U.S. patent application number 12/665652 was filed with the patent office on 2011-05-05 for microfabrication methods for the optimal patterning of substrates.
This patent application is currently assigned to ILLUMINA, INC.. Invention is credited to David Barker, David L. Heiner, Michael Lebl, Chanfeng Zhao.
Application Number | 20110105366 12/665652 |
Document ID | / |
Family ID | 40156962 |
Filed Date | 2011-05-05 |
United States Patent
Application |
20110105366 |
Kind Code |
A1 |
Lebl; Michael ; et
al. |
May 5, 2011 |
MICROFABRICATION METHODS FOR THE OPTIMAL PATTERNING OF
SUBSTRATES
Abstract
The invention is directed to a method of fabricating a
microarray. The method includes: (a) providing a substrate having
at least two layers of different chemical reactivity, wherein a
well in an outer layer exposes an inner layer; (b) contacting the
substrate with a first reagent specifically reactive with the outer
layer to produce a first modified layer; (c) contacting the
substrate with a second reagent specifically reactive with the
inner layer of the substrate to produce a modified inner layer,
wherein the modified inner layer has a higher affinity for a
biopolymer than the modified outer layer, and (d) depositing the
biopolymer onto the modified inner layer within the well, wherein
the higher affinity of the modified inner layer facilitates
localization of the biopolymer onto the well. Methods of
fabricating a microarray which include polishing a substrate or
functionalizing a plurality of features with a reactive reagent
also are provided. A method of fabricating a microarray which
includes loading a plurality of discrete nanochannels is
additionally provided.
Inventors: |
Lebl; Michael; (San Diego,
CA) ; Heiner; David L.; (San Diego, CA) ;
Zhao; Chanfeng; (San Diego, CA) ; Barker; David;
(Del Mar, CA) |
Assignee: |
ILLUMINA, INC.
SAN DIEGO
CA
|
Family ID: |
40156962 |
Appl. No.: |
12/665652 |
Filed: |
June 18, 2008 |
PCT Filed: |
June 18, 2008 |
PCT NO: |
PCT/US08/67402 |
371 Date: |
January 14, 2011 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
60936287 |
Jun 18, 2007 |
|
|
|
Current U.S.
Class: |
506/32 |
Current CPC
Class: |
B01J 2219/00677
20130101; B01J 19/0046 20130101; B01J 2219/00596 20130101; B01J
2219/00725 20130101; C40B 50/14 20130101; B01J 2219/00605 20130101;
B01J 2219/00497 20130101; B01J 2219/00585 20130101; B01J 2219/00648
20130101; C40B 50/06 20130101; B01J 2219/00317 20130101; B01J
2219/00722 20130101; B01J 2219/00659 20130101; C40B 40/06
20130101 |
Class at
Publication: |
506/32 |
International
Class: |
C40B 50/18 20060101
C40B050/18 |
Claims
1. A method of fabricating a microarray, comprising: (a) providing
a substrate having at least two layers of different chemical
reactivity, wherein a well in an outer layer exposes an inner
layer; (b) contacting said substrate with a first reagent
specifically reactive with said outer layer to produce a first
modified layer; (c) contacting said substrate with a second reagent
specifically reactive with said inner layer of said substrate to
produce a modified inner layer, wherein said modified inner layer
has a higher affinity for a biopolymer than said modified outer
layer, and (d) depositing said biopolymer onto said modified inner
layer within said well, wherein said higher affinity of said
modified inner layer facilitates localization of said biopolymer
onto said well.
2. The method of claim 1, wherein said inner layer comprises
silicon and said outer layer comprises silicon oxide.
3. The method of claim 2, wherein said first reagent comprises a
chlorosilane having a moiety that repels or is inert to said
biopolymer.
4. The method of claim 3, wherein said silicon is converted to
silicon oxide after step (b) and prior to step (c).
5. The method of claim 4, wherein said second reagent comprises a
chlorosilane having a moiety with affinity for said biopolymer.
6. The method of claim 1, wherein said deposited biopolymer is
covalently attached to said modified inner layer.
7. The method of claim 1, wherein said biopolymer comprises a
nucleic acid clonal ball or a nucleic acid attached to a
microsphere.
8. A method of fabricating a microarray, comprising: (a) contacting
a substrate having wells with a reagent reactive with said
substrate to produce a surface modification within said wells and a
surface modification surrounding said wells; (b) polishing said
substrate to produce a polished surface surrounding said wells ,
whereby said surface modification surrounding said wells is removed
and said surface modification within said wells is retained, and
(c) depositing a biopolymer onto said substrate, wherein higher
affinity of said surface modification within said wells compared to
said polished surface facilitates localization of said biopolymer
within said wells.
9. The method of claim 8, wherein said polishing comprises removing
a first portion of a layer to expose a second portion of the
layer.
10. The method of claim 8, wherein said substrate comprises two or
more layers.
11. The method of claim 10, wherein a first layer comprises
silicon.
12. The method of claim 10, wherein a second layer comprises
silicon oxide.
13. The method of claim 11, wherein said first layer comprises an
inner layer of said substrate corresponding to a bottom of a
well.
14. The method of claim 12, wherein said second layer comprises an
outer layer of said substrate corresponding to at least a portion
of the sides of a well.
15. The method of claim 8, wherein said deposited biopolymer is
covalently attached to said modified substrate within said
wells.
16. A method of fabricating a microarray, comprising: (a)
functionalizing a plurality of features on a substrate to create
discrete single biopolymer anchor sites, said functionalization
comprising: (1) contacting said substrate with a reagent reactive
with said substrate to produce a modified substrate; (2) applying a
protecting reagent to discrete sites on said modified substrate,
said discrete sites having an area of between about 5-40 nm.sup.2;
(3) modifying said reagent located in unprotected regions of said
modified substrate surrounding said discrete sites, thereby
rendering said regions unreactive to a target biopolymer, and (4)
removing said protecting reagent to produce a substrate having a
plurality of discrete functionalized features, and (b) attaching a
single target biopolymer to one or more of said discrete
functionalized features.
17. The method of claim 16, wherein a target biopolymer having a
different target nucleotide sequence is attached at each of said
discrete functionalized features.
18. The method of claim 17, wherein each different single target
biopolymer further comprises a common nucleotide priming site.
19. The method of claims 17, further comprising, (c) attaching a
plurality of secondary biopolymers to said regions surrounding said
discrete sites, wherein said secondary biopolymers comprise a
common nucleotide primer sequence complementary to said common
nucleotide priming sequence.
20. The method of claim 19, further comprising amplifying each of
said target biopolymers, thereby forming a plurality of biopolymers
comprising said target nucleotide sequence at said region
surrounding each of said discrete functionalized features.
21. A method of fabricating a microarray, comprising: (a)
contacting a substrate having a plurality of discrete nanochannels
with a plurality of biopolymers, said nanochannels having a length
and diameter sufficient for entry of only a single biopolymer
molecule; (b) applying an electric potential to said substrate
sufficient to translocate said single biopolymer molecules into
said nanochannels to produce a substrate containing a plurality of
single biopolymer molecules each in said plurality of discrete
nanochannels, and (c) transferring said plurality of single
biopolymer molecules contained in said plurality of discrete
nanochannels to a solid support.
22. The method of claim 21, wherein said transferring step
comprises reversing the polarity of said electric potential.
23. The method of claim 21, wherein said transferring step
comprises subjecting said substrate containing a plurality of
single biopolymers each in said plurality of discrete nanochannels
to centrifugal force.
Description
BACKGROUND OF THE INVENTION
[0001] The present invention relates generally to genomics
analysis, and more specifically to methods for producing arrays for
high throughput genomics analysis.
[0002] The task of cataloguing human genetic variation and
correlating this variation with susceptibility to disease is
daunting and expensive. A single genome sequence has a price tag of
approximately $10-20 million using traditional methods. A drastic
reduction in this cost is imperative for advancing the
understanding of health and disease. The near term goal in genomics
analysis is to resequence the human genome at a cost 3-4 orders of
magnitude less, or about $100,000 dollars. The ultimate goal is to
reduce this cost to $1000 dollars per genome. A reduction in
sequencing costs to less than $100,000 per genome will require a
number of technical advances in the field. Fortunately, the same
basic principles of readout parallelization and sample multiplexing
that proved so powerful for gene expression and SNP genotyping
analysis are also being successfully applied to large-scale
sequencing. Technical advances that stand to facilitate the
$100,000 genome analysis, or less, include: (1) library generation;
(2) highly-parallel clonal amplification and analysis; (3)
development of robust cycle sequencing biochemistry; (4)
development of ultrafast imaging technology; and (5) development of
algorithms for sequence assembly from short reads.
[0003] The ability to specify the content of the DNA library in a
targeted manner is extremely useful for a number of applications.
In particular, the ability to resequence all exons in the cancer
genome would greatly facilitate the discovery of new cancer genes.
The comprehensive resequencing of cancer genomes is a major
objective of the Cancer Genome Atlas Project
(cancergenome.nih.gov/index.asp) and would greatly benefit from a
reduction in sequencing price. Given the near term objective of the
$100,000 genome, it should be feasible to resequence all
approximately 250,000 exons in the genome for about $1000 per
sample. A good method for creating a targeted library of the
250,000 exons from the genome is important. The approach of
single-plex PCR for each exon is clearly cost prohibitive. As such,
parallelization of the sample preparation is of paramount
importance in reducing sequencing costs.
[0004] In addition to library generation, the creation of clonal
amplifications in a highly-parallel manner is also essential to
cost-effective sequencing. Sequencing is generally performed on
clonal populations of DNA molecules traditionally prepared from
plasmids grown from picking individual bacterial colonies. In the
human genome project, each clone was individually picked, grown-up,
and the DNA extracted or amplified out of the clone. In recent
years, there have been a number of innovations to enable
highly-parallelized analysis of DNA clones particularly using
array-based approaches. In the simplest approach, the library can
be analyzed at the single molecule level which by its very nature
is clonal. Generally, DNA molecules are captured on a solid phase
surface such that individual species are spatially separated from
each other and distinguishable in subsequent cycles of sequencing.
Current capture methods are random in nature and rely, at least in
part, on precise control of conditions to allow an optimal density
of DNA molecules to attach to the surface. Improper conditions can
lead to overcrowding such that individual species are not
distinguishable or, alternatively, high vacancy rates that can
reduce the information gained per run to a level that wastes
expensive sequencing reagents.
[0005] Thus, there exists a need to develop methods to improve
nucleic acid capture for genomics analysis and provide more cost
effective methods for sequence analysis. The present invention
satisfies this need and provides related advantages as well.
SUMMARY OF THE INVENTION
[0006] The invention is directed to a method of fabricating a
microarray. The method includes: (a) providing a substrate having
at least two layers of different chemical reactivity, wherein a
well in an outer layer exposes an inner layer; (b) contacting the
substrate with a first reagent specifically reactive with the outer
layer to produce a first modified layer; (c) contacting the
substrate with a second reagent specifically reactive with the
inner layer of the substrate to produce a modified inner layer,
wherein the modified inner layer has a higher affinity for a
biopolymer than the modified outer layer, and (d) depositing the
biopolymer onto the modified inner layer within the well, wherein
the higher affinity of the modified inner layer facilitates
localization of the biopolymer onto the well. Methods of
fabricating a microarray which include polishing a substrate or
functionalizing a plurality of features with a reactive reagent
also are provided. A method of fabricating a microarray which
includes loading a plurality of discrete nanochannels is
additionally provided.
BRIEF DESCRIPTION OF THE DRAWINGS
[0007] FIG. 1 is a schematic diagram showing one embodiment of a
polishing method for surface pattern microfabrication of substrate
features having different biopolymer affinities compared to the
surrounding substrate.
DETAILED DESCRIPTION OF THE INVENTION
[0008] This invention is directed to methods for producing optimal
substrates for analysis of populations of molecules such as nucleic
acids. The substrates of the invention increase the parallelization
of current substrates allowing for increased efficiency in
multiplexing and the simultaneous analysis of a large number of
different species of molecules. The methods employ substrate
treatments or procedures that allow for the placement of molecular
species at defined sites. Thus, the substrate is ordered with
respect to the positions of sites available for attachment of
nucleic acids or other desired molecules. Molecules can be
contacted with the substrate under conditions that allow individual
molecular species to populate the attachment sites in a
predetermined or random manner. As such, there are at least two
types of order for a substrate described herein, the first relating
to the spacing and relative location of attachment sites and the
second relating to predetermined knowledge of the particular
species of molecule that attaches at a particular site. For
example, a substrate can be contacted with a population of nucleic
acids under conditions where the nucleic acids attach at sites that
are ordered with respect to their relative locations but random
with respect to knowledge of the sequence for the nucleic acid
species present at any particular site. A particularly useful
characteristic of controlled placement of single molecular species
is that it allows for optimal packing of substrates with target
probes or samples while minimizing overlap of different molecular
species.
[0009] In one specific embodiment, the invention utilizes
differential treatments of substrate features and surrounding areas
to promote the deposition of single molecular species at each
feature site. The features include planar surfaces or surfaces
having different physical shapes such as substrate wells or raised
features. The treatment in the center of the feature is selected to
promote deposition of target molecules whereas the treatment
surrounding the center, such as the sides of wells, substantially
precludes deposition of target molecules. The differential chemical
and/or physical treatments include chemical modifications of the
surface to produce different affinities for target molecules at the
feature compared to the surrounding area. In another specific
embodiment, the invention utilizes nanomaterials to efficiently
produce controlled patterns having single molecular species at
defined placements.
[0010] As used herein, the term "substrate" is intended to mean a
solid support. The term includes any material that can serve as a
solid or semi-solid foundation for creation of features such as
wells for the deposition of biopolymers, including nucleic acids,
polypeptide and/or other polymers. A substrate of the invention is
modified, for example, or can be modified to accommodate attachment
of biopolymers by a variety of methods well known to those skilled
in the art. Exemplary types of substrate materials include glass,
modified glass, functionalized glass, inorganic glasses,
microspheres, including inert and/or magnetic particles, plastics,
polysaccharides, nylon, nitrocellulose, ceramics, resins, silica,
silica-based materials, carbon, metals, an optical fiber or optical
fiber bundles, a variety of polymers other than those exemplified
above and multiwell microtier plates. Specific types of exemplary
plastics include acrylics, polystyrene, copolymers of styrene and
other materials, polypropylene, polyethylene, polybutylene,
polyurethanes and Teflon.TM.. Specific types of exemplary
silica-based materials include silicon and various forms of
modified silicon.
[0011] Those skilled in the art will know or understand that the
composition and geometry of a substrate of the invention can vary
depending on the intended use and preferences of the user.
Therefore, although planar substrates such as slides, chips or
wafers are exemplified herein in reference to microarrays for
illustration, given the teachings and guidance provided herein,
those skilled in the art will understand that a wide variety of
other substrates exemplified herein or well known in the art also
can be used in the methods and/or compositions of the
invention.
[0012] As used herein, the term "feature" is intended to mean a
discrete physical element or discrete physical trait of a
substrate. A feature includes a location, position or site
occupied, or available for occupancy on a substrate, or a
distinguishable physical, structural or chemical trait of
substrate. Therefore, a feature is a component of a substrate that
provides physical or functional separability. A feature separates a
biopolymer deposited at a first feature from a biopolymer deposited
at a second feature. Examples of features include spots contained
on a slide, chip or other planar substrate, a patterned substrate
and separable chemical moieties or reactive groups.
[0013] A patterned substrate can include, for example, wells etched
into a slide or chip. The pattern of the etchings and geometry of
the wells can take on a variety of different shapes and sizes so
long as such features are physically or functionally separable from
each other. Particularly useful substrates having such structural
features are patterned substrates that can select the size of solid
support particles such as microspheres. An exemplary patterned
substrate having these characteristics is the etched substrate used
in connection with BeadArray technology (Illumina, Inc., San Diego,
Calif.). Further examples, are described in U.S. Pat. No.
6,770,441, which is incorporated herein by reference.
[0014] As used herein, the term "nanochannel" is intended to mean a
cylindrical structure having an inner diameter measured on a
nanometer (nm) scale. Exemplary internal diameters applicable to
the methods of the invention include, for example, ranges between
about 0.5-500 nm. Useful internal diameters include, for example,
ranges between 1.0-100 nm. Particularly, useful internal diameters
for the methods of fabricating a microarray of the invention
include, for example, ranges between about 5.0-20 nm. Internal
diameters of about 10 nm are exemplified herein with reference to
nucleic acid biopolymers. In particular embodiments, nanochannels
have a diameter that accommodates no more than a single biopolymer
of a particular type such as a single stranded nucleic acid or
double stranded nucleic acid. Nanochannels can be composed of a
wide variety of materials well known in the art of material science
and include, for example, silica, plastic, glass, metals, cladding
of a fiber optic and/or polymers.
[0015] As used herein, the term "affinity" or a grammatical
equivalent thereof, is intended to mean the attractive force
exerted between substances that causes them to enter into and/or
remain in combination. Therefore, when used in reference to the
attraction of a modified substrate layer to a biopolymer the term
is intended to refer to the strength at which a modified substrate
layer and a biopolymer associate. The measure of the strength of
association can be, for example, qualitative, relative, or
quantitative. The type of association can include, for example,
non-covalent interactions, covalent interactions. Specific examples
of non-covalent interactions include electrostatic forces, hydrogen
bonding and/or van der waal's forces. A specific example of a
covalent interaction includes chemical bond formation.
[0016] As used herein, the term "functionalize" or a grammatical
equivalent thereof, when used in reference to a substrate or
feature thereof is intended to mean a modification that changes a
chemical property of the referenced substrate or feature.
Modifications can include, for example, chemical and/or physical
alterations that confer a desired property, performance or
activity. Predetermined properties include, for example, chemical
or reactive specificity that confers a new activity onto the
referenced substrate or feature. Specific examples of chemical
modifications that can be used to functionalize a substrate or
feature to change the chemical or reactive specificity include
reacting silicon dioxide with trichloro alkyl silane or
trichlorosilane derivatized with a moiety other than alkyl such as
a hydrophilic moiety or a moiety that is reactive with nucleic
acids or other biopolymer. Further examples include, but are not
limited to, reacting a substrate with ammonia gas to create
diaminotriazine, reacting amorphous silicon with Grignard reagents
to substitute surface hydrogens with organic groups such as a
hydrophilic moiety or a moiety that is reactive with nucleic acids
or other biopolymer (see Ehara et al., Chemistry Letters 30:616
(2001). Self assembly of thiols on a gold surface can be used for
surface patterning including, for example, use of
6-(Ferrocenyl)hexanethiol for self-assembly on a gold surface.
Other modifications to substrates or features thereof well known in
the art such as chemical vapor deposition or atomic layer
deposition methods used for semiconductor and thin film research
can also be used and are included within the meaning of the term as
it is used herein.
[0017] As used herein, the term "polishing" is intended to mean
mechanical or chemical treatment of a substrate, or a portion
thereof, to remove a part of the substrate. Therefore, the term
includes removing a coat of a substrate, including a coat of a
layer of a substrate. Removal can be uniform or non-uniform. The
term includes, for example, rubbing, chafing, smoothing, or
otherwise treating a surface by the motion of applied pressure or
other frictional forces as well as developing, finishing or
refining the substrate to produce an altered surface of the
substrate. The resultant surface is referred to herein as a
"polished" surface. A direct polishing method can be used such that
an abrasive surface contacts the surface to be polished or indirect
polishing can be used such that a slurry or suspended aggregate is
contacted with the surface in a lapping process. Specific examples
of mechanical polishing include sanding, grinding or lapping.
Chemical polishing methods can also be used such as treatment with
acids such as hydrofluoric acid or bases such as sodium hydroxide.
Other methods well know in the art that can remove a part of a
substrate, including a part of a layer of a substrate, also are
included within the meaning of the term as it is used herein. One
exemplary polishing method for removing part of a substrate, and
reactive groups thereon, to yield either a deep or shallow well
patterned substrate is illustrated in FIG. 1. In particular
embodiments, polishing can exclude mechanical and chemical methods
of removing a photoresist.
[0018] As used herein, the term "microsphere," "bead" or "particle"
is intended to mean a small discrete particle as a solid support of
the invention. Populations of microspheres can be used for
attachment of populations of biopolymers such as nucleic acid
probes, other nucleic acids, polypeptides, ligands, and the like.
The composition of a microsphere can vary, depending on, for
example, the format, chemistry and/or method of attachment and/or
on the method of biopolymer synthesis, including nucleic acid
synthesis. Exemplary microsphere compositions include solid
supports, and chemical functionalities imparted thereto, used in
polynucleotide, polypeptide and/or organic moiety synthesis. Such
compositions include, for example, plastics, ceramics, glass,
polystyrene, methylstyrene, acrylic polymers, paramagnetic
materials, thoria sol, carbon graphite, titanium dioxide, latex or
cross-linked dextrans such as Sepharose, cellulose, nylon,
cross-linked micelles and Teflon.TM., as well as any other
materials that can be found described in, for example, "Microsphere
Detection Guide" from Bangs Laboratories, Fishers Ind.
[0019] The geometry of a microsphere also can correspond to a wide
variety of different forms and shapes. For example, microspheres
used as solid supports of the invention can be spherical,
cylindrical or any other geometrical shape and/or irregularly
shaped particles. In addition, microspheres can be, for example,
porous, thus increasing the surface area of the microsphere
available for probe or other nucleic acid attachment. Exemplary
sizes for microspheres used as solid supports in the methods and
compositions of the invention can range from nanometers to
millimeters or from about 10 nm-1 mm. Particularly useful sizes
include microspheres from about 0.2 .mu.m to about 200 .mu.m, with
from about 0.5 .mu.m to about 5 .mu.m being particularly
useful.
[0020] As used herein, the term "biopolymer" is intended to mean a
polymer corresponding to a chemical compound or composite of
chemical compounds formed by polymerization of monomeric subunits
in a biological system. Biopolymers include high or low molecular
weight polymer such as a macromolecule consisting of a few or of
many repeating monomers of relatively low molecular weight.
Particular classes of biopolymers include, for example, nucleic
acids, polypeptides, polysaccharides and lipids. Monomers of
macromolecules include, for example, nucleotides as the repeating
building blocks or subunits of nucleic acids, amino acids for
polypeptides, and carbohydrates for polysaccharide. Biopolymers can
be composed of naturally occurring monomers as well as
non-naturally occurring monomers including, for example, analogs,
derivatives and mimetics thereof. Accordingly, specific biopolymers
can be formed biosynthetically or by chemical synthesis. Polymers
formed by biosynthesis well known in the art other than those
exemplified above also are included within the definition of the
term as it is used herein. The invention is exemplified by specific
reference to nucleic acid biopolymers. However, those skilled in
the art will understand that the methods and processes of the
invention are equally applicable for producing microarrays having
optimal characteristics to any type of biopolymer well known in the
art.
[0021] As used herein, the term "nucleic acid" is intended to mean
a ribonucleic or deoxyribonucleic acid or analog thereof, including
a nucleic acid analyte presented in any context; for example, a
probe, target or primer. Particular forms of nucleic acids of the
invention include all types of nucleic acids found in an organism
as well as synthetic nucleic acids such as polynucleotides produced
by chemical synthesis. Particular examples of nucleic acids that
are applicable for analysis through incorporation into microarrays
produced by methods of the invention include genomic DNA (gDNA),
expressed sequence tags (ESTs), DNA copied messenger RNA (cDNA),
RNA copied messenger RNA (cRNA), mitochondrial DNA or genome, RNA,
messenger RNA (mRNA) and/or other populations of RNA. Fragments
and/or portions of these exemplary nucleic acids also are included
within the meaning of the term as it is used herein.
[0022] The compositions and methods set forth herein are equally
useful for analysis of large genome nucleic acid analytes, such as
those typically found in eukaryotic unicellular and multicellular
organisms, shorter nucleic acids such as cDNA as well as for
synthetic polynucleotides. Exemplary eukaryotic nucleic acids that
can be used in a method of the invention include, without
limitation, nucleic acids obtained from a mammal such as a rodent,
mouse, rat, rabbit, guinea pig, ungulate, horse, sheep, pig, goat,
cow, cat, dog, primate, human or non-human primate; a plant such as
Arabidopsis thaliana, corn, sorghum, oat, wheat, rice, canola, or
soybean; an algae such as Chlamydomonas reinhardtii; a nematode
such as Caenorhabditis elegans; an insect such as Drosophila
melanogaster, mosquito, fruit fly, honey bee or spider; a fish such
as zebrafish; a reptile; an amphibian such as a frog or Xenopus
laevis; a dictyostelium discoideum; a fungi such as pneumocystis
carinii, Takifugu rubripes, yeast, Saccharamoyces cerevisiae or
Schizosaccharomyces pombe; or a plasmodium falciparum. The methods
of the invention also can be used with nucleic acids from organisms
having smaller genomes such as those from a prokaryote such as a
bacterium, Escherichia coli, staphylococci or mycoplasma
pneumoniae; an archae; a virus such as Hepatitis C virus or human
immunodeficiency virus; or a viroid.
[0023] A nucleic acid can be isolated from one or more cells,
bodily fluids or tissues. Methods well known in the art can be used
to obtain a bodily fluid such as blood, sweat, tears, lymph, urine,
saliva, semen, cerebrospinal fluid, feces or amniotic fluid.
Similarly known biopsy methods can be used to obtain cells or
tissues such as buccal swab, mouthwash, surgical removal, biopsy
aspiration or the like. Nucleic acids also can be obtained from one
or more cell or tissue in primary culture, in a propagated cell
line, a fixed archival sample, forensic sample, fresh frozen
paraffin embedded sample or archeological sample.
[0024] Exemplary cell types from which nucleic acids can be
obtained include, without limitation, a blood cell such as a B
lymphocyte, T lymphocyte, leukocyte, erythrocyte, macrophage, or
neutrophil; a muscle cell such as a skeletal cell, smooth muscle
cell or cardiac muscle cell; germ cell such as a sperm or egg;
epithelial cell; connective tissue cell such as an adipocyte,
fibroblast or osteoblast; neuron; astrocyte; stromal cell; kidney
cell; pancreatic cell; liver cell; or keratinocyte. A cell from
which gDNA is obtained can be at a particular developmental level
including, for example, a hematopoietic stem cell or a cell that
arises from a hematopoietic stem cell such as a red blood cell, B
lymphocyte, T lymphocyte, natural killer cell, neutrophil,
basophil, eosinophil, monocyte, macrophage, or platelet. Other
cells include a bone marrow stromal cell (mesenchymal stem cell) or
a cell that develops therefrom such as a bone cell (osteocyte),
cartilage cells (chondrocyte), fat cell (adipocyte), or other kinds
of connective tissue cells such as one found in tendons; neural
stem cell or a cell it gives rise to including, for example, a
nerve cells (neuron), astrocyte or oligodendrocyte; epithelial stem
cell or a cell that arises from an epithelial stem cell such as an
absorptive cell, goblet cell, Paneth cell, or enteroendocrine cell;
skin stem cell; epidermal stem cell; or follicular stem cell.
Generally any type of stem cell can be used including, without
limitation, an embryonic stem cell, adult stem cell, or pluripotent
stem cell.
[0025] Methods for synthesizing polynucleotides are well known in
the art. Such methods can be found described in, for example,
Oligonucleotide Synthesis: A Practical Approach, Gate, ed., IRL
Press, Oxford (1984); Weiler et al., Anal. Biochem. 243:218 (1996);
Maskos et al., Nucleic Acids Res. 20(7):1679 (1992); Atkinson et
al., Solid Phase Synthesis of Oligodeoxyribonucleotides by the
Phosphitetriester Method, in Oligonucleotide Synthesis 35 (M. J.
Gait ed., 1984); Blackburn and Gait (eds.), Nucleic Acids in
Chemistry and Biology, Second Edition, New York: Oxford University
Press (1996), and in Ansubel et al., Current Protocols in Molecular
Biology, John Wiley and Sons, Baltimore, Md. (1999).
[0026] As used herein, the term "species" when used in reference to
a biopolymer is intended to mean one or more biopolymer molecules
having substantially the same monomer sequence. Therefore, a
biopolymer species includes a single biopolymer molecule or a
population of biopolymer molecules where all molecules within the
population have substantially the same primary sequence. The term
also is intended to refer to a biopolymer having concatenated
copies of substantially the same nucleotide sequence.
[0027] Biopolymer species or single molecules, for example, can be
attached to a substrate of the invention using any of the methods
exemplified herein as well as a variety of other methods well known
in the art. Such methods include for example, attachment by direct
chemical synthesis onto the solid support, chemical attachment,
photochemical attachment, thermal attachment, enzymatic attachment,
enzymatic synthesis and/or absorption. These and other methods are
will known in the art and are applicable for attachment of
biopolymers, including nucleic acids, in any of a variety of
formats and configurations. The resulting biopolymer species or
molecules can be attached to a substrate via a covalent linkage or
via non-covalent interactions as exemplified herein or through
other methods well known in the art.
[0028] As used herein, the terms "nucleic acid clonal ball,"
"nucleic acid ball" or "DNA ball" are intended to mean a concatemer
of a nucleic acid sequence collapsed into a random coil
configuration. Therefore, a nucleic acid ball refers to multiple
copies of a DNA or RNA sequence linked end to end in a tandem
series that assumes a compacted configuration relative to its
linear or other extended configuration. Nucleic acid balls and
methods of preparing them are well known in the art and can
utilize, for example, enzymatic amplification reactions of a wide
variety of nucleic acid sources to produce concatenated repeats of
the amplification template. One particularly useful amplification
reaction is rolling circle amplification (RCA), which is used to
amplify a circular nucleic acid template. Such methods can be found
described in, for example, Shendure et al., Nature Rev. 5:335-344
(2004); Baner et al., Nucl. Acids Res. 26:5073-5078 (1998); Furuqi
et al., BMC Genomics 2:4 (2001); U.S. Patent application Ser. No.
60/878,792, and in U.S. Pat. No. 6,355,431, each of which is
incorporated herein by reference. The product of amplification is a
single concatemer having multiple copies of circle sequence
complements. This concatemer will collapse into a random coil
configuration or other more condensed structure to form a nucleic
acid ball when placed in, for example, a high salt buffer. It will
be understood that these "balls" need not be perfectly spherical
and can include other globular or packed conformations. One
particular form of a nucleic acid ball useful in the methods of the
invention is a ball having a desired or predetermined dimension.
The number of amplified template copies can be modulated to obtain
a desired size or to generate, for example, a sufficient number of
copies for efficient subsequent analysis such as for sequencing,
for example.
[0029] The number of template copies or amplicons that can be
produced can be modulated by appropriate modification of the
amplification reaction including, for example, varying the number
of amplification cycles run, using polymerases of varying
processivity in the amplification reaction and/or varying the
length of time that the amplification reaction is run, as well as
modification of other conditions known in the art to influence
amplification yield. Generally, the number of copies of a nucleic
acid template is at least 1, 10, 100, 200, 500, 1000, 2000, 3000,
4000, 5000, 6000, 7000, 8000, 9000 and 10,000 copies, and can be
varied depending on the particular application.
[0030] Methods for generating circular templates from linear or
other non-circular template nucleic acids for RCA amplification,
for example are will known in the art. One exemplary method is to
enzymatically circularize a template nucleic acid molecule using,
for example, a ligase. Exemplary ligases include a single stranded
DNA ligase, such as CircLigase.TM. (Epicentre), a double stranded
DNA ligase and an RNA ligase, which can be selected based on the
type of nucleic acid molecule to be circularized, for example,
single or double stranded DNA or RNA. A splint ligation reaction in
which a splint acts as a template to hybridize two ends of a sample
nucleic acid such that the ends are juxtaposed for ligation to
circularize the sample nucleic acid molecule can also be used.
[0031] Amplicons can be compacted prior to deposition on a
substrate of the invention, such as an array. Methods of compacting
amplicons are known in the art (for example, as described by
Bloomfield, Curr Opin Struct Biol 6(3): 334-41 (1996)) and
exemplified further herein. For example, an alcohol or polyamine
such as spermine or spermidine can be used. A compacted nucleic
acid will have a structure that is more densely packed than the
structure of the nucleic acid in the absence of a compacting agent
or compacting condition and the structure will typically resemble a
ball or globule. The generation of such compacted nucleic acid
balls is useful for distribution at discrete locations on an array,
as discussed further below in more detail. Various methods can be
used to generate balls of a desired size, for example, using
various compacting techniques and/or varying the number of copies
in an amplicon. Generally, the compacted amplicons have an average
diameter or width ranging from about 0.1-5.0 for example, about 0.1
.mu.m, about 0.2 .mu.m, 0.3 .mu.m, about 0.4 .mu.m, about 0.5
.mu.m, about 0.6 .mu.m, about 0.7 .mu.m, about 0.8 .mu.m, about 0.9
.mu.m, about 1 .mu.m, about 1.5 .mu.m, about 2 .mu.m, about 2.5
.mu.m, about 3 .mu.m, about 3.5 .mu.m, about 4 .mu.m, about 4.5
.mu.m and about 5 .mu.m or more. Those skilled in the art will
understand that nucleic acid balls of the invention also include
all sizes in between and outside of the above exemplary size range.
Such nucleic acid balls can then be manipulated and analyzed using
a number of technology formats, as exemplified further herein.
[0032] If desired, the amplicons compacted as nucleic acid balls
can be opened after or during use in a method of the invention,
including opened following deposition on a substrate in an array.
As used herein, an amplicon or DNA ball that is "opened" is one
that has been treated to allow access of reagents for subsequent
reactions, such as by partial or complete de-compaction. For
example, the methods of the invention can be particularly useful
for parallel sequence analysis of multiple nucleic acid molecules
distributed on an array. In this specific embodiment, the amplicons
distributed on an array should be accessible to reagents such as
primers, nucleotides, buffers and enzymes such as polymerases or
ligases as used in a particular sequencing method, so that a
sequencing reaction can be carried out. Thus, a compacted amplicon
that is inaccessible or partially accessible due to being in the
form of a DNA ball or other compacted structure can be rendered
more accessible by "opening" the compacted amplicon. Methods for
"opening" nucleic acid molecules are well known in the art and
include, for example, removal of compacting agents. Such an
"opening" of an amplicon is analogous to, although not limited to
the same mechanism as, the melting of regions of chromatin for
expression of a particular region of a chromosome. It is understood
that such methods of "opening" a compacted nucleic acid molecule
need not result in a detectably different size of the compacted
amplicon, only that the amplicon be rendered more accessible to
reagents for a subsequent reaction.
[0033] As used herein, the term "plurality" is intended to mean a
population of two or more different members. Pluralities can range
in size from small, medium, large, to very large. The size of small
plurality can range, for example, from a few members to tens of
members. Medium sized pluralities can range, for example, from tens
of members to about 100 members or hundreds of members. Large
pluralities can range, for example, from about hundreds of members
to about 1000 members, to thousands of members and up to tens of
thousands of members. Very large pluralities can range, for
example, from tens of thousands of members to about hundreds of
thousands, a million, millions, tens of millions and up to or
greater than hundreds of millions of members. Therefore, a
plurality can range in size from two to well over one hundred
million members as well as all sizes, as measured by the number of
members, in between and greater than the above exemplary ranges. An
Exemplary number of features within a microarray includes a
plurality of about 500,000 or more discrete features within 1.28
cm.sup.2. Exemplary nucleic acid pluralities include, for example,
populations of about 1.times.10.sup.5, 5.times.10.sup.5 and
1.times.10.sup.6 or more different nucleic acid species.
Accordingly, the definition of the term is intended to include all
integer values greater than two. An upper limit of a plurality of
the invention can be set, for example, by the theoretical diversity
of nucleotide sequences in a nucleic acid sample of the
invention.
[0034] The term "each," when used in reference to individual
members within a plurality, is intended to recognize one or more
members in a population. Unless explicitly stated otherwise the
term "each" when used in this context is not intended to require or
necessarily recognize all of the members in a plurality. Thus,
"each" is intended to be an open term.
[0035] In one specific embodiment a welled substrate is
differentially treated such that the wells favor deposition of
target biopolymers and the well sides and substrate surface are
refractory to deposition. In this specific embodiment, the
substrate includes a silicon BeadChip (Illumina, Inc., San Diego,
Calif.) having a silicon layer covered by a silicon dioxide layer.
In this embodiment, the silicon layer can be treated so as to
produce sites for the ligation of the 3' end of the DNA target
sequence and the SiO.sub.2 layer can be treated so as to repel or
resist attachment or binding of DNA. This provides for the top
surface of the substrate as well as the side wall of the individual
well cores to repel DNA attachment while the bottom of the well
cores can attach to DNA molecules. A SiO.sub.2 depth of about 0.5
to 1.0 .mu.m is used to achieve a mean well diameter of about 1
.mu.m. Such a substrate is uniquely suited for favorable deposition
of amplicons such as nucleic acid balls to produce an array having
a single biopolymer species at each feature of the substrate.
[0036] Accordingly, the invention provides a method of fabricating
a microarray. The method includes: (a) providing a substrate having
at least two layers of different chemical reactivity, wherein a
well in a second layer exposes a first layer; (b) contacting the
substrate with a first reagent specifically reactive with the first
layer to produce a first modified layer; (c) contacting the
substrate with a second reagent specifically reactive with the
second layer of the substrate to produce a second modified layer,
wherein the first and second modified layers have different
affinities for a biopolymer, and (d) depositing the biopolymer onto
the first modified layer within the well, wherein the different
affinities of the first and second modified layers facilitate
localization of the biopolymer onto the first modified layer.
[0037] In one aspect of the invention, the methods of fabricating a
microarray generate a substrate feature having a higher affinity
for a biopolymer than the substrate surface or area surrounding the
feature. Deposition of biopolymer on substrates prepared according
to this method of the invention inherently concentrates the
biopolymer at each feature of the substrate due to differential
attraction at the feature compared to its surrounding. Additional
procedures also can be implemented to further facilitate
localization of deposited biopolymers at each feature while
maintaining the surrounding substrate devoid of biopolymer.
Following the teachings and guidance provided herein, a variety of
different substrates and modifying reagents can be employed to
achieve a substrate having a differential affinity at one or more
features for a biopolymer compared to the surrounding area. As
described further below, the surrounding area of a substrate
feature can be, for example, the perimeter of a feature, including
the perimeter of a planar feature, a structural feature and/or the
sides of a well or cylindrical feature. These surrounding areas can
be maintained or modified to have, for example, hydrophobic
characteristics and, therefore, a lower affinity for a biopolymer
compared to substrate feature.
[0038] The methods of invention for fabricating a microarray can
employ a wide variety of substrate types and/or substrate forms.
Various different types of substrate materials have been
exemplified previously. These and other materials well known in the
art can be used in the methods of fabrication of the invention. For
illustration purposes, various methods are described herein with
reference to silicon or glass substrates. However, given the
teachings and guidance provided herein, those skilled in the art
will understand that the methods are equally applicable to other
substrates having similar functional characteristics and a
compatible chemistry for producing differential reactivities or, as
with other embodiments of the invention, differential
functionalization, albeit such other substrates can have a
different structure and/or material makeup.
[0039] In this aspect of the invention, a substrate is provided for
construction of a microarray having at least two layers of
different chemical reactivity. In particular embodiments, the first
layer has an affinity for the biopolymer type that is to be
deposited onto the substrate. It is understood that reference to a
first layer or to a second layer refers to different layers of the
substrate and is not intended to connote a particular order or
orientation of the referenced layers unless such order or
orientation is explicitly described herein. Similarly, reference to
a first layer or to a second layer also is not intended to connote
a particular location or require a particular order for performance
of a step in a method of the invention unless such location or
order of performance is explicitly described herein. Therefore,
reference to a first, second or other substrate layer is intended
to distinguish one referenced layer from another referenced layer,
or layers, with respect to the reference activity or any referenced
relative orientation. The term "layer" as it is used herein is
intended to have its plain and ordinary meaning, namely, a
thickness of a substance lying over, under, surrounding or adjacent
to another substance. The substances of two or more layers can be
the same and the demarcation between layers empirical, or the
substances of two or more layers can differ, thus producing an
actual boundary between each layer.
[0040] The substrate can be, for example, silicon or other silica
or silicates, glass, plastics, polysaccharides, nylon,
nitrocellulose resins, carbon and metals or other solid support as
exemplified previously and includes a first layer having a first
chemical reactivity. As described below, a substrate of the
invention having at least two layers of different chemical
reactivity can be fabricated anew or obtained from sources well
known in the art. In this aspect of microfabrication of the
invention, the substrate should have at least two layers of
different chemical reactivity or the substrate should be amenable
to modification to exhibit at least two layers having different
chemical reactivities. Therefore, substrates of the invention
include solid supports employed in the art as microarrays.
[0041] For fabrication of a microarray, features of the selected
substrate can be in the form of a well, an etched feature or other
concave, conical or cylindrical feature in a substrate. Methods for
preparing concave substrate features such as wells are well known
in the art. One particularly useful method is acid etching a
substrate to produce micrometer scale wells as described in, for
example, Michael et al., Anal. Chem. 70:1242-48 (1998) and in U.S.
Pat. Nos 6,023,540, and 6,327,410. Other methods for creating wells
in a substrate include, for example, a variety of other etching
methods, imprinting, stamping, ablating, ion bombardment and the
like such as described in, for example, U.S. Pat. Nos. 6,942,968;
6,770,441, and 3,666,527. Substrates available in the art having
etched features or wells include, for example, the etched substrate
used in connection with the fiber optic Sentrix array matrix and
the BeadChip (both from Illumina, Inc., San Diego, Calif.),
injection molded plastic surfaces or etched substrates made by
methods available from Boehringer Ingelheim microParts GmbH
(Dortmund, Germany).
[0042] Concave features such as etched wells can be constructed in
a substrate to various desired sizes including, for example, well
diameter, feature width, feature length and/or depth using methods
well known in the art. Particularly useful sizes are wells having
sufficient breadth and depth to hold a single microsphere of a
certain size or a single nucleic acid ball of a certain size. Wells
designed and constructed to hold single microspheres and/or nucleic
acid balls allows for the placement of a single target biopolymer
species at each feature of the microarray. The number of copies of
each deposited biopolymer can be varied depending on the need of
the user. Alternatively, wells also can be constructed to hold
multiple microspheres and/or nucleic acid balls of particular
dimensions.
[0043] Generally, concave features such as wells will have sizes
ranging from, for example, about 0.05-20 .mu.m in depth although
wells depths larger that 20 .mu.m can routinely be constructed
using the methods exemplified above. Concave features such as wells
having depths between, for example, 0.1-10 .mu.m are particularly
useful for depositing single microspheres or nucleic acid
molecules. Accordingly, the depth of a feature or well on a
substrate of the invention can include, for example, an average
depth of, for example, about 0.1 .mu.m, about 0.2 .mu.m, 0.3 .mu.m,
about 0.4 .mu.m, about 0.5 .mu.m, about 0.6 .mu.m, about 0.7 .mu.m,
about 0.8 .mu.m, about 0.9 .mu.m, about 1 .mu.m, about 1.5 .mu.m,
about 2 .mu.m, about 2.5 .mu.m, about 3 .mu.m, about 3.5 .mu.m,
about 4 .mu.m, about 4.5 .mu.m, about 5 .mu.m, about 5.5 .mu.m,
about 6.0 .mu.m, about 6.5 .mu.m, about 7.0 .mu.m, about 7.5 .mu.m,
about 8.0 .mu.m, about 8.5 .mu.m, about 9.0 .mu.m, about 9.5 .mu.m
and 10.0 .mu.m or more. Those skilled in the art will understand
that well depths of the invention also include all sizes smaller,
in between and greater than the above exemplary depths.
[0044] Well breadth such as diameter, width and/or length,
depending on the shape of the feature, also generally will be
within the above size ranges. Particularly useful well breadths for
holding single microspheres or nucleic acid balls include diameters
of, for example, about 0.1-10 .mu.m as described above. As with the
well depths exemplified above, the well breadth also can be altered
depending on the need and particular application. For example, it
may be desirable to use microspheres larger than the above
exemplified depths and/or breadths. In general, microspheres can
range from, for example, about 0.1 .mu.m to about 1.0 mm although
microspheres of about 0.2-200 .mu.m can be more useful, with
microspheres of about 0.5-5 .mu.m being particularly useful. Given
the teachings and guidance provided herein, those skilled in the
art will understand that the depth, diameter, width and/or length
of concave features such as wells can be generated to suit any size
microsphere and/or nucleic acid. Similarly, those skilled in the
art also will understand that the depth, diameter, width and/or
length of a concave feature such as a well can be generated to
have, for example, a microsphere and/or nucleic acid ball,
completely within the well, partially protruding above the well rim
or mostly protruding above the well rim.
[0045] In addition to the first layer and concave features of a
substrate, in this particular embodiment a substrate also will
contain at least a second layer. This second layer can form a
coating on a first layer or can be an outer layer covering a first
layer that is configured as an inner layer. In general, the second
layer will consist of a substance having a different chemical
reactivity compared to the first layer. Useful second layers are
substances that have, or can be modified to have, a lower affinity
for the biopolymer to be deposited onto the microarray compared to
the first layer. Particularly useful second layers are substances
that are refractory or repulsive to the deposited biopolymer.
Methods for generating layers having different chemical
reactivities and methods of differentially modifying one layer
compared to other layers for creating different affinities for
deposited biopolymers are described further below.
[0046] Wells are constructed or oriented in a substrate to
penetrate through one layer so as to expose the other layer. For
example, a substrate having at least two layers will have wells of
sufficient depth or length to extend through the second layer into
the first layer. Extension and exposure of the first layer unmasks
and allows access to the different chemical reactivity of the first
layer. It will be understood by those skilled in the art that the
geometry and/or shape of the well in the second layer need not be
identical to the geometry or shape of the well at its intersection
or boundary with the first layer or at any portion of the well
extending or terminating in the first layer. Similarly, the area of
an exposed first layer need not be symmetrical and/or proportional
to, for example, the circumference, diameter, shape, length or
volume of the well penetrating the second layer. Rather, exposure
is sufficient if there is sufficient surface area of the first
layer to allow access of at least one chemical reactivity different
from the second layer. In certain embodiments, efficiency in
construction and increased stability of the resultant microarray
can be enhanced by exposing a larger first layer surface area, up
to proportions approximate in size to the circumference or surface
area of the well at the first/second layer interface. Those skilled
in the art will understand that smaller first layer exposures can
be useful where single biopolymer molecules are to be deposited
compared to larger first layer exposures where, for example,
nucleic acid balls or biopolymer-containing microspheres are to be
deposited.
[0047] In various embodiments, the orientation of the substrate
wells is orthogonal to the at least first and second layers.
However, such an orientation is not required to achieve
modification of the first and second layers of the substrate to
produce different affinity characteristics for a biopolymer.
Rather, exposure of at least the different chemical reactivity of
the first layer is well sufficient irrespective of orientation.
Therefore, substrate wells can be any desired orientation with
respect to the at least first and second layers so long as a
biopolymer can be deposited in the well and contact the first layer
following modification as described further below.
[0048] Substrates of the invention also can employ more than two
layers resulting in multilayer microarrays. In such fabrications,
some or all of the different layers can be used to produce a
distinct chemical reactivity. Multiple layers of more than two are
particularly useful because they impart flexibility in selection of
the chemistry and type of biopolymer that can be applied to a
microarray. More than two layers having different chemical
reactivities also allow for the fabrication of a single general
microarray that can be instantly tailored to accommodate a
particular need without needing to design and fabricate new
substrates having particular chemical reactivities. For example, a
desired chemical reactivity can be generated in such general
utility multilayered substrates by simply creating wells having a
depth that will expose a layer having the desired chemical
reactivity.
[0049] Therefore, in other embodiments of the invention, substrates
having more than at least two layers of different chemical
reactivity can be employed for the fabrication of microarrays. For
example, microarrays can be constructed to contain 2, 3, 4 or 5 or
more layers. The number of different chemical reactivities can
range from at least two to the number of layers in the substrate or
more. Achieving a number of different chemical reactivities greater
than the number of substrates can be accomplished by, for example,
partitioning one or more layers into two or more different chemical
reactivities. For example, a first portion of a first layer can
exhibit a first chemical reactivity and a second portion of the
first layer can exhibit a second chemical reactivity due to
selective treatment of one portion over another. Layering and
apportioning different chemical reactivities can be used to achieve
variety of different functionalities in one substrate. Any or all
of the different functionalities can be selected for fabrication
using the methods of the invention to yield a versatile microarray
having a wide variety of different biopolymer applications.
[0050] Where more than two layers are utilized in a substrate of
the invention, the actual depth or degree of intrusion into one or
more different layers is determined by the user. Well depths can be
differentially fabricated to intrude into different layers for
alternative modifications on the same substrate. Accordingly, in
this exemplary embodiment it is possible to construct a microarray
exhibiting a variety of different affinities for one type of
biopolymer or construct a microarray exhibiting a variety of
different affinities each for a different type of biopolymer. A
wide variety of formats for such a multifunctional microarray
exist. For example, such substrates can be used to produce
microarrays having the same type of biopolymer deposited at each
well. Alternatively, a multifunctional substrate can be used to
efficient construction of microarrays capable of attaching
multiple, different types of biopolymers.
[0051] Methods for generating layers having different chemical
reactivities and methods of differentially modifying one layer
compared to other layers for creating different affinities for
deposited biopolymers are applicable to the particular substrates
of the invention as described herein. For example, in the specific
embodiment of a two layer substrate, one layer can consist of
silicon (Si) and the second layer can be silicon oxide (SiO.sub.2).
Each layer exhibits a different chemical reactivity which can be
used to differentially modify the first layer compared to the
second layer. Alternatively, a substrate having a single layer of
the same chemical composition can be employed where a portion of
the substrate is first modified to create at least a second layer.
For example, a single silicon substrate can be employed where a
portion such as the top, bottom, side or a subregion of one or more
of these edges is modified to silicon dioxide. This preparatory
modification thus produces a two layer substrate for use in a
fabrication method of the invention. In like fashion, any of a
variety of such preparatory procedures can be implemented to
generate a substrate having the desired number of layers and/or
different chemical reactivities.
[0052] The methods of the invention produce a microarray where at
least one of the two layers is modified to facilitate deposition of
a biopolymer at a feature. Various modification designs and
chemistries can be implemented to achieve differential affinity of
first and second layers of the substrate that will facilitate
biopolymer deposition. The substrates having at least two layers
allow for such implementation because a reagent having reactivity
with one layer and not the other allows for such differential
modifications. For example, a substrate which has low affinity for
a selected biopolymer can be modified at a first layer, such as a
layer found in a well or other feature, with moieties having higher
affinities compared to a second layer surrounding the first layer.
The different affinities will concentrate deposited biopolymers
onto the modified first layer whether it be wells or another type
of feature. In contrast, wells or other features on a substrate
which exhibit a favorable affinity for the deposited biopolymer can
be left unmodified at the first layer and a second layer, for
example, one found at the periphery of the well or feature, can be
modified with moieties having lower affinity for the biopolymer
compared to the first layer. This latter single modification design
also will result in two layers having different affinities that
will preferentially localize biopolymers into the wells or other
features at the first, unmodified layer.
[0053] In addition, a substrate having at least two layers of
different chemical reactivity also can be modified at each layer to
generate different affinities or to enhance different affinities at
the first layer. In this specific embodiment, each layer will be
modified and the resultant microarray will exhibit greater affinity
for a selected biopolymer at the well or other feature at the first
layer compared to the affinity for the selected biopolymer at the
second feature. Other modification designs include, for example,
starting with a single substrate composition and sequentially
modifying one layer with a reagent to produce a reactive layer and
modifying that reactive layer to generate a desired biopolymer
affinity. The second layer can then be modified similarly or
differently compared to the first layer to produce a second
reactive layer. Subsequent modification to generate a second
modified layer having an affinity different from the first modified
layer can then be implemented to generate first and second layers
that will facilitate deposition of a selected biopolymer into the
well or other feature.
[0054] When performed sequentially as exemplified above, the second
reagent and/or reactive layer can be the same or different compared
to the first reagent and/or reactive layer. However, the final
modified first and second layers will differ in affinity for a
deposited biopolymer. A specific example where first and second
reagents and/or reactive layers are the same is the conversion of
layers of silicon to silicon dioxide on a substrate. In this
embodiment, a first silicon layer can be converted to silicon
dioxide under conditions where a second silicon layer is not
converted. The silicon dioxide layer can then be modified to have a
desired affinity for a biopolymer. The second silicon layer can
subsequently be converted to silicon dioxide and further modified
to a layer having a different biopolymer affinity than the first
layer such as a reduced affinity or even repulsion of the
biopolymer. Modifications also can be performed where the first
layer is converted to the same or similar chemical specificity as
the second layer on substrates having separable first and second
layers that allow separate modifications of each layer. A specific
example is the modification of a substrate having a first silicon
layer and a second silicon dioxide layer. The first silicon layer
can be converted to silicon dioxide layer and subsequently reacted
with a first reagent specifically reactive, where the first layer
is a well. Placing the first reagent in the well to modify the
silicon dioxide layer leaves the second silicon dioxide layer
unaffected. The second silicon dioxide layer can then be modified
with a second reagent to produce a second modification.
[0055] With reference for purposes of illustration to the above two
layer substrate corresponding to silicon and silicon dioxide,
generation of first and second modified layers having different
affinities for a biopolymer will be exemplified using trichloro
alkyl silanes as a modifying reagent. Trichloro alkyl silane, or
chlorosilane, is specifically reactive with silicon dioxide and can
contain a wide variety of different chemical moieties to confer a
different chemical characteristic onto the silicon dioxide layer;
for example, reactivity with a biopolymer or other molecule, a
hydrophobic characteristic or, alternatively, a hydrophilic
characteristic. Conditions can be used in which silicon is inert or
substantially unreactive to trichloro alkyl silane or chlorosilane.
Thus, a layer of silicon is protected from modification by
trichloro alkyl silane or chlorosilane. If desired, the silicon
layer can first be converted to silicon dioxide and then
specifically reacted with a trichloro alkyl silane having a
chemical characteristic different from the first modified
layer.
[0056] Modification of a welled substrate having at least two
layers of different chemical reactivity to generate two modified
layers having different affinity for a target biopolymer can be
achieved by the utilization of inherent reactivity difference
between silicon and silicon oxide. A layer of silicon dioxide will
react with trichloro alkyl silanes to create a non-reactive
hydrophobic surface which will naturally repel hydrophilic nucleic
acids, polypeptides and other biopolymers. For example, a substrate
can include an outer layer of silicon dioxide that surrounds a well
that exposes an inner layer of the substrate. In this embodiment,
the hydrophobic surface surrounding the well promotes localization
of the target biopolymer into the welled inner layer having
characteristics that attract or bind the biopolymer such as
characteristics resulting from modification of the well as
described below to contain hydrophilic characteristics.
[0057] For example, the remaining silicon surface, or untreated
first layer inside the etched wells of the substrate, can be
converted to silicon dioxide to produce a reactive layer. The
reactive wells can then be treated with a second specifically
reactive reagent containing moieties having hydrophilic
characteristics or moieties specifically reactive with the target
biopolymer. The second specifically reactive reagent also can be a
trichloro alkyl silane albeit having chemical functional groups
different from the trichloro alkyl silane reagent used to modify
the second layer above which are attractive to the target
biopolymer and/or reactive with the target biopolymer. In the
former example, chlorosilane can have hydrophilic moieties which
will promote localization of the target biopolymer at the well
features due to non-covalent affinity interactions, particularly
where the modified surface second layer exhibits a hydrophobic
character. In the latter example, the chlorosilane can have, for
example, any of a variety of chemical moieties that can covalently
bond to the target biopolymer.
[0058] A variety of first and second reagents well known in the art
can be employed with various first and second substrate layers
having different chemical reactivities to produce first and second
modified layers having different affinities for a biopolymer. The
invention has been exemplified above with reference to a silicon or
silicon/silicon dioxide two layer substrate and first and second
trichloro alkyl silane reagents having different chemical moieties,
respectively, however, other first and second layers having
different chemical reactivities can include, for example, layers
produced by sequential reactions of a substrate with chlorosilanes
having different attached moieties, whereby differences in the
moieties confer different properties to each layer. Given the
teachings and guidance provided herein, those skilled in the art
will understand that these and other substrate layers and
compatible reagents specifically reactive thereto, such as those
well known in the art, are equally applicable in the fabrication
methods of the invention.
[0059] A variety of chemical moieties and functional groups can be
employed with, for example, the above-exemplified substrate layers
having various different chemical reactivities as well as others
well known in the art to generate modified layers having the
differential affinities for a target biopolymer as described
herein. Examples of hydrophilic moieties that can be attached to or
associated with trichloro alkyl silanes to confer, for example,
non-covalent affinity interactions onto the modified first layer
include, for example, positively charged moieties or negatively
charged moieties. Examples of hydrophobic moieties that can be
attached to or associated with trichloro alkyl silanes to confer,
for example, repulsive affinity interactions onto the modified
second layer include, for example, hydrocarbons or other noncharged
moieties.
[0060] In addition to the above-exemplified moieties conferring
non-covalent affinities onto a modified first or second layer,
reactive moieties for covalent attachment of biopolymers also can
be included or associated with a modifying reagents for creating
the at least two modified layers having different affinities.
Numerous reactive groups which are or can be associated with
modifying reagents of the invention are well known in the art that
have compatible chemistry for covalent bonding to a variety of
biopolymers. Particularly useful reactive groups can be activated
by, for example, light or other external stimulus such as exposure
to gas or a liquid reagent for controlled linkage to the substrate
at the first layer of a feature. Irradiation or other stimulation
of the substrate after placing biopolymers including, for example,
nucleic acid balls in the wells will immobilize the biopolymer on
the bottom of the wells at the first modified layer of the
substrate. Exemplary reactive groups include, for example, a
reactive silane group, a reactive vinyl group, amino group,
carboxylic acid, ester, thiol, aldehyde, hydrazine, carbodiimide,
maleimide, photoreactive group or the like.
[0061] Biopolymers can be deposited into substrates generated as
described above for the production of microarrays that are random
or ordered with respect to the identity of the biopolymer(s) at
each site. Multiple different species can be deposited at each
feature or, alternatively, single biopolymer species can be
deposited. Deposition of single species is particularly useful
because it allows for enhanced performance in a wide variety of
assays including, for example, sequencing by synthesis (SBS) of
nucleic acids. Deposition of biopolymers will be exemplified
further below with reference to multiple copies of a single nucleic
acid species at each feature.
[0062] As described previously, deposition of single nucleic acid
species can be accomplished by a variety of methods well known to
those skilled in the art. Particularly useful approaches include,
for example, deposition of microspheres having a single species of
attached nucleic acid sequence or the deposition of nucleic acid
balls. Placement of such complexes into the wells can be
facilitated by, for example, moving the nucleic acid balls using
alternating electrical charge applied at the ends of the substrate
covered with a thin layer of aqueous solution to suspend the balls.
A magnetic force can analogously be used when employing magnetic
microspheres, for example.
[0063] Nucleic acid balls and microspheres are particularly useful
vehicles for depositing single biopolymer species to a welled
substrate feature because they can be readily generated using well
known methods to create a physical structure of an individual
target species that can be physically manipulated to form an
ordered or random array. For example, rolling circle amplification
(RCA) of a DNA sequence can be used to form a concatemer of length
approximately 1000 times the length of the originating sequence,
and consisting of approximately 1000 complimentary repeats of the
originating sequence to form a molecular complex approximately 1
.mu.m in diameter. Such complexes are referred to herein as
"nucleic acid balls" and include, but are not limited to, those
described in U.S. Ser. No. 60/878,792, which is incorporated herein
by reference. Similarly, multiple copies of a target biopolymer
species can be synthesized on, or chemically attached to a
microsphere of a desired dimension. Any desired number of such
complexes including, for example, tens of thousands of such
complexes, can be prepared in parallel to each have a unique
biopolymer species. Subsequent deposition onto a substrate of the
invention will produce a microarray having a like number of
biopolymer species, each located at a single feature, to produce a
microarray of the invention. Because the biopolymer complexes will
have approximately the same charge, they will repel each other and
not clump together, instead assembling into the wells constructed
to be of similar size.
[0064] With respect to microspheres, once deposited into the wells
or, perhaps, deposited into the wells and covalently attached, they
are directly usable in any of a variety of analysis methods.
Nucleic acid balls similarly can be used directly in various
analysis methods. However, in some embodiments it is beneficial to
relax their compact structure and/or produce fragments containing a
single copy of the repeated sequence of the concatamer in the
nucleic acid balls (or the fragments can contain more than one copy
of the repeated sequence but fewer copies than the number found in
the unfragmented concatamer). Relaxing or fragmenting or both
relaxing and fragmenting condensed repeated structures can allow
greater access of for example, reagents and enzymes for subsequent
analysis.
[0065] For example, once nucleic acid balls are situated in wells,
with one biopolymer species per well, an enzyme can be introduced
to the microarray to cleave the balls into individual repeat
strands of the target biopolymer sequence. In addition to allowing
reagent access, this procedure will free 3' ends of the resultant
nucleic acid fragments and allow a further means for covalent
attachment to the bottom of the wells. The extent of fragmentation
of the target sequences can be controlled by, for example, varying
the temperature, time or reaction conditions of the digestion.
Following attachment, free, unattached biopolymer can be removed
by, for example, washing the microarray. Such immobilized nucleic
acids on the microarray can be employed in a wide variety of
nucleic acid analysis and detection procedures, including
sequencing by synthesis.
[0066] In another specific embodiment, the invention is directed to
a method for achieving optimal close packing of single biopolymer
species such as DNA clones, DNA balls or microspheres. As with the
previous exemplified method, substrates produced by this method
also are particularly useful in high throughput parallel analysis
such as DNA sequencing or digital gene expression. This specific
embodiment employs microfabrication methods to pattern a surface
with wells, specific biopolymer binding chemistry or both. In
particular, chemical functional groups can be introduced to defined
locations on a substrate surface, or in the interior of well
features during the fabrication processes. Following
microfabrication, the substrate is altered by chemical or
mechanical polishing as illustrated in FIG. 1 to generate a
non-reactive surface or a surface having lower affinity for the
biopolymer compared to the affinity of the feature. Also as
illustrated in FIG. 1, the amount of polishing can be metered to
maintain, for example, deep well features or to achieve
progressively shallower well features proportional to the amount of
polishing. For example, polishing can be performed to produce a
hydrophobic surface refractory to nucleic acids while the feature
retains its original hydrophilic character.
[0067] Therefore, the invention provides a method of fabricating a
microarray. The method includes: (a) contacting a substrate having
wells with a reagent reactive with said substrate to produce a
surface modification within said wells and a surface modification
surrounding said wells; (b) polishing said substrate to produce a
polished surface modification surrounding said wells, wherein said
surface modification surrounding said wells is removed and said
surface modification within said wells is retained, and (c)
depositing a biopolymer onto said substrate, wherein different
affinities of said surface modification within said wells and said
polished surface facilitate localization of said biopolymer within
said wells.
[0068] In this specific embodiment, polishing a surface of a
substrate can be employed to produce a substrate having features
exhibiting a different affinity for a biopolymer compared to the
surrounding substrate surface or perimeter of the feature.
Polishing is an efficient method to produce two different affinity
layers in lieu of, or in addition to, differentially reacting
layers having different chemical reactivities with modifying
reagents as described previously.
[0069] In one embodiment, a substrate can be composed of a single
material, without any initial layers. The substrate also can be
created without the need to design at least two layers having
different chemical reactivities. Rather, in this specific
embodiment a single layer substrate can be modified throughout all
surfaces, including well interiors (i.e., bottom and/or sides).
Following modification the outermost surface of the substrate is
polished to then remove the chemical modifications. This polishing
results in return of the surface surrounding the well features to
its original chemical reactivity while leaving the interior of the
well features having reactive characteristics corresponding the
chemical modifications.
[0070] The material of the unmodified substrate can exhibit, for
example, a lower affinity or a neutral affinity toward the
deposited biopolymer compared to the affinity of well surface areas
following modification. By way of exemplification, the substrate
can be silicon and the silicon can be converted to, for example,
silicon dioxide. A reagent such as trichloro alkyl silane having
hydrophilic moieties or reactive groups for covalent attachment of
biopolymers can be reacted with the silicon dioxide to produce a
modified substrate. The well bottom and sides as well as the
surrounding surface will exhibit a hydrophilic character or have
reactive groups toward the biopolymer. Alternatively, substrate
having at least two layers can be used or produced as described
previously. The first and second layers can have different chemical
reactivities. Both layers layer can be reacted with a reagent to
result in two modified layers having the same or different
affinities for a deposited biopolymer.
[0071] Polishing the surrounding surface will remove such
hydrophilic moieties or reactive attachment groups and result in
the return of the surrounding surface to the original silicon. At
least a portion of the well interiors will remain hydrophilic or
have reactive groups for covalent attachment of biopolymers. For
example, at least the bottom of the wells and a bottom portion of
the sides of the wells will remain modified whereas the top portion
of the well sides will be removed by polishing. Accordingly, in
this specific embodiment of the invention, all surface areas of the
substrate can be made to exhibit a favorable affinity toward the
deposited biopolymer. Polishing at least a portion of the surface
sufficient to remove the modification will result in a first layer,
corresponding to the polished portion, and a second layer,
corresponding to the concave features, having different affinity
for the target biopolymer since they concave features were not
polished. Similarly, where a two layer substrate is employed as
exemplified above, polishing of a portion of either the first or
second layer will result in removal of the modification of that
layer with concomitant removal of the affinity for the
biopolymer.
[0072] Accordingly, the invention further provides a method of
fabricating a microarray where the polishing includes removing a
first portion of a layer such that a second portion of the layer
remains on the substrate. The second portion of the layer is
exposed by the removing of the first portion of the layer. The
substrate can include two or more layers. A first layer can be, for
example, silicon and a second layer can be, for example, silicon
oxide. The first layer can correspond to a lower layer which is the
bottom of a well and the second layer can correspond to an upper
layer which surrounds the wells and forms at least a portion of the
sides of a well.
[0073] Polishing can be accomplished using a variety of physical or
chemical methods well known in the art. One particularly useful
method is to apply friction by mechanical motion. Typically, a
brush or other device that contacts the surface of a substrate will
have a contact area that is substantially larger than the width or
diameter of wells in the substrate. Thus, the brush or contact
device will be precluded from entering into the well, thereby
leaving the bottom of the well unpolished. Various means can be
employed to mechanically polish a substrate including, for example,
rubbing, chafing, smoothing or other uses of frictional forces as
exemplified above. Physical or mechanical methods well known in the
art other than those exemplified above also can be employed in the
methods of the invention for polishing a substrate so as to confer
a different biopolymer affinity onto at least a portion of one
layer of the substrate.
[0074] Chemical methods for polishing a substrate are equally
applicable in the methods of the invention and include, for
example, treatment with acids such as hydrofluoric acid or bases
such as sodium hydroxide. Chemical methods well known in the art
other than those exemplified above also can be employed in the
methods of the invention for polishing a substrate so as to confer
a different biopolymer affinity onto at least a portion of one
layer of the substrate. Particular features, such as wells, can be
masked from being polished when a chemical polishing method is
used. Removal of the mask then allows those features that were
protected from polishing to be subsequently available for
modification such as modification to attach a biopolymer.
[0075] The initial substrate, well features and the like can be
obtained or generated as described previously. Once prepared the
substrate having at least one layer with a first polished portion
and a second, unpolished modified portion can be used for
depositing single species of biopolymers into each well feature.
For example, deposition or deposition and covalent attachment of
microspheres or nucleic acid balls as described previously are two
exemplary means for generating a microarray having a single
biopolymer species at each feature on a polished array.
[0076] In yet another specific embodiment, the invention is
directed to methods for capturing individual molecules, to the
exclusion of other molecules in the targeted vicinity, on the
surface of a substrate. As with other methods and substrates of the
invention, such single molecule capture is particularly useful for
enhancing nucleic acid sequencing methods that benefit from the
amplification of isolated molecules to build up localized
populations in order to generate greater signal strength. In this
specific embodiment, the methods of the invention allow for the
capture of a single nucleic acid molecule or other biopolymer
molecule within a total circular area having a diameter of about
0.1 .mu.m and employs differential treatment of the center of the
substrate feature compared to the area outside the
circumference.
[0077] Additionally provided is a method of fabricating a
microarray. The method includes: (a) functionalizing a plurality of
features on a substrate to create discrete single biopolymer anchor
sites, said functionalization comprising: (1) contacting said
substrate with a reagent reactive with said substrate to produce a
modified substrate; (2) applying a protecting reagent to discrete
sites on said modified substrate, said discrete sites having an
area of between about 5-40 nm.sup.2; (3) modifying said reagent
located in unprotected regions of said modified substrate
surrounding said discrete sites, thereby rendering said regions
unreactive to a target biopolymer, and (4) removing said protecting
reagent to produce a substrate having a plurality of discrete
functionalized features, and (b) attaching a single target
biopolymer to one or more of said discrete functionalized
features.
[0078] In this particular embodiment, microarray features utilize
differential chemical functionalization to generate discrete
features for attachment of a single nucleic acid molecule at each
feature. Although well features can be used, in certain embodiments
this variation of the methods of the invention is particularly
useful with, for example, planar substrates because both the steps
of well fabrication and microsphere or nucleic acid ball
preparation can be omitted. The method includes functionalizing a
plurality of discrete features on a substrate. The substrates and
design of features can be those described previously.
[0079] Functionalization can be performed using any method well
known in the art to create anchor sites for a target biopolymer. An
anchor site refers to a substrate site that immobilizes a deposited
biopolymer. Immobilization can occur through, for example, covalent
or non-covalent interactions. Functionalization of discrete sites
for covalent immobilization can be performed as described
previously by, for example, modifying the surface of the substrate
to contain desirable reactive groups. Functionalization of discrete
sites for non-covalent immobilization can be performed by, for
example, modifying the surface of the substrate to confer a
chemical characteristic such as a hydrophilic characteristic or to
localize agents that can be used to indirectly anchor the deposited
biopolymer. A particular example of a covalent interaction includes
those reactive functional groups described previously that form
chemical bonds between the reactive group and the biopolymer. A
particular example of a non-covalent interaction includes a ligand
affinity interaction with its binding partner; for example, biotin
and streptavidin or two complementary nucleic acid sequences.
Various other non-covalent interactions are well known in the art
and can be equally employed in the methods of the invention.
[0080] One useful method for functionalization of discrete sites
includes contacting the substrate with a reagent specifically
reactive with the substrate to produce a modified substrate as
described previously. The functionalization can be across the
entire surface of the substrate or areas covering the positions
where the discrete sites are to be produced. In a particular
embodiment, functionalization can occur by treating the entire
substrate through submersion or exposure to the modifying reagent.
Following modification, positions where discrete sites are to be
fabricated are masked by applying a protecting reagent.
Alternatively, discrete site positions can be chemically masked by,
for example, chemical blocking groups activated by stimuli such as
focused irradiation using a laser. The applied or chemical
protecting reagent should cover an area of about 5-40 nm.sup.2
since this range is particularly useful for anchoring single
biopolymer molecules.
[0081] For example, an area covering about 5-40 nm.sup.2 is
sufficient to form a discrete feature for attachment of a single
biopolymer molecule, being of such a size that, once a biopolymer
is attached, the site is too small to allow attachment of another
biopolymer molecule. Generally, a protected area will have a size
ranging from, for example, the above range to as small as about
5-10 nm.sup.2. Protected areas between, for example, 10-30 nm.sup.2
are particularly useful for anchoring single biopolymer molecules
at the exclusion of other molecules. Accordingly, the size of a
protected feature on a substrate of the invention can include, for
example, an average area of, for example, about 5 nm.sup.2, 6
nm.sup.2, 7 nm.sup.2, 8 nm.sup.2, 9 nm.sup.2, 10 nm.sup.2, 11
nm.sup.2, 12 nm.sup.2, 13 nm.sup.2, 14 nm.sup.2, 15 nm.sup.2, 16
nm.sup.2, 17 nm.sup.2, 18 nm.sup.2, 19 nm.sup.2, 20 nm.sup.2, 21
nm.sup.2, 22 nm.sup.2, 23 nm.sup.2, 24 nm.sup.2, 25 nm.sup.2, 26
nm.sup.2, 27 nm.sup.2, 28 nm.sup.2, 29 nm.sup.2, 30 nm.sup.2, 31
nm.sup.2, 32 nm.sup.2, 33 nm.sup.2, 34 nm.sup.2, 35 nm.sup.2, 36
nm.sup.2, 37 nm.sup.2, 38 nm.sup.2, 39 nm.sup.2 or 40 nm.sup.2.
Those skilled in the art will understand that protected areas of
the invention also include all sizes larger than, smaller than or
in between the above exemplary areas.
[0082] A variety of protecting reagents are well known in the art
and can be equally employed in the methods of the invention.
Particularly useful protecting reagents include, for example, wax
or oil droplets applied from a micro dispenser, chrome or other
light absorbing or reflecting masks such as those described in U.S.
Pat. No. 6,949,638, which is incorporated herein by reference, tape
or other adhesives, solder, or polymeric compounds.
[0083] In order to provide for targeted immobilization to the
anchor sites it is beneficial to modify the unprotected substrate
area. A range of modifications can be employed to, for example,
make these substrate regions unreactive or confer a lower affinity
toward biopolymers (e.g., confer a hydrophobic character) or to
impart the same or a different chemical reactivity for attachment
of secondary or other biopolymers. In one embodiment, the
unprotected regions of the modified substrate surrounding each
protected discrete anchor site are further modified to render these
regions unreactive to a target biopolymer. The protecting reagent
is then removed to yield a substrate having discrete functionalized
sites and a surrounding area of each feature that is unreactive to
deposited biopolymers. A population of biopolymers can be applied
to the substrate for construction of a microarray of the invention.
Each anchor site will immobilize a single biopolymer as described
above. The surrounding area will be devoid of biopolymer due to its
non-reactivity or affinity to the biopolymer. Depositing and/or
localization of single biopolymer molecules to each functionalized
discrete feature can be performed as described previously with
other substrate configurations of the invention.
[0084] In another embodiment, the unprotected regions of the
modified substrate surrounding each protected discrete anchor site
are further modified to render these regions reactive to a
secondary biopolymer. Further modification can be performed as an
independent modification step as exemplified below. Alternatively,
such further modification can be performed on, for example, the
non-reactive surrounding areas simultaneous with attachment of, for
example, secondary biopolymers. Given the teachings and guidance
provided herein, those skilled in the art will understand that the
selection of which route to proceed will depend on the substrate
modification and the available chemistry that is compatible with
the modified substrate and the target biopolymer.
[0085] With reference to further modification of the unprotected
substrate areas for purposes of exemplification, each protected
single anchor site can be further surrounded by, for example, a
region of secondary anchor sites modified for attachment of
secondary biopolymers. The secondary anchor sites can have a
chemical reactivity different from the primary anchor site such
that once the protecting reagent is removed the primary and
secondary biopolymers can be differentially attached to the primary
and secondary anchor sites, respectively. Alternatively, the
secondary anchor sites can be modified, for example, with reactive
groups that can be activated by an external stimulus as described
previously (i.e. made chemically reactive by an additional step
such as exposure to light). Employing differential chemical
reactivity or activation, for example, this surrounding area can
capture a wide variety of secondary biopolymers for use in
conjunction with the single biopolymer molecule once immobilized at
some or all of the discrete features.
[0086] Any of the methods described previously for producing
different chemical reactivities or for modifying layers to impart
different affinities for a biopolymer also can be employed for
producing the discrete features or areas surrounding the protected
discrete features. Another particularly useful method for producing
a modified substrate with a reactive reagent and further modifying
the reagent to render unprotected surrounding regions unreactive
includes covering the substrate with a trichlorotriazine layer.
Following protection of discrete sites, the substrate can be
treated, for example, with ammonia gas to destroy any reactive
molecules in the unprotected areas by creating diaminotriazine. The
diaminotriazine will still be available to be reactivated for
attachment of secondary biopolymers. In this exemplary embodiment
of the invention, the protected sites contain trichlorotriazine as
the modified substrate component while the unprotected regions have
an unreactive diaminotriazine modified substrate. Biopolymers can
be generated having aldehyde moieties specifically reactive with
either the trichlorotriazine or diaminotriazine moieties using
compatible chemistry well known in the art. An alternative method
includes further activation of the diaminotriazine regions with
bromoacetic acid. The bromoacetic acid in turn can bind to the
secondary biopolymers for immobilization at secondary anchor
sites.
[0087] Secondary biopolymers can be employed in conjunction with
the primary single biopolymer molecule for a variety of different
analyses and procedures. Particularly useful purposes for such
secondary biopolymers include anchoring primers and or templates
for amplification of nucleic acid biopolymers. Accordingly, in some
embodiments of the invention secondary biopolymers include, for
example, probes, templates and/or short pieces of nucleic acid used
for a variety of nucleic acid amplification processes including,
bridge amplification. Bridge amplification localizes the target and
one or more primers within sufficient proximity so that
complementary sequences hybridize. Following hybridization, the
single stranded regions are extended with, for example, a template
directed nucleic acid polymerase to modify each molecule to include
the sequence of the extension product. Multiple rounds of this
extension procedure will result in the synthesis of a population of
amplicons. Bridge amplification can be carried out using methods
set forth in further detail below or otherwise known in the art
such as those described in U.S. Pat. No. 7,115,400, which is
incorporated herein by reference. Because the target nucleic acid
and the probe or primer is immobilized at a feature and its
adjacent surrounding area, the amplicons become highly localized
and concentrated at the area of the discrete feature.
[0088] One useful variation of bridge amplification includes
incorporating a common priming region sequence into the target
biopolymer and incorporating a complementary common primer sequence
into a plurality of secondary nucleic acids immobilized to the
secondary regions surrounding each of the discrete functionalized
features of the substrate. The common priming region is typically
the same for a plurality of different features on an array thereby
allowing for priming of all bridge amplification reactions using a
single primer and extension into at least a portion of each of the
plurality of different target nucleic acid molecules. Utilization
of a common primer sequence complementary to a portion of the
secondary nucleic acids is particularly useful because it requires
a population of only a single secondary nucleic acid species for
amplification of the entire plurality of different primary
biopolymers, having different target nucleotide sequences, each
attached at a different discrete feature. Accordingly, microarrays
can be constructed having a single species of secondary nucleic
acids and a diverse plurality of target nucleic acids. However,
each of the different single molecules can be amplified by
extending the common primer sequence into each of the different
species of primary nucleic acids to generate a diverse population
of amplicons discretely located at different features throughout an
array of the invention. As a result each feature will have a many
copies of a particular species of nucleic acid and the species at
each site will differ from the species at other sites.
[0089] Bridge amplification is particularly useful with the
microarrays of the invention because primers can be captured in
specific locations for the production of higher density arrays as
compared to random spacing or localization of capture locations.
Bridge amplification employing the substrates and microarrays of
the invention also significantly reduces background noise in
subsequent detection and analysis methods. Typically, bridge
amplification results in outward spreading of amplicons away from
the location where the initial target nucleic acid is attached. If
the locations of the initial targets are randomly spaced then a
fraction of these locations will be so close that this outward
spreading causes unwanted overlap of amplified features. However,
ordered spacing of the features where initial target nucleic acids
are attached reduces the possibility of merging of amplicon
colonies through growth.
[0090] Given the teaching and guidance provided herein, those
skilled in the art will understand that there is no particular
order for attachment of the primary biopolymer at a functionalized
feature and attachment of secondary biopolymers, if any, at the
surrounding areas. In some embodiments, it may be beneficial to
attach the target biopolymer at discrete features first to reduce
possible loss through specific or non-specific interactions with
the secondary biopolymers. However, any of a variety of procedures
well known in the art can be utilized to minimize such interactions
including, for example, performing the attachment under conditions
that disfavor such interactions such as utilizing stringent
temperature and solute conditions. Generally, following
modification of the secondary areas surrounding the functionalized
features, the protective reagent such as wax, oil or other mask is
removed using methods well known in the art. One exemplary method
includes, for example, washing the protected substrate with an
organic solvent. The substrate is now ready for deposition of
target biopolymers and immobilization. Immersion into a solution of
biopolymer molecules can be performed to achieve this step. Due to
the size of the now unprotected and active discrete features on the
surface, only about one molecule will be captured at each feature.
The captured target biopolymer molecules can be covalently
immobilized by chemical coupling. The microarray is now available
for use directly in an assay procedure or, alternatively, secondary
biopolymers can be attached to the secondary anchor sites for
employment in procedures such as described above.
[0091] Following the above teachings and guidance, those skilled in
the art can employ the methods of the invention to fabricate a
microarray having plurality of biopolymer molecules each attached
at a discrete feature on a substrate. As with the previously
exemplified substrates of the invention, the functionalization of
discrete sites to produce discrete features also can be employed to
generate substrates having optimally packed features to maximize
the used surface area and the signal-to-noise ratio when a
microarray produced therefrom is employed in a subsequent assay. In
this respect, a large plurality of biopolymers can be deposited and
immobilized throughout optimally spaced discrete features such that
maximum signal can be generated with minimum noise derived from
adjacent features.
[0092] In yet a further specific embodiment, nucleic acid molecules
are positioned in precise locations on a substrate using
nanomaterials that allow for the controlled manipulation of many
single molecules in parallel. In this particular embodiment, a high
density sample of nucleic acids can be simultaneously guided into
nanochannels by, for example, application of an electric potential
to achieve a single molecule per channel. Nanochannels having a
diameter of about 100 nm can physically exclude more than one
nucleic acid from entering. Once encased by the nanochannel, a
substrate is placed on the initial opening and the single molecules
are deposited in a pattern mirroring the pattern of nanochannels
upon application of a force in the reverse direction.
[0093] Also provided by the invention is another method of
fabricating a microarray. The method includes: (a) contacting a
substrate having a plurality of discrete nanochannels and an
electrode material orthogonal to said nanochannels with a plurality
of biopolymers, said nanochannels having a length and diameter
sufficient for entry of only a single biopolymer molecule; (b)
applying an electric potential to said substrate sufficient to
translocate said single biopolymer molecules into said nanochannels
to produce a substrate containing a plurality of single biopolymers
each in said plurality of discrete nanochannels, and (c)
transferring said plurality of single biopolymers contained in said
plurality of discrete nanochannels to a solid support.
[0094] In this specific embodiment of the invention, microarrays
can be fabricated by loading individual biopolymer molecules into a
plurality of discrete nanochannels. The nanochannels can be
organized at essentially any desired spacing and density, limited
only by their outer diameter. The pattern and density of the
nanochannels will be a template to microarrays produced therefrom.
Particularly useful patterns and densities include nanochannels
optimally spaced to achieve a maximum density of biopolymer
molecules and a maximum signal-to-noise ratio with minimum
bleed-over of signal from adjacent features within the microarray
as described previously.
[0095] A variety of different methods can be employed to load
single biopolymer molecules into a plurality of discrete
nanochannels. One method includes, for example, applying an
electric potential at the distal end of the plurality to produce a
force that translocates the biopolymers into the nanochannels.
Depending on the selected biopolymer the electric potential can
differ. For example, nucleic acids exhibit a net negative charge
due to their phosphodiester backbone. Therefore, the anode of an
electrolytic cell can be placed at the distal end of the plurality
with the cathode at the proximal or entrance for loading. Applying
such an electric potential will draw the plurality of nucleic acid
biopolymers toward the distal end of the nanochannels. Each opening
of a nanochannel will allow a single molecule to enter until
translocation to the distal end is complete. Once complete, the
electric potential is terminated and the loaded plurality of
nanochannels is ready to be transferred onto a substrate of choice
for fabrication of a microarray.
[0096] Voltage applicable to apply a sufficient electric potential
to draw a biopolymer into a nanochannel will typically include
those voltage ranges employed in electrophoresis methods. Such
electrophoresis methods include, for example, agarose gel
electrophoresis, polyacrylamide gel electrophoresis, capillary gel
electrophoresis and pulse-field gel electrophoresis. Voltages can
range from between about 5-400 volts per cm of path length.
Particular voltage ranges include, for example, between about 1-5,
6-10, 11-15, 16-20, 21-25, 26-30, 31-35, 36-40, 41-45, 46-50,
51-55, 56-60, 61-65, 66-70, 71-75, 76-80, 81-85, 86-90, 91-95,
96-100, 101-105, 106-110, 111-115, 116-120, 121-125, 126-130,
131-135, 136-140, 141-145, 146-150, 151-155, 156-160, 161-165,
166-170, 171-175, 176-180, 181-185, 186-190, 191-195, 196-200 and
201 or more volts per cm of path length. All values within, above
and below these exemplary ranges also can be used to load a
biopolymer into a nanochannel of the invention. The electric
potential is influenced by solutes within a buffer. For a review of
the relationship between electrical parameters in electrophoresis
see, for example, "Physical Chemistry: Applications to Biochemistry
and molecular Biology" Freifelder, D., 2nd Edition, W. H. Freeman
and Co., 1982. Buffers that can be employed for loading a
biopolymer into a nanochannel of the invention also include, for
example, those typically employed for electrophoresis. Exemplary
buffers include Tris-Acetate EDTA (TAE) and Tris-Borate EDTA (TBE),
for example.
[0097] Other methods that can be utilized to load a single
biopolymer molecule within a population each into a single
nanochannel within a plurality include, for example, centrifugation
such that the g force is parallel to the length of the nanochannels
or fluid flow in a direction through the nanochannels.
[0098] The internal diameter of each nanochannel should be
sufficient to allow passage of a single biopolymer into a
nanochannel and exclude, by size limitation, entrance of additional
biopolymers. Generally, nanochannels useful for fabrication of
microarrays containing nucleic acid biopolymers will have an
average internal diameter ranging from about 0.005-0.05 .mu.m.
Particularly useful internal diameters include, for example, about
0.006 .mu.m, 0.007 .mu.m, about 0.008 .mu.m, about 0.009 .mu.m,
about 0.01 .mu.m, about 0.02 .mu.m, about 0.03 .mu.m, about 0.04
.mu.m or about 0.045 or more. Those skilled in the art will
understand that internal diameters of nanochannels used in the
invention also include all sizes in between, smaller and greater
than the above exemplary sizes. Given the teachings and guidance
provided herein, those skilled in the art also will understand that
the average internal diameter of nanochannels can vary depending on
the type of biopolymer to be applied. For example, polypeptide
biopolymers can require somewhat larger internal diameters for
efficient translocation due to bulky side chains on some amino
acids. Accordingly, the diameters exemplified above can be adjusted
based on such known or other empirically determined
characteristics.
[0099] Similarly, the average length of each nanochannel should be
sufficient to allow entrance of an entire single biopolymer into a
nanochannel and exclude, by preclusion through occupation, entrance
of additional biopolymers. Generally, nanochannels useful for
fabrication of microarrays containing nucleic acid biopolymers will
have lengths ranging from about 1.0-3.0 .mu.m. Particularly useful
lengths include, for example, about 1.1 .mu.m, 1.2 .mu.m, 1.3
.mu.m, 1.4 .mu.m, 1.5 .mu.m, 1.6 .mu.m, 1.7 .mu.m, 1.8 .mu.m, 1.9
.mu.m, 2.0 .mu.m, 2.1 .mu.m, 2.2 .mu.m, 2.3 .mu.m, 2.4 .mu.m, 2.5
.mu.m, 2.6 .mu.m, 2.7 .mu.m, 2.8 .mu.m, 2.9 .mu.m, 3.0 .mu.m or
more. Those skilled in the art will understand that nanochannel
lengths of the invention also include all sizes in between, smaller
and greater than the above exemplary sizes. Given the teachings and
guidance provided herein, those skilled in the art also will
understand that the actual length of a nanochannel can vary
depending on the size of biopolymer to be applied. For example,
longer nucleic acid sequences can be used with channels having
larger lengths compared to shorter nucleic acids for partial or
complete translocation into a nanochannel. Similarly, shorter
nucleic acids can be used with channels having shorter lengths to
avoid loading a second molecule in tandem into nanochannels longer
than the first molecule. Accordingly, the lengths exemplified above
can be adjusted based on such known or other empirically determined
characteristics.
[0100] By way of exemplification for applying a translocation force
as described above, a plurality of discrete nanochannels is
configured to contain an electrode material orthogonal to the long
axis of each nanochannel within the plurality. Such configurations
can include, for example, metal or other conductive material bonded
to the distal end of each nanochannel. Bonding includes, for
example, temporary or permanent attachment of the electrode
material. The electrode material should span the opening of the
nanochannel so as to preclude complete translocation of the
biopolymer through the nanochannel and passage into solution at the
distal end. Alternatively, a conductive or semi-porous material can
be placed in between the electrode and opening to stop
translocation at the distal end.
[0101] Transfer of biopolymers from their loaded state within a
nanochannel to a substrate can be performed using a variety of
methods well known in the art. For example, forces applied using
electrical potential, centrifugal force, vacuum applied and
pressure can be employed to pull or force each of the plurality of
biopolymers out of its nanochannel and onto a substrate. By way of
exemplification, a substrate can be placed or affixed onto the
proximal or loading end of the plurality of nanochannels and an
electric potential can be applied in the opposite orientation to
that used for loading biopolymers into the nanochannels. Once
applied, the biopolymers will translocate in the opposite direction
and proceed out of the nanochannels. The affixed substrate will
catch each biopolymer in a location corresponding to the terminus
of each nanochannel. The biopolymers applied to the substrate can
be used directly or covalently attached prior to use as a
microarray of the invention.
[0102] Nanochannels can be constructed using a variety of materials
and methods well known in the material science art. For example,
nanochannels can be bored, grooved or constructed out of, silicon,
using chemical or abrasive methods described previously herein in
regard to polishing. A particularly useful method for constructing
a plurality of nanochannels applicable for fabricating the
microarrays of the invention includes boring out a plurality of
fiber optic fibers to leave only the cladding. The inner region can
be bored out by acid treatment as described, for example, in U.S.
Pat. No. 6,859,570 or U.S. Pat. No. 6,266,459, each of which is
incorporated herein by reference. Physical boring can also be used.
Each plurality of cylindrical cladding devoid of its optical fiber
can be cut to a desired length and utilized as a plurality of
nanochannels. Another particularly useful means to produce a
plurality of discrete nanochannels is to employ capillary arrays.
Capillary arrays are well known in the art and, given the teachings
and guidance provided herein, can be readily modified by, for
example, attachment of an electrode material at one set of termini.
Loading and unloading of bored out fiber optic cladding or
capillary arrays can be performed as described previously.
[0103] It is understood that modifications which do not
substantially affect the activity of the various embodiments of
this invention are also included within the definition of the
invention provided herein. Accordingly, the following examples are
intended to illustrate but not limit the present invention.
[0104] Throughout this application various publications have been
referenced within parentheses. The disclosures of these
publications in their entireties are hereby incorporated by
reference in this application in order to more fully describe the
state of the art to which this invention pertains.
[0105] Although the invention has been described with reference to
the disclosed embodiments, those skilled in the art will readily
appreciate that the specific examples and studies detailed above
are only illustrative of the invention. It should be understood
that various modifications can be made without departing from the
spirit of the invention. Accordingly, the invention is limited only
by the following claims.
* * * * *