U.S. patent application number 10/357679 was filed with the patent office on 2003-09-11 for replicable probe array.
This patent application is currently assigned to SurModics, Inc. Invention is credited to Guire, Patrick E., Swanson, Melvin J..
Application Number | 20030170914 10/357679 |
Document ID | / |
Family ID | 22906652 |
Filed Date | 2003-09-11 |
United States Patent
Application |
20030170914 |
Kind Code |
A1 |
Guire, Patrick E. ; et
al. |
September 11, 2003 |
Replicable probe array
Abstract
A system for producing substantially identical specific binding
ligand (e.g., nucleic acid) probe arrays, for instance, by
preparing and replicating an original master array and/or by
providing a reusable assay array that is capable of being
regenerated. In one embodiment the system includes the preparation
and use of a) a master array surface having address sequences
immobilized in the form of a patterned, and optionally random,
array, b) a multi-ligand conjugate having a binding domain
complementary to an address sequence, a binding domain
complementary to a target sequence, and a third ligand for use in
forming (e.g., by binding or polymerization) the conjugates into or
onto the surface of assay array, which can be used with or upon
disassociation of the address and its complementary sequences.
Inventors: |
Guire, Patrick E.; (Eden
Prairie, MN) ; Swanson, Melvin J.; (Carver,
MN) |
Correspondence
Address: |
Attention: Mark T. Skoog
MERCHANT & GOULD P.C.
P.O. Box 2903
Minneapolis
MN
55402-0903
US
|
Assignee: |
SurModics, Inc
Eden Prairie
MN
|
Family ID: |
22906652 |
Appl. No.: |
10/357679 |
Filed: |
February 3, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10357679 |
Feb 3, 2003 |
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09240466 |
Jan 29, 1999 |
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6514768 |
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Current U.S.
Class: |
436/518 ;
435/6.11; 435/6.12 |
Current CPC
Class: |
Y10S 436/809 20130101;
B01J 2219/00722 20130101; B01J 2219/005 20130101; C12Q 1/6837
20130101; B01J 2219/00585 20130101; C40B 40/06 20130101; Y10S
977/849 20130101; B01J 2219/00527 20130101; B01J 2219/00605
20130101; B01J 2219/00637 20130101; B01J 2219/00612 20130101; B01J
2219/00626 20130101; B01J 19/0046 20130101; B01J 2219/00648
20130101; B01J 2219/00596 20130101; Y10S 436/807 20130101; B01J
2219/00659 20130101 |
Class at
Publication: |
436/518 ;
435/6 |
International
Class: |
C12Q 001/68; G01N
033/543 |
Claims
What is claimed is:
1. A system for replicating a specific binding ligand probe array,
the system comprising: a) a master array comprising a support
surface having a plurality of address ligands immobilized thereon
in the form of a patterned array; b) a plurality of multi-ligand
conjugates, each multi-ligand conjugate comprising a core having
attached thereto: (i) at least one molecule of a first ligand
binding domain, comprising a ligand selected to bind in a specific
manner with a particular address ligand of the master array, (ii)
at least one molecule of a second ligand binding domain, comprising
a ligand selected to bind in a specific manner with a target
ligand, and (iii) at least one molecule of a third ligand, selected
from the group consisting of binding ligands and polymerizable
groups; and c) an assay array support comprising a support surface
for the replicate array.
2. A system according to claim 1 wherein the address ligands, and
the first and second binding ligands each, independently, comprise
a nucleic acid.
3. A system according to claim 2 wherein the nucleic acids each
comprise a synthetic oligonucleotide.
4. A system according to claim 1 further comprising a linking
agent, the linking agent providing attachment of the address ligand
with the master array support surface.
5. A system according to claim 4 wherein the linking agent
comprises a micro-bead.
6. A system according to claim 1 wherein the first ligand binding
domain, second ligand binding domain and third ligand are
independently attached to the core of the multi-ligand
conjugate.
7. A system according to claim 1 wherein the binding ligands
comprise biotin derivatives.
8. A system according to claim 1 wherein the polymerizable groups
are selected from the group consisting of acrylic and vinyl
groups.
9. A system according to claim 1 wherein the assay array support is
coated with attachment sites for the third ligand.
10. A system according to claim 9 wherein the third ligand
comprises a binding ligand, and the attachment sites comprise
molecules of a binding partner specific for the third ligand.
11. A method for replicating a specific binding ligand probe array,
the method comprising: a) providing a master array comprising a
support surface having a plurality of address ligands immobilized
thereon; b) providing a plurality of multi-ligand conjugates, each
multi-ligand conjugate comprising a core having attached thereto:
(i) at least one molecule of a first ligand binding domain,
comprising a ligand selected to bind in a specific manner with a
particular address ligand of the master array, (ii) at least one
molecule of a second ligand binding domain, comprising a ligand
selected to bind in a specific manner with a target ligand, and
(iii) at least one molecule of a third ligand, selected from the
group consisting of binding ligands and polymerizable groups; c)
attaching the multi-ligand conjugates to the master array by
allowing the first ligand binding domains to bind in a specific
manner with complementary address ligands; d) providing an assay
array support comprising a support surface for the replicate array;
e) bringing the assay array support into sufficient proximity with
the master array, under conditions suitable to permit the attached
multi-ligand conjugates to attach to the assay array support; and
f) disassociating the bound complementary address ligand and first
ligand binding domain under conditions suitable to permit the assay
array support to be recovered and used.
12. A method according to claim 11, wherein the third ligand
comprises a polymerizable group, wherein the polymerizable groups
can be reacted to form a polymer film.
13. A method according to claim 11 wherein the address ligands, and
the first and second binding ligands each, independently, comprise
a nucleic acid.
14. A method according to claim 13 wherein the nucleic acids each
comprise a synthetic oligonucleotide.
15. A method according to claim 11 further comprising a linking
agent, the linking agent providing attachment of the address ligand
with the master array support surface.
16. A method according to claim 15 wherein the linking agent
comprises a micro-bead.
17. A method according to claim 11 wherein the first ligand binding
domain, second ligand binding domain and third ligand are
independently attached to the core of the multi-ligand
conjugate.
18. A method according to claim 11 wherein the binding ligands
comprise biotin derivatives.
19. A method according to claim 11 wherein the polymerizable groups
are selected from the group consisting of acrylic and vinyl
groups.
20. A method according to claim 11 wherein the assay array support
is coated with attachment sites for the third ligand.
21. A method according to claim 20 wherein the third ligand
comprises a binding ligand, and the attachment sites comprise
molecules of a binding partner specific for the third ligand.
22. A system for preparing a replicable array, in the form of a
reusable array, the system comprising: a) a master array comprising
a plurality of optical fibers, each optical fiber having a support
surface located at the distal end of the fiber; and b) a plurality
of oligonucleotide binding domains, each oligonucleotide binding
domain comprising an oligonucleotide sequence selected to bind in a
specific manner with a target ligand.
Description
TECHNICAL FIELD
[0001] The present invention relates to the immobilization of
specific binding ligands, such as nucleic acids and other ligands,
in a known spatial arrangement. In another aspect, the invention
relates to solid supports, such as oligonucleotide chips,
incorporating such nucleic acids. In yet another aspect, the
invention relates to photoreactive groups, to molecules and/or
surfaces derivatized with such groups, and to the attachment of
such molecules to support surfaces by the activation of such
groups.
BACKGROUND OF THE INVENTION
[0002] The development of oligonucleotide probe arrays, more
commonly known as "DNA chips" and "Gene Chip" (a registered
trademark of Affymetrix, Inc.), has made significant advances over
the past few years, and is becoming the center of ever-increasing
attention and heightened significance. See, for instance, Stipp,
D., Fortune, p.56, Mar. 31, 1997. See also Borman, S., C&EN,
p.42, Dec. 9, 1996, and Travis, J., Science News 151:144-145
(1997). These 2- or 3-cm square chips are capable of containing
tens of thousands to hundreds of thousands of immobilized
oligonucleotides, allowing researchers to witness for the first
time the behavior of thousands of genes acting in concert. DNA
chips are useful for observing unique gene expression patterns,
gauging the success of drug treatment, tailoring medications to
patients based upon their genetic makeup, sequencing genes, and
conducting research in the area of genetic medicine. See also,
"Microchip Arrays Put DNA on the Spot", R. Service, Science
282(5388):396-399, 16, Oct. 1998; and "Fomenting a Revolution, in
Miniature", I. Amato, Science 282(5388): 402-405, 16, Oct.
1998.
[0003] Typically, oligonucleotide probe arrays display specific
oligonucleotide sequences at precise locations in an information
rich format. In use, the hybridization pattern of a fluorescently
labeled nucleic acid target is used to gain primary structure
information for the target. This format can be applied to a broad
range of nucleic acid sequence analysis problems including pathogen
identification, forensic applications, monitoring mRNA expression
and de novo sequencing. See, for instance, Lipshutz, R. J., et al.,
BioTechniques 19(3):442-447 (1995). Such arrays sometimes need to
carry several tens of thousands, or even hundreds of thousands of
individual probes. The chips also need to provide a broad range of
sensitivities in order to detect sequences that can be expressed at
levels anywhere from 1 to 10,000 copies per cell.
[0004] A variety of approaches have been developed for the
fabrication and/or use of oligonucleotide probe arrays. See, for
instance, Weaver, et al. (WO 92/10092) which describes a synthetic
strategy for the creation of large scale chemical diversity on a
solid-phase support. The system employs solid-phase chemistry,
photolabile protecting groups and photolithography to achieve
light-directed, spatially addressable, parallel chemical synthesis.
Using the proper sequence of masks and chemical stepwise reactions,
a defined set of oligonucleotides can be constructed, each in a
predefined position on the surface of the array.
[0005] Using this technology, Affymetrix, Inc. (Santa Clara,
Calif.), has developed libraries of unimolecular, double-stranded
oligonucleotides on a solid support. See, for instance, U.S. Pat.
No. 5,770,722 which describes arrays containing oligonucleotides
from 4 to 100 nucleotides in length. The arrays comprise a solid
support, an optional spacer, a first oligonucleotide, a second
oligonucleotide that is complementary to the first, and a flexible
linker or probe. The libraries described are useful for screening
for such receptors as proteins, RNA or other molecules which bind
double-stranded DNA. Another array developed by Affymetrix is
described in U.S. Pat. No. 5,837,832. This reference describes
methods for making high-density arrays of oligonucleotide probes on
silica chips. The oligonucleotide probes are 9 to 20 nucleotides in
length and are synthesized directly on a solid support. The arrays
comprise oligonucleotide probes that are complementary to a section
of the reference sequence.
[0006] Synteni (Palo Alto, Calif.) produces arrays of cDNA by
applying polylysine to glass slides, followed by printing cDNA onto
the coated slides. The arrays are then exposed to UV light, in
order to crosslink the DNA with the polylysine. Unreacted
polylysine is then blocked by reaction with succinic anhydride.
These arrays, called "Gene Expression Microarrays" (GEM.TM.) are
used by labeling mRNA prepared from a normal cell with a
fluorescent dye, then labeling mRNA from an abnormal cell with a
fluorescent dye of a different color. These two labeled mRNA
molecules are simultaneously applied to the microarray, where they
competitively bind to the immobilized cDNA molecules. This two
color coding technique is used to identify the differences in gene
expression between two cell samples. (Heller, R. A., et al., Proc.
Natl. Acad. Sci. USA, 94:2150-2155 (1997)).
[0007] At least one group, Cantor, et al. (U.S. Pat. No.
5,795,714), describes methods for replicating arrays of probes
which are said to be useful for the large scale manufacture of
diagnostic aids. The patent includes a method for replicating an
array of single-stranded probes on a solid support comprising the
steps of:
[0008] a) synthesizing an array of nucleic acids each comprising a
non-variant sequence of length C at a 3'-terminus and a variable
sequence of length R at a 5'-terminus;
[0009] b) fixing the array to a first solid support;
[0010] c) synthesizing a set of nucleic acids each comprising a
sequence complementary to the non-variant sequence;
[0011] d) hybridizing the nucleic acids of the set to the
array;
[0012] e) enzymatically extending the nucleic acids of the set
using the variable sequences of the array as templates;
[0013] f) denaturing the set of extended nucleic acids; and
[0014] g) fixing the denatured nucleic acids of the set to a second
solid support to create the replicated array of single-stranded
probes.
[0015] On a separate subject, the assignee of the present invention
has previously described a variety of applications for the use of
photochemistry, and in particular, photoreactive groups, e.g., for
attaching polymers and other molecules to support surfaces. See,
for instance, U.S. Pat. Nos. 4,722,906, 4,979,959, 5,217,492,
5,512,329, 5,563,056, 5,637,460, and 5,714,360 and International
Patent Application Nos. PCT/US96/08797 (Virus Inactivating
Coatings), PCT/US96/07695 (Capillary Endothelialization), and
PCT/US97/05344 (Chain Transfer Agents).
[0016] In spite of the various developments to date, there remains
a need for methods and reagents that improve the immobilization of
nucleic acids onto a variety of support materials, e.g., in order
to form oligonucleotide probe arrays. What is clearly needed are
new and improved methods and reagents for reproducibly preparing
specific binding ligand (e.g., nucleic acid) arrays in a
cost-effective and efficient manner, while maintaining an accurate,
sensitive product.
SUMMARY OF THE INVENTION
[0017] The present invention, in one preferred embodiment, provides
a system for producing substantially identical specific binding
ligand (e.g., nucleic acid) probe arrays, for instance, by
preparing and replicating an original "master" array and/or by
providing a reusable assay array that is capable of being
regenerated. The present approach can be contrasted with the
traditional approach of separately and individually preparing each
probe array anew, without the efficiency of replicating a master
array or the flexibility of regenerating an array for subsequent
use. The present method can be adapted for use with conventional
arrays, in order to provide replicates thereof, but is preferably
used with a master array (and other components) specifically
designed for such purposes, in the manner described herein.
[0018] In such a preferred embodiment, the present invention
provides a method and system for reproducibly preparing an assay
array, the system comprising:
[0019] a) a master array comprising a support surface having a
plurality of "address" ligands (e.g., oligonucleotide sequences)
immobilized thereon, for instance, directly or via respective
linking agents, such as "micro-beads" or other suitable linkers,
the ligands being immobilized in the form of a patterned, and
optionally random, array;
[0020] b) a plurality of multi-ligand conjugates, each multi-ligand
conjugate comprising a core (e.g., molecular or solid) having
(preferably independently) attached thereto: (i) at least one
molecule of a first ligand (e.g., oligonucleotide) binding domain,
comprising a ligand selected to bind in a complementary manner to a
particular address ligand of the master array, (ii) at least one
molecule of a second ligand (e.g., oligonucleotide) binding domain,
comprising a ligand selected to bind in a complementary manner to a
characteristic ligand of a target nucleic acid sequence (e.g., a
gene and/or gene fragment), and (iii) at least one molecule of a
third (and preferably non-oligonucleotide) ligand, selected from
the group consisting of binding ligands and polymerizable groups
(e.g., acrylic or vinyl groups); and
[0021] c) an assay array support comprising a support surface for
the replicate array, e.g., coated with attachment sites for the
third ligand (such as, immobilized molecules of a corresponding
binding partner specific for the third ligand).
[0022] A corresponding preferred method of the invention, wherein
the address and target ligands are both nucleic acids, preferably
oligonucleotides, involves the steps of: (1) providing a master
array as described above, (2) attaching the multi-ligand conjugates
thereto, by allowing their respective oligonucleotide binding
domains to hybridize to the complementary address sequences of the
master array, (3) bringing the assay array support into sufficient
proximity with the master array, under conditions suitable to
permit the attached multi-ligand conjugates to attach to the assay
array support (e.g., by binding between the third ligands and the
corresponding attachment sites present upon the assay support
surface), and (4) disassociating the hybridized complementary
oligonucleotides under conditions suitable to permit the assay
array support to be recovered and used. The resulting assay array
comprises an assay array support having attached thereto a
plurality of multi-ligand conjugates (preferably still having
complementary target oligonucleotides thereon) present in a pattern
established by the master array.
[0023] In an alternative preferred embodiment, where the conjugate
includes a polymerizable group (e.g., in addition to the third
binding ligand, or in place of the third binding ligand), the
multi-ligand conjugates can be maintained in their oriented
positions upon the master array surface by polymerizing those
groups in situ, in order to form a polymeric backing sufficient to
permit the resultant polymerized layer to be supported and used,
e.g., transferred to an assay array support, while retaining the
spatial arrangement established by the master array addresses.
[0024] Once transferred to an assay array support, the second
oligonucleotide binding domains, in turn, can be used in a
conventional manner to determine the presence (e.g., in absolute or
relative amounts) of one or more target nucleic acids in a sample.
The order and arrangement of the second binding domains is
predetermined and maintained in the course of the replicating
method set forth herein. For instance, the resultant assay array
can be used in a conventional manner, e.g., by contacting the array
with a sample suspected of containing the target nucleic acid,
under conditions suitable to permit any target nucleic acid to be
hybridized and detected.
[0025] In other aspects, the invention provides a method of using
such a system; the various components for use in such a system,
including a kit or combination of one or more components; as well
as an assay array formed by the method of the invention.
DETAILED DESCRIPTION
[0026] The present invention provides a method and system for
reproducibly preparing a specific binding ligand array, such as a
nucleic acid array. As used herein, "nucleic acids" include
polymeric molecules such as deoxyribonucleic acid (DNA),
ribonucleic acid (RNA), peptide nucleic acid (PNA), or any sequence
of what are commonly referred to as bases joined by a chemical
backbone that have the ability to form base pairs or hybridize with
a complementary chemical structure. The nucleic acid can be
provided in any suitable form, e.g., isolated from natural sources,
recombinantly produced, or artificially synthesized. The word
"oligonucleotide," in turn, will be used interchangeably with the
term "nucleic acid" to refer generally to short chain (e.g., less
than about 100 nucleotides in length, and typically 20 to 50
nucleotides in length) nucleic acid sequences, e.g., as prepared
using synthetic techniques presently available in the art, such as
solid support nucleic acid synthesis. While the invention will be
described with particular reference to nucleic acids (and their
ability to specifically "bind" via hybridization), it is understood
that the invention has applicability to other specific binding
ligands as well, such as immunological binding pairs or other
ligand/anti-ligand binding pairs.
[0027] In one embodiment, the present invention provides a master
array having immobilized thereon a plurality of address
oligonucleotides, the pattern of which can be controlled and/or
determined by any suitable means. Thereafter, a composition
comprising a plurality of multi-ligand conjugates is brought into
binding proximity with the master array, under conditions suitable
to permit a first (e.g., oligonucleotide) binding domain of the
multi-ligand conjugate to bind (e.g., hybridize) with complementary
address oligonucleotides on the master array support.
[0028] In a particularly preferred embodiment, the third ligand is
provided in the form of a binding ligand (e.g., biotin). The master
array and multi-ligand conjugate can be prepared and combined in
substantially the manner described above, with the third binding
ligand then serving to transfer the resultant conjugate complex to
the assay array support. The master array containing bound
conjugate molecules can be brought into binding proximity with an
assay array support. The assay array support, in turn, bears
attachment sites (e.g., binding partner molecules) adapted to bind
(e.g., non-covalently) with the third binding ligand of the
multi-ligand conjugate. The assay array support can be brought into
binding proximity under conditions suitable to permit the binding
partner molecules of the assay array support to bind to
corresponding binding ligands on the multi-ligand conjugate, thus
forming a transitory "sandwich" structure in the form of master
array--multi-ligand conjugate--assay array support.
[0029] In an optional preferred embodiment, the multi-ligand
conjugate of the present invention further comprises a
polymerizable group (e.g., an acrylic or vinyl group). In this
embodiment, once the address oligonucleotides of the master array
have each hybridized with their respective first binding domains of
the multi-ligand conjugates, the polymerizable group can be
reacted, using reagents and conditions within the skill of those in
the art, to allow the formation of a polymer film. The
polymerizable groups are copolymerized into the film backing while
substantially maintaining their respective positions. The film can
be used as is, or optionally stabilized (e.g., gelled, laminated
with another layer, or otherwise solidified) such that it is
sufficiently stable (e.g., self supporting) to maintain the
spatially-defined locations of each "address." Simultaneously or
thereafter, this polymeric backing can be transferred to, or
otherwise incorporated into, an assay array surface in a manner
that maintains the desired orientation of conjugates.
[0030] Once the "sandwich" structure has been formed, the base
pairing, between address oligonucleotide (of the master array) and
complementary oligonucleotides (of the conjugate) can then be
disassociated, in order to permit the assay array to be removed
with the multi-ligand conjugates attached thereto. The conjugates
are attached in a spatial relationship that is sufficiently true to
that established by their initial binding to the master array
surface, thereby permitting the location of target nucleic acids to
be maintained and determined. The master array, in turn, can be
reused repeatedly and in a similar manner to provide additional,
and substantially identical, assay arrays. In another aspect, the
present invention provides a plurality of identical assay arrays,
e.g., formed by replicating a common master array or sequentially
replicating one or more assay arrays.
[0031] In yet another aspect, the present invention provides a
method and system for directly preparing reusable nucleic acid
arrays, without the need to first prepare or replicate a master
array, e.g., wherein the assay array surface is provided in the
form of an optical fiber array. In one such embodiment, each of the
fibers is provided with a nucleic acid adapted to hybridize to its
complementary nucleic acid in a detectable fashion. Suitable fiber
arrays for use in such an embodiment are described, for instance,
in (K. Michael et al.), Analytical Chem. 70(7):1242 (1998).
[0032] The assay arrays of this invention, in turn, are preferably
adapted to detect a wide variety of nucleic acids in a biological
sample. In the course of its use, an assay array can be exposed to
a test solution suspected of containing one or more target nucleic
acids (e.g., genes or gene fragments), under conditions suitable to
permit the target nucleic acids to be bound through hybridization
to their corresponding oligonucleotide binding domain complement on
the assay array. The presence or absence of the target nucleic acid
on the assay array can be determined with the chosen signal
generation and detection system.
[0033] A system of the present invention provides a number of
advantages over current methods, including, for instance, an
optimal combination of such properties as nucleic acid probe
density, ease of use, reproducibility, and reduced cost. The
present invention can, for instance, provide a higher nucleic acid
probe density than commercial approaches that currently rely on
photolithography. Such probe density is achieved, for instance, by
the use of micro-beads that are smaller than the wavelength of
photolithography light systems. Further, the present invention can
provide improved assay specificity, e.g., by the use of
oligonucleotides that are longer (and therefore more specific as to
binding) than those currently available through photolithographic
synthesis.
[0034] Moreover, the present assay array can be used with
conventional array readers to provide a variety of target nucleic
acid capacities, depending, for instance, upon the area density of
the immobilized array. The present system can also provide a
significant reduction in the cost of manufacture of high-density
array slides. The cost of preparing the master array can itself be
reduced, for instance, through the use of patterned deposition and
immobilization of address oligonucleotide sequences, optionally
with the use of micro-beads.
[0035] In a preferred embodiment, the system employs the use of a
dedicated master array comprising a plurality of different address
oligonucleotides. The support surface of such an array can be
fabricated from any suitable material to provide an optimal
combination of such desired properties as stability, dimensions,
shape, and surface smoothness. The address oligonucleotides can be
attached to the support surface directly or via linkers to the
surface (e.g., by providing and/or derivatizing either the surface,
the oligonucleotide, or both, with mutually reactive groups). In a
particularly preferred embodiment, the master array support surface
is coated with a plurality of linking micro-beads that, in turn,
have oligonucleotides attached thereto. Hence, when used in
combination with linking micro-beads, the master array support
surface is preferably provided with sufficient smoothness such that
topographical irregularities are smaller than the radius of the
linking micro-bead (e.g., a roughness no greater than 50% the
diameter of the linking micro-beads of the master array).
[0036] A master array support surface of this sort is preferably
provided in the form of a planar, non-porous solid support having
at least a first surface. A plurality of different oligonucleotides
can be attached to the first surface of the solid support at a
density exceeding 100 different oligonucleotides/cm.sup.2, and
preferably exceeding 1000 different oligonucleotides/cm.sup.2,
wherein each of the different oligonucleotides is attached to the
surface of the solid support in a different predefined region, has
a different determinable sequence, and is at least 4 nucleotides in
length, and preferably at least 10 nucleotides, and more preferably
30 nucleotides in length. The oligonucleotides are provided in
sufficient length to provide suitable diversity of "addresses" on
the master array, while at the same time avoiding substantial
complementarity to target nucleic acid (e.g., naturally-occurring)
sequences to be detected by the assay array.
[0037] In a preferred embodiment, the master array includes at
least 1,000, and more preferably at least 10,000, different address
oligonucleotides attached to the first surface of the solid
support, each located (e.g., spotted) in a predefined region
physically separated from other regions. Given the preference for
multiple sites of each oligonucleotide, the total number of
oligonucleotide sites in a single array can often exceed
10,000.
[0038] The master array can be fabricated from any suitable
material, including, for example, biological or nonbiological,
organic or inorganic materials. For example, the master array can
be fabricated from any suitable plastic or polymer, silicon, glass,
ceramic, or metal, and can be provided in the form of a solid,
resin, gel, rigid film, or flexible membrane. Suitable polymers
include polystyrene, poly(alkyl)methacrylate,
poly(vinylbenzophenone), and polycarbonate. Preferred materials
include glass and silicon. In a particularly preferred embodiment,
the master array is provided with planar dimensions of between
about one-half cm and about 7.5 cm in length, and between about
one-half cm and 7.5 cm, and preferably between about 1 and 2 cm, in
width. Arrays can also be singly or multiply positioned on other
supports, such as microscope slides (e.g., having dimensions of
about 7.5 cm by 2.5 cm).
[0039] The support surface of the master array can also be prepared
by uniformly distributing photoreactive bi- or poly-functional
reagents on the surface and thereafter coupling these reagents to
the surface. In one embodiment, for example, a silicon chip with
sufficient smoothness is cleaned and reacted with a photoreactive
organosiloxane reagent. This reagent can be prepared, for instance,
by bonding a commercial organosiloxane reagent (such as
aminopropyldimethylmethoxy siloxane) with a thermochemically
reactive photoreagent such as the acid chloride of benzoylbenzoic
acid. The resulting photoreactive siloxane reagent can then be
reacted with the silicon oxide surface of the master array support
to provide a smooth surface, primed with photoactivatible groups.
The photoreactive groups on the resulting primed surface can be
activated and used to coat the oligonucleotides, directly or
indirectly by means of linkers, onto the surface.
[0040] In an alternative embodiment, a melt or concentrated
solution of a reagent having both latent reactive (e.g.,
photoreactive) groups and ligand-attractive (e.g., charged,
cationic) groups can be used. For instance, a polystyrene
derivative containing one or more of both types of groups (e.g.,
thiocholine derivatives of polyvinylbenzoic acid) can be deposited
on the surface of the master array to provide a reactive surface
film for immobilization of the address oligonucleotides. The
surface of the master array can be illuminated to generate a smooth
photoreactive surface. Optionally, the ligand-attractive group
(e.g., cation/thiocholine) is removed, if desired.
[0041] As used herein, the term "address oligonucleotides" will be
used to refer to nucleic acid sequences that are selected to be
non-complementary with target nucleic acid sequences present in a
biological sample to be tested. As a result, the address
oligonucleotides, are expected to not form base pairs or otherwise
hybridize with any target nucleic acids found in a particular
biological test sample, thereby avoiding or minimizing the
possibility of background or false positive readings. Preferably,
these address oligonucleotides are between 10 and 100 nucleotides
in length, and most preferably between 20 and 50 nucleotides in
length.
[0042] Deposition of the address oligonucleotides onto the master
array surface can be accomplished in any suitable manner, including
for instance, by direct attachment or indirect attachment through a
linker (e.g., a linking micro-bead). For example, if the address
oligonucleotide is bound to a micro-bead, which is in turn to be
bound to the master array, deposition can be accomplished by a
gravity-based method in which the specific gravity of the
micro-bead causes the individual micro-bead carrying the address
oligonucleotide to contact the master array surface. This
deposition forms an ordered but random pattern on the master array
surface. The resultant surface can then be illuminated under
conditions suitable to activate the photoreactive groups and cause
bond formation between the micro-bead carrying address
oligonucleotide and the master array support surface.
[0043] In a particularly preferred embodiment, oligonucleotides are
immobilized onto the master array surface using photoactivatable
compounds to form bonds between the address oligonucleotide and the
master array support surface. These photoactivatable compounds can
be provided in any suitable manner, e.g., by the master array
support surface or the address oligonucleotides themselves. In one
embodiment, for instance, the master array support surface is
coated with a photoreactive (e.g., photosiloxane) reagent in order
to provide a latent reactive surface for the formation of bonds
with the address oligonucleotides. Thereafter, a solution
containing a plurality of address oligonucleotides can be applied
to the master array support surface. The master array is
illuminated with long-wavelength ultraviolet or visible light to
photochemically fix the address oligonucleotides onto the master
array support surface. In an alternative embodiment, the address
oligonucleotides themselves are photoreactive.
[0044] Preferably, address oligonucleotides of the present
invention include one or more pendent latent reactive (preferably
photoreactive) groups covalently attached, directly or indirectly,
thereto. Photoreactive groups are defined herein, and preferred
groups are sufficiently stable to be stored under conditions in
which they retain such properties. See, e.g., U.S. Pat. No.
5,002,582, the disclosure of which is incorporated herein by
reference. Latent reactive groups can be chosen that are responsive
to various portions of the electromagnetic spectrum, with those
responsive to ultraviolet and visible portions of the spectrum
(referred to herein as "photoreactive") being particularly
preferred.
[0045] Photoreactive groups respond to specific applied external
stimuli to undergo active specie generation with resultant covalent
bonding to an adjacent chemical structure, e.g., as provided by the
same or a different molecule. Photoreactive groups are those groups
of atoms in a molecule that retain their covalent bonds unchanged
under conditions of storage but that, upon activation by an
external energy source, form covalent bonds with other
molecules.
[0046] The photoreactive groups generate active species such as
free radicals and particularly nitrenes, carbenes, and excited
states of ketones upon absorption of electromagnetic energy.
Photoreactive groups may be chosen to be responsive to various
portions of the electromagnetic spectrum, and photoreactive groups
that are responsive to e.g., ultraviolet and visible portions of
the spectrum are preferred and may be referred to herein
occasionally as "photochemical group" or "photogroup".
[0047] Photoreactive aryl ketones are preferred, such as
acetophenone, benzophenone, anthraquinone, anthrone, and
anthrone-like heterocycles (i.e., heterocyclic analogs of anthrone
such as those having N, O, or S in the 10-position), or their
substituted (e.g., ring substituted) derivatives. Examples of
preferred aryl ketones include heterocyclic derivatives of
anthrone, including acridone, xanthone, and thioxanthone, and their
ring substituted derivatives. Particularly preferred are
thioxanthone, and its derivatives, having excitation energies
greater than about 360 nm.
[0048] The functional groups of such ketones are preferred since
they are readily capable of undergoing the
activation/inactivation/reactivation cycle described herein.
Benzophenone is a particularly preferred photoreactive moiety,
since it is capable of photochemical excitation with the initial
formation of an excited singlet state that undergoes intersystem
crossing to the triplet state. The excited triplet state can insert
into carbon-hydrogen bonds by abstraction of a hydrogen atom (from
a support surface, for example), thus creating a radical pair.
Subsequent collapse of the radical pair leads to formation of a new
carbon-carbon bond. If a reactive bond (e.g., carbon-hydrogen) is
not available for bonding, the ultraviolet light-induced excitation
of the benzophenone group is reversible and the molecule returns to
ground state energy level upon removal of the energy source.
Photoactivatible aryl ketones such as benzophenone and acetophenone
are of particular importance inasmuch as these groups are subject
to multiple reactivation in water and hence provide increased
coating efficiency.
[0049] The azides constitute a preferred class of photoreactive
groups and include arylazides (C.sub.6R.sub.5N.sub.3) such as
phenyl azide and particularly 4-fluoro-3-nitrophenyl azide, acyl
azides (--CO--N.sub.3) such as benzoyl azide and p-methylbenzoyl
azide, azido formates (--O--CO--N.sub.3) such as ethyl
azidoformate, phenyl azidoformate, sulfonyl azides
(--SO.sub.2--N.sub.3) such as benzenesulfonyl azide, and phosphoryl
azides (RO).sub.2PON.sub.3 such as diphenyl phosphoryl azide and
diethyl phosphoryl azide. Diazo compounds constitute another class
of photoreactive groups and include diazoalkanes (--CHN.sub.2) such
as diazomethane and diphenyldiazomethane, diazoketones
(--CO--CHN.sub.2) such as diazoacetophenone and
1-trifluoromethyl-1-diazo-2-pentanone, diazoacetates
(--O--CO--CHN.sub.2) such as t-butyl diazoacetate and phenyl
diazoacetate, and beta-keto-alpha-diazoacetates
(--CO--CN.sub.2--CO--O--) such as t-butyl alpha diazoacetoacetate.
Other photoreactive groups include the diazirines (--CHN.sub.2)
such as 3-trifluoromethyl-3-phenyldiazirine, and ketenes
(--CH.dbd.C.dbd.O) such as ketene and diphenylketene.
[0050] Upon activation of the photoreactive groups, the reagent
molecules are covalently bound to each other and/or to the material
surface by covalent bonds through residues of the photoreactive
groups. Exemplary photoreactive groups, and their residues upon
activation, are shown as follows.
1 Photoreactive Group Residue Functionality aryl azides amine
R--NH--R' acyl azides amide R--CO--NH--R' azidoformates carbamate
R--O--CO--NH--R' sulfonyl azides sulfonamide R--SO.sub.2--NH--R'
phosphoryl azides phosphoramide (RO).sub.2PO--NH--R' diazoalkanes
new C--C bond diazoketones new C--C bond and ketone diazoacetates
new C--C bond and ester beta-keto-alpha-diazoacetates new C--C bond
and beta-ketoester aliphatic azo new C--C bond diazirines new C--C
bond ketenes new C--C bond photoactivated ketones new C--C bond and
alcohol
[0051] The photoactivatable nucleic acids of the invention can be
applied to any surface having carbon-hydrogen bonds, with which the
photoreactive groups can react to immobilize the nucleic acids to
surfaces. Examples of appropriate substrates include, but are not
limited to, polypropylene, polystyrene, poly(vinyl chloride),
polycarbonate, poly(methyl methacrylate), parylene and any of the
numerous organosilanes used to pretreat glass or other inorganic
surfaces. The photoactivatable nucleic acids can be printed onto
surfaces in arrays, then photoactivated by uniform illumination to
immobilize them to the surface in specific patterns. They can also
be sequentially applied uniformly to the surface, then
photoactivated by illumination through a series of masks to
immobilize specific sequences in specific regions. Thus, multiple
sequential applications of specific photoderivatized nucleic acids
with multiple illuminations through different masks and careful
washing to remove uncoupled photo-nucleic acids after each
photocoupling step can be used to prepare arrays of immobilized
nucleic acids. The photoactivatable nucleic acids can also be
uniformly immobilized onto surfaces by application and
photoimmobilization.
[0052] Immobilization of the address oligonucleotide onto the
master array support surface can also be accomplished, for example,
by placing address oligonucleotide-loaded micro-beads at specific
addresses on a photoreactive surface. The micro-beads can be
placed, for instance, by such techniques as electrostatic
attraction, electrode or magnetic probes, or optical tweezers.
[0053] More preferably, the linking micro-beads containing address
oligonucleotides are immobilized in a spaced relationship onto the
master array support surface. In such an embodiment, a woven screen
having spaces only slightly larger than the bead diameter can be
used as, or positioned upon, the surface of the master array, in
order to promote deposition of micro-beads onto the surface in a
separated, but ordered, configuration.
[0054] Other suitable methods for immobilization of the address
oligonucleotide include chemical modification of the
oligonucleotide (e.g., thiol modification), and non-covalent
attachment by high-affinity binding agent (e.g., avidin-biotin).
The surface of the master array support, in turn, can be
derivatized (e.g., by amine groups) in order to link a
corresponding reactive group provided by the oligonucleotide itself
or a suitable cross-linking reagent. Alternatively, a binding
partner (e.g., streptavidin) can be directly attached to the master
array support surface, allowing interaction between the address
oligonucleotide and the support surface of the master array. Other
methods include adsorption of unmodified or modified
oligonucleotides, as well as immobilization of the oligonucleotides
onto the support surface by ink jet or "needle" printing with
thermochemical immobilization of soluble probes on an activated
master array support surface.
[0055] The typical master array of the present invention includes a
large number of address oligonucleotides immobilized onto its
surface, each in a discrete location or address. The word
"address," as used in this sense, refers to a spatially-defined
location upon the support surface that is sufficiently distinct to
permit an oligonucleotide bound thereto to be identified and
distinguished. The result of this immobilized crystalline array
provides a master array containing a large number of unique
"addresses" on its surface.
[0056] In a particularly preferred embodiment of the invention, the
master array support surface contains redundant address
oligonucleotides immobilized thereon, such that a particular
address appears at a plurality of spatially-defined locations on
the master array. As a result of this redundancy, a particular
multi-ligand conjugate containing the complementary sequence to a
specific address oligonucleotide will be able to bind at any of a
plurality of locations on the master array. This redundancy of any
particular address on the master array serves to increase the
sensitivity of the assay for detection of corresponding target
nucleic acid within a sample. For example, in one embodiment, the
master array will contain an address sequence that is immobilized
to the support surface at between 1 and 100, preferably between 2
and 10, different locations. In such a preferred embodiment
therefore, detection of the target nucleic acid associated with the
redundant address will take place at a number of different
locations on the master array.
[0057] In one embodiment described above, the address
oligonucleotides can be provided in immobilized form, e.g., in the
form of individual linking micro-beads, each bearing a plurality of
identical "address" oligonucleotide sequences. In one such
embodiment, an organized array of linking micro-beads loaded with
address oligonucleotides can be deposited on a smooth master array
support surface primed with photoreactive groups.
[0058] The linking micro-beads can be procured from commercial
sources and loaded with oligonucleotides by the use of commercially
available processes and materials. Preferably, the linking
micro-beads are selected to have a specific gravity sufficient to
allow them to settle onto the surface of the master array by
gravity (while avoiding the effects of Brownian motion). The
attachment of the address oligonucleotides to the linking
micro-bead is preferably accomplished by the formation of covalent
bonds between the oligonucleotide and the bead. In one particularly
preferred embodiment, the surface of the linking micro-bead is
reacted with an organosiloxane reagent containing a
thermochemically reactive group (e.g., an epoxy).
Glycidoxypropyltriethoxy siloxane, for instance, can be reacted
with the micro-beads to provide an epoxy surface. The 5' terminus
of the address oligonucleotide is, in turn, modified to produce a
terminus containing alkylamine groups on spacers. The epoxy
siloxane of the micro-bead surface reacts with the alkyl amine
positioned at the terminus of the address oligonucleotide, forming
a bond between the address oligonucleotide and the micro-bead
surface.
[0059] An array of micro-beads can be deposited from an aqueous
suspension, optionally containing block copolymers having both
photoreactive groups and ligand-attractive groups (e.g., ionic
groups such as quaternary amines) thereon. Monodisperse micro-beads
(e.g., between about one-half micron and 5 microns in diameter),
previously loaded with address oligonucleotides, are applied to the
master array support surface from suspension in an aqueous solution
of the copolymers. The cationic reactive group serves to associate
with the oligonucleotide sequences on the beads, while the aromatic
block (e.g., oligostyrene) serves to associate with and couple to
the photoreactive surface. In a particularly preferred embodiment,
the linking micro-beads are provided in slight excess.
[0060] The smooth flat photoreactive master array support surface
is exposed to a slurry of linking micro-beads with address
oligonucleotide sequences immobilized thereon, in sufficient number
to provide at least a monolayer of linking micro-beads on the
master array support surface. This slurry is comprised of
approximately equal numbers of beads with each of the desired
address oligonueleotide sequences, plus a multiple (e.g., at least
3-fold) of beads containing no oligonucleotides. The number of
oligonucleotide-coated beads is sufficient to provide a small
redundancy (e.g., at least 3-fold) of each target nucleic
acid-specific site.
[0061] The master array is incubated in the solution containing
linking micro-beads in the dark or dim light, so as to avoid
premature activation of the photogroups. As the solvent (preferably
aqueous) evaporates from the slurry pool on the photoreactive
surface, the uniform-sized beads are packed into a two-dimensional
"crystalline" arrangement, optionally being spaced apart and
positioned, e.g., using a micro-screen as described herein.
[0062] This ordered arrangement can then be "fixed" in place, e.g.,
by illumination in the dry state to form carbon-carbon bonds
between the photoreactive groups on the master array support
surface and the organic groups (mostly oligonucleotides) on the
linking micro-beads. A patterned array of beads, having settled by
gravity onto the photoreactive surface, can be photochemically
fixed to the surface by illumination with long-wave ultraviolet or
visible light. This illumination activates the photoreactive
organosiloxane reagent, which in turn, forms bonds with the address
oligonucleotides. Optionally, once the arrayed linking micro-beads
have been photocoupled to the master array support surface, the
unbound block copolymer can be rinsed from the master array. This
rinsing removes subsequent interference (e.g., during hybridization
steps) with the oligonucleotide sequences on the outer side of the
beads.
[0063] The spatially-defined location of each immobilized address
oligonucleotide can then be determined using any suitable means,
e.g., by fluorescent hybridization involving the sequential
addition of fluorescent-labeled, complementary sequences to the
array, followed by rinsing and scanning between each addition.
Fluorescent-labeled oligonucleotides complementary to each of the
address oligonucleotide sequences are available or can be
synthesized to contain one of several (e.g., ten or more) different
fluorescent compounds commonly known to be distinguishable from
each other. A plurality (e.g., ten) different oligonucleotides can
be prepared, each having a different, known sequence complementary
to an address sequence on the surface and each having a
corresponding distinguishable fluorophore. The labeled probes can
be simultaneously applied to the surface and hybridized, e.g., at
55.degree. C. for 30 minutes. The surface is then rinsed and
scanned to identify the locations of each of the ten sequences.
Another set of ten sequences is then hybridized to the surface and
scanned to identify the second set of ten sequences. This process
is continued until all of the addresses are identified, after which
the master array is stripped of hybridized oligonucleotides, e.g.,
by immersing in boiling water for two minutes, and used for
preparing replicate assay arrays.
[0064] A preferred system of the present invention further includes
a multi-ligand conjugate containing a plurality of active (e.g.,
binding or polymerizable) domains. In accordance with the
invention, the individual ligands can be attached to a core atom or
molecule in any suitable manner and/or order, e.g., individually or
together (e.g., in linear sequence and at a single location or at a
plurality of locations). For example, in one embodiment, the
ligands are attached to the core simultaneously, e.g., under
similar reaction conditions. Optionally, the ligand serving as the
third ligand is attached to the core after hybridization between
the address oligonucleotide of the master array and the
complementary oligonucleotide provided by the multi-ligand
conjugate. In this embodiment, the third ligand is present as a
separate reagent; however, attachment of the third ligand is
capable of taking place under the same or similar reaction
conditions as the first and second binding domains. In yet another
embodiment, each of the ligands is attached to the transfer core
under separate conditions of separate reactions.
[0065] The sequence of attachment of these ligands to the core of
the multi-ligand conjugate is not crucial to the present invention,
and so long as the individual active (e.g., binding) domains are
available for binding their specific complement, they can be
attached to the core or to one another in any order. In a
particularly preferred embodiment, each individual binding domain
is attached to the core individually, at a plurality of locations
on the surface.
[0066] Optionally, the core of the multi-ligand conjugate is itself
provided in the form of a transfer micro-bead. The micro-beads
useful in preparing a multi-ligand conjugate can be of the same
variety as those described above with respect to the "linking
micro-beads." Preferred beads are desirably spherical, homogeneous
in size, with a specific gravity greater than that of water. Also,
preferred beads have a chemical composition, or at least surface,
that is subject to bonding with organic reagents. For example,
homogeneous silica spheres of between about 0.5 micron and about 5
microns (.mu.) diameter can be reacted with an organosiloxane
reagent containing a thermochemically reactive group (e.g., epoxy),
in order to couple the reagent to corresponding groups of a binding
ligand.
[0067] In one such embodiment, for instance,
glycidoxypropyltriethoxy siloxane is reacted with glass micro-beads
under published reaction conditions, to provide epoxy-glass
surfaces. The three binding domains of the present invention, each
terminated with alkylamine groups on spacers, can be readily
coupled with the epoxy groups on the surface of the bead.
[0068] First and second oligonucleotide binding domains can be
attached to the transfer micro-bead in any suitable fashion, e.g.,
alkylamine-terminated binding domains can be synthesized and
thermochemically attached. For the first and second oligonucleotide
binding domains, the lengths of the oligonucleotides are optimized
for desired hybridization strength and kinetics (usually in the
20-50 nucleotide range). The first binding domain comprises an
oligonucleotide that is complementary to the address
oligonucleotide immobilized onto the master array support surface,
while the second binding domain is complementary to a chosen target
nucleic acid suspected or known to be in a sample to be tested.
Preferably, the sequences of the first binding domain are not
complementary either to one another or to any known natural gene
sequence and/or gene fragment with significant probability of being
present in the biological sample. As a result, the first binding
domain oligonucleotide sequences will hybridize only with their
respective complementary address oligonucleotide sequences
immobilized onto the master array support surface. This avoids
cross-reactivity (e.g., cross-hybridization) between the first
binding domain, which is responsible for placing the multi-ligand
conjugate at a spatially-defined location on the assay array
support surface, and the target nucleic acid sequences screened in
the test sample.
[0069] Oligonucleotides are selected for the first binding domain
by preparing the complementary sequence to the random sequences
comprising the address oligonucleotides of the master array. This
preparation can be accomplished using any suitable methods known in
the art, for example, by solid phase DNA synthesis. Once the
oligonucleotides for the first binding domain have been
synthesized, a terminus of each oligonucleotide can be modified to
contain an alkylamine group on a spacer.
[0070] Oligonucleotides are selected for the second binding domain
by first selecting the target nucleic acids (e.g., genes and/or
gene fragments) to be detected by the microarray to be fabricated
in accordance with the invention. The sequences of these genes
and/or gene fragments may be known, or may be determined using
techniques well known in the art (e.g., Sanger dideoxy method, PCR
sequencing, etc.). Once the second binding domains have been
chosen, a terminus of each oligonucleotide can be modified to
contain an alkylamine group on a spacer.
[0071] The third ligand (e.g., binding domain or polymerizable
group) can be of any suitable type, and can be attached to the core
or transfer micro-bead in any suitable fashion. In a preferred
embodiment, for instance, the third ligand is a binding ligand
provided in the form of a biotin-poly(alkyleneoxide) amine
derivative. Such a derivative can be prepared, for instance, by
reacting an N-oxysuccinimide ester of biotin with a
poly(ethyleneoxide) diamine of approximately the same molecular
length as the oligonucleotide derivatives to be placed on the same
core or micro-bead.
[0072] The multi-ligand conjugates can be fabricated to include
oligonucleotides that are complementary to, and thus will hybridize
with, any desired target nucleic acid found in a biological sample,
and which has a known, or determinable, sequence. In one
embodiment, the various second binding domains of the multi-ligand
conjugates of the present invention can comprise a wide array of
oligonucleotides that are complementary to various target nucleic
acids found in a biological sample.
[0073] In an alternative embodiment, the second binding domains of
the multi-ligand conjugates will be fabricated such that the
resulting assay array will locate target nucleic acids of a
particular family of naturally-occurring genes. One example of this
particular embodiment would be a set of multi-ligand conjugates
containing second binding domains complementary to the 50-60 known
target sequences of cystic fibrosis. Thus, the resulting assay
array would be fabricated such that it detects the presence of the
sequences known to be associated with this disorder only.
[0074] The third ligand of the multi-ligand conjugate of the
present invention optionally comprises a binding ligand adapted to
form bonds with a corresponding binding partner. The third ligand
can be attached directly to the core or transfer micro-bead of the
multi-ligand conjugate, or indirectly by the use of a spacer, in a
manner that allows the ligand to be free of steric hindrance (e.g.,
steric hindrance caused by proximity of the ligand to the transfer
micro-bead). In other words, use of a spacer to attach the third
ligand will ensure that the ligand is sufficiently distant from the
core or surface of the transfer micro-bead to allow it to perform
its desired function, e.g., polymerize or bind with a corresponding
binding partner contained on the assay array surface. In a
particularly preferred embodiment, the third ligand is attached to
the core or transfer micro-bead via a hydrophilic spacer, e.g., a
polyethylene glycol spacer, of sufficient length to allow the
binding domain to be free of steric hindrance.
[0075] In an alternative embodiment, the third ligand is provided
in solution, rather than attached to the micro-bead or core of the
multi-ligand conjugate. In this embodiment, the third ligand is
incorporated after hybridization of the master array address
oligonucleotide with its complementary first binding domain of the
multi-ligand conjugate. Therefore, in this embodiment, the third
ligand is incorporated into the complex formed on the master array
after attachment of the multi-ligand conjugate, and before the
assay array is brought into binding proximity to the master array.
Preferably, the addition of the third ligand does not require
different reaction conditions from that of the first and second
binding domains; rather, the addition of the third ligand can take
place under the same conditions.
[0076] In a preferred embodiment, the third ligand is attached to
the multi-ligand conjugate using photoreactive chemistry. In this
embodiment, the surface of a micro-bead is pretreated, for example,
with an organosiloxane in order to prepare the surface for reacting
with photoreactive compounds. In this embodiment, the third ligand
is attached to a hydrophilic spacer, which is, in turn, attached to
a photoreactive group. The photoreactive group allows bond
formation between the spacer (attached to the binding ligand) and
the transfer micro-bead.
[0077] In an alternative embodiment, wherein the invention includes
the use of porous linking micro-beads immobilized onto the surface
of the master array, such micro-beads having attached thereto
address oligonucleotides, the transfer micro-bead of the
multi-ligand conjugate is not required. In this embodiment, the
porous linking micro-beads can be immobilized onto the surface of
the master array, each porous micro-bead having a plurality of
address oligonucleotides attached to its surface. In this
embodiment, the multi-ligand conjugates comprise a first binding
domain complementary to the address oligonucleotide, a second
binding domain complementary to a target nucleic acid sequence
found in a biological sample, and a third ligand comprising a
binding ligand. The multi-ligand conjugate does not contain a
transfer micro-bead, but rather a chemical (e.g., atomic or
molecular) core.
[0078] A plurality of multi-ligand conjugates are applied to the
master array and incubated under conditions suitable to allow
hybridization of the address sequence of the master array with its
complementary oligonucleotide contained within the multi-ligand
conjugate. Those skilled in the art will be able to determine
suitable conditions of temperature, salt concentration, and buffer
composition. For a discussion of hybridization conditions and
methods for applying them, see Nucleic Acid Hyrbridization: A
Practical Approach, Hames and Higgins, eds., 1985, the disclosure
of which is incorporated herein by reference.
[0079] The third ligand of the multi-ligand conjugate can be
provided in the form of a polymerizable group, e.g., to allow the
conjugates to form into a stable film while in position on the
master array. Suitable polymerizable groups include, for example,
acrylic and vinyl groups. In a preferred embodiment, the
polymerizable groups are adapted to undergo addition
polymerization, in the presence of added monomers and other
reagents (e.g., initiators), to form a stable film. Polymerization
can take place via any suitable reaction, including aqueous
free-radical solution polymerization, exposure to heat, high energy
radiation, ultrasonic waves, ultraviolet radiation, and ionic
polymerization catalysts to produce water-soluble or swellable
polymers. Further, polymerization of the groups can take place via
base-catalyzed hydrogen-transfer polymerization. Those skilled in
the art, given the present description, will appreciate the manner
in which such groups can be used to copolymerize the ligands with
other monomers, and in turn their corresponding conjugates, into
the form of an integral polymeric structure, e.g., a film backing,
which can in turn be used as or transferred to an assay array
support surface.
[0080] The assay array support of the present invention can be
fabricated from any suitable material to provide an optimal
combination of such properties as stability of dimension, shape,
and smoothness. Suitable materials for use in fabricating the assay
array include those described as being useful for the master array
itself, and preferably include silicon and glass. In a particularly
preferred embodiment, the assay support is provided with dimensions
measuring 50 square .mu.m. Preferably, the assay array support is
of comparable dimensions to the master array, in order to provide
direct spatial relationships.
[0081] The surface of a discrete assay array support can be
pretreated to facilitate attachment of the oriented conjugate
layer. In a preferred embodiment, the assay array is provided in
the form of a silicon chip that has been cleaned, polished and
treated to provide a smooth silicon oxide surface. The silicon
oxide surface can then be treated with an organosiloxane reagent
(as described herein) to provide a smooth photoactivatable
surface.
[0082] In one embodiment, the assay array support of the present
invention can be coated with binding partner molecules adapted to
form bonds with the third ligand of the multi-ligand conjugate. The
assay array support surface can be prepared by coating the surface
with a photoactivatable compound (e.g., photoreactive siloxane
reagent) to render the surface photoreactive. Thereafter, the assay
array support surface can be exposed to a solution containing
binding partner molecules (e.g., avidin), and the solution
illuminated, in order to allow bond formation between the binding
partner and surface of the assay array. Optionally, the surface of
the assay array is passivated prior to and/or after exposure to the
binding partner, e.g., with a surfactant (e.g., a biotin triblock
copolymer), to prevent non-specific binding of molecules found in
the test sample to the assay array support surface.
[0083] In a particularly preferred embodiment, the binding partner
is provided in the form of a monolayer upon the assay array support
surface. Optionally, the binding partner can be provided in the
form of a three-dimensional layer, e.g., an avidin-hydrogel
composite of up to a micron thickness. Such a structure can be
obtained, for instance, by loading the photoreactive assay array
surface with a solution of avidin plus photoreactive hydrogel
(e.g., copolymer of benzoylbenzamidopropyl methacrylamide with
vinylpyrollidone or acrylamide) and illuminating while wet.
[0084] In another particularly preferred embodiment, a thin film of
photopolymer is applied to a dimensionally stable assay array
support surface. A self-assembling monolayer film of passivating
surfactant containing a high surface density (e.g., 0.1
pmol/mm.sup.2) of high-affinity ligand group (e.g., biotin) is
applied to the photoreactive surface. The monolayer surfactant film
is then photocoupled to the surface, and any unbound surfactant is
rinsed away. The passivated, ligand-loaded surface is saturated
with high-affinity binding partner molecule (e.g., X-avidin), and
any excess binding partner is rinsed away. Suitable high-affinity
binding partner molecules include, for example, x-avidin film, or
high-affinity antibody (e.g., anti-digoxygenin).
[0085] Optionally, the system further includes other components as
well, including for instance, means (e.g., reagents) for use in
disassociating complementary, hybridized oligonucleotides (e.g.,
the address oligonucleotide of the master array and its
complementary oligonucleotide carried by the multi-ligand
conjugate). For example, suitable means for disassociating the
hybridized DNA include altering the temperature, pH, or salt
concentration of the system.
[0086] Optionally, the system further comprises a scanner, as
commercially available (e.g., a confocal fluorescence microscope as
available from Molecular Dynamics) to detect the hybridization of
nucleic acid targets to specific addresses.
[0087] In another aspect, the present invention provides a method
for reproducibly preparing a nucleic acid array, the method
comprising:
[0088] a) providing a master array having a support surface;
[0089] b) immobilizing (e.g., depositing and fixing, e.g., directly
or via respective linkers or "linking" micro-beads) a plurality of
address oligonucleotides on the master array support surface in the
form of a patterned and optionally random array;
[0090] c) determining and recording the pattern of the immobilized
address oligonucleotides (e.g., using fluorophore-coupled
oligonucleotides for hybridization to the address
oligonucleotide);
[0091] d) providing a plurality of free (i.e., nonimmobilized)
multi-ligand conjugates, each multi-ligand conjugate comprising a
core having (preferably independently) attached thereto: (i) at
least one molecule of a first oligonucleotide binding domain,
comprising an oligonucleotide sequence selected to be complementary
to a particular address oligonucleotide sequence of the master
array, (ii) at least one molecule of a second oligonucleotide
binding domain, comprising an oligonucleotide sequence selected to
be complementary to a characteristic oligonucleotide sequence of a
target nucleic acid sequence (e.g., a gene and/or gene fragment),
and (iii) at least one molecule of a third (and preferably
non-oligonucleotide) ligand, selected from the group consisting of
binding ligands and polymerizable groups (e.g., acrylic or vinyl
groups);
[0092] e) contacting and incubating the multi-ligand conjugates
with the master array support surface under conditions suitable to
allow each address oligonucleotide to hybridize with its
complementary first oligonucleotide binding domain of the
multi-ligand conjugates (and optionally removing non-hybridized
multi-ligand conjugates);
[0093] f) providing an assay array support surface,
[0094] g) contacting the hybridized multi-ligand conjugates with
the assay support surface under conditions suitable to permit the
conjugates to be transferred to the assay support in a pattern
corresponding to their pattern on the master array, e.g., either by
polymerizing a third ligand (provided in the form of a
polymerizable group), or by binding a third ligand (provided in the
form of a third binding ligand) with a corresponding attachment
site provided by the assay support surface; and
[0095] h) disassociating the first binding domain from the master
array support in a manner that permits the conjugates to remain
upon the assay support surface in the desired pattern.
[0096] In use, the core (e.g., transfer micro-beads) bearing three
or more ligands can be mixed in equal numbers and suspended in
aqueous buffer. This suspension can contain an excess (over the
hybridization capacity of the micro-beads on the master array) of
each transfer micro-bead carrying the oligonucleotide sequence
complementary to a characteristic oligonucleotide sequence of a
target nucleic acid expected to be detected in a sample.
[0097] The master array surface can be flooded with this suspension
of the chosen mixture of multi-ligand conjugates and solid-state
hybridization can be allowed to proceed to near equilibrium. The
excess multi-ligand conjugates can then be recovered from the
hybridized master array by rinsing, and the bound conjugates either
copolymerized to form a stable backing or bound to corresponding
binding partners on the assay array. The master array is then
available for a repeat replication process.
[0098] The master array can be used repeatedly to prepare
corresponding assay arrays, which in turn can be packaged, stored
and transported, e.g., in the wet or dry state. Upon removal from
its package, an assay array will be ready to use in a routine
hybridization assay protocol with a scanner. Moreover, the present
invention provides a master array that is versatile in use--the
master array can be used and reused, to create identical arrays or
to change the function of the array (e.g., to alter the
oligonucleotide sequence complementary to a characteristic
oligonucleotide sequence of a target nucleic acid, such that the
user can alter the target nucleic acid to be detected in a
sample).
[0099] Those skilled in the art, given the present description,
will appreciate the manner in which commercially available
microarrays can themselves be used as master arrays of the present
invention, to be replicated one or more times to form corresponding
assay arrays. In this case, the individual nucleic acids of the
commercial array, regardless of their original specificity, can
each be considered as mere address sequences, and used in the
manner described herein to provide assay arrays of any desired
specificity.
[0100] In yet a further aspect, and particularly where the third
ligand is provided in the form of a third binding ligand, the
present invention provides a sandwich array comprising:
[0101] a) a master array comprising a support surface having a
plurality of address oligonucleotide sequences immobilized thereon
(e.g., directly or via respective linkers or "linking"
micro-beads), the oligonucleotide sequences being immobilized in
the form of a patterned, and optionally random array;
[0102] b) a plurality of multi-ligand conjugates, each multi-ligand
conjugate comprising a core having (preferably independently)
attached thereto: (i) molecules of a first oligonucleotide binding
domain, each molecule comprising an oligonucleotide sequence
complementary to a particular address oligonucleotide sequence of
the master array, (ii) molecules of a second oligonucleotide
binding domain, each molecule comprising an oligonucleotide
sequence complementary to a characteristic oligonucleotide sequence
of a target nucleic acid (e.g., a gene and/or gene fragment), and
(iii) at least one molecule of a third (and preferably
non-oligonucleotide) binding ligand selected from the group
consisting of binding ligands and polymerizable groups (e.g.,
acrylic or vinyl groups), wherein the first oligonucleotide binding
domain is hybridized to the corresponding address oligonucleotide
immobilized onto the master array; and
[0103] c) an assay array support comprising a support surface
coated with attachment sites for the third binding ligand (such as,
immobilized molecules of a corresponding binding partner specific
for the third binding ligand), wherein the attachment site of the
assay array is bound to the corresponding binding ligand of the
multi-ligand conjugates.
[0104] Optionally, the sandwich array further comprises a linking
micro-bead immobilized onto the surface of the master array, the
linking micro-bead having attached thereto a plurality of address
oligonucleotides.
[0105] In yet another alternative embodiment, the present invention
provides a corresponding kit comprising one or more components for
reproducibly preparing a nucleic acid array, the kit components
selected from the group consisting of:
[0106] a) a master array comprising a support surface having a
plurality of "address" oligonucleotide sequences immobilized
thereon (e.g., directly or via respective linkers or "linking
micro-beads"), the oligonucleotide sequences being immobilized in
the form of a patterned, and optionally random, array;
[0107] b) a plurality of multi-ligand conjugates, each multi-ligand
conjugate comprising a core having (preferably independently)
attached thereto: (i) molecules of a first oligonucleotide binding
domain, each molecule comprising an oligonucleotide sequence
complementary to a particular address oligonucleotide sequence of
the master array, (ii) molecules of a second oligonucleotide
binding domain, each molecule comprising an oligonucleotide
sequence complementary to a characteristic oligonucleotide sequence
of a target nucleic acid sequence (e.g., a gene and/or gene
fragment), and (iii) at least one molecule of a third (and
preferably non-oligonucleotide) ligand, selected from the group
consisting of a binding ligands and polymerizable groups (e.g.,
acrylic or vinyl groups);
[0108] c) an assay array support comprising a support surface for
the replicate array, e.g., coated with attachment sites for the
third ligand (such as, immulbilized molecules of a corresponding
binding partner specific for the third binding ligand); and
[0109] d) an array reader for determining the location of address,
oligonucleotides on the master array and the presence of hybridized
target nucleic acids on the assay array.
[0110] In yet another alternative embodiment, the present invention
provides a method and system for directly preparing a replicable
array in the form of a reusable nucleic acid array. In this
embodiment, the surface to which the nucleic acid is immobilized is
provided by an optical fiber array, the assay array support surface
being provided by the distal ends of the fibers themselves.
Optionally, and preferably, micro-wells are etched into the distal
ends of the optical fibers.
[0111] In such an embodiment, a conventional optical fiber bundle
can be adapted for use as a specific binding array biosensor system
for detecting a variety of target nucleic acids in a sample.
Commercial sources provide optical imaging fiber bundles comprising
thousands (e.g., 3,000-100,000) of hexagonally packed,
individually-coated optical fibers. Each fiber in the array carries
its own, isolated optical signal from one end of the fiber to the
other. Individual specific binding ligands can be bound to one end
of each fiber and the other end is functionally connected to a
optical signal analyzer (e.g., a CCD camera).
[0112] The assay end (containing specific-binding ligands) of the
fiber bundle is exposed to a sample solution containing the unknown
target and other necessary reagents (if any) of the optical assay
system. The optical signal pattern is recorded and analyzed at the
other end of the bundle, using the image analyzer system.
[0113] Applicant has found that oligonucleotide binding domains,
complementary to target sequences, can be attached to the optical
assay array support surface in any suitable manner, e.g., either
directly or by means of reversibly or irreversibly bound linking
molecules or micro-beads. In turn, the ultimate location of the
oligonucleotide binding domains can be determined (e.g., by optical
means and assay), preferably controlled (e.g., by the use of
reusable address domains), and optionally replicated (e.g., by the
use of reversible linkers).
[0114] In one preferred embodiment, the system comprises a
plurality of multi-ligand conjugates, each multi-ligand conjugate
comprising molecules of a) a first oligonucleotide binding domain
adapted to bind with a corresponding address ligand, and b) a
second oligonucleotide binding domain complementary to a
characteristic oligonucleotide sequence of a target nucleic acid
(e.g., a gene and/or gene fragment). The address ligands are
immobilized onto the distal end of the master array, either
directly, or by the use of a linker or micro-bead. The multi-ligand
conjugates would then load each of the addressed fibers with a
known analyte-specific ligand. The set of analyte-specific ligands
on the fibers in the bundle can be changed at will.
[0115] In another embodiment, attachment of the oligonucleotide
binding domain is accomplished using a micro-bead. In this
embodiment, the micro-bead is prepared such that it is attached to
the oligonucleotide binding domain. The micro-bead and binding
domain are then attached to the assay array support surface.
Attachment of the micro-bead can be accomplished using any suitable
binding pair, such as, for example, avidin-biotin. In an
alternative embodiment, photoreactive reagents can be used to
attach the micro-bead to the assay array support surface.
[0116] Optionally, and preferably, the system further comprises an
image analyzer. In this embodiment, the bundle of fibers is
connected at one end to an optical sensor that collects signals and
processes information from these multiple sensors (e.g.,
fibers).
[0117] In such an approach the invention provides a reusable
nucleic acid array, in which the user is able to change the
function of the nucleic acid array for each particular use. For
example, the linker or micro-bead can be coated with iminobiotin,
and the assay array support surface can be coated with biotin. The
linker or micro-bead can thus be reversibly attached to the assay
array support surface. The linker or micro-bead can be dissociated
by altering the pH such that the lower pH causes the linker or
micro-bead to dissociate with the assay array support surface.
[0118] In one embodiment, micro-wells can be etched into the distal
end of each optical fiber using techniques known in the art. For
example, a wet chemical etching procedure can be used to
selectively etch the cores of the individual fibers. This technique
takes advantage of the difference in etch rates between the core
and cladding materials of the fiber. By controlling the etching
time, those of skill in the art will appreciate the manner in which
high-density, ordered micro-well arrays of known shape and well
volume are obtained. Well architecture is determined by the
preformed imagining fiber. Since each micro-well is contained at
the end of its own optical fiber, each well can serve as an
individual sensor. Preferably, the diameter of the etched
micro-wells is slightly larger than that of individual linking
micro-beads used in connection therewith.
[0119] In one embodiment, for instance, a solution containing a
plurality of micro-beads having one or more oligonucleotide binding
domains complementary to a target nucleic acid attached thereto is
added to the surface of a bundle of optical fibers. The individual
linking micro-beads randomly settle into each micro-well as the
solution is allowed to evaporate from the optical fiber bundle
surface. Optionally, and in the event a photoreactive compound is
used in connection with this embodiment, the optical fibers are
illuminated, to allow the linking micro-bead to photocouple to the
surface of each optical fiber. Optionally, excess micro-beads are
then washed from the surface of the fiber bundle.
[0120] For instance, immobilization of a bead or oligonucleotide
binding domain onto the distal end of the fiber, within the
micro-well, can be accomplished using photoactivatable compounds to
form bonds with the well surface. In one embodiment, the well
surface of the optical fiber is coated with a photoreactive
siloxane reagent, and thereafter, a solution containing free (i.e.,
unbound) oligonucleotide binding domain is applied to the well
surface. The solution is then illuminated, activating the
photoreactive groups and causing bond formation between the
oligonucleotide binding domain and well surface. Illumination is
accomplished by passing activating light through the fiber into the
well.
[0121] As discussed above, each individual fiber of the fiber
bundle can be etched at one end to provide a concave receptacle for
a micro-bead. A photoreactive organosiloxane reagent can be
prepared by bonding a commercial organosiloxane reagent, such as
aminopropyldimethyl methoxy siloxane, with a thermochemically
reactive photoreagent, such as the acid chloride of benzoylbenzoic
acid. The photoreactive siloxane reagent is reacted with the
concave silicon oxide surface of the etched optical fibers in the
image analyzer bundle. In an alternative embodiment, each
micro-well is coated with X-avidin to create a surface that will
react with biotin. In this embodiment, the micro-beads are coated
with biotin or iminobiotin derivatives.
[0122] Preferably, the linking micro-beads are spherical, and of a
diameter approximately equal to (or slightly less than) that of the
individual optical fibers. The linking micro-beads of the present
invention are fabricated from any suitable material, including, for
example, glass, polystyrene, polyvinylbenzophenone,
photopolysiloxane-coated glass. Preferably, the micro-beads have a
specific gravity greater than that of water, and have a chemical
composition that is subject to bonding with organic reagents.
[0123] The oligonucleotide derivatives can be synthesized according
to published and commercially utilized methods, as described in
more detail herein. The length of the oligonucleotide sequences are
optimized for desired hybridization strength and speed (usually in
the 20-50 nucleotide range). The oligonucleotide sequences are
complementary to the chosen target nucleic acid.
[0124] The "address oligonucleotide" derivatives can be coupled to
the epoxy-activated linking micro-beads for the optical fiber ends.
In a preferred embodiment, for example, homogeneous silica spheres
of approximately the fiber diameter are reacted with a commercial
organosiloxane reagent containing a thermochemically reactive group
(e.g., epoxy) useful for coupling to amine groups of the
oligonucleotide derivatives in this invention.
Glycidoxypropyltriethoxy siloxane is reacted with the glass
micro-beads under published reaction conditions, to provide an
epoxy-glass surface. The address oligonucleotide derivatives of
this invention terminate with alkylamine groups on spacers to
couple readily with the epoxy groups on the surface of the linking
micro-beads.
[0125] The bundle of etched photoreactive optical fibers is exposed
to a slurry of linking micro-beads (in sufficient number to provide
at least one type of address oligonucleotide per fiber), each
micro-bead having address oligonucleotide sequences immobilized on
its surface. Preferably, one oligonucleotide binding domain is
bound to each optical fiber well. In this embodiment, the master
array is reusable. This slurry is comprised of approximately equal
numbers of beads with each of the desired address oligonucleotide
sequences. The number of beads providing oligonucleotide sequences
is sufficient to provide a redundancy (at least 3-fold) of each
target nucleic acid specific site. As the solvent (preferably
aqueous) evaporates from the slurry pool on the photoreactive
surface in the dark or dim light, the uniform-sized beads are left
in the concave pockets at the ends of the ordered arrangement of
fibers. The linking micro-beads providing oligonucleotide sequences
are then "fixed" by illumination in the dry state to form
carbon-carbon bonds between the photoreactive groups on the optical
fiber surface and the organic groups (mostly oligonucleotides) on
the address oligonucleotide beads. Excess micro-beads are rinsed
from the system.
[0126] The resultant optical fiber bundle will have a stable and
ordered arrangement of the oligonucleotides complementary to target
nucleic acid sequences on one end of its fibers, and the locations
of the oligonucleotide sequences can be then determined by
conventional methods of fluorescent hybridization, comprising
sequential addition of fluorescent-labeled, complementary sequences
to the array, with rinsing and array reading between each addition,
as discussed in detail herein.
[0127] In one preferred embodiment, complementary sequences are
labeled with fluorophores. Fluorescent-labeled oligonucleotides
complementary to each of the sequences are synthesized, each
containing several (e.g., one of ten) different fluorescent
compounds that can be distinguished from each other. Each
oligonucleotide, therefore, has a known sequence and a known
identifiable label. The several oligonucleotides, each having a
different, known sequence complementary to an oligonucleotide
sequence on the surface and one of a comparable number of
distinguishable fluorophores, are simultaneously applied to the
surface and hybridized at 55.degree. C. for 30 minutes.
[0128] The surface is then rinsed and analyzed to identify the
locations of each of the sequences. Another set of sequences is
then hybridized to the surface and scanned to identify the second
set of sequences. This process is continued until all of the
locations are identified, after which the master array is stripped
by immersing in boiling water for two minutes. It is then ready for
use to detect the presence of target nucleic acids in a sample.
[0129] In use, the master array is exposed to a test solution
containing a plurality of target nucleic acid sequences (e.g.,
genes and/or gene fragments). The test solution is labeled via a
fluorescent label to permit visualization of the results. The test
solution is incubated under conditions to allow the oligonucleotide
binding domain to hybridize to the target nucleic acid contained
within the sample. Unbound solution is then rinsed from the master
array. The hybridized nucleic acid sequences are visualized using
an assay array reader, such as that mentioned above from Molecular
Dynamics.
[0130] The assay arrays are washed with phosphate buffered saline
containing 0.05% Tween 20 (PBS/Tween), then blocked with
hybridization buffer which consists of 4.times.SCC (0.6 M NaCl,
0.06 M citrate, pH 7.0), 0.1% (w/v) lauroylsarcosine, and 0.02%
(w/v) sodium dodecyl sulfate, at 55.degree. C. for 30 minutes. A
sample containing DNA having a sequence complementary to one of the
immobilized oligonucleotide probes on the array is applied to the
support surface of the fiber and incubated for one hour at
55.degree. C. in a sealed hybridization chamber. The support
surface is then washed with 2.times.SSC containing 0.1% SDS (sodium
dodecylsulfate) for 5 minutes at 55.degree. C., after which 50
fmole of a fluorescent-labeled detection probe that is
complementary to another sequence on the target DNA is applied to
the support surface and incubated for one hour at 55.degree. C. The
support surface is then washed with 2.times.SSC containing 0.1% SDS
for 5 minutes at 55.degree. C., followed by a wash with
0.1.times.SSC, then dried and the fluorescence pattern recorded by
the image analysis instrument, e.g., an array reader (e.g., as
commercially available from Molecular Dynamics).
[0131] One of skill in the art will appreciate that the present
invention can provide a variety of oligonucleotide sequences in
association with a particular fiber bundle. In one embodiment, for
example, the fiber wells are coated individually with specific
binding agents. In another embodiment, the fiber wells are coated
simultaneously with a general binding agent (e.g., iminobiotin). In
this embodiment, the fiber wells are loaded from a mixture of
specific binding agent micro-beads each containing the complement
to the general binding agent (e.g., X-avidin).
[0132] Optionally, the system further includes an array reader,
e.g., an assay array reader such as that commercially available
from Molecular Dynamics. The resultant assay array can be used in a
conventional manner, e.g., the assay array can be read by a
standard array reader, while the master array can be re-used to
prepare more assay arrays by repeating some or all of the steps set
forth above.
* * * * *