set relative to the
substrate to couple a second layer of monomers on the first layer,
the second layer of monomers including different types of monomers,
wherein a plurality of probes including two monomers are formed on
the substrate
[0016] In another embodiment, a method for determining the layout
of a reticle for synthesizing probes on a substrate comprises the
steps of receiving input of a target sequence of monomers,
selecting a type of monomer in the target sequence, and designing a
reticle with monomer addition regions corresponding to each monomer
in the target sequence that is the selected type of monomer.
[0017] In another embodiment, a method of synthesizing probes on a
substrate comprises the steps of coupling a plurality of first
monomers on the substrate at locations specified by a set of
monomer addition regions of a reticle, shifting the reticle
relative to the substrate, and coupling at least one second monomer
on one of the first monomers at a location specified by one of the
set of monomer addition regions of the reticle, wherein a probe
including the first and second monomers is formed on the
substrate.
[0018] In another embodiment, a computer-implemented method for
determining the layout of a reticle for synthesizing probes on a
substrate comprises the steps of receiving input of a target
sequence of monomers, selecting a type of monomer in the target
sequence, and designing a reticle with monomer addition regions
specified by n*(i-1)+1 wherein n=the number of different types of
monomers and i=a position of a first monomer in the target
sequence.
[0019] In another embodiment, a method for specifying the layout of
a substrate including probes synthesized on the substrate,
comprises the steps of defining the probes to be synthesized on the
substrate as a sequential list of analysis regions, each analysis
region including probes, receiving input as to a characteristic of
the sequential list of analysis regions, and designing at least one
reticle to synthesize the probes of each analysis region on the
substrate with the input characteristic. Typically the input
characteristic includes the location, scale or orientation of the
analysis regions.
[0020] In another embodiment, a method of synthesizing rows of
probes including an interrogation position on a substrate,
comprising the steps of coupling non-interrogation position
monomers on the substrate in a first region having a first width,
and coupling rows of interrogation position monomers on the
substrate in a second region having a second width, the second
region being within the first region and the second width being
less than the first width. Hybridization data from the probes may
be more accurate because the "edge effect" between adjacent probe
regions or cells is reduced.
[0021] In other embodiments, shift masks may be utilized to
synthesize diverse probes for interrogating a base position in a
target. For example, probes of a specific length may be synthesized
that include every possible interrogation position in the probes.
Additionally, probes of different lengths with different
interrogation positions may be synthesized on a chip at the same
time.
[0022] A further understanding of the nature and advantages of the
inventions herein may be realized by reference to the remaining
portions of the specification and the attached drawings.
BRIEF DESCRIPTION OF THE DRAWINGS
[0023] FIG. 1 illustrates an example of a computer system used to
execute software embodiments of the present invention,
[0024] FIG. 2 shows a system block diagram of a typical computer
system used to execute software embodiments of the present
invention,
[0025] FIG. 3A illustrates a probe set that may be utilized to
detect a target sequence and FIG. 3B shows a layout of the probe
set on a substrate in one embodiment,
[0026] FIG. 4 shows prior art reticles that produce the probe set
of FIG. 3A,
[0027] FIG. 5 shows the prior art addition of monomers to produce
the probe set of FIG. 3A,
[0028] FIG. 6 shows a high level flow of a process of generating
reticles according to one embodiment of the present invention,
[0029] FIG. 7 shows shift reticles that produce the probe set of
FIG. 3A,
[0030] FIGS. 8A-8C shows the addition of monomers using the shift
reticles to produce the probe set,
[0031] FIGS. 9A-9B show reticles for producing multiple probe
sets,
[0032] FIG. 10 shows the transformation of a linear reticle into a
rectangular reticle,
[0033] FIG. 11A shows a reticle for adding monomers at an
interrogation position, FIG. 11B shows probes on a chip that vary
at an interrogation position, and FIG. 11C is an image of a chip
produced with a reticle similar to the one in FIG. 11A,
[0034] FIG. 12A shows another reticle for adding monomers at an
interrogation position, FIG. 12B shows probes on a chip that vary
at an interrogation position, and FIG. 12C is an image of a chip
produced with a reticle similar to the one in FIG. 12A,
[0035] FIG. 13A shows a reticle for producing multiple probe set of
different lengths and FIG. 13B shows a chip with multiple length
probe sets,
[0036] FIG. 14A is a mask including multiple reticles, FIG. 14B
shows the layout of a reticle in one embodiment, FIG. 14C shows
reticles for synthesizing probes on two chips simultaneously, FIG.
14D shows a mask for synthesizing varying length probes on two
chips simultaneously, and FIG. 14E shows a sample chip layout,
[0037] FIG. 15A shows a layout of a chip in another embodiment,
FIG. 15B shows a shift reticle for coupling a particular monomer on
a chip in pairs of rows, FIG. 15C shows a shift reticle for
coupling a particular monomer on a chip in a single lane, and FIG.
15D shows a shift reticle for forming control lanes,
[0038] FIG. 16 shows a high level flow of a process of generating
reticles according to another embodiment of the present
invention,
[0039] FIGS. 17A-17D show the formation of a single shift
reticle,
[0040] FIG. 18 shows a single reticle that produces the probe set
of FIG. 3A and the addition of monomers using this reticle,
[0041] FIG. 19 shows a reticle for producing multiple probe
sets,
[0042] FIG. 20A shows a shift reticle for another embodiment, FIGS.
20B-20D shows interrogation position reticles, and FIG. 20E shows a
chip including perfectly complementary, interrogation position and
deletion probes,
[0043] FIG. 21A shows a shift reticle for synthesizing related
probes of varying lengths and FIG. 21B shows an example of the
layout of the probes that may be synthesized on the substrate,
[0044] FIG. 22 is a simple example of speckle masks,
[0045] FIG. 23 shows that the shift reticles of FIG. 7 are speckle
masks,
[0046] FIG. 24 shows the packing of speckle masks,
[0047] FIG. 25 shows the layout of a chip utilizing post chip
synthesis,
[0048] FIG. 26 shows the layout of another chip utilizing post chip
synthesis,
[0049] FIG. 27A shows an active region of a chip that has tightly
packed lanes, FIG. 27B shows a subregion from FIG. 27A, and FIG.
27C shows how the subregion of FIG. 27B may be synthesized to
minimize edges,
[0050] FIG. 28 shows shift reticles that produce equal length
probes with different interrogation positions,
[0051] FIG. 29 illustrates the probes that may be produced by the
reticles of FIG. 28,
[0052] FIG. 30 shows a shift reticle for producing probes with
different lengths and interrogation positions,
[0053] FIG. 31 illustrates the probes that may be produced by the
shift reticles according to FIG. 30, and
[0054] FIG. 32 shows a shift reticle for producing probes that
interrogate every ninth base position in a target
DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS
Terminology
[0055] As used herein, the following terms are intended to have the
following meanings.
[0056] "Mask" refers to a lithographic member, usually a plate of
glass, with a number of apertures therein that allow for selective
passage of light. A mask may contain one or more reticles.
[0057] "Reticle" refers to all or a particular portion of a mask
that is used to direct light to a substrate during an exposure.
Introduction
[0058] High density, miniaturized arrays of molecular probes are
made herein using light directed synthesis techniques. Such arrays
may be arrays of oligonucleotides, peptides, small molecules (such
as benzodiazapines, prostoglandins, beta-turn mimetics),
non-natural ligands, enzymes, or any of a wide variety of other
molecules synthesized in a "building block" fashion, such as
oligosaccharides, and the like. Oligonucleotide probe arrays are
representative of the arrays that may be used according to specific
aspects of the invention herein.
[0059] The design and fabrication of oligonucleotide probe arrays
relies on VLSIPS technology according to a specific aspect of the
invention. The first step in fabricating a oligonucleotide probe
array involves choosing a set of oligonucleotide probes to be
synthesized on the chip. Suppose, for example, it is desirable to
detect a base change mutation at a single position in gene. The
techniques herein would provide a set of four probes that are
complementary to a short region around the single position. The
first probe would be exactly complementary to the wild-type
(normal) sequence for that region of the gene. The other three
probes would be identical to the first, except they would
substitute the three bases that are not complementary to the
wild-type sequence at the position being interrogated (i.e., the
interrogation position).
[0060] In this way regardless of the base change mutation, one of
the probes will be perfectly complementary to the target
oligonucleotide sequence. To detect any such mutation in the gene,
i.e., to resequence the gene, one may define similar sets of probes
for each position in the gene. For example, to resequence the 1040
bases of HIV necessary to detect drug resistance related mutations,
4160 probes are generally required. Such techniques are described
in greater detail in PCT WO95/11995 which is incorporated herein by
reference for all purposes. Of course, arrays such as peptide
arrays will provide for different techniques of probe
selection.
[0061] Once a set of probes is chosen, the layout of the probes on
the chip is determined. The layout is used to design the
photolithographic masks used in chip synthesis process. These
designs in general are produced in electronic form and are used to
fabricate the masks in a mask fabrication shop such as those widely
used in the semiconductor industry.
[0062] FIG. 1 illustrates an example of a computer system used to
execute software embodiments of the present invention to generate
masks for chip synthesis. FIG. 1 shows a computer system 1 which
includes a monitor 3, screen 5, cabinet 7, keyboard 9, and mouse
11. Mouse 11 may have one or more buttons such as mouse buttons 13.
Cabinet 7 houses a. CD-ROM drive 15 and a hard drive (not shown)
that may be utilized to store and retrieve software programs
including computer code incorporating the present invention.
Although a. CD-ROM 17 is shown as the computer readable medium,
other computer readable media including floppy disks, DRAM, hard
drives, flash memory, tape, and the like may be utilized. Cabinet 7
also houses familiar computer components (not shown) such as a
processor, memory, and the like.
[0063] FIG. 2 shows a system block diagram of computer system 1
used to execute software embodiments of the present invention. As
in FIG. 1, computer system 1 includes monitor 3 and keyboard 9.
Computer system 1 further includes subsystems such as a central
processor 102, system memory 104, I/O controller 106, display
adapter 108, removable disk 112, fixed disk 116, network interface
118, and speaker 120. Removable disk 112 is representative of
removable computer readable media like floppies, tape, CD-ROM,
removable hard drive, flash memory, and the like. Fixed disk 116 is
representative of an internal hard drive or the like. Other
computer systems suitable for use with the present invention may
include additional or fewer subsystems. For example, another
computer system could include more than one processor 102 (i.e., a
multi-processor system) or memory cache.
[0064] Arrows such as 122 represent the system bus architecture of
computer system 1. However, these arrows are illustrative of any
interconnection scheme serving to link the subsystems. For example,
display adapter 108 may be connected to central processor 102
through a local bus or the system may include a memory cache.
Computer system 1 shown in FIG. 2 is but an example of a computer
system suitable for use with the present invention. Other
configurations of subsystems suitable for use with the present
invention will be readily apparent to one of ordinary skill in the
art. For example, software embodiments of the invention may be
implemented on an IBM compatible computer, workstations from Sun
Microsystems, and the like.
[0065] Light-directed chemical synthesis combines
semiconductor-based photolithography and solid phase chemical
synthesis. To begin the process, linkers modified with
photochemically removable protecting groups are attached to a solid
substrate or chip surface. Light is directed through a
photolithographic mask or reticle to specific areas of the
synthesis surface, activating those areas for chemical coupling.
The first of a series of chemical building blocks (A, C, G, U or T)
is incubated with the chip, and chemical coupling occurs at those
sites which have been illuminated in the preceding step. Next,
light is directed to a different region of the substrate through a
new mask, and the chemical cycle is repeated.
[0066] The patterns of light and the order of chemical reagents
dictate the identity of each oligonucleotide probe on the chip
surface. Using combinatorial synthesis methods, millions of
chemical compounds can be created rapidly in very few process
steps.
[0067] Oligonucleotide probe arrays contain thousands or millions
of oligonucleotide probes that can be used to recognize longer
target oligonucleotide sequences (for example, from patient
samples). The recognition of sample oligonucleotide by the set of
oligonucleotide probes on the chip takes place through the
mechanism of oligonucleotide hybridization. Oligonucleotide
hybridization is the simple process in which two complementary
strands of oligonucleotide join together (A pairs with T and G
pairs with C). When an oligonucleotide target hybridizes with an
array of oligonucleotide probes, the target will bind to those
probes that are complementary to a part of the target
oligonucleotide sequence.
[0068] Information about the sequence of the target oligonucleotide
may be determined according to which probes hybridized with the
target. Such arrays have applications for oligonucleotide probe
arrays in oligonucleotide sequence analysis, oligonucleotide
sequence checking, mutational analysis, mRNA expression monitoring,
and medical diagnostic research.
[0069] The invention herein provides a technique for synthesizing
probe arrays in which the cost of mask manufacturing is reduced. In
preferred embodiments of the invention, mask costs are reduced by
designing one or a few shift reticles that may be used to
synthesize arrays of probes on a substrate. Accordingly, the shift
reticle(s) may be used to synthesize "custom" arrays of probes, but
the cost of making the mask set for such custom probes is greatly
reduced on a per chip basis.
[0070] FIG. 3A illustrates a probe set that would be desirable for
the evaluation of nucleic acid samples expected to contain the
sequence TGACAT. To evaluate a sample to determine if its sequence
is, in fact, TGACAT a set of 3-mer probes as shown in FIG. 3A may
be synthesized on a substrate. If a particular sample did have the
sequence. TGACAT (the "target" sequence), it would be expected to
hybridize to each of the probes ACT, CTG, TGT, and GTA as they are
complementary. If, however, there was a variation in the second
base position ("G"), lower hybridization would likely be observed
in the ACT probe region as there is a single base mismatch to the
target. Suppose, for example, a particular sample had the sequence.
TAACAT. Hybridization would not likely be observed in the ACT probe
region since. ACT is not perfectly complementary to the sequence.
TAACAT at any position.
[0071] As disclosed PCT WO95/11995, additional probes may be
synthesized to determine which variation is present at a particular
position. For example, in addition to the ACT probe, the probes
AAT, ATT, and AGT may be synthesized on the substrate (the
interrogation position is underlined). The strong hybridization of
the probe. ATT, for example, would indicate that the sample is
likely to be TAACAT.
[0072] FIG. 3B illustrates probe sets that would be desirable for
determining mutations in samples expected to contain the sequence
TGACAT. Each column contains a set of four probes for determining a
nucleotide in the sample corresponding to the interrogation
position. As shown, each of the four probes in a column differ at
the interrogation position. In a preferred embodiment, probes with
the same nucleotide at their respective interrogation position are
placed in a row, thereby forming an A-lane, C-lane, T-lane, and
G-lane. The wild-type probes from FIG. 3A are designated with a "*"
within the probe region. Typically, there are many multiples (e.g.,
hundreds or thousands) of identical probes within a probe region or
cell.
[0073] FIG. 4 shows prior art reticles that would be utilized to
synthesize the ACT, CTG, TGT, and GTA probes of FIG. 3A. Reticles 1
and 6 are for adding the nucleotide. A onto the substrate. Reticle
2 is for adding nucleotide C onto the substrate. Reticle 4 is for
adding nucleotide G onto the substrate. Lastly, reticles 3 and 5
are for adding the nucleotide T onto the substrate. Utilizing these
reticles, the synthesis can be viewed as repetitive additions of A,
C, T, and. Then G, with unnecessary addition steps skipped.
[0074] In the figures depicting reticles, shaded portions represent
openings through the reticle through which light will deprotect
areas on the substrate. Monomers (e.g., nucleotides) will then be
washed over the substrate so that the monomers may bind in the
deprotected regions. Although in preferred embodiments, the monomer
addition regions of the reticles are openings, the monomer addition
regions may be closed on the reticles in a similar matter.
[0075] FIG. 5 shows the prior art addition of nucleotide monomers
to produce the probe set. Reticles 1-6 are utilized to sequentially
add monomers to a substrate 202. At the top of FIG. 5, reticle 1 is
utilized to add the nucleotide A to the substrate at a location
specified by the monomer addition region of the reticle. Then,
reticle 2 is utilized to add the nucleotide C to the substrate as
specified by the reticle. The process continues through reticle 6
which results in four probes that may be utilized to analyze the
target sequence.
[0076] Although the process in FIG. 5 can be viewed as repetitive
additions of A, C, T, and G, in some instances, a particular
monomer addition step may be "skipped". For example, in FIG. 5, the
additions of A and C after the G addition are skipped in the second
cycle of A, C, T, G additions. Thus, six reticles are needed for
the synthesis of these probes. In the worst case, an addition of A,
C, T, and G would be needed for each of the n monomers in the
probes. Accordingly, in the worst case, n*4 reticles would be
needed to synthesize a probe set. In many cases, this number is
reduced to some number in where the sequence allows, as in the
above example, where in would be 6 which is better than the worst
case of 12 (3*4) reticles. As the number of monomers in the probes
grows, however, the number of required reticles can become quite
large thereby increasing costs.
[0077] The present invention provides techniques for synthesizing
probe arrays using far fewer reticles, which greatly reduces costs.
With one embodiment of the invention, as few as one reticle may be
used to make, for example, the exact complement probe set. An
additional reticle may be utilized to make probes with nucleotide
variations at an interrogation position and other reticles may be
utilized to fabricate different probe sets on the substrate (e.g.,
probe sets with different probe lengths)
Set of Shift Reticles
[0078] In one embodiment, the present invention utilizes a set of
shift reticles to synthesize desired probes on a substrate. The set
of shift reticles includes a single reticle for each monomer that
is to be added to the substrate. Utilizing these reticles, the
length and interrogation position of the probes may be specified at
synthesis time, e.g., after the reticles have been generated.
[0079] FIG. 6 shows a high level flow of a process of generating a
shift reticle set. At step 252, the nucleotides in the perfect
complement of the target sequence are numbered. Thus, for the
target sequence shown in FIG. 3A, the nucleotides may be numbered
1-6. Four 1.times.6 reticles will subsequently be formed for
synthesizing probes to detect the target sequence. Each of the four
reticles will be utilized to add a different monomer (e.g., A, C,
G, T) onto the substrate.
[0080] At step 254, a reticle for adding the nucleotide A to the
substrate is created. The reticle is designed with openings
corresponding to each A in the perfect complement to the target
sequence. Thus, for the perfect complement. ACTGTA, the reticle
would have openings corresponding to nucleotides 1 and 6. Reticle 1
of FIG. 7 shows a reticle produced in this manner.
[0081] At step 256, a reticle for adding the nucleotide C is
created in a similar manner. The reticle is designed with openings
corresponding to each C in the perfect complement to the target
sequence. Therefore, the reticle is designed with an opening
corresponding to nucleotide 2 in the target. Reticle 2 of FIG. 7
shows a reticle for adding nucleotide C. Steps 258 and 260 create
reticles 3 and 4 shown in FIG. 7 in a similar manner for
nucleotides G and T, respectively.
[0082] At step 262, a computer file containing the design of the
masks is output. This file may be utilized by a mask generating
system to produce the masks used in synthesis. A system for
designing masks is described in U.S. Pat. No. 5,571,639, which is
hereby incorporated by reference for all purposes.
[0083] FIGS. 8A-8C show the addition of monomers using the shift
reticles to produce the probe set. In FIG. 8A, the shift reticles
are used to produce a single "layer" of monomers on a substrate
302. By a "layer" of monomers, it is meant that each synthesis
cycle of monomer addition steps uniformly adds monomers to
specified locations on the substrate. As shown in FIG. 8A, the
specified locations may include the entire active region of the
substrate. However, as will be shown in FIG. 18, the specified
locations may include only a subset of locations of the active
region.
[0084] Referring still to FIG. 8A, reticle 1 is initially used to
add the nucleotide. A onto the substrate. Reticle 2 is subsequently
utilized to add the nucleotide C to the substrate, which is
followed by the addition of nucleotides. G and T utilizing reticles
3 and 4, respectively. With one synthesis cycle of A, C, G, and
then T, a single layer of monomers has been added to the active
region of the substrate which is shown as the four centermost
positions on the substrate. The number in parenthesis indicates the
reticle being used. The arrow indicates where the reticles are
aligned with the substrate (i.e., the first possible opening in
each reticle). The reticles will all be shifted one position after
each synthesis cycle.
[0085] In FIG. 8B, the reticles are shifted one position to the
left relative to the substrate as indicated by the arrow in the
figure (compare to FIG. 8A). Each reticle is then cycled through to
add each of the different monomers onto the substrate at locations
specified by the reticles. Again, the reticles add a single layer
of monomers to the active region of the substrate. Although the
reticles are shown shifted or translated to the left, the reticles
may, of course, be shifted to the right or any other direction.
[0086] In FIG. 8C, the reticles are again left shifted by one
position as indicated by the arrow in the figure. The reticles are
cycled through adding nucleotides A, C, G, and T to the substrate
as specified by the openings in the reticles. After the last
reticle is utilized, four probes that are perfectly complementary
to the target sequence have been synthesized at the centermost
positions of the substrate as shown at the bottom of FIG. 8C. These
four probes represent the active region of the substrate. The
probes shown that are not in the active region of the substrate
will be called the "edge" of the substrate. These edge probes are
typically ignored during analysis or sequencing of a sample.
[0087] For simplicity, the monomer addition regions of a reticle
have been shown to add a single monomer onto the subject substrate.
In practice, each monomer addition region of a reticle adds
hundreds or thousands of monomers to the area specified by the
opening. Similarly, the reticles typically synthesize hundreds of
rows of probes on the substrate. In a preferred embodiment, the
probes are synthesized in multiples of four rows where the probes
in each row differ from the other by a single nucleotide at an
interrogation position.
[0088] FIGS. 9A-9B show reticles that may be used to produce
multiple probe sets for the target sequence shown in FIG. 3. FIG.
9A shows a reticle similar to reticle 1 in FIG. 7. However, this
reticle would be utilized to produce two sets of four rows of
probes. Likewise, FIG. 9B shows a reticle similar to reticle 2 in
FIG. 7 that would be utilized to produce two sets of four rows of
probes. The reticles for nucleotides G and T are not shown but
would be similarly produced.
[0089] Each of these four shift reticles would be utilized to
produce two sets of four rows of probes on the substrate that would
be complementary to the target sequence. Each synthesis cycle in
the synthesis produces a set of n-mer complementary probes to this
target. Thus, after three cycles through the shift reticles, the
substrate contains a set of 3-mer complementary probes. The length
of complementary probes may be selected at synthesis tune by the
number of synthesis cycles of monomer addition steps that are used,
where a "synthesis cycle" is defined as cycling through each of the
monomers to be added to the substrate. One synthesis cycle results
in adding a layer of monomers to specified locations on the
substrate, typically the active region of the substrate.
[0090] FIG. 10 shows the transformation of a linear reticle into a
rectangular reticle. Although the shift reticles have been shown as
long linear reticles, the linear reticles may be transformed into a
rectangular shift reticle. A linear reticle 302 includes sixteen
active cells and three extra cells. The extra cells are the cells
added to the reticle for shifting relative to the substrate.
Typically the number of cells in the linear shift reticle will be
substantially greater but the simple reticle is shown for
illustration purposes.
[0091] When transforming the linear reticle into a rectangular
reticle, the sixteen active cells are placed in a rectangular
region with the appropriate extra cells at the edge. Thus, a
rectangular reticle 304 was formed by placing cells 1-16 into a
square region of the reticle. Each row of the rectangular reticle
ended with the same number of extra cells as was in the linear
reticle, these extra cells continuing sequentially after the active
cells. The resulting rectangular reticle is a shift reticle that
forms rows of probes for a target sequence.
[0092] In order to add differing monomers at an interrogation
position, reticles such as those shown in FIGS. 11A and 12A are
utilized. FIG. 11A shows a reticle that is used to couple
interrogation position monomers on the substrate. The reticle
includes multiple rows of openings that are utilized to add a
monomer to the probes. These rows are perpendicular to the reticles
shown in FIGS. 9A and 9B.
[0093] The reticle in FIG. 11A is first utilized to add a monomer
like nucleotide. A to a row of probes (i.e., the A-lane). The
reticle is then shifted downwards to the next row of probes and a
different monomer like nucleotide. C is then added to this row of
probes (i.e., the C-lane). This process is continued until a
different monomer is added to the interrogation position for each
row in a set of probes. For a deletion, a monomer is not added to a
row of probes.
[0094] Thus, in order to produce 5-mer probes with an interrogation
position at the third position in the probes, one performs two
synthesis cycles of monomer addition steps with the shift reticles
to produce 2-mer probes in the active region of the substrate. Then
the interrogation position reticle is utilized to add a different
monomer to each row of probes by shifting the mask in a direction
perpendicular to the direction that the shift reticles are shifted.
Except in the case of deletions, the interrogation position reticle
also adds a layer of monomers in the active region of the substrate
with one synthesis cycle. Then, after shifting the shift reticles
two positions (the extra position accounts for the synthesis cycle
utilized by the interrogation position reticle), the shift reticles
are utilized to add the last two monomers to the probes by
performing two synthesis cycles of monomer addition steps.
[0095] FIG. 11B shows probes on a chip that vary at an
interrogation position. The top half of the chip includes 4-mer
probes with the interrogation position at the third nucleotide in
the probe (the interrogation nucleotide is circled for easy
identification). As shown, there are 4-mer probes in the active
region of this half of the chip. The bottom half of the chip
includes 3-mer probes in the active region that were synthesized
using the same shift reticles and the interrogation position
reticle at the same time with the use of one additional reticle
which allows different length probes to be synthesized on the chip
at the same time. This reticle will be described in more detail in
reference to FIG. 13A
[0096] The shift reticles of the present invention are target
structure specific but not sequence specific. For example, the
shift reticles may be utilized to synthesize probes complementary
to the sense or anti-sense strands of DNA. Additionally, shift
reticles that produce probes complementary to TGACAT may also be
used to produce probes complementary to AGTCTA by switching the A
and T nucleotides utilized with the shift reticles. Accordingly, at
synthesis time, one may specify characteristics of the probes by
selecting the order of shift reticles in a synthesis cycle, the
monomers in a synthesis cycle, the monomers associated with each of
the shift reticles, and the interrogation position.
[0097] FIG. 11C is an image of a chip that was produced in the
manner described above. The chip has 20-mer probes with an
interrogation position at a central position. The chip is for
sequencing a 1000 base-pair sequence of HIV. It should be
understood that the examples described above are simplified to aid
the readers understanding of the invention. The chip images show
actual chips and are therefore more complex, but they nevertheless
utilize the principles of the present invention extended to a
larger scale.
[0098] The probes in the edge regions of the substrate will still
bind to the labeled target with varying hybridization intensities
as shown on the right side of the chip in FIG. 11C. However, it is
difficult for one to visually identify where the active region of
the chip begins or ends. In a preferred embodiment, an
interrogation position reticle as shown in FIG. 12A is utilized.
This reticle has openings that only correspond to or overly the
active region of the substrate. Because the openings will not be
above the edge regions of the substrate, the probes at the edge
regions of the substrate will not receive the interrogation
position nucleotides. In this manner, the probes in each column in
the edge regions will be identical and therefore, the edge region
will appear as stripes in the image of the chip so the start of the
active region may be more easily identified. This is shown on the
left side of the chip in FIG. 11C
[0099] FIG. 12B shows probes on a chip that vary at an
interrogation position but were synthesized utilizing the reticle
shown in FIG. 12A. The top half of the chip includes 4-mer probes
with the interrogation position at the third nucleotide in the
probe. As shown, there are 4-mer probes in the active region lane
of this half of the chip. The circled interrogation nucleotides
were only added in the active region. Thus, the probes in each
column outside the active region (edges) are identical. As they are
identical, the resulting stripes may be utilized to identify the
edges of the chip after hybridization and scanning. The bottom half
of the chip includes 3-mer probes that were synthesized using the
reticle shown in FIG. 13A.
[0100] A chip synthesized as described above is shown in FIG. 12C
where the edge regions may be easily identified by the stripes. The
chip has 20-mer probes with an interrogation position at a central
position. The chip is for sequencing a 2,500 base-pair sequence of
HIV.
[0101] Utilizing these shift reticles and the interrogation
position reticle, any length probe with any substitution position
may be synthesized for a target sequence limited only by the size
of the reticles. Typically, the size of the reticles is equal to
the size of the target along a row of the substrate plus the
desired length of the synthesized probes minus one. For example, if
there are 100 columns of cells on the chip and the target sequence
is equal to or longer than 100 monomers, the reticles may be 111
cells (or possible monomer addition regions) wide for 12-mer probes
(i.e., 100+12-1).
[0102] With the present invention, five reticles may be utilized to
sequence any length probe with any interrogation position for the
target sequence. Furthermore, the length of the probes and the
interrogation position need not be determined before synthesis.
After the reticles are produced, the specific probes that are
produced on the substrate may be determined at synthesis time by
indicating the number of cycles of monomer addition steps and the
cycle where the interrogation position reticle will be
utilized.
[0103] A reticle as shown in FIG. 13A may be utilized to generate
probes of varying length with the lengths being determined at
synthesis time if desired. The reticle includes an opening which
will allow light to strike a set of probes. The reticle may be
utilized in conjunction with the shift reticles so that only the
top half of the chip is deprotected. For example, the reticle may
be utilized to add a first layer of nucleotides only to the top
half of the chip. After the first layer of nucleotides has been
added, synthesis may continue but now by adding nucleotides to the
whole chip. It is in this manner that the 4-mer and 3-mer probes of
FIGS. 11B and 12B were produced.
[0104] Alternatively, the reticle of FIG. 13A may be utilized to
stop synthesis. After probes have been synthesized in a region, the
region specified by the reticle is deprotected and a capping
reagent may be added to the substrate so that subsequent exposure
to light will not deprotect the probes in this region. The capping
region may be DMT or any other known capping reagent. Utilizing
this reticle, one region of the substrate may contain probes that
are of a different length than another area of the substrate. For
example, the substrate shown in FIG. 13B includes 8-mer probes and
12-mer probes. The 8-mer probes were first formed on the whole chip
and then the reticle of FIG. 13A was utilized to cap the probes on
the top half so that subsequent exposure to light would not result
in the addition of subsequent monomers. The region for the 12-mer
probes was not capped so the subsequent addition of monomers
resulted in 12-mer probes.
[0105] Additionally, the reticle shown in FIG. 13A may be utilized
to synthesize probes with nucleotide deletions. For example, by
utilizing the reticle to skip a cycle of nucleotide additions,
probes with deletions may be synthesized.
[0106] FIG. 14A is a mask including multiple reticles. A mask 500
includes shift reticles 502, 504, 506, and 508, one for each
nucleotide monomer. The mask also includes an interrogation
position reticle 510 which adds monomers at an interrogation
position over the active region of the substrate. Additionally, the
mask includes a reticle 508 which is utilized to deprotect the
entire surface of the substrate in order to cap the probes after
synthesis. Although FIG. 14A shows a mask that includes multiple
reticles, the present invention may be advantageously utilized in
those systems where each mask includes a single reticle.
[0107] FIG. 14B shows the layout of a reticle in one embodiment.
The majority of the reticle includes rows with monomer addition
regions for repeating groups of A, C, G, and T lanes of the
substrate. The monomer addition regions for each group of lanes are
typically the same as shown in FIGS. 9A and 9B. Each group of rows
corresponding to the A, C, G, and T lanes may differ in order to
synthesize probes complementary to different sections of the
target. For example, one group may be for synthesizing probes for
identifying nucleotides at positions 500-599 while the next group
is for synthesizing probes for identifying nucleotides at positions
600-699.
[0108] The top and bottom rows of the reticle in FIG. 14B are for
producing probes complementary to a control oligonucleotide
sequence (i.e., control probe lanes). The control sequence is a
known sequence that is added to the target to allow easier
identification and/or alignment of the active region of the chip
after scanning.
[0109] FIG. 14C shows reticles for synthesizing probes on two chips
simultaneously. As shown, there are identical A reticles, C
reticles, G reticles, T reticles, and interrogation position
reticles for each chip (denoted chip 1 and chip 2). These reticles
reside on the same piece of glass so that two identical chips may
be produced simultaneously. Thus, if the synthesis cycle begins
with A, the two A reticles would be utilized. The glass would then
be shifted so that the next nucleotide reticle is over the chips to
add the next nucleotide in the synthesis cycle, and so forth. At
the next synthesis cycle, the reticles would be positioned over the
chip at a position shifted horizontally relative to the chip.
Accordingly, the nucleotide reticles are wider than the chips.
[0110] The interrogation position reticles may be utilized to
synthesize nucleotides at an interrogation position in the probes
on the chips. During the synthesis cycle which is designated to add
the interrogation position nucleotides, the glass is shifted
vertically relative to the chip. One should understand that
although the nucleotide reticles are described as being shifted
horizontally and the interrogation position reticles as being
shifted vertically, the reticles may be shifted any direction.
Also, the reticles for chip 1 and chip 2 need not be identical, nor
limited to two chips. Accordingly, multiple different chips may be
synthesized with the present invention simultaneously.
[0111] FIG. 14D shows a mask for synthesizing varying length probes
on two chips simultaneously. Ways have been described for utilizing
reticles for selecting regions of the chip in order to synthesize
probes of varying length (see, e.g., FIGS. 13A and 13B). Another
way of achieving this objective is illustrated with the mask in
FIG. 14D.
[0112] The mask includes reticles similar to the reticles described
in FIG. 14C, and in fact, the reticles in the left bottom of the
mask are identical to these reticles. The underlying chip has five
groups of A, C, G, and T lanes of the chip. As shown, the reticles
in the left bottom of the mask have rows of monomer addition
regions that correspond to each of the five groups of A, C, G, and
T lanes. Each group of A, C, G, and T lanes are identical, however,
not all of the reticles have the same number of groups. There are
other reticles with one, two, three, and four groups of A, C, G,
and T lanes.
[0113] In order to synthesize chips with varying length probes, one
selects the reticles that will add monomers at desired regions on
the chip. For example, if one desires to synthesize 3, 5, 7, 9, and
11-mer probes on two chips simultaneously with interrogation
positions at the center of the probes, one could first use the
reticles with the single group of A, C, G, and T lanes for one
synthesis cycle. This would couple monomers on the top portion of
the chip.
[0114] Next, one could use the reticles with the two groups of A,
C, G, and T lanes for one synthesis cycle. This would synthesize a
top region with two layers of monomers (i.e., 2-mer probes) and an
adjacent region with one layer of monomers. This process may be
repeated utilizing the reticles with three, four and five groups of
A, C, G, and T lanes until there are regions on the chip with five,
four, three, two and one layer monomers (from top to bottom of the
chip).
[0115] The interrogation position reticle at the lower middle of
the mask may then be utilized to add interrogation position
nucleotides to all of the probes on the chip. After the
interrogation position reticle has been utilized, the previous
process of adding nucleotides may be reversed. After synthesis,
open chip reticles may be utilized to cap the probes thereby
generating two chips with 3, 5, 7, 9, and 11-mer probes with
interrogation positions at the center of the probes. The layout of
one of these chips is shown in FIG. 14E.
[0116] FIG. 15A shows a layout of a chip in another embodiment
which is typically utilized for genotyping or gene expression
applications. As shown in FIG. 15A, a chip 550 has perfect
complement lanes 552, mutation lanes 554 and control lanes 556. The
perfect complement lane has probes that are perfectly complementary
to the target sequence. The mutation lane has probes that are
complementary to the target sequence except for a mutation
position. The mutation lanes are utilized to check the validity of
the data. Thus, hybridization intensities in the perfect complement
lane are compared to the hybridization intensities in the mutation
lane.
[0117] FIG. 15B shows a shift reticle for coupling a particular
monomer on a chip in pairs of rows. The reticle may be utilized to
add nucleotides to both the perfect complement and mutation rows.
Typically, there will be four reticles, one for each nucleotide
(see, e.g., FIG. 7), that are used in each synthesis cycle.
However, only one reticle is shown.
[0118] FIG. 15C shows a shift reticle for coupling a particular
monomer on a chip in a single lane. In order to produce probes in
the mutation lane that differ from the probes in the perfect
complement, a shift reticle as shown in FIG. 15C may be utilized.
As before, there will typically be four reticles, one for adding
each of the nucleotides, but one is shown for simplicity. In one
synthesis cycle, these shift reticles may be utilized to add
nucleotides that are perfectly complementary to the target sequence
in the perfect complement lane as was done with the reticle shown
in FIG. 15B.
[0119] With the invention, the same shift reticle shown in FIG. 15C
may be utilized to add mutation nucleotides to the probes in the
mutation lanes. The shift reticles are shifted vertically so that
the monomer addition regions overly the mutation lanes. In order to
add mutation nucleotides to the probes in the mutation lanes, one
may change the order of the nucleotide addition steps in the
synthesis cycle. For example, if the nucleotides A, C, G, and then
T are added in a synthesis cycle, one can instead add T, G, C, and
then A, which is reverse order. Thus, each probe in the mutation
lanes will have a mutation nucleotide added.
[0120] Alternatively, one may keep the order of the nucleotide
addition steps but switch the order of the shift reticles that are
utilized. As should be apparent, this has the same effect of adding
a mutation nucleotide to the probes in the mutation lanes.
[0121] FIG. 15D shows a shift reticle for forming control lanes
that include control probes. The control probes may be perfectly
complementary to a known oligonucleotide that is hybridized with
the chip in order to aid in analyzing the scanning results. Again,
one shift reticle is shown but there will typically be one for each
monomer.
[0122] The above embodiment provides shift reticles which may be
utilized to form probes of varying lengths which are complementary
to the target sequence. These shift reticles may be utilized with
one or more masks in order to produce probes with interrogation
position nucleotides or probes of varying length on the same
substrate as described. The cost for producing probes on a
substrate are reduced because the number of reticles may be greatly
reduced (e.g., down to five reticles or less). Flexibility is
increased as one may specify characteristics of the probes at
synthesis time.
Single Shift Reticle
[0123] In another embodiment, the present invention provides a
single shift reticle that may be utilized to synthesize probes
complementary to the target sequence. FIG. 16 shows a high level
flow of a process of generating reticles according to thus
embodiment of the invention. At step 602, the nucleotides in the
perfect complement of the target sequence are numbered. If the
target sequence is TGACAT as shown in FIG. 3A, the perfect
complement will be ACTGTA. Thus, the nucleotides in the perfect
complement are numbered 1-6 with the first A being 1, C being 2,
the first T being 3, and so forth.
[0124] A single shift reticle is then produced according to steps
604-610. It should be noted that these steps do not need to be
performed in any specific order and in fact, they may be performed
in parallel. Furthermore, each equation is not specific to the
nucleotide shown. However, the steps will be described as being
performed sequentially for each nucleotide A, C, G, and T for ease
of illustration.
[0125] At step 604, openings are created in the single reticle for
each A in the perfect complement by the equation n*(i-1)+1, where n
is equal to the number of different types of monomers (e.g.,
nucleotides) and i is equal to a position of the monomer in the
perfect complement (or desired probe). As the nucleotide. A is at
base positions 1 and 6 in the perfect complement, openings will be
created in the single reticle at position 1 and 21 because n is
equal to 4 for the four nucleotides A, C, G, and T, and i is equal
to 1 for the first A and 6 for the second. A FIG. 17A shows the
resulting single reticle.
[0126] At step 606, openings are created in the single reticle for
each C in the perfect complement by the equation n*(i-1)+2, where n
is equal to the number of different types of monomers and i is
equal to a position of the monomer in the perfect complement. As
the nucleotide C is at base position 2 in the perfect complement,
an opening will be created in the single reticle at position 6
because n is equal to 4 and i is equal to 2. FIG. 17B shows the
single reticle with openings for both A and C.
[0127] Openings are created in the single reticle for each G in the
perfect complement by the equation n*(i-1)+3 at step 608. As the
nucleotide G is at base position 4 in the perfect complement, an
opening will be created in the single reticle at position 15
because n is equal to 4 and i is equal to 2. FIG. 17C shows the
single reticle with openings for A, C and G.
[0128] At step 610, openings are created in the single reticle for
each T in the perfect complement by the equation n*(i-1)+4. As the
nucleotide T is at base positions 3 and 5 in the perfect
complement, openings will be created in the single reticle at
positions 12 and 20 because n is equal to 4 and i is equal to 2.
FIG. 17D shows the single reticle with openings for A, C, G, and
T
[0129] At step 612, a mask file for generating a mask including the
single reticle is output. This mask file is typically utilized by a
computer operated system to generate the mask.
[0130] FIG. 18 shows a single shift reticle that produces the probe
set of FIG. 3A and the addition of monomers using this reticle.
Reticle 652 is produced according to the process described in
reference to FIG. 16. As shown, the reticle includes six cycles of
A, C, G, and T (denoted 1-6 above the reticle). Each cycle includes
a single opening at various positions as shown.
[0131] Initially, the mask is utilized to add the nucleotide A to
the substrate at the regions specified by the mask. With each
subsequent synthesis step, the reticle is shifted by one position
or cell with each step, resulting in four shifts for each synthesis
cycle of nucleotides. This process is shown in a table 654
underneath the reticle with the nucleotide addition steps
sequentially listed on the left side of the table. The dashed line
in the table represents the rightmost border of the active region
of the substrate. In other words, nucleotides to the right of the
dashed line would not be coupled to the substrate.
[0132] The table is typically not utilized during synthesis but is
shown to aid in understanding how the probes on the substrate in
this embodiment are formed. Each column in the table represents a
probe on the substrate. However, as the table grows downward as
monomers are added, the first nucleotide from the top in each
column is nearer the substrate.
[0133] A substrate 656 results with the desired 3-mer probes
indicated by the four arrows underneath the substrate. The desired
probes are formed by a uniform addition of nucleotides at these
specified regions because each cycle adds one nucleotide to each
desired probe. Accordingly, an interrogation position reticle may
be utilized that is similar to the ones shown in FIGS. 11A and 12A
in order to add interrogation position nucleotides. After the
interrogation position nucleotides are added, a synthesis cycle of
the single shift reticle is then skipped so the reticle is shifted
four positions or cells (e.g., to the left in FIG. 18).
[0134] As shown, there are a number of "junk" probes surrounding
the desired probes. Typically these probes will be ignored during
sequencing of the target. For simplicity, the single reticle has
been shown as a linear reticle. However, a reticle may be utilized
for producing two sets of four rows of probes as shown in FIG. 19.
More rows of probes may be generated by an extension of the
principles of the invention.
[0135] Although the single shift reticle has been shown as a long
linear reticle, the linear reticle may be transformed into a
rectangular shift reticle as shown in FIG. 10. As the single shift
reticle is shifted with each monomer addition step, the number of
extra cells at the end of each row in the resulting rectangular
reticle may be substantially higher.
[0136] This embodiment of the present invention allows probes
perfectly complementary to the target sequence to be synthesized on
the substrate with a single shift reticle. Additional reticles may
be utilized to synthesize probes with interrogation position
nucleotides or probes of varying lengths as described above. By
reducing the number of reticles needed down to possibly one, this
embodiment greatly reduces the cost of generating masks for probe
array synthesis. Additionally, flexibility is increased because
characteristics of the desired probes may be specified at synthesis
time.
Other Shift Reticle Embodiments
[0137] In another embodiment, the present invention provides shift
reticles that may be utilized to synthesize probes for detecting
mutations, deletions, and the like. These shift reticles are not
target sequence structure specific so the target sequence may be
specified at synthesis time. In other words, a set of "generic"
shift reticles may be utilized to synthesize probes for analyzing
any target sequence. Additionally, these probes may be generated
with very few reticles.
[0138] FIGS. 20A-20D show shift reticles for synthesizing probes of
including multiple monomers for detecting mutations and a deletion.
In order to illustrate how the shift reticles work it may be
beneficial to discuss an example. Suppose it is desired to
synthesize probes that would detect a mutation or deletion at the
middle (or 8th) position in a 15-mer target. It should be
understood that typically target sequences are much longer but this
example will be utilized to illustrate the invention.
[0139] If the target sequence is. TACCGTGAAGCTACG (SEQ ID NO 1)
then it would be desirable to synthesize the following probes.
ATGGCACTTCGATGC (SEQ ID NO 2), ATGGCACGTCGATGC (SEQ ID NO 3),
ATGGCAC CTCGATGC (SEQ ID NO 4), ATGGCACATCGATGC (SEQ ID NO 5), and
ATGGCACTCGATGC (SEQ ID NO 6). The interrogation position
nucleotides are underlined which illustrates that the first probe
is the perfect complement to the target sequence. The next three
probes have a mutation at the interrogation position and the last
probe has a deletion at the interrogation position.
[0140] Four shift reticles (or less) may be utilized to synthesize
these probes. The shift reticle in FIG. 20A is utilized for
coupling non-interrogation position monomers to the substrate.
Nucleotide addition steps are cycled through that correspond to the
complement of the target sequence. As indicated by the nucleotides
above the shift reticle, first A is added, then T, then G, and so
forth. After each monomer addition step, the shift reticle is
shifted one position to the left. As the shift reticle is shown as
being five rows high, five identical probes will be generated up to
the 8th monomer addition step.
[0141] At the 8th monomer addition step, which corresponds to T in
the perfect complement, only one monomer addition region overlies
the probes. Accordingly, T will be only added to one of the probes,
which is the top probe in the FIG. 20A.
[0142] FIG. 20B shows a reticle that has a single monomer addition
region. The reticle may be utilized to add a G at the 8th position
of the second probe from the top. Similarly, the reticles in FIGS.
20C and 20D may be utilized to add a C and A at the 8th position of
the probes corresponding to the one monomer addition region of the
reticles. The probe at the bottom does not have a monomer added at
the 8th position so that a deletion at this position may be
detected. Although FIGS. 20B-20D show three shift reticles, a
single shift reticle may be utilized that is shifted
vertically.
[0143] After the interrogation position nucleotides are added, the
shift reticle of FIG. 20A is utilized to add the rest of the
nucleotides to the probes. FIG. 20E shows a chip including these
probes. A chip 800 includes a perfectly complementary probe,
interrogation position probes, and a probe for detecting a deletion
(SEQ ID NO 2, SEQ ID NO 3, SEQ ID NO 4, SEQ ID NO 5, and SEQ ID NO
6).
[0144] The shift reticle described above may be modified to produce
probes of varying lengths. FIG. 21A shows a shift reticle for
synthesizing related probes of varying lengths on a substrate. The
shift reticle has monomer addition regions that vary in width to
produce 9, 11, 13 15, 17, 19, and 21-mer probes. As the shift
reticle is shifted along the direction indicated by the arrow in
FIG. 21A, monomers are added to the substrate.
[0145] The varying widths of the monomer addition regions may be
designed to result in varying length probes that are centered
around the same position of a target sequence. For example, as
shown in FIG. 21A, each monomer addition step is labeled from 1-21.
At step 1, only one region will have a monomer added. Each
subsequent step adds a monomer to this region and the region above
it. In this way, the stair-step design of the shift reticle allows
varying length probes to be synthesized on the substrate. By
utilizing a stair-step design at each end of the shift reticle, the
probes vary by two monomers instead of one which would be achieved
with a single stair-step.
[0146] FIG. 21B shows an example of the layout of the probes that
may be synthesized on the substrate. A chip 850 includes regions
that include 9, 11, 13, 15, 17, 19, and 21-mer probes. Although it
has only been described that there is one row for each length
probe, there may be multiple rows for each length probe. For
example, there may be four rows for each length probe, one for
nucleotide at an interrogation position. At the interrogation
position, an interrogation position reticle as was described in
reference to FIGS. 11A and 12A may be utilized to add the
interrogation position nucleotides instead of utilizing the shift
reticle. An advantage of the invention is that the sequence of the
probes, the length of the probes, and interrogation position may be
selected at synthesis time.
[0147] This embodiment of the present invention allows probes of
varying lengths and that are centered around a position in the
target sequence to be synthesized. The shift reticle may also be
utilized with an interrogation position reticle to produce varying
length probes that detect mutations.
[0148] These embodiments of the invention have the significant
advantage that the shift reticles are not target sequence structure
specific. Accordingly, the sequence of the target may be specified
at synthesis time and a "generic" set of shift reticles utilized to
synthesize probes for analyzing the target sequence. As with the
other embodiments of the invention, the number of reticles needed
is significantly reduced which lowers the cost of producing the
chips. Also, flexibility is increased because characteristics
(e.g., interrogation position) of the desired probes may be
specified at synthesis time.
Speckle Masks
[0149] Some embodiments of the present invention utilize speckle
masks. A "speckle mask" is a set of reticles that when taken
together have an opening at each location, thus they, in effect,
can be said to form a full open mask. FIG. 22 is a simple example
of speckle masks. As shown, the three reticles have a single
monomer addition opening at a different location. When the openings
are added together, a full open mask is generated. This is a
property of speckle masks.
[0150] Another example of a speckle mask is the set of reticles (or
masks) shown in FIG. 7. These masks, together include one and only
one opening for each monomer addition region in the reticles. FIG.
23 shows how the openings of the shift reticles add up to form a
full open mask.
[0151] A fundamental property of speckle masks is that if all the
reticles are used in a synthesis cycle, exactly one monomer is
added to each of the probes in the active region of the substrate.
This property is used to great effect in allowing construction of
probes of any length and interrogation position at synthesis
time.
[0152] Another application of speckle masks is to generate a number
of distinct chips from a single speckle set. Take a grid and
construct a speckle set by assigning random numbers from 1-4 (or
whatever the number of monomers happens to be) in each cell. The
number indicates which reticle will have an opening at that
location. If all four reticles are cycled through with some
permutation of A, C, G, and T in a synthesis cycle, a set of
"random" nucleotides are added to each probe on the substrate. If
some arbitrary (x and y) offset is utilized in each step, very
little correlation between the nucleotides added to each probe is
expected. For each district set of offsets, radically different
sets of probes may be generated. Thus, "random" chips with probes
of uniform length (neglecting probes on the edges of the chip) may
be generated.
[0153] A further application of a speckle set is to generate a
chosen set of uniform length probes. A shift mask may be generated
that produces a specific set of probes by picking a sequence
containing that set of probes, and generating a shift mask to that
sequence. However, the sequence containing some set of probes will
in general be very much longer than the total number of probes.
Since a shift mask contains a number of cells approximating the
total length of the sequence, this may be an inefficient way of
generating some sets of probes.
[0154] A shift mask uses one-dimensional offsets to generate the
probes. A way of looking at this is that each probe must be encoded
on the mask in a strip 1.times.n, where n is the length of the
probe. The strips are packed onto the mask set to produce the set
of probes. Any pair of strips may only interact in O(n) ways,
corresponding to the number of ways the rectangles may overlap.
[0155] A better method of packing probes onto a speckle set is to
use two-dimensional offsets. With 2-dimensional offsets, probes are
encoded on the mask in "speckles"--some arrangement of n cells
(where n is the length of the probe). In general, there are
O(n.sup.2) ways for two speckles to interact. This suggests that
two-dimensional offsets may be used to pack probes efficiently in a
speckle set. However, this problem appears computationally very
difficult, given the degrees of freedom to choose offsets, base
permutation used at each synthesis cycle, and probe location. Some
form of simulated annealing could be used to choose locations,
given the chosen set of offsets and base permutation.
[0156] FIG. 24 shows the packing of speckle masks. Two sequence
ACTGT and ATCTG may be packed by taking advantage of the common
subsequence CTG. The packing of the speckle masks involves both an
x and y offset as shown.
[0157] Several possible generalizations of speckle sets exist. One
may use a number of masks greater than the set of bases used to
increase the number of degrees of freedom. One may also generate
sets of masks that add up to several open masks (each cell is open
exactly k times, when the full set of masks is taken together).
Additionally, one may generate sets of masks that have many
different subsets that add up to an open mask.
Post Chip Synthesis
[0158] In the embodiments described above, the reticles were
designed as rectangular grids. The rectangular grids are utilized
as it lends itself well to switch matrix representation. Switch
matrices provide an excellent generalization of combinational
masks, but they generally require that the chips include an array
of rectangular cells, where all of the cells are the same size.
These chips may include wasted space as the blank lanes (lanes
including no probes) are the same size as lanes which include
probes.
[0159] With post chip synthesis, each set of related probes (e.g.,
probes varying by a single base at an interrogation position) are
treated as a character in a text document. A set of related probes
will be referred to as an "analysis region" Just as characters are
not restricted to rectangular grids in modern printers, analysis
regions are also not limited but instead may be scaled, rotated,
stretched or manipulated. Accordingly, the analysis regions may be
input as a sequential list.
[0160] FIG. 25 shows the layout of a chip in one post chip
synthesis embodiment. An analysis region 900 includes four cells
denoted G, T, A, and C to indicate the nucleotide at the
interrogation position of the probes in each cell. As shown, the
analysis regions are placed in a circular pattern. Although only
one ring of analysis regions is shown, more rings may be generated
around a center. This pattern of analysis regions may be extended
to resemble data that is stored on hard drives, including sectors
and tracks, for reading by a computer controlled device.
[0161] FIG. 26 shows the layout of another chip utilizing post chip
synthesis. In this embodiment, the analysis regions are synthesized
in a spiral pattern, similar to a phonographic record.
Additionally, the masks that synthesized the probes on the chips
did not add the nucleotides in a rectangular cell. Instead, the
probes were synthesized on the chip in an outline of the
interrogation nucleotide letter A, C, G, or T. As expected, the
probes that best hybridize with the sample sequence shown generate
the highest intensity, which will form the brightest outline of one
of these characters. In other words, a person may be able to just
read the bases right off the scan image.
[0162] Alternatively, a computer system may utilize optical
character recognition techniques to read the characters indicative
of the interrogation base from the scan image. This process may be
further added by the spiral placement of the analysis regions.
[0163] With post chip synthesis, analysis regions may be placed in
differing orientations, spirals, or with variable spacing between
the analysis regions. Flexibility in laying out the chip is
provided which may prove to be very beneficial in many
applications.
Edge Minimization
[0164] In order to maximize the utilization of the active region of
the substrate, it may be beneficial to pack groups of A, C, G, and
T-lanes together with no blank lanes in between FIG. 27A shows an
active region 1000 of a substrate that includes multiple groups of
A, C, G, and T-lanes 1002. As shown, there are no blank lanes
separating each group of lanes 1002. Although this may appear to be
the best utilization of the real estate of the active region, the
synthesis of the probes in the T-lane of one group may adversely
affect the synthesis of probes in an adjacent A-lane of another
group.
[0165] In order to show how the synthesis of one group may affect
another, FIG. 27B shows a subregion of eight cells 1004 from FIG.
27A. As shown, four of the cells 1050, 1052, 1054, and 1056 are
from a first group of A, C, G, and T-lanes and four cells 1058,
1060, 1062, and 1064 are from an adjacent group. Within each cell
are 5-mer probes with an interrogation position at the third
position (underlined). In practice, the probes are typically longer
than 5-mers but shorter probes are shown to benefit the reader.
[0166] When the cells on the substrate are tightly packed, data
from cells (e.g., cells 1056 and 1058) that are adjacent to another
group of cells is not as accurate. The reason for thus is that the
probe AGTAT from cell 1056 and the probe GCAAA from cell 1058 only
have one base in common, the fourth base in both probes is an A.
Therefore, during synthesis, many of the masks will have an opening
for only one of these cells, which creates an "edge" on the mask
between the two cells. Accordingly, it can be said that there are
four edges on the reticles utilized to generate the probes in cells
1058 and 1060.
[0167] In stark contrast, the probe AGGAT from cell 1054 and the
probe. AGTAT from cell 1056 have four bases in common. As these two
probes are from the same group of probes, only the interrogation
position bases differ. Thus, it can be said that there is only one
edge on the reticles utilized to generate the probes in cells 1056
and 1058. The significance of the number of edges is described
below.
[0168] Light tends to diffuse somewhat around an edge of a reticle
so the more edges that are present between two cells, the more it
is that the cells will have incorrect probes near the edge. As
described above, there were four edges between cells 1056 and 1058,
whereas there was only one edge between cells 1054 and 1056.
Accordingly, the data from probes near the border between cells
1056 and 1058 will likely be less accurate. Although synthesizing a
blank lane between the groups of A, C, G, and T-lanes reduces this
"edge effect," the reduction is only approximately one half since
there will still be edges for the generation of the blank lane.
[0169] The present invention reduces the number of edges by
utilizing shift reticles that synthesize non-interrogation position
bases in an area that is wider that the area in which interrogation
position bases are synthesized. For example, the shift reticles
shown in FIGS. 9A and 9B have monomer addition regions that are
four cells wide. The monomer addition regions may be widened to
five cells wide so that non-interrogation position bases are
synthesized in an area on the chip that is five cells wide. An
interrogation shift reticle, such as shown in FIGS. 11A and 12A,
may then be utilized to synthesize interrogation position reticles
in an area that is narrower (e.g., four cells wide) than the area
occupied by the non-interrogation position bases.
[0170] In order to more clearly see how the invention provides a
reduction in edges, FIG. 27C shows a subregion of FIG. 27B that may
be synthesized with reduced edges. A subregion 1004' includes eight
cells 1050, 1052, 1054, 1056, 1058, 1060, 1062, and 1064 that are
the same as those in FIG. 27B that have the same reference
numerals. However, in subregion 1004, the non-interrogation
position bases are synthesized five cells wide. Accordingly, there
are half cells 1070 and 1072 surrounding each of the multiple
groups of A, C, G, and T-lanes 1002.
[0171] Half cells 1070 include the same bases as the probes in
cells 1050 and 1056 except for a single additional base, the
interrogation base. Therefore, there is only one edge difference
between half cells 1070 and the full cells they border. As
described above, there is only a one edge difference between, e.g.,
cells 1054 and 1056. Therefore, each of cells 1050, 1052, 1054, and
1056 have the same number of edges at their borders so they should
provide more accurate data.
[0172] Although in preferred embodiments, the non-interrogation
position bases are synthesized in an area five cells wide, this
exact size is not required. Edges may be reduced when the
non-interrogation position bases are synthesized in an area that is
wider than the area in which the interrogation position bases are
synthesized. It may seem that having unused space between groups of
lanes would waste real estate in the active area on the chip.
However, it has been found that because the data is more accurate,
the feature sizes may be reduced more so that the density of cells
may actually be increased.
Probe Optimization
[0173] In some instances, it may be beneficial to synthesize
various probes that interrogate a specific base position in a
target. For example, one may only be interested in specific point
mutations in a gene. In order to fully interrogate the specific
base, it would be beneficial to have many different probes (e.g.,
length and/or interrogation position in the probe) that interrogate
the position.
[0174] An embodiment of the invention allows one to synthesize
different probes for interrogating a specific base position.
Conceptually, the invention combines the non-interrogation base
reticles with the interrogation position reticle. FIG. 28 shows
shift reticles that produce equal length probes with different
interrogation positions. It should be understood that in this
instance, "interrogation position" means the position in the probe
that interrogates a position in the target. The "interrogation
position" may also refer to the position in the target that is
being interrogated.
[0175] Assume a target was AGCGATANCTGCGTA (SEQ ID NO 7), where the
underlined N designates an unknown base at an interrogation
position. The shift reticles of FIG. 28 may be created as described
in reference to FIG. 7. The bases shown on top of the shift
reticles are merely a reference to the corresponding base in the
target and an asterisk 1102 indicates the interrogation position.
The cells at this location in the reticles will be formed similar
to the interrogation position reticles of FIG. 11A or 12A. As
shown, at the interrogation position, a different monomer addition
region is generated for each reticle. For example, Reticle 1 (for
A) has a monomer addition region in the A-lane, Reticle 2 (for C)
has a monomer addition region in the C-lane, and so forth.
[0176] When the shift reticles of FIG. 28 are utilized, there is no
need for an interrogation position reticle. After eight cycles
through the addition of A, C, G, and T with the shift reticles,
8-mer probes would be synthesized that have all the possible
interrogation positions in the probes.
[0177] FIG. 29 illustrates an example of the position of the 8-mer
probes. A chip 1150 has eight different sets of four probes in its
active region. The number below (1-8) indicates the position of the
interrogation position in the probes if the shift reticles of FIG.
28 are shifted to the left after each monomer addition step. A "1"
indicates that interrogation position is nearer the chip in the
probes, whereas an "8" indicates that the interrogation position is
farther from the chip in the probes.
[0178] By utilizing the shift masks in FIG. 28, one may synthesize
probes of a specific length with every possible interrogation
position. Although 8-mers have been described as an example, the
invention is not limited to any specific probe length.
Additionally, probes may be further optimized by having probes of
different lengths synthesized on the chip at the same time as
follows.
[0179] FIG. 30 shows a shift reticle 1200 for producing probes with
different lengths and interrogation positions. The top half of the
shift reticle 1202 is the same as Reticle 1 of FIG. 28.
Accordingly, it may be utilized to form 8-mer probes with different
interrogation positions. The bottom half of the shift reticle 1204
is similar to the top half except that it has two "blank" positions
1206. These blank positions will not have any monomer addition
regions in any of the shift reticles. Because there are two base
positions that are blank, the bottom half of the shift reticle does
not cover as much of the target as the top half. For simplicity,
only one shift reticle is shown but it should be readily understood
that for nucleic acid applications, there will be three other shift
reticles for the other three bases.
[0180] FIG. 31 illustrates the probes that may be produced by shift
reticles according to FIG. 30. A chip 1250 has two probe regions
corresponding to the different halves of the shift reticle of FIG.
30. A first region 1252 has eight different sets of four probes
where the number below (1-8) indicates the position of the
interrogation position in the probes. As before, a "1" indicates
that interrogation position is nearer the chip in the probes,
whereas an "8" indicates that the interrogation position is farther
from the chip in the probes.
[0181] A second region 1254 has eight different sets of four
probes, but as indicated by the number below (1-7), there are two
sets of probes with an interrogation position at the fifth base in
the probes. The duplicate set of probes was generated because of a
blank position in the shift reticle. Additionally, the probes in
region 1254 will be 7-mers and include probes with interrogation
positions at each possible position in the probes. Therefore,
probes of different lengths and different interrogation positions
may be synthesized on a chip at the same time with an embodiment of
the shift reticles of the invention.
[0182] The formation of duplicate sets of probes may be also
utilized to isolate problems during synthesis and/or to increase
the accuracy of the resulting data. For example, although the two
sets of probes in region 1254 that have an interrogation position
at the fifth base may be identical in terms of sequence, the bases
were synthesized during different monomer addition steps.
Accordingly, if the fourth monomer addition step that adds an A is
faulty, this may affect one set of probes but not the other.
Therefore, by analyzing the accuracy of the data from the duplicate
set of probes, one can identify synthesis problems and since there
may be duplicate sets of probes, the synthesis problems may be
accounted for by utilizing another probe set.
[0183] In some embodiments, blank probes are placed in the shift
reticles at various locations so that duplicate probe sets will be
formed. As discussed above, the duplicate probe sets may be
utilized to isolate problems during synthesis and possibly even
accounting for the errors.
[0184] The shift reticles may also be longer to synthesize probes
that interrogate multiple base positions in the target. FIG. 32
shows a shift reticle for producing probes that interrogate every
ninth base position in the target. The first part of the shift
reticle is the same as shown in FIG. 28 (Reticle 1). However, this
shift reticle is longer and may be utilized to interrogate the base
positions indicated by the asterisks 1275. Probes similar to the
one in FIG. 32 may be utilized to form, e.g., 8-mer probes that
interrogate every ninth position in the target with probes that
have every possible interrogation position. Although the
interrogation positions in the target that are being interrogated
is fixed in the design of the shift reticles, other shift reticles
may be produced to interrogate other positions in the target.
[0185] One may also reduce the number of probes by utilizing one
set of shift reticles for the even interrogation positions and one
set of shift reticles for the odd interrogation positions. Both
sets of probes are utilized and then shifted. In this manner,
probes that have interrogation positions at every other possible
location may be synthesized. Since there are less probes
synthesized on the chip, more base positions in the target may be
interrogated on the chip. Although two sets of shift reticles have
been described (one for even positions and one for odd positions),
more sets of shift reticles may be utilized. For example, one may
utilize a different set of shift reticles for each base position in
the target where (base position mod 3=0), (base position mod 3=1),
and (base position mod 3=2).
[0186] The above description is illustrative and not restrictive.
Many variations of the invention will become apparent to those of
skill in the art upon review of this disclosure. Merely by way of
example, while the invention is illustrated primarily with regard
to the synthesis of oligonucleotide or RNA, the invention will find
application to the synthesis of many other molecules. Further,
while the invention is primarily illustrated in relation to the
fabrication of small numbers of identical arrays, the invention may
also be applied to situations where a large number of identical
arrays is to be synthesized. The scope of the invention should,
therefore, be determined not with reference to the above
description, but instead should be determined with reference to the
appended claims along with their full scope of equivalents
Sequence CWU 1
1
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