U.S. patent application number 13/533594 was filed with the patent office on 2012-10-18 for oxide layers on silicone substrates for effective confocal laser microscopy.
This patent application is currently assigned to Affymetrix, Inc.. Invention is credited to Zihui Chen, Robert G. Kuimelis, Glenn H. McGall.
Application Number | 20120264644 13/533594 |
Document ID | / |
Family ID | 38575799 |
Filed Date | 2012-10-18 |
United States Patent
Application |
20120264644 |
Kind Code |
A1 |
Kuimelis; Robert G. ; et
al. |
October 18, 2012 |
Oxide Layers on Silicone Substrates for Effective Confocal Laser
Microscopy
Abstract
Methods of performing confocal laser microscopy on a polymer
array disposed on a silicon wafer substrate, the method comprising
the steps of providing a silicon wafer substrate having a top side
and a bottom side, coating the top side of the silicon wafer with
an oxide coating to provide an oxide coated wafer, covalently
coupling a plurality of probes to the top side of the coated wafer
to provide a fixed polymer array, hybridizing the fixed polymer
array with a plurality of labeled ligands, and assaying for one or
more hybridized ligands using confocal laser fluorescence
microscopy to detect hybridization are provided.
Inventors: |
Kuimelis; Robert G.; (Palo
Alto, CA) ; Chen; Zihui; (Mountain View, CA) ;
McGall; Glenn H.; (Palo Alto, CA) |
Assignee: |
Affymetrix, Inc.
Santa Clara
CA
|
Family ID: |
38575799 |
Appl. No.: |
13/533594 |
Filed: |
June 26, 2012 |
Related U.S. Patent Documents
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Application
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Filing Date |
Patent Number |
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13015041 |
Jan 27, 2011 |
8227253 |
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13533594 |
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11614896 |
Dec 21, 2006 |
7951601 |
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13015041 |
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60754534 |
Dec 28, 2005 |
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Current U.S.
Class: |
506/9 |
Current CPC
Class: |
Y10T 436/14 20150115;
G01N 21/6458 20130101; G01N 21/6452 20130101; Y10T 436/142222
20150115; Y10T 436/25 20150115; G01N 21/6428 20130101; Y10T
436/143333 20150115 |
Class at
Publication: |
506/9 |
International
Class: |
C40B 30/04 20060101
C40B030/04 |
Claims
1. A method of performing confocal laser microscopy on a polymer
array disposed on a silicon wafer substrate, said method comprising
the steps of: providing a silicon wafer substrate having a top side
and a bottom side; coating said top side of said silicon wafer with
an oxide coating to provide an oxide coated wafer; covalently
coupling a plurality of probes to said top side of said coated
wafer to provide a fixed polymer array; hybridizing said fixed
polymer array with a plurality of labeled ligands; and assaying for
one or more hybridized ligands using confocal laser fluorescence
microscopy to detect hybridization.
2. The method of claim 1, further comprising applying BisB to said
oxide coating.
3. A method of performing confocal laser microscopy on a polymer
array disposed on a silicon wafer substrate, said method comprising
the steps of: providing a silicon wafer substrate having a top side
and a bottom side; coating said top side of said substrate with a
transparent oxide layer to provide an oxide coated wafer;
depositing a reactive functional group comprising a labile
protecting group substantially uniformly across the transparent
oxide layer; selectively removing one or more of said labile
protecting groups from predefined regions of said wafer to provide
exposed functional groups in said predefined regions; reacting said
exposed functional groups with a monomer comprising a reactive
functional group and a labile protecting group; repeating the steps
of selectively removing and reacting to produce said polymer array;
hybridizing said polymer array with a plurality of ligands; and
assaying for one or more hybridized ligands using a confocal laser
fluorescence microscopy to detect hybridization.
4. The method of claim 3, wherein said oxide layer has a thickness
of at least 3,500 angstroms.
5. The method of claim 3, wherein said oxide layer has a thickness
of at least 35,000 angstroms.
6. The method of claim 3, wherein said labile protecting group is
an acid labile protecting group.
7. The method of claim 6, wherein said acid labile protecting group
is a dimethoxytrityl group.
8. The method of claim 6, wherein said acid labile protecting group
is removed by activating a photoacid generator with light of an
appropriate wavelength to produce acid.
9. The method of claim 8, wherein said photoacid generator is an
ionic photoacid generator or a non-ionic photoacid generator.
10. The method of claim 9, wherein said photoacid generator is an
ionic photoacid generator.
11. The method of claim 9, wherein said photoacid generator is a
non-ionic photoacid generator.
12. The method of claim 11, wherein said non-ionic photoacid
generator is 2,6-dinitrobenzyl tosylate.
13. The method of claim 10, wherein said ionic photoacid generator
is an onium salt.
14. The method of claim 13, wherein said onium salt is
bis-(4-t-butyl phenyl) iodonium PF.sub.6.sup.-.
15. The method of claim 3, wherein said labile protective group is
a photolabile protecting group.
16. The method of claim 3, wherein said monomer is selected from
the group consisting of a nucleotide, a nucleic acid, an amino acid
and a peptide.
17. The method of claim 3, wherein said monomer is a nucleic acid
and said labile protecting group is MeNPOC.
18. The method of claim 3, wherein said monomer is a nucleic acid
and said labile protecting group is NNPOC or MBPMOC.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to coated silicon substrates
useful for array synthesis and subsequent fluorescence analysis of
arrays.
BACKGROUND OF THE INVENTION
[0002] Methods for synthesizing a variety of different types of
polymers are well known in the art. For example, the "Merrifield"
method, described in Atherton et a., "Solid Phase Peptide
Synthesis," IRL Press, 1989, has been used to synthesize peptides
on a solid support. In the Merrifield method, an amino acid is
covalently bonded to a support made of an insoluble polymer or
other material. Another amino acid with an alpha protecting group
is reacted with the covalently bonded amino acid to form a
dipeptide. After washing, the protecting group is removed and a
third amino acid with an alpha protecting group is added to the
dipeptide. This process is continued until a peptide of a desired
length and sequence is obtained.
[0003] Methods have also been developed for producing large arrays
of polymer sequences on solid substrates. These large "array" of
polymer sequences have wide ranging applications and are of
substantial importance to the pharmaceutical, biotechnology and
medical industries. For example, the arrays may be used in
screening large numbers of molecules for biological activity, i.e.,
receptor binding capability. Alternatively, arrays of
oligonucleotide probes can be used to identify mutations in known
sequences, as well as in methods for de novo sequencing of target
nucleic acids.
SUMMARY OF THE INVENTION
[0004] Embodiments of the present invention are based in part on
the discovery that a variety of silicon substrates comprising an
oxide layer are suitable for array synthesis and subsequent
fluorescence analysis. A variety of silicon substrates were
investigated and determined to be suitable to support silanation
and non-photochemical methods of phosphoramidite-based probe
synthesis, generating results that were comparable to results
obtained using fused silica.
[0005] The present invention provides methods of performing
confocal laser microscopy on a polymer array disposed on a silicon
wafer substrate. In certain embodiments, a method of the invention
includes providing a silicon wafer substrate having a top side and
a bottom side, coating the top side of the silicon wafer with an
oxide coating to provide an oxide coated wafer, covalently coupling
a plurality of probes to the top side of the coated wafer to
provide a fixed polymer array, hybridizing the fixed polymer array
with a plurality of labeled ligands, and assaying for one or more
hybridized ligands using confocal laser fluorescence microscopy to
detect hybridization. Certain aspects of the invention include
applying BisB to the oxide coating.
[0006] In other embodiments, the present invention provides a
method of performing confocal laser microscopy on a polymer array
disposed on a silicon wafer substrate including the steps of
providing a silicon wafer substrate having a top side and a bottom
side, coating the top side of the substrate with a transparent
oxide layer to provide an oxide coated wafer, depositing a reactive
functional group comprising a labile protecting group substantially
uniformly across the transparent oxide layer, selectively removing
one or more of the labile protecting groups from predefined regions
of the wafer to provide exposed functional groups in said
predefined regions, reacting the exposed functional groups with a
monomer comprising a reactive functional group and a labile
protecting group, repeating the steps of selectively removing and
reacting to produce said polymer array, hybridizing the polymer
array with a plurality of ligands, and assaying for one or more
hybridized ligands using a confocal laser fluorescence microscopy
to detect hybridization.
[0007] Certain aspects of the invention provide an oxide layer
having a thickness of at least 3,500 angstroms or having a
thickness of at least 35,000 angstroms. Other aspects of the
invention provide that a labile protecting group is an acid labile
protecting group such as a dimethoxytrityl group. Other aspects of
the invention provide that an acid labile protecting group is
removed by activating a photoacid generator with light of an
appropriate wavelength to produce acid. A photoacid generator
includes an ionic photoacid generator such as an onium salt such as
bis-(4-t-butyl phenyl) iodonium PF.sub.6.sup.-, or a non-ionic
photoacid generator such as 2,6-dinitrobenzyl tosylate.
[0008] Certain aspects of the invention provide that a labile
protective group is a photolabile protecting group. Labile
protecting groups include MeNPOC. In accordance with an aspect of
the present invention, more efficient photolabile protecting groups
can also be used. It has been discovered in accordance with the
present invention that to achieve suitable primer purity and
quantity, a highly-efficient photogroup (>90% average stepwise
coupling efficiency) is preferred, such as NPPOC or MBPMOC:
##STR00001##
[0009] Both NNPOC and MBPMOC give greater than 90% stepwise
coupling. Other aspects of the invention provide that a monomer is
a nucleotide, a nucleic acid, an amino acid or a peptide.
BRIEF DESCRIPTION OF THE DRAWINGS
[0010] The foregoing and other features and advantages of the
present invention will be more fully understood from the following
detailed description of illustrative embodiments taken in
conjunction with the accompanying drawings in which:
[0011] FIG. 1 depicts the site density of silicon wafers coated
with BisB. The density of the silicon wafers was 2.33 g/cm.sup.3,
and the thickness of the silicon wafers was approximately 400 to
700 .mu.m.
[0012] FIG. 2 depicts TCA 6-mers on fused silica and oxide
silicone.
[0013] FIG. 3 depicts dosage response curves of a photoacid
generator (PAG) on oxide silicon BisB.
[0014] FIG. 4 depicts T-6-mers (PAG) with no base on oxide silicon
and fused silica.
[0015] FIG. 5 depicts 100 mm Si wafer testing (BisB) using Cy3
stain galvo scans.
[0016] FIG. 6 depicts 100 mm Si wafer testing (BisB) using Cy3
stain Axon scans.
[0017] FIG. 7 depicts 100 mm Si wafer testing (BisB) using Cy3
stain galvo scans showing oxide thickness.
[0018] FIG. 8 depicts a 2 nM dual label target (BisB oxide-Si
substrate) fluorescein channel, front side scan.
[0019] FIG. 9 depicts 100 mm Si wafer testing (BisB) using MeNPOC
hexamers. MeNPOC stepwise deprotection was reduced to 55% on native
oxide. Other surfaces compared well to a fused-silica (FS) control,
and, without intending to be bound by theory, each surface tested
should produce good single-MeNPOC experiments.
DETAILED DESCRIPTION OF THE INVENTION
[0020] The present invention has many preferred embodiments and
relies on many patents, applications and other references for
details known to those of the art. Therefore, when a patent,
application, or other reference is cited or repeated below, it
should be understood that it is incorporated by reference in its
entirety for all purposes as well as for the proposition that is
recited.
[0021] As used herein, the singular forms "a," "an," and "the"
include, but are not limited to, plural references unless the
context clearly dictates otherwise. For example, the term "an
agent" includes, but is not limited to, a plurality of agents,
including mixtures thereof.
[0022] Throughout this disclosure, various aspects of this
invention can be presented in a range format. It should be
understood that the description in range format is merely for
convenience and brevity and should not be construed as an
inflexible limitation on the scope of the invention. Accordingly,
the description of a range should be considered to have
specifically disclosed all the possible subranges as well as
individual numerical values within that range. For example,
description of a range such as from 1 to 6 should be considered to
have specifically disclosed subranges such as from 1 to 3, from 1
to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as
well as individual numbers within that range, for example, 1, 2, 3,
4, 5 and 6. This applies regardless of the breadth of the
range.
[0023] The practice of the present invention may employ, unless
otherwise indicated, conventional techniques and descriptions of
organic chemistry, polymer technology, molecular biology (including
recombinant techniques), cell biology, biochemistry and immunology,
which are within the skill of the art. Such conventional techniques
include polymer array synthesis, hybridization, ligation, and
detection of hybridization using a label. Specific illustrations of
suitable techniques can be had by reference to the description
provided below. However, other equivalent conventional procedures
can, of course, also be used. Such conventional techniques and
descriptions can be found in standard laboratory manuals such as
Genome Analysis: A Laboratory Manual Series (Vols. I-IV), Using
Antibodies: A Laboratory Manual, Cells: A Laboratory Manual, PCR
Primer: A Laboratory Manual, and Molecular Cloning: A Laboratory
Manual (all from Cold Spring Harbor Laboratory Press), Stryer, L.
(1995) Biochemistry (4th Ed.) Freeman, New York, Gait,
"Oligonucleotide Synthesis: A Practical Approach" 1984, IRL Press,
London, Nelson and Cox (2000), Lehninger, Principles of
Biochemistry 3rd Ed., W. H. Freeman Pub., New York, N.Y. and Berg
et al. (2002) Biochemistry, 5th Ed., W. H. Freeman Pub., New York,
N.Y., all of which are herein incorporated in their entirety by
reference for all purposes.
[0024] The present invention can employ solid substrates, including
arrays in certain embodiments. Methods and techniques applicable to
polymer array synthesis have been described in U.S. Ser. No.
09/536,841, WO 00/58516, U.S. Pat. Nos. 5,143,854, 5,242,974,
5,252,743, 5,324,633, 5,384,261, 5,405,783, 5,424,186, 5,451,683,
5,482,867, 5,491,074, 5,527,681, 5,550,215, 5,571,639, 5,578,832,
5,593,839, 5,599,695, 5,624,711, 5,631,734, 5,795,716, 5,831,070,
5,837,832, 5,856,101, 5,858,659, 5,936,324, 5,968,740, 5,974,164,
5,981,185, 5,981,956, 6,025,601, 6,033,860, 6,040,193, 6,090,555,
6,136,269, 6,269,846 and 6,428,752, in PCT Applications Nos.
PCT/US99/00730 (International Publication No. WO 99/36760) and
PCT/US01/04285 (International Publication No. WO 01/58593), each of
which is incorporated herein by reference in its entirety for all
purposes.
[0025] Patents that describe synthesis techniques in specific
embodiments include U.S. Pat. Nos. 5,412,087, 6,147,205, 6,262,216,
6,310,189, 5,889,165 and 5,959,098, each of which is incorporated
herein by reference in its entirety for all purposes. Nucleic acid
arrays are described in many of the above patents, but the same
techniques are applied to polypeptide arrays.
[0026] The present invention also contemplates many uses for
polymers attached to solid substrates. These uses include gene
expression monitoring, profiling, library screening, genotyping and
diagnostics. Gene expression monitoring, and profiling methods can
be shown in U.S. Pat. Nos. 5,800,992, 6,013,449, 6,020,135,
6,033,860, 6,040,138, 6,177,248 and 6,309,822, which are all
incorporated by reference in their entirety for all purposes.
Genotyping and uses therefore are shown in U.S. Ser. Nos.
60/319,253, 10/013,598 (U.S. Patent Application Publication
20030036069), and U.S. Pat. Nos. 5,856,092, 6,300,063, 5,858,659,
6,284,460, 6,361,947, 6,368,799 and 6,333,179, which are
incorporated by reference in their entirety for all purposes. Other
uses are embodied in U.S. Pat. Nos. 5,871,928, 5,902,723,
6,045,996, 5,541,061, and 6,197,506, which are incorporated by
reference in their entirety for all purposes.
[0027] The present invention also contemplates sample preparation
methods in certain preferred embodiments. Prior to or concurrent
with genotyping, the genomic sample may be amplified by a variety
of mechanisms, some of which may employ PCR. See, e.g., PCR
Technology: Principles and Applications for DNA Amplification (Ed.
H. A. Erlich, Freeman Press, NY, N.Y., 1992); PCR Protocols: A
Guide to Methods and Applications (Eds. Innis, et al., Academic
Press, San Diego, Calif., 1990); Mattila et al., Nucleic Acids Res.
19, 4967 (1991); Eckert et al., PCR Methods and Applications 1, 17
(1991); PCR (Eds. McPherson et al., IRL Press, Oxford); and U.S.
Pat. Nos. 4,683,202, 4,683,195, 4,800,159 4,965,188, and 5,333,675,
each of which is incorporated herein by reference in its entirety
for all purposes. The sample may be amplified on the array. See,
for example, U.S. Pat. No. 6,300,070 and U.S. Ser. No. 09/513,300,
which are incorporated herein by reference in their entirety for
all purposes.
[0028] Other suitable amplification methods include the ligase
chain reaction (LCR) (e.g., Wu and Wallace (1989) Genomics 4:560,
Landegren et al. (1988) Science 241:1077 and Barringer et al.
(1990) Gene 89:117), transcription amplification (Kwoh et al.
(1989) Proc. Natl. Acad. Sci. USA 86:1173 and WO88/10315),
self-sustained sequence replication (Guatelli et al. (1990) Proc.
Nat. Acad. Sci. USA, 87:1874 and WO90/06995), selective
amplification of target polynucleotide sequences (U.S. Pat. No
6,410,276), consensus sequence primed polymerase chain reaction
(CP-PCR) (U.S. Pat. No. 4,437,975), arbitrarily primed polymerase
chain reaction (AP-PCR) (U.S. Pat. Nos. 5,413,909, 5,861,245) and
nucleic acid based sequence amplication (NABSA). Each of the above
references is incorporated herein by reference in its entirety for
all purposes. (See, U.S. Pat. Nos. 5,409,818, 5,554,517, and
6,063,603, each of which is incorporated herein by reference in its
entirety for all purposes). Other amplification methods that may be
used are described in, U.S. Pat. Nos. 5,242,794, 5,494,810,
4,988,617 and in U.S. Ser. No. 09/854,317. Each of the above
references is incorporated herein by reference in its entirety.
[0029] Additional methods of sample preparation and techniques for
reducing the complexity of a nucleic sample are described in Dong
et al. (2001) Genome Research 11:1418, in U.S. Pat. Nos. 6,361,947,
6,391,592 and U.S. Ser. Nos. 09/916,135, 09/920,491 (U.S. Patent
Application Publication 20030096235), Ser. No. 09/910,292 (U.S.
Patent Application Publication 20030082543), and Ser. No.
10/013,598, each of which is incorporated herein by reference in
its entirety.
[0030] Numerous methods for conducting polynucleotide hybridization
assays have been well developed. Hybridization assay procedures and
conditions will vary depending on the application and are selected
in accordance with the general binding methods known including
those referred to in: Maniatis et al. Molecular Cloning: A
Laboratory Manual (2.sup.nd Ed. Cold Spring Harbor, N.Y, 1989);
Berger and Kimmel Methods in Enzymology, Vol. 152, Guide to
Molecular Cloning Techniques (Academic Press, Inc., San Diego,
Calif., 1987); Young and Davism, Proc. Natl. Acad. Sci. USA,
80:1194 (1983). Methods and apparatus for carrying out repeated and
controlled hybridization reactions have been described in U.S. Pat.
Nos. 5,871,928, 5,874,219, 6,045,996 and 6,386,749, 6,391,623 each
of which is hereby incorporated by reference in its entirety.
[0031] The present invention contemplates detection of
hybridization between a ligand and its corresponding receptor by
generation of specific signals. See U.S. Pat. Nos. 5,143,854,
5,578,832; 5,631,734; 5,834,758; 5,936,324; 5,981,956; 6,025,601;
6,141,096; 6,185,030; 6,201,639; 6,218,803; and 6,225,625, in U.S.
Ser. No. 60/364,731 and in PCT Application PCT/US99/06097
(published as WO99/47964), each of which also is hereby
incorporated by reference in its entirety. Each of these references
is incorporated herein by reference in its entirety.
[0032] Methods and apparatus for signal detection and processing of
intensity data are disclosed in, for example, U.S. Pat. Nos.
5,143,854, 5,547,839, 5,578,832, 5,631,734, 5,800,992, 5,834,758;
5,856,092, 5,902,723, 5,936,324, 5,981,956, 6,025,601, 6,090,555,
6,141,096, 6,185,030, 6,201,639; 6,218,803; and 6,225,625, in U.S.
Ser. No. 60/364,731 and in PCT Application PCT/US99/06097
(published as WO99/47964), each of which also is hereby
incorporated by reference in its entirety.
[0033] The practice of the present invention may also employ
conventional biology methods, software and systems. Computer
software products of the invention typically include computer
readable medium having computer-executable instructions for
performing the logic steps of the method of the invention. Suitable
computer readable medium include floppy disk, CD-ROM/DVD/DVD-ROM,
hard-disk drive, flash memory, ROM/RAM, magnetic tapes and etc. The
computer executable instructions may be written in a suitable
computer language or combination of several languages. Basic
computational biology methods are described in, e.g. Setubal and
Meidanis et al., Introduction to Computational Biology Methods (PWS
Publishing Company, Boston, 1997); Salzberg, Searles, Kasif, (Ed.),
Computational Methods in Molecular Biology, (Elsevier, Amsterdam,
1998); Rashidi and Buehler, Bioinformatics Basics: Application in
Biological Science and Medicine (CRC Press, London, 2000) and
Ouelette and Bzevanis Bioinformatics: A Practical Guide for
Analysis of Gene and Proteins (Wiley & Sons, Inc., 2.sup.nd
ed., 2001). See U.S. Pat. No. 6,420,108. Each of these references
is incorporated herein by reference in its entirety.
[0034] The present invention may also make use of various computer
program products and software for a variety of purposes, such as
probe design, management of data, analysis, and instrument
operation. See, U.S. Pat. Nos. 5,593,839, 5,795,716, 5,733,729,
5,974,164, 6,066,454, 6,090,555, 6,185,561, 6,188,783, 6,223,127,
6,229,911 and 6,308,170. Each of references is incorporated herein
by reference in its entirety.
[0035] Light patterns can also be generated using Digital
Micromirrors, Light Crystal on Silicon (LCOS), light valve arrays,
laser beam patterns and other devices suitable for direct-write
photolithography. See. e.g., U.S. Pat. Nos. 6,271,957 and
6,480,324, incorporated herein by reference in their entirety for
all purposes.
[0036] Additionally, the present invention may have preferred
embodiments that include methods for providing biological
information over networks such as the internet as shown in U.S.
Ser. Nos. 10/197,621, 10/063,559 (United States Publication No.
20020183936), Ser. Nos. 10/065,856, 10/065,868, 10/328,818,
10/328,872, 10/423,403, and 60/482,389, each of which is
incorporated herein by reference in its entirety for all
purposes.
[0037] The following definitions are used, unless otherwise
described.
[0038] An "array," as defined herein, includes but is not limited
to a preselected collection of different polymer sequences or
probes which are associated with a surface of a substrate. An array
may include polymers of a given length having all possible monomer
sequences made up of a specific basis set of monomers, or a
specific subset of such an array. For example, an array of all
possible oligonucleotides of length 8 includes 65,536 different
sequences. However, as noted above, an oligonucleotide array also
may include only a subset of the complete set of probes. Similarly,
a given array may exist on more than one separate substrate, e.g.,
where the number of sequences necessitates a larger surface area in
order to include all of the desired polymer sequences.
[0039] A "functional group," as used herein, includes but is not
limited to a reactive chemical moiety present on a given monomer,
polymer or substrate surface. Examples of functional groups
include, e.g., the 3' and 5' hydroxyl groups of nucleotides and
nucleosides, as well as the reactive groups on the nucleobases of
the nucleic acid monomers, e.g., the exocyclic amine group of
guanosine, as well as amino and carboxyl groups on amino acid
monomers.
[0040] A "monomer" or "building block," as used herein, includes
but is not limited to a member of the set of smaller molecules
which can be joined together to form a larger molecule or polymer.
The set of monomers includes but is not restricted to, for example,
the set of common L-amino acids, the set of D-amino acids, the set
of natural or synthetic amino acids, the set of nucleotides (both
ribonucleotides and deoxyribonucleotides, natural and unnatural)
and the set of pentoses and hexoses. As used herein, monomer refers
to any member of a basis set for synthesis of a larger molecule. A
selected set of monomers forms a basis set of monomers. For
example, the basis set of nucleotides includes A, T (or U), G and
C. In another example, dimers of the 20 naturally occurring L-amino
acids form a basis set of 400 monomers for synthesis of
polypeptides. Different basis sets of monomers may be used in any
of the successive steps in the synthesis of a polymer. Furthermore,
each of the sets may include protected members which are modified
after synthesis.
[0041] A "feature," as used herein, includes but is not limited to
a selected region on a surface of a substrate in which a given
polymer sequence is contained. Thus, where an array contains, e.g.,
100,000 different positionally distinct polymer sequences on a
single substrate, there will be 100,000 features.
[0042] An "edge," as used herein, includes but is not limited to a
boundary between two features on a surface of a substrate. The
sharpness of this edge, in terms of reduced bleed-over from one
feature to another, is termed the "contrast" between the two
features.
[0043] A "protecting group," as used herein, includes but is not
limited to a material which is chemically bound to a reactive
functional group on a monomer unit or polymer and which protective
group may be removed upon selective exposure to an activator such
as a chemical activator, or another activator, such as
electromagnetic radiation or light, especially ultraviolet and
visible light. Protecting groups that are removable upon exposure
to electromagnetic radiation, and in particular light, are termed
"photolabile protecting groups."
[0044] Halo is fluoro, chloro, bromo, or iodo. Alkyl, alkoxy,
aralkyl, alkylaryl, and the like denote both straight and branched
alkyl groups, but reference to an individual radical such as
"propyl" embraces only the straight chain radical, a branched chain
isomer such as "isopropyl" being specifically referred to. Aryl
includes a phenyl radical or an ortho-fused, bicyclic, carbocyclic
radical having about nine to ten ring atoms in which at least one
ring is aromatic. Heteroaryl encompasses a radical attached via a
ring carbon of a monocyclic aromatic ring containing five or six
ring atoms consisting of carbon and one to four heteroatoms each
selected from the group consisting of non-peroxide oxygen, sulfur,
and N(X) wherein X is absent or is H, O, (C,--C.sub.4)alkyl, phenyl
or benzyl, as well as a radical of an ortho-fused, bicyclic
heterocycle of about eight to ten ring atoms derived therefrom,
particularly a benz-derivative or one derived by fusing a
propylene, trimethylene or tetramethylene diradical thereto.
[0045] An "alkyl," as used herein, refers without limitation to a
straight chain, branched or cyclic chemical groups containing only
carbon and hydrogen. Alkyl groups include, without limitation,
ethyl, propyl, butyl, pentyl, cyclopentyl and 2-methylbutyl. Alkyl
groups are unsubstituted or substituted with one or more
substituents (e.g., halogen, alkoxy, amino).
[0046] An "alkylene," as used herein, refers without limitation to
a straight chain, branched or cyclic chemical group containing only
carbon and hydrogen. Alkyl groups include, without limitation,
ethylene, propylene, butylene, pentylene, and 2-methylbutylene.
Alkyl groups are unsubstituted or substituted with one or more
substituents (e.g., halogen, alkoxy, amino).
[0047] An "aryl," as used herein, refers without limitation to a
monovalent, unsaturated, aromatic carbocyclic group. Aryl groups
include, without limitation, phenyl, naphthyl, anthryl and
biphenyl. Aryl groups are unsubstituted or substituted with 1 or
more substituents (e.g. halogen, alkoxy, amino). "Arylene" refers
to a divalent aryl group.
[0048] An "amido," as used herein, refers without limitation to a
chemical group having the structure --C(O)NR.sub.3--, wherein
R.sub.3 is hydrogen, alkyl or aryl. Preferably, the amido group is
of the structure --C(O)NR.sub.3-- where R.sub.3 is hydrogen or
alkyl having from about 1 to about 6 carbon atoms. More preferably,
the amido alkyl group is of the structure --C(O)NH--.
[0049] An "alkanoyl," as used herein, refers without limitation to
a chemical group having the structure --(CH.sub.2).sub.nC(O)--,
wherein n is an integer ranging from 0 to about 10. Preferably, the
alkanoyl group is of the structure --(CH.sub.2).sub.nC(O)--,
wherein n is an integer ranging from about 2 to about 10. More
preferably, the alkanoyl group is of the structure
--(CH.sub.2).sub.n(O)--, wherein n is an integer ranging from about
2 to about 6. Most preferably, the alkanoyl group is of the
structure --CH.sub.2C(O)--.
[0050] An "alkyl amido," as used herein, refers without limitation
to a chemical group having the structure --R.sub.4C(O)NR.sub.3--,
wherein R.sub.3 is hydrogen, alkyl or aryl, and R.sub.4 is alkylene
or arylene. Preferably, the alkyl amido group is of the structure
--(CH.sub.2).sub.nC(O)NH--, wherein n is an integer ranging from
about 1 to about 10. More preferably, n is an integer ranging from
about 1 to about 6. Most preferably, the alkyl amido group has the
structure --(CH.sub.2).sub.2C(O)NH-- or the structure
--CH.sub.2C(O)NH--.
[0051] An "N-amido alkyl," as used herein, refers without
limitation to a chemical group having the structure
--C(O)NR.sub.3R.sub.4--, wherein R.sub.3 is hydrogen, alkyl or
aryl, and R.sub.4 is alkylene or arylene. Preferably, the N-amido
alkyl group is of the structure --C(O)NH(CH.sub.2).sub.nR.sub.5--,
wherein n is an integer ranging from about 2 to about 10, and
R.sub.5 is O, NR.sub.6, or C(O), and wherein R.sub.6 is hydrogen,
alkyl or aryl. More preferably, the N-amido alkyl group is of the
structure --C(O)NH(CH.sub.2).sub.nN(H)--, wherein n is an integer
ranging from about 2 to about 6. Most preferably, the N-amido alkyl
group is of the structure --C(O)NH(CH.sub.2).sub.4N(H)--.
[0052] An "alkynyl alkyl," as used herein, refers without
limitation to a chemical group having the structure
--CC--R.sub.4--, wherein R.sub.4 is alkyl or aryl. Preferably, the
alkynyl alkyl group is of the structure
--CC--(CH.sub.2).sub.nR.sub.5--, wherein n is an integer ranging
from 1 to about 10, and R.sub.5 is O, NR.sub.6 or C(O), wherein
R.sub.6 is hydrogen, alkyl or aryl. More preferably, the alkynyl
alkyl group is of the structure --CC--(CH.sub.2).sub.nN(H)--,
wherein n is an integer ranging from 1 to about 4. Most preferably,
the alkynyl alkyl group is of the structure
--CC--CH.sub.2N(H)--.
[0053] An "alkenyl alkyl," as used herein, refers without
limitation to a chemical group having the structure
--CH.dbd.CH--R.sub.4--, wherein R.sub.4 is a bond, alkyl or aryl.
Preferably, the alkenyl alkyl group is of the structure
--CH.dbd.CH--(CH.sub.2)nR.sub.5--, wherein n is an integer ranging
from 0 to about 10, and R.sub.5 is O, NR.sub.6, C(O) or
C(O)NR.sub.6, wherein R.sub.6 is hydrogen, alkyl or aryl. More
preferably, the alkenyl alkyl group is of the structure
--CH.dbd.CH-- (CH.sub.2).sub.nC(O)NR.sub.6--, wherein n is an
integer ranging from 0 to about 4. Most preferably, the alkenyl
alkyl group is of the structure --CH.dbd.CH--C(O)N(H)--.
[0054] A "functionalized alkyl," as used herein, refers without
limitation to a chemical group of the structure
--(CH.sub.2).sub.nR.sub.7--, wherein n is an integer ranging from 1
to about 10, and R.sub.7 is O, S, NH or C(O). Preferably, the
functionalized alkyl group is of the structure
--(CH.sub.2).sub.nC(O)--, wherein n is an integer ranging from 1 to
about 4. More preferably, the functionalized alkyl group is of the
structure --CH.sub.2C(O)--.
[0055] An "alkoxy," as used herein, refers without limitation to a
chemical group of the structure --O(CH.sub.2).sub.nR.sub.8--,
wherein n is an integer ranging from 2 to about 10, and R.sub.8 is
a bond, O, S, NH or C(O). Preferably, the alkoxy group is of the
structure --O(CH.sub.2)n, wherein n is an integer ranging from 2 to
about 4. More preferably, the alkoxy group is of the structure
--OCH.sub.2CH.sub.2--.
[0056] An "alkyl thio," as used herein, refers without limitation
to a chemical group of the structure --S(CH.sub.2).sub.nR.sub.8--,
wherein n is an integer ranging from 1 to about 10, and R.sub.8 is
a bond, O, S, NH or C(O). Preferably, the alkyl thio group is of
the structure --S(CH.sub.2).sub.n, wherein n is an integer ranging
from 2 to about 4. More preferably, the thio group is of the
structure --SCH.sub.2CH.sub.2C(O)--.
[0057] An "amino alkyl," as used herein, refers without limitation
to a chemical group having an amino group attached to an alkyl
group. Preferably an amino alkyl is of the structure
--(CH).sub.nNH--, wherein n is an integer ranging from about 2 to
about 10. More preferably it is of the structure
--(CH.sub.2).sub.nNH--, wherein n is an integer ranging from about
2 to about 4. Most preferably, the amino alkyl group is of the
structure --(CH.sub.2).sub.2NH--.
[0058] The term "nucleic acid," as used herein, includes, but is
not limited to, a polymer comprising two or more nucleotides and
includes single-, double- and triple stranded polymers. As used
herein, the term "nucleotide" refers without limitation to both
naturally occurring and non-naturally occurring compounds and
comprises a heterocyclic base, a sugar, and a linking group,
preferably a phosphate ester. As used herein, the term "nucleoside"
refers to both naturally occurring and non-naturally occurring
compounds and comprises a heterocyclic base and a sugar.
[0059] Structural groups may be added to the ribosyl or
deoxyribosyl unit of the nucleotide, such as a methyl or allyl
group at the 2'-O position or a fluoro group that substitutes for
the 2'-O group. The linking group, such as a phosphodiester, of the
nucleic acid may be substituted or modified, for example with
methyl phosphonates or O-methyl phosphates. Bases and sugars can
also be modified, as is known in the art. "Nucleic acid," for the
purposes of this disclosure, also includes "peptide nucleic acids"
in which native or modified nucleic acid bases are attached to a
polyamide backbone.
[0060] The term "oligonucleotide," sometimes referred to as
"polynucleotide," includes, but is not limited to, a nucleic acid
ranging from at least 5, 10, or 20 bases long and may be up to 20,
50, 100, 1,000, or 5,000 bases long and/or a compound that
specifically hybridizes to a polynucleotide. Oligonucleotides of
the present invention include sequences of deoxyribonucleic acid
(DNA) or ribonucleic acid (RNA) and mimetics thereof which may be
isolated from natural sources, recombinantly produced or
artificially synthesized. A further example of a polynucleotide of
the present invention is a peptide nucleic acid (PNA). See U.S.
Pat. No. 6,156,501, incorporated herein by reference in its
entirety for all purposes. The invention also encompasses
situations in which there is a nontraditional base pairing such as
Hoogsteen base pairing which has been identified in certain tRNA
molecules and postulated to exist in a triple helix.
"Polynucleotide" and "oligonucleotide" are used interchangeably in
this application.
[0061] The phrase "coupled to a support" includes, but is not
limited to, being bound directly or indirectly thereto including
attachment by covalent binding, hydrogen bonding, ionic
interaction, hydrophobic interaction, or otherwise.
[0062] A "probe," as defined herein, includes but is not limited to
a surface-immobilized molecule that is recognized by a particular
target. These may also be referred to as ligands. Examples of
probes encompassed by the scope of this invention include, but are
not limited to, agonists and antagonists of cell surface receptors,
toxins and venoms, viral epitopes, hormone receptors, peptides,
peptidomimetics, enzymes, enzyme substrates, cofactors, drugs,
lectins, sugars, oligonucleotides, nucleic acids, oligosaccharides,
proteins or monoclonal antibodies, natural or modified, e.g.,
reshaped, chimeric, etc.
[0063] The terms "solid support," "support," and "substrate" as
used herein are used interchangeably and include, but are not
limited to, a material or group of materials having a rigid or
semi-rigid surface or surfaces. In many embodiments, at least one
surface of the solid support will be substantially flat or planar,
although in some embodiments it may be desirable to physically
separate synthesis regions for different compounds with, for
example, wells, raised regions, pins, etched trenches, or the like.
According to other embodiments, the solid support(s) will take the
form of beads, resins gels, microspheres, or other geometric
configurations. Preferred substrates generally comprise planar
crystalline substrates used in, e.g., the semiconductor and
microprocessor industries, such as silicon, gallium arsenide and
the like, or crystalline substrates such as silica based substrates
(e.g. glass, quartz, or the like). These substrates are generally
resistant to the variety of synthesis and analysis conditions to
which they may be subjected. See U.S. Pat. No. 5,744,305 and U.S.
Patent Appln. Pub. 20040105932, each of which is incorporated
herein by reference in its entirety for all purposes, for exemplary
substrates.
[0064] Individual planar substrates generally exist as wafers which
can have varied dimensions. As used herein, the term "wafer"
generally refers without limitation to a substantially flat sample
of substrate from which a plurality of individual arrays or chips
may be fabricated. The terms "array" or "chip" are used without
limitation to refer to the final product of the individual array of
polymer sequences, having a plurality of different positionally
distinct polymer sequences coupled to the surface of the substrate.
The size of a substrate wafer is generally defined by the number
and nature of arrays that will be produced from the wafer. For
example, more complex arrays, e.g., arrays having all possible
polymer sequences produced from a basis set of monomers and having
a given length, will generally utilize larger areas and thus employ
larger substrates, whereas simpler arrays may employ smaller
surface areas, and thus, less substrate.
[0065] In certain aspects of the invention, silicon wafers can be
used to fabricate high-density arrays of oligonucleotides using the
techniques described herein. Certain advanced lithography equipment
(e.g., a stepper) is designed around the industry-standard round
silicon wafer substrate. Commercially available substrates are
typically 5, 6 and 8 inches in diameter. In contrast to fused
silica, silicon wafers are thinner and non-transparent. The opacity
of silicon requires that photochemistry and also scanning be
performed "front side." A number of bulk physical properties, such
as crystal lattice orientation, dopant, resistivity and insulating
oxide layer thickness have been discovered in accordance with the
instant invention to not be critical to silanation or array
synthesis. It was also discovered in accordance with the present
invention that a variety of silicon substrates support silanation
and non-photochemical methods of phosphoramidite-based probe
synthesis with results comparable to fused silica. However, in
accordance with the present invention, it has been discovered that
effective confocal laser scanning has been found to be surprisingly
dependent upon a suitable coating such as a layer of transparent
oxide.
[0066] Typically, the substrate wafer will range in size of from
about 1''.times.1'' to about 12''.times.12'', and will have a
thickness of from about 0.5 mm to about 5 mm. Individual substrate
segments which include the individual arrays, or in some cases a
desired collection of arrays, are typically much smaller than the
wafers, measuring from about 0.2 cm..times.0.2 cm to about 5
cm.times.5 cm. In particularly preferred aspects, the substrate
wafer is about 5''.times.5'' whereas the substrate segment is
approximately 1.28 cm.times.1.28 cm. Although a wafer can be used
to fabricate a single large substrate segment, typically, a large
number of substrate segments will be prepared from a single wafer.
For example, a wafer that is 5''.times.5'' can be used to fabricate
upwards of 49 separate 1.28 cm.times.1.28 cm substrate segments.
The number of segments prepared from a single wafer will generally
vary depending upon the complexity of the array, and the desired
feature size.
[0067] Although primarily described in terms of flat or planar
substrates, the present invention may also be practiced with
substrates having substantially different conformations. For
example, the substrate may exist as particles, strands,
precipitates, gels, sheets, tubing, spheres, containers,
capillaries, pads, slices, films, plates, slides, and the like. In
a preferred alternate embodiment, the substrate is a glass tube or
microcapillary. The capillary substrate provides advantages of
higher surface area to volume ratios, reducing the amount of
reagents necessary for synthesis. Similarly, the higher surface to
volume ratio of these capillary substrates imparts more efficient
thermal transfer properties. Additionally, preparation of the
polymer arrays may be simplified through the use of these capillary
based substrates. For example, minimizing differences between the
regions on the array, or "cells," and their "neighboring cells" is
simplified in that there are only two neighboring cells for any
given cell (see discussion below for edge minimization in chip
design). Spatial separation of two neighboring cells on an array
merely involves the incorporation of a single blank cell, as
opposed to full blank lanes as generally used in a flat substrate
conformation. This substantially conserves the surface area
available for polymer synthesis. Manufacturing design may also be
simplified by the linear nature of the substrate. In particular,
the linear substrate may be moved down a single mask in a direction
perpendicular to the length of the capillary. As it is moved, the
capillary will encounter linear reticles (translucent regions of
the mask), one at a time, thereby exposing selected regions within
the capillary or capillary. This can allow bundling of parallel
capillaries during synthesis wherein the capillaries are exposed to
thicker linear reticles, simultaneously, for a batch processing
mode, or individual capillaries may be placed on a mask having all
of the linear reticles lined up so that the capillary can be
stepped down the mask in one direction. Subsequent capillaries may
be stepped down the mask at least one step behind the previous
capillary. This employs an assembly line structure to the substrate
preparation process.
[0068] As an example, a standard optimization chip for detecting 36
simultaneous mutations using a flat geometry chip and an
optimization tiling strategy, is 44.times.45 features (1980 probes
and blanks), with 36 blocks of 40 probes each (1440 probes), plus
15 blanks per block (540 blank probes). A capillary format,
however, can incorporate the same number of test probes in a
smaller space. Specifically, in a capillary substrate, 36 strings
of 40 probes will have only one blank space separating each probe
group (35 blank probes), for a total of 1475 features.
[0069] Finally, linear capillary based substrates can provide the
advantage of reduced volume over flat geometries. In particular,
typical capillary substrates have a volume in the 1-10 .mu.l range,
whereas typical flow cells for synthesizing or screening flat
geometry chips have volumes in the range of 100 .mu.l.
A. Stripping and Rinsing
[0070] In one aspect of the present invention, oxide-coated wafers
are silanated as supplied from the wafer vendor without prior
stripping. In other aspects, in order to ensure maximum efficiency
and accuracy in synthesizing polymer arrays, it is desirable to
provide a clean substrate surface upon which the various reactions
are to take place. Accordingly, in some processing embodiments of
the present invention, the substrate is stripped to remove any
residual dirt, oils or other fluorescent materials which may
interfere with the synthesis reactions, or subsequent analytical
use of the array.
[0071] The process of stripping the substrate typically involves
applying, immersing or otherwise contacting the substrate with a
stripping solution. Stripping solutions may be selected from a
number of commercially available, or readily prepared chemical
solutions used for the removal of dirt and oils, which solutions
are well known in the art. Particularly preferred stripping
solutions are composed of a mixture of concentrated H.sub.2SO.sub.4
and H.sub.2O.sub.2. Such solutions are generally available from
commercial sources, e.g., NANOSTRIPT.TM. from Cyantek Corp.
(Fremont, Calif.). After stripping, the substrate is rinsed with
water and in preferred aspects, is then contacted with a solution
of NaOH, which results in regeneration of an even layer of hydroxyl
functional groups on the surface of the substrate. In this case,
the substrate is again rinsed with water, followed by a rinse with
HCl to neutralize any remaining base, followed again by a water
rinse. The various stripping and rinsing steps may generally be
carried out using a spin-rinse-drying apparatus of the type
generally used in the semiconductor manufacturing industry.
[0072] Gas phase cleaning and preparation methods may also be
applied to the substrate wafers using, e.g., H.sub.2O or O.sub.2
plasma or reactive ion etching (RIE) techniques that are well known
in the art.
B. Coating Layer
[0073] Embodiments of the present invention are based on the
unexpected finding that there is a strong correlation between array
signal detection and oxide layer thickness. In accordance with a
preferred embodiment, silicon wafers coated with a coating layer,
e.g., an oxide layer, are used as substrates for array synthesis
and subsequent fluorescence analysis. An oxide layer can be
composed of conventional oxide materials such as silicon oxide
(SiO), silicon dioxide (SiO.sub.2), borophosphosilicate glass
(BPSG), borosilicate glass (BSG), fluorosilicate glass (FSG),
tetraethoxysilane (TEOS), and the like. Oxide layers are described
in U.S. Pat. No. 6,191,046, incorporated herein by reference in its
entirety for all purposes.
[0074] In a certain embodiments, the coating layer, e.g., an oxide
layer, has a thickness of approximately at least 3,500 angstroms,
4,000 angstroms, 4,500 angstroms, 5,000 angstroms, 5,500 angstroms,
6,000 angstroms, 6,500 angstroms, 7,000 angstroms, 7,500 angstroms,
8,000 angstroms, 8,500 angstroms, 9,000 angstroms, 9,500 angstroms,
10,000 angstroms, 11,000 angstroms, 12,000 angstroms, 13,000
angstroms, 14,000 angstroms, 15,000 angstroms, 16,000 angstroms,
17,000 angstroms, 18,000 angstroms, 19,000 angstroms, 20,000
angstroms, 21,000 angstroms, 22,000 angstroms, 23,000 angstroms,
24,000 angstroms, 25,000 angstroms, 26,000 angstroms, 27,000
angstroms, 28,000 angstroms. 29,000 angstroms, 30,000 angstroms,
40,000 angstroms, 45,000 angstroms, 50,000 angstroms, 55,000
angstroms, 60,000 angstroms, 65,000 angstroms, 70,000 angstroms,
75,000 angstroms, 80,000 angstroms, 85,000 angstroms, 90,000
angstroms, 95,000 angstroms, 100,000 angstroms, or more. In a
preferred embodiment, a coating layer (e.g., an oxide layer) is
approximately at least 3,500 angstroms thick. In a particularly
preferred embodiment, a coating layer (e.g., an oxide layer) is
approximately at least 35,000 angstroms thick.
[0075] In accordance with preferred aspects of the present
invention, it is contemplated that antireflective or adsorptive
coatings can substitute for an oxide coating to attain a robust
fluorescence signal. Antireflective and/or adsorptive coatings are
known in the art and described in U.S. Pat. No. 6,156,149,
incorporated herein by reference in its entirety for all
purposes.
[0076] In certain aspects of the invention, a coating comprising a
silicon compound can be added to the substrates described herein.
Such coatings can be added to the substrate itself or to another
coating layer. Suitable silicon compounds are described in U.S.
Patent Appl. Pub. No. 20010027187, incorporated herein by reference
in its entirety for all purposes.
[0077] The use of a fluorophore in front of a reflecting surface
and the resulting interaction of standing waves as a function of
the distance to the reflector and the wavelength(s) of light is
described in Lambacher and Fromherz (1996) Appl. Phys. A 63:207;
Braun and Fromherz (1997) Appl. Phys. A 65:341; Braun and Fromherz
(1998) Phys. Rev. Lett. 81:5241; and Drexhage (1974) Prog. In
Optics XII:163, each of which is incorporated herein by reference
in its entirety for all purposes.
C. Derivatization
[0078] Following the optional step of cleaning and stripping of the
substrate surface and the addition of a coating layer, the surface
is derivatized to provide sites or functional groups on the
substrate surface for synthesizing the various polymer sequences on
that surface. In particular, derivatization provides reactive
functional groups, e.g., hydroxyl, carboxyl, amino groups or the
like, to which the first monomers in the polymer sequence may be
attached. In preferred aspects, the substrate surface is
derivatized using silane in either water or ethanol. Preferred
silanes include mono- and dihydroxyalkylsilanes, which provide a
hydroxyl functional group on the surface of the substrate. Also
preferred are aminoalkyltrialkoxysilanes which can be used to
provide the initial surface modification with a reactive amine
functional group. Particularly preferred are
3-aminopropyltriethoxysilane and 3-aminopropyltrimethoxysilane
("APS"). Derivatization of the substrate using these latter amino
silanes provides a linkage that is stable under synthesis
conditions and final deprotection conditions (for oligonucleotide
synthesis, this linkage is typically a phosphoramidate linkage, as
compared to the phosphodiester linkage where hydroxyalkylsilanes
are used). Additionally, this amino silane derivatization provides
several advantages over derivatization with hydroxyalkylsilanes.
For example, the aminoalkyltrialkoxysilanes are inexpensive and can
be obtained commercially in high purity from a variety of sources,
the resulting primary and secondary amine functional groups are
more reactive nucleophile than hydroxyl groups, the
aminoalkyltrialkoxysilanes are less prone to polymerization during
storage, and they are sufficiently volatile to allow application in
a gas phase in a controlled vapor deposition process.
[0079] Additionally, silanes can be prepared having protected or
"masked" hydroxyl groups and which possess significant volatility.
As such, these silanes can be readily purified by, e.g.,
distillation, and can be readily employed in gas-phase deposition
methods of silanating substrate surfaces. After coating these
silanes onto the surface of the substrate, the hydroxyl groups may
be deprotected with a brief chemical treatment, e.g., dilute acid
or base, which will not attack the substrate-silane bond, so that
the substrate can then be used for polymer synthesis. Examples of
such silanes include acetoxyalkylsilanes, such as
acetoxyethyltrichlorosilane, acetoxypropyltrimethoxysilane, which
may be deprotected after application using, e.g., vapor phase
ammonia and methylamine or liquid phase aqueous or ethanolic
ammonia and alkylamines. Epoxyalkylsilanes may also be used, such
as glycidoxypropyltrimethoxysilane which may be deprotected using,
e.g., vapor phase HCl, trifluoroacetic acid or the like, or liquid
phase dilute HCl.
[0080] The physical operation of silanation of the substrate
generally involves dipping or otherwise immersing the substrate in
the silane solution. Following immersion, the substrate is
generally spun as described for the substrate stripping process,
i.e., laterally, to provide a uniform distribution of the silane
solution across the surface of the substrate. This ensures a more
even distribution of reactive functional groups on the surface of
the substrate. Following application of the silane layer, the
silanated substrate may be baked to polymerize the silanes on the
surface of the substrate and improve the reaction between the
silane reagent and the substrate surface. Baking typically takes
place at temperatures in the range of from 90.degree. C. to
120.degree. C., with 110.degree. C. being most preferred, for a
time period of from about 1 minute to about 10 minutes, with 5
minutes being preferred.
[0081] In alternative aspects, as noted above, the silane solution
may be contacted with the surface of the substrate using controlled
vapor deposition methods or spray methods. These methods involve
the volatilization or atomization of the silane solution into a gas
phase or spray, followed by deposition of the gas phase or spray
upon the surface of the substrate, usually by ambient exposure of
the surface of the substrate to the gas phase or spray. Vapor
deposition typically results in a more even application of the
derivatization than simply immersing the substrate into the
solution.
[0082] The efficacy of the derivatization process, e.g., the
density and uniformity of functional groups on the substrate
surface, may generally be assessed by adding a fluorophore which
binds the reactive groups, e.g., a fluorescent phosphoramidite such
as FLUOREPRIME.TM. from Pfizer Inc. (New York, N.Y.),
FLUOREDITE.TM. from Millipore Corp. (Billerica, Mass.) or FAM, and
ascertaining the relative fluorescence across the surface of the
substrate.
D. Synthesis
[0083] General methods for the solid phase synthesis of a variety
of polymer types have been previously described. Methods of
synthesizing arrays of large numbers of polymer sequences,
including oligonucleotides and peptides, on a single substrate have
also been described. See U.S. Pat. Nos. 5,143,854 and 5,384,261 and
Published PCT Application No WO 92/10092, each of which is
incorporated herein by reference in its entirety for all
purposes.
[0084] The synthesis of oligonucleotides on the surface of a
substrate can be carried out using light directed methods as
described in, e.g., U.S. Pat. Nos. 5,143,854 and 5,384,261 and
Published PCT Application No WO 92/10092, or mechanical synthesis
methods as described in U.S. Pat. No. 5,384,261 and Published PCT
Application No. 93/09668, each of which is incorporated herein by
reference in its entirety for all purposes. In preferred
embodiments, photochemical steps, and in particular photoacid
generator (PAG) synthesis techniques are preformed "front side" on
a substrate having a front side and a back side. In particular,
these light-directed or photolithographic synthesis methods involve
a photolysis step and a chemistry step. The substrate surface,
prepared as described herein, comprises functional groups on its
surface. These functional groups are protected by photolabile
protecting groups, i.e., "photoprotected," also as described
herein. During the photolysis step, portions of the surface of the
substrate are exposed to light or other activators to activate the
functional groups within those portions, i.e., to remove
photoprotecting groups. The substrate is then subjected to a
chemistry step in which chemical monomers that are photoprotected
at one or more functional groups are then contacted with the
surface of the substrate. These monomers bind to the activated
portion of the substrate through an unprotected functional
group.
[0085] Subsequent activation and coupling steps couple monomers to
other preselected regions, which may overlap with all or part of
the first region. The activation and coupling sequence at each
region on the substrate determines the sequence of the polymer
synthesized thereon. In particular, light is shown through the
photolithographic masks which are designed and selected to expose
and thereby activate a first particular preselected portion of the
substrate. Monomers are then coupled to all or part of this portion
of the substrate. The masks used and monomers coupled in each step
can be selected to produce arrays of polymers having a range of
desired sequences, each sequence being coupled to a distinct
spatial location on the substrate which location also dictates the
polymer's sequence. The photolysis steps and chemistry steps are
repeated until the desired sequences have been synthesized upon the
surface of the substrate.
[0086] Basic strategy for light directed synthesis of
oligonucleotides on a VLSIPS.TM. Array is described in U.S. Patent
Appl. Pub. No. 20040105932, incorporated herein by reference in its
entirety for all purposes. Briefly, the surface of a substrate or
solid support, modified with photosensitive protecting groups is
illuminated through a photolithographic mask, yielding reactive
hydroxyl groups in the illuminated regions. A selected nucleotide,
typically in the form of a 3'-O-phosphoramidite-activated
deoxynucleoside (protected at the 5' hydroxyl with a photosensitive
protecting group), is then presented to the surface and coupling
occurs at the sites that were exposed to light. Following capping
and oxidation, the substrate is rinsed and the surface is
illuminated through a second mask, to expose additional hydroxyl
groups for coupling. A second selected nucleotide (e.g.,
5'-protected, 3'-O-phosphoramidite-activated deoxynucleoside) is
presented to the surface. The selective deprotection and coupling
cycles are repeated until the desired set of products is obtained
(Pease et al. (1994) Proc. Natl. Acad. Sci. USA 91:5022,
incorporated herein by reference in its entirety for all purposes.
Since photolithography is used, the process can be readily
miniaturized to generate high density arrays of oligonucleotide
probes. Furthermore, the sequence of the oligonucleotides at each
site is known. Such photolithographic methods are also described in
U.S. Pat. Nos. 5,143,854 and 5,489,678 and Published PCT
Application No. WO 94/10128 each of which is incorporated herein by
reference in its entirety for all purposes. In the large scale
processes of the present invention, it is typically preferred to
utilize photolithographic synthesis methods.
[0087] Using the above described methods, arrays may be prepared
having all polymer sequences of a given length which are composed
of a basis set of monomers. Such an array of oligonucleotides, made
up of the basis set of four nucleotides, for example, would contain
up to 4.sup.n oligonucleotides on its surface, where n is the
desired length of the oligonucleotide probe. For an array of 8-mer
or 10-mer oligonucleotides, such arrays could have upwards of about
65,536 and 1,048,576 different oligonucleotides, respectively.
Generally, where it is desired to produce arrays having all
possible polymers of length n, a simple binary mashing strategy can
be used, as described in U.S. Pat. No. 5,143,854, incorporated
herein by reference in its entirety for all purposes.
[0088] Alternate masking strategies can produce arrays of probes
which contain a subset of polymer sequences, i.e., polymers having
a given subsequence of monomers, but are systematically substituted
at each position with each member of the basis set of monomers. In
the context of oligonucleotide probes, these alternate synthesis
strategies may be used to lay down or "tile" a range of probes that
are complementary to, and span the length of a given known nucleic
acid segment. The tiling strategy will also include substitution of
one or more individual positions within the sequence of each of the
probe groups with each member of the basis set of nucleotides.
These positions are termed "interrogation positions." By reading
the hybridization pattern of the target nucleic acid, one can
determine if and where any mutations lie in the sequence, and also
determine what the specific mutation is by identifying which base
is contained within the interrogation position. Tiling methods and
strategies are discussed in substantial detail in U.S. patent
application Ser. No. 08/143,312 filed Oct. 26, 1993, and
incorporated herein by reference in its entirety for all
purposes.
[0089] Tiled arrays may be used for a variety of applications, such
as identifying mutations within a known oligonucleotide sequence or
"target." Specifically, the probes on the array will have a
subsequence which is complementary to a known nucleic acid
sequence, but wherein at least one position in that sequence has
been systematically substituted with the other three
nucleotides.
[0090] Use of photolabile protecting groups during polymer
synthesis has been previously reported, as described above.
Preferred photolabile protecting groups generally have the
following characteristics: they prevent selected reagents from
modifying the group to which they are attached; they are stable to
synthesis reaction conditions (that is, they remain attached to the
molecule); they are removable under conditions that minimize
potential adverse effects upon the structure to which they are
attached; and, once removed, they do not react appreciably with the
surface or surface bound oligomer. In some embodiments, liberated
byproducts of the photolysis reaction can be rendered non-reactive
toward the growing oligomer by adding a reagent that specifically
reacts with the byproduct.
[0091] The removal rate of the photolabile protecting groups
generally depends upon the wavelength and intensity of the incident
radiation, as well as the physical and chemical properties of the
protecting group itself. Preferred protecting groups are removed at
a faster rate and with a lower intensity of radiation. Generally,
photoprotecting groups that undergo photolysis at wavelengths in
the range from 300 nm to approximately 450 nm are preferred.
[0092] Generally, photolabile or photosensitive protecting groups
include ortho-nitrobenzyl and ortho-nitrobenzyloxycarbonyl
protecting groups. The use of these protecting groups has been
proposed for use in photolithography for electronic device
fabrication (see, e.g., Reichmanis et al. (1985) J. Polymer Sci.
Polymer Chem. Ed. 23:1, incorporated herein by reference in its
entirety for all purposes).
[0093] Examples of additional photosensitive protecting groups
which may be used in the light directed synthesis methods herein
described, include, e.g., 1-pyrenylmethyloxycarbonyl,
.alpha.,.alpha.-dimethyl-3,5-dimethoxybenzyloxycarbonyl,
4-methoxyphenacyloxycarbonyl, 3'-methoxybenzoinyloxycarbonyl,
3',5'-dimethoxybenzoinyloxycarbonyl
2',3'-dimethoxy-benzoinyloxycarbonyl,
2',3'-(methylenedioxy)benzoinyloxycarbonyl,
N-(5-bromo-7-nitroindolinyl)carbonyl
3,5-dimethoxybenzyloxycarbony-1,
.alpha.-(2-methylene-anthraquinone)oxycarbonyl and the like.
[0094] Particularly preferred photolabile protecting groups for
protection of either the 3' or 5'-hydroxyl-groups of nucleotides or
nucleic acid polymers include the o-nitrobenzyl protecting groups
described in Published PCT Application No. WO 92/10092,
incorporated herein by reference in its entirety for all purposes.
These photolabile protecting groups include, e.g.,
(2-nitro-naphthalen-1-yl)-phenylmethylcarbonyl (NNPOC),
94'-methoxy-3-nitro-biphenyl-4-yl)-phenylmethylcarbonyl (MBPMOC),
nitroveratryloxycarbonyl (NVOC), nitropiperonyl oxycarbonyl (NPOC),
.alpha.-methyl-nitroveratryloxycarbonyl (MeNVOC),
.alpha.-methyl-nitropiperonyloxycarbonyl (MeNPOC),
1-pyrenylmethyloxycarbonyl (PYMOC), and the benzylic forms of each
of these (i.e., NNP, MBPM, NV, NP, MeNV, MeNP and PYM,
respectively), with MeNPOC, NPPOC and MBPMOC being most
preferred.
[0095] Protection strategies may be optimized for different
phosphoramidite nucleosides to enhance synthesis efficiency.
Examples of such optimized synthesis methods are reported in, e.g.,
U.S. patent application Ser. No. 08/445,332 filed May 19, 1995,
incorporated herein by reference in its entirety for all purposes.
Generally, these optimization methods involve selection of
particular protecting groups for protection of the O.sup.6 group of
guanosine, which can markedly improve coupling efficiencies in the
synthesis of guanosine containing oligonucleotides. Similarly,
selection of the appropriate protecting group for protection of the
N.sup.2 group of guanosine can also result in such an improvement
in the absence of protection of the O.sup.6 group. For example,
suitable protecting groups for protection of the N.sup.2 group,
where the O.sup.6 group is also protected, include, e.g., mono- or
diacyl protecting groups, triarylmethyl protecting groups, e.g.,
DMT and MMT, and amidine type protecting groups, e.g.,
N,N-dialkylformamidines. Particularly preferred protecting groups
for the N.sup.2 group include, e.g., DMT, DMF, PAC, Bz and Ibu.
[0096] Protection of the O.sup.6 group will generally be carried
out using carbamate protecting groups such as --C(O)NX.sub.2, where
X is alkyl, or aryl; or the protecting group --CH.sub.2CH.sub.2Y,
where Y is an electron withdrawing group such as cyano,
p-nitrophenyl, or alkyl- or aryl-sulfonyl; and aryl protecting
groups. In a particularly preferred embodiment, the O.sup.6 group
is protected using a diphenylcarbamoyl protecting group (DPC).
[0097] Alternatively, improved coupling efficiencies may be
achieved by selection of an appropriate protecting group for only
the N.sup.2 group. For example, where the N.sup.2-PAC protecting
group is substituted with an Ibu protecting group, a substantial
improvement in coupling efficiency is seen, even without protection
of the O.sup.6 group.
[0098] A variety of modifications can be made to the
above-described synthesis methods. For example, in some
embodiments, it may be desirable to directly transfer or add
photolabile protecting groups to functional groups, e.g., NH.sub.2,
OH, SH or the like, on a solid support. For these methods,
conventional peptide or oligonucleotide monomers or building blocks
having chemically removable protecting groups are used instead of
monomers having photoprotected functional groups. In each cycle of
the synthesis procedure, the monomer is coupled to reactive sites
on the substrate, e.g., sites deprotected in a prior photolysis
step. The protecting group is then removed using conventional
chemical techniques and replaced with a photolabile protecting
group prior to the next photolysis step.
[0099] A number of reagents will effect this replacement reaction.
Generally, these reagents will have the following generic
structure:
##STR00002##
[0100] where R.sub.1 is a photocleavable protecting group and X is
a leaving group, i.e., from the parent acid HX. The stronger acids
typically correspond to better leaving groups and thus, more
reactive acylating agents.
[0101] Examples of suitable reagents are described in U.S. Patent
Appl. Pub. No. 20040105932, incorporated herein by reference in its
entirety for all purposes.
[0102] Conditions for carrying out this transfer are similar to
those used for coupling reaction in solid phase peptide synthesis,
or for the capping reaction in solid phase oligonucleotide
synthesis. The solid phase amine, hydroxyl or thiol groups are
exposed to a solution of the protecting group coupled to the
leaving group, e.g., MeNPOC-X in a non-nucleophilic organic
solvent, e.g., DMF, NMP, DCM, THF, ACN, and the like, in the
presence of a base catalysts, such as pyridine, 2,6-lutidine, TEA,
DIEA and the like. In cases where acylation of surface groups is
less efficient under these conditions, nucleophilic catalysts such
as DMAP, NMI, HOBT, HOAT and the like, may also be included to
accelerate the reaction through the in situ generation of more
reactive acylating agents. This would typically be the case where a
derivative is preferred for its longer term stability in solution,
but is not sufficiently reactive without the addition of one or
more of the catalysts mentioned above. On automated synthesizers,
it is generally preferable to choose a reagent which can be stored
for longer terms as a stable solution and then activated with the
catalysts only when needed, i.e., in the reactor system flow cell,
or just prior to the addition of the reagent to the flow cell.
[0103] In addition to the protection of amine groups and hydroxyl
groups in peptide and oligonucleotide synthesis, the reagents and
methods described herein may be used to transfer photolabile
protecting groups directly to any nucleophilic group, either
tethered to a solid support or in solution.
E. Individual Processing
Flow Cell/Reactor System
[0104] In one embodiment, the substrate preparation process of the
present invention is performed in a single unit operation. In this
embodiment, the substrate wafer is mounted in a flow cell during,
for example, both the photolysis and chemistry or monomer addition
steps. In particular, the substrate is mounted in a reactor system
that allows for the photolytic exposure of the synthesis surface of
the substrate to activate the functional groups thereon. Solutions
containing chemical monomers are then introduced into the reactor
system and contacted with the synthesis surface, where the monomers
can bind with the active functional groups on the substrate
surface. The monomer containing solution is then removed from the
reactor system, and another photolysis step is performed, exposing
and activating different selected regions of the substrate surface.
This process is repeated until the desired polymer arrays are
created.
[0105] Reactor systems and flow cells that are particularly suited
for the combined photolysis/chemistry process include those
described in, e.g., U.S. Pat. No. 5,424,186 and U.S. Patent Appl.
Pub. 20040105932, each of which is incorporated herein by reference
in its entirety for all purposes.
Photolysis
[0106] As described above, photolithographic methods can be used to
activate selected regions on the surface of the substrate.
Specifically, functional groups on the surface of the substrate or
present on growing polymers on the surface of the substrate, are
protected with photolabile protecting groups. Activation of
selected regions of the substrate is carried out by exposing
selected regions of the substrate surface to activation radiation,
e.g., light within the effective wavelength range, as described
previously. Selective exposure is typically carried out by shining
a light source through a photolithographic mask. Alternate methods
of exposing selected regions may also be used, e.g., fiber optic
faceplates and the like.
[0107] Because the individual feature sizes on the surface of the
substrate prepared according to the processes described herein can
typically range as low as 1-10 the effects of reflected or
refracted light at the surface of the substrate can have
significant effects upon the ability to expose and activate
features of this size. One method of reducing the occurrence of
reflected light is to incorporate a light absorptive material as
the back surface of the flow cell, as described above. Refraction
of the light as it enters the flow cell can also result in a loss
in feature resolution at the synthesis surface of the substrate
resulting from refraction and reflection. To alleviate this
problem, during the photolysis step, it is generally desirable to
fill the flow cell with an index matching fluid ("IMF") to match
the refractive index of the substrate, thereby reducing refraction
of the incident light and the associated losses in feature
resolution. The index matching fluid will typically have a
refractive index that is close to that of the substrate. Typically,
the refractive index of the IMF will be within about 10% that of
the substrate, and preferably within about 5% of the refractive
index of the substrate. Refraction of the light entering the flow
cell, as it contacts the interface between the substrate and the
IMF is thereby reduced. Where synthesis is being carried out on,
e.g., a silica substrate, a particularly preferred IMF is dioxane
which has a refractive index roughly equivalent to the silica
substrate.
[0108] The light source used for photolysis is selected to provide
a wavelength of light that is photolytic to the particular
protecting groups used, but which will not damage the forming
polymer sequences. Typically, a light source which produces light
in the UV range of the spectrum will be used. For example, in
oligonucleotide synthesis, the light source typically provides
light having a wavelength above 340 nm to effect photolysis of the
photolabile protecting groups without damaging the forming
oligonucleotides. This light source is generally provided by a
Hg-Arc lamp employing a 340 nm cut-off filter (i.e., passing light
having a wavelength greater than 340-350 nm). Typical photolysis
exposures are carried out at from about 6 to about 10 times the
exposed half-life of the protecting group used, with from 8-10
times the half-life being preferred. For example, MeNPOC, a
preferred photolabile protecting group, has an exposed half-life of
approximately 6 seconds, which translates to an exposure time of
approximately 36 to 60 seconds.
[0109] Photolithographic masks used during the photolysis step
typically include transparent regions and opaque regions, for
exposing only selected portions of the substrate during a given
photolysis step. Typically, the masks are fabricated from glass
that has been coated with a light-reflective or absorptive
material, e.g., a chrome layer. The light-reflective or absorptive
layer is etched to provide the transparent regions of the mask.
These transparent regions correspond to the regions to be exposed
on the surface of the substrate when light is shown through the
mask.
[0110] In general, it is desirable to produce arrays with smaller
feature sizes, allowing the incorporation of larger amounts of
information in a smaller substrate area, allowing interrogation of
larger samples, more definitive results from an interrogation and
greater possibility of miniaturization. Alternatively, by reducing
feature size, one can obtain a larger number of arrays, each having
a given number of features, from a single substrate wafer. The
result is substantially higher product yields for a given process.
This technique, generally referred to as "die shrinking" is
commonly used in the semiconductor industry to enhance product
outputs or to reduce chip sizes following a over-sized test run of
a manufacturing process.
[0111] In seeking to reduce feature size, it is important to
maximize the contrast between the regions of the substrate exposed
to light during a given photolysis step, and those regions which
remain dark or are not exposed. By "contrast" is meant the
sharpness of the line separating an exposed region and an unexposed
region. For example, the gradient of activated to non-activated
groups running from an activated or exposed region to a non-exposed
region is a measure of the contrast. Where the gradient is steep,
the contrast is high, while a gradual gradient indicates low or
poor contrast. One cause of reduced contrast is "bleed-over" from
exposed regions to non-exposed regions during a particular
photolysis step. In certain embodiments, contrast between features
is enhanced through the front side exposure of the substrate. Front
side exposure reduces effects of diffraction or divergence by
allowing the mask to be placed closer to the synthesis surface.
Additionally, and perhaps more importantly, refractive effects from
the light passing through the substrate surface prior to exposure
of the synthesis surface are also reduced or eliminated by front
side exposure.
[0112] Contrast between features may also be enhanced using a
number of other methods. For example, the level of contrast
degradation between two regions generally increases as a function
of the number of differential exposures or photolysis steps between
the two regions, i.e., incidences where one region is exposed while
the other is not. The greater the number of these incidences, the
greater the opportunity for bleed-over from one region to the other
during each step and the lower the level of contrast between the
two regions. Translated into sequence information, it follows that
greater numbers of differences between polymers synthesized in
adjacent regions on a substrate can result in reduced contrast
between the regions. Namely, the greater the number of differences
in two polymer sequences, the greater the number of incidences of a
region bearing the first polymer being exposed while the other was
not. These effects are termed "edge" effects as they generally
occur at the outer edges of the feature.
[0113] It is thus desirable to minimize these edge effects to
enhance contrast in synthesis. Accordingly, in one aspect, the
present invention provides a method of enhancing contrast by
reducing the number of differential synthesis/photolysis steps
between adjacent polymer sequence containing regions throughout an
array.
[0114] One method of edge minimization is to divide the polymers to
be sequenced into blocks of related polymers, leaving blank lanes
between the blocks to prevent bleed-over into other blocks. While
this method is effective in reducing edge effects, it requires the
creation of a specific algorithm for each new tiling strategy. That
is, the layout of each block in terms of probe location will depend
upon the tiled sequence. In one aspect, the present invention
provides methods for aligning polymer synthesis steps on an array
whereby the number of differential synthesis steps is reduced,
and/or the syntheses in adjacent regions of the array are optimized
for similarity. An example of a typical photolysis synthesis
strategy is set forth in U.S. Patent Appl. Pub. No. 20040105932,
incorporated herein by reference in its entirety for all
purposes.
Photo Acid Generator and Acid Scavenger
[0115] One embodiment of the present invention includes a
photochemical amplification method wherein photon radiation signals
are converted into chemical signals in a manner that increases the
effective quantum yield of the photon in the desired reaction. The
use of photochemical amplification in methods of synthesizing
patterned arrays (PASPA) is particularly advantageous since the
time and the intensity of irradiation required to remove protective
groups is decreased relative to known direct photochemical methods.
Additionally, photoacid generators (PAG) generate acid directly
upon radiation to remove protecting groups.
[0116] In general, radiation signals are detected by a catalyst
system including, for example, a photo activated catalyst (PAC).
The catalyst activates an enhancer, which increases the effective
quantum yield of the photons in subsequent chemical reactions. Such
subsequent reactions include the removal of protective groups in
the synthesis of patterned arrays. It is desirable to remove all
the protecting groups in a very precise location without removing
protecting groups outside of the desired location. To prevent
removal of protective groups in undesirable locations, a catalyst
scavenger in some cases may be added but is not necessary to
compete for the catalyst, thus enabling the user to more
specifically define the area effected by the radiation signals.
[0117] In certain embodiments, a photo activated acid catalyst
(PAAC) is irradiated. The resulting acid produced from the PAAC
activates an enhancer to undergo an acid-catalyzed reaction to
itself produce an acid that removes acid labile protecting groups
from a linker molecule or synthesis intermediate. The combination
of PACs and enhancers converts and amplifies the photon signal
irradiated on the surface of the substrate. Because of the
amplification, the effective quantum yield of the radiation
directed at the surface of the substrate is much larger than one,
resulting in high sensitivity.
[0118] One way of controlling acid catalyst "bleed-over" is the
addition of an acid scavenger which serves to soak up the acid
catalyst in competition with the photo activation reaction.
Adjusting the concentration of acid catalyst aids in fine tuning
the area in which the protecting groups are removed.
[0119] According to one embodiment of the present invention, linker
molecules having reactive functional groups protected by protecting
groups are provided on the surface of a substrate. A catalyst
system including a PAC and an enhancer are also provided on the
surface. In some embodiments, an acid catalyst scavenger may also
be added. A set of selected regions on the surface of the substrate
is exposed to radiation using well-known lithographic methods as
discussed herein (See Thompson et al. (1994) American Chemical
Society, 1994:212, incorporated herein by reference in its entirety
for all purposes).
[0120] The PAC catalyst activated by the region-selective
irradiation discussed above acts to initiate a reaction of the
enhancer. The enhancer produces at least one product that removes
the protecting groups from the linker molecules in the first
selected regions. Preferably, the enhancer is capble of removing
protective groups in a catalytic manner. In some cases an acid
scavenger may be added to react with the acid catalyst, limiting
the amount of acid catalyst available to react with the enhancer.
The substrate is then washed or otherwise contacted with a first
monomer that reacts with exposed functional groups on the linker
molecules. Those bound monomers are termed first-bound
monomers.
[0121] A second set of selected regions is, thereafter, exposed to
radiation. The radiation-initiated reactions remove the protecting
groups on molecules in the second set of selected regions, i.e.,
the linker molecules and the first-bound monomers. The substrate is
then contacted with a second monomer containing a removable
protective group for reaction with exposed functional groups. This
process is repeated to selectively apply monomers until polymers of
a desired length and desired chemical sequence are obtained.
Protective groups are then optionally removed and the sequence is,
thereafter, optionally capped. Side chain protective groups, if
present, are also optionally removed.
[0122] In one preferred embodiment, the mononomer is a
2'-deoxynucleoside phosphoramidite containing an acid removable
protecting group at its 5' hydroxyl group. In an alternate
embodiment, the protecting group is present at the 3' hydroxyl
group if synthesis of the polynucleotide is from the 5' to 3'
direction. The nucleoside phosphoramidite is represented by the
following formula:
##STR00003##
[0123] wherein the base is adenine, guanine, thymine, cytosine or
any other nucleobase analog; R.sub.1 is a protecting group which
makes the 5' hydroxyl group unavailable for reaction and includes
dimethoxytrityl, MeNPOC, tert-butyloxycarbonyl or any of the
protecting groups previously identified; R.sub.2 is cyanoethyl,
methyl, t-butyl, trimethylsilyl and the like; R.sub.3 and R4 are
isopropyl, cyclohexone and the like; and R.sub.5 is hydrogen,
NR'R'', OR, SR, CRR'R'', or OSi(R''').sub.3 wherein R, R', R'', and
R''' are hydrogen, alkyl and the like. Exocyclic amines present on
the bases can also be protected with acyl protecting groups such as
benzoyl, isobutyryl, phenoxyacetyl and the like. The linker
molecule contains an acid- or base-removable protecting group.
Useful linker molecules are well known to those skilled in the art
and representative examples include oligo ethers such as
hexaethylene glycol, oligomers of nucleotides, esters, carbonates,
amides and the like. Useful protecting groups include those
previously listed and others known to those skilled in the art.
[0124] In another preferred embodiment, the monomer is an amino
acid containing an acid- or base-removable protecting group at its
amino or carboxy terminus and the linker molecule terminates in an
amino or carboxy acid group bearing an acid- or base removable
protecting group. Protecting groups include tert-butyloxycarbonyl,
9-fluorenylmethyloxycarbonyl, and any of the protective groups
previously mentioned and others known to those skilled in the
art.
[0125] In a preferred embodiment the catalyst scavenger may be an
acid scavenger such as an amine and more specifically may be
trioctylamine or 2,5-di-tertbutylanaline. Other acid scavengers
include carboxylate salts and hydroxides. See, e.g. Huang (1999)
Proc. SPIE-Int. Soc. Opt. Eng. 3678:1040, incorporated herein by
reference in its entirety for all purposes. Those of skill in the
art will be familiar with other acid scavengers which will be
appropriate for the present invention.
[0126] In another preferred embodiment, the catalyst scavenger may
be a base scavenger such as acetic acid or trichloro acetic acid.
Other base scavengers include phosphoric acid, sulfuric acid or any
other carboxylic acid. Care should be taken to chose a base
scavenger which will not interfere with or destroy the monomer.
Those of skill in the art will be familiar with other base
scavengers which will be appropriate for the present invention.
[0127] It is apparent to those skilled in the art that
photochemically amplified radiation-based activation is not limited
to photo activated enhancers or catalysts or to acid or base
production cascades. Various compounds or groups can produce
catalysts or enhancers in response to radiation exposure.
Non-limiting examples include photogeneration of radicals using
diphenylsulfide, benzoylperoxide, 2,2'-azobis(butyronitrile),
benzoin and the like; cations such as triarylsulfonium salts,
diaryl iodonium salts and the like; and anions. Furthermore, it is
apparent to those of skill in the art that the catalyst scavengers
are not limited to acid or base scavengers but may include any
other compound which will interfere with the catalysts' ability to
interact with the enhancer.
[0128] In a preferred embodiment, the catalyst and catalyst
scavenger are capable of engaging in a cyclic reaction. For
example, a compound X comprises subcompound Y which is capable of
acting as a catalyst and subcompound Z which is capable of acting
as a catalyst scavenger. Compound X is capable of entering into an
excited state after exposure to radiation. During this excited
state the subcompounds Y and Z separate and subcompound Y is free
to catalyze removal of protecting groups. In a further preferred
embodiment, the subcompounds Y and Z are capable of remaining in
this excited state for only a very short period of time. This time
period may be from between a few nanoseconds to a few milliseconds.
After the time period lapses, subcompounds Y and Z are free to
interact with one another once again forming compound X Exposure to
radiation may then initiate another cycle. In a preferred
embodiment, compound X is very stable prior to exposure to
radiation, and only capable of interacting with other molecules
during the excited state.
[0129] The selection of radiation sources is based upon the
sensitivity spectrum of the compound to be irradiated. Potential
damage to synthesis substrates, intermediates, or products is also
considered. In some preferred embodiments, the radiation could be
ultraviolet (UV), infrared (IR), or visible light. In a specific
embodiment, the radiation source is a light beam with a wavelength
in the range of from 190-500 nm, preferably from 250-450 nm, more
preferably from 365-400 nm. Specific radiation wavelengths include
193 nm, 254 nm, 313 nm, 340 nm, 365 nm, 396 nm, 413 nm, 436 nm, and
500 nm. Suitable light sources include high pressure mercury arc
lamps and are readily commercially available from Oriel, OAI,
Cannon, A-B Manufacturing and the like. In embodiments utilizing
the catalytic system, the sensitivity spectrum of the RAC can be
altered by providing radiation sensitizers. The energy of the
sensitizer must be matched to the PAC and include
2-ethyl-9,10-dimethoxy-anthracene, perylene, phenothiazine,
xanthone and the like. Many radiation sensitizers are known to
those skilled in the art and include those previously mentioned. It
is to be understood that one of ordinary skill in the art will be
able to readily identify additional radiation sensitizers based
upon the present disclosure.
[0130] In addition to the foregoing, aspects of the invention
include additional methods which can be used to generate an array
of oligonucleotides on a single substrate are described in U.S.
Pat. Nos. 5,677,195 and 5,384,261, and in PCT Publication No. WO
93/09668, each of which is incorporated herein by reference in its
entirety for all purposes. In the methods disclosed in these
applications, reagents are delivered to the substrate by either (1)
flowing within a channel defined on predefined regions or (2)
"spotting" on predefined regions or (3) through the use of
photoresist. However, other approaches, as well as combinations of
spotting and flowing, may be employed. In each instance, certain
activated regions of the substrate are mechanically separated from
other regions when the monomer solutions are delivered to the
various reaction sites.
[0131] In one aspect, a typical "flow channel" method is applied to
the compounds and libraries of the present invention, and can
generally be described as follows. Diverse polymer sequences are
synthesized at selected regions of a substrate or solid support by
forming flow channels on a surface of the substrate through which
appropriate reagents flow or in which appropriate reagents are
placed. For example, assume a monomer "A" is to be bound to the
substrate in a first group of selected regions. If necessary, all
or part of the surface of the substrate in all or a part of the
selected regions is activated for binding by, for example, flowing
appropriate reagents through all or some of the channels, or by
washing the entire substrate with appropriate reagents. After
placement of a channel block on the surface of the substrate, a
reagent having the monomer A flows through or is placed in all or
some of the channel(s). The channels provide fluid contact to the
first selected regions, thereby binding the monomer A on the
substrate directly or indirectly (via a spacer) in the first
selected regions.
[0132] Thereafter, a monomer B is coupled to second selected
regions, some of which may be included among the first selected
regions. The second selected regions will be in fluid contact with
a second flow channel(s) through translation, rotation, or
replacement of the channel block on the surface of the substrate;
through opening or closing a selected valve; or through deposition
of a layer of chemical or photoresist. If necessary, a step is
performed for activating at least the second regions. Thereafter,
the monomer B is flowed through or placed in the second flow
channel(s), binding monomer B at the second selected locations. In
this particular example, the resulting sequences bound to the
substrate at this stage of processing will be, for example, A, B,
and AB. The process is repeated to form a vast array of sequences
of desired length at known locations on the substrate.
[0133] After the substrate is activated, monomer A can be flowed
through some of the channels, monomer B can be flowed through other
channels, a monomer C can be flowed through still other channels,
etc. In this manner, many or all of the reaction regions are
reacted with a monomer before the channel block must be moved or
the substrate must be washed and/or reactivated. By making use of
many or all of the available reaction regions simultaneously, the
number of washing and activation steps can be minimized.
[0134] One of skill in the art will recognize that there are
alternative methods of forming channels or otherwise protecting a
portion of the surface of the substrate. For example, according to
some embodiments, a protective coating such as a hydrophilic or
hydrophobic coating (depending upon the nature of the solvent) is
utilized over portions of the substrate to be protected, sometimes
in combination with materials that facilitate wetting by the
reactant solution in other regions. In this manner, the flowing
solutions are further prevented from passing outside of their
designated flow paths.
[0135] In another aspect, the "spotting" methods of preparing
compounds and libraries of the present invention can be implemented
in much the same manner as the flow channel methods. For example, a
monomer A can be delivered to and coupled with a first group of
reaction regions which have been appropriately activated.
Thereafter, a monomer B can be delivered to and reacted with a
second group of activated reaction regions. Unlike the flow channel
embodiments described above, reactants are delivered by directly
depositing (rather than flowing) relatively small quantities of
them in selected regions. In some steps, of course, the entire
substrate surface can be sprayed or otherwise coated with a
solution. In preferred embodiments, a dispenser moves from region
to region, depositing only as much monomer as necessary at each
stop. Typical dispensers include a micropipette to deliver the
monomer solution to the substrate and a robotic system to control
the position of the micropipette with respect to the substrate. In
other embodiments, the dispenser includes a series of tubes, a
manifold, an array of pipettes, or the like so that various
reagents can be delivered to the reaction regions
simultaneously.
F. Assembly of Probe Array Cartridges
[0136] Following synthesis, final dcprotection and other finishing
steps, e.g. polymer coat removal where necessary, the substrate
wafer can be assembled for use as individual substrate segments.
Assembly typically employs the steps of separating the substrate
wafer into individual substrate segments, and inserting or
attaching these individual segments to a housing which includes a
reaction chamber in fluid communication with the front surface of
the substrate segment, e.g., the surface having the polymers
synthesized thereon.
[0137] Methods of separating and packaging substrate wafers are
described in substantial detail in Published PCT Application No.
95/33846, which is hereby incorporated herein by reference in its
entirety for all purposes.
[0138] Typically, the arrays are synthesized on the substrate wafer
in a grid pattern, with each array being separated from each other
array by a blank region where no compounds have been synthesized.
These separating regions are termed "streets." The wafer typically
includes a number of alignment marks located in these streets.
These marks serve a number of purposes, including aligning the
masks during synthesis of the arrays as described above, separation
of the wafer into individual chips and placement of each chip into
its respective housing for subsequent use, which are both described
in greater detail below.
[0139] Generally, the substrate wafer can be separated into a
number of individual substrates using scribe and break methods that
are well known in the semiconductor manufacturing industry. For
example, well known scribe and break devices may be used for
carrying out the separation steps, e.g., a fully programmable
computer controlled scribe and break devices, such as a DX-III
Scriber-Breaker manufactured by Dynatex International (Santa Rosa,
Calif.), or the LCD-1 scriber/dicer manufactured by Loomis
Industries Inc. (St. Helena, Calif.). The steps typically involve
scribing along the desired separation points, e.g., between the
individual synthesized arrays on the substrate wafer surface,
followed by application of a breaking force along the scribe line.
For example, typical scribe and break devices break the wafer by
striking the bottom surface of the wafer along the scribe lines
with an impulse bar, or utilizing a three point beam substrate
bending operation. The shock from the impulse bar fractures the
wafer along the scribe line. Because the majority of force applied
by the impulse bar is dissipated along the scribe line, the device
is able to provide high breaking forces without exerting
significant force on the substrate itself, allowing separation of
the wafer without damaging the individual chips.
[0140] In alternative methods, the wafer may be separated into
individual segments by, e.g., sawing methods, such as those
described in U.S. Pat. No. 4,016,855, incorporated herein by
reference in its entirety for all purposes.
[0141] Once the wafer is separated into individual segments, these
segments may be assembled in a housing that is suited for the
particular analysis for which the array will be used. Examples of
methods and devices for assembling the substrate segments or arrays
in cartridges are described in, e.g., U.S. Pat. No. 5,945,334,
incorporated herein by reference in its entirety for all purposes.
Typically, the housing includes a body having a cavity disposed
within it. The substrate segment is mounted over the cavity on the
body such that the front side of the segment, e.g., the side upon
which the polymers have been synthesized, is in fluid communication
with the cavity. The bottom of the cavity may optionally include a
light absorptive material, such as a glass filter or carbon dye, to
prevent impinging light from being scattered or reflected during
imaging by detection systems. This feature improves the
signal-to-noise ratio of such systems by significantly reducing the
potential imaging of undesired reflected light.
[0142] The cartridge also typically includes fluid inlets and fluid
outlets for flowing fluids into and through the cavity. A septum,
plug, or other seal may be employed across the inlets and/or
outlets to seal the fluids in the cavity. The cartridge also
typically includes alignment structures, e.g., alignment pins,
bores, and/or an asymmetrical shape to ensure correct insertion
and/or alignment of the cartridge in the assembly devices,
hybridization stations, and reader devices. Example of certain
embodiments of cartridges are described in U.S. Patent Appl. Pub.
No. 20040105932, incorporated herein by reference in its entirety
for all purposes.
[0143] In a preferred embodiment, the bottom casing with selected
cavity dimensions may be removed from the middle and top casings,
and replaced with another bottom casing with different cavity
dimensions. This allows a user to attach a chip having a different
size or shape by changing the bottom casing, thereby providing ease
in using different chip sizes, shapes, and the like. Of course, the
size, shape, and orientation of the cavity will depend upon the
particular application. The body of the cartridge may generally be
fabricated from one or more parts made using a number of
manufacturing techniques. In preferred aspects, the cartridge is
fabricated from two or more injection molded plastic parts.
Injection molding enables the casings to be formed inexpensively.
Also, assembling the cartridge from two parts simplifies the
construction of various features, such as the internal channels for
introducing fluids into the cavity. As a result, the cartridges may
be manufactured at a relatively low cost.
[0144] The substrate segment may be attached to the body of the
cartridge using a variety of methods. In preferred aspects, the
substrate is attached using an adhesive. Preferred adhesives are
resistant to degradation under conditions to which the cartridge
will be subjected. In particularly preferred aspects, an
ultraviolet cured adhesive attaches the substrate segment to the
cartridge. Devices and methods for attaching the substrate segment
are described in Published PCT Application No. 95/33846,
incorporated herein by reference in its entirety for all purposes.
Particularly preferred adhesives are commercially available from a
variety of commercial sources, including Loctite Corp. (Irvine,
Calif.) and Dymax Corp. (Torrington, Conn.).
[0145] A variety of modifications can be incorporated in the
assembly methods and devices that are generally described herein,
and these too are outlined in greater detail in published PCT
Application No. 95/33846, incorporated herein by reference in its
entirety for all purposes.
[0146] Upon completion, the cartridged substrate will have a
variety of uses. For example, the cartridge can be used in a
variety of sequencing by hybridization ("SBH") methods, sequence
checking methods, diagnostic methods and the like. Arrays which are
particularly suited for sequence checking and SBH methods are
described in, e.g., U.S. patent application Ser. Nos. 08/505,919,
filed Jul. 24, 1995, 08/441,887, filed May 16 1995, 07/972,007,
filed Nov. 5, 1992, each of which is incorporated herein by
reference in its entirety for all purposes.
[0147] Typically, in carrying out these methods, the cartridged
substrate is mounted on a hybridization station where it is
connected to a fluid delivery system. The fluid delivery system is
connected to the cartridge by inserting needles into the inlet and
outlet ports through the septa disposed therein. In this manner,
various fluids are introduced into the cavity for contacting the
probes synthesized on the front side of the substrate segment,
during the hybridization process.
[0148] Usually, hybridization is performed by first exposing the
sample with a pre-hybridization solution. Next, the sample is
incubated under binding conditions for a suitable binding period
with a sample solution that is to be analyzed. The sample solution
generally contains a target molecule, e.g., a target nucleic acid,
the presence or sequence of which is of interest to the
investigator. Binding conditions will vary depending on the
application and are selected in accordance with the general binding
methods known including those referred to in: Maniatis et al
Molecular Cloning: A Laboratory Manual (1989), 2nd Ed., Cold Spring
Harbor, N.Y.; Berger and Kimmel, Methods in Enzymology, Volume 152,
Guide to Molecular Cloning Techniques (1987), Academic Press, Inc.,
San Diego, Calif.; Laboratory Techniques in Biochemistry and
Molecular Biology, Vol. 24: Hybridization With Nucleic Acid Probes,
P. Tijssen, ed. Elsevier, N.Y., (1993); and Young and Davis (1983)
Proc. Natl. Acad. Sci. USA 80:1194, each of which is incorporated
herein by reference in its entirety for all purposes. In certain
embodiments, the solution may contain about 1 M salt and about 1 to
50 nM targets. Optionally, the fluid delivery system includes an
agitator to improve mixing in the cavity, which shortens the
incubation period. Finally, the sample is washed with a buffer,
which may be 6.times. SSPE buffer, to remove the unbound targets.
In some embodiments, the cavity is filled with the buffer after
washing the sample.
[0149] Following hybridization and appropriate rinsing/washing, the
cartridged substrate may be aligned on a detection or imaging
system, such as those disclosed in U.S. Pat. Nos. 5,143,854 and
5,631,734, and U.S. patent application Ser. Nos. 08/465,782, filed
Jun. 6, 1995, and 08/456,598, filed Jun. 1, 1995, each of which is
incorporated herein by reference in its entirety for all purposes.
Such detection systems may take advantage of the cartridge's
asymmetry (i.e., non-flush edge) by employing a holder to match the
shape of the cartridge specifically. Thus, the cartridge is assured
of being properly oriented and aligned for scanning. The imaging
systems are capable of qualitatively analyzing the reaction between
the probes and targets. Based on this analysis, sequence
information of the targets is extracted. In accordance with a
preferred embodiment of the present invention, confocal
fluorescence scanning is conducted front side, since the excitation
light would otherwise be blocked by the substrate.
[0150] While the foregoing invention has been described in some
detail for purposes of clarity and understanding, it will be clear
to one skilled in the art from a reading of this disclosure that
various changes in form and detail can be made without departing
from the true scope of the invention. All publications and patent
documents cited in this application are incorporated by reference
in their entirety for all purposes to the same extent as if each
individual publication or patent document were so individually
denoted.
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