U.S. patent number 10,385,335 [Application Number 15/101,168] was granted by the patent office on 2019-08-20 for modified surfaces.
This patent grant is currently assigned to CENTRILLION TECHNOLOGY HOLDINGS CORPORATION. The grantee listed for this patent is CENTRILLION TECHNOLOGY HOLDINGS CORPORATION. Invention is credited to Glenn McGall.
United States Patent |
10,385,335 |
McGall |
August 20, 2019 |
Modified surfaces
Abstract
Provided herein are methods and compositions for coating
surfaces with polymers. The methods and compositions are suited for
conducting biological reactions.
Inventors: |
McGall; Glenn (Palo Alto,
CA) |
Applicant: |
Name |
City |
State |
Country |
Type |
CENTRILLION TECHNOLOGY HOLDINGS CORPORATION |
Grand Cayman |
N/A |
KY |
|
|
Assignee: |
CENTRILLION TECHNOLOGY HOLDINGS
CORPORATION (Grand Cayman, KY)
|
Family
ID: |
53274198 |
Appl.
No.: |
15/101,168 |
Filed: |
December 5, 2014 |
PCT
Filed: |
December 05, 2014 |
PCT No.: |
PCT/US2014/068947 |
371(c)(1),(2),(4) Date: |
June 02, 2016 |
PCT
Pub. No.: |
WO2015/085268 |
PCT
Pub. Date: |
June 11, 2015 |
Prior Publication Data
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Document
Identifier |
Publication Date |
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US 20160298110 A1 |
Oct 13, 2016 |
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Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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61979431 |
Apr 14, 2014 |
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61912027 |
Dec 5, 2013 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12Q
1/6874 (20130101); C12N 15/1093 (20130101); C12Q
1/6834 (20130101); C12Q 1/686 (20130101); C40B
50/18 (20130101); C08F 220/58 (20130101); C12Q
1/6834 (20130101); C12Q 2523/101 (20130101); C12Q
2527/153 (20130101); C12Q 2531/113 (20130101); C12Q
1/686 (20130101); C12Q 2523/101 (20130101); C12Q
2527/153 (20130101); B01J 2219/00659 (20130101); B01J
2219/00637 (20130101); C08F 220/56 (20130101); C09D
4/00 (20130101); B01J 2219/00722 (20130101); B01J
2219/00626 (20130101) |
Current International
Class: |
C12Q
1/68 (20180101); C12Q 1/6874 (20180101); C12Q
1/6834 (20180101); C12Q 1/686 (20180101); C40B
50/18 (20060101); C08F 220/58 (20060101); C12N
15/10 (20060101); C08F 220/56 (20060101); C09D
4/00 (20060101) |
References Cited
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Primary Examiner: Priest; Aaron A
Attorney, Agent or Firm: Wilson Sonsini Goodrich &
Rosati
Parent Case Text
CROSS-REFERENCE
This application claims the benefit of U.S. Provisional Application
No. 61/912,027, filed Dec. 5, 2013, and U.S. Provisional
Application No. 61/979,431, filed Apr. 14, 2014, which applications
are incorporated herein by reference.
Claims
What is claimed is:
1. A method for performing an enzymatic reaction, comprising: (a)
providing a substrate comprising surface-bonded initiator species
comprising a silane; (b) conducting surface initiated
polymerization of a mixture of (i) first monomers comprising
acrylamide and/or ethoxylated acrylamide, each of which has no
biomolecule attached to, and (ii) second monomers comprising
acrydite, each of which has a biomolecule attached to, wherein said
surface initiated polymerization starting from said initiator
species, and coupling a plurality of said biomolecules to a polymer
brush coating during said surface initiated polymerization, thereby
producing said polymer brush coating with said plurality of said
biomolecules coupled to said polymer brush coating; and (c)
performing one or more enzymatic reactions with said biomolecules
on said substrate; wherein the biomolecules are selected from the
group consisting of: oligonucleotides and polynucleotides, wherein
the one enzymatic reaction is selected from the group consisting
of: polymerase chain reaction, sequencing reaction, sequencing by
synthesis reaction, ligation reaction, extension reaction, and
transcription reaction.
2. The method of claim 1, further comprising applying heat to said
substrate.
3. The method of claim 1, wherein said polymer brush coating
coupled with said plurality of biomolecules exhibits robustness
wherein at least 90% of said biomolecules are retained on said
surface after 40 cycles of sequencing by synthesis reactions.
4. The method of claim 1, wherein the substrate comprises at least
1,000,000 different types of biomolecules, and wherein each
biomolecule is an oligonucleotide.
5. The method of claim 4, wherein said enzymatic reaction is an
extension reaction.
6. A method for making a modified surface, comprising: (a)
providing a surface; (b) covalently bonding initiator species to
said surface, wherein said initiator species comprises a silane;
and (c) conducting surface initiated polymerization of a mixture of
(i) first monomers comprising acrylamide and/or ethoxylated
acrylamide, each of which has no biomolecule attached to, and (ii)
second monomers comprising acrydite, each of which has a
biomolecule attached, wherein said surface initiated polymerization
starting polymer from said initiator species, thereby producing a
polymer brush coating comprising a plurality of polymer chains,
each of which has said biomolecule attached.
7. The method of claim 6, wherein said initiator species comprises
the molecule shown below: ##STR00005##
8. The method of claim 6, wherein said second monomers are 5'
acrydite modified oligonucleotides.
9. The method of claim 1, wherein said polymer brush coating
coupled with said plurality of biomolecules exhibits robustness in
said polymerase chain reactions and wherein said polymerase chain
reactions comprise the following reaction conditions: (a) a
denaturation step at a temperature of at least 85.degree. C. for at
least 15 seconds; (b) an annealing step at a temperature of at
least 50.degree. C. for at least 15 seconds; and (c) an extension
step at a temperature of at least 70.degree. C. for at least 30
seconds.
10. The method of claim 1, wherein said first monomers comprise
acrylamide.
11. The method of claim 10, wherein said first monomers further
comprise N-(2-hydroxyethyl)acrylamide.
12. The method of claim 1, wherein said surface-bonded initiator
species comprises
2-bromo-2-methyl-N,N-bis-(3-trimethoxysilanylpropyl)propionamid-
e.
13. The method of claim 1, wherein said surface initiated
polymerization comprises atom-transfer radical polymerization
(ATRP).
14. The method of claim 1, wherein said second monomers are 5'
acrydite modified oligonucleotides or polynucleotides.
15. The method of claim 1, wherein said first monomers comprise
ethoxylated acrylamide.
16. The method of claim 6, wherein said first monomers comprise
ethoxylated acrylamide.
17. The method of claim 1, wherein robustness of said polymer brush
coating coupled with said plurality of biomolecules is exhibited in
said polymerase chain reaction: at least 90% of said biomolecules
are retained on said surface after 25 cycles of said polymerase
chain reactions.
Description
BACKGROUND
In many sequencing by synthesis (SBS) systems, clonal amplification
and SBS are performed in glass flow cell channels. PCR primers are
attached to the inner surface of the channels via a passively bound
polymer coating. Weakly bound polymer chains are washed away prior
to use, but the remaining polymer can become depleted to varying
extents during extensive cycles of SBS, causing progressive loss of
signal. This is a particular concern when high pH and elevated
temperature conditions are employed.
SUMMARY
Methods and compositions are provided for fabricating polymer
coatings by surface initiated polymerization incorporating
biomolecules. In some cases, the compositions and methods are
useful in performing nucleic acid reactions and sequencing by
synthesis. In some cases, the compositions and methods are useful
in providing coatings that are robust.
An aspect of the present disclosure provides a composition,
comprising: a surface with a 10 or more nucleic acid molecules
coupled thereto, wherein at least 90% of the nucleic acid molecules
remain intact and coupled to the surface after at least 30 PCR
cycles, wherein each PCR cycle comprises the following reaction
conditions: (a) a denaturation step at a temperature of at least
85.degree. C. for at least 15 seconds; (b) an annealing step at a
temperature of at least 50.degree. C. for at least 15 seconds; and
(c) an extension step at a temperature of at least 70.degree. C.
for at least 30 seconds.
In some embodiments of aspects provided herein, the surface is
covered with a polymer brush. In some embodiments of aspects
provided herein, the polymer brush comprises acrylamide. In some
embodiments of aspects provided herein, the polymer brush further
comprises N-(2-hydroxyethyl)acrylamide. In some embodiments of
aspects provided herein, at least 1,000 different nucleic acid
molecules are coupled to the surface. In some embodiments of
aspects provided herein, at least 100,000 different nucleic acid
molecules are coupled to the surface. In some embodiments of
aspects provided herein, at least 1,000,000 different nucleic acid
molecules are coupled to the surface.
An aspect of the present disclosure provides a method for
performing an enzymatic reaction, comprising: (a) providing a
substrate having a polymer brush coating and a plurality of
biomolecules coupled to the polymer brush; and (b) performing one
or more enzymatic reactions with the biomolecules on the
substrate.
In some embodiments of aspects provided herein, the biomolecules
are selected from the group consisting of: oligonucleotides,
polynucleotides, aptamers, proteins, and antibodies. In some
embodiments of aspects provided herein, the enzymatic reaction is
selected from the group consisting of: polymerase chain reaction,
sequencing reaction, ligation reaction, extension reaction, and
transcription reaction. In some embodiments of aspects provided
herein, further comprises applying heat to the substrate. In some
embodiments of aspects provided herein, at least 90% of the
biomolecules are retained with at least 90% integrity after 40
cycles of sequencing by synthesis reactions. In some embodiments of
aspects provided herein, at least 90% of the biomolecules are
retained with at least 90% integrity after 25 cycles of polymerase
chain reactions. In some embodiments of aspects provided herein,
the substrate comprises at least 1,000,000 different types of
biomolecules, and wherein each biomolecule is an oligonucleotide.
In some embodiments of aspects provided herein, the enzymatic
reaction is an extension reaction.
An aspect of the present disclosure provides a method for making a
modified surface, comprising: (a) providing a surface; (b)
covalently bonding initiator species to the surface; (c) conducting
surface initiated polymerization of a polymer from the initiator
species, thereby producing a polymer coating comprising a plurality
of polymer chains; and (d) coupling two or more different
biomolecules to the polymer coating.
An aspect of the present disclosure provides a method for making a
modified surface, comprising: (a) providing a surface; (b)
covalently bonding initiator species to the surface; (c) conducting
surface initiated polymerization of a mixture two or more different
types of acrylamide monomers from the initiator species, thereby
producing a polymer coating comprising a plurality of polymer
chains; and (d) coupling biomolecules to the polymer coating.
In some embodiments of aspects provided herein, the biomolecules
are selected from the group consisting of: oligonucleotides,
polynucleotides, aptamers, proteins, and antibodies. In some
embodiments of aspects provided herein, the two or more different
biomolecules are two different oligonucleotides. In some
embodiments of aspects provided herein, the two or more different
types of acrylamide monomers are selected from the group consisting
of: acrylamide, N-(2-hydroxyethyl)acrylamide, ethylene glycol
acrylamide, and hydroxyethylmethacrylate (HEMA). In some
embodiments of aspects provided herein, the surface is selected
from the group consisting of glass, silica, titanium oxide,
aluminum oxide, indium tin oxide (ITO), silicon,
polydimethylsiloxane (PDMS), polystyrene, polycyclicolefins,
polymethylmethacrylate (PMMA), titanium, and gold. In some
embodiments of aspects provided herein, the surface comprises
glass. In some embodiments of aspects provided herein, the surface
comprises silicon. In some embodiments of aspects provided herein,
the surface is selected from the group consisting of: flow cells,
sequencing flow cells, flow channels, microfluidic channels,
capillary tubes, piezoelectric surfaces, wells, microwells,
microwell arrays, microarrays, chips, wafers, non-magnetic beads,
magnetic beads, ferromagnetic beads, paramagnetic beads,
superparamagnetic beads, and polymer gels. In some embodiments of
aspects provided herein, the initiator species comprises an
organosilane. In some embodiments of aspects provided herein, the
initiator species comprises the molecule shown in FIG. 1. In some
embodiments of aspects provided herein, the surface initiated
polymerization comprises atom-transfer radical polymerization
(ATRP). In some embodiments of aspects provided herein, the surface
initiated polymerization comprises reversible addition
fragmentation chain-transfer (RAFT). In some embodiments of aspects
provided herein, the biomolecules comprise 5' acrydite modified
oligonucleotides. In some embodiments of aspects provided herein,
the biomolecules comprise antibodies. In some embodiments of
aspects provided herein, the biomolecules comprise peptides. In
some embodiments of aspects provided herein, the biomolecules
comprise aptamers. In some embodiments of aspects provided herein,
the coupling of the biomolecules comprises incorporation of
acrydite-modified biomolecules during polymerization. In some
embodiments of aspects provided herein, the biomolecules comprises
reaction at bromoacetyl sites. In some embodiments of aspects
provided herein, the coupling of the biomolecules comprises
reaction at azide sites. In some embodiments of aspects provided
herein, the coupling of the biomolecules comprises azide-alkyne
Huisgen cycloaddition.
An aspect of the present disclosure provides a composition,
comprising: (a) a surface; (b) a polymer coating covalently bound
to the surface, formed by surface-initiated polymerization, wherein
the polymer coating comprises 2 or more different types of
acrylamide monomers; and (c) a biomolecule coupled to the polymer
coating.
An aspect of the present disclosure provides a composition,
comprising: (a) a surface; (b) a polymer coating covalently bound
to the surface, formed by surface-initiated polymerization; and (c)
at least two different biomolecules coupled to the polymer
coating.
In some embodiments of aspects provided herein, the biomolecule
comprises an oligonucleotide. In some embodiments of aspects
provided herein, the oligonucleotide is coupled to the polymer at
its 5' end. In some embodiments of aspects provided herein, the
oligonucleotide is coupled to the polymer at its 3' end. In some
embodiments of aspects provided herein, the biomolecule comprises
an antibody. In some embodiments of aspects provided herein, the
biomolecule comprises an aptamer. In some embodiments of aspects
provided herein, the at least two different biomolecules comprise
oligonucleotides. In some embodiments of aspects provided herein,
the oligonucleotides are coupled to the polymer coating at their 5'
ends. In some embodiments of aspects provided herein, the
oligonucleotides are coupled to the polymer coating at their 3'
ends. In some embodiments of aspects provided herein, the at least
two different biomolecules comprise antibodies. In some embodiments
of aspects provided herein, the at least two different biomolecules
comprise aptamers. In some embodiments of aspects provided herein,
the surface comprises glass. In some embodiments of aspects
provided herein, the surface comprises silicon. In some embodiments
of aspects provided herein, the polymer coating comprises
polyacrylamide. In some embodiments of aspects provided herein, the
polymer coating comprises PMMA. In some embodiments of aspects
provided herein, the polymer coating comprises polystyrene. In some
embodiments of aspects provided herein, the surface-initiated
polymerization comprises atom-transfer radical polymerization
(ATRP). In some embodiments of aspects provided herein, the
surface-initiated polymerization comprises reversible addition
fragmentation chain-transfer (RAFT).
INCORPORATION BY REFERENCE
All publications, patents, and patent applications mentioned in
this specification are herein incorporated by reference to the same
extent as if each individual publication, patent, or patent
application was specifically and individually indicated to be
incorporated by reference.
BRIEF DESCRIPTION OF THE DRAWINGS
The novel features of the invention are set forth with
particularity in the appended claims. A better understanding of the
features and advantages of the present invention will be obtained
by reference to the following detailed description that sets forth
illustrative embodiments, in which the principles of the invention
are utilized, and the accompanying drawings of which:
FIG. 1 shows an example of an initiator silane.
FIG. 2 shows an example of a phosphorylcholine-acrylamide
monomer.
FIG. 3 shows an example of a betaine-acrylamide monomer.
FIG. 4 shows an example of a process for producing a polyacrylamide
surface coating with oligonucleotides.
DETAILED DESCRIPTION OF THE INVENTION
Overview
This disclosure provides methods and compositions for improved
polymer coatings on surfaces. The polymer coatings can be generated
via surface-initiated polymerization (SIP) via initiator species
bound to a surface. The polymer coatings can incorporate modified
monomers to modulate physicochemical properties of the coatings.
The polymer coatings can incorporate oligonucleotides.
Surfaces
The methods and compositions provided in this disclosure can
comprise creating a polymer coating on a surface. The surface can
comprise glass, silica, titanium oxide, aluminum oxide, indium tin
oxide (ITO), silicon, polydimethylsiloxane (PDMS), polystyrene,
polyolefins, such as Poly(methylpentene) (PMP) and Zeonor.TM.,
cyclic olefin copolymer such as Topas.TM., polymethylmethacrylate
(PMMA), other plastics, titanium, gold, other metals, or other
suitable materials. The surface can be flat or round, continuous or
non-continuous, smooth or rough. Examples of surfaces include flow
cells, sequencing flow cells, flow channels, microfluidic channels,
capillary tubes, piezoelectric surfaces, wells, microwells,
microwell arrays, microarrays, chips, wafers, non-magnetic beads,
magnetic beads, ferromagnetic beads, paramagnetic beads,
superparamagnetic beads, and polymer gels.
Initiator Species Attachment
The methods and compositions provided in this disclosure can
comprise initiator species for bonding to a support surface. In
some cases, the initiator species comprises at least one
organosilane. The organosilane can comprise one surface-bonding
group, resulting in a mono-pedal structure. The organosilane can
comprise two surface-bonding groups, resulting in a bi-pedal
structure. The organosilane can comprise three surface-bonding
groups, resulting in a tri-pedal structure. The surface bonding
group can comprise MeO.sub.3Si (e.g. see FIG. 1, item [0100]). The
surface bonding group can comprise (MeO).sub.3Si. The surface
bonding group can comprise (EtO).sub.3Si. The surface bonding group
can comprise (AcO).sub.3Si. The surface bonding group can comprise
(Me.sub.2N).sub.3Si. The surface bonding group can comprise
(HO).sub.3Si. For cases where the organosilane comprises multiple
surface bonding groups, the surface bonding groups can be the same
or can be different. The organosilane can comprise the silane
reagent shown in FIG. 1. In some cases, the initiator species
comprises at least one organophosphonic acid, wherein the surface
bonding group comprises (HO).sub.2P(.dbd.O). The organophosphonic
acid can comprise one surface-bonding group, resulting in a
mono-pedal structure. The organophosphonic acid can comprise two
surface-bonding groups, resulting in a bi-pedal structure. The
organophosphonic acid can comprise three surface-bonding groups,
resulting in a tri-pedal structure.
Silane treatment of substrates (e.g., glass substrates) can be
performed with a silane solution, such as a solution of silane in
ethanol, water, or a mixture thereof. Prior to treatment with a
silane solution, a substrate can be cleaned. Cleaning can be
performed by immersion in sulfuric-peroxide solution. For
attachment of an initiator species to a plastic substrate, a thin
film of silica can be applied to the surface. Silica can be
deposited by a variety of methods, such as vacuum deposition
methods including but not limited to chemical vapor deposition
(CVD), sputtering, and electron-beam evaporation. Silane treatment
can then be performed on the deposited silica layer.
Surface-Initiated Polymerization (SIP)
The methods and compositions provided in this disclosure can
comprise forming a polymer coating from surface-bound initiator
species. The resulting polymer coatings can comprise linear chains.
The resulting polymer coatings can comprise lightly branched
chains. The polymer coatings can form polymer brush thin-films. The
polymer coatings can include some cross-linking. The polymer
coatings can form a graft structure. The polymer coatings can form
a network structure. The polymer coatings can form a branched
structure. The polymers can comprise homogenous polymers. The
polymers can comprise block copolymers. The polymers can comprise
gradient copolymers. The polymers can comprise periodic copolymers.
The polymers can comprise statistical copolymers.
Polymer coatings can comprise polymer molecules of a particular
length or range of lengths. Polymer molecules can have a length of
at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,
18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90,
95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250,
300, 350, 400, 450, or 500 backbone atoms or molecules (e.g.,
carbons). Polymer molecules can have a length of at most 2, 3, 4,
5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,
23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95,
100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300,
350, 400, 450, or 500 backbone atoms or molecules (e.g., carbons).
Polymer molecules can have a length of at least 2, 3, 4, 5, 6, 7,
8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,
25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100,
110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300, 350,
400, 450, or 500 monomer units (e.g., acrylamide molecules).
Polymer molecules can have a length of at most 2, 3, 4, 5, 6, 7, 8,
9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,
30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110,
120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300, 350, 400,
450, or 500 monomer units (e.g., acrylamide molecules).
The polymer can comprise polyacrylamide (PA). The polymer can
comprise polymethylmethacrylate (PMMA). The polymer can comprise
polystyrene (PS). The polymer can comprise polyethylene glycol
(PEG). The polymer can comprise polyacrylonitrile (PAN). The
polymer can comprise poly(styrene-r-acrylonitrile) (PSAN). The
polymer can comprise a single type of polymer. The polymer can
comprise multiple types of polymer. The polymer can comprise any of
the polymers described in "Ayres, N. (2010). Polymer brushes:
Applications in biomaterials and nanotechnology. Polymer Chemistry,
1(6), 769-777," or in "Barbey, R., Lavanant, L., Paripovic, D.,
Schuwer, N., Sugnaux, C., Tugulu, S., & Klok, H. A. (2009).
Polymer brushes via surface-initiated controlled radical
polymerization: synthesis, characterization, properties, and
applications. Chemical reviews, 109(11), 5437-5527."
The polymerization can comprise methods to control polymer chain
length, coating uniformity, or other properties. The polymerization
can comprise controlled radical polymerization (CRP). The
polymerization can comprise atom-transfer radical polymerization
(ATRP). The polymerization can comprise reversible addition
fragmentation chain-transfer (RAFT). The polymerization can
comprise living polymerization processes, including those described
in "Ayres, N. (2010). Polymer brushes: Applications in biomaterials
and nanotechnology. Polymer Chemistry, 1(6), 769-777," or in
"Barbey, R., Lavanant, L., Paripovic, D., Schuiwer, N., Sugnaux,
C., Tugulu, S., & Klok, H. A. (2009). Polymer brushes via
surface-initiated controlled radical polymerization: synthesis,
characterization, properties, and applications. Chemical reviews,
109(11), 5437-5527."
Incorporation of Biomolecules
Biomolecules can be coupled to the polymer coatings described in
this disclosure. The biomolecules can comprise antibodies. The
biomolecules can comprise proteins. The biomolecules can comprise
peptides. The biomolecules can comprise enzymes. The biomolecules
can comprise aptamers. The biomolecules can comprise
oligonucleotides.
Oligonucleotides can be coupled to the polymer coatings described
in this disclosure. The oligonucleotides can comprise primers. The
oligonucleotides can comprise cleavable linkages. Cleavable
linkages can be enzymatically cleavable. The oligonucleotides can
comprise at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,
15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, or 60 bases.
The oligonucleotides can vary in length, such as from 3 to 5 bases,
from 1 to 50 bases, from 6 to 12 bases, from 8 to 12 bases, from 15
to 25 bases, from 25 to 35 bases, from 35 to 45 bases, or from 45
to 55 bases. The individual oligonucleotides coupled to the
coatings can differ from each other in length.
Biomolecules (e.g., oligonucleotides) can be incorporated into the
polymer coatings during the polymerization process. For example,
5'-acrydite-modified oligonucleotides chains can be added during
the polymerization process to allow the incorporation of the
oligonucleotides into the polymerizing polyacrylamide structure. In
some cases, oligonucleotides are coupled to the polymer coating at
the 5' end. In some cases, oligonucleotides are coupled to the
polymer coating at the 3' end. In some cases, some oligonucleotides
are coupled to the polymer coating at the 3' end and some
oligonucleotides are coupled to the polymer coating at the 5'
end.
Biomolecules (e.g., oligonucleotides) can be incorporated into the
polymer coatings after the polymerization process. For example,
reactive sites can be added to the polymer structure during the
polymerization process. Biomolecules can be incorporated at the
reactive sites subsequent to the polymerization. The reactive sites
can comprise bromoacetyl sites. The reactive sites can comprise
azides. The reactive sites can comprise sites compatible with
azide-alkyne Huisgen cycloaddition.
Biomolecules (e.g., oligonucleotides) can be incorporated into the
polymer coatings in a controlled manner, with particular
biomolecules located at particular regions of the polymer coatings.
Biomolecules can be incorporated into the polymer coatings at
random, with particular biomolecules randomly distributed
throughout the polymer coatings.
In some instances a composition of the invention comprises a
surface, a polyacrylamide coating covalently bound to said surface;
and at least one oligonucleotide coupled to said polyacrylamide
coating. In other instances, the surface includes at least 1, 10,
100, 10,000, 100,000, 1,000,000, 10,000,000, 100,000,000, or
1,000,000,000 oligonucleotides coupled to the polyacrylamide
coating.
Modification of Physicochemical Characteristics of Polymer
Coating
The polymer coatings described in this disclosure can have their
physicochemical characteristics modulated. This modulation can be
achieved by incorporating modified acrylamide monomers during the
polymerization process.
In some cases, ethoxylated acrylamide monomers can be incorporated
during the polymerization process. Ethoxylated acrylamide monomers
can be incorporated by being present in the polymerization
solution. The ethoxylated acrylamide monomers can comprise monomers
of the form CH.sub.2.dbd.CH--CO--NH(--CH.sub.2--CH2-O--).sub.nH.
The ethoxylated acrylamide monomers can comprise hydroxyethyl
acrylamide monomers. The ethoxylated acrylamide monomers can
comprise ethylene glycol acrylamide monomers. The ethoxylated
acrylamide monomers can comprise hydroxyethylmethacrylate (HEMA).
The ethoxylated acrylamide monomers can comprise
N-(2-hydroxyethyl)acrylamide. The incorporation of ethoxylated
acrylamide monomers can result in a more hydrophobic polyacrylamide
surface coating.
In some cases, phosphorylcholine acrylamide monomers can be
incorporated during the polymerization process. The
phosphorylcholine acrylamide monomers can comprise monomers of the
structure shown in FIG. 2. The phosphorylcholine acrylamide
monomers can comprise other phosphorylcholine acrylamide monomers.
Phosphorylcholine acrylamide monomers can be incorporated by being
present in the polymerization solution.
In some cases, betaine acrylamide monomers can be incorporated
during the polymerization process. The betaine acrylamide monomers
can comprise monomers of the structure shown in FIG. 3. Betaine
acrylamide monomers can be incorporated by being present in the
polymerization solution.
The polymer coating can be of uniform thickness. The polymer
coating can be of varying thickness over its area. The polymer
coating can be, on average, at least 1 .mu.m thick. The polymer
coating can be at least 2 .mu.m thick. The polymer coating can be
at least 3 .mu.m thick. The polymer coating can be at least 5 .mu.m
thick. The polymer coating can be at least 10 .mu.m thick. The
polymer coating can be at least 15 .mu.m thick. The polymer coating
can be at least 20 .mu.m thick. The polymer coating can be at least
25 .mu.m thick. The polymer coating can be at least 30 .mu.m thick.
The polymer coating can be at least 40 .mu.m thick. The polymer
coating can be at least 50 .mu.m thick. The polymer coating can be
at least 75 .mu.m thick. The polymer coating can be at least 100
.mu.m thick. The polymer coating can be at least 150 .mu.m thick.
The polymer coating can be at least 200 .mu.m thick. The polymer
coating can be at least 300 .mu.m thick. The polymer coating can be
at least 400 .mu.m thick. The polymer coating can be at least 500
.mu.m thick. The polymer coating can be between about 1 .mu.m and
about 10 .mu.m thick. The polymer coating can be between about 5
.mu.m and about 15 .mu.m thick. The polymer coating can be between
about 10 .mu.m and about 20 .mu.m thick. The polymer coating can be
between about 30 .mu.m and about 50 .mu.m thick. The polymer
coating can be between about 10 .mu.m and about 50 .mu.m thick. The
polymer coating can be between about 10 .mu.m and about 100 .mu.m
thick. The polymer coating can be between about 50 .mu.m and about
100 .mu.m thick. The polymer coating can be between about 50 .mu.m
and about 200 .mu.m thick. The polymer coating can be between about
100 .mu.m and about 30 .mu.m thick. The polymer coating can be
between about 100 .mu.m and about 500 .mu.m thick.
Reactions
The polymer coatings described in this disclosure can be used in
performing reactions. The reactions performed can be enzymatic. The
reagents for the reactions performed can comprise nucleic acids.
The reactions can comprise digestion reactions. The reactions can
comprise extension reactions such as primer extension, or overlap
extension. The reactions can comprise amplification reactions, such
as polymerase chain reaction (PCR) and variants thereof (such as
multiplex PCR, nested PCR, reverse transcriptase PCR (RT-PCR),
semi-quantitative PCR, quantitative PCR (qPCR) or real time PCR,
touchdown PCR, or assembly PCR), nucleic acid sequence based
amplification (NASBA) (see e.g., "Compton, J (1991). Nucleic acid
sequence-based amplification. Nature 350 (6313): 91-2."), strand
displacement assay (SDA) (see e.g., U.S. Pat. No. 5,712,124,
"Strand displacement amplification"), and loop mediated isothermal
amplification (LAMP) (see e.g., U.S. Pat. No. 6,410,278, "Process
for synthesizing nucleic acid"). The reactions can comprise
transcription reactions, such as in vitro transcription. The
reactions can comprise sequencing reactions, such as BAC-based
sequencing, pyrosequencing, sequencing by synthesis, or any method
described in "Mardis, E. R. (2008). Next-generation DNA sequencing
methods. Annu. Rev. Genomics Hum. Genet., 9, 387-402."
The polymer coatings described in this disclosure can be robust.
The robustness of the polymer coatings can be exhibited by the
durability, the resistance to degradation, or the level of
attachment of the coating after being subjected to certain
conditions. The robustness of the polymer coatings can be exhibited
by the number or percentage of biomolecules (e.g.,
oligonucleotides) molecules coupled to the polymer coating which
remain coupled to the polymer coating after being subjected to
certain conditions. Conditions can include but are not limited to
duration of time, a temperature or set of temperatures, presence of
chemicals (e.g., acids, bases, reducing agents, oxidizing agents),
mechanical forces (e.g. stress, strain, vibrations, high pressures,
vacuums), combinations of conditions, or repeated cycles of
conditions or combinations of conditions (e.g. reaction cycles
comprising temperatures and use of chemicals). Durations of time
can comprise at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, or
50 minutes, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,
15, 16, 17, 18, 19, 20, 21, 22, or 23 hours, at least 1, 2, 3, 4,
5, 6, 7, 8, 9, 10, 11, 12, or 13 days, or at least 2, 3, 4, 5, 6,
7, 8, 9, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 40, 50, or 60
weeks. Temperatures can comprise at least 0, 5, 10, 15, 20, 25, 30,
35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100.degree.
C. Temperatures can comprise at most 0, 5, 10, 15, 20, 25, 30, 35,
40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100.degree. C.
Chemicals can comprise strong acids, weak acids, strong bases, weak
bases, strong oxidizers, weak oxidizers, strong reducers, weak
reducers, enzymes, monomers, polymers, buffers, solvents, or other
reagents. Cycles of conditions can comprise at least 1, 2, 3, 4, 5,
6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,
24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40,
41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85,
90, 95, 100, 125, 150, 175, 200, 250, 300, 350, 400, 450, 500, 600,
700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000,
9000, or 10,000 cycles. In some embodiments, the polymer coatings
herein are used to perform at least 10, 20, 30, 40, 50, 60, 70, 80,
90, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 cycles of
conditions, and wherein at least 50, 60, 70, 80, 90, 91, 92, 93,
94, 95, 96, 97, 98, 99, 99.5 or 99.9% the polymer chains remain
completely intact and bonded to said surface after the cycles.
In some embodiments, the polymer coatings herein are used as a
solid support to perform sequencing by synthesis (SBS). In SBS, a
target polynucleotide sequence can be determined by generating its
complement using the polymerase reaction to extend a suitable
primer, and characterizing the successive incorporation of bases
that generate the complement. The target sequence is, typically,
immobilized on a solid support. Each of the different bases A, T, G
or C is then brought, by sequential addition, into contact with the
target, and any incorporation events detected via a suitable label
attached to the base. In contrast to the prior art methods, the
present invention requires the presence of a polymerase enzyme that
retains a 3' to 5' exonuclease function, which is induced to remove
an incorporated labeled base after detection of incorporation. A
corresponding non-labeled base can then be incorporated into the
complementary strand to allow further sequence determinations to be
made. Repeating the procedure allows the sequence of the complement
to be identified, and thereby the target sequence also. In some
embodiments, the polymer coatings herein are used to perform at
least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500,
600, 700, 800, 900, or 1000 cycles of sequencing by synthesis
(SBS), for example as described by the methods of U.S. Pat. No.
6,833,246, and wherein at least 50, 60, 70, 80, 90, 91, 92, 93, 94,
95, 96, 97, 98, 99, 99.5 or 99.9% the polymer chains remain
completely intact and bonded to said surface after the SBS. Prior
to the SBS cycles, the polymer coating can have coupled to it at
least 10, 20, 50, 100, 200, 500, 1,000, 2,000, 5,000, 10,000,
20,000, 50,000 or 100,000, 200,000, 500,000, 1,000,000, 2,000,000,
5,000,000, 10,000,000, 20,000,000, 100,000,000, 200,000,000,
500,000,000, or a billion nucleic acid molecules. Prior to the SBS
cycles, the polymer coating can have nucleic acid molecules
arranged on it at an areal density of at least about 10, 20, 50,
100, 200, 500, 1,000, 2,000, 5,000, 10,000, 20,000, 50,000,
100,000, 1,000,000, 1.times.10.sup.7, 5.times.10.sup.7,
1.times.10.sup.8, 5.times.10.sup.8, 1.times.10.sup.9,
5.times.10.sup.9, 1.times.10.sup.10, 5.times.10.sup.10, or
1.times.10.sup.11 molecules per square micrometer. In some cases,
prior to the SBS cycles, the polymer coating has nucleic acid
molecules arranged on it at an areal density of about
1.times.10.sup.2 to about 1.times.10.sup.6 per square micrometer.
In some cases, prior to the SBS cycles, the polymer coating has
nucleic acid molecules arranged on it at an areal density of about
5.times.10.sup.2 to about 5.times.10.sup.4 per square micrometer.
In some cases, prior to the SBS cycles, the polymer coating has
nucleic acid molecules arranged on it at an areal density of about
1.times.10.sup.3 to about 1.times.10.sup.4 per square
micrometer.
In some embodiments, the polymer coatings herein are used to
perform PCR on nucleic acid polymer chains bound to the coating.
PCR, for example, can include multiple cycles, wherein each cycle
includes a denaturation step, an annealing step, and an extension
or elongation step. The denaturation step can comprise subjecting
the nucleic acids to a temperature of at least about 85.degree. C.,
86.degree. C., 87.degree. C., 88.degree. C., 89.degree. C.,
90.degree. C., 91.degree. C., 92.degree. C., 93.degree. C.,
94.degree. C., 95.degree. C., 96.degree. C., 97.degree. C., or 98
OC. The denaturation step can comprise duration of at least about
15 seconds, 20 seconds, 25 seconds, 30 seconds, 35 seconds, 40
seconds, or 45 seconds. The annealing step can comprise subjecting
the nucleic acids to a temperature of at least about 50.degree. C.,
51.degree. C., 52.degree. C., 53.degree. C., 54.degree. C.,
55.degree. C., 56.degree. C., 57.degree. C., 58.degree. C.,
59.degree. C., 60.degree. C., 61.degree. C., 62.degree. C.,
63.degree. C., 64.degree. C., or 65 OC. The annealing step can
comprise duration of at least about 15 seconds, 20 seconds, 25
seconds, 30 seconds, 35 seconds, 40 seconds, or 45 seconds. The
extension or elongation step can comprise a temperature of at least
about 70.degree. C., 71.degree. C., 72.degree. C., 73.degree. C.,
74.degree. C., 75.degree. C., 76.degree. C., 77.degree. C.,
78.degree. C., 79.degree. C., or 80.degree. C. The extension or
elongation step can comprise duration of at least about 30 seconds,
40 seconds, 50 seconds, 60 seconds, 70 seconds, 80 seconds, 90
seconds, 100 seconds, 110 seconds, or 120 seconds. The polymer
coatings herein can be used to perform at least 10, 20, 30, 40, 50,
60, 70, 80, 90, or, 100 cycles of polymerase chain reaction (PCR),
and wherein at least 50, 60, 70, 80, 90, 91, 92, 93, 94, 95, 96,
97, 98, 99, 99.5 or 99.9% the polymer chains remain completely
intact and bonded to said surface after the final PCR cycle. Prior
to the PCR cycles, the polymer coating can have coupled to it at
least 10, 20, 50, 100, 200, 500, 1,000, 2,000, 5,000, 10,000,
20,000, 50,000 or 100,000, 200,000, 500,000, 1,000,000, 2,000,000,
5,000,000, 10,000,000, 20,000,000, 100,000,000, 200,000,000,
500,000,000, or a billion nucleic acid molecules. Prior to the PCR
cycles, the polymer coating can have nucleic acid molecules
arranged on it at a density of at least 10, 20, 50, 100, 200, 500,
1,000, 2,000, 5,000, 10,000, 20,000, 50,000, 100,000, 1,000,000,
1.times.10.sup.7, 5.times.10.sup.7, 1.times.10.sup.8,
5.times.10.sup.8, 1.times.10.sup.9, 5.times.10.sup.9,
1.times.10.sup.10, 5.times.10.sup.0, or 1.times.10.sup.1 molecules
per square micrometer. In some cases, prior to the PCR cycles, the
polymer coating has nucleic acid molecules arranged on it at an
areal density of about 1.times.10.sup.2 to about 1.times.10.sup.6
per square micrometer. In some cases, prior to the PCR cycles, the
polymer coating has nucleic acid molecules arranged on it at an
areal density of about 5.times.10.sup.2 to about 5.times.10.sup.4
per square micrometer. In some cases, prior to the PCR cycles, the
polymer coating has nucleic acid molecules arranged on it at an
areal density of about 1.times.10.sup.3 to about 1.times.10.sup.4
per square micrometer.
Advantages
Use of initiator species, such as silanes, with multiple bonding
groups can provide high thermal and hydrolytic stability (see,
e.g., U.S. Pat. No. 6,262,216). Such stability can increase the
durability of the coating through repeated cycles of reactions or
other processing.
Use of surface coatings as described herein can provide a more
enzymatically compatible or favorable environment than that
provided by an uncoated surface. Surface coatings with modulated
physicochemical characteristics as described herein can provide
advantages to use for conducting enzymatic reactions on, near, or
on molecules bound to the surfaces. The advantages can comprise a
reduction in non-specific binding to the surface. The advantages
can comprise an optimal environment for enzymes, such as
polymerases. For example, neutral hydrophilic polymers and linking
groups can provide favorable environments for enzymes.
EXAMPLES
Example 1--Production of a Flat Surface Array
Initiator silanes of the structure shown in FIG. 1 are bound to a
flat silica substrate in the presence of EtOH, forming di-podal
surface polymer initiation sites. A mixture of acrylamide and
ethoxylated acrylamide, together with acrydite-modified
oligonucleotides, undergoes atom-transfer radical polymerization
(ATRP) on the substrate in the presence of CuBr, PMDETA, and
H.sub.2O. This forms a covalently-bonded, lightly-crosslinked
polyacrylamide surface coating bound to the surface initiator
sites, with thickness between about 50 nm and about 200 nm, with
oligonucleotides incorporated into the structure (see FIG. 4).
Example 2--Use of a Flat Surface Array in Sequencing
A polyacrylamide coated substrate is prepared as described in
Example 1. DNA to be sequenced is bound to the oligonucleotides
incorporated into the polymer structure. Sequencing by synthesis
reagents are added to the substrate and sequencing by synthesis is
performed for 40 cycles. At least 90% of polymer chains remain
intact and bonded to the surface.
Example 3--Use of a Flat Surface Array in DNA Amplification
A polyacrylamide coated substrate is prepared as described in
Example 1. DNA to be amplified is bound to the oligonucleotides
incorporated into the polymer structure. Polymerase chain reaction
(PCR) reagents are added to the substrate and PCR is performed for
30 cycles. At least 90% of polymer chains remain intact and bonded
to the surface.
Example 4--Synthesis of
Azido-PEG4-N,N-Bis(3-(Trimethoxysilyl)Propyl)Carbamate
Azido-PEG4-alcohol (BroadPharm, 220 mg; 1.0 mmol) was dried by
co-evaporating twice with 2 ml CH.sub.3CN, then combined with
diphosgene (200 mg; 1.0 mmol) in 1 ml of CH.sub.2Cl.sub.2 under N2.
After standing overnight at ambient temperature, the solvent was
evaporated to obtain 280 mg of the product as a pale yellow oil,
which was used without further purification. .sup.1H-NMR
(CDCl.sub.3): .delta. (ppm) 4.46 (2H, t J=2.8 Hz; CH.sub.2OC(O)Cl);
3.79 (2H, t J=4.5 Hz; CH.sub.2CH.sub.2N.sub.3); 3.68-3.70 (10H, m,
CH.sub.2OCH.sub.2); 3.41 (2H, t J=5.2 Hz, CH.sub.2N.sub.3).
##STR00001##
Bis(trimethoxysilylpropyl)amine (342 mg/320 uL; 1.0 mmol) and DIEA
(136 mg/182 uL; 1.05 mmol) were combined in 1 ml dry ether under
N.sub.2 and cooled on ice to 0-4.degree. C. The azido-PEG4
chloroformate (280 mg; 1.0 mmol) was dissolved in 1 ml dry ether
and added dropwise via syringe, and then stirring was continued at
ambient temperature overnight. Another 2 ml of dry ether was added,
and the solution was quickly filtered and evaporated to yield the
silane as a light yellow oil (.about.550 mg). .sup.1H-NMR
(CD.sub.3OD): .delta. (ppm) 4.20-4.24 (2H, br m,
CH.sub.2OC(O)N<); 3.67-3.74 (13H, m, CH.sub.2OCH.sub.2); 3.39
(2H, t J.about.5.0 Hz, CH.sub.2N.sub.3); 3.35 (21H, s,
CH.sub.3OSi); 3.22-3.28 (4H, br m, --CH.sub.2NC(O)O--); 1.60-1.70
(4H, br m, C--CH.sub.2--C); 0.55-0.65 (4H, br m,
C--CH.sub.2--Si).
##STR00002##
Example 5--Synthesis of
N-(3-(Bromoacetamido)propyl)methacrylamide
N-(3-aminopropyl)methacrylamide hydrochloride (Polysciences; 360
mg; 2.0 mmol) and N-(bromoacetoxy)succinimide (Broad Pharm; 570 mg;
2.4 mmol) were combined in 10 mL dry CH.sub.2Cl.sub.2 under N.sub.2
and cooled to -10.degree. C. with ice-MeOH. Diisopropylethylamine
(Aldrich, 800 uL; 4.2 mmol) was then added dropwise while stirring.
The solution was stirred for another 30 min cold, then for 3 h at
rm temp. The solution was diluted with 40 ml ethyl acetate, and
washed successively with 12 ml each of 1M HCl; 0.1M NaOH; and then
brine. The organic phase was dried with MgSO.sub.4 and evaporated
to yield 220 mg (.about.40%) of 3:1 mixture of bromo-, and
chloroacetylated products as an off-white solid. .sup.1H-NMR
(acetone-d.sub.6): .delta. (ppm) 7.70 (1H, br s, NH.sub.a); 7.40
(1H, br s, NH.sub.b); 5.71-5.73 (1H, br m, CH.dbd.C); 5.30-5.32
(1H, m, CH'.dbd.C); 4.08 (0.5H, s, CH.sub.2Cl); 3.89 (1.5H, s,
CH.sub.2Br); 3.24-3.32 (4H, m, CH.sub.2N); 1.91-1.93 (3H, br m,
CH.sub.3); 1.68 (2H, br qnt, J=6.4 Hz;
H.sub.2'CCH.sub.2CH.sub.2''). LC-MS (ESI): 5.7 min: 242, 243, 244
(10:1:3; M.Na.sup.+/chloro); 219, 220, 221 (10:1:3;
M.H.sup.+/chloro); 134, 135, 136 (10:0.6:3;
M--CH.sub.2.dbd.C(Me)CONH/chloro); 126, 127 (10:1;
M-Cl/BrCH.sub.2CCONH.sup.-). 5.9 min: 286, 287, 288, 289
(10:1:10:1; M.Na.sup.-/bromo); 263, 265, 266 (10:10:1;
M.H.sup.+/bromo); 178, 179, 180, 181 (10:0.6:10:0.6;
M--CH.sub.2.dbd.C(Me)CONH.sup.-/bromo); 126, 127 (10:1;
M--Cl/BrCH.sub.2CCONH.sup.-).
##STR00003##
Example 6--Synthesis of N-(4-Azidobutyl)methacrylamide
4-Azido-1-butylamine (Synthonix; 1.1 g; 8.75 mmol)) was combined
with DIEA (1.22 g; 9.5 mmol) in 15 mL of dry ethyl acetate in a 50
mL flask equipped w/ stirbar & dropping funnel and flushed with
dry N.sub.2. The solution was cooled to 2.degree. C. on an
ice-waterbath, and a solution of methacryoyl chloride (0.96 g; 9.2
mmol) in 5 ml dry ether was added dropwise with stirring over 30
min. The ice bath was removed, another 15 ml of dry ethyl acetate
was added, and stirring was continued at ambient temperature
overnight. The solids were removed by filtration and the combined
filtrates were washed twice w/ 10 ml water, once w/ brine, then
dried (MgSO.sub.4) evaporated in vacuo to obtain 1.50 g (93%)
product as an orange liquid. .sup.1H-NMR (CDCl.sub.3): .delta.
(ppm) 5.92-5.83 (1H, br s, NH); 5.68 (1H, t J=0.8 Hz;
.dbd.CH.sub.a); 5.68 (1H, m, .dbd.CH.sub.b); 3.45 (4H, br m,
NCH.sub.2); 1.97 (3H, t J=1.4 Hz, CH.sub.3); 1.69-1.60 (4H, br m,
C--CH.sub.2--C). MS (ESI): 126.2 (M-CH.sub.2N.sub.3); 183.2 (M.H+);
205.2 (M.Na+). The product was used within 10 days, as
decomposition with evolution of N.sub.2 was noted after 2-3 weeks
storage at 4.degree. C. by NMR.
##STR00004##
Example 7--Silanation of Flowcell Surfaces
For most experiments, the flowcells used were flat "capillary micro
glass slides" made from Corning.RTM. 7740 borosilicate, low
expansion, type I glass (p/n 63825-05, EM Sciences, Hatfield, Pa.).
A short length of 0.5 mm ID heat-shrink PTFE tubing was sealed to
both ends of the capillaries to provide leak-proof connection to
manifolds, syringes, etc. For some experiments, "refurbished"
Illumina MiSeq.TM. flowcells were employed. These were stripped of
indigenous surface coatings with 200 mM sodium persulfate at
65.degree. C. for 18 hr, followed by 1M KOH/65.degree. C./6 hr,
rinsing with deionized water and drying with a stream of
nitrogen.
Prior to silanation, all capillary flowcell surfaces were cleaned
by immersion in sulfuric-peroxide solution (Nanostrip, Cyantek
Corp., Fremont Calif.) for 16-18 hr at 25.degree. C., then rinsed
thoroughly with deionized water and dried with a stream of
nitrogen. The cleaned flowcells were stored under nitrogen and
silanated within 48 hours. Silanation was performed by filling the
flowcell with a freshly prepared 2% (wt/vol) solution of the
appropriate silane in 95:5 ethanol-water, and incubating for 4-18
hours at room temperature. The flowcells were then rinsed
thoroughly with ethanol and deionized water; dried with nitrogen,
and stored at ambient temperature.
Example 8--Oligonucleotide Primer Immobilization by
Surface-Initiated Acrylamide ATRP
Flowcells for SI-ATRP were silanated as described in Example 7,
with
2-Bromo-2-methyl-N,N-bis-(3-trimethoxysilanylpropyl)propionamide
(see, e.g., US 2011/0143967).
Dry-down Primers: Equivalent amounts of 5'-acrydite modified
primers FWD (4 uL, 1 mM) and REV (4 uL, 1 mM) were combined in a
0.9 mL conical-tip HPLC vial. The solutions were reduced to dryness
on a Speed-Vac evaporator at ambient temperature (10-15 minutes).
The vial containing dried primers was tightly closed with a
septum-sealed screw cap and connected to a vacuum/N.sub.2 manifold
via an 18-gauge syringe needle. The vial was deoxygenated 5 cycles
of alternating vacuum/nitrogen refill through a syringe needle.
Deoxygenate Flowcell: The flowcell to be used for SI-ATRP was
deoxygenated by purging with dry nitrogen.
Deoxygenate Solvent: In another vial, a solvent mixture composed of
28% methanol in water (v:v) was deoxygenated by sparging
continuously with nitrogen for 30 minutes,
Preparation of Catalyst/Acrylamide Solution: CuBr (6.8 mg, 47.4
umol) and CuBr.sub.2 (3.9 mg, 17.5 umol) were weighed and placed in
a 20 mL septum-capped vial containing a magnetic stirring bar. The
vial was connected to a vacuum/nitrogen manifold and deoxygenated
carefully with three cycles of evacuation-nitrogen back-fill. Then
a portion of the deoxygenated solution (14.5 mL) was transferred to
the vial containing the copper salts via gas-tight syringe.
Finally, acrylamide (42.5 mg, 600 umol) and PMDETA (14 uL, 67.2
umol) were added, and the solution was stirred vigorously while
sparging with nitrogen for another 15 minutes. It was occasionally
necessary to sonicate the solution briefly to disperse the CuBr
solid to obtain a light blue homogeneous solution.
Transfer Polymerization Solution to Flowcell: The dried-down
primers were reconstituted in deoxygenated catalyst/acrylamide
solution (20 uL), which was transferred via gas tight syringe. The
resulting solution was transferred to the pre-purged flowcell from
step 3, filling it completely. The ends of the flowcell were sealed
with parafilm, and the flowcell was maintained at ambient
temperature for 24-48 hours in an anaerobic environment.
Wash and Storage: The flowcell was flushed with 28%
methanol-water), and 1.times.TE buffer (.about.1 mL/ea) and stored
at 4.degree. C.
Example 9--Oligonucleotide Primer Immobilization Via
Solution-Initiated FRP Grafting of
Acrylamide/Bromoacetyl-Acrylamide
Flowcell surfaces were silanated with
3-(acrylamido)propyltrimethoxysilane (Gelest, Inc).
Purge Flowcell: The flowcell to be used for FRP was deoxygenated by
purging with dry nitrogen.
Solution Preparation and Polymerization: A solution of acrylamide
(0.0713 g, 1 mmol) and N-(3-bromoacetamidopropyl)methacrylamide
(6.4 mg, 0.024 mmol) in Milliq water (5 g) in a vial was capped
with rubber septum-sealed cap. The solution was deoxygenated by
sparging with nitrogen for 30 minutes. Polymerization was initiated
by adding a solution of potassium persulfate (2.5 mg, 0.0093 mmol
in degassed water 50 uL) and neat tetramethylenediamine (4.45 mg,
0.038 mmol). The resulting solution was transferred immediately
into the flowcell, filling it completely. The ends of the flowcell
were sealed with parafilm, and the flowcell was maintained at
ambient temperature for 60-80 minutes in an anaerobic environment.
Polymerization was terminated by purging the flowcell with 4-6 mL
of water, followed by 1 mL of 6.times.SSPE to remove unbound
polymer. The flowcell was stored in 6.times.SSPE at 4.degree.
C.
Primer Conjugation: A combined solution of FWD (2.5 uL, 1 mM) and
REV (2.5 uL, 1 mM) 5'-phosphorothioate-modified primers was placed
in a 0.9 mL conical-tip HPLC vial. The solution was reduced to
dryness on a Speed-Vac evaporator at ambient temperature (10-15
minutes) and then redissolved in 6.times.SSPE (20 uL). The storage
solution was removed from the flowcell and replaced with the primer
solution via a gas-tight syringe. The ends of the flowcell were
sealed tightly with parafilm, and the flowcell was maintained at
55.degree. C. for 2 hours. The flowcell was allowed to cool to
ambient temperature and then rinsed with Milliq water,
6.times.SSPE, and 1.times.TE (1 mL per rinse). The flowcell
containing 1.times.TE was sealed with parafilm and stored at
4.degree. C.
Example 10--Direct Immobilization of Primers on Silanated Flowcell
Surface Using Click Chemistry
Flowcell surfaces were cleaned by immersion in sulfuric-peroxide
solution (Nanostrip, Cyantek Corp., Fremont Calif.) for 16-18 hr at
25.degree. C., then rinsed thoroughly with deionized water and
dried with a stream of nitrogen. Flowcells were stored under
nitrogen and silanated within 48 hours with a freshly prepared 2%
(wt/vol) solution of
Azido-PEG4-N,N-bis(3-(trimethoxysilyl)propyl)carbamate in 95:5
ethanol-water for 18 hours. The flowcells were then rinsed
thoroughly with ethanol and deionized water; and dried with
nitrogen. A solution containing 100 uM each of the
5'-alkynyl-modified oligonucleotide primers FWD and REV, 5 mM CuI,
and 10 mM tris-(3-hydroxypropyltriazolylmethyl)amine (THPTA) in
0.1M Tris buffer (pH 7.0) was added and maintained at 22.degree. C.
for 18 hours, after which the oligonucleotide solution was removed
and the flowcell was rinsed with deionized water, dried &
stored at 4.degree. C.
Example 11--Immobilization Analysis by Hybridization
Successful primer attachment was confirmed with a 5'-CY3-labeled
oligonucleotide hybridization target complimentary to the FWD
primer ("FWD"): the flowcell was filled with 250 nM target oligo in
6.times.SSPE buffer pH 7.4, incubated for 1 h at 55.degree. C.,
cooling to 25.degree. C., and then washed with 4-5 volumes
6.times.SSPE. Surface fluorescence was measured with a CCD-based
imaging fluorescence microsope (LED bb excitation; >640 nm
emission filter). The hybridization target solution was then
removed and the flowcell was washed out with 20 volumes of
formamide at 55.degree. C., and stored at 4.degree. C. in
nuclease-free water.
Example 12--Solid Phase DNA Amplification and Cluster
Generation
Prepared flowcells (e.g., those prepared in previous examples) were
placed on a programmable thermo-fluidic station (purpose built
CentiPD). An actively cooled Peltier thermoelectric module (Laird),
NTC thermistor temperature sensors and a programmable PID
Controller (Laird) provided thermal control. The range of
achievable temperatures was 20-100.degree. C. On the fluidic side,
a 250 ul syringe pump (Cavro) pulled a programmed volume of reagent
at a specified speed through the capillary flowcell. The
appropriate reagent was selected via a 24-way selector valve (VICI)
with sippers leading to each of the reagent tubes. The prepared
reagents Eppendorf tubes were sitting in an aluminum cooling block
placed in an ice bath (to maintain them at 4.degree. C. during the
protocol time period).
A solution of 10 mM dNTPs was prepared as follows: combine 300
.mu.L of each dNTP stock solution (stock solution concentration:
100 mM) to make 25 mM stock, then add 1000 .mu.L of 25 mM stock to
1500 .mu.L of 10 mM Tris pH 8.0.
An HB1 solution was prepared in 1.times.(.about.10 mL aliquot) and
5.times. amounts, shown in Table 1:
TABLE-US-00001 TABLE 1 HB1 solution Reagent Stock Final 1 RXN H2O
7400 ul 20X SSC 20X 5X 2500 ul Tween-20 10% 0.1% 100 ul Total 10
ml
A Wash Buffer (W2) solution was prepared in 5.times. and 1.times.
amounts (.about.10 mL aliquot), as shown in Table 2:
TABLE-US-00002 TABLE 2 W2 solution Reagent Stock Final 1 RXN 5 RXNS
H2O 9750 ul 48750 ul 20X SSC 20X 0.3X 150 ul 750 ul Tween-20 10%
0.1% 100 ul 500 ul Total 10 ml 50000 ul
Labeled Primer (FP) solution was prepared at a concentration of 5
.mu.M by adding 15 .mu.L of 500 .mu.M primer stock solution to 1485
.mu.L of HB1 solution as shown in Table 3:
TABLE-US-00003 TABLE 3 FP solution Reagent Stock Final 1 RXN 16
RXNS Cost HB1 360 ul 5760 ul $0 5 uM Primer 5.0 uM 0.5 uM 40 ul 640
ul $0 Total 400 ul 6400 ul $0
An Amplification Premix (APM) solution was prepared as shown in
Table 4:
TABLE-US-00004 TABLE 4 APM Buffer solution Reagent Stock Final 1
RXN 32 RXNS H2O 687 ul 21984 ul 10X Thermopol 10X 1X 100 ul 3200 ul
5M Betaine 5M 1M 200 ul 6400 ul DMSO 100% 1.3% 13 ul 416 ul Total
1000 ul 32000 ul
An Amplification Mix (AM) was prepared as shown in Table 5:
TABLE-US-00005 TABLE 5 AM Buffer solution Reagent Stock Final 1 RXN
16 RXNS Cost H2O 1756 ul 28090 ul $0 10X 10X 1X 280 ul 4480 ul $0
Thermopol 5M Betaine 5M 1M 560 ul 8960 ul $128 DMSO 100% 1.3% 36 ul
582 ul $0 10 mM dNTPs 10 mM .sup. 0.2 mM 56 ul 896 ul $0 Bst Lg. 8
U/ul 0.32 U/ul 112 ul 1792 ul $444 Fragment Total 2800 ul 44800 ul
$573
An Linearization Mix (LM) solution was prepared as shown in Table
6:
TABLE-US-00006 TABLE 6 LM solution Reagent Stock Final 1 RXN 16
RXNS Cost H2O 356 ul 5696 ul $0 10X Thermopol 10X 1X 40 ul 640 ul
$0 USER 1 U/ul 0.01 U/ul 4 ul 64 ul $83 Total 400 ul 6400 ul
$83
A Library Dilution Buffer was prepared which comprises 10 mM
Tris-Cl at pH 8.5 with 0.1% Tween-20.
A dilute library was prepared as follows: 1) Stock 2 N NaOH
solution was diluted to 0.1 N NaOH solution, as shown in Table 7.
2) Stock 10 nM PhiX solution was diluted to 2 nM by adding 2 .mu.L
of PhiX to 8 .mu.L of Library Dilution Buffer. 3) The sample was
denatured by adding 10 .mu.L of 0.1 N NaOH to 10 .mu.L of 2 nM
sample solution and incubating for 5 minutes at room temperature.
4) The denatured sample was diluted to 20 .mu.M by adding 980 .mu.L
of pre-chilled HB1 solution to 20 .mu.L of sample. 5) The diluted
sample was further diluted to 7 .mu.M by adding 650 .mu.L of
pre-chilled HB1 solution to 350 .mu.L of 20 .mu.M sample solution.
6) The diluted sample was saved on ice until later use.
TABLE-US-00007 TABLE 7 0.1N NaOH solution Reagent 1 RXN 4 RXNS H2O
475 ul 1900 ul 2N NaOH 25 ul 100 ul Total 500 ul 2000 ul
A reagent plate was loaded with solutions in 2 mL Eppendorf tubes,
with reagent tubes matched to appropriate CentPD sippers, as
follows: Reagent 1: 950 ul HB1; Reagent 2: 950 ul APM; Reagent 3:
1300 ul AM1; Reagent 4: 1100 ul FM (Formamide 100%); Reagent 5:
1300 ul AM2; Reagent 6: 1100 ul W2; Reagent 7: 350 ul LM; Reagent
8: 400 ul NAOH (0.1 N NaOH); Reagent 9: 400 ul FP.
A prepared flowcell, such as described in previous examples, was
placed on a thermo-fluidic station and a clustering protocol was
initiated and run on a CentPD as described in Table 9:
TABLE-US-00008 TABLE 9 CentPD clustering protocol Heat Step Wait
Flow Step Temp Rate Time Time Volume Rate Time Flow Check Repeats
[.degree. C.] [.degree. C./s] [s] [s] Chem [.mu.L] [.mu.L/s] [s]
Initial Prime 25 60 HB1 60 4 15 W2 60 4 15 NAOH 60 4 15 APM 60 4 15
AM1 60 4 15 AM2 60 4 15 FM 60 1 60 HB1 120 1 120 TMP 90 Library 150
1 150 Introduction W2 20 1 20 TMP 40 0.05 1120 80 Rampdown TMP
Buffer W2 100 0.5 200 wash First W2 100 0.5 200 Extension AIR 3 0.5
6 AM1 100 1 100 AIR 3 0.5 6 W2 40 1 40 FE Wait 40 90 Amp- TempRamp
Template 25 NAOH 150 0.5 300 Strip 60 W2 150 1 150 Amplification 1
16X FM 28 8.5 8 APM 28 1 28 AM1 72 4 18 Amplification 2 16X FM 28
3.5 8 APM 28 1 28 AM2 72 4 18 Amplification 25 W2 120 2 60 wash HB1
95 4 23.75 Linearization 38 300 LM 150 1 150 Start Linearization 5X
300 LM 20 1 Cycle Linearization 25 W2 150 4 37.5 Finish Read 1 60
300 HB1 95 4 23.75 Preparation 40 NAOH 200 1 200 25 W2 200 1 200 FP
200 1 200 W2 150 1 150 HB1 150 1 150
1) All the reagents were primed (60 .mu.L, 4 .mu.L/s 25.degree.
C.), last of which were HB1 and W2 buffers (Illumina nomenclature).
2) 150 .mu.L of template was introduced at 90.degree. C. at a rate
of 1 .mu.L/s. The template was a PhiX DNA library (7 pM in HB1,
denatured, insert size 450 bp). 3) After incubating for 30 seconds,
the temperature was slowly reduced to 40.degree. C. over 18 minutes
at a rate of 0.05 deg/s. 4) The excess template was washed out with
200 .mu.L of W2 at 0.5 .mu.L/sec, also at 40.degree. C. 5) First
extension of the grafted primers was achieved by infusing 150 .mu.L
of amplification mix (AM1) at 1 .mu.L/s, book ended with 3 .mu.L
air bubbles in order to prevent mixing of reagents that may occur
in the line in transit to the flowcell. A 90 second incubation step
allows plentiful time for full template replication by Bst enzyme.
6) The flowcell is cooled to 25.degree. C., and the template is
stripped with 150 .mu.L of 0.1N NaOH pumped at rate of 0.5 .mu.L/s,
followed by 150 .mu.L of buffer (W2). 7) The flowcell is heated to
60.degree. C. in preparation for isothermal amplification. 8) 32
cycles of isothermal amplification are performed by repeating these
3 steps: (a) denaturation in 100% formamide (FM) 28 .mu.L at 3.5
.mu.L/s; (b) pre-amplification buffer without the enzyme (APM) to
remove formamide & allow for re-hybridization, 28 .mu.L at 1
.mu.L/s; and (c) extension of the primer with amplification mix
(AM), 72 .mu.L at 4 .mu.L/s. 9) The amplification reagents are
washed out with 120 .mu.L of W2 and 95 .mu.L of HB1). 10) 150 .mu.L
linearization reagent (LM) is introduced at 1 .mu.L/s, temp
25.degree. C. (to cut half of the amplified strands). 11) The
flowcell is heated to 38.degree. C., and incubated for 5 min (USER
treatment, cutting of dU via Uracil DNA Glycosylase). 12) Fresh 20
.mu.L of the LM solution is moved into the flowcell and incubating
for 5 min, repeated five times. 13) After linearization, the
temperature is reduced to 25.degree. C., and washed with 150 .mu.L
W2 and 95 .mu.L HB1. 14) The flowcell is denatured again with 200
.mu.L of 0.1N NaOH and washed with 200 .mu.L of W2. 15) Cy3 5'
labeled sequencing primer (FP) complimentary to the remaining
strand is introduced 200 .mu.L at 1 .mu.L/s. 16) The temperature is
raised to 60.degree. C. and the solution is incubated for 5 min to
allow for hybridization. 17) After reducing the temperature to
40.degree. C., excess primer is washed away with 150 .mu.L W2. 18)
After further reducing the temperature to 25.degree. C., the
flowcell is further washed with 150 .mu.L of W2.
Images of clustered colonies were taken on a custom
epi-fluorescence microscope with an Alta U-4000 CCD camera
(Apogee). Since the hybridized primers were labeled on the 5' end
with Cy3 fluorophore, we used Cy3-4040C filter cube (Semrock) and a
532 nm LED as the excitation light source. The images were
magnified 40.times. with an ELWD Nikon 0.6 NA objective, rendering
a field of view 375.times.375 um in size.
While preferred embodiments of the present invention have been
shown and described herein, it will be obvious to those skilled in
the art that such embodiments are provided by way of example only.
Numerous variations, changes, and substitutions will now occur to
those skilled in the art without departing from the invention. It
should be understood that various alternatives to the embodiments
of the invention described herein can be employed in practicing the
invention. It is intended that the following claims define the
scope of the invention and that methods and structures within the
scope of these claims and their equivalents be covered thereby.
* * * * *
References