U.S. patent application number 17/398315 was filed with the patent office on 2021-12-02 for multipart reagents having increased avidity for polymerase binding.
The applicant listed for this patent is Element Biosciences, Inc.. Invention is credited to Sinan ARSLAN, Molly HE, Matthew KELLINGER, Jake LEVIEUX, Tyler LOPEZ, Michael PREVITE, Su ZHANG, Junhua ZHAO.
Application Number | 20210373000 17/398315 |
Document ID | / |
Family ID | 1000005782740 |
Filed Date | 2021-12-02 |
United States Patent
Application |
20210373000 |
Kind Code |
A1 |
ARSLAN; Sinan ; et
al. |
December 2, 2021 |
MULTIPART REAGENTS HAVING INCREASED AVIDITY FOR POLYMERASE
BINDING
Abstract
Multivalent binding compositions including a particle-nucleotide
conjugate having a plurality of copies of a nucleotide attached to
the particle are described. The multivalent binding compositions
allow one to localize detectable signals to active regions of
biochemical interaction, e.g., sites of protein-protein
interaction, protein-nucleic acid interaction, nucleic acid
hybridization, or enzymatic reaction, and can be used to identify
sites of base incorporation in elongating nucleic acid chains
during polymerase reactions and to provide improved base
discrimination for sequencing and array based applications.
Inventors: |
ARSLAN; Sinan; (San Diego,
CA) ; HE; Molly; (San Diego, CA) ; KELLINGER;
Matthew; (San Diego, CA) ; LEVIEUX; Jake; (San
Diego, CA) ; PREVITE; Michael; (San Diego, CA)
; ZHAO; Junhua; (San Diego, CA) ; ZHANG; Su;
(San Diego, CA) ; LOPEZ; Tyler; (San Diego,
CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Element Biosciences, Inc. |
San Diego |
CA |
US |
|
|
Family ID: |
1000005782740 |
Appl. No.: |
17/398315 |
Filed: |
August 10, 2021 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
17063608 |
Oct 5, 2020 |
|
|
|
17398315 |
|
|
|
|
PCT/US2020/031161 |
May 1, 2020 |
|
|
|
17063608 |
|
|
|
|
16543351 |
Aug 16, 2019 |
|
|
|
PCT/US2020/031161 |
|
|
|
|
16936121 |
Jul 22, 2020 |
|
|
|
17063608 |
|
|
|
|
16579794 |
Sep 23, 2019 |
10768173 |
|
|
16936121 |
|
|
|
|
62841541 |
May 1, 2019 |
|
|
|
62897172 |
Sep 6, 2019 |
|
|
|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
G01N 33/5308 20130101;
C12Q 1/6874 20130101; G01N 33/582 20130101 |
International
Class: |
G01N 33/53 20060101
G01N033/53; G01N 33/58 20060101 G01N033/58; C12Q 1/6874 20060101
C12Q001/6874 |
Claims
1. A method of determining the identity of a nucleotide in a target
nucleic acid comprising: a. providing a composition comprising: i.
a target nucleic acid comprising two or more repeats of an
identical sequence; ii. two or more primer nucleic acids
complementary to one or more regions of said target nucleic acid;
and iii. two or more polymerase molecules; b. contacting said
composition with binding composition comprising a nucleotide moiety
under conditions sufficient to allow a binding complex to be formed
between said nucleotide moiety and the composition of step (a); and
c. detecting said binding complex, thereby establishing the
identity of said nucleotide in the target nucleic acid.
2. The method of claim 1, wherein the target nucleic acid is
DNA.
3. The method of claim 1 wherein the detection of the binding
complex is performed in the absence of unbound or solution-borne
nucleotide moieties.
4. The method of claim 1 wherein the target nucleic acid has been
replicated or amplified or has been produced by replication or
amplification.
5. The method of claim 1 wherein the detectable label is a
fluorescent label.
6. The method of claim 1 wherein detecting the complex comprises a
fluorescence measurement.
7. The method of claim 1 wherein the binding composition comprises
one type of nucleotide moieties.
8. The method of claim 1 wherein the binding composition comprises
two or more types of nucleotide moieties.
9. The method of claim 8, wherein each type of the two or more
types of nucleotide moieties comprises a different type of
nucleotide.
10. The method of claim 9 wherein the binding composition consists
of three types of nucleotide moieties and wherein each type of the
three types of nucleotide moiety comprises a different type of
nucleotide.
11. The method of claim 1 wherein the binding complex further
comprises a blocked nucleotide.
12. The method of claim 11 wherein the blocked nucleotide is a
3'-O-azidomethyl, 3'-O-methyl nucleotide, or 3'-O-alkyl
hydroxylamine.
13. The method of claim 1 wherein said contacting occurs in the
presence of an ion that stabilizes said binding complex, said
complex comprising a nucleotide moiety, two or more polymerase
molecules, and two or more binding sites within the target nucleic
acid.
14. The method of claim 1 wherein the contacting is done in the
presence of strontium, magnesium, calcium ions, or any combination
thereof.
15. The method of claim 1 wherein the polymerase molecule is
catalytically inactive.
16. The method of claim 1 wherein the binding complex has a
persistence time of greater than 2 seconds.
17. The method of claim 1, further comprising hybridizing the two
or more primer nucleic acids to the one or more regions of said
target nucleic acid by bringing the two or more primer nucleic
acids into contact with a hybridizing composition comprising said
target nucleic acid at a concentration of 1 nanomolar or less under
conditions sufficient for said target nucleic acid to hybridize to
the two or more primer nucleic acids in 30 minutes or less.
18. The method of claim 17, wherein the two or more primer nucleic
acids are coupled to a hydrophilic polymer surface having a water
contact angle of less than 45 degrees.
19. The method of claim 17, wherein the hybridization composition
further comprises: (a) at least one organic solvent having a
dielectric constant of no greater than about 115 as measured at 68
degrees Fahrenheit; and (b) a pH buffer.
20. The method of claim 17, wherein the hybridization composition
further comprises: (c) at least one organic solvent that is polar
and aprotic; and (d) a pH buffer.
Description
CROSS-REFERENCE
[0001] This application is a continuation of U.S. patent
application Ser. No. 17/063,608, filed Oct. 5, 2020, which is a
continuation-in-part of PCT/US2020/031161, filed May 1, 2020, which
is a continuation-in part of U.S. patent application Ser. No.
16/543,351, filed Aug. 16, 2019, which claims the benefit of U.S.
Provisional Application No. 62/841,541, filed May 1, 2019; and of
U.S. patent application Ser. No. 16/936,121, filed Jul. 22, 2020,
which is a continuation of U.S. patent application Ser. No.
16/579,794, filed on Sep. 23, 2019, now U.S. patent Ser. No.
10/768,173, which claims the benefit of U.S. Provisional
Application No. 62/897,172 filed on Sep. 6, 2019, each of which is
hereby incorporated by reference in its entirety.
SEQUENCE LISTING
[0002] The instant application contains a Sequence Listing which
has been submitted electronically in ASCII format and is hereby
incorporated by reference in its entirety. Said ASCII copy, created
on Aug. 6, 2021, is named 52933_733_301_SL.txt and is 4,476 bytes
in size.
BACKGROUND
[0003] This disclosure herein relates to the field of molecular
biology, such as compositions, methods, and systems for nucleic
acid hybridization and nucleic acid sequencing. In particular, it
relates to compositions and methods for nucleic acid hybridization
to nucleic acid molecules coupled to a surface and sequencing of
those nucleic acid molecules.
[0004] Nucleic acid hybridization protocols constitute an important
part of many different nucleic acid amplification and analysis
techniques. The limited specificity and reaction rates achieved
through the use of existing nucleic acid hybridization protocols
can have detrimental effects on the throughput and accuracy of
downstream nucleic acid analysis methods. Methods of stringency
control often involve conditions causing a significant decrease in
the number of hybridized complexes. Therefore, there is a need for
an improved method to achieve a high stringency of hybridization
during the sequencing analysis.
[0005] Nucleic acid sequencing can be used to obtain information in
a wide variety of biomedical contexts, including diagnostics,
prognostics, biotechnology, and forensic biology. Various
sequencing methods have been developed including Maxam-Gilbert
sequencing and chain-termination methods, or de novo sequencing
methods including shotgun sequencing and bridge PCR, or
next-generation methods including polony sequencing, 454
pyrosequencing, Illumina sequencing, SOLiD sequencing, Ion Torrent
semiconductor sequencing, HeliScope single molecule sequencing,
SMRT.RTM. sequencing, and others. Despite advances in DNA
sequencing, many challenges to cost effective, high throughput
sequencing remain unaddressed. The present disclosure provides
novel solutions and approaches to addressing many of the
shortcomings of existing technologies.
SUMMARY
[0006] Provided herein are methods of determining the identity of a
nucleotide in a target nucleic acid comprising: (a) providing a
composition comprising: (i) a target nucleic acid comprising two or
more repeats of an identical sequence; (ii) two or more primer
nucleic acids complementary to one or more regions of said target
nucleic acid; and (iii) two or more polymerase molecules; (b)
contacting said composition with a multivalent binding composition
comprising a polymer-nucleotide conjugate under conditions
sufficient to allow a binding complex to be formed between said
polymer-nucleotide conjugate and the composition of step (a),
wherein the polymer-nucleotide conjugate comprises two or more
copies of a nucleotide and optionally one or more detectable
labels; and (d) detecting said binding complex, thereby
establishing the identity of said nucleotide in the target nucleic
acid. In some embodiments, the target nucleic acid is DNA. In some
embodiments, the detection of the binding complex is performed in
the absence of unbound or solution-borne polymer nucleotide
conjugates. In some embodiments, the target nucleic acid has been
replicated or amplified or has been produced by replication or
amplification. In some embodiments, the detectable label is a
fluorescent label. In some embodiments, detecting the complex
comprises a fluorescence measurement. In some embodiments, the
multivalent binding composition comprises one type of
polymer-nucleotide conjugate. In some embodiments, the multivalent
binding composition comprises two or more types of
polymer-nucleotide conjugates. In some embodiments, each type of
the two or more types of polymer-nucleotide conjugates comprises a
different type of nucleotide. In some embodiments, the multivalent
binding composition consists of three types of polymer-nucleotide
conjugates and wherein each type of the three types of
polymer-nucleotide conjugates comprises a different type of
nucleotide. In some embodiments, the binding complex further
comprises a blocked nucleotide. In some embodiments, the blocked
nucleotide is a 3'-O-azidomethyl, 3'-O-methyl nucleotide, or
3'-O-alkyl hydroxylamine. In some embodiments, said contacting
occurs in the presence of an ion that stabilizes said binding
complex, said complex comprising a polymer nucleotide conjugate,
two or more polymerase molecules, and two or more binding sites
within the target nucleic acid. In some embodiments, the contacting
is done in the presence of strontium, magnesium, calcium ions, or
any combination thereof. In some embodiments, the polymerase
molecule is catalytically inactive. In some embodiments, the
binding complex has a persistence time of greater than 2 seconds.
In some embodiments, methods further comprise hybridizing the two
or more primer nucleic acids to the one or more regions of said
target nucleic acid by bringing the two or more primer nucleic
acids into contact with a hybridizing composition comprising said
target nucleic acid at a concentration of 1 nanomolar or less under
conditions sufficient for said target nucleic acid to hybridize to
the two or more primer nucleic acids in 30 minutes or less. In some
embodiments, the two or more primer nucleic acids are coupled to a
hydrophilic polymer surface having a water contact angle of less
than 45 degrees. In some embodiments, the hybridization composition
further comprises: (a) at least one organic solvent having a
dielectric constant of no greater than about 115 as measured at 68
degrees Fahrenheit; and (b) a pH buffer. In some embodiments, the
hybridization composition further comprises: (a) at least one
organic solvent that is polar and aprotic; and (b) a pH buffer.
INCORPORATION BY REFERENCE
[0007] All publications, patents, and patent applications mentioned
in this specification are herein incorporated by reference in their
entirety to the same extent as if each individual publication,
patent, or patent application was specifically and individually
indicated to be incorporated by reference in its entirety. In the
event of a conflict between a term herein and a term in an
incorporated reference, the term herein controls.
BRIEF DESCRIPTION OF THE DRAWINGS
[0008] The patent or application file contains at least one drawing
executed in color. Copies of this patent or patent application
publication with color drawing(s) will be provided by the Office
upon request and payment of the necessary fee.
[0009] Some novel features of the methods and compositions
disclosed herein are set forth in the present disclosure. A better
understanding of the features and advantages of the methods and
compositions disclosed herein will be obtained by reference to the
following detailed description that sets forth illustrative
embodiments, in which the principles of the disclosed compositions
and methods are utilized, and the accompanying drawings of
which:
[0010] FIGS. 1A-1B provide non-limiting examples of image data that
demonstrate the improvements in hybridization stringency, speed,
and efficacy that may be achieved through the reformulation of the
hybridization buffer used for solid-phase nucleic acid
amplification, as described herein. FIG. 1A provides examples of
image data for two different hybridization buffer formulations and
protocols. FIG. 1B provides an example of the corresponding image
data obtained using a standard hybridization buffer and
protocol.
[0011] FIG. 2 illustrates a workflow for nucleic acid sequencing
using the disclosed hybridization methods on low binding surfaces
and non-limiting examples of the processing times that may be
achieved.
[0012] FIG. 3 shows the surface template hybridization images (NASA
results at 100 pM) of the samples corresponding to the compositions
used for hybridization.
[0013] FIG. 4 shows a table with hybridization design of experiment
spot counts.
[0014] FIG. 5 shows the post nucleic acid surface amplification PCR
images of the samples.
[0015] FIG. 6 shows a work flow according to various embodiments
disclosed herein.
[0016] FIG. 7 shows a work flow for a sequence reaction according
to various embodiments described herein.
[0017] FIG. 8 shows a sample nucleic acid hybridization workflow
according to various embodiments described herein.
[0018] FIG. 9A-9B show how sample nucleic acids hybridized to the
nucleic acid molecules coupled to the low-non-specific binding
surface is visualized (FIG. 9A) or amplified (FIG. 9B) according to
various embodiments described herein.
[0019] FIG. 10 schematically depicts an example computer control
system.
[0020] FIG. 11 shows a workflow of purification and isolation of
sample nucleic acids from a biological sample, library preparation,
and hybridization according to various embodiments described
herein.
[0021] FIGS. 12A-12H illustrate the steps for sequencing a target
nucleic acid utilizing a non-limiting example of a multivalent
binding composition: FIG. 12A illustrates non-limiting example 8 of
attaching a target nucleic acid to a surface; FIG. 12B illustrates
a non-limiting example of clonally amplifying the target nucleic
acid to form clusters of amplified target nucleic acid molecules;
FIG. 12C illustrates a non-limiting example of priming the target
nucleic acid to produce a primed target nucleic acid; FIG. 12D
illustrates a non-limiting example of contacting the primed target
nucleic acid with the multivalent binding composition and
polymerase to form a binding complex; FIG. 12E illustrates a
non-limiting example of the images of the binding complex captured
on the surface; FIG. 12F illustrates a non-limiting example of
extending a primer strand by one nucleotide; FIG. 12G illustrates a
non-limiting example of another cycle of contacting the primed
target nucleic acid to the multivalent binding composition and
polymerase to form a binding complex; and FIG. 12H illustrates
non-limiting examples of images of a binding complex captured on
the surface in subsequent sequencing cycles.
[0022] FIG. 13 shows a flow chart outlining steps for sequencing a
target nucleic acid and extending a primer strand through a single
base addition according to various embodiments described
herein.
[0023] FIG. 14 shows a flow chart outlining steps for sequencing a
target nucleic acid and extending a primer strand through
incorporating the nucleotide on the particle-nucleotide conjugate
according to various embodiments described herein.
[0024] FIGS. 15A-15B illustrate a non-limiting example of detecting
a target nucleic acid using the polymer-nucleotide conjugates. FIG.
15A shows the step of contacting the polymerase and
polymer-nucleotide conjugates to some nucleic acid molecules; FIG.
15B shows the binding complex formed between the polymerase,
polymer-nucleotide conjugates, and the target nucleic acid
molecules.
[0025] FIGS. 16A-16C show schematic representations of non-limiting
examples of varying configurations of the polymer-nucleotide
conjugates: FIG. 16A shows polymer-nucleotide conjugates having
various multi-arm configurations; FIG. 16B shows a
polymer-nucleotide conjugate having the polymer branch radiating
from the center; and FIG. 16C shows polymer-nucleotide conjugates
having the binding moiety biotin.
[0026] FIG. 17 shows a generalized graphical depiction of the
increase in signal intensity that has been observed during binding,
persistence, and washing and removal of multivalent substrates.
[0027] FIGS. 18A-18J show fluorescence images of the steps in a
sequencing reaction using multivalent PEG-substrate compositions.
FIG. 18A. shows red and green fluorescent images post exposure of
DNA RCA templates (G and A first base) to 500 nM base labeled
nucleotides (A-Cy3 and G-Cy5) in exposure buffer containing 20 nM
Klenow polymerase and 2.5 mM Sr+2. Images were collected after
washing with imaging buffer with the same composition as the
exposure buffer but containing no nucleotides or polymerase.
Contrast was scaled to maximize visualization of the dimmest
signals, but no signals persisted following washing with imaging
buffer (FIG. 18A, inset). FIGS. 18B-18 show fluorescence images
showing multivalent PEG-nucleotide (base-labeled) ligands PB1 (FIG.
18B), PB2 (FIG. 18C), PB3 (FIG. 18D), and PB5 (FIG. 18E) having an
effective nucleotide concentration of 500 nM after mixing in the
exposure buffer and imaging in the imaging buffer as described
above. FIG. 18F shows fluorescence image showing multivalent
PEG-nucleotide (base-labeled) ligand PB5 at 2.5 .mu.M after mixing
in the exposure buffer and imaging in the imaging buffer as above.
FIGS. 18G-18I show fluorescence images showing further base
discrimination by exposure of the multivalent binding composition
to inactive mutants of klenow polymerase (FIG. 18G. D882H; FIG.
18H. D882E; FIG. 18I. D882A) vs. the wild type Klenow (control)
enzyme (FIG. 18J).
[0028] FIGS. 19A-19B show the efficacy of the multivalent reporter
compositions in determining the base sequence of a DNA sequence
over 5 sequencing cycles: FIG. 19A shows images and expected
sequences for templates taken after each sequencing cycle; and FIG.
19B shows aligned sequencing results utilizing the images taken in
FIG. 19A.
[0029] FIGS. 20A-20G show fluorescence images of multivalent
polyethylene glycol (PEG) polymer-nucleotide (base-labeled)
conjugates having an effective nucleotide concentration of 500 nM
and varying PEG branch length, after contacting to a support
surface comprising DNA templates (comprising G or A as the first
base; prepared using rolling circle amplification (RCA)) in an
exposure buffer comprising 20 nM Klenow polymerase and 2.5 mM Sr+2.
Images were acquired after washing with an imaging buffer having
the same composition as the exposure buffer but lacking nucleotides
and polymerase. Panels show images obtained using multivalent
PEG-nucleotide ligands with arm lengths as follows: FIG. 20A: 1K
PEG. FIG. 20B: 2K PEG. FIG. 20C: 3K PEG. FIG. 20D: 5K PEG. FIG.
20E: 10K PEG. FIG. 20F: 20K PEG. FIG. 20G shows images obtained
using 10K PEG and an inactive klenow polymerase comprising the
mutation D882H.
[0030] FIG. 21 shows a quantitative representation of the
fluorescence intensities in the images shown in FIGS. 20A-20F,
separated by color value, with orange trace corresponding to the
red label (Cy3 label; A bases) and blue trace corresponding to the
green label (Cy5 label; G bases).
[0031] FIG. 22 shows normalized fluorescence from multivalent
substrates bound to DNA clusters as described for FIGS. 18A-18J,
with the substrate complexes formed in the presence (condition B)
and absence (condition A) of Triton-X100 (0.016%).
[0032] FIGS. 23A-23B show plots of normalized fluorescence
intensity measured for multivalent polymer-nucleotide conjugates
and free nucleotides. FIG. 23A shows two replicates of a
multivalent polymer-nucleotide conjugate bound to DNA clusters
(Conditions A and B) vs. binding complexes formed using labeled
free nucleotides (Condition C) after 1 minute. FIG. 23B shows the
time course of fluorescence from multivalent substrate complexes
over the course of 60 min.
DETAILED DESCRIPTION
[0033] Disclosed herein are methods, compositions, systems, and
kits for nucleic acid hybridization to nucleic acid molecules
coupled to a surface. The methods, compositions, systems, and kits
described herein are particularly useful for nucleic acid
amplification, nucleic acid sequencing, or a combination thereof.
The methods, compositions, systems, and kits described herein
enable superior nucleic acid hybridization performance. For
example, nucleic acid hybridization according to the methods,
compositions, systems, and kits described herein can be performed
for a fraction of the cost and/or in a fraction of the time as
compared to nucleic acid hybridization methods requiring high
temperature (e.g., 90 degrees Celsius) incubations, long incubation
times (e.g., 1-2 hours), and large amounts of input nucleic acid
(e.g., 10 nanomolar). This is accomplished by utilizing optimized
hybridization compositions (e.g., buffers, organic solvents) in
combination with low non-specific binding surfaces that are
hydrophilic.
[0034] Nucleic acid hybridization methods requiring high
temperature incubations, long incubation times, and large amounts
of input nucleic acid are complex, time consuming, and lack the
specificity and efficiency needed for cost-effective high
throughput applications. At least one reason such nucleic acid
hybridization methods lack specificity and efficiency is that the
surfaces used are prone to non-specific binding of proteins or
nucleic acids, contributing to increased background signal.
[0035] The methods, compositions, systems, and kits described
herein provide superior hybridization specificity and efficiency of
target nucleic acid molecules to surface-bound nucleic acid
molecules, as compared to nucleic acid hybridization methods using
surfaces prone to non-specific binding reactions. Described herein,
are methods and systems utilizing a low non-specific binding
surface, thereby reducing background signal. The low non-specific
binding surfaces described herein are engineered so that proteins,
nucleic acids, and other biomolecules do not "stick" to the
substrate of the surface. The low non-specific binding surfaces
described herein are hydrophilic. In some instances, the low
non-specific binding surfaces have a water contact angle of less
than or equal to about 50 degrees.
[0036] The methods described herein comprise hybridizing a target
nucleic acid to a nucleic acid molecule coupled to a hydrophilic
surface (e.g., a low non-specific binding surface) in the presence
of the hybridization compositions described herein. The methods
described herein are useful for nucleic acid hybridization,
amplification, sequencing, or a combination thereof. In some
instances, the methods described herein achieve superior
hybridization performance on a low non-specific binding surface. In
addition, in some instances, the methods described herein achieve a
non-specific cyanine dye-3 (Cy3) dye absorption of less than about
0.25 molecules/.mu.m.sup.2.
[0037] Optimized hybridization compositions described herein, for
example, when used with low non-specific binding surfaces, enable
isothermal hybridization reactions to be performed at 60 degrees
Celsius for as few as 2 minutes, using as little as 50 picomolar
concentration of input nucleic acid. Methods described herein
provide (i) superior hybridization rates, (ii) superior
hybridization specificity, (iii) superior hybridization stringency,
(iv) superior hybridization efficiency (or yield), (v) reduced
requirements for the amount of starting material necessary, (vi)
lowered temperature requirements for isothermal or thermal ramping
amplification protocols, (vii) increased annealing rates, and
(viii) a yield having a low percentage of the total number target
nucleic acid molecules (or amplified clusters of target nucleic
acid molecules) being associated with the surface without
hybridizing to the surface bound nucleic acid, as compared to
hybridization reactions using non-specific binding surfaces. The
increased performance and reduced cost and time required to perform
a hybridization reaction make the methods, compositions, systems,
and kits described herein ideally suited for high throughput
nucleic acid hybridization, amplification, and sequencing
applications.
[0038] Hybridization formulations using, for example, saline sodium
citrate buffer achieve poor hybridization specificity or efficiency
when used with hybridization protocols using the non-specific
binding surfaces described herein. The hybridization reaction or
annealing interaction between target nucleic acid molecules in the
solution and nucleic acid molecules coupled to the low non-specific
binding surfaces can be impacted by several factors, including the
availability of hydrogen bonding partners in the solution and the
polarity of the solution. In general, nucleic acids preferentially
inhabit bulk solution where possible in order to take advantage of
the additional entropic stabilization presented by the ability to
access dynamic states in three, rather than two, dimensions such as
would be available on a solid surface. At equilibrium, in a system
comprising a nucleic acid, a solution, and a hydrophilic surface
(e.g., low non-specific binding surface), a nucleic acid molecule
will be preferentially stabilized in solution, rather than in a
surface-bound state when the solvent is aqueous.
[0039] Hybridization compositions and methods utilizing protic
solvents (e.g., saline sodium citrate buffer) are disadvantageous
for nucleic acid hybridization reactions with the low non-specific
binding surfaces described herein, because aprotic solvents provide
a favorable environment for the target nucleic acid molecules to
stay in solution, rather than binding to the low non-specific
binding surface. This is due to the ability of the protic solvent
to provide sufficient hydrogen bonding partners of sufficient size
and distribution such that hydrogen bonding interactions between
the exposed hydrogen bond donors and acceptors along the nucleic
acid backbone, or, any exposed sidechain moieties, occur.
[0040] By contrast, the hybridization compositions described herein
drive the target nucleic acid molecule to the low non-specific
binding surface while in solution, by utilizing an aprotic organic
solvent, such as, for example, formamide. The aprotic solvents
described herein reduce the proportion of solvent molecules capable
of satisfying the hydrogen bonding requirements of the nucleic acid
chain, and make it possible to create an entropic penalty in the
bulk solution, which will drive the system toward stabilization by
depositing the nucleic acid on the surface (e.g., the entropic
penalty caused by ordering the bulk solution to accommodate the
unbonded hydrogen bonding elements in the nucleic acid becomes
greater than the entropic penalty caused by loss of the third
dimension of dynamic freedom when the polymer is adsorbed to the
surface). Furthermore, introduction of an aprotic organic solvent
into the solution may help drive down the entropy and in turn
provides a more favorable environment for the nucleic acid to bind
to the hydrophilic surface. For example, addition of aprotic
solvent acetonitrile helps to drive the nucleic acid in the
solution towards a surface bound state.
[0041] The hybridization compositions described herein may further
comprise concentrations of protic and aprotic organic solvents, in
order to prevent precipitation of the target nucleic acid from
solution that can be caused by high concentrations of aprotic
solvent in the solution. In this manner, hybridization compositions
described herein may cause the nucleic acids to selectively
associate with hydrophilic surfaces (e.g., low non-specific binding
surfaces), while remaining substantially solvated.
[0042] The hybridization compositions described herein, may
comprise crowding agents, which are capable of modulating
interactions of nucleic acids with the bulk solution. In some
instances, the hybridization compositions comprise relaxing agents,
divalent cations, or intercalating agents, which are capable of
modulating the dynamics of the polymer itself and may also modulate
the interactions of nucleic acids with surfaces in the presence of
partially aprotic bulk solvents. Providing such agents in
combination with buffers containing some fraction of aprotic or
non-hydrogen-bonding components can, in some instances, provide
superior control over the interaction of nucleic acid molecules
with hydrophilic surfaces.
[0043] Various aspects of the disclosed nucleic acid hybridization
methods may be applied to solution-phase or solid-phase nucleic
acid hybridization, and also to any other type of nucleic acid
amplification, or, analysis applications (e.g., nucleic acid
sequencing), or any combination thereof. It shall be understood
that different aspects of the disclosed methods, devices, and
systems can be appreciated individually, collectively, or in
combination with each other.
[0044] The methods, compositions, systems, and kits described
herein are useful for a wide range of applications beyond those
involving nucleic acid-surface interactions, because the same
thermodynamic parameters optimized by the methods and compositions
described herein govern a number of interactions between polymers
and biomolecules, as well as polymer and surface interactions and
biomolecule and surface interactions. Thus, the methods
compositions, systems and kits described herein may be applied to
tune the polarity, or the hydrogen bonding potential, or a
combination thereof, of a solvent in other systems involving these
interactions.
[0045] Solution-based hybridization is the foundation for many
solution-based molecular biology and solution-phase DNA
manipulation applications, most notably the polymerase chain
reaction (PCR) (L. Garibyan and N. Avashia, J. Invest. Dermatol.,
2013, 133, e6; Z. Xiao, D. Shangguan, Z. Cao, X. Fang, and W. Tan,
2008, DNA guided drug delivery, Chemistry 14, 1769-75; and F. Wei,
C. Chen, L. Zhai, N. Zhang, and X. S. Zhao, 2005, DNA based
biosensors, J. Am. Chem. Soc., 127, 5306-5307; and S. Tyagi and F.
R. Kramer, Nat. Biotechnol., 1996, 14, 303-308). The diffusion
rates in many of these reactions are sufficient to drive efficient
hybridization and the formation of a functional double-stranded
form, which can be analyzed kinetically as a second order kinetic
reaction, whereby the forward reaction of duplex formation is
second order and the reverse reaction comprising the dissociation
of the duplex structure to form the two single stranded complements
(strands A and B) is first order (Han, C., Improvement of the Speed
and Sensitivity of DNA Hybridization Using Isotachophoresis,
Stanford Thesis. 2015). These reactions may be written as:
A + B .times. .revreaction. k off k on .times. AB ##EQU00001## d
.function. [ AB ] dt = k on .function. [ A ] .function. [ B ] - k
off .function. [ AB ] ##EQU00001.2##
[0046] Various approaches have been deployed to increase not only
the speed of the hybridization reaction but also the reaction
specificity in the wake of confounding DNA non-complementary
fragments. Such approaches include, but are not limited to, the
addition of MgCl.sub.2 and higher salt concentrations, and lower
temperatures to accelerate the reactions (H. Kuhn, V. V Demidov, J.
M. Coull, M. J. Fiandaca, B. D. Gildea, and M. D.
Frank-Kamenetskii, J. Am. Chem. Soc., 2002, 124, 1097-1103; N. A.
Straus and T. I. Bonner, Biochim. Biophys. Acta, Nucleic Acids
Protein Synth., 1972, 277, 87-95). The trade-off for accelerated
reaction rates is often reaction specificity (J. M. S. Bartlett and
D. Stirling, PCR protocols, Humana Press, 2003; W. Rychlik, W. J.
Spencer, and R. E. Rhoads, Nucleic Acids Res., 1990, 18).
Additional methods are sometimes employed that yield potential
improvements of reaction specificity through the use of volume
exclusion, or, molecular crowding techniques, or a combination
thereof that utilize inert polymers as hybridization buffer
additives (R. Wieder and J. G. Wetmur, Biopolymers, 1981, 20,
1537-1547, J. G. Wetmur, Biopolymers, 1975, 14, 2517-2524). In
addition, organic solvents have been employed as additives to
accelerate hybridization kinetics and maintain reaction specificity
(N. Dave and J. Liu, J. Phys. Chem. B, 2010, 114, 15694-15699).
[0047] While hybridization improvements in solution may be
translated to surface-based hybridization techniques, surface-based
hybridization needs have far ranging implications for many critical
bioassays, such as gene expression analysis (D. T. Ross, U. Scherf,
M. B. Eisen, C. M. Perou, C. Rees, P. Spellman, V. Iyer, S. S.
Jeffrey, M. Van de Rijn, M. Waltham, A. Pergamenschikov, J. C. Lee,
D. Lashkari, D. Shalon, T. G. Myers, J. N. Weinstein, D. Botstein,
and P. O. Brown, Nat. Genet., 2000, 24, 227-235; A. Adomas, G.
Heller, A. Olson, J. Osborne, M. Karlsson, J. Nahalkova, L. Van
Zyl, R. Sederoff, J. Stenlid, R. Finlay, and F. O. Asiegbu, Tree
Physiol., 2008, 28, 885-897; M. Schena, D. Shalon, R. W. Davis, and
P. O. Brown, Science, 1995, 270, 467-470), diagnosis of disease (J.
Marx, Science, 2000, 289, 1670-1672), genotyping and SNP detection
(J. G. Hacia, J. B. Fan, O. Ryder, L. Jin, K. Edgemon, G. Ghandour,
R. A. Mayer, B. Sun, L. Hsie, C. M. Robbins, L. C. Brody, D. Wang,
E. S. Lander, R. Lipshutz, S. P. Fodor, and F. S. Collins, Nat.
Genet., 1999, 22, 164-167), rapid nucleic acid based pathogen
screening, next generation sequencing (NGS) and a host of other
genomics based applications (M. J. Heller, Annu. Rev. Biomed. Eng.,
2002, 4, 129-53). The common necessity of all of these reactions is
high reaction specificity in a highly multiplexed solution of
target sequences that may range from thousands to billions of
different sequences, such that the targets are quickly tethered on
a solid surface for subsequent probing, or, amplification, or a
combination thereof to enable DNA (or other nucleic acid)
interrogation for applications such as sequencing or array-based
analysis. The efficiencies of surface-based hybridization reactions
were found to be much less than that of in solution reactions,
e.g., about an order of magnitude less efficient. A great deal of
work has been done in past attempts to create a hybridization
method for solid surface that provides high specificity and
accelerated hybridization reaction rates (D. Y. Zhang, S. X. Chen,
and P. Yin, Nat. Chem., 2012, 4, 208-14).
[0048] Disclosed herein are innovative combinations of approaches
gleaned from studies of surface- and solution-based hybridization
as outlined above, as well as from other fields of study that
include DNA hydration and quadruplex studies, which lead to
substantial improvements in hybridization kinetics and specificity.
The disclosed hybridization compositions provide for highly
specific (e.g., >2 orders of magnitude improvement over
traditional approaches) and accelerated hybridization (e.g.,
>1-2 orders of magnitude improvement over traditional
approaches) when used with low non-specific binding surfaces for
applications such as next generation sequencing (NGS) and other
bioassays that require highly specific nucleic acid hybridization
in a multiplexed pool comprised of a large number of target
sequences.
Hybridization Methods
[0049] Provided herein are methods for nucleic acid hybridization
between a sample nucleic acid molecule and a capture nucleic acid
molecule. Referring to FIG. 11, the sample nucleic acid molecule is
isolated and purified from a biological sample obtained from a
subject 1110. A library of isolated and purified sample nucleic
acid molecules is prepared 1111. The library of sample nucleic acid
molecules is hybridized to nucleic acid molecules coupled to a low
non-specific binding surface described herein in the presence of a
hybridization composition 1112.
[0050] Biological Sample. The biological samples disclosed herein
may comprise nucleic acid molecules, amino acids, polypeptides,
proteins, carbohydrates, fats, or viruses. In an example, a
biological sample is a nucleic acid sample including one or more
nucleic acid molecules. Exemplary biological samples may include
polynucleotides, nucleic acids, oligonucleotides, cell-free nucleic
acid (e.g., cell-free DNA (cfDNA)), circulating cell-free nucleic
acid, circulating tumor nucleic acid (e.g., circulating tumor DNA
(ctDNA)), circulating tumor cell (CTC) nucleic acids, nucleic acid
fragments, nucleotides, DNA, RNA, peptide polynucleotides,
complementary DNA (cDNA), double stranded DNA (dsDNA), single
stranded DNA (ssDNA), plasmid DNA, cosmid DNA, chromosomal DNA,
genomic DNA (gDNA), viral DNA, bacterial DNA, mtDNA (mitochondrial
DNA), ribosomal RNA, cell-free DNA, cell free fetal DNA (cffDNA),
mRNA, rRNA, tRNA, nRNA, siRNA, snRNA, snoRNA, scaRNA, microRNA,
dsRNA, viral RNA, and the like.
[0051] Any substance that comprises nucleic acid may be the source
of the biological sample. The substance may be a fluid, e.g., a
biological fluid. A fluidic substance may include, but is not
limited to, blood, cord blood, saliva, urine, sweat, serum, semen,
vaginal fluid, gastric and digestive fluid, spinal fluid, placental
fluid, cavity fluid, ocular fluid, serum, breast milk, lymphatic
fluid, or combinations thereof. The substance may be solid, for
example, a biological tissue. The substance may comprise normal
healthy tissues, diseased tissues, or a mix of healthy and diseased
tissues.
[0052] Biological samples described herein are obtained from
various subjects. A subject may be a living subject or a dead
subject. Examples of subjects may include, but are not limited to,
humans, mammals, non-human mammals, rodents, amphibians, reptiles,
canines, felines, bovines, equines, goats, ovines, hens, avines,
mice, rabbits, insects, slugs, microbes, bacteria, parasites, or
fish. The subject, in some cases, is a patient who is having,
suspected of having, or at a risk of developing a disease or
disorder. In some instances, the subject may be a pregnant woman.
In some instances, the subject may be a normal healthy pregnant
woman. In some instances, the subject may be a pregnant woman who
is at a risking of carrying a baby with certain birth defect.
[0053] A sample may be obtained from a subject by various
approaches. For example, a sample may be obtained from a subject
through accessing the circulatory system (e.g., intravenously or
intra-arterially via a syringe or other apparatus), collecting a
secreted biological sample (e.g., saliva, sputum urine, feces),
surgically (e.g., biopsy) acquiring a biological sample (e.g.,
intra-operative samples, post-surgical samples), swabbing (e.g.,
buccal swab, oropharyngeal swab), or pipetting.
[0054] Biological Sample Processing. The biological sample
described herein, in some instances, is processed. Processing
comprises filtering a sample, binding a component of the sample
that contains an analyte, binding the analyte, stabilizing the
analyte, purifying the analyte, or a combination thereof.
Non-limiting examples of sample components are cells, viral
particles, bacterial particles, exosomes, and nucleosomes. In some
instances, blood plasma or serum is isolated from a whole blood
sample. In some instances, the whole blood is obtained from venous
blood or capillary blood of a subject described herein.
[0055] Library Preparation of Sample Nucleic Acids. The sample
nucleic acids described herein, in some cases, are converted to a
library by labeling the sample nucleic acids with a label, barcode
or tag. The library of sample nucleic acids are amplified in some
instances, for example, by isothermal amplification. Non-limiting
examples of amplification methods include, but are not limited to,
loop mediated isothermal amplification (LAMP), nucleic acid
sequence based amplification (NASBA), strand displacement
amplification (SDA), multiple displacement amplification (MDA),
rolling circle amplification (RCA), ligase chain reaction (LCR),
helicase dependent amplification (HDA), nicking enzyme
amplification reaction (NEAR), recombinase polymerase amplification
(RPA), and ramification amplification method (RAM).
[0056] In some instances, isothermal amplification is used. In some
instances, amplification is isothermal with the exception of an
initial heating step before isothermal amplification begins. A
number of isothermal amplification methods, each having different
considerations and providing different advantages, are known in the
art and have been discussed in the literature, e.g., by Zanoli and
Spoto, 2013, "Isothermal Amplification Methods for the Detection of
Nucleic Acids in Microfluidic Devices," Biosensors 3: 18-43, and
Fakruddin, et al., 2013, "Alternative Methods of Polymerase Chain
Reaction (PCR)," Journal of Pharmacy and Bioallied Sciences 5(4):
245-252, each incorporated herein by reference in its entirety.
[0057] In some instances, the amplification method is Rolling
Circle Amplification (RCA). RCA is an isothermal nucleic acid
amplification method which allows amplification of the probe DNA
sequences by more than 10.sup.9-fold at a single temperature,
typically about 30.degree. C. Numerous rounds of isothermal
enzymatic synthesis are carried out by O29 DNA polymerase, which
extends a circle-hybridized primer by continuously progressing
around the circular DNA probe. In some instances, the amplification
reaction is carried out using RCA, at about 28.degree. C. to about
32.degree. C. Suitable methods of RCA are described in U.S. Pat.
No. 6,558,928.
[0058] In some instances, amplifying comprises targeted
amplification. In some instances, amplifying a nucleic acid
comprises contacting a nucleic acid with at least one primer having
a sequence corresponding to a target chromosome sequence.
Amplification may be multiplexed, involving contacting the nucleic
acid with multiple sets of primers, wherein each of a first pair in
a first set and each of a pair in a second set are all
different.
[0059] Hybridization. Methods described herein comprise bringing a
sample nucleic acid molecule into contact with a capture nucleic
acid molecule that is optionally coupled to a low non-specific
binding surface in the presence of a hybridization composition
described herein. In some cases, the capture nucleic acid molecule
is coupled to the low non-specific binding surface and
hybridization occurs on the surface. In some cases, the capture
nucleic acid molecules are not coupled to the low non-specific
binding surface and hybridization occurs in solution. Methods
provided herein further comprising hybridizing the sample nucleic
acid molecule with the capture nucleic acid molecule.
[0060] Methods described herein comprise hybridizing at least a
portion of the sample nucleic acid molecule comprising a nucleic
acid sequence that is sufficiently complementary to a portion of
the capture nucleic acid molecule. The portion of the capture
nucleic acid molecule and the sample nucleic acid molecule can be
at least or equal to about 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, or 50 nucleotides. The portion of the capture nucleic acid
molecule and the sample nucleic acid molecule can be between 4 and
50, 5 and 49, 6 and 48, 7 and 47, 8 and 46, 9 and 45, 10 and 44, 11
and 43, 12 and 42, 13 and 41, 14 and 40, 15 and 39, 16 and 38, 17
and 37, 18 and 36, 19 and 35, 20 and 34, 21 and 33, 22 and 32, 23
and 31, 24 and 30, 25 and 29, 26 and 28 nucleotides. The portion of
the capture nucleic acid molecule and the sample nucleic acid
molecule can be between 8 and 20 nucleotides. In some instances, at
least 90% of the nucleic acids in the portion of the sample nucleic
acid molecule and the portion of the capture nucleic acid molecule
hybridize completely. In some instances, at least 95% of the
nucleic acids in the portion of the sample nucleic acid molecule
and the portion of the capture nucleic acid molecule hybridize
completely. In some instances, between 95-100% of the nucleic acids
in the portion of the sample nucleic acid molecule and the portion
of the capture nucleic acid molecule hybridize completely.
[0061] A non-limiting example provided in FIG. 8 shows one or more
sample nucleic acid molecules 801 to be circularized 802 using
ligation (e.g., splint ligation) 802, and introduced to one or more
nucleic acid molecules 808 coupled to a hydrophilic substrate 807
of a low non-specific binding surface 806 in the presence of a
hybridization composition 805. In this example, the
low-non-specific binding surface is submerged in the hybridization
composition. In another example, the one or more sample nucleic
acid molecules is introduced to the hybridization composition
before introduction to the one or more nucleic acid molecules 808
coupled to the hydrophilic substrate 807 of the low non-specific
binding surface 806. Hybridization occurs between the sample
nucleic acid molecule and the surface-coupled nucleic acid molecule
809.
[0062] Sample Nucleic Acids. The one or more sample nucleic acid
molecules described herein is derived from a biological sample
described herein. The sample nucleic acid molecules may be a
deoxyribonucleic acid (DNA) molecule or a ribonucleic acid (RNA)
molecule. In some instances, the DNA is selected from cell-free DNA
(cfDNA)), circulating cell-free nucleic acid, circulating tumor
nucleic acid (e.g., circulating tumor DNA (ctDNA)), circulating
tumor cell (CTC) nucleic acids, nucleic acid fragments,
nucleotides, DNA, complementary DNA (cDNA), double stranded DNA
(dsDNA), single stranded DNA (ssDNA), plasmid DNA, cosmid DNA,
chromosomal DNA, genomic DNA (gDNA), viral DNA, bacterial DNA, and
mtDNA (mitochondrial DNA). In some instances, the RNA is selected
from ribosomal RNA, cell-free DNA, cell free fetal DNA (cffDNA),
mRNA, rRNA, tRNA, nRNA, siRNA, snRNA, snoRNA, scaRNA, microRNA,
dsRNA, viral RNA, and the like.
[0063] Coupling the Capture Nucleic Acids to the Surface. The
nucleic acid molecules coupled to the surface (e.g., capture
molecules) may be coupled to the surface by a number of suitable
options. In some instances, the nucleic acid molecules are coupled
to the surface through covalent bonds. In some instances, the
nucleic acid molecules are coupled to the surface through
noncovalent bonds. In some instances, the nucleic acid molecules
are attached to the surface through a bio-interaction. Non-limiting
examples of bio-interaction surface chemistry include
biotin-streptavidin interactions (or variations thereof),
polyhistidine (his) tag--Ni/NTA conjugation chemistries, methoxy
ether conjugation chemistries, carboxylate conjugation chemistries,
amine conjugation chemistries, NHS esters, maleimides, thiol,
epoxy, azide, hydrazide, alkyne, isocyanate, and silane.
Compositions
[0064] Provided herein are hybridization compositions. The
hybridization compositions of the present disclosure comprise at
least one organic solvent, which in some cases is polar and aprotic
(e.g., having a dielectric constant of less than or equal to about
115 as measured at 68 degrees F.). The hybridization compositions
comprise a pH buffer. Optionally, the hybridization compositions
comprise one or more molecular crowding/volume exclusion agents,
one or more additives that impact DNA melting temperatures, one or
more additives that impact DNA hydration, or any combination
thereof. The hybridization compositions described herein can be
used with the low non-specific binding surfaces described herein,
such as, for example, silicon dioxide coated with low binding
polymers (e.g., polyethylene glycol (PEG)), for genotyping or
sequencing related technologies. Genotyping and sequencing may be
achieved using any or a combination of the following hybridization
composition components.
[0065] Organic Solvent: An organic solvent is a solvent or solvent
system comprising carbon-based or carbon-containing substance
capable of dissolving or dispersing other substances. An organic
solvent may be miscible or immiscible with water.
[0066] Polar Solvent: A polar solvent, as included in the
hybridization composition described herein, is a solvent or solvent
system comprising one or more molecules characterized by the
presence of a permanent dipole moment, e.g., a molecule having a
spatially unequal distribution of charge density. A polar solvent
may be characterized by a dielectric constant of 20, 25, 30, 35,
40, 45, 50, 55, 60 or higher, or by a value or a range of values
incorporating any of the aforementioned values. For example, a
polar solvent may have a dielectric constant of higher than 100,
higher than 110, higher than 111, or higher than 115. In some
cases, the dielectric constant is measured at a temperature of
greater than or equal to about 20, 25, 30, 35, 40, 45, 50, 55, 60,
65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450,
or 500 degrees Fahrenheit (F). In some cases, the dielectric
constant is measured at a temperature of less than or equal to
about -20, -25, -30, -35, -40, -45, -50, -55, -60, -65, -70, -75,
-80, -85, -90, -95, -100, -150, -200, -250, -300, -350, -400, -450,
or -459 degrees F. In some cases, the dielectric constant is
measured at a temperature of at 68 degrees F. In some cases, the
dielectric constant is measured at a temperature of at 20 degrees
F.
[0067] A polar solvent as described herein may comprise a polar
aprotic solvent. A polar aprotic solvent as described herein may
further contain no ionizable hydrogen in the molecule. In addition,
polar solvents or polar aprotic solvents may be preferably
substituted in the context of the presently disclosed compositions
with a strong polarizing functional groups such as nitrile,
carbonyl, thiol, lactone, sulfone, sulfite, and carbonate groups so
that the underlying solvent molecules have a dipole moment. Polar
solvents and polar aprotic solvents can be present in both
aliphatic and aromatic or cyclic form. In some embodiments, the
polar solvent is acetonitrile.
[0068] The organic solvent described herein can have a dielectric
constant that is the same as or close to acetonitrile. The
dielectric constant of the organic solvent can be in the range of
about 20-60, about 25-55, about 25-50, about 25-45, about 25-40,
about 30-50, about 30-45, or about 30-40. The dielectric constant
of the organic solvent can be greater than or equal to about 20,
25, 30, 35, or 40. The dielectric constant of the organic solvent
can be lower than 30, 40, 45, 50, 55, or 60. The dielectric
constant of the organic solvent can be about 35, 36, 37, 38, or
39.
[0069] Dielectric constant may be measured using a test capacitor.
Representative polar aprotic solvents having a dielectric constant
between 30 and 120 may be used. Such solvents may particularly
include, but are not limited to, acetonitrile, diethylene glycol,
N,N-dimethylacetamide, dimethyl formamide, dimethyl sulfoxide,
ethylene glycol, formamide, hexamethylphosphoramide, glycerin,
methanol, N-methyl-2-pyrrolidinone, nitrobenzene, or
nitromethane.
[0070] The organic solvent described herein can have a polarity
index that is the same as or close to acetonitrile. The polarity
index of the organic solvent can be in the range of about 2-9, 2-8,
2-7, 2-6, 3-9, 3-8, 3-7, 3-6, 4-9, 4-8, 4-7, or 4-6. The polarity
index of the organic solvent can be greater than, or equal to,
about 2, 3, 4, 4.5, 5, 5.5, or 6. The polarity index of the organic
solvent can be lower than about 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8,
8.5, 9, or 10. The polarity index of the organic solvent can be
about 5.5, 5.6, 5.7, or 5.8.
[0071] The Snyder Polarity Index may be calculated according to the
methods disclosed in Snyder, L. R., Journal of Chromatography A,
92(2):223-30 (1974), which is incorporated by reference herein in
its entirety. Representative polar aprotic solvents having a Snyder
polarity index between 6.2 and 7.3 may be used. Such solvents may
particularly include, but are not limited to, acetonitrile,
dimethyl acetamide, dimethyl formamide, N-methyl pyrrolidone,
N,N-dimethyl sulfoxide, methanol, or formamide.
[0072] Relative polarity may be determined according to the methods
given in Reichardt, C., Solvents and Solvent Effects in Organic
Chemistry, 3rd ed., 2003, which is incorporated herein by reference
in its entirety, and especially with respect to its disclosure of
polarities and methods of determining or assessing the same for
solvents and solvent molecules. Polar aprotic solvents having a
relative polarity between 0.44 and 0.82 may be used. Such solvents
may particularly include, but are not limited to,
dimethylsulfoxide, acetonitrile, 3-pentanol, 2-pentanol, 2-butanol,
Cyclohexanol, 1-octanol, 2-propanol, 1-heptanol, i-butanol,
1-hexanol, 1-pentanol, acetyl acetone, ethyl acetoacetate,
1-butanol, benzyl alcohol, 1-propanol, 2-aminoethanol, Ethanol,
diethylene glycol, methanol, ethylene glycol, glycerin, or
formamide.
[0073] The Solvent Polarity (ET(30)) may be calculated according to
the methods disclosed in Reichardt, C., Molecular Interactions,
Volume 3, Ratajczak, H. and Orville, W. J., Eds (1982), which is
incorporated by reference herein in its entirety.
[0074] Some examples of organic solvents include, but are not
limited to, acetonitrile, dimethylformamide (DMF),
dimethylsulfoxide (DMSO), acetanilide, N-acetyl pyrrolidone,
4-amino pyridine, benzamide, benzimidazole, 1,2,3-benzotriazole,
butadienedioxide, 2,3-butylene carbonate, .gamma.-butyrolactone,
caprolactone (epsilon), chloro maleic anhydride,
2-chlorocyclohexanone, chloroethylene carbonate,
chloronitromethane, citraconic anhydride, crotonlactone,
5-cyano-2-thiouracil, cyclopropylnitrile, dimethyl sulfate,
dimethyl sulfone, 1,3-dimethyl-5-tetrazole, 1,5-dimethyl tetrazole,
1,2-dinitrobenzene, 2,4-dinitrotoluene, dipheynyl sulfone,
1,2-dinitrobenzene, 2,4-dinitrotoluene, dipheynyl sulfone,
epsilon-caprolactam, ethanesulfonylchloride, ethyl ethyl
phosphinate, N-ethyl tetrazole, ethylene carbonate, ethylene
trithiocarbonate, ethylene glycol sulfate, ethylene glycol sulfite,
furfural, 2-furonitrile, 2-imidazole, isatin, isoxazole,
malononitrile, 4-methoxy benzonitrile, 1-methoxy-2-nitrobenzene,
methyl alpha bromo tetronate, 1-methyl imidazole, N-methyl
imidazole, 3-methyl isoxazole, N-methyl morpholine-N-oxide, methyl
phenyl sulfone, N-methyl pyrrolidinone, methyl sulfolane,
methyl-4-toluenesulfonate, 3-nitroaniline, nitrobenzimidazole,
2-nitrofuran, 1-nitroso-2-pyrolidinone, 2-nitrothiophene,
2-oxazolidinone, 9,10-phenanthrenequinone, N-phenyl sydnone,
phthalic anhydride, picolinonitrile (2-cyanopyridine), 1,3-propane
sultone, .beta.-propiolactone, propylene carbonate,
4H-pyran-4-thione, 4H-pyran-4-one (.gamma.-pyrone), pyridazine,
2-pyrrolidone, saccharin, succinonitrile, sulfanilamide, sulfolane,
2,2,6,6-tetrachlorocyclohexanone, tetrahydrothiapyran oxide,
tetramethylene sulfone (sulfolane), thiazole, 2-thiouracil,
3,3,3-trichloro propene, 1,1,2-trichloro propene, 1,2,3-trichloro
propene, trimethylene sulfide-dioxide, or trimethylene sulfite.
[0075] Polar aprotic solvents having a solvent polarity between 44
and 60 may be used. Such solvents may particularly include, but are
not limited to, dimethyl sulfoxide, 2-methoxycarbonylphenol,
triethyl phosphite, 3-pentanol, acetonitrile, nitromethane,
cyclohexanol, 2-pentanol, 4-methyl-1,3, dioxolan-2-one, propylene
carbonate, acrylonitrile, 1-phenylethanol, 1-dodecanol, 2-butanol,
2-methylcyclohexanol, 2,6,dimethylphenol, 2,6-xylenol, 1-decanol,
cyclopentanol, dimethyl sulfone, 1-octanoldiethylene glycol mono
n-butyl ether, butyl digol, 1-heptanol, 3-phenyl-1-propanol,
1,3-dioxolane-2-one, ethylene carbonate, 1-hexanol,
4-chlorobutyronitrile, 5-methyl-2-isopropylphenol, thymol,
3,5,5-trimethyl-1-hexanol, 3-methyl-1-butanol, isoamyl alcohol,
2-methyl-1-propanol, isobutyl alcohol, 2-(tert-butyl)phenol,
1-pentanol, 2-phenylethanol, 2-methylpentane-2,4-diol, dipropylene
glycol, 2-isopropylphenol, 2-n-butoxyethanol, ethylene glycol
mono-n-butyl ether, 1-butanol, 2-hydroxymethyl-tetrahydrofuran,
tetrahydrofurfuryl alcohol, 2-hydroxymethylfuran, furfuryl alcohol,
1-propanol, 2,4-dimethylphenol, 2,4-xylenol, benzyl alcohol,
2-ethoxyphenol, 2-ethoxyethanol, 1,5-pentanediol,
1-bromo-2-propanol, 2-methyl-5-isopropylphenol, carvacrol,
2-aminoethanol, ethanol, n-methyl acetamide, 3-chloropropionitrile,
2-propen-1-ol, allyl alcohol, 2-methoxy ethanol, 2-methylphenol,
o-cresol, 1,3-butanediol, 2-propyn-1-ol, propargyl alcohol,
3-methylphenol, m-cresol, triethylene glycol, diethylene glycol,
n-methylformamide, 1,2-propanediol, 1,3-propanediol,
2-chlorophenol, methanol, 1,2-ethanediol, glycol, formamide,
2,2,2-trichloroethanol, 1,2,3-propanetriol, glycerol,
2,2,3,3-tetrafluoro-1-propanol, 2,2,2-trifluoroethanol,
4-n-butylphenol, 4-methylphenol, or p-cresol.
[0076] Polar aprotic solvents having a dielectric constant in the
range of about 30-115 may be used. Such solvents may particularly
include, but are not limited to, dimethyl sulfoxide,
2-methoxycarbonylphenol, triethyl phosphite, 3-pentanol,
acetonitrile, nitromethane, cyclohexanol, 2-pentanol, 4-methyl-1,3,
dioxolan-2-one, propylene carbonate, acrylonitrile,
1-phenylethanol, 1-dodecanol, 2-butanol, 2-methylcyclohexanol,
2,6,dimethylphenol, 2,6-xylenol, 1-decanol, cyclopentanol, dimethyl
sulfone, 1-octanoldiethylene glycol mono n-butyl ether, butyl
digol, 1-heptanol, 3-phenyl-1-propanol, 1,3-dioxolane-2-one,
ethylene carbonate, 1-hexanol, 4-chlorobutyronitrile,
5-methyl-2-isopropylphenol, thymol, 3,5,5-trimethyl-1-hexanol,
3-methyl-1-butanol, isoamyl alcohol, 2-methyl-1-propanol, isobutyl
alcohol, 2-(tert-butyl)phenol, 1-pentanol, 2-phenylethanol,
2-methylpentane-2,4-diol, dipropylene glycol, 2-isopropylphenol,
2-n-butoxyethanol, ethylene glycol mono-n-butyl ether, 1-butanol,
2-hydroxymethyl-tetrahydrofuran, tetrahydrofurfuryl alcohol,
2-hydroxymethylfuran, furfuryl alcohol, 1-propanol,
2,4-dimethylphenol, 2,4-xylenol, benzyl alcohol, 2-ethoxyphenol,
2-ethoxyethanol, 1,5-pentanediol, 1-bromo-2-propanol,
2-methyl-5-isopropylphenol, carvacrol, 2-aminoethanol, ethanol,
n-methylacetamide, 3-chloropropionitrile, 2-propen-1-ol, allyl
alcohol, 2-methoxyethanol, 2-methylphenol, o-cresol,
1,3-butanediol, 2-propyn-1-ol, propargyl alcohol, 3-methylphenol,
m-cresol, triethylene glycol, diethylene glycol, n-methylformamide,
1,2-propanediol, 1,3-propanediol, 2-chlorophenol, methanol,
1,2-ethanediol, glycol, formamide, 2,2,2-trichloroethanol,
1,2,3-propanetriol, glycerol, 2,2,3,3-tetrafluoro-1-propanol,
2,2,2-trifluoroethanol, 4-n-butylphenol, 4-methylphenol, or
p-cresol.
[0077] Organic solvent addition: In some instances, the disclosed
hybridization buffer formulations include the addition of an
organic solvent. Examples of suitable solvents include, but are not
limited to, acetonitrile, ethanol, DMF, and methanol, or any
combination thereof at varying percentages (for example >5%). In
some instances, the percentage of organic solvent (by volume)
included in the hybridization buffer may range from about 1% to
about 20%. In some instances, the percentage by volume of organic
solvent may be at least 1%, at least 2%, at least 3%, at least 4%,
at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at
least 10%, at least 15%, or at least 20%. In some instances, the
percentage by volume of organic solvent may be at most 20%, at most
15%, at most 10%, at most 9%, at most 8%, at most 7%, at most 6%,
at most 5%, at most 4%, at most 3%, at most 2%, or at most 1%. Any
of the lower and upper values described in this paragraph may be
combined to form a range included within the present disclosure,
for example, the percentage by volume of organic solvent may range
from about 4% to about 15%. The percentage by volume of organic
solvent may have any value within this range, e.g., about 7.5%.
[0078] When the organic solvent comprises a polar aprotic solvent,
the amount of the polar aprotic solvent is present in an amount
effective to denature a double stranded nucleic acid. In some
instances, the amount of the polar aprotic solvent is greater than,
or equal to, about 10% by volume based on the total volume of the
formulation. In some instances, the amount of the polar aprotic
solvent is about, or more than about, 5%, 10%, 15%, 20%, 25%, 30%,
35%, 40%, 50%, 60%, 70%, 80%, 90%, or higher, by volume based on
the total volume of the formulation. In some instances, the amount
of the polar aprotic solvent is lower than about 15%, 20%, 25%,
30%, 35%, 40%, 50%, 60%, 70%, 80%, 90%, or higher, by volume based
on the total volume of the formulation. In some embodiments, the
amount of the polar aprotic solvent is in the range of about 10% to
90% by volume based on the total volume of the formulation. In some
instances, the amount of the polar aprotic solvent is in the range
of about 25% to 75% by volume based on the total volume of the
formulation. In some instances, the amount of the polar aprotic
solvent is in the range of about 10% to 95%, 10% to 85%, 20% to
90%, 20% to 80%, 20% to 75%, or 30% to 60% by volume based on the
total volume of the formulation. In some instances, the polar
aprotic solvent is formamide.
[0079] When the organic solvent comprises a polar aprotic solvent,
the amount of the aprotic solvent is present in an amount effective
to denature a double stranded nucleic acid. In some instances, the
amount of the aprotic solvent is greater than, or equal to, about
10% by volume based on the total volume of the formulation. In some
instances, the amount of the aprotic solvent is about, or more than
about, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 60%, 70%, 80%,
90%, or higher, by volume based on the total volume of the
formulation. In some instances, the amount of the aprotic solvent
is lower than about 15%, 20%, 25%, 30%, 35%, 40%, 50%, 60%, 70%,
80%, 90%, or higher, by volume based on the total volume of the
formulation. In some instances, the amount of the aprotic solvent
is in the range of about 10% to 90% by volume based on the total
volume of the formulation. In some instances, the amount of the
aprotic solvent is in the range of about 25% to 75% by volume based
on the total volume of the formulation. In some instances, the
amount of the aprotic solvent is in the range of about 10% to 95%,
10% to 85%, 20% to 90%, 20% to 80%, 20% to 75%, or 30% to 60% by
volume based on the total volume of the formulation.
[0080] Addition of molecular crowding/volume exclusion agents: The
composition described herein can include one or more crowding
agents that enhances molecular crowding. In some instances, the
crowding agent is selected from the group consisting of
polyethylene glycol (PEG), dextran, hydroxypropyl methyl cellulose
(HPMC), hydroxyethyl methyl cellulose (HEMC), hydroxybutyl methyl
cellulose, hydroxypropyl cellulose, methylcellulose, and hydroxyl
methyl cellulose, and combinations thereof. An example crowding
agent may comprise one or more of polyethylene glycol (PEG),
dextran, proteins, for example, ovalbumin or hemoglobin, or
Ficoll.
[0081] A suitable amount of a crowding agent in the composition
allows for, enhances, or facilitates molecular crowding. In some
instances, the amount of the crowding agent is about, or more than
about, 1%, 2%, 3%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 60%,
or higher, by volume based on the total volume of the formulation.
In some instances, the amount of the molecular crowding agent is
greater than or equal to about 5% by volume based on the total
volume of the formulation. In some instances, the amount of the
crowding agent is lower than about 3%, 5%, 10%, 12.5%, 15%, 20%,
25%, 30%, 35%, 40%, 50%, 60%, 70%, 80%, 90%, or higher, by volume
based on the total volume of the formulation. In some instances,
the amount of the molecular crowding agent is less than or equal to
about 30% by volume based on the total volume of the formulation.
In some instances, the amount of the organic solvent is in the
range of about 25% to 75% by volume based on the total volume of
the formulation. In some instances, the amount of the organic
solvent is in the range of about 1% to 40%, 1% to 35%, 2% to 50%,
2% to 40%, 2% to 35%, 2% to 30%, 2% to 25%, 2% to 20%, 2% to 10%,
5% to 50%, 5% to 40%, 5% to 35%, 5% to 30%, 5% to 25%, or 5% to 20%
by volume based on the total volume of the formulation. In some
instances, the amount of the molecular crowding agent is in the
range of about 5% to about 20% by volume based on the total volume
of the formulation. In some instances, the amount of the crowding
agent is in the range of about 1% to 30% by volume based on the
total volume of the formulation.
[0082] One example of the crowding agent in the composition is
polyethylene glycol (PEG). In some instances, the PEG used can have
a molecular weight sufficient to enhance or facilitate molecular
crowding. In some instances, the PEG used in the composition has a
molecular weight in the range of about 5 k-50 k Da. In some
instances, the PEG used in the composition has a molecular weight
in the range of about 10 k-40 k Da. In some instances, the PEG used
in the composition has a molecular weight in the range of about 10
k-30 k Da. In some instances, the PEG used in the composition has a
molecular weight in the range of about 20 k Da.
[0083] In some instances, the disclosed hybridization buffer
formulations may include the addition of a molecular crowding or
volume exclusion agent. Molecular crowding or volume exclusion
agents are, for example, macromolecules (e.g., proteins) which,
when added to a solution in high concentrations, may alter the
properties of other molecules in solution by reducing the volume of
solvent available to the other molecules. In some instances, the
percentage by volume of the molecular crowding or volume exclusion
agent included in the hybridization buffer formulation may range
from about 1% to about 50%. In some instances, the percentage by
volume of the molecular crowding or volume exclusion agent may be
at least 1%, at least 5%, at least 10%, at least 15%, at least 20%,
at least 25%, at least 30%, at least 35%, at least 40%, at least
45%, or at least 50%. In some instances, the percentage by volume
of the molecular crowding or volume exclusion agent may be at most
50%, at most 45%, at most 40%, at most 35%, at most 30%, at most
25%, at most 20%, at most 15%, at most 10%, at most 5%, or at most
1%. Any of the lower and upper values described in this paragraph
may be combined to form a range included within the present
disclosure, for example, the percentage by volume of molecular
crowding or volume exclusion agent may range from about 5% to about
35%. The percentage by volume of molecular crowding or volume
exclusion agent may have any value within this range, e.g., about
12.5%.
[0084] PH buffer system: The compositions described herein include
a pH buffer system that maintains the pH of the compositions in a
range suitable for hybridization process. The pH buffer system can
include one or more buffering agents selected from the group
consisting of Tris, HEPES, TAPS, Tricine, Bicine, Bis-Tris, NaOH,
KOH, TES, EPPS, MES, and MOPS. The pH buffer system can further
include a solvent. An example pH buffer system includes MOPS,
IVIES, TAPS, phosphate buffer combined with methanol, acetonitrile,
ethanol, isopropanol, butanol, t-butyl alcohol, DMF, DMSO, or any
combination therein.
[0085] The amount of the pH buffer system is effective to maintain
the pH of the formulation in a range suitable for hybridization. In
some instances, the pH may be at least 3, at least 4, at least 5,
at least 6, at least 7, at least 8, at least 9, or at least 10. In
some instances, the pH may be at most 10, at most 9, at most 8, at
most 7, at most 6, at most 5, at most 4, or at most 3. Any of the
lower and upper values described in this paragraph may be combined
to form a range included within the present disclosure, for
example, the pH of the hybridization buffer may range from about 4
to about 8. The pH of the hybridization buffer may have any value
within this range, e.g., about pH 7.8. In some cases, the pH range
is about 3 to about 10. In some instances, the disclosed
hybridization buffer formulations may include adjustment of pH over
the range of about pH 3 to pH 10, with a narrower buffer range of
5-9.
[0086] Additives that impact DNA melting temperatures: The
compositions described herein can include one or more additives to
allow for better control of the melting temperature of the nucleic
acid and enhance the stringency control of the hybridization
reaction. Hybridization reactions are usually carried out under
stringent conditions in order to achieve hybridization specificity.
In some cases, the additive for controlling melting temperature of
nucleic acid is formamide.
[0087] The amount of the additive for controlling melting
temperature of nucleic acid can vary depending on other agents used
in the compositions. In some instances, the amount of the additive
for controlling melting temperature of the nucleic acid is about,
or more than about, 1%, 2%, 3%, 5%, 10%, 15%, 20%, 25%, 30%, 35%,
40%, 50%, 60%, or higher by volume based on the total volume of the
formulation. In some instances, the amount of the additive for
controlling the melting temperature of the nucleic acid is greater
than or equal to about 2% by volume based on the total volume of
the formulation. In some instances, the amount of the additive for
controlling the melting temperature of the nucleic acid is greater
than or equal to about 5% by volume based on the total volume of
the formulation. In some instances, the amount of the additive for
controlling the melting temperature of the nucleic acid is lower
than about 3%, 5%, 10%, 12.5%, 15%, 20%, 25%, 30%, 35%, 40%, 50%,
60%, 70%, 80%, 90%, or higher, by volume based on the total volume
of the formulation. In some instances, the amount of the additive
for controlling the melting temperature of the nucleic acid is in
the range of about 1% to 40%, 1% to 35%, 2% to 50%, 2% to 40%, 2%
to 35%, 2% to 30%, 2% to 25%, 2% to 20%, 2% to 10%, 5% to 50%, 5%
to 40%, 5% to 35%, 5% to 30%, 5% to 25%, or 5% to 20% by volume
based on the total volume of the formulation. In some instances,
the amount of the additive for controlling melting temperature of
the nucleic acid is in the range of about 2% to 20% by volume based
on the total volume of the formulation. In some instances, the
amount of the additive for controlling melting temperature of the
nucleic acid is in the range of about 5% to 10% by volume based on
the total volume of the formulation.
[0088] In some instances, the disclosed hybridization buffer
formulations may include the addition of an additive that alters
nucleic acid duplex melting temperature. Examples of suitable
additives that may be used to alter nucleic acid melting
temperature include, but are not limited to, formamide. In some
instances, the percentage by volume of a melting temperature
additive included in the hybridization buffer formulation may range
from about 1% to about 50%. In some instances, the percentage by
volume of a melting temperature additive may be at least 1%, at
least 5%, at least 10%, at least 15%, at least 20%, at least 25%,
at least 30%, at least 35%, at least 40%, at least 45%, or at least
50%. In some instances, the percentage by volume of a melting
temperature additive may be at most 50%, at most 45%, at most 40%,
at most 35%, at most 30%, at most 25%, at most 20%, at most 15%, at
most 10%, at most 5%, or at most 1%. Any of the lower and upper
values described in this paragraph may be combined to form a range
included within the present disclosure, for example, the percentage
by volume of a melting temperature additive may range from about
10% to about 25%. The percentage by volume of a melting temperature
additive may have any value within this range, e.g., about
22.5%.
[0089] Additives that impact DNA hydration: In some instances, the
disclosed hybridization buffer formulations include the addition of
an additive that impacts nucleic acid hydration. Examples include,
but are not limited to, betaine, urea, glycine betaine, or any
combination thereof. In some instances, the percentage by volume of
a hydration additive included in the hybridization buffer
formulation ranges from about 1% to about 50%. In some instances,
the percentage by volume of a hydration additive is at least 1%, at
least 5%, at least 10%, at least 15%, at least 20%, at least 25%,
at least 30%, at least 35%, at least 40%, at least 45%, or at least
50%. In some instances, the percentage by volume of a hydration
additive is at most 50%, at most 45%, at most 40%, at most 35%, at
most 30%, at most 25%, at most 20%, at most 15%, at most 10%, at
most 5%, or at most 1%. Any of the lower and upper values described
in this paragraph may be combined to form a range included within
the present disclosure. For example, the percentage by volume of a
hydration additive may range from about 1% to about 30%. The
percentage by volume of a melting temperature additive may have any
value within this range, e.g., about 6.5%.
Systems
[0090] Provided herein are systems comprising the hybridization
compositions described herein and a low non-specific binding
surface. The systems described herein, in some instances, comprise
a flow cell device. The systems described herein further comprise,
in some instances, an imaging system (e.g., a camera and an
inverted fluorescent microscope). Systems may further comprise one
or more computer control systems to perform computer-implemented
methods of nucleic acid analysis.
[0091] Low non-specific binding surface: Disclosed herein is a low
non-specific binding surface that enables improved nucleic acid
hybridization and amplification performance. The disclosed surface
may comprise one or more layers of covalently or non-covalently
attached low-binding chemical modification layers, e.g., silane
layers or polymer films, and one or more covalently or
non-covalently attached primer sequences that may be used for
tethering single-stranded template oligonucleotides to the surface.
In some instances, the formulation of the surface, e.g., the
chemical composition of the one or more layers, the coupling
chemistry used to cross-link the one or more layers to the surface,
or, to each other or a combination thereof, and the total number of
layers, may be varied such that non-specific binding of proteins,
nucleic acid molecules, and other hybridization and amplification
reaction components to the surface is minimized or reduced relative
to a comparable monolayer. The formulation of the surface may be
varied such that non-specific hybridization on the surface is
minimized or reduced relative to a comparable monolayer. The
formulation of the surface may be varied such that non-specific
amplification on the surface is minimized or reduced relative to a
comparable monolayer. The formulation of the surface may be varied
such that specific amplification rates, or, yields, or a
combination thereof on the surface are maximized. Amplification
levels suitable for detection are achieved in no more than 2, 3, 4,
5, 6, 7, 8, 9, 10, 15, or 30 amplification cycles in some cases
disclosed herein.
[0092] Non-limiting examples of low non-specific binding surfaces
are provided in co-pending U.S. patent application Ser. No.
16/739,007, which is hereby incorporated by reference in its
entirety. The terms, "low non-specific binding surface" and "low
binding surface" are used interchangeably to refer to hydrophilic
surfaces that exhibit a low amount of non-specific binding to
proteins or nucleic acids, as compared with a surface that is not
hydrophilic. In some instances, the low non-specific binding
surface is passivated, meaning it is coated with a substrate that
is hydrophilic.
[0093] Examples of materials from which the substrate or support
structure may be fabricated include, but are not limited to, glass,
fused-silica, silicon, a polymer (e.g., polystyrene (PS),
macroporous polystyrene (MPPS), polymethylmethacrylate (PMMA),
polycarbonate (PC), polypropylene (PP), polyethylene (PE), high
density polyethylene (HDPE), cyclic olefin polymers (COP), cyclic
olefin copolymers (COC), polyethylene terephthalate (PET)), or any
combination thereof. Various compositions of both glass and plastic
substrates are contemplated.
[0094] The substrate or support structure may be rendered in any of
a variety of geometries and dimensions, and may comprise any of a
variety of materials. For example, in some instances, the substrate
or support structure is locally planar (e.g., comprising a
microscope slide or the surface of a microscope slide). Globally,
the substrate or support structure may be cylindrical (e.g.,
comprising a capillary or the interior surface of a capillary),
spherical (e.g., comprising the outer surface of a non-porous
bead), or irregular (e.g., comprising the outer surface of an
irregularly-shaped, non-porous bead or particle). In some
instances, the surface of the substrate or support structure used
for nucleic acid hybridization and amplification may be a solid,
non-porous surface. In some instances, the surface of the substrate
or support structure used for nucleic acid hybridization and
amplification may be porous, such that the coatings described
herein penetrate the porous surface, and nucleic acid hybridization
and amplification reactions performed thereon may occur within the
pores.
[0095] The substrate or support structure that comprises the one or
more chemically-modified layers, e.g., layers of a low non-specific
binding polymer, may be independent or integrated into another
structure or assembly. For example, in some instances, the
substrate or support structure comprises one or more surfaces
within an integrated or assembled microfluidic flow cell. The
substrate or support structure may comprise one or more surfaces
within a microplate format, e.g., the bottom surface of the wells
in a microplate. As noted above, in some instances, the substrate
or support structure comprises the interior surface (such as the
lumen surface) of a capillary. In another example, the substrate or
support structure comprises the interior surface (such as the lumen
surface) of a capillary etched into a planar chip.
[0096] The chemical modification layers may be applied uniformly
across the surface of the substrate or support structure. In
another example, the surface of the substrate or support structure
may be non-uniformly distributed or patterned, such that the
chemical modification layers are confined to one or more discrete
regions of the substrate. For example, the substrate surface may be
patterned using photolithographic techniques to create an ordered
array or random pattern of chemically-modified regions on the
surface. The substrate surface may be patterned using contact
printing, or, ink-jet printing techniques, or a combination
thereof. In some instances, an ordered array or random patter of
chemically-modified discrete regions may comprise at least 1, 5,
10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600,
700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000,
9000, or 10,000 or more discrete regions, or any intermediate
number spanned by the range herein.
[0097] In order to achieve low non-specific binding surfaces (also
referred to herein as "low binding" or "passivated" surfaces),
hydrophilic polymers may be non-specifically adsorbed or covalently
grafted to the substrate or support surface. For example,
passivation can be performed utilizing poly(ethylene glycol) (PEG,
also known as polyethylene oxide (PEO) or polyoxyethylene),
poly(vinyl alcohol) (PVA), poly(vinyl pyridine), poly(vinyl
pyrrolidone) (PVP), poly(acrylic acid) (PAA), polyacrylamide,
poly(N-isopropylacrylamide) (PNIPAM), poly(methyl methacrylate)
(PMA), poly(2-hydroxylethyl methacrylate) (PHEMA),
poly(oligo(ethylene glycol) methyl ether methacrylate) (POEGMA),
polyglutamic acid (PGA), poly-lysine, poly-glucoside, streptavidin,
dextran, or other hydrophilic polymers with different molecular
weights and end groups that are linked to a surface using, for
example, silane chemistry. The end groups distal from the surface
can include, but are not limited to, biotin, methoxy ether,
carboxylate, amine, NHS ester, maleimide, and bis-silane. In some
instances, two or more layers of a hydrophilic polymer, e.g., a
linear polymer, branched polymer, or multi-branched polymer, may be
deposited on the surface. In some instances, two or more layers may
be covalently coupled to each other or internally cross-linked to
improve the stability of the resulting surface. In some instances,
oligonucleotide primers with different base sequences and base
modifications (or other biomolecules, e.g., enzymes or antibodies)
may be tethered to the resulting surface layer at various surface
densities. In some instances, for example, both surface functional
group density and oligonucleotide concentration may be varied to
target a certain primer density range. Additionally, primer density
can be controlled by diluting the oligonucleotides with other
molecules that carry the same functional group. For example,
amine-labeled oligonucleotides can be diluted with amine-labeled
polyethylene glycol in a reaction with an NETS-ester coated surface
to reduce the final primer density. Primers with different lengths
of linker between the hybridization region and the surface
attachment functional group can also be applied to control surface
density. Examples of suitable linkers include poly-T and poly-A
strands at the 5' end of the primer (e.g., 0 to 20 bases), PEG
linkers (e.g., 3 to 20 monomer units), and carbon-chains (e.g., C6,
C12, C18, etc.). To measure the primer density,
fluorescently-labeled primers may be tethered to the surface and a
fluorescence reading corresponding to the fluorescently-labeled
primers may then be compared with that for a dye solution of known
concentration.
[0098] As a result of the surface passivation techniques disclosed
herein, proteins, nucleic acids, and other biomolecules do not
"stick" to substrates, that is, they exhibit low non-specific
binding (non-specific binding). Examples are shown below using
standard monolayer surface preparations with varying glass
preparation conditions. Hydrophilic surfaces that have been
passivated to achieve ultra-low non-specific binding for proteins
and nucleic acids require novel reaction conditions to improve
primer deposition reaction efficiencies and hybridization
performance, and to induce effective amplification. All of these
processes require oligonucleotide attachment and subsequent protein
binding and delivery to a low binding surface. As described below,
the combination of a new primer surface conjugation formulation
(Cy3 oligonucleotide graft titration) and resulting ultra-low
non-specific background (non-specific binding functional tests
performed using red and green fluorescent dyes) yielded results
that demonstrate the viability of the disclosed approaches. Some
surfaces disclosed herein exhibit a ratio of specific (e.g.,
hybridization to a tethered primer or probe) to non-specific
binding (e.g., B.sub.inter) of a fluorophore such as Cy3 of at
least 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1,
13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, 25:1, 30:1, 35:1,
40:1, 50:1, 75:1, 100:1, or greater than 100:1, or any intermediate
value spanned by the range herein. Some surfaces disclosed herein
exhibit a ratio of specific to non-specific fluorescence signal
(e.g., for specifically-hybridized to non-specifically bound
labeled oligonucleotides, or for specifically-amplified to
non-specifically-bound (B.sub.inter) or non-specifically amplified
(B.sub.intra) labeled oligonucleotides or a combination thereof
(B.sub.inter+B.sub.intra)) for a fluorophore such as Cy3 of at
least 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1,
13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, 25:1, 30:1, 35:1,
40:1, 50:1, 75:1, 100:1, or greater than 100:1, or any intermediate
value spanned by the range herein.
[0099] In order to scale primer surface density and add additional
dimensionality to hydrophilic or amphoteric surfaces, substrates
comprising multi-layer coatings of PEG and other hydrophilic
polymers have been developed. By using hydrophilic and amphoteric
surface layering approaches that include, but are not limited to,
the polymer/co-polymer materials described below, it is possible to
increase primer loading density on the surface significantly.
Traditional PEG coating approaches use monolayer primer deposition,
which has been generally reported as successful for single molecule
applications, but does not yield high copy numbers for nucleic acid
amplification applications. As described herein, "layering" can be
accomplished using traditional crosslinking approaches with any
compatible polymer or monomer subunits, such that a surface
comprising two or more highly crosslinked layers can be built
sequentially. Examples of suitable polymers include, but are not
limited to, streptavidin, poly acrylamide, polyester, dextran,
poly-lysine, and copolymers of poly-lysine and PEG. In some
instances, the different layers may be attached to each other
through any of a variety of conjugation reactions including, but
not limited to, biotin-streptavidin binding, azide-alkyne click
reaction, amine-NETS ester reaction, thiol-maleimide reaction, and
ionic interactions between positively charged polymers and
negatively charged polymers. In some instances, high primer density
materials may be constructed in solution and subsequently layered
onto the surface in multiple operations.
[0100] The attachment chemistry used to graft a first
chemically-modified layer to a support surface will generally be
dependent on both the material from which the support is fabricated
and the chemical nature of the layer. In some instances, the first
layer may be covalently attached to the support surface. In some
instances, the first layer may be non-covalently attached, e.g.,
adsorbed to the surface through non-covalent interactions such as
electrostatic interactions, hydrogen bonding, or van der Waals
interactions between the surface and the molecular components of
the first layer. In either case, the substrate surface may be
treated prior to attachment or deposition of the first layer. Any
of a variety of surface preparation techniques may be used to clean
or treat the support surface. For example, glass or silicon
surfaces may be acid-washed using a Piranha solution (a mixture of
sulfuric acid (H.sub.2SO.sub.4) and hydrogen peroxide
(H.sub.2O.sub.2)), or, cleaned using an oxygen plasma treatment
method, or a combination thereof.
[0101] Silane chemistries constitute one non-limiting approach for
covalently modifying the silanol groups on glass or silicon
surfaces to attach more reactive functional groups (e.g., amines or
carboxyl groups), which may then be used in coupling linker
molecules (e.g., linear hydrocarbon molecules of various lengths,
such as C6, C12, C18 hydrocarbons, or linear polyethylene glycol
(PEG) molecules) or layer molecules (e.g., branched PEG molecules
or other polymers) to the surface. Examples of suitable silanes
that may be used in creating any of the disclosed low binding
support surfaces include, but are not limited to, (3-Aminopropyl)
trimethoxysilane (APTMS), (3-Aminopropyl) triethoxysilane (APTES),
any of a variety of PEG-silanes (e.g., comprising molecular weights
of 1K, 2K, 5K, 10K, 20K, etc.), amino-PEG silane (e.g., comprising
a free amino functional group), maleimide-PEG silane, biotin-PEG
silane, and the like.
[0102] Any of a variety of molecules including, but not limited to,
amino acids, peptides, nucleotides, oligonucleotides, other
monomers or polymers, or combinations thereof may be used in
creating the one or more chemically-modified layers on the support
surface, where the choice of components used may be varied to alter
one or more properties of the support surface, e.g., the surface
density of functional groups, or, tethered oligonucleotide primers,
or a combination thereof; the hydrophilicity/hydrophobicity of the
support surface, or the three three-dimensional nature (e.g.,
"thickness") of the support surface. Examples of polymers that may
be used to create one or more layers of low non-specific binding
material in any of the disclosed support surfaces include, but are
not limited to, polyethylene glycol (PEG) of various molecular
weights and branching structures, streptavidin, polyacrylamide,
polyester, dextran, poly-lysine, and poly-lysine copolymers, or any
combination thereof. Examples of conjugation chemistries that may
be used to graft one or more layers of material (e.g., polymer
layers) to the support surface, or, to cross-link the layers to
each other, or a combination thereof include, but are not limited
to, biotin-streptavidin interactions (or variations thereof), his
tag--Ni/NTA conjugation chemistries, methoxy ether conjugation
chemistries, carboxylate conjugation chemistries, amine conjugation
chemistries, NHS esters, maleimides, thiol, epoxy, azide,
hydrazide, alkyne, isocyanate, and silane.
[0103] One or more layers of a multi-layered surface may comprise a
branched polymer or may be linear. Examples of suitable branched
polymers include, but are not limited to, branched PEG, branched
poly(vinyl alcohol) (branched PVA), branched poly(vinyl pyridine),
branched poly(vinyl pyrrolidone) (branched PVP), branched),
poly(acrylic acid) (branched PAA), branched polyacrylamide,
branched poly(N-isopropylacrylamide) (branched PNIPAM), branched
poly(methyl methacrylate) (branched PMA), branched
poly(2-hydroxylethyl methacrylate) (branched PHEMA), branched
poly(oligo(ethylene glycol) methyl ether methacrylate) (branched
POEGMA), branched polyglutamic acid (branched PGA), branched
poly-lysine, branched poly-glucoside, and dextran.
[0104] In some instances, the branched polymers used to create one
or more layers of any of the multi-layered surfaces disclosed
herein may comprise at least 4 branches, at least 5 branches, at
least 6 branches, at least 7 branches, at least 8 branches, at
least 9 branches, at least 10 branches, at least 12 branches, at
least 14 branches, at least 16 branches, at least 18 branches, at
least 20 branches, at least 22 branches, at least 24 branches, at
least 26 branches, at least 28 branches, at least 30 branches, at
least 32 branches, at least 34 branches, at least 36 branches, at
least 38 branches, or at least 40 branches. Molecules often exhibit
a `power of 2` number of branches, such as 2, 4, 8, 16, 32, 64, or
128 branches.
[0105] PEG multilayers comprising PEG (8,16,8) on PEG-amine-APTES
exposed to two layers of 7 uM primer pre-loading exhibited a
concentration of 2,000,000 to 10,000,000 on the surface. Similar
concentrations were observed for 3-layer multi-arm PEG (8,16,8) and
(8,64,8) on PEG-amine-APTES exposed to 8 uM primer, and 3-layer
multi-arm PEG (8,8,8) using star-shaped PEG-amine to replace
dumbbell-shaped 16mer and 64mer. PEG multilayers having comparable
first, second and third PEG levels are also contemplated.
[0106] Linear, branched, or multi-branched polymers used to create
one or more layers of any of the multi-layered surfaces disclosed
herein may have a molecular weight of at least 500, at least 1,000,
at least 2,000, at least 3,000, at least 4,000, at least 5,000, at
least 10,000, at least 15,000, at least 20,000, at least 25,000, at
least 30,000, at least 35,000, at least 40,000, at least 45,000, or
at least 50,000 daltons.
[0107] In some instances, e.g., wherein at least one layer of a
multi-layered surface comprises a branched polymer, the number of
covalent bonds between a branched polymer molecule of the layer
being deposited and molecules of the underlying layer may range
from about one covalent linkage per molecule to about 32 covalent
linkages per molecule. In some instances, the number of covalent
bonds between a branched polymer molecule of the new layer and
molecules of the underlying layer may be at least 1, at least 2, at
least 3, at least 4, at least 5, at least 6, at least 7, at least
8, at least 9, at least 10, at least 12, at least 14, at least 16,
at least 18, at least 20, at least 22, at least 24, at least 26, at
least 28, at least 30, or at least 32 or more than 32 covalent
linkages per molecule.
[0108] Any reactive functional groups that remain following the
coupling of a material layer to the support surface may optionally
be blocked by coupling a small, inert molecule using a high yield
coupling chemistry. For example, in the case that amine coupling
chemistry is used to attach a new material layer to the underlying
one, any residual amine groups may subsequently be acetylated or
deactivated by coupling with a small amino acid such as
glycine.
[0109] The number of layers of low non-specific binding material,
e.g., a hydrophilic polymer material, deposited on the surface of
the disclosed low binding supports may range from 1 to about 10. In
some instances, the number of layers is at least 1, at least 2, at
least 3, at least 4, at least 5, at least 6, at least 7, at least
8, at least 9, or at least 10. In some instances, the number of
layers may be at most 10, at most 9, at most 8, at most 7, at most
6, at most 5, at most 4, at most 3, at most 2, or at most 1. Any of
the lower and upper values described in this paragraph may be
combined to form a range included within the present disclosure,
for example, in some instances the number of layers may range from
about 2 to about 4. In some instances, all of the layers may
comprise the same material. In some instances, each layer may
comprise a different material. In some instances, the plurality of
layers may comprise a plurality of materials. In some instances, at
least one layer may comprise a branched polymer. In some instances,
all of the layers may comprise a branched polymer.
[0110] One or more layers of low non-specific binding material may,
in some cases, be deposited on, or, conjugated to the substrate
surface, or a combination thereof, using a polar protic solvent, a
polar aprotic solvent, a nonpolar solvent, or any combination
thereof. In some instances, the solvent used for layer deposition,
or, coupling, or a combination thereof may comprise an alcohol
(e.g., methanol, ethanol, propanol, etc.), another organic solvent
(e.g., acetonitrile, dimethyl sulfoxide (DMSO), dimethyl formamide
(DMF), etc.), water, an aqueous buffer solution (e.g., phosphate
buffer, phosphate buffered saline, 3-(N-morpholino)propanesulfonic
acid (MOPS), etc.), or any combination thereof. In some instances,
an organic component of the solvent mixture used may comprise at
least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%,
60%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% of the total, or any
percentage spanned or adjacent to the range herein, with the
balance made up of water or an aqueous buffer solution. In some
instances, an aqueous component of the solvent mixture used may
comprise at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%,
50%, 55%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% of the
total, or any percentage spanned or adjacent to the range herein,
with the balance made up of an organic solvent. The pH of the
solvent mixture used may be less than or equal to about 5, 5.5, 6,
6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, or any value spanned or adjacent
to the range described herein. The pH of the solvent mixture may be
greater than or equal to about 10.
[0111] In some instances, one or more layers of low non-specific
binding material may be deposited on, or, conjugated to the
substrate surface, or a combination thereof, using a mixture of
organic solvents, wherein the dielectric constant of at least once
component is less than 40 and constitutes at least 50% of the total
mixture by volume. In some instances, the dielectric constant of
the at least one component may be less than 10, less than 20, less
than 30, or less than 40. In some instances, the at least one
component constitutes at least 20%, at least 30%, at least 40%, at
least 50%, at least 60%, at least 70%, or at least 80% of the total
mixture by volume.
[0112] As noted, the low non-specific binding supports of the
present disclosure exhibit reduced non-specific binding of
proteins, nucleic acids, and other components of the hybridization,
or, amplification formulation, or a combination thereof, used for
solid-phase nucleic acid amplification. The degree of non-specific
binding exhibited by a given support surface may be assessed either
qualitatively or quantitatively. For example, in some instances,
exposure of the surface to fluorescent dyes (e.g., Cy3, Cy5, etc.),
fluorescently-labeled nucleotides, fluorescently-labeled
oligonucleotides, or, fluorescently-labeled proteins (e.g.,
polymerases), or a combination thereof, under a standardized set of
conditions, followed by a specified rinse protocol and fluorescence
imaging may be used as a qualitative tool for comparison of
non-specific binding on supports comprising different surface
formulations. In some instances, exposure of the surface to
fluorescent dyes, fluorescently-labeled nucleotides,
fluorescently-labeled oligonucleotides, or, fluorescently-labeled
proteins (e.g., polymerases), or a combination thereof, under a
standardized set of conditions, followed by a specified rinse
protocol and fluorescence imaging may be used as a quantitative
tool for comparison of non-specific binding on supports comprising
different surface formulations--provided that care has been taken
to ensure that the fluorescence imaging is performed under
conditions where fluorescence signal is linearly related (or
related in a predictable manner) to the number of fluorophores on
the support surface (e.g., under conditions where signal
saturation, or, self-quenching of the fluorophore, or a combination
thereof, is not an issue) and suitable calibration standards are
used. In some instances, other techniques, for example,
radioisotope labeling and counting methods may be used for
quantitative assessment of the degree to which non-specific binding
is exhibited by the different support surface formulations of the
present disclosure.
[0113] Some surfaces disclosed herein exhibit a ratio of specific
to non-specific binding of a fluorophore such as Cy3 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, 50, 75, 100, or greater than 100, or any
intermediate value spanned by the range herein. Some surfaces
disclosed herein exhibit a ratio of specific to non-specific
fluorescence of a fluorophore such as Cy3 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, 50, 75, 100, or greater than 100, or any intermediate value
spanned by the range herein.
[0114] As noted, in some instances, the degree of non-specific
binding exhibited by the disclosed low-binding supports may be
assessed using a standardized protocol for contacting the surface
with a labeled protein (e.g., bovine serum albumin (BSA),
streptavidin, a DNA polymerase, a reverse transcriptase, a
helicase, a single-stranded binding protein (SSB), etc., or any
combination thereof), a labeled nucleotide, a labeled
oligonucleotide, etc., under a standardized set of incubation and
rinse conditions, followed by detection of the amount of label
remaining on the surface and comparison of the signal resulting
therefrom to an appropriate calibration standard. In some
instances, the label may comprise a fluorescent label. In some
instances, the label may comprise a radioisotope. In some
instances, the label may comprise any other detectable label. In
some instances, the degree of non-specific binding exhibited by a
given support surface formulation may thus be assessed in terms of
the number of non-specifically bound protein molecules (or other
molecules) per unit area. In some instances, the low-binding
supports of the present disclosure may exhibit non-specific protein
binding (or non-specific binding of other specified molecules,
e.g., Cy3 dye) of less than or equal to about 0.001 molecule per
.mu.m.sup.2, less than or equal to about 0.01 molecule per
.mu.m.sup.2, less than or equal to about 0.1 molecule per
.mu.m.sup.2, less than or equal to about 0.25 molecule per
.mu.m.sup.2, less than or equal to about 0.5 molecule per
.mu.m.sup.2, less than or equal to about 1 molecule per
.mu.m.sup.2, less than or equal to about 10 molecules per
.mu.m.sup.2, less than or equal to about 100 molecules per
.mu.m.sup.2, or less than or equal to about 1,000 molecules per
.mu.m.sup.2. A given support surface of the present disclosure may
exhibit non-specific binding falling anywhere within this range,
for example, of less than 86 molecules per .mu.m.sup.2.
[0115] In some instances, the surfaces disclosed herein exhibit a
ratio of specific to non-specific binding of a fluorophore such as
Cy3 of at least or equal to about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,
12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 50, 75, 100, or
any intermediate value spanned by the range herein. In some
instances, the surfaces disclosed herein exhibit a ratio of
specific to non-specific binding of fluorophore such as Cy3 of
greater than or equal to about 100. In some instances, the surfaces
disclosed herein exhibit a ratio of specific to non-specific
fluorescence signals for a fluorophore such as Cy3 of at least or
equal to about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,
17, 18, 19, 20, 25, 30, 35, 40, 50, 75, 100, or any intermediate
value spanned by the range herein. In some instances, the surfaces
disclosed herein exhibit a ratio of specific to non-specific
fluorescence signals for a fluorophore such as Cy3 of greater than
or equal to about 100.
[0116] The low-background surfaces consistent with the disclosure
herein may exhibit specific dye attachment (e.g., Cy3 attachment)
to non-specific dye adsorption (e.g., Cy3 dye adsorption) ratios of
at least 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 15:1, 20:1, 30:1,
40:1, 50:1, or more than 50 specific dye molecules attached per
molecule non-specifically adsorbed. Similarly, when subjected to an
excitation energy, low-background surfaces consistent with the
disclosure herein to which fluorophores, e.g., Cy3, have been
attached may exhibit ratios of specific fluorescence signal (e.g.,
arising from Cy3-labeled oligonucleotides attached to the surface)
to non-specific adsorbed dye fluorescence signals of at least 4:1,
5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 15:1, 20:1, 30:1, 40:1, 50:1, or
more than 50:1.
[0117] In some instances, the degree of hydrophilicity (or
"wettability" with aqueous solutions) of the disclosed support
surfaces may be assessed, for example, through the measurement of
water contact angles in which a small droplet of water is placed on
the surface and its angle of contact with the surface is measured
using, e.g., an optical tensiometer. In some instances, a static
contact angle may be determined. In some instances, an advancing or
receding contact angle may be determined. In some instances, the
water contact angle for the hydrophilic, low-binding support
surfaced disclosed herein may range from about 0 degrees to about
30 degrees. In some instances, the water contact angle for the
hydrophilic, low-binding support surfaced disclosed herein may be
no more than 50 degrees, 45 degrees, 40 degrees, 30 degrees, 25
degrees, 20 degrees, 18 degrees, 16 degrees, 14 degrees, 12
degrees, 10 degrees, 8 degrees, 6 degrees, 4 degrees, 2 degrees, or
1 degree. In many cases the contact angle is no more than 40
degrees. A given hydrophilic, low-binding support surface of the
present disclosure may exhibit a water contact angle having a value
of anywhere within this range.
[0118] In some instances, the hydrophilic surfaces disclosed herein
facilitate reduced wash times for bioassays, often due to reduced
non-specific binding of biomolecules to the low-binding surfaces.
In some instances, adequate washes may be performed in less than or
equal to about 60, 50, 40, 30, 20, 15, 10, or less than 10 seconds.
For example, in some instances adequate washes may be performed in
less than 30 seconds.
[0119] Some low-binding surfaces of the present disclosure exhibit
significant improvement in stability or durability to prolonged
exposure to solvents and elevated temperatures, or to repeated
cycles of solvent exposure or changes in temperature. For example,
in some instances, the stability of the disclosed surfaces may be
tested by fluorescently labeling a functional group on the surface,
or a tethered biomolecule (e.g., an oligonucleotide primer) on the
surface, and monitoring fluorescence signal before, during, and
after prolonged exposure to solvents and elevated temperatures, or
to repeated cycles of solvent exposure or changes in temperature.
In some instances, the degree of change in the fluorescence used to
assess the quality of the surface may be less than or equal to
about 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, or 25% over a time period
of 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 10
minutes, 20 minutes, 30 minutes, 40 minutes, 50 minutes, 60
minutes, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8
hours, 9 hours, 10 hours, 15 hours, 20 hours, 25 hours, 30 hours,
35 hours, 40 hours, 45 hours, 50 hours, or 100 hours of exposure to
solvents, or, elevated temperatures, or a combination thereof (or
any combination of these percentages as measured over these time
periods). In some instances, the degree of change in the
fluorescence used to assess the quality of the surface may be less
than or equal to about 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, or 25%
over 5 cycles, 10 cycles, 20 cycles, 30 cycles, 40 cycles, 50
cycles, 60 cycles, 70 cycles, 80 cycles, 90 cycles, 100 cycles, 200
cycles, 300 cycles, 400 cycles, 500 cycles, 600 cycles, 700 cycles,
800 cycles, 900 cycles, or 1,000 cycles of repeated exposure to
solvent changes, or changes in temperature, or a combination
thereof (or any combination of these percentages as measured over
this range of cycles).
[0120] In some instances, the surfaces disclosed herein may exhibit
a high ratio of specific signal to non-specific signal or other
background. For example, when used for nucleic acid amplification,
some surfaces may exhibit an amplification signal that is at least
4-, 5-, 6-, 7-, 8-, 9-, 10-, 15-, 20-, 30-, 40-, 50-, 75-, 100-, or
greater than 100-fold greater than a signal of an adjacent
unpopulated region of the surface. In some instances, the surfaces
exhibit an amplification signal that is at least 4-, 5-, 6-, 7-,
8-, 9-, 10-, 15-, 20-, 30-, 40-, 50-, 75-, 100-, or greater than
100-fold greater than a signal of an adjacent amplified nucleic
acid population region of the surface.
[0121] Fluorescence excitation energies vary among particular
fluorophores and protocols, and may range in excitation wavelength,
consistent with fluorophore selection or other parameters of use of
a surface disclosed herein. In some instances, the wavelength is
less than or equal to about 400 nanometers (nm). In some instances,
the wavelength is more than or equal to about 800 nm. In some
instances, the wavelength is between 400 nm and 800 nm.
[0122] Accordingly, low background surfaces as disclosed herein
exhibit low background fluorescence signals or high contrast to
noise (CNR) ratios. For example, in some instances, the background
fluorescence of the surface at a location that is spatially
distinct or removed from a labeled feature on the surface (e.g., a
labeled spot, cluster, discrete region, sub-section, or subset of
the surface) comprising a hybridized cluster of nucleic acid
molecules, or a clonally-amplified cluster of nucleic acid
molecules produced by 20 cycles of nucleic acid amplification via
thermocycling, may be no more than 20.times., 10.times., 5.times.,
2.times., 1.times., 0.5.times., 0.1.times., or less than 0.1.times.
greater than the background fluorescence measured at that same
location prior to performing said hybridization or said 20 cycles
of nucleic acid amplification.
[0123] In some instances, fluorescence images of the disclosed low
background surfaces when used in nucleic acid hybridization or
amplification applications to create clusters of hybridized or
clonally-amplified nucleic acid molecules (e.g., that have been
directly or indirectly labeled with a fluorophore) exhibit
contrast-to-noise ratios (CNRs) of at least 10, 20, 30, 40, 50, 60,
70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 20,
210, 220, 230, 240, 250, or greater than 250.
[0124] The surface that comprises the one or more
chemically-modified layers, e.g., layers of a low non-specific
binding polymer, may be independent or integrated into another
structure or assembly. The chemical modification layers may be
applied uniformly across the surface. Alternately, the surface may
be patterned, such that the chemical modification layers are
confined to one or more discrete regions of the substrate. For
example, the surface may be patterned using photolithographic
techniques to create an ordered array or random pattern of
chemically-modified regions on the surface. The substrate surface
may be patterned using, e.g., contact printing, or, ink-jet
printing techniques, or a combination thereof. In some instances,
an ordered array or random patter of chemically-modified regions
may comprise at least 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90,
100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000,
4000, 5000, 6000, 7000, 8000, 9000, or 10,000 or more discrete
regions.
[0125] In order to achieve low non-specific binding surfaces (also
referred to herein as "low binding" or "passivated" surfaces),
hydrophilic polymers may be non-specifically adsorbed or covalently
grafted to the surface. For example, passivation can be performed
utilizing poly(ethylene glycol) (PEG, also known as polyethylene
oxide (PEO) or polyoxyethylene) or other hydrophilic polymers with
different molecular weights and end groups that are linked to a
surface using, for example, silane chemistry. The end groups distal
from the surface can include, but are not limited to, biotin,
methoxy ether, carboxylate, amine, NHS ester, maleimide, and
bis-silane. In some instances, two or more layers of a hydrophilic
polymer, e.g., a linear polymer, branched polymer, or
multi-branched polymer, may be deposited on the surface. In some
instances, two or more layers may be covalently coupled to each
other or internally cross-linked to improve the stability of the
resulting surface. In some instances, oligonucleotide primers with
different base sequences and base modifications (or other
biomolecules, e.g., enzymes or antibodies) may be tethered to the
resulting surface layer at various surface densities. In some
instances, for example, both surface functional group density and
oligonucleotide concentration may be varied to target a certain
primer density range. Additionally, primer density can be
controlled by diluting oligonucleotides with other molecules that
carry the same functional group. For example, amine-labeled
oligonucleotides can be diluted with amine-labeled polyethylene
glycol in a reaction with an NETS-ester coated surface to reduce
the final primer density. Primers with different lengths of linker
between the hybridization region and the surface attachment
functional group can also be applied to control surface density.
Examples of suitable linkers include poly-T and poly-A strands at
the 5' end of the primer (e.g., 0 to 20 bases), PEG linkers (e.g.,
3 to 20 monomer units), and carbon-chain (e.g., C6, C12, C18,
etc.). To measure the primer density, fluorescently-labeled primers
may be tethered to the surface and a fluorescence reading
corresponding to the primers may then be compared with that for a
dye solution of known concentration.
[0126] As noted, the low non-specific binding surfaces described
herein exhibit reduced non-specific binding of nucleic acids, and
other components of the hybridization, or, amplification
formulation, or a combination thereof used for solid-phase nucleic
acid amplification. The degree of non-specific binding exhibited by
a given surface may be assessed either qualitatively or
quantitatively. For example, in some instances, exposure of the
surface to fluorescent dyes (e.g., Cy3, Cy5, etc.),
fluorescently-labeled nucleotides, fluorescently-labeled
oligonucleotides, or, fluorescently-labeled proteins (e.g.,
polymerases), or a combination thereof under a standardized set of
conditions, followed by a specified rinse protocol and fluorescence
imaging may be used as a qualitative tool for comparison of
non-specific binding surfaces comprising different surface
formulations. In some instances, exposure of the surface to
fluorescent dyes, fluorescently-labeled nucleotides,
fluorescently-labeled oligonucleotides, or, fluorescently-labeled
proteins (e.g., polymerases), or combination thereof under a
standardized set of conditions, followed by a specified rinse
protocol and fluorescence imaging may be used as a quantitative
tool for comparison of non-specific binding on surfaces comprising
different surface formulations--provided that care has been taken
to ensure that the fluorescence imaging is performed under
conditions where fluorescence signal is linearly related (or
related in a predictable manner) to the number of fluorophores on
the surface (e.g., under conditions where signal saturation, or,
self-quenching of the fluorophore, or a combination thereof is not
an issue) and suitable calibration standards are used. In some
instances, other techniques, for example, radioisotope labeling and
counting methods may be used for quantitative assessment of the
degree to which non-specific binding is exhibited by the different
surface formulations of the present disclosure.
[0127] As noted, in some instances, the degree of non-specific
binding exhibited by the disclosed low-binding surfaces may be
assessed using a standardized protocol for contacting the surface
with a labeled protein (e.g., bovine serum albumin (BSA),
streptavidin, a DNA polymerase, a reverse transcriptase, a
helicase, a single-stranded binding protein (SSB), etc., or any
combination thereof), a labeled nucleotide, a labeled
oligonucleotide, etc., under a standardized set of incubation and
rinse conditions, followed by detection of the amount of label
remaining on the surface and comparison of the signal resulting
therefrom to an appropriate calibration standard. In some
instances, the label may comprise a fluorescent label. In some
instances, the label may comprise a radioisotope. In some
instances, the label may comprise any other detectable label. In
some instances, the degree of non-specific binding exhibited by a
given surface formulation may thus be assessed in terms of the
number of non-specifically bound protein molecules (or other
molecules) per unit area. In some instances, the low-binding
surfaces of the present disclosure may exhibit non-specific protein
binding (or non-specific binding of other specified molecules,
e.g., Cy3 dye) of less than or equal to about 0.001 molecule per
.mu.m.sup.2, less than or equal to about 0.01 molecule per
.mu.m.sup.2, less than or equal to about 0.1 molecule per
.mu.m.sup.2, less than or equal to about 0.25 molecule per
.mu.m.sup.2, less than or equal to about 0.5 molecule per
.mu.m.sup.2, less than or equal to about 1 molecule per
.mu.m.sup.2, less than or equal to about 10 molecules per
.mu.m.sup.2, less than or equal to about 100 molecules per
.mu.m.sup.2, or less than or equal to about 1,000 molecules per
.mu.m.sup.2. A given surface of the present disclosure may exhibit
non-specific binding falling anywhere within this range, for
example, of less than or equal to about 86 molecules per
.mu.m.sup.2. For example, some modified surfaces disclosed herein
exhibit non-specific protein binding of less than or equal to about
0.5 molecule/.mu.m.sup.2 following contact with a 1 .mu.M solution
of bovine serum albumin (BSA) in phosphate buffered saline (PBS)
buffer for 30 minutes, followed by a 10 minute PBS rinse. In
another example, some modified surfaces disclosed herein exhibit
non-specific protein binding of less than or equal to about 0.5
molecule/.mu.m.sup.2 following contact with a 1 .mu.M solution of
Cyanine 3 dye-labeled streptavidin (GE Amersham) in phosphate
buffered saline (PBS) buffer for 15 minutes, followed by 3 rinses
with deionized water. Some modified surfaces disclosed herein
exhibit non-specific binding of Cy3 dye molecules of less than or
equal to about 0.25 molecules per .mu.m.sup.2.
[0128] The low-background surfaces consistent with the disclosure
herein may exhibit specific dye attachment (e.g., Cy3 attachment)
to non-specific dye adsorption (e.g., Cy3 dye adsorption) ratios of
at least 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 15:1, 20:1, 30:1,
40:1, 50:1, or more than 50 specific dye molecules attached per
molecule non-specifically adsorbed. Similarly, when subjected to an
excitation energy, low-background surfaces consistent with the
disclosure herein to which fluorophores, e.g., Cy3, have been
attached may exhibit ratios of specific fluorescence signal (e.g.,
arising from Cy3-labeled oligonucleotides attached to the surface)
to non-specific adsorbed dye fluorescence signals of at least 4:1,
5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 15:1, 20:1, 30:1, 40:1, 50:1, or
more than 50:1.
[0129] In some instances, the degree of hydrophilicity (or
"wettability" with aqueous solutions) of the disclosed surfaces may
be assessed, for example, through the measurement of water contact
angles in which a small droplet of water is placed on the surface
and its angle of contact with the surface is measured using, e.g.,
an optical tensiometer. In some instances, a static contact angle
may be determined. In some instances, an advancing or receding
contact angle may be determined. In some instances, the water
contact angle for the hydrophilic, low-binding surfaces disclosed
herein may range from about 0 degrees to about 30 degrees. In some
instances, the water contact angle for the hydrophilic, low-binding
surfaced disclosed herein may be no more than 50 degrees, 40
degrees, 30 degrees, 25 degrees, 20 degrees, 18 degrees, 16
degrees, 14 degrees, 12 degrees, 10 degrees, 8 degrees, 6 degrees,
4 degrees, 2 degrees, or 1 degree. In many cases the contact angle
is no more than 40 degrees. A given hydrophilic, low-binding
surface of the present disclosure may exhibit a water contact angle
having a value of anywhere within this range.
[0130] In some instances, the low-binding surfaces of the present
disclosure may exhibit significant improvement in stability or
durability to prolonged exposure to solvents and elevated
temperatures, or to repeated cycles of solvent exposure or changes
in temperature. For example, in some instances, the stability of
the disclosed surfaces may be tested by fluorescently labeling a
functional group on the surface, or a tethered biomolecule (e.g.,
an oligonucleotide primer) on the surface, and monitoring
fluorescence signal before, during, and after prolonged exposure to
solvents and elevated temperatures, or to repeated cycles of
solvent exposure or changes in temperature. In some instances, the
degree of change in the fluorescence used to assess the quality of
the surface may be less than or equal to about 1%, 2%, 3%, 4%, 5%,
10%, 15%, 20%, or 25% over a time period of 1 minute, 2 minutes, 3
minutes, 4 minutes, 5 minutes, 10 minutes, 20 minutes, 30 minutes,
40 minutes, 50 minutes, 60 minutes, 2 hours, 3 hours, 4 hours, 5
hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 15 hours, 20
hours, 25 hours, 30 hours, 35 hours, 40 hours, 45 hours, 50 hours,
or 100 hours of exposure to solvents, or, elevated temperatures, or
a combination thereof (or any combination of these percentages as
measured over these time periods). In some instances, the degree of
change in the fluorescence used to assess the quality of the
surface may be less than or equal to about 1%, 2%, 3%, 4%, 5%, 10%,
15%, 20%, or 25% over 5 cycles, 10 cycles, 20 cycles, 30 cycles, 40
cycles, 50 cycles, 60 cycles, 70 cycles, 80 cycles, 90 cycles, 100
cycles, 200 cycles, 300 cycles, 400 cycles, 500 cycles, 600 cycles,
700 cycles, 800 cycles, 900 cycles, or 1,000 cycles of repeated
exposure to solvent changes, or, changes in temperature, or a
combination thereof (or any combination of these percentages as
measured over this range of cycles).
[0131] In some instances, the surfaces disclosed herein may exhibit
a high ratio of specific signal to non-specific signal or other
background. For example, when used for nucleic acid amplification,
some surfaces may exhibit an amplification signal that is at least
4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 75, 100, or greater than
100 fold greater than a signal of an adjacent unpopulated region of
the surface. Similarly, some surfaces exhibit an amplification
signal that is at least 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50,
75, 100, or greater than 100 fold greater than a signal of an
adjacent amplified nucleic acid population region of the
surface.
[0132] Accordingly, low background surfaces as disclosed herein
exhibit low background fluorescence signals or high contrast to
noise (CNR) ratios.
[0133] Flow Cell Devices: The low non-specific binding surfaces
described herein, in some examples, are surfaces of a flow device
described herein. Flow devices described herein can include a first
reservoir housing a first solution and having an inlet end and an
outlet end, wherein the first agent flows from the inlet end to the
outlet end in the first reservoir; a second reservoir housing a
second solution and having an inlet end and an outlet end, wherein
the second agent flows from the inlet end to the outlet end in the
second reservoir; a central region having an inlet end fluidically
coupled to the outlet end of the first reservoir and the outlet end
of the second reservoir through at least one valve. In the flow
cell device, the volume of the first solution flowing from the
outlet of the first reservoir to the inlet of the central region is
less than the volume of the second solution flowing from the outlet
of the second reservoir to the inlet of the central region.
[0134] The reservoirs described in the device can be used to house
different reagents. In some examples, the first solution housed in
the first reservoir is different from the second solution that is
housed in the second reservoir. The second solution comprises at
least one reagent common to a plurality of reactions occurring in
the central region. In some examples, the second solution comprises
at least one reagent selected from the list consisting of a
solvent, a polymerase, and a dNTP. In some examples, the second
solution comprise low cost reagents. In some examples, the first
reservoir is fluidically coupled to the central region through a
first valve and the second reservoir is fluidically coupled to the
central region through a second valve. The valve can be a diaphragm
valve or other suitable valves.
[0135] The central region can include a capillary tube or
microfluidic chip having one or more microfluidic channels. In some
examples, the capillary tube is an off-shelf product. The capillary
tube or the microfluidic chip can also be removable from the
device. In some examples, the capillary tube or microfluidic
channel comprises an oligonucleotide population directed to
sequence a eukaryotic genome. In some examples, the capillary tube
or microfluidic channel in the central region is removable.
[0136] Disclosed herein are single capillary flow cell devices that
comprise a single capillary and one or two fluidic adapters affixed
to one or both ends of the capillary, where the capillary provides
a fluid flow channel of specified cross-sectional area and length,
and where the fluidic adapters are configured to mate with standard
tubing to provide for convenient, interchangeable fluid connections
with an external fluid flow control system. In general, the
capillary used in the disclosed flow cell devices (and flow cell
cartridges to be described below) will have at least one internal,
axially-aligned fluid flow channel (or "lumen") that runs the full
length of the capillary. In some examples, the capillary may have
two, three, four, five, or more than five internal, axially-aligned
fluid flow channels (or "lumens").
[0137] A number specified cross-sectional geometries for a single
capillary (or a lumen thereof) are consistent with the disclosure
herein, including, but not limited to, circular, elliptical,
square, rectangular, triangular, rounded square, rounded
rectangular, or rounded triangular cross-sectional geometries. In
some examples, the single capillary (or lumen thereof) may have any
specified cross-sectional dimension or set of dimensions. For
example, in some examples, the largest cross-sectional dimension of
the capillary lumen (e.g., the diameter, if the lumen is circular
in shape, or the diagonal, if the lumen is square or rectangular in
shape) may range from about 10 .mu.m to about 10 mm. The length of
the one or more capillaries used to fabricate the disclosed single
capillary flow cell devices or flow cell cartridges may range from
about 5 mm to about 5 cm or greater. Capillaries, in some examples,
have a gap height of about or exactly 50, 75, 100, 125, 150, 175,
200, 225, 250, 275, 300, 350, 400, or 500 um, or any value falling
within the range defined thereby.
[0138] Disclosed herein also are flow cell devices that comprise
one or more microfluidic chips and one or two fluidic adapters
affixed to one or both ends of the microfluidic chips, where the
microfluidic chip provides one or more fluid flow channels of
specified cross-sectional area and length, and where the fluidic
adapters are configured to mate with the microfluidic chip to
provide for convenient, interchangeable fluid connections with an
external fluid flow control system.
[0139] The microfluidic chip described herein includes one or more
microfluidic channels etched on the surface of the chip. The
microfluidic channels are defined as fluid conduits with at least
one minimum dimension from <1 nm to 1000 .mu.m. The microfluidic
channel system, fabricated on either a glass or silicon substrate,
has channel heights and widths on the order of <1 nm to 1000
.mu.m. The channel length can be in the micrometer range.
[0140] The capillaries or microfluidic chip used for constructing
the disclosed flow cell devices may be fabricated from any of a
variety of materials known to those of skill in the art including,
but not limited to, glass (e.g., borosilicate glass, soda lime
glass, etc.), fused silica (quartz), polymer (e.g., polystyrene
(PS), macroporous polystyrene (MPPS), polymethylmethacrylate
(PMMA), polycarbonate (PC), polypropylene (PP), polyethylene (PE),
high density polyethylene (HDPE), cyclic olefin polymers (COP),
cyclic olefin copolymers (COC), polyethylene terephthalate (PET),
polydimethylsiloxane (PDMS), etc.), polyetherimide (PEI) and
perfluoroelastomer (FFKM) as more chemically inert examples. PEI is
somewhere between polycarbonate and PEEK in terms of both cost and
compatibility. FFKM is also known as Kalrez.
[0141] In some examples, a flow cell device described herein (e.g.,
a microfluidic chip or capillary flow cell) is operatively coupled
to an imaging system described herein to capture or detect signals
of DNA bases for applications such as nucleic acid sequencing,
analyte capture and detection, and the like.
[0142] Oligonucleotide primers and adapter sequences: In general,
at least one layer of the one or more layers of low non-specific
binding material may comprise functional groups for covalently or
non-covalently attaching oligonucleotide adapter or primer
sequences, or the at least one layer may already comprise
covalently or non-covalently attached oligonucleotide adapter or
primer sequences at the time that it is deposited on the support
surface. In some instances, the oligonucleotides tethered to the
polymer molecules of at least one third layer may be distributed at
a plurality of depths throughout the layer.
[0143] One or more types of oligonucleotide primer may be attached
or tethered to the support surface. In some instances, the one or
more types of oligonucleotide adapters or primers may comprise
spacer sequences, adapter sequences for hybridization to
adapter-ligated template library nucleic acid sequences, forward
amplification primers, reverse amplification primers, sequencing
primers, or, molecular barcoding sequences, or any combination
thereof. In some instances, 1 primer or adapter sequence may be
tethered to at least one layer of the surface. In some instances,
at least 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 different
primer or adapter sequences may be tethered to at least one layer
of the surface.
[0144] In some instances, the tethered oligonucleotide adapter, or,
primer sequences, or a combination thereof may range in length from
about 10 nucleotides to about 100 nucleotides. In some instances,
the tethered oligonucleotide adapter, or, primer sequences, or a
combination thereof may be at least 10, at least 20, at least 30,
at least 40, at least 50, at least 60, at least 70, at least 80, at
least 90, or at least 100 nucleotides in length. In some instances,
the tethered oligonucleotide adapter, or, primer sequences, or a
combination thereof may be at most 100, at most 90, at most 80, at
most 70, at most 60, at most 50, at most 40, at most 30, at most
20, or at most 10 nucleotides in length. Any of the lower and upper
values described in this paragraph may be combined to form a range
included within the present disclosure, for example, in some
instances the length of the tethered oligonucleotide adapter, or,
primer sequences, or combination thereof may range from about 20
nucleotides to about 80 nucleotides. The length of the tethered
oligonucleotide adapter, or, primer sequences, or combination
thereof may have any value within this range, e.g., about 24
nucleotides.
[0145] In some instances, the tethered primer sequences may
comprise modifications designed to facilitate the specificity and
efficiency of nucleic acid amplification as performed on the
low-binding supports. For example, in some instances the primer may
comprise polymerase stop points such that the stretch of primer
sequence between the surface conjugation point and the modification
site is always in single-stranded form and functions as a loading
site for 5' to 3' helicases in some helicase-dependent isothermal
amplification methods. Other examples of primer modifications that
may be used to create polymerase stop points include, but are not
limited to, an insertion of a PEG chain into the backbone of the
primer between two nucleotides towards the 5' end, insertion of an
abasic nucleotide (e.g., a nucleotide that has neither a purine nor
a pyrimidine base), or a lesion site which can be bypassed by the
helicase.
[0146] As will be discussed further in the examples below, the
surface density of tethered primers on the support surface, or, the
spacing of the tethered primers away from the support surface
(e.g., by varying the length of a linker molecule used to tether
the primers to the surface), or a combination thereof, may be
varied in order to "tune" the support for optimal performance when
using a given amplification method. As noted below, adjusting the
surface density of tethered primers may impact the level of
specific, or, non-specific amplification, or a combination thereof,
observed on the support in a manner that varies according to the
amplification method selected. In some instances, the surface
density of tethered oligonucleotide primers may be varied by
adjusting the ratio of molecular components used to create the
support surface. For example, in an example where an
oligonucleotide primer--PEG conjugate is used to create the final
layer of a low-binding support, the ratio of the oligonucleotide
primer--PEG conjugate to a non-conjugated PEG molecule may be
varied. The resulting surface density of tethered primer molecules
may then be estimated or measured using any of a variety of
techniques. Examples include, but are not limited to, the use of
radioisotope labeling and counting methods, covalent coupling of a
cleavable molecule that comprises an optically-detectable tag
(e.g., a fluorescent tag) that may be cleaved from a support
surface of defined area, collected in a fixed volume of an
appropriate solvent, and then quantified by comparison of
fluorescence signals to that for a calibration solution of known
optical tag concentration, or using fluorescence imaging techniques
provided that care has been taken with the labeling reaction
conditions and image acquisition settings to ensure that the
fluorescence signals are linearly related to the number of
fluorophores on the surface (e.g., that there is no significant
self-quenching of the fluorophores on the surface).
[0147] In some instances, the resultant surface density of
oligonucleotide primers on the low binding support surfaces of the
present disclosure may range from about 1,000 primer molecules per
.mu.m.sup.2 to about 1,000,000 primer molecules per .mu.m.sup.2. In
some instances, the surface density of oligonucleotide primers may
be at least 1,000, at least 10,000, at least 100,000, or at least
1,000,000 molecules per .mu.m.sup.2. In some instances, the surface
density of oligonucleotide primers may be at most 1,000,000, at
most 100,000, at most 10,000, or at most 1,000 molecules per
.mu.m.sup.2. Any of the lower and upper values described in this
paragraph may be combined to form a range included within the
present disclosure, for example, in some instances the surface
density of primers may range from about 10,000 molecules per
.mu.m.sup.2 to about 100,000 molecules per .mu.m.sup.2. The surface
density of primer molecules may have any value within this range,
e.g., about 455,000 molecules per .mu.m.sup.2. In some instances,
the surface density of template library nucleic acid sequences
initially hybridized to adapter or primer sequences on the support
surface may be less than or equal to that indicated for the surface
density of tethered oligonucleotide primers. In some instances, the
surface density of clonally-amplified template library nucleic acid
sequences hybridized to adapter or primer sequences on the support
surface may span the same range as that indicated for the surface
density of tethered oligonucleotide primers.
[0148] Local densities as listed above do not preclude variation in
density across a surface, such that a surface may comprise a region
having an oligo density of, for example, 500,000 per .mu.m.sup.2,
while also comprising at least a second region having a
substantially different local density.
[0149] Imaging Systems. Imaging systems described herein are
utilized to detect hybridization between one or more sample nucleic
acid molecules and capture nucleic acid molecules coupled to a low
non-specific binding surface. In some examples, the imaging systems
comprise a camera. In some examples, the imaging systems comprise a
microscope, such as a fluorescence microscope. An inverted
fluorescence microscope in combination with a camera may be used to
capture an image of the low non-specific binding surface and
visualize hybridization between one or more sample nucleic acid
molecules and capture nucleic acid molecules. A non-limiting
example of an imaging system described herein is an Olympus IX83
microscope (Olympus Corp., Center Valley, Pa.) with a total
internal reflectance fluorescence (TIRF) objective (100.times., 1.5
NA, Olympus), a CCD camera (e.g., an Olympus EM-CCD monochrome
camera, Olympus XM-10 monochrome camera, or an Olympus DP80 color
and monochrome camera), an illumination source (e.g., an Olympus
100 W Hg lamp, an Olympus 75 W Xe lamp, or an Olympus U-HGLGPS
fluorescence light source), and excitation wavelengths of 532 nm or
635 nm. Dichroic mirrors were purchased from Semrock (IDEX Health
& Science, LLC, Rochester, N.Y.), e.g., 405, 488, 532, or 633
nm dichroic reflectors/beamsplitters, and band pass filters were
chosen as 532 LP or 645 LP concordant with the appropriate
excitation wavelength.
[0150] Computer Control Systems. The present disclosure provides
computer systems that are programmed or otherwise configured to
implement methods provided herein, such as, for example, methods
for nucleic sequencing, storing reference nucleic acid sequences,
conducting sequence analysis and/or comparing sample and reference
nucleic acid sequences as described herein. An example of such a
computer system is shown in FIG. 10. As shown in FIG. 10, the
computer system 1001 includes a central processing unit (CPU, also
"processor" and "computer processor" herein) 1005, which can be a
single core or multi core processor, or a plurality of processors
for parallel processing. The computer system 1001 also includes
memory or memory location 1010 (e.g., random-access memory,
read-only memory, flash memory), an electronic storage unit 1015
(e.g., hard disk), a communication interface 1020 (e.g., network
adapter) for communicating with one or more other systems, and
peripheral devices 1025, such as cache, other memory, data storage
and/or electronic display adapters. The memory 1010, storage unit
1015, interface 1020 and peripheral devices 1025 are in
communication with the CPU 1005 through a communication bus (solid
lines), such as a motherboard. The storage unit 1015 can be a data
storage unit (or data repository) for storing data. The computer
system 1001 can be operatively coupled to a computer network
("network") 1030 with the aid of the communication interface 1020.
The network 1030 can be the Internet, an internet and/or extranet,
or an intranet and/or extranet that is in communication with the
Internet. The network 1030 in some cases is a telecommunication
and/or data network. The network 1030 can include one or more
computer servers, which can enable distributed computing, such as
cloud computing. The network 1030, in some instances, with the aid
of the computer system 1001, can implement a peer-to-peer network,
which may enable devices coupled to the computer system 1001 to
behave as a client or a server.
[0151] The CPU 1005 can execute a sequence of machine-readable
instructions, which can be embodied in a program or software. The
instructions may be stored in a memory location, such as the memory
1010. Examples of operations performed by the CPU 1005 can include
fetch, decode, execute, and writeback.
[0152] The storage unit 1015 can store files, such as drivers,
libraries and saved programs. The storage unit 1015 can store user
data, e.g., user preferences and user programs. The computer system
1001 in some instances can include one or more additional data
storage units that are external to the computer system 1001, such
as located on a remote server that is in communication with the
computer system 1001 through an intranet or the Internet.
[0153] The computer system 1001 can communicate with one or more
remote computer systems through the network 1030. For instance, the
computer system 1001 can communicate with a remote computer system
of a user (e.g., operator). Examples of remote computer systems
include personal computers (e.g., portable PC), slate or tablet
PC's (e.g., Apple.RTM. iPad, Samsung.RTM. Galaxy Tab), telephones,
Smart phones (e.g., Apple.RTM. iPhone, Android-enabled device,
Blackberry.RTM.), or personal digital assistants. The user can
access the computer system 1001 via the network 1030.
[0154] Methods as described herein can be implemented by way of
machine (e.g., computer processor) executable code stored on an
electronic storage location of the computer system 1001, such as,
for example, on the memory 1010 or electronic storage unit 1015.
The machine executable or machine readable code can be provided in
the form of software. During use, the code can be executed by the
processor 1005. In some cases, the code can be retrieved from the
storage unit 1015 and stored on the memory 1010 for ready access by
the processor 1005. In some situations, the electronic storage unit
1015 can be precluded, and machine-executable instructions are
stored on memory 1010.
[0155] The code can be pre-compiled and configured for use with a
machine comprising a processer adapted to execute the code or can
be compiled during runtime. The code can be supplied in a
programming language that can be selected to enable the code to
execute in a pre-compiled or as-compiled fashion.
[0156] Aspects of the systems and methods provided herein, such as
the computer system 1001, can be embodied in programming. Various
aspects of the technology may be thought of as "products" or
"articles of manufacture" typically in the form of machine (or
processor) executable code and/or associated data that is carried
on or embodied in a type of machine readable medium.
Machine-executable code can be stored on an electronic storage
unit, such memory (e.g., read-only memory, random-access memory,
flash memory) or a hard disk. "Storage" type media can include any
or all of the tangible memory of the computers, processors or the
like, or associated modules thereof, such as various semiconductor
memories, tape drives, disk drives and the like, which may provide
non-transitory storage at any time for the software programming.
All, or portions, of the software may at times be communicated
through the Internet or various other telecommunication networks.
Such communications, for example, may enable loading of the
software from one computer or processor into another, for example,
from a management server or host computer into the computer
platform of an application server. Thus, another type of media that
may bear the software elements includes optical, electrical and
electromagnetic waves, such as those used across physical
interfaces between local devices, through wired and optical
landline networks and over various air-links. The physical elements
that carry such waves, such as wired or wireless links, optical
links or the like, also may be considered as media bearing the
software. As used herein, unless restricted to non-transitory,
tangible "storage" media, terms such as computer or machine
"readable medium" refer to any medium that participates in
providing instructions to a processor for execution.
[0157] Hence, a machine readable medium, such as
computer-executable code, may take many forms, including but not
limited to, a tangible storage medium, a carrier wave medium or
physical transmission medium. Non-volatile storage media include,
for example, optical or magnetic disks, such as any of the storage
devices in any computer(s) or the like, such as may be used to
implement the databases, etc. shown in the drawings. Volatile
storage media include, for example, dynamic memory, such as main
memory of such a computer platform. Tangible transmission media
include, for example, coaxial cables; copper wire and fiber optics,
including the wires that comprise a bus within a computer system.
Carrier-wave transmission media may take the form of electric or
electromagnetic signals, or acoustic or light waves such as those
generated during radio frequency (RF) and infrared (IR) data
communications. Common forms of computer-readable media therefore
include, for example: a floppy disk, a flexible disk, a hard disk,
magnetic tape, any other magnetic medium, a CD-ROM, DVD or DVD-ROM,
any other optical medium, punch cards paper tape, any other
physical storage medium with patterns of holes, a RAM, a ROM, a
PROM and EPROM, a FLASH-EPROM, any other memory chip or cartridge,
a carrier wave transporting data or instructions, cables or links
transporting such a carrier wave, or any other medium from which a
computer may read programming code and/or data. Many of these forms
of computer readable media may be involved in carrying one or more
sequences of one or more instructions to a processor for
execution.
[0158] The computer system 1001 can include, or be in communication
with, an electronic display 1035 that comprises a user interface
(UI) for providing, for example, an output or readout of a nucleic
acid sequencing instrument coupled to the computer system 1001.
Such readout can include a nucleic acid sequencing readout, such as
a sequence of nucleic acid bases that comprise a given nucleic acid
sample. The UI may also be used to display the results of an
analysis making use of such readout. Examples of UI's include,
without limitation, a graphical user interface (GUI) and web-based
user interface. The electronic display 1035 can be a computer
monitor, or a capacitive or resistive touchscreen.
Performance of Compositions and Systems
[0159] Improvements in hybridization rate: In some instances, the
use of the buffer formulations disclosed herein (optionally, used
in combination with a low non-specific binding surface) yield
relative hybridization rates that range from about 2.times. to
about 20.times. faster than that for a standard hybridization
protocol. In some instances, the relative hybridization rate may be
at least 2.times., at least 3.times., at least 4.times., at least
5.times., at least 6.times., at least 7.times., at least 8.times.,
at least 9.times., at least 10.times., at least 12.times., at least
14.times., at least 16.times., at least 18.times., or at least
20.times. that for a standard hybridization protocol.
[0160] The method and compositions described herein can help
shorten the time required for completing hybridization. In some
embodiments, the hybridization time can be in the range of about 1
seconds (s) to 2 hours (h), about 5 s to 1.5 h, about 15 s to 1 h,
or about 15 s to 0.5 h. In some embodiments, the hybridization time
can be in the range of about 15 s to 1 h. In some embodiments, the
hybridization time can be shorter than 15 s, 30 s, 1 minutes (min),
1.5 min, 2 min, 2.5 min, 3 min, 4 min, 5 min, 6 min, 7 min, 8 min,
9 min, 10 min, 15 min, 20 min, 25 min, 30 min, 40 min, 50 min, 60
min, 70 min, 80 min, 90 min, 100 min, 110 min, or 120 min. In some
embodiments, the hybridization time can be longer than 1 s, 5 s, 10
s, 15 s, 30 s, 1 min, 1.5 min, 2 min, 2.5 min, 3 min, 4 min, or 5
min.
[0161] The annealing methods described herein can significantly
shorten the annealing time. In some instances, at least 90% of the
target nucleic acid anneals to the surface bound nucleic acid in
less than or equal to about 15 s, 30 s, 1 min, 1.5 min, 2 min, 2.5
min, 3 min, 4 min, 5 min, 6 min, 7 min, 8 min, 9 min, 10 min, 15
min, 20 min, 25 min, 30 min, 40 min, 50 min, 60 min, 70 min, 80
min, 90 min, 100 min, 110 min, or 120 min. In some instances, at
least 80% of the target nucleic acid anneals to the surface bound
nucleic acid in less than or equal to about 15 s, 30 s, 1 min, 1.5
min, 2 min, 2.5 min, 3 min, 4 min, 5 min, 6 min, 7 min, 8 min, 9
min, 10 min, 15 min, 20 min, 25 min, 30 min, 40 min, 50 min, 60
min, 70 min, 80 min, 90 min, 100 min, 110 min, or 120 min. In some
instances, at least 90% of the target nucleic acid anneals to the
surface bound nucleic acid in greater than or equal to about 1 s, 5
s, 10 s, 15 s, 30 s, 1 min, 1.5 min, 2 min, 2.5 min, 3 min, 4 min,
or 5 min. In some instances, at least 90% of the target nucleic
acid anneals to the surface bound nucleic acid in the range of
about 10 s to about 1 hour, about 30 s to about 50 min, about 1 min
to about 50 min, or about 1 min to about 30 min. In some instances,
at least 90% of the target nucleic acid anneals to the surface
bound nucleic acid in between 2-25, 3-24, 4-23, 5-23, 6-22, 7-21,
8-20, 9-19, 10-18, 11-17, 12-16, or 13-15 min.
[0162] Improvements in hybridization efficiency: As used herein,
hybridization efficiency (or yield) is a measure of the percentage
of total available tethered adapter sequences on a solid surface,
primer sequences, or oligonucleotide sequences in general that are
hybridized to complementary sequences. In some instances, the use
of optimized buffer formulations disclosed herein (optionally, used
in combination with a low non-specific binding surface) yield
improved hybridization efficiency compared to that for a standard
hybridization protocol. In some instances, the hybridization
efficiency that may be achieved is better than 80%, 85%, 90%, 95%,
98%, or 99% in any of the hybridization reaction times specified
above.
[0163] The methods and compositions described herein can be used in
an isothermal annealing conditions. In some embodiments, the
methods described herein can eliminate the cooling required for
most hybridizations. In some embodiments, the annealing methods
described herein can be performed at a temperature in the range of
about 10.degree. C. to 95.degree. C., about 20.degree. C. to
80.degree. C., or about 30.degree. C. to 70.degree. C. In some
embodiments, the temperature can be lower than about 40.degree. C.,
50.degree. C., 60.degree. C., 70.degree. C., 80.degree. C., or
90.degree. C.
[0164] Improvements in hybridization specificity: Methods, systems,
compositions, and kits described herein provide for improved
hybridization specificity, as compared to, for example, a
hybridization reaction performed on a low-non-specific binding
surface described herein at 90 degrees Celsius for 5 minutes
followed by cooling for 120 minutes to reach a final temperature of
37 degrees Celsius in a buffer comprising saline-sodium citrate. In
some instances, the hybridization specificity that may be achieved
is better than 1 base mismatch in 10 hybridization events, 1 base
mismatch in 100 hybridization events, 1 base mismatch in 1,000
hybridization events, or 1 base mismatch in 10,000 hybridization
events. Hybridization specificity may be measured using techniques
described herein
[0165] In some examples, at least or about 70%, 80%, or 90% of the
sample nucleic acid molecules correctly hybridize to the capture
nucleic acid molecules (e.g., adapter sequences, primer sequences,
or oligonucleotide sequences) with a complementary sequence. In
some examples, more than 90% of the sample nucleic acid molecules
correctly hybridize to the capture nucleic acid molecules. In some
examples, between 90%-99% of the sample nucleic acid molecules
correctly hybridize to the capture nucleic acid molecules. In some
examples, 100% of the sample nucleic acid molecules correctly
hybridize to the capture nucleic acid molecules.
[0166] Hybridization specificity can be measured, by hybridizing
labeled (e.g., Cy3) complementary oligos to surface bound nucleic
acid molecules immobilized to the surface, dehybridizing and
collecting the hybridized oligos, measuring a fluorescent signal
from the collected oligos using a fluorescence plate reader at the
appropriate excitation and emission wavelengths (e.g., 532, peak
570/30). The results can be used to develop standard curves for
accurately measuring concentration. This assay can be repeated with
oligos that show varying degrees of complementarity and the
respective specificities.
[0167] Hybridization specificity as measured on the surface, may be
measured by dividing the nonspecific background counts (e.g.,
calculated using methods provided in example 3) by the nonspecific
probe hybridization-nonspecific background counts (also may be
calculated using methods in example 3). Calibration curves can be
built using the hybridization specificity measurements. Experiments
with oligos having varying degrees of complementarity can be used
to calculate respective specificities more accurately.
[0168] The specificity of a given nucleic acid probe, p, can be
quantified by the relative sensitivity when a p spot is exposed to
a perfectly matched target, t, or to a mismatch, m,
S e t S e m = c 50 0 m c 50 0 t = K t K m . ##EQU00002##
[0169] The specificity of the assay can be quantified by
considering the fraction of incorrectly hybridized probes,
P m . .times. P m = y x + y = c m .times. K m c m .times. K m + c t
.times. K t . ##EQU00003##
In this case, y=x(c.sub.m/c.sub.t)(K.sub.m/K.sub.t).
[0170] Improvements in hybridization sensitivity. "Hybridization
sensitivity" refers to a concentration range of sample (or target)
nucleic molecules in which hybridization occurs with a target
hybridization specificity. In some instances, the target
hybridization specificity is 90%, or more. In some instances, the
methods, systems, compositions, and kits described herein utilize
less than a 10 nanomolar concentration of sample nucleic acid
molecules to hybridize the sample nucleic acid molecules to capture
nucleic acid molecules with high specificity. In some instances,
between a 10 nanomolar and 50 picomolar concentration of sample
nucleic acid molecules is used. In some instances, between a 9
nanomolar and 100 picomolar concentration of sample nucleic acid
molecules is used. In some instances, between a 9 nanomolar and 150
picomolar concentration of sample nucleic acid molecules is used.
In some instances, between a 7 nanomolar and 200 picomolar
concentration of sample nucleic acid molecules is used. In some
instances, between a 6 nanomolar and 250 picomolar concentration of
sample nucleic acid molecules is used. In some instances, between a
5 nanomolar and 250 picomolar concentration of sample nucleic acid
molecules is used. In some instances, between a 4 nanomolar and 300
picomolar concentration of sample nucleic acid molecules is used.
In some instances, between a 3 nanomolar and 350 picomolar
concentration of sample nucleic acid molecules is used. In some
instances, between a 2 nanomolar and 400 picomolar concentration of
sample nucleic acid molecules is used. In some instances, between a
1 nanomolar and 500 picomolar concentration of sample nucleic acid
molecules is used. In some instances, less than or equal to about a
1 nanomolar concentration of sample nucleic acid molecules is used.
In some instances, less than or equal to about a 250 picomolar
concentration of sample nucleic acid molecules is used. In some
instances, less than or equal to about a 200 picomolar
concentration of a sample nucleic acid molecules is used. In some
instances, less than or equal to about a 150 picomolar
concentration of sample nucleic acid molecules is used. In some
instances, less than or equal to about a 100 picomolar
concentration of sample nucleic acid molecules is used. In some
instances, less than or equal to about a 50 picomolar concentration
of sample nucleic acid molecules is used.
[0171] In some instances, the hybridization sensitivity is
calculated using the International Union of Pure and Applied
Chemistry (IUPAC) analytical techniques, which identify the
sensitivity, S.sub.e, with the slope of the calibration curve. The
calibration curve describes the measured response, R, to a target
concentration, c.sub.t, R(c.sub.t), and S.sub.e=dR/dc.sub.t.
The quantitative resolution of the assay, Act, is then specified by
.DELTA.c.sub.t=.di-elect cons..sub.r(c.sub.t)/S.sub.e(c.sub.t),
where .di-elect cons..sub.r is the measurement error as given by
its standard deviation. The detection limit, the lowest detectable
c.sub.t, is determined by .DELTA.c.sub.t(c.sub.t=0), because when
the concentration c.sub.t is lower than .DELTA.c.sub.t(c.sub.t=0),
the error is larger than the signal; and assuming that R(ct) is
proportional to the equilibrium hybridization fraction at the
surface, x; i.e., R(ct)=.kappa.x+const where .kappa. is a constant.
This assumption is justified when the following conditions are
fulfilled: (1) nonspecific adsorption is negligible and R is due
only to hybridization at the surface; (2) the duration of the
experiment is sufficiently long to allow the hybridization to reach
equilibrium; and (3), the measured signal depends linearly on the
amount of oligonucleotides at the surface.
Nucleic Acid Sequencing Applications
[0172] Nucleic acid sequencing is among the many applications for
which the methods, compositions, systems, and kits described herein
may be useful. Referring to FIG. 2, the methods disclosed herein,
in some embodiments, comprise preparing a library of sample nucleic
acid molecules for sequencing, hybridizing the library of sample
nucleic acids to nucleic acid molecules coupled to a low
non-specific binding surface in the presence of the hybridizing
compositions described herein, amplifying the library of sample
nucleic acids in situ, optionally linearizing the amplified sample
nucleic acids in situ, de-hybridizing the linearized and amplified
sample nucleic acids from the nucleic acid molecules coupled to the
low non-specific binding surface, hybridizing a primer sequence to
the sample nucleic acids, and sequencing the sample nucleic
acids.
[0173] FIG. 6 provides an example of a workflow of the methods
described herein, wherein a library of sample nucleic acid
molecules is prepared 601, for example by a split ligation
protocol, the library of sample nucleic acid molecules is
hybridized to nucleic acid molecules coupled to a low non-specific
binding surface in the presence of a hybridization composition
described herein 602, hybridization of the sample nucleic acid
molecules to the nucleic acid molecules coupled to the low
non-specific binding surface occurs 603, sequencing primers are
hybridized to complementary primer binding sequences on sample
nucleic acids 604, and sequencing of the sample nucleic acids is
performed 605.
[0174] FIG. 7 provides an example sequencing workflow of the
methods described herein, wherein a labeled deoxyribonucleotide
triphosphate (dNTP) binds to the sample nucleic acid molecule to
determine the identity of the complementary nucleotide in the
nucleic acid sequence of the sample nucleic acid molecule 701. In
some instances, the dNTP is labeled with a fluorophore (e.g., Cy3),
either directly or by interaction with a labeled detection reagent.
The surface is optionally washed, to remove the unbound labeled
dNTP. The surface is imaged to detect the presence of the labeled
dNTP 702. The labeled dNTP is unbound from the sample nucleic acid
molecule, and a blocked unlabeled dNTP is incorporated into the
sample nucleic acid molecule 703. The blocked unlabeled nucleotide
is cleaved 704. Steps 701-704 are repeated for the next nucleotide
in the sample nucleic acid molecule 705.
[0175] The methods, compositions, systems, and kits described
herein provide at least the following advantages, particularly in a
nucleic acid sequencing process: (i) decreased fluidic wash times
(due to reduced non-specific binding, and thus faster sequencing
cycle times), (ii) decreased imaging times (and thus faster
turnaround times for assay readout and sequencing cycles), (iii)
decreased overall work flow time requirements (due to decreased
cycle times), (iv) decreased detection instrumentation costs (due
to the improvements in contrast-to-noise ratio), (v) improved
readout (base-calling) accuracy (due to improvements in
contrast-to-noise ratio), (vi) improved reagent stability and
decreased reagent usage requirements (and thus reduced reagents
costs), and (vii) fewer run-time failures due to nucleic acid
amplification failures.
[0176] Methods of Analyzing a Target Nucleic Acid Utilizing
Multivalent Binding or Incorporation Compositions. Disclosed herein
are multivalent binding or incorporation compositions and uses of
said compositions in analyzing nucleic acid molecules, including in
sequencing or other bioassay applications. An increase in binding
or incorporation of a nucleotide to an enzyme (e.g., polymerase) or
an enzyme complex can be affected by increasing the effective
concentration of the nucleotide. The increase can be achieved by
increasing the concentration of the nucleotide in free solution, or
by increasing the amount of the nucleotide in proximity to the
relevant binding or incorporation site. The increase can also be
achieved by physically restricting a number of nucleotides to a
limited volume, thus resulting in a local increase in
concentration, and, resultingly, in the nucleotides binding or
incorporating to a binding or incorporation site with a higher
apparent avidity than would be observed with unconjugated,
untethered, or otherwise unrestricted individual nucleotides. One
non-limiting mechanism of effecting such a restriction is a
multivalent binding or incorporation composition in which multiple
nucleotides are bound to a particle such as a polymer, a branched
polymer, a dendrimer, a micelle, a liposome, a microparticle, a
nanoparticle, a quantum dot, or other suitable particle known in
the art.
[0177] The multivalent binding or incorporation composition
disclosed herein can include at least one particle-nucleotide
conjugate, wherein the particle-nucleotide conjugate comprises a
plurality of copies of the same nucleotide attached to a particle.
When the nucleotide is complementary to the target nucleic acid,
the particle-nucleotide conjugate forms a binding or incorporation
complex with the polymerase and the target nucleic acid, and the
binding or incorporation complex exhibits increased stability and
longer persistence time than the binding or incorporation complex
formed using a single unconjugated or untethered nucleotide. Each
of the nucleotide moieties of the multivalent binding composition
may bind to a complementary N+1 nucleotide of a primed target
nucleic acid molecule, thereby forming a multivalent binding
complex comprising two or more target nucleic acid molecules, two
or more polymerase (or other enzyme) molecules, and the multivalent
binding composition (e.g., the polymer-nucleotide conjugate). Each
of the nucleotide moieties of the multivalent binding composition
may bind to a complementary N nucleotide of a primed target nucleic
acid molecule, thereby forming a multivalent binding complex
comprising two or more target nucleic acid molecules, two or more
polymerase (or other enzyme) molecules, and the multivalent binding
composition (e.g., the polymer-nucleotide conjugate). From this
bound complex the nucleotide can interrogate the complementary base
prior to incorporation of a modified reversibly blocked nucleotide
that elongates the replicating strand by 1 base. In addition, it is
possible to imagine interrogation of the N nucleotide with a bound
complex, stepping forward with a reversibly terminated nucleotide,
and subsequently probing the N+1 base to pre- and post-deblocking.
In this way you could perform error checking and improve the
overall accuracy of base-calling by reading the interrogated base
twice. The important discriminating factor from traditional methods
is the binding is used to interrogate the matched base, while the
stepping or incorporation step is used only to move forward on the
elongating strand.
[0178] The multivalent binding or incorporation composition
described herein can be used to localize detectable signals to
active regions of biochemical interactions, such as sites of
protein-nucleic acid interactions, nucleic acid hybridization
reactions, or enzymatic reactions, such as polymerase reactions.
For instance, the multivalent binding or incorporation composition
described herein can be utilized to identify sites of base
incorporation in elongating nucleic acid chains during polymerase
reactions and to provide base discrimination for sequencing and
array-based applications. The increased binding or incorporation
between the target nucleic acid and the nucleotide in the
multivalent binding or incorporation composition, when the
nucleotide is complementary to the target nucleic acid, provides
enhanced signal that greatly improve base call accuracy and
shortens imaging time.
[0179] In addition, the use of multivalent binding composition
described herein allows sequencing signals from a given sequence to
originate within cluster regions containing multiple copies of the
target sequence. Sequencing methods incorporating multiple copies
of a target sequence are advantageous in that signals can be
amplified due to the presence of multiple simultaneous sequencing
reactions within the defined region, each providing its own signal.
The presence of multiple signals within a defined area also reduces
the impact of any single skipped cycle, due to the fact that the
signal from a large number of correct base calls can overwhelm the
signal from a smaller number of skipped or incorrect base calls,
therefore providing methods for reducing phasing errors and/or
improving read length in sequencing reactions.
[0180] The multivalent binding compositions and their uses
disclosed herein lead to one or more of: (i) a stronger signal for
better base-calling accuracy compared to nucleic acid amplification
and sequencing methodologies using, for example, protic solvents;
ii) greater discrimination of sequence-specific signal from
background signals; (iii) reduced requirements for the amount of
starting material necessary, (iv) increased sequencing rate and
shortened sequencing time; (v) reducing phasing errors, and (vi)
improving read length in sequencing reactions.
[0181] In some examples, the target nucleic acid refers to a target
nucleic acid sample having one or more nucleic acid molecules. In
some examples, the target nucleic acid includes a plurality of
nucleic acid molecules. In some examples, the target nucleic acid
includes two or more nucleic acid molecules. In some examples, the
target nucleic acid includes two or more nucleic acid molecules
having the same sequences.
Sequencing Target Nucleic Acid
[0182] FIG. 12A-12H illustrate a non-limiting example of a method
in which the multivalent binding composition is used for sequencing
a target nuclei acid. As shown in FIG. 12A, the target nucleic acid
1202 can be tethered to a solid support surface 1201. The target
nucleic acid can be attached to the surface either directly or
indirectly. Although not shown in FIG. 12A, the target nucleic acid
1202 can be hybridized to an adapter, which is attached to the
surface through a covalent or noncovalent bond. When one or more
adapters are used to attach the target nucleic acid to the surface,
the target surface can comprise a fragment that is complementary to
the adapter and thus hybridize to the adaptor. In some instances,
one adapter sequence may be tethered to the surface. In some
instances, a plurality of adapter sequences may be tethered to the
surface. In some instances, the target nucleic acid 1202 can also
be attached directly to the solid-support surface without the use
of an adapter. The solid support can be a low non-specific binding
surface.
[0183] In FIG. 12B, after the initial step of attaching the target
nucleic acid to the surface of a solid support surface (e.g.,
through hybridization to adapters), the target nucleic acid is then
clonally-amplified to form clusters of amplified nucleic acids.
When the target nucleic acid is attached to the surface through an
adapter, the surface density of clonally-amplified nucleic acid
sequences hybridized to adapter on the support surface may span the
same range as the surface density of tethered adapters (or
primers). The clonal amplification may be performed using a
polymerase chain reaction (PCR), multiple displacement
amplification (MDA), transcription-mediated amplification (TMA),
nucleic acid sequence-based amplification (NASBA), strand
displacement amplification (SDA), real-time SDA, bridge
amplification, isothermal bridge amplification, rolling circle
amplification, circle-to-circle amplification, helicase-dependent
amplification, recombinase-dependent amplification, single-stranded
binding (SSB) protein-dependent amplification, or any combination
thereof.
[0184] FIG. 12C illustrates a non-limiting step of annealing a
primer 1203 to the target nucleic acid 1202 to form a primed target
nucleic acid 1204. FIG. 12B only shows one primer being used in the
annealing step, but more than one primer can be used depending on
the types of target nucleic acid. In some instances, the adapter
that is used to attach the target nucleic acid to the surface has
the same sequence as the primer used to prepare the primed target
nucleic acid. The primer may comprise forward amplification
primers, reverse amplification primers, sequencing primers, and/or
molecular barcoding sequences, or any combination thereof. In some
instances, one primer sequence may be used in the hybridization
step. In some instances, a plurality of different primer sequences
may be used in the hybridization step.
[0185] As shown in FIG. 12D, the primed target nucleic acid 1204 is
combined with a multivalent binding or incorporation composition
and a polymerase 1206 to form a binding or incorporation complex.
The non-limiting example of a multivalent binding or incorporation
composition in FIG. 12D comprises four particle-nucleotide
conjugates 1205a, 1205b, 1205c, and 1205d. Each particle-nucleotide
conjugate has multiple copies of a nucleotide attached to the
particle, and the four particle-nucleotide conjugates cover four
types of nucleotide respectively. The particle-nucleotide conjugate
having a nucleotide that is complementary to the next base on the
primed target nucleic acid will form a binding or incorporation
complex with the polymerase and the target nucleic acid. In some
instances, the multivalent binding or incorporation composition may
include one, two or three particle-nucleotide conjugates. In some
instances, each different type of particle-nucleotide conjugate can
be labeled with a separate label. In some instances, three of four
types of nucleotide conjugates can be labeled, with a fourth either
unlabeled or conjugated to an undetectable label. In some
instances, 1, 2, 3, or 4 particle-nucleotide conjugates can be
labeled, either with the same label, or each with a label
corresponding to the identity of its conjugated nucleotide, with,
respectively, 3, 2, 1, or no particle-nucleotide conjugates that
may be either left unlabeled or conjugated to an undetectable
label. In some examples, detection of a polymerase complex
incorporating a particle-nucleotide conjugate may be carried out
using four-color detection, such that conjugates corresponding to
all four nucleotides are present in a sample, each conjugate having
a separate label corresponding to the nucleotide conjugated
thereto. In some examples, the four particle-nucleotide conjugates
may be exposed to or contacted with the target nucleic acid at the
same time; in some other examples, the four particle-nucleotide
conjugates may be exposed to or contacted with the target nucleic
acid sequentially, either individually, or in groups of two or
three. In some examples, detection of a polymerase complex
incorporating a particle-nucleotide conjugate may be carried out
using three-color detection, such that conjugates corresponding to
three of the four nucleotides are present in a sample, with three
conjugates having a separate label corresponding to the nucleotide
conjugated thereto and one conjugate having no label or being
conjugated to an undetectable label. In some examples, only three
types of conjugates are provided, such that conjugates
corresponding to three of the four nucleotides are present in a
sample, with three conjugates having a separate label corresponding
to the nucleotide conjugated thereto and one conjugate being
absent. In some examples, the identity of nucleotides corresponding
to an unlabeled or absent nucleotide conjugate can be determined
with respect to the location and/or identity of "dark" spots or
locations of known target nucleic acids showing no fluorescence
signal. In some methods provided in the present disclosure, the
detection of the binding or incorporation complex is performed in
the absence of unbound or solution-borne polymer nucleotide
conjugates.
[0186] In some examples, where three of the four
particle-nucleotide conjugates are labeled, or where only three of
the four particle-nucleotide conjugates are present, the identity
of the nucleotide corresponding to the unlabeled or absent
conjugate may be established by the absence of a signal or by
monitoring of the presence of unlabeled complexes such as by the
identification of "dark" spots or unlabeled regions in a sequencing
reaction. In some examples, detection of a polymerase complex
incorporating a particle-nucleotide conjugate may be carried out
using two-color detection, such that conjugates corresponding to
two of the four nucleotides are present in a sample, with two
conjugates having a separate label corresponding to the nucleotide
conjugated thereto and two conjugates having no label or being
conjugated to an undetectable label. In some examples, only two of
the four particle-nucleotide conjugates are labeled. In some
examples, where two of the four particle-nucleotide conjugates are
labeled, the identity of the nucleotide corresponding to the
unlabeled conjugate or conjugates may be established by the absence
of a signal or by monitoring of the presence of unlabeled complexes
such as by the identification of "dark" spots or unlabeled regions
in a sequencing reaction. In some examples, where two of the four
particle-nucleotide conjugates are labeled, the four
particle-nucleotide conjugates may be exposed to, or contacted
with, the target nucleic acid sequentially, either individually, or
in groups of two or three. In some examples, two of the four
particle-nucleotide conjugates may share a common label, and the
four particle-nucleotide conjugates may be exposed to or contacted
with the target nucleic acid sequentially, either individually, or
in groups of two or three, wherein each contacting step shows the
distinction between two or more different bases, such that after
two, three, four, or more such contacting steps the identities of
all unknown bases have been determined.
[0187] FIG. 12E illustrates the images captured on the surface
after the binding or incorporation complex is formed between the
polymerase, the target nucleic acid, and the particle-nucleotide
conjugate having a nucleotide commentary to the next base of the
primed target nucleic acid. The captured image includes four
binding or incorporation complexes 1207a, 1207b, 1207c, and 1207d
formed on the surface, and each binding or incorporation complex
has a different nucleotide which can be distinguished based on the
label (e.g., fluorescence emission color) on the
particle-nucleotide conjugate. Because use of the
particle-nucleotide conjugate allows binding or incorporation
signals from a given sequence to originate within cluster regions
containing multiple copies of the target sequence, the sequencing
signals are greatly enhanced. Although FIG. 12E involves four
particle-nucleotide conjugates, each having a different type of
nucleotide, some methods can use one, two, or three
particle-nucleotide conjugates, each having a different type of
nucleotide and label. In some examples, each different type of
particle-nucleotide conjugate can be labeled either with the same
label, or each with a label corresponding to the identity of its
conjugated nucleotide. In some examples, three of four types of
nucleotide conjugates can be labeled, with a fourth either
unlabeled or conjugated to an undetectable label. In some examples,
1, 2, 3, or 4 particle-nucleotide conjugates can be labeled with a
separate label, with, respectively, 3, 2, 1, or no
particle-nucleotide conjugates either unlabeled or conjugated to an
undetectable label. In some examples, a detection step can comprise
simultaneous and/or serial excitation of up to 4 different
excitation wavelengths, such as wherein the fluorescence imaging is
carried out by detecting single and/or multiple fluorescence
emission bands that uniquely classify each of the possible base
pairings (A, G, C, or T). In some examples, four different nucleic
acid binding or incorporation compositions, each comprising a
different nucleotide or nucleotide analog, may be used to determine
the identity of the terminal nucleotide, wherein one of the four
different nucleic acid binding or incorporation compositions is
labeled with a first fluorophore, one is labeled with a second
fluorophore, one is labeled with both the first and second
fluorophore, and one is not labeled, and wherein the detecting step
comprises simultaneous excitation at a first excitation wavelength
and a second excitation wavelength and images are acquired at a
first fluorescence emission wavelength and a second fluorescence
emission wavelength.
[0188] When the multivalent binding or incorporation composition is
used in replacement of single unconjugated or untethered
nucleotides to form a binding or incorporation complex with the
polymerase and the primed target nucleic acid, the local
concentration of the nucleotide is increased many-fold, which in
turn enhances the signal intensity. The formed binding or
incorporation complex also has a longer persistence time which in
turn helps shorten the imaging step. The high signal intensity
results from the high binding or incorporation avidity of the
polymer nucleotide conjugate (which may also comprise multiple
fluorophores or other labels) which thus forms a complex which
remains stable for the entire binding or incorporation and imaging
step. The strong binding or incorporation between the polymerase,
the primed target strand, and the polymer-nucleotide or nucleotide
analog conjugate also means that the multivalent binding or
incorporation complex thus formed will remain stable during washing
steps, and the signal intensity will remain high when other
reaction mixture components and unmatched nucleotide analogs are
washed away. After the imaging step, the binding or incorporation
complex can be destabilized (e.g., by changing the buffer
composition) and the primed target nucleic acid can then be
extended for one base.
[0189] The sequencing method may further comprise incorporating the
N+1 or terminal nucleotide into the primed strand as shown in FIG.
12F. In FIG. 12F, the primer strand of the primed target nucleic
acid 1208 can be extended for one base to form an extended nucleic
acid 1209. The extension step can occur after or concurrently with
the destabilization of the multivalent binding or incorporation
complex. The primed target nucleic acid 1208 can be extended using
a complementary nucleotide that is attached to the particle in the
particle-nucleotide conjugate or using an unconjugated or
untethered free nucleotide that is provided after the multivalent
binding or incorporation composition has been removed.
[0190] After the extension step, the contacting step as shown in
FIG. 12G can be performed again to form binding or incorporation
complexes and imitate the next sequencing cycle. The contacting,
detecting, and extension steps can be repeated for one or more
cycles, thereby determining the sequence of the target nucleic acid
molecule. For example, FIG. 12H illustrates the surface images
obtained after performing multiple sequencing cycles, and the
images can then be processed to determine the sequences of the
target nucleic acid molecules.
[0191] The extension of the primed target nucleic acid may be
prevented or inhibited due to a blocked nucleotide on the strand or
the use of polymerase that is catalytically inactive. When the
nucleotide in the polymer-nucleotide conjugate has a blocking group
that prevents the extension of the nucleic acid, incorporation of a
nucleotide may be achieved by the removal of a blocking group from
said nucleotide (such as by detachment of said nucleotide from its
polymer, branched polymer, dendrimer, particle, or the like). When
the extension of the primed target nucleic acid is inhibited due to
the use of polymerase that is catalytically inactive, incorporation
of a nucleotide may be achieved by the provision of a cofactor or
activator such as a metal ion.
[0192] Also disclosed herein are systems configured for performing
any of the disclosed nucleic acid sequencing or nucleic acid
analysis methods. The system may comprise a fluid flow controller
and/or fluid dispensing system configured to sequentially and
iteratively contact the primed target nucleic acid molecules
attached to a solid support with the disclosed polymerase and
multivalent binding or incorporation compositions and/or reagents.
The contacting may be performed within one or more flow cells. In
some instances, said flow cells may be fixed components of the
system. In some instances, said flow cells may be removable and/or
disposable components of the system.
[0193] The sequencing system may include an imaging module, i.e.,
one or more light sources, one or more optical components, and one
or more image sensors for imaging and detection of binding or
incorporation of the disclosed nucleic acid binding or
incorporation compositions to target nucleic acid molecules
tethered to a solid support or the interior of a flow cell. The
disclosed compositions, reagents, and methods may be used for any
of a variety of nucleic acid sequencing and analysis applications.
Examples include, but are not limited to, DNA sequencing, RNA
sequencing, whole genome sequencing, targeted sequencing, exome
sequencing, genotyping, and the like.
[0194] The sequencing system may also include computer control
systems that are programmed to implement methods of the disclosure.
The computer system is programmed or otherwise configured to
implement methods of the disclosure including, for example, nucleic
acid sequencing methods, interpretation of nucleic acid sequencing
data and analysis of cellular nucleic acids, such as RNA (e.g.,
mRNA), or characterization of cells from sequencing data. The
computer system can be an electronic device of a user or a computer
system that is remotely located with respect to the electronic
device. The electronic device can be a mobile electronic
device.
[0195] FIG. 13 is a flowchart outlining a non-limiting example of
the steps in sequencing a target nucleic acid. 1301 describes a
step of attaching target library sequences to a solid support
surface by hybridizing the target nucleic acid molecules to
complementary adapters on a substrate surface. The target nucleic
acid molecules can be single stranded or partially double stranded.
Prior to 1301, the nucleic acid molecules in the target library may
have been prepared to contain fragments complementary to the
adaptor sequences through ligation or other methods. 1302 describes
the step of clonal amplification to generate clusters of target
nucleic acid molecules on the surface. 1303 describes hybridizing
sequencing primers to complementary primer binding or incorporation
sequences on the target nucleic acid to form the primed target
nucleic acid. 1304 describes combining the polymerase, the
multivalent binding or incorporation composition, which contains
labeled (e.g., fluorescently-labeled) particle-nucleotide
conjugates, and the primed target nucleic acid. 1304 may also
include a step of washing or removing the unbound reagents
including polymerase and particle-nucleotide conjugate.
[0196] Again referring to FIG. 13, when the nucleotide on the
particle-nucleotide conjugate is complementary to the next base of
the primed target nucleic acid (1305), the particle-nucleotide
conjugate, polymerase, and primed target nucleic acid form a
ternary binding or incorporation complex, which can be detected by
detection methods (e.g., florescence imaging) compatible with the
label on the particle-nucleotide conjugate. 1305 can also include
measuring the persistence time of the ternary binding or
incorporation complex. In 1306, the binding or incorporation
complex is destabilized to remove the binding or incorporation of
the particle-nucleotide conjugate and polymerase. The dissociation
can be achieved by placing the binding or incorporation complex in
a condition (e.g., adding Strontium ions) that will change the
conformation of the polymerase and destabilize the binding or
incorporation. 1306 may also include a step of washing or removing
the dissociated particle-nucleotide conjugate and/or polymerase.
1307 describes the step of extending the primed strand of the
primed target nucleic acid by a single base addition reaction.
After the single base extension, steps 1304, 1305, 1306, and 1307
can be repeated in multiple cycles to determine the sequences of
the target nucleic acid.
[0197] FIG. 14 is another flowchart outlining a non-limiting
example of the steps in sequencing a target nucleic acid, which
includes cleaving a nucleotide from the particle-nucleotide
conjugate and incorporating the cleaved nucleotide. 1401 describes
a step of attaching target library sequences to a solid support
surface by hybridizing the target nucleic acid molecules to
complementary adapters on substrate surface. The target nucleic
acid molecules can be single stranded or partially double stranded.
Prior to 1401, the nucleic acid molecules in the target library may
have been prepared to contain fragments complementary to the
adaptor sequences through ligation or other methods. 1402 describes
the step of clonal amplification to generate clusters of target
nucleic acid molecules on the surface. 1403 describes hybridizing
sequencing primers to complementary primer binding or incorporation
sequences on the target nucleic acid to form the primed target
nucleic acid. 1404 describes combining the polymerase, the
multivalent binding or incorporation composition, which contains
labeled (e.g., fluorescently-labeled) particle-nucleotide
conjugates, and the primed target nucleic acid. In the
particle-nucleotide conjugates, the nucleotides are attached to the
particle through chemical bonds or interactions that can be later
severed. 1404 may also include a step of washing or removing the
unbound reagents including polymerase and particle-nucleotide
conjugate.
[0198] Again referring to FIG. 14, when the nucleotide on the
particle-nucleotide conjugate is complementary to the next base of
the primed target nucleic acid (1405), the particle-nucleotide
conjugate, polymerase, and primed target nucleic acid form a
ternary binding or incorporation complex, which can be detected by
detection methods (e.g., florescence imaging) compatible with the
label on the particle-nucleotide conjugate. 1405 can also include
measuring the persistence time of the ternary binding or
incorporation complex. In 1406, the polymerase is placed in a
condition that would make it catalytically active to incorporate a
nucleotide. The condition can include exposing the polymerase to Mg
or Mn ions in the reaction solution. The nucleotide that is bound
to the polymerase and the primed target nucleic acid is then
cleaved from the particle and then incorporated into the primed
strand of the primed target nucleic acid. The binding or
incorporation complex is destabilized. 1406 may also include a step
of washing or removing the dissociated particle-nucleotide
conjugate and/or polymerase. After the extension, steps 1404, 1405,
and 1406 can be repeated in multiple cycles to determine the
sequences of the target nucleic acid.
[0199] Detecting Target Nucleic Acid Molecules. FIGS. 15A-15B
illustrate one exemplified method in which the multivalent binding
or incorporation composition is used for detecting a target nucleic
acid. As shown in FIG. 15A, the polymer-nucleotide conjugate 1501
is placed in contact with polymerase 1506, a first nucleic acid
molecule 1504, and a second nucleic acid molecule 1505. The
polymer-nucleotide conjugate 1501 has multiple polymer branches
radiating from the core, and some branches are attached to a
nucleotide or oligonucleotide 1502, and some branches are attached
to a label 1503. When the nucleotide or oligonucleotide 1502 on the
polymer-nucleotide conjugate 1501 is complementary to at least a
fraction of the first nucleic acid 1504, a multivalent binding or
incorporation complex is formed as shown in FIG. 15B, and the
strong binding or incorporation signal can help detect target
nucleic acid with sequences complementary or partially
complementary to the nucleotide or oligonucleotide on the
polymer-nucleotide conjugate. In some instances, at least one of
the polymerase, nucleic acid molecules, and polymer-nucleotide
conjugates is attached to a solid support.
[0200] The multivalent binding or incorporation composition
described herein can be used in a method of detecting a target
nucleic acid in a sample. Also disclosed herein are systems
configured for performing any of the disclosed nucleic acid
analysis methods. The systems may comprise a fluid flow controller
and/or fluid dispensing system configured to sequentially and
iteratively contact the nucleic acid molecules with the disclosed
polymerase and multivalent binding or incorporation compositions
and/or reagents. The contacting may be performed within one or more
flow cells. In some instances, said flow cells may be fixed
components of the system. In some instances, said flow cells may be
removable and/or disposable components of the system. The system
may also include a cartridge comprising a sample collection unit
and an assay assembly, wherein the sample collection unit is
configured to collect a sample, and wherein the assay assembly
comprises at least one reaction site containing a multivalent
binding or incorporation composition adapted to interact with said
analyte, allowing the predetermined portion of sample to react with
assay reagents contained within the assay assembly to yield a
signal indicative of the presence of the analyte in the sample, and
detecting the signal generated from the analyte.
[0201] Multivalent Binding or incorporation Composition. The
present disclosure relates to multivalent binding or incorporation
compositions having a plurality of nucleotides conjugated to a
particle (e.g., a polymer, branched polymer, dendrimer, or
equivalent structure). Contacting the multivalent binding or
incorporation composition with a polymerase and multiple copies of
a primed target nucleic acid may result in the formation of a
ternary complex which may be detected and in turn achieve a more
accurate determination of the bases of the target nucleic acid.
[0202] When the multivalent binding or incorporation composition is
used in replacement of a single unconjugated or untethered
nucleotide to form a complex with the polymerase and one or more
copies of the target nucleic acid, the local concentration of the
nucleotide as well as the binding avidity of the complex (in the
case that a complex comprising two or more target nucleic acid
molecules is formed) is increased many fold, which in turn enhances
the signal intensity, particularly the correct signal versus
mismatch. The multivalent binding or incorporation composition
described herein can include at least one particle-nucleotide
conjugate (each particle-nucleotide conjugate comprising multiple
copies of a single nucleotide moiety) for interacting with the
target nucleic acid. The multivalent composition can also include
two, three, or four different particle-nucleotide conjugates, each
having a different nucleotide conjugated to the particle.
[0203] The multivalent binding or incorporation composition can
comprise 1, 2, 3, 4, or more types of particle-nucleotide
conjugates, wherein each particle-nucleotide conjugate comprises a
different type of nucleotide. A first type of the
particle-nucleotide conjugate can comprise a nucleotide selected
from the group consisting of ATP, ADP, AMP, dATP, dADP, and dAMP. A
second type of the particle-nucleotide conjugate can comprise a
nucleotide selected from the group consisting of TTP, TDP, TMP,
dTTP, dTDP, dTMP, UTP, UDP, UMP, dUTP, dUDP, and dUMP. A third type
of the particle-nucleotide conjugate can comprise a nucleotide
selected from the group consisting of CTP, CDP, CMP, dCTP, dCDP,
and dCMP. A fourth type of the particle-nucleotide conjugate can
comprise a nucleotide selected from the group consisting of GTP,
GDP, GMP, dGTP, dGDP, and dGMP. In some instances, each
particle-nucleotide conjugate comprises a single type of nucleotide
respectively corresponding to one or more nucleotides selected from
the group consisting of ATP, ADP, AMP, dATP, dADP, dAMP TTP, TDP,
TMP, dTTP, dTDP, dTMP, UTP, UDP, UMP, dUTP, dUDP, dUMP, CTP, CDP,
dCTP, dCDP, dCMP, GTP, GDP, GMP, dGTP, dGDP, and dGMP. Each
multivalent binding or incorporation composition may further
comprise one or more labels corresponding to the particular
nucleotide conjugated to each respective conjugate. Non-limiting
examples of labels include fluorescent labels, colorimetric labels,
electrochemical labels (such as, for example, glucose or other
reducing sugars, or thiols or other redox active moieties),
luminescent labels, chemiluminescent labels, spin labels,
radioactive labels, steric labels, affinity tags, or the like.
[0204] Particle-Nucleotide Conjugate. In a particle-nucleotide
conjugate, multiple copies of the same nucleotide may be covalently
bound to or noncovalently bound to the particle. Examples of the
particle can include a branched polymer; a dendrimer; a cross
linked polymer particle such as an agarose, polyacrylamide,
acrylate, methacrylate, cyanoacrylate, methyl methacrylate
particle; a glass particle; a ceramic particle; a metal particle; a
quantum dot; a liposome; an emulsion particle, or any other
particle (e.g, nanoparticles, microparticles, or the like) known in
the art. In one example, the particle is a branched polymer.
[0205] In some instances, the particle-nucleotide conjugate (e.g.,
a polymer-nucleotide conjugate) may comprise 1, 2, 3, 4, 5, 6, 7,
8, 9, 10, or more than 10 copies of a nucleotide, nucleotide
analog, nucleoside, or nucleoside analog tethered to the
particle.
[0206] The nucleotide can be linked to the particle through a
linker, and the nucleotide can be attached to one end or location
of the polymer. The nucleotide can be conjugated to the particle
through the 5' end of the nucleotide. In some particle-nucleotide
conjugates, one nucleotide is attached to one end or location of a
polymer. In some particle-nucleotide conjugate, multiple
nucleotides are attached to one end or location of a polymer. The
conjugated nucleotide is sterically accessible to one or more
proteins, one or more enzymes, and nucleotide binding or
incorporation moieties. In some examples, a nucleotide may be
provided separately from a nucleotide binding or incorporation
moiety such as a polymerase. In some examples, the linker does not
comprise a photo emitting or photo absorbing group.
[0207] The particle can also have a binding or incorporation
moiety. In some examples, particles may self-associate without the
use of a separate interaction moiety. In some examples, particles
may self-associate due to buffer conditions or salt conditions,
e.g., as in the case of calcium-mediated interactions of
hydroxyapatite particles, lipid or polymer mediated interactions of
micelles or liposomes, or salt-mediated aggregation of metallic
(such as iron or gold) nanoparticles.
[0208] The particle-nucleotide conjugate can have one or more
labels. Examples of the labels include, but are not limited to,
fluorophores, spin labels, metals or metal ions, colorimetric
labels, nanoparticles, PET labels, radioactive labels, or other
such labels as may render said composition detectable by such
methods as are known in the art of the detection of macromolecules
or molecular interactions. The label may be attached to the
nucleotide (e.g., by attachment to the 5' phosphate moiety of a
nucleotide), to the particle itself (e.g., to the PEG subunits), to
an end of the polymer, to a central moiety, or to any other
location within said polymer-nucleotide conjugate which would be
recognized by one of skill in the art to be sufficient to render
said composition, such as a particle, detectable by such methods as
are known in the art or described elsewhere herein. In some
examples, one or more labels are provided so as to correspond to or
differentiate a particular particle-nucleotide conjugate.
[0209] In some examples, the label is a fluorophore. Non-limiting
examples of fluorescent moieties include, but are not limited to,
fluorescein and fluorescein derivatives such as carboxyfluorescein,
tetrachlorofluorescein, hexachlorofluorescein,
carboxynapthofluorescein, fluorescein isothiocyanate,
NHS-fluorescein, iodoacetamidofluorescein, fluorescein maleimide,
SAMSA-fluorescein, fluorescein thiosemicarbazide,
carbohydrazinomethylthioacetyl-amino fluorescein, rhodamine and
rhodamine derivatives such as TRITC, TMR, lissamine rhodamine,
Texas Red, rhodamine B, rhodamine 6G, rhodamine 10, NHS-rhodamine,
TMR-iodoacetamide, lissamine rhodamine B sulfonyl chloride,
lissamine rhodamine B sulfonyl hydrazine, Texas Red sulfonyl
chloride, Texas Red hydrazide, coumarin and coumarin derivatives
such as AMCA, AMCA-NHS, AMCA-sulfo-NHS, AMCA-HPDP, DCIA,
AMCE-hydrazide, BODIPY and derivatives such as BODIPY FL C3-SE,
BODIPY 530/550 C3, BODIPY 530/550 C3-SE, BODIPY 530/550 C3
hydrazide, BODIPY 493/503 C3 hydrazide, BODIPY FL C3 hydrazide,
BODIPY FL IA, BODIPY 530/551 IA, Br-BODIPY 493/503, Cascade Blue
and derivatives such as Cascade Blue acetyl azide, Cascade Blue
cadaverine, Cascade Blue ethylenediamine, Cascade Blue hydrazide,
Lucifer Yellow and derivatives such as Lucifer Yellow
iodoacetamide, Lucifer Yellow CH, cyanine and derivatives such as
indolium based cyanine dyes, benzo-indolium based cyanine dyes,
pyridium based cyanine dyes, thiozolium based cyanine dyes,
quinolinium based cyanine dyes, imidazolium based cyanine dyes, Cy
3, Cy5, lanthanide chelates and derivatives such as BCPDA, TBP,
TMT, BHHCT, BCOT, Europium chelates, Terbium chelates, Alexa Fluor
dyes, DyLight dyes, Atto dyes, LightCycler Red dyes, CAL Flour
dyes, JOE and derivatives thereof, Oregon Green dyes, WellRED dyes,
IRD dyes, phycoerythrin and phycobilin dyes, Malachite green,
stilbene, DEG dyes, NR dyes, near-infrared dyes and others known in
the art such as those described in Haugland, Molecular Probes
Handbook, (Eugene, Oreg.) 6th Edition; Lakowicz, Principles of
Fluorescence Spectroscopy, 2nd Ed., Plenum Press New York (1999),
or Hermanson, Bioconjugate Techniques, 2nd Edition, or derivatives
thereof, or any combination thereof. Cyanine dyes may exist in
either sulfonated or non-sulfonated forms, and consist of two
indolenin, benzo-indolium, pyridium, thiozolium, and/or quinolinium
groups separated by a polymethine bridge between two nitrogen
atoms. Commercially available cyanine fluorophores include, for
example, Cy3, (which may comprise
1-[6-(2,5-dioxopyrrolidin-1-yloxy)-6-oxohexyl]-2-(3-{1-[6-(2,5-dioxopyrro-
lidin-1-yloxy)-6-oxohexyl]-3,3-dimethyl-1,3-dihydro-2H-indol-2-ylidene}pro-
p-1-en-1-yl)-3,3-dimethyl-3H-indolium or
1-[6-(2,5-dioxopyrrolidin-1-yloxy)-6-oxohexyl]-2-(3-{1-[6-(2,5-dioxopyrro-
lidin-1-yloxy)-6-oxohexyl]-3,3-dimethyl-5-sulfo-1,3-dihydro-2H-indol-2-yli-
dene}prop-1-en-1-yl)-3,3-dimethyl-3H-indolium-5-sulfonate), Cy5
(which may comprise
1-(6-((2,5-dioxopyrrolidin-1-yl)oxy)-6-oxohexyl)-2-((1E,3E)-5-((-
E)-1-(6-((2,5-dioxopyrrolidin-1-yl)oxy)-6-oxohexyl)-3,3-dimethyl-5-indolin-
-2-ylidene)penta-1,3-dien-1-yl)-3,3-dimethyl-3H-indol-1-ium or
1-(6-((2,5-dioxopyrrolidin-1-yl)oxy)-6-oxohexyl)-2-((1E,3E)-5-((E)-1-(6-(-
(2,5-dioxopyrrolidin-1-yl)oxy)-6-oxohexyl)-3,3-dimethyl-5-sulfo
indolin-2-ylidene)penta-1,3-dien-1-yl)-3,3-dimethyl-3H-indol-1-ium-5-sulf-
onate), and Cy7 (which may comprise
1-(5-carboxypentyl)-2-[(1E,3E,5E,7Z)-7-(1-ethyl-1,3-dihydro-2H-indol-2-yl-
idene)hepta-1,3,5-trien-1-yl]-3H-indolium or
1-(5-carboxypentyl)-2-[(1E,3E,5E,7Z)-7-(1-ethyl-5-sulfo-1,3-dihydro-2H-in-
dol-2-ylidene)hepta-1,3,5-trien-1-yl]-3H-indolium-5-sulfonate),
where "Cy" stands for `cyanine`, and the first digit identifies the
number of carbon atoms between two indolenine groups. Cy2, which is
an oxazole derivative rather than indolenin, and the
benzo-derivatized Cy3.5, Cy5.5 and Cy7.5 are exceptions to this
rule.
[0210] In some embodiments, the detection label can be a FRET pair,
such that multiple classifications can be performed under a single
excitation and imaging step. As used herein, FRET may comprise
excitation exchange (Forster) transfers, or electron-exchange
(Dexter) transfers.
[0211] Polymer Nucleotide Conjugate. One example of the
particle-nucleotide conjugate is a polymer-nucleotide conjugate.
Some non-limiting examples of the polymer-nucleotide conjugates are
shown in FIGS. 16A-16C. For example, FIG. 16A shows
polymer-nucleotide conjugates having various configurations, e.g.,
a "starburst" configuration comprising a fluorescently-labeled
streptavidin core and four nucleotides bound to the core via
biotinylated, linear PEG linkers of molecular weight ranging from
1K Dalton to 10K Daltons; FIG. 16B shows a polymer-nucleotide
conjugate having a dendrimer core of, for example, 12, 24, 48, or
96 arms, and linear PEG linkers of molecular weight ranging from 1K
Dalton to 10K Daltons radiating from the center; and FIG. 16C shows
an example of polymer-nucleotide conjugates comprising a network
of, e.g., streptavidin cores, linked together by branched PEG
linkers comprising a binding or incorporation moiety such as a
biotin.
[0212] Examples of suitable linear or branched polymers include
linear or branched polyethylene glycol (PEG), linear or branched
polypropylene glycol, linear or branched polyvinyl alcohol, linear
or branched polylactic acid, linear or branched polyglycolic acid,
linear or branched polyglycine, linear or branched polyvinyl
acetate, a dextran, or other such polymers, or copolymers
incorporating any two or more of the foregoing or incorporating
other polymers as are known in the art. In one example, the polymer
is a PEG. In another embodiment, the polymer can have PEG
branches.
[0213] Suitable polymers may be characterized by a repeating unit
incorporating a functional group suitable for derivatization such
as an amine, a hydroxyl, a carbonyl, or an allyl group. The polymer
can also have one or more pre-derivatized substituents such that
one or more particular subunits will incorporate a site of
derivatization or a branch site, whether or not other subunits
incorporate the same site, substituent, or moiety. A
pre-derivatized substituent may comprise or may further comprise,
for example, a nucleotide, a nucleoside, a nucleotide analog, a
label such as a fluorescent label, radioactive label, or spin
label, an interaction moiety, an additional polymer moiety, or the
like, or any combination of the foregoing.
[0214] In the polymer-nucleotide conjugate, the polymer can have a
plurality of branches. The branched polymer can have various
configurations, including, but not limited to, stellate
("starburst") forms, aggregated stellate ("helter skelter") forms,
bottle brush, or dendrimer. The branched polymer can radiate from a
central attachment point or central moiety, or may incorporate
multiple branch points, such as, for example, 2, 3, 4, 5, 6, 7, 8,
9, 10, or more branch points. In some instances, each subunit of a
polymer may optionally constitute a separate branch point.
[0215] The length and size of the branch can differ based on the
type of polymer. In some branched polymers, the branch may have a
length of between 1 and 1,000 nm, between 1 and 100 nm, between 1
and 200 nm, between 1 and 300 nm, between 1 and 400 nm, between 1
and 500 nm, between 1 and 600 nm, between 1 and 700 nm, between 1
and 800 nm, or between 1 and 900 nm, or more, or may have a length
falling within or between any of the values disclosed herein.
[0216] In some polymer-nucleotide conjugates, the polymer core may
have a size corresponding to an apparent molecular weight of 1K Da,
2K Da, 3K Da, 4K Da, 5K Da, 10K Da, 15K Da, 20K Da, 30K Da, 50K Da,
80K Da, 100K Da, or any value within a range defined by any two of
the foregoing. The apparent molecular weight of a polymer may be
calculated from the known molecular weight of a representative
number of subunits, as determined by size exclusion chromatography,
as determined by mass spectrometry, or as determined by any other
method as is known in the art.
[0217] In some branched polymers, the branch may have a size
corresponding to an apparent molecular weight of 1K Da, 2K Da, 3K
Da, 4K Da, 5K Da, 10K Da, 15K Da, 20K Da, 30K Da, 50K Da, 80K Da,
100K Da, or any value within a range defined by any two of the
foregoing. The apparent molecular weight of a polymer may be
calculated from the known molecular weight of a representative
number of subunits, as determined by size exclusion chromatography,
as determined by mass spectrometry, or as determined by any other
method as is known in the art. The polymer can have multiple
branches. The number of branches in the polymer can be 2, 3, 4, 5,
6, 7, 8, 12, 16, 24, 32, 64, 128 or more, or a number falling
within a range defined by any two of these values.
[0218] For polymer-nucleotide conjugates comprising a branched
polymer of, for example, a branched PEG comprising 4, 8, 16, 32, or
64 branches, the polymer nucleotide conjugate can have nucleotides
attached to the ends of the PEG branches, such that each end has
attached thereto 0, 1, 2, 3, 4, 5, 6 or more nucleotides. In one
non-limiting example, a branched PEG polymer of between 3 and 128
PEG arms may have attached to the ends of the polymer branches one
or more nucleotides, such that each end has attached thereto 0, 1,
2, 3, 4, 5, 6 or more nucleotides or nucleotide analogs. In some
embodiments, a branched polymer or dendrimer has an even number of
arms. In some embodiments, a branched polymer or dendrimer has an
odd number of arms.
[0219] In some instances, the length of the linker (e.g., a PEG
linker) may range from about 1 nm to about 1,000 nm. In some
instances, the length of the linker may be at least 1 nm, at least
10 nm, at least 25 nm, at least 50 nm, at least 75 nm, at least 100
nm, at least 200 nm, at least 300 nm, at least 400 nm, at least 500
nm, at least 600 nm, at least 700 nm, at least 800 nm, at least 900
nm, or at least 1,000 nm. In some instances, the length of the
linker may range between any two of the values in this paragraph.
For example, in some instances, the length of the linker may range
from about 75 nm to about 400 nm. Those of skill in the art will
recognize that in some instances, the length of the linker may have
any value within the range of values in this paragraph, e.g., 834
nm.
[0220] In some instances, the length of the linker is different for
different nucleotides (including deoxyribonucleotides and
ribonucleotides), nucleotide analogs (including deoxyribonucleotide
analogs and ribonucleotide analogs), nucleosides (including
deoxyribonucleosides or ribonucleosides), or nucleoside analogs
(including deoxyribonucleoside analogs or ribonucleoside analogs).
In some instances, one of the nucleotides, nucleotide analogs,
nucleosides, or nucleoside analogs comprises, for example,
deoxyadenosine, and the length of the linker is between 1 nm and
1,000 nm. In some instances, one of the nucleotides, nucleotide
analogs, nucleosides, or nucleoside analogs comprises, for example,
deoxyguanosine, and the length of the linker is between 1 nm and
1,000 nm. In some instances, one of the nucleotides, nucleotide
analogs, nucleosides, or nucleoside analogs comprises, for example,
thymidine, and the length of the linker is between 1 nm and 1,000
nm. In some instances, one of the nucleotides, nucleotide analogs,
nucleosides, or nucleoside analogs comprises, for example,
comprises deoxyuridine, and the length of the linker is between 1
nm and 1,000 nm. In some instances, one of the nucleotides,
nucleotide analogs, nucleosides, or nucleoside analogs comprises,
for example, deoxycytidine, and the length of the linker is between
1 nm and 1,000 nm. In some instances, one of the nucleotides,
nucleotide analogs, nucleosides, or nucleoside analogs comprises,
for example, adenosine, and the length of the linker is between 1
nm and 1,000 nm. In some instances, one of the nucleotides,
nucleotide analogs, nucleosides, or nucleoside analogs comprises,
for example, guanosine, and the length of the linker is between 1
and 1,000 nm. In some instances, one of the nucleotides, nucleotide
analogs, nucleosides, or nucleoside analogs comprises, for example,
5-methyl-uridine, and the length of the linker is between 1 nm and
1,000 nm. In some instances, one of the nucleotides, nucleotide
analogs, nucleosides, or nucleoside analogs comprises, for example,
uridine, and the length of the linker is between 1 nm and 1,000 nm.
In some instances, one of the nucleotides, nucleotide analogs,
nucleosides, or nucleoside analogs comprises, for example,
cytidine, and the length of the linker is between 1 nm and 1,000
nm.
[0221] In the polymer-nucleotide conjugate, each branch or a subset
of branches of the polymer may have attached thereto a moiety
comprising a nucleotide (e.g., an adenine, a thymine, a uracil, a
cytosine, or a guanine residue or a derivative or mimetic thereof),
and the moiety is capable of binding or incorporation to a
polymerase, reverse transcriptase, or other nucleotide binding or
incorporation domain. Optionally, the moiety may be capable of
being incorporated into an elongating nucleic acid chain during a
polymerase reaction. In some instances, said moiety may be blocked
such that it is not capable of being incorporated into an
elongating nucleic acid chain during a polymerase reaction. In some
other instances, said moiety may be reversibly blocked such that it
is not capable of being incorporated into an elongating nucleic
acid chain during a polymerase reaction until such block is
removed, after which said moiety is then capable of being
incorporated into an elongating nucleic acid chain during a
polymerase reaction.
[0222] The nucleotide can be conjugated to the polymer branch
through the 5' end of the nucleotide. In some instances, the
nucleotide may be modified so as to inhibit or prevent
incorporation of the nucleotide into an elongating nucleic acid
chain during a polymerase reaction. By way of example, the
nucleotide may include a 3' deoxyribonucleotide, a 3'
azidonucleotide, a 3'-methyl azido nucleotide, or another such
nucleotide as is or may be known in the art, so as to not be
capable of being incorporated into an elongating nucleic acid chain
during a polymerase reaction. In some instances, the nucleotide can
include a 3'-O-azido group, a 3'-O-azidomethyl group, a
3'-phosphorothioate group, a 3'-O-malonyl group, a 3'-O-alkyl
hydroxylamino group, or a 3'-O-benzyl group. In some instances, the
nucleotide lacks a 3' hydroxyl group.
[0223] The polymer can further have a binding or incorporation
moiety in each branch or a subset of branches. Some examples of the
binding or incorporation moiety include but are not limited to
biotin, avidin, strepavidin or the like, polyhistidine domains,
complementary paired nucleic acid domains, G-quartet forming
nucleic acid domains, calmodulin, maltose-binding protein,
cellulase, maltose, sucrose, glutathione-S-transferase,
glutathione, O-6-methylguanine-DNA methyltransferase, benzylguanine
and derivatives thereof, benzylcysteine and derivatives thereof, an
antibody, an epitope, a protein A, a protein G. The binding or
incorporation moiety can be any interactive molecules or fragment
thereof known in the art to bind to or facilitate interactions
between proteins, between proteins and ligands, between proteins
and nucleic acids, between nucleic acids, or between small molecule
interaction domains or moieties.
[0224] In some embodiments, a composition as provided herein may
comprise one or more elements of a complementary interaction
moiety. Non-limiting examples of complementary interaction moieties
include, for example, biotin and avidin; SNAP-benzylguanosine;
antibody or FAB and epitope; IgG FC and Protein A, Protein G,
ProteinA/G, or Protein L; maltose binding protein and maltose;
lectin and cognate polysaccharide; ion chelation moieties,
complementary nucleic acids, nucleic acids capable of forming
triplex or triple helical interactions; nucleic acids capable of
forming G-quartets, and the like. One of skill in the art will
readily recognize that many pairs of moieties exist and are
commonly used for their property of interacting strongly and
specifically with one another; and thus any such complementary pair
or set is considered to be suitable for this purpose in
constructing or envisioning the compositions of the present
disclosure. In some examples, a composition as disclosed herein may
comprise compositions in which one element of a complementary
interaction moiety is attached to one molecule or multivalent
ligand, and the other element of the complementary interaction
moiety is attached to a separate molecule or multivalent ligand. In
some examples, a composition as disclosed herein may comprise
compositions in which both or all elements of a complementary
interaction moiety are attached to a single molecule or multivalent
ligand. In some examples, a composition as disclosed herein may
comprise compositions in which both or all elements of a
complementary interaction moiety are attached to separate arms of,
or locations on, a single molecule or multivalent ligand. In some
examples, a composition as disclosed herein may comprise
compositions in which both or all elements of a complementary
interaction moiety are attached to the same arm of, or locations
on, a single molecule or multivalent ligand. In some examples,
compositions comprising one element of a complementary interaction
moiety and compositions comprising another element of a
complementary interaction moiety may be simultaneously or
sequentially mixed. In some examples, interactions between
molecules or particles as disclosed herein allow for the
association or aggregation of multiple molecules or particles such
that, for example, detectable signals are increased. In some
examples, fluorescent, colorimetric, or radioactive signals are
enhanced. In other examples, other interaction moieties as
disclosed herein, or as are known in the art, are contemplated. In
some examples, a composition as provided herein may be provided
such that one or more molecules comprising a first interaction
moiety such as, for example, one or more imidazole or pyridine
moieties, and one or more additional molecules comprising a second
interaction moiety such as, for example, histidine residues, are
simultaneously or sequentially mixed. In some examples, said
composition comprises 1, 2, 3, 4, 5, 6, or more imidazole or
pyridine moieties. In some examples, said composition comprises 1,
2, 3, 4, 5, 6, or more histidine residues. In such examples,
interaction between the molecules or particles as provided may be
facilitated by the presence of a divalent cation such as nickel,
manganese, magnesium, calcium, strontium, or the like. For example,
a (His)3 group may interact with a (His)3 group on another molecule
or particle via coordination of a nickel or manganese ion.
[0225] The multivalent binding or incorporation composition may
comprise one or more buffers, salts, ions, or additives.
Representative additives may include, but are not limited to,
betaine, spermidine, detergents such as Triton X-100, Tween 20,
SDS, or NP-40, ethylene glycol, polyethylene glycol, dextran,
polyvinyl alcohol, vinyl alcohol, methylcellulose, heparin, heparan
sulfate, glycerol, sucrose, 1,2-propanediol, DMSO,
N,N,N-trimethylglycine, ethanol, ethoxyethanol, propylene glycol,
polypropylene glycol, block copolymers such as the Pluronic.RTM.
series polymers, arginine, histidine, imidazole, or any combination
thereof, or any substance known in the art as a DNA "relaxer"
(i.e., a compound, with the effect of altering the persistence
length of DNA, altering the number of within-polymer junctions or
crossings, or altering the conformational dynamics of a DNA
molecule such that the accessibility of sites within the strand to
DNA binding or incorporation moieties is increased).
[0226] The multivalent binding or incorporation composition may
include zwitterionic compounds as additives. Further representative
additives may be found in Lorenz, T. C. J. Vis. Exp. (63), e3998,
doi:10.3791/3998 (2012), which is hereby incorporated by reference
with respect to its disclosure of additives for the facilitation of
nucleic acid binding or dynamics, or the facilitation of processes
involving the manipulation, use, or storage of nucleic acids. In
some instances, representative cations include, but are not limited
to, sodium, magnesium, strontium, potassium, manganese, calcium,
lithium, nickel, cobalt, or other such cations as are known in the
art to facilitate nucleic acid interactions, such as
self-association, secondary or tertiary structure formation, base
pairing, surface association, peptide association, protein binding,
or the like.
[0227] Binding Between Target Nucleic Acid and Multivalent Binding
or Incorporation Composition. When the multivalent binding or
incorporation composition is used in replacement of a single
unconjugated or untethered nucleotide to form a complex with the
polymerase and one or more copies of the target nucleic acid, the
local concentration of the nucleotide as well as the binding
avidity of the complex (in cases where a complex comprising two or
more target nucleic acid molecules is formed) is increased
many-fold, which in turn enhances the signal intensity,
particularly the correct signal versus mismatch. The present
disclosure contemplates contacting the multivalent binding or
incorporation composition with a polymerase and a primed target
nucleic acid to determine the formation of a ternary binding or
incorporation complex.
[0228] FIG. 17 illustrates the use of the disclosed
polymer-nucleotide conjugates for achieving increased signal
intensity during binding, persistence, and washing/removal steps.
Because of the increased local concentration of the nucleotide on
the polymer-nucleotide conjugate and/or the formation of
non-covalent bonds with two or more primed target nucleic acid
molecules, the binding between the polymerase, the primed target
strand, and the polymer-conjugated nucleotide, when the nucleotide
is complementary to the next base of the target nucleic acid,
becomes more favorable. The formed binding complex has a longer
persistence time, which in turn helps increase signal and shorten
the imaging step. The high signal intensity resulting from the use
of the disclosed polymer nucleotide conjugates remains stable for
the entire binding and imaging steps. The strong binding between
the polymerase, the primed target strand, and the
polymer-conjugated nucleotide or nucleotide analog also means that
the binding complex thus formed will remain stable during wash
steps as other reaction mixture components and unmatched nucleotide
analogs are washed away. After the imaging step, the binding
complex can be destabilized (e.g., by changing the buffer
composition) and the primed target nucleic acid can then be
extended for one base. After the extension, the binding and imaging
steps can be repeated with the use of the disclosed polymer
nucleotide conjugates to determine the identity of the next
base.
[0229] As an example, a graphical depiction of the increase in
signal intensity during binding, persistence, and washing/removal
of a multivalent substrate as described herein is provided in FIG.
17, which is representative of the changes in signal intensity that
have been observed experimentally. Therefore, the compositions and
methods of the present disclosure provide a robust and controllable
means of establishing and maintaining a ternary enzyme complex, as
well as providing vastly improved means by which the presence of
said complex may be identified and/or measured, and a means by
which the persistence of said complex may be controlled. This
provides important solutions to problems such as that of
determining the identity of the N+1 base in nucleic acid sequencing
applications.
[0230] Without intending to be bound by any particular theory, it
has been observed that multivalent binding compositions disclosed
herein associate with polymerase nucleotide complexes in order to
form a ternary binding complexes with a rate that is
time-dependent, though substantially slower than the rate of
association known to be obtainable by nucleotides in free solution.
Thus, the on-rate (Kon) is substantially and surprisingly slower
than the on rate for single nucleotides or nucleotides not attached
to multivalent ligand complexes. Importantly, however, the off rate
(Koff) of the multivalent ligand complex is substantially slower
than that observed for nucleotides in free solution. Therefore, the
multivalent ligand complexes of the present disclosure provide a
surprising and beneficial improvement of the persistence of ternary
polymerase-polynucleotide-nucleotide complexes (especially over
such complexes that are formed with free nucleotides) allowing, for
example, significant improvements in imaging quality for nucleic
acid sequencing applications over currently available methods and
reagents. Importantly, this property of the multivalent binding
compositions disclosed herein renders the formation of visible
ternary complexes controllable, such that subsequent visualization,
modification, or processing steps may be undertaken essentially
without regard to the dissociation of the complex--that is, the
complex can be formed, imaged, modified, or used in other ways as
necessary, and will remain stable until a user carries out an
affirmative dissociation step, such as exposing the complexes to a
dissociation buffer.
[0231] In some instances, the persistence times for the multivalent
binding complexes formed using the disclosed particle-nucleotide or
polymer-nucleotide conjugates may range from about 0.1 second to
about 600 second under non-destabilizing conditions. In some
instances, the persistence time may be at least 0.1 second, at
least 1 second, at least 2 seconds, at least 3 second, at least 4
second, at least 5 seconds, at least 6 seconds, at least 7 seconds,
at least 8 seconds, at least 9 seconds, at least 10 seconds, at
least 20 seconds, at least 30 second, at least 40 second, at least
50 seconds, at least 60 seconds, at least 120 seconds, at least 180
seconds, at least 240 seconds, at least 300 seconds, at least 360
seconds, at least 420 seconds, at least 480 seconds, at least 540
seconds, or at least 600 seconds. In some instances, the
persistence time may range between any two of the values specified
in this paragraph. For example, in some instances, the persistence
time may range from about 10 seconds to about 360 seconds. Those of
skill in the art will recognize that in some instances, the
persistence time may have any value within the range of values
specified in this paragraph, e.g., 78 seconds.
[0232] In various examples, polymerases suitable for the binding or
incorporation interaction describe herein include may include any
polymerase as is or may be known in the art. It is, for example,
known that every organism encodes within its genome one or more DNA
polymerases. Examples of suitable polymerases may include but are
not limited to: Klenow DNA polymerase, Thermus aquaticus DNA
polymerase I (Taq polymerase), KlenTaq polymerase, and
bacteriophage T7 DNA polymerase; human alpha, delta and epsilon DNA
polymerases; bacteriophage polymerases such as T4, RB69 and phi29
bacteriophage DNA polymerases, Pyrococcus furiosus DNA polymerase
(Pfu polymerase); Bacillus subtilis DNA polymerase III, and E. coli
DNA polymerase III alpha and epsilon; 9 degree N polymerase,
reverse transcriptases such as HIV type M or O reverse
transcriptases, avian myeloblastosis virus reverse transcriptase,
or Moloney Murine Leukemia Virus (MMLV) reverse transcriptase, or
telomerase. Further non-limiting examples of DNA polymerases can
include those from various Archaea genera, such as, Aeropyrum,
Archaeglobus, Desulfurococcus, Pyrobaculum, Pyrococcus, Pyrolobus,
Pyrodictium, Staphylothermus, Stetteria, Sulfolobus, Thermococcus,
and Vulcanisaeta and the like or variants thereof, including such
polymerases as are known in the art such as Vent.TM., Deep
Vent.TM., Pfu, KOD, Pfx, Therminator.TM., and Tgo polymerases. In
some examples, the polymerase is a klenow polymerase.
[0233] The ternary complex has longer persistence time when the
nucleotide on the polymer-nucleotide conjugate is complementary to
the target nucleic acid than when it is non-complementary to the
target nucleic acid. The ternary complex also has longer
persistence time when the nucleotide on the polymer-nucleotide
conjugate is complementary to the target nucleic acid than a
complementary nucleotide that is not conjugated or tethered. For
example, in some embodiments, said ternary complexes may have a
persistence time of less than 1 s, greater than 1 s, greater than 2
s, greater than 3 s, greater than 5 s, greater than 10 s, greater
than 15 s, greater than 20 s, greater than 30 s, greater than 60 s,
greater than 120 s, greater than 360 s, greater than 3600 s, or
more, or for a time lying within a range defined by any two or more
of these values.
[0234] The persistence time can be measured, for example, by
observing the onset and/or duration of a binding complex, such as
by observing a signal from a labeled component of the binding
complex. For example, a labeled nucleotide or a labeled reagent
comprising one or more nucleotides may be present in a binding
complex, thus allowing the signal from the label to be detected
during the persistence time of the binding complex.
[0235] It has been observed that different ranges of persistence
times are achievable with different salts or ions, showing, for
example, that complexes formed in the presence of, for example,
magnesium ions (Mg2+) form more quickly than complexes formed with
other ions. It has also been observed that complexes formed in the
presence of, for example, strontium ions (Sr2+), form readily and
dissociate completely or with substantial completeness upon
withdrawal of the ion or upon washing with buffer lacking one or
more components of the present compositions, such as, e.g., a
polymer and/or one or more nucleotides, and/or one or more
interaction moieties, or a buffer containing, for example, a
chelating agent which may cause or accelerate the removal of a
divalent cation from the multivalent reagent containing complex.
Thus, in some examples, a composition of the present disclosure
comprises Mg2+. In some examples, a composition of the present
disclosure comprises Ca2+. In some examples, a composition of the
present disclosure comprises Sr2+. In some examples, a composition
of the present disclosure comprises cobalt ions (Co2+). In some
examples, a composition of the present disclosure comprises MgCl2.
In some examples, a composition of the present disclosure comprises
CaCl2. In some examples, a composition of the present disclosure
comprises SrCl2. In some examples, a composition of the present
disclosure comprises CoCl2. In some examples, the composition
comprises no, or substantially no magnesium. In some examples, the
composition comprises no, or substantially no calcium. In some
examples, the methods of the present disclosure provide for the
contacting of one or more nucleic acids with one or more of the
compositions disclosed herein, wherein said composition lacks
either one of calcium or magnesium or lacks both calcium or
magnesium.
[0236] The dissociation of ternary complexes can be controlled by
changing the buffer conditions. After the imaging step, a buffer
with increased salt content is used to cause dissociation of the
ternary complexes such that labeled polymer-nucleotide conjugates
can be washed out, providing a means by which signals can be
attenuated or terminated, such as in the transition between one
sequencing cycle and the next. This dissociation may be affected,
in some embodiments, by washing the complexes with a buffer lacking
a necessary metal or cofactor. In some instances, a wash buffer may
comprise one or more compositions for the purpose of maintaining pH
control. In some instances, a wash buffer may comprise one or more
monovalent cations, such as sodium. In some instances, a wash
buffer lacks or substantially lacks a divalent cation, for example,
having no or substantially no strontium, calcium, magnesium, or
manganese. In some instances, a wash buffer further comprises a
chelating agent, such as, for example, EDTA, EGTA, nitrilotriacetic
acid, polyhistidine, imidazole, or the like. In some instances, a
wash buffer may maintain the pH of the environment at the same
level as for the bound complex. In some instances, a wash buffer
may raise or lower the pH of the environment relative to the level
seen for the bound complex. In some instances, the pH may be within
a range from 2-4, 2-7, 5-8, 7-9, 7-10, or lower than 2, or higher
than 10, or a range defined by any two of the values provided
herein.
[0237] Addition of a particular ion may affect the binding of the
polymerase to a primed target nucleic acid, the formation of a
ternary complex, the dissociation of a ternary complex, or the
incorporation of one or more nucleotides into an elongating nucleic
acid such as during a polymerase reaction. In some instances,
relevant anions may comprise chloride, acetate, gluconate, sulfate,
phosphate, or the like. In some instances, an ion may be
incorporated into the compositions of the present disclosure by the
addition of one or more acids, bases, or salts, such as NiCl2,
CoCl2, MgCl2, MnCl2, SrCl2, CaCl2, CaSO4, SrCO3, BaCl2 or the like.
Representative salts, ions, solutions and conditions may be found
in Remington: The Science and Practice of Pharmacy, 20th. Edition,
Gennaro, A. R., Ed. (2000), which is hereby incorporated by
reference in its entirety, and especially with respect to Chapter
17 and related disclosure of salts, ions, salt solutions, and ionic
solutions.
[0238] The present disclosure contemplates contacting the
multivalent binding or incorporation composition comprising at
least one particle-nucleotide conjugate with one or more
polymerases. The contacting can be optionally done in the presence
of one or more target nucleic acids. In some examples, said target
nucleic acids are single stranded nucleic acids. In some examples,
said target nucleic acids are primed single stranded nucleic acids.
In some examples, said target nucleic acids are double stranded
nucleic acids. In some examples, said contacting comprises
contacting the multivalent binding or incorporation composition
with one polymerase. In some examples, said contacting comprises
the contacting of said composition comprising one or more
nucleotides with multiple polymerases. The polymerase can be bound
to a single nucleic acid molecule.
[0239] The binding between target nucleic acid and multivalent
binding composition may be provided in the presence of a polymerase
that has been rendered catalytically inactive. In one example, the
polymerase may have been rendered catalytically inactive by
mutation. In one example, the polymerase may have been rendered
catalytically inactive by chemical modification. In some examples,
the polymerase may have been rendered catalytically inactive by the
absence of a necessary substrate, ion, or cofactor. In some
examples, the polymerase enzyme may have been rendered
catalytically inactive by the absence of magnesium ions.
[0240] The binding between a target nucleic acid and multivalent
binding composition described herein may occur in the presence of a
polymerase wherein the binding solution, reaction solution, or
buffer lacks magnesium or manganese. In another example, the
binding between the target nucleic acid and multivalent binding
composition occurs in the presence of a polymerase wherein the
binding solution, reaction solution, or buffer comprises calcium or
strontium.
[0241] In some instances, when catalytically inactive polymerases
are used to help a nucleic acid interact with a multivalent binding
composition, the interaction between said composition and said
polymerase stabilizes a ternary complex so as to render the complex
detectable by fluorescence or by other methods as disclosed herein
or otherwise known in the art. Unbound polymer-nucleotide
conjugates may optionally be washed away prior to detection of the
ternary binding complex.
[0242] Contacting of one or more nucleic acids with the
polymer-nucleotide conjugates disclosed herein may occur in a
solution containing either one of calcium or magnesium or
containing both calcium and magnesium. In another example, the
contacting of one or more nucleic acids with the polymer-nucleotide
conjugates disclosed herein occurs in a solution lacking either one
of calcium or magnesium, or lacking both calcium or magnesium, and
in a separate step, without regard to the order of the steps, one
of calcium or magnesium, or both calcium and magnesium, may be
added to the solution. In some embodiments, the contacting of one
or more nucleic acids with the polymer-nucleotide conjugates
disclosed herein occurs in a solution lacking strontium, and
comprises in a separate step, without regard to the order of the
steps, adding to the solution strontium.
Illustrative Embodiment 1
[0243] The disclosed methods of determining the sequence of a
target nucleic acid comprise: a) contacting a double-stranded or
partially double-stranded target nucleic acid molecule comprising
the template strand to be sequenced and a primer strand to be
elongated with one or more of the disclosed nucleic acid binding
compositions; and b) detecting the binding of a nucleic acid
binding composition to the nucleic acid molecule, thereby
determining the presence of one of said one or more nucleic acid
binding compositions on said nucleic acid molecule and the identity
of the next nucleotide (i.e., the N+1 or terminal nucleotide) to be
incorporated into the complementary strand.
[0244] The sequencing method may further comprise incorporating the
N+1 or terminal nucleotide into the primer strand, and then
repeating the contacting, detecting, and incorporating steps for
one or more additional iterations, thereby determining the sequence
of the template strand of the nucleic acid molecule. After the step
of detecting the ternary binding complex, the primed strand of the
primed target nucleic acid is extended for one base before another
round of analysis is performed. The primed target nucleic acid can
be extended using the conjugated nucleotide that is attached to the
polymer in the multivalent binding composition or using an
unconjugated or untethered free nucleotide that is provided after
the multivalent binding composition has been removed.
[0245] The extension of the primed target nucleic acid may be
prevented or inhibited due to a blocked nucleotide on the strand or
the use of polymerase that is catalytically inactive. When the
nucleotide in the polymer-nucleotide conjugate has a blocking group
that prevents the extension of the nucleic acid, incorporation of a
nucleotide may be achieved by the removal of a blocking group from
said nucleotide (such as by detachment of said nucleotide from its
polymer, branched polymer, dendrimer, particle, or the like). When
the extension of the primed target nucleic acid is inhibited due to
the use of polymerase that is catalytically inactive, incorporation
of a nucleotide may be achieved by the provision of a cofactor or
activator such as a metal ion.
[0246] Detection of the ternary complex is achieved prior to,
concurrently with, or following the incorporation of the nucleotide
residue. In some instances, a primed target nucleic acid may
comprise a target nucleic acid with multiple primed locations for
the attachment of polymerases and/or nucleic acid binding moieties.
In some instances, multiple polymerases may be attached to a single
target nucleic acid molecule, such as at multiple sites within a
target nucleic acid molecule. In some instances, multiple
polymerases may be bound to a multivalent binding composition
disclosed herein comprising multiple nucleotides. In some
instances, a target nucleic acid molecule may be a product of a
strand displacement synthesis, a rolling circle amplification, a
concatenation or fusion of multiple copies of a query sequence, or
other such methods as are known in the art or as are disclosed
elsewhere herein to produce nucleic acid molecules comprising
multiple copies of an identical sequence. Therefore, in some
instances, multiple polymerases may be attached at multiple
identical or substantially identical locations within a target
nucleic acid, which comprises multiple identical or substantially
identical copies of a query sequence. In some instances, said
multiple polymerases may then be involved in interactions with one
or more multivalent binding complexes; however, in some examples,
the number of binding sites within a target nucleic acid is at
least two, and the number of nucleotides or substrate moieties
present on a particle-nucleotide conjugate such as a
polymer-nucleotide conjugate is also greater than or equal to
two.
[0247] In some examples, the multivalent binding compositions are
provided in combination with other elements such as to provide
optimized signals, for example to provide identification of a
nucleotide at a particular position in a nucleic acid sequence. In
some instances, the compositions disclosed herein are provided in
combination with a surface providing low background binding or low
levels of protein binding, such as, for example, a hydrophilic or
polymer coated surface. Representative surfaces may be found, for
example, in U.S. patent application Ser. No. 16/363,842, the
contents of which are hereby incorporated by reference in their
entirety.
[0248] In some instances, the nucleic acid molecule is tethered to
the surface of a solid support, e.g., through hybridization of the
template strand to an adapter nucleic acid sequence or primer
nucleic acid sequence that is tethered to the solid support. In
some instances, the solid support comprises a glass, fused-silica,
silicon, or polymer substrate. In some instances, the solid support
comprises a low non-specific binding coating comprising one or more
hydrophilic polymer layers (e.g., PEG layers) where at least one of
the hydrophilic polymer layers comprises a branched polymer
molecule (e.g., a branched PEG molecule comprising 4, 8, 16, or 32
branches).
[0249] The solid support comprises oligonucleotide adapters or
primers tethered to at least one hydrophilic polymer layer at a
surface density ranging from about 1,000 primer molecules per
.mu.m.sup.2 to about 1,000,000 primer molecules per .mu.m.sup.2. In
some instances, the surface density of oligonucleotide primers may
be at least 1,000, at least 10,000, at least 100,000, or at least
1,000,000 molecules per .mu.m.sup.2. In some instances, the surface
density of oligonucleotide primers may be at most 1,000,000, at
most 100,000, at most 10,000, or at most 1,000 molecules per
.mu.m.sup.2. Any of the lower and upper values described in this
paragraph may be combined to form a range included within the
present disclosure, for example, in some instances the surface
density of primers may range from about 10,000 molecules per
.mu.m.sup.2 to about 100,000 molecules per .mu.m.sup.2. Those of
skill in the art will recognize that the surface density of primer
molecules may have any value within this range, e.g., about 455,000
molecules per .mu.m.sup.2.
[0250] One of ordinary skill would recognize that in a series of
iterative sequencing reactions, occasionally one or more sites will
fail to incorporate a nucleotide during a given cycle, thus leading
one or more sites to be unsynchronized with the bulk of the
elongating nucleic acid chains. Under conditions in which
sequencing signals are derived from reactions occurring on single
copies of a target nucleic acid, these failures to incorporate will
yield discrete errors in the output sequence. It is an object of
the present disclosure to describe methods for reducing this type
of error in sequencing reactions. For example, the use of
multivalent substrates that are capable of incorporation into the
elongating strand, by providing increased probabilities of
rebinding upon premature dissociation of a ternary polymerase
complex, can reduce the frequency of "skipped" cycles in which a
base is not incorporated. Thus, in some examples, the present
disclosure contemplates the use of multivalent substrates as
disclosed herein in which the nucleoside moiety is comprised within
a nucleotide having a free, or reversibly modified, 5' phosphate,
diphosphate, or triphosphate moiety, and wherein the nucleotide is
connected to the particle or polymer as disclosed herein, through a
labile or cleavable linkage. In some examples, the present
disclosure contemplates a reduction in the intrinsic error rate due
to skipped incorporations as a result of the use of the multivalent
substrates disclosed herein.
[0251] The present disclosure also contemplates sequencing
reactions in which sequencing signals from or relating to a given
sequence are derived from or originate within definable regions
containing multiple copies of the target sequence. Sequencing
methods incorporating multiple copies of a target sequence are
advantageous in that signals can be amplified due to the presence
of multiple simultaneous sequencing reactions within the defined
region, each providing its own signal. The presence of multiple
signals within a defined area also reduces the impact of any single
skipped cycle, due to the fact that the signal from a large number
of correct base calls can overwhelm the signal from a smaller
number of skipped or incorrect base calls. The present disclosure
further contemplates the inclusion of free, unlabeled nucleotides
during elongation reactions, or during a separate part of the
elongation cycle, in order to provide incorporation at sites that
may have been skipped in previous cycles. For example, during or
following an incorporation cycle, unlabeled blocked nucleotides may
be added such that they may be incorporated at skipped sites. The
unlabeled blocked nucleotides may be of the same type or types as
the nucleotide attached to the multivalent binding substrate or
substrates that are or were present during a particular cycle, or a
mixture of 1, 2, 3, 4 or more types of unlabeled blocked
nucleotides may be included.
[0252] When each sequencing cycle proceeds perfectly, each reaction
within the defined region will provide an identical signal.
However, as noted elsewhere herein, in a series of iterative
sequencing reactions, occasionally one or more sites will fail to
incorporate a nucleotide during a given cycle, thus leading one or
more sites to be unsynchronized with the bulk of the elongating
nucleic acid chains. This issue, referred to as "phasing," leads to
degradation of the sequencing signal as the signal is contaminated
with spurious signals from sites having skipped one or more cycles.
This, in turn, creates the potential for errors in base
identification. The progressive accumulation of skipped cycles
through multiple cycles also reduces the effective read length, due
to progressive degradation of the sequencing signal with each
cycle. It is a further object of this disclosure to provide methods
for reducing phasing errors and/or to improve read length in
sequencing reactions.
[0253] The sequencing method can include contacting a target
nucleic acid or multiple target nucleic acids, comprising multiple
linked or unlinked copies of a target sequence, with the
multivalent binding compositions described herein. Contacting said
target nucleic acid, or multiple target nucleic acids comprising
multiple linked or unlinked copies of a target sequence, with one
or more particle-nucleotide conjugates may provide a substantially
increased local concentration of the correct nucleotide being
interrogated in a given sequencing cycle, thus suppressing signals
from improper incorporations or phased nucleic acid chains (i.e.,
those elongating nucleic acid chains which have had one or more
skipped cycles).
[0254] Methods of obtaining nucleic acid sequence information can
include contacting a target nucleic acid, or multiple target
nucleic acids, wherein said target nucleic acid or multiple target
nucleic acids comprise multiple linked or unlinked copies of a
target sequence, with one or more particle-nucleotide conjugates.
This method results in a reduction in the error rate of sequencing
as indicated by reduction in the misidentification of bases, the
reporting of nonexistent bases, or the failure to report correct
bases. In some embodiments, said reduction in the error rate of
sequencing may comprise a reduction of 5%, 10%, 15%, 20% 25%, 50%,
75%, 100%, 150%, 200%, or more compared to the error rate observed
using monovalent ligands, including free nucleotides, labeled free
nucleotides, protein or peptide bound nucleotides, or labeled
protein or peptide bound nucleotides.
[0255] The method of obtaining nucleic acid sequence information
can include contacting a target nucleic acid, or multiple target
nucleic acids, wherein said templet nucleic acid or multiple target
nucleic acids comprise multiple linked or unlinked copies of a
target sequence, with one or more particle-nucleotide conjugates.
This method results in an increase in average read length of 5%,
10%, 15%, 20%, 25%, 50%, 75%, 100%, 150%, 200%, 300%, or more
compared to the average read length observed using monovalent
ligands, including free nucleotides, labeled free nucleotides,
protein or peptide bound nucleotides, or labeled protein or peptide
bound nucleotides.
[0256] Disclosed herein are methods of obtaining nucleic acid
sequence information, said methods comprising contacting a target
nucleic acid, or multiple target nucleic acids, wherein said target
nucleic acid or multiple target nucleic acids comprise multiple
linked or unlinked copies of a target sequence, with one or more
particle-nucleotide conjugates. Such methods may result in an
increase in average read length of 10 nucleotides (NT), 20 NT, 25
NT, 30 NT, 50 NT, 75 NT, 100 NT, 125 NT, 150 NT, 200 NT, 250 NT,
300 NT, 350 NT, 400 NT, 500 NT, or more compared to the average
read length observed using monovalent ligands, including free
nucleotides, labeled free nucleotides, protein or peptide bound
nucleotides, or labeled protein or peptide bound nucleotides.
[0257] In some instances, the disclosed compositions and methods
may result in average read lengths for sequencing applications that
range from 100 nucleotides to 1,000 nucleotides. In some instances,
the average read length may be at least 100 nucleotides, at least
200 nucleotides, at least 225 nucleotides, at least 250
nucleotides, at least 275 nucleotides, at least 300 nucleotides, at
least 325 nucleotides, at least 350 nucleotides, at least 375
nucleotides, at least 400 nucleotides, at least 425 nucleotides, at
least 450 nucleotides, at least 475 nucleotides, at least 500
nucleotides, at least 525 nucleotides, at least 550 nucleotides, at
least 575 nucleotides, at least 600 nucleotides, at least 625
nucleotides, at least 650 nucleotides, at least 675 nucleotides, at
least 700 nucleotides, at least 725 nucleotides, at least 750
nucleotides, at least 775 nucleotides, at least 800 nucleotides, at
least 825 nucleotides, at least 850 nucleotides, at least 875
nucleotides, at least 900 nucleotides, at least 925 nucleotides, at
least 950 nucleotides, at least 975 nucleotides, or at least 1,000
nucleotides. In some instances, the average read length may be a
range bounded by any two of the values within this range, e.g., an
average read length ranging from 375 nucleotides to 825
nucleotides. Those of skill in the art will recognize that in some
instances, the average read length may have any value within the
range specified in this paragraph, e.g., 523 nucleotides.
[0258] In some instances, the use of multivalent binding
compositions described herein for sequencing effectively shortens
the sequencing time. The sequencing reaction cycle comprising the
contacting, detecting, and incorporating steps is performed in a
total time ranging from about 5 minutes to about 60 minutes. In
some instances, the sequencing reaction cycle is performed in at
least 5 minutes, at least 10 minutes, at least 20 minutes, at least
30 minutes, at least 40 minutes, at least 50 minutes, or at least
60 minutes. In some instances, the sequencing reaction cycle is
performed in at most 60 minutes, at most 50 minutes, at most 40
minutes, at most 30 minutes, at most 20 minutes, at most 10
minutes, or at most 5 minutes. Any of the lower and upper values
described in this paragraph may be combined to form a range
included within the present disclosure, for example, in some
instances the sequencing reaction cycle may be performed in a total
time ranging from about 10 minutes to about 30 minutes. Those of
skill in the art will recognize that the sequencing cycle time may
have any value within this range, e.g., about 16 minutes.
[0259] In some instances, the disclosed compositions and methods
for nucleic acid sequencing will provide an average base-calling
accuracy of at least 80%, at least 85%, at least 90%, at least 92%,
at least 94%, at least 96%, at least 98%, at least 99%, at least
99.5%, at least 99.8%, or at least 99.9% correct over the course of
a sequencing run. In some instances, the disclosed compositions and
methods for nucleic acid sequencing will provide an average
base-calling accuracy of at least 80%, at least 85%, at least 90%,
at least 92%, at least 94%, at least 96%, at least 98%, at least
99%, at least 99.5%, at least 99.8%, or at least 99.9% correct per
every 1,000 bases, 10,0000 bases, 25,000 bases, 50,000 bases,
75,000 bases, or 100,000 bases called.
[0260] In some instances, the use of multivalent binding
compositions disclosed herein for sequencing provides more accurate
base readout. In some instances, the disclosed compositions and
methods for nucleic acid sequencing will provide an average Q-score
for base-calling accuracy over a sequencing run that ranges from
about 20 to about 50. In some instances, the average Q-score is at
least 20, at least 25, at least 30, at least 35, at least 40, at
least 45, or at least 50. Those of skill in the art will recognize
that the average Q-score may have any value within this range,
e.g., about 32.
[0261] In some instances, the disclosed compositions and methods
for nucleic acid sequencing will provide a Q-score of greater than
30 for at least 50%, at least 60%, at least 70%, at least 80%, at
least 85%, at least 90%, at least 95%, at least 98%, or at least
99% of the terminal (or N+1) nucleotides identified. In some
instances, the disclosed compositions and methods for nucleic acid
sequencing will provide a Q-score of greater than 35 for at least
50%, at least 60%, at least 70%, at least 80%, at least 85%, at
least 90%, at least 95%, at least 98%, or at least 99% of the
terminal (or N+1) nucleotides identified. In some instances, the
disclosed compositions and methods for nucleic acid sequencing will
provide a Q-score of greater than 40 for at least 50%, at least
60%, at least 70%, at least 80%, at least 85%, at least 90%, at
least 95%, at least 98%, or at least 99% of the terminal (or N+1)
nucleotides identified. In some instances, the disclosed
compositions and methods for nucleic acid sequencing will provide a
Q-score of greater than 45 for at least 50%, at least 60%, at least
70%, at least 80%, at least 85%, at least 90%, at least 95%, at
least 98%, or at least 99% of the terminal (or N+1) nucleotides
identified. In some instances, the disclosed compositions and
methods for nucleic acid sequencing will provide a Q-score of
greater than 50 for at least 50%, at least 60%, at least 70%, at
least 80%, at least 85%, at least 90%, at least 95%, at least 98%,
or at least 99% of the terminal (or N+1) nucleotides
identified.
[0262] The disclosed low non-specific binding supports and
associated nucleic acid hybridization and amplification methods may
be used for the analysis of nucleic acid molecules derived from any
of a variety of different cell, tissue, or sample types known to
those of skill in the art. For example, nucleic acids may be
extracted from cells, or tissue samples comprising one or more
types of cells, derived from eukaryotes (such as animals, plants,
fungi, or protista), archaebacteria, or eubacteria. In some
instances, nucleic acids may be extracted from prokaryotic or
eukaryotic cells, such as adherent or non-adherent eukaryotic
cells. Nucleic acids are variously extracted from, for example,
primary or immortalized rodent, porcine, feline, canine, bovine,
equine, primate, or human cell lines. Nucleic acids may be
extracted from any of a variety of different cell, organ, or tissue
types (e.g., white blood cells, red blood cells, platelets,
epithelial cells, endothelial cells, neurons, glial cells,
astrocytes, fibroblasts, skeletal muscle cells, smooth muscle
cells, gametes, or cells from the heart, lungs, brain, liver,
kidney, spleen, pancreas, thymus, bladder, stomach, colon, or small
intestine). Nucleic acids may be extracted from normal or healthy
cells. In another example, or in combination, nucleic acids are
extracted from diseased cells, such as cancerous cells, or from
pathogenic cells that are infecting a host. Some nucleic acids may
be extracted from a distinct subset of cell types, e.g., immune
cells (such as T cells, cytotoxic (killer) T cells, helper T cells,
alpha beta T cells, gamma delta T cells, T cell progenitors, B
cells, B-cell progenitors, lymphoid stem cells, myeloid progenitor
cells, lymphocytes, granulocytes, Natural Killer cells, plasma
cells, memory cells, neutrophils, eosinophils, basophils, mast
cells, monocytes, dendritic cells, and/or macrophages, or any
combination thereof), undifferentiated human stem cells, human stem
cells that have been induced to differentiate, rare cells (e.g.,
circulating tumor cells (CTCs), circulating epithelial cells,
circulating endothelial cells, circulating endometrial cells, bone
marrow cells, progenitor cells, foam cells, mesenchymal cells, or
trophoblasts). Nucleic acids may further comprise nucleic acids
derived from viral samples and from subviral pathogens, such as
viroids and infectious RNAs. Nucleic acids may be derived from
clinical or other samples, such as sputum, saliva, ocular fluid,
synovial fluid, blood, feces, urine, tissue exudate, sweat, pus,
drainage fluid or the like. Nucleic acids may further be derived
from plant or fungal samples, such as leaf, cambium, root,
meristem, pollen, ovum, seed, spore, inflorescence, mycelium, or
the like. Nucleic acids may also be derived from environmental or
industrial samples, such as water, air, dust, food, or the like.
Other cells, tissues, and samples are contemplated and consistent
with the disclosure herein.
[0263] Nucleic acid extraction from cells or other biological
samples may be performed using any of a number of techniques known
to those of skill in the art. For example, a DNA extraction
procedure may comprise (i) collection of the cell sample or tissue
sample from which DNA is to be extracted, (ii) disruption of cell
membranes (i.e., cell lysis) to release DNA and other cytoplasmic
components, (iii) treatment of the lysed sample with a concentrated
salt solution to precipitate proteins, lipids, and RNA, followed by
centrifugation to separate out the precipitated proteins, lipids,
and RNA, and (iv) purification of DNA from the supernatant to
remove detergents, proteins, salts, or other reagents used during
the cell membrane lysis step.
[0264] A variety of suitable commercial nucleic acid extraction and
purification kits are consistent with the disclosure herein.
Examples include, but are not limited to, the QIAamp kits (for
isolation of genomic DNA from human samples) and DNAeasy kits (for
isolation of genomic DNA from animal or plant samples) from Qiagen
(Germantown, Md.), or the Maxwell.RTM. and ReliaPrep.TM. series of
kits from Promega (Madison, Wis.).
Illustrative Embodiment 2
[0265] Provided herein are methods for attaching a target nucleic
acid molecule to a surface, the methods comprising: bringing a
mixture comprising said target nucleic acid molecule at a
concentration of 1 nanomolar or less in contact with a hydrophilic
surface comprising a capture probe coupled thereto under conditions
sufficient for said target nucleic acid molecule to be captured by
said capture probe in a time period of less than 30 minutes.
[0266] In some instances, said mixture comprises a polar aprotic
solvent. In some instances, the polar aprotic solvent comprises
formamide. In some instances, said capture probe is a nucleic acid
molecule. In some instances, said concentration is 0.50 nanomolar
or less. In some instances, said concentration is 250 picomolar or
less. In some instances, said concentration is 100 picomolar or
less. In some instances, said time period is less than or equal to
20 minutes. In some instances, said time period is less than or
equal to 15 minutes. In some instances, said time period is less
than or equal to 10 minutes. In some instances, said time period is
less than or equal to 5 minutes.
[0267] In some instances, said hydrophilic surface is maintained at
a temperature of about 30 degrees Celsius to about 70 degrees
Celsius. In some instances, said hydrophilic surface is maintained
at a substantially constant temperature. In some instances, methods
further comprise hybridizing the target nucleic acid molecule to
the capture probe at a hybridization efficiency that is increased
as compared to a comparable hybridization reaction performed for
120 minutes at 90 degrees Celsius for 5 minutes followed by cooling
for 120 minutes to reach a final temperature of 37 degrees Celsius
in a buffer composition comprising saline-sodium citrate. In some
instances, methods further comprise hybridizing the target nucleic
acid molecule to the capture probe with a hybridization stringency
of at least 80%.
[0268] In some instances, the hydrophilic surface exhibits a level
of non-specific Cyanine 3 dye absorption of less than about 0.25
molecules per square micrometer. In some instances, the mixture
further comprises a pH buffer comprising
2-(N-morpholino)ethanesulfonic acid, acetonitrile,
3-(N-morpholino)propanesulfonic acid, methanol, or a combination
thereof. In some instances, the mixture further comprises a
crowding agent selected from the group consisting of polyethylene
glycol, dextran, hydroxypropyl methyl cellulose, hydroxyethyl
methyl cellulose, hydroxybutyl methyl cellulose, hydroxypropyl
cellulose, methyl cellulose, and hydroxyl methyl cellulose, and any
combination thereof. In some instances, the hydrophilic surface
comprises one or more hydrophilic polymer layers. In some
instances, the one or more hydrophilic polymer layers comprises a
molecule selected from the group consisting of polyethylene glycol
(PEG), poly(vinyl alcohol) (PVA), poly(vinyl pyridine), poly(vinyl
pyrrolidone) (PVP), poly(acrylic acid) (PAA), polyacrylamide,
poly(N-isopropylacrylamide) (PNIPAM), poly(methyl methacrylate)
(PMA), poly(2-hydroxylethyl methacrylate) (PHEMA),
poly(oligo(ethylene glycol) methyl ether methacrylate) (POEGMA),
polyglutamic acid (PGA), poly-lysine, poly-glucoside, streptavidin,
and dextran. In some instances, the one or more hydrophilic polymer
layers comprises at least one dendrimer.
[0269] Provided herein are methods for hybridizing a target nucleic
acid molecule to a nucleic acid molecule coupled to a hydrophilic
polymer surface, the method comprising: (a) providing at least one
nucleic acid molecule that is coupled to a hydrophilic polymer
surface; and (b) bringing the at least one nucleic acid molecule
coupled to the polymer surface into contact with a hybridizing
composition comprising a target nucleic acid molecule at a
concentration of 1 nanomolar or less under conditions sufficient
for said target nucleic acid molecule to hybridize to the at least
one nucleic acid molecule coupled to the polymer surface in 30
minutes or less. In some instances, said conditions are maintained
at a substantially constant temperature.
[0270] In some instances, the hydrophilic polymer surface has a
water contact angle of less than 45 degrees. In some instances, the
target nucleic acid molecule is present in the hybridizing
composition at a concentration of 0.50 nanomolar or less. In some
instances, the target nucleic acid molecule is present in the
hybridizing composition at a concentration of 250 picomolar or
less. In some instances, the target nucleic acid molecule is
present in the hybridizing composition at a concentration of 100
picomolar or less. In some instances, bringing the at least one
nucleic acid molecule coupled to the polymer surface into contact
with the hybridization composition is performed for a time period
of less than 30 minutes. In some instances, the time period is less
than 20 minutes. In some instances, the time period is less than 15
minutes. In some instances, the time period is less than 10
minutes. In some instances, the time period is less than 5
minutes.
[0271] In some instances, methods further comprise hybridizing the
target nucleic acid molecule to the at least one nucleic molecule
coupled to the polymer surface at a hybridization efficiency that
is increased as compared to a comparable hybridization reaction
performed for 120 minutes at 90 degrees Celsius for 5 minutes
followed by cooling for 120 minutes to reach a final temperature of
37 degrees Celsius in a buffer comprising saline-sodium citrate. In
some instances, the temperature is from about 30 degrees Celsius to
70 degrees Celsius. In some instances, the temperature is about 50
degrees Celsius. In some instances, methods further comprise
hybridizing the target nucleic acid molecule to the at least one
nucleic acid molecule with a hybridization stringency of at least
80%. In some instances, the hydrophilic polymer surface exhibits a
level of non-specific Cyanine 3 dye absorption of less than about
0.25 molecules per square micrometer.
[0272] In some instances, the hybridization composition further
comprises: (a) at least one organic solvent having a dielectric
constant of no greater than about 115 as measured at 68 degrees
Fahrenheit; and (b) a pH buffer. In some instances, the
hybridization composition further comprises: (a) at least one
organic solvent that is polar and aprotic; and (b) a pH buffer. In
some instances, the at least one organic solvent comprises at least
one functional group selected from hydroxy, nitrile, lactone,
sulfone, sulfite, and carbonate. In some instances, the at least
one organic solvent comprises formamide. In some instances, the at
least one organic solvent is miscible with water. In some
instances, the at least one organic solvent is at least about 5% by
volume based on the total volume of the hybridizing composition. In
some instances, the at least one organic solvent is at most about
95% by volume based on the total volume of the hybridizing
composition.
[0273] In some instances, the pH buffer is at most about 90% by
volume of the total volume of the hybridizing composition. In some
instances, the pH buffer comprises 2-(N-morpholino)ethanesulfonic
acid, acetonitrile, 3-(N-morpholino)propanesulfonic acid, methanol,
or a combination thereof. In some instances, the pH buffer further
comprises a second organic solvent. In some instances, the pH
buffer is present in the hybridizing composition in an amount that
is effective to maintain the pH of the hybridizing composition in a
range of about 3 to about 10.
[0274] In some instances, the hybridizing composition further
comprises a molecular crowding agent. In some instances, the
molecular crowding agent is selected from the group consisting of
polyethylene glycol, dextran, hydroxypropyl methyl cellulose,
hydroxyethyl methyl cellulose, hydroxybutyl methyl cellulose,
hydroxypropyl cellulose, methyl cellulose, and hydroxyl methyl
cellulose, and any combination thereof. In some instances, the
molecular crowding agent is polyethylene glycol. In some instances,
the molecular crowding agent has a molecular weight in the range of
about 5,000 to 40,000 Daltons. In some instances, an amount of the
molecular crowding agent is at least about 5% by volume based on
the total volume of the hybridizing composition. In some instances,
an amount of the molecular crowding agent is at most about 50% by
volume based on the total volume of the hybridizing composition. In
some instances, the at least one nucleic acid molecule coupled to
the polymer surface is coupled to the polymer surface through
covalent bonding.
[0275] In some instances, the hydrophilic polymer surface comprises
one or more hydrophilic polymer layers, and the at least one
nucleic acid molecule is coupled to the one or more hydrophilic
polymer layers. In some instances, the one or more hydrophilic
polymer layers comprises a molecule selected from the group
consisting of polyethylene glycol (PEG), poly(vinyl alcohol) (PVA),
poly(vinyl pyridine), poly(vinyl pyrrolidone) (PVP), poly(acrylic
acid) (PAA), polyacrylamide, poly(N-isopropylacrylamide) (PNIPAM),
poly(methyl methacrylate) (PMA), poly(2-hydroxylethyl methacrylate)
(PHEMA), poly(oligo(ethylene glycol) methyl ether methacrylate)
(POEGMA), polyglutamic acid (PGA), poly-lysine, poly-glucoside,
streptavidin, and dextran. In some instances, the one or more
hydrophilic polymer layers comprises at least one dendrimer.
[0276] Provided herein are methods of attaching a target nucleic
acid to a surface, comprising: (a) providing at least one surface
bound nucleic acid that is attached to a polymer surface having a
water contact angle of less than 45 degrees; and (b) bringing the
surface bound nucleic acid into contact with a hybridizing
composition under isothermal conditions, wherein the hybridizing
composition comprises: (i) the target nucleic acid; (ii) at least
one organic solvent having a dielectric constant of no greater than
about 115 when measured at 68 degrees Fahrenheit; and (iii) a pH
buffer.
[0277] In some instances, the organic solvent is a polar aprotic
solvent. In some instances, the organic solvent is an organic
solvent having a dielectric constant of no greater than 40 when
measured at 68 degrees Fahrenheit. In some instances, the organic
solvent is acetonitrile, alcohol, or formamide. In some instances,
the organic solvent comprises at least one functionality selected
from hydroxy, nitrile, lactone, sulfone, sulfite, and carbonate. In
some instances, the organic solvent is miscible with water. In some
instances, the organic solvent is present in an amount effective to
denature a double stranded nucleic acid. In some instances, an
amount of the organic solvent is at least about 5% by volume based
on the total volume of the hybridizing composition. In some
instances, an amount of the organic solvent is in the range of
about 5% to 95% by volume based on the total volume of the
hybridizing composition. In some instances, an amount of the pH
buffer is no greater than 90% by volume based on the total volume
of the hybridizing composition. In some instances, the hybridizing
composition further comprises a molecular crowding agent. In some
instances, the molecular crowding agent is selected from the group
consisting of polyethylene glycol (PEG), dextran, hydroxypropyl
methyl cellulose (HPMC), hydroxyethyl methyl cellulose (HEMC),
hydroxybutyl methyl cellulose, hydroxypropyl cellulose,
methylcellulose, and hydroxyl methyl cellulose, and any combination
thereof. In some instances, the molecular crowding agent is
polyethylene glycol (PEG). In some instances, the molecular
crowding agent has a molecular weight in the range of about 5,000
to 40,000 Daltons. In some instances, an amount of the molecular
crowding agent is at least about 5% by volume based on the total
volume of the hybridizing composition. In some instances, an amount
of the molecular crowding agent is less than 50% by volume based on
the total volume of the hybridizing composition. In some instances,
methods further comprise an additive for controlling a melting
temperature of the target nucleic acid. In some instances, an
amount of the additive for controlling melting temperature of the
target nucleic acid is at least about 2% by volume based on the
total volume of the hybridizing composition. In some instances, an
amount of the additive for controlling melting temperature of the
nucleic acid is in the range of about 2% to 50% by volume based on
the total volume of the hybridizing composition. In some instances,
the pH buffer comprises at least one buffering agent selected from
the group consisting of Tris, HEPES (e.g.,
4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), TAPS (e.g.,
[tris(hydroxymethyl)methylamino]propanesulfonic acid), Tricine,
Bicine, Bis-Tris, sodium hydroxide (NaOH), potassium hydroxide
(KOH), TES (e.g.,
2-[[1,3-dihydroxy-2-(hydroxymethyl)propan-2-yl]amino]ethanesulfonic
acid), EPPS (e.g., 4-(2-Hydroxyethyl)-1-piperazinepropanesulfonic
acid, 4-(2-Hydroxyethyl)piperazine-1-propanesulfonic acid,
N-(2-Hydroxyethyl)piperazine-N'-(3-propanesulfonic acid)), and MOPS
(e.g., 3-(N-morpholino)propanesulfonic acid). In some instances,
the pH buffer further comprises a second organic solvent. In some
instances, the pH buffer comprises MOPS and methanol. In some
instances, an amount of the pH buffer is effective to maintain the
pH of the hybridizing composition to be in the range of about 3 to
about 10.
[0278] In some instances, the surface bound nucleic acid is coupled
to the surface through covalent or noncovalent bonding. In some
instances, the polymer surface comprises one or more hydrophilic
polymer layers, and the surface bound nucleic acid is coupled to
the one or more hydrophilic polymer layers. In some instances, no
more than 10% of the target nucleic acid is associated with the
surface without hybridizing to the polymer surface bound nucleic
acid. In some instances, the polymer surface exhibits a level of
non-specific cyanine 3 (Cy3) dye absorption of less than about 0.25
molecules per micrometer squared (.mu.m.sup.2). In some instances,
the one or more hydrophilic polymer layers comprises a molecule
selected from the group consisting of polyethylene glycol (PEG),
poly(vinyl alcohol) (PVA), poly(vinyl pyridine), poly(vinyl
pyrrolidone) (PVP), poly(acrylic acid) (PAA), polyacrylamide,
poly(N-isopropylacrylamide) (PNIPAM), poly(methyl methacrylate)
(PMA), poly(2-hydroxylethyl methacrylate) (PHEMA),
poly(oligo(ethylene glycol) methyl ether methacrylate) (POEGMA),
polyglutamic acid (PGA), poly-lysine, poly-glucoside, streptavidin,
and dextran. In some instances, the one or more hydrophilic polymer
layers comprise at least one dendrimer.
[0279] In some instances, bringing the surface bound nucleic acid
into contact with the hybridizing composition is performed for a
period of no more than 25 minutes. In some instances, bringing the
surface bound nucleic acid into contact with the hybridizing
composition is performed for a period of no more than 15 minutes.
In some instances, bringing the surface bound nucleic acid into
contact with the hybridizing composition is performed for a period
between 2-25 minutes. In some instances, the isothermal conditions
are at a temperature in the range of about 30 to 70 degrees
Celsius. In some instances, attaching the target nucleic acid
molecule to the surface comprises hybridizing the target nucleic
acid to the surface bound nucleic acid with a hybridization
stringency of at least 80%. In some instances, attaching the target
nucleic acid molecule to the surface comprises hybridizing the
target nucleic acid to the surface bound nucleic with an increased
hybridization efficiency, as compared to a comparable hybridization
reaction wherein the organic buffer is a saline-sodium citrate and
hybridizing is performed for 120 minutes at 90 degrees Celsius for
5 minutes followed by cooling for 120 minutes to reach a final
temperature of 37 degrees Celsius. In some instances, the target
nucleic acid is present in the hybridizing composition at a 1
nanomolar concentration or less. In some instances, the target
nucleic acid is present in the hybridizing composition at a 250
picomolar concentration or less. In some instances, the target
nucleic acid is present in the hybridizing composition at a 100
picomolar concentration or less. In some instances, the target
nucleic acid is present in the hybridizing composition at a 50
picomolar concentration or less. In some instances, methods further
comprise hybridizing at least a portion of the surface bound
nucleic acid to at least a portion of the target nucleic acid in
the hybridizing composition, wherein hybridizing does not consist
of cooling.
[0280] Provided herein are methods of hybridization, the methods
comprising: (a) providing at least one surface bound nucleic acid
molecule coupled to a surface; and (b) bringing the at least one
surface bound nucleic acid molecule into contact with a hybridizing
composition comprising a target nucleic acid molecule, wherein the
hybridizing composition comprises: (i) at least one organic
solvent; and (ii) a pH buffer. In some instances, the surface
exhibits a level of non-specific Cy3 dye absorption corresponding
to less than about 0.25 molecules/.mu.m.sup.2 when measured by a
fluorescence imaging system under non-signal saturating conditions.
In some instances, no more than 5% of a total number of the target
nucleic acid molecule is associated with the surface without
hybridizing to the surface bound nucleic acid molecule.
[0281] In some instances, the surface bound nucleic acid molecule
is coupled to the surface by being tethered to the surface. In some
instances, the surface is a hydrophilic polymer surface. In some
instances, the surface has a water contact angle of less than 45
degrees. In some instances, the at least one organic solvent has a
dielectric constant of no greater than about 115 when measured at
68 degrees Fahrenheit. In some instances, the organic solvent is a
polar aprotic solvent. In some instances, the organic solvent is an
organic solvent having a dielectric constant of no greater than 40
when measured at 68 degrees Fahrenheit. In some instances, the
organic solvent is acetonitrile, alcohol, or formamide. In some
instances, the organic solvent comprises at least one functionality
selected from hydroxy, nitrile, lactone, sulfone, sulfite, and
carbonate. In some instances, the organic solvent is miscible with
water. In some instances, the organic solvent is present in an
amount effective to denature a double stranded nucleic acid. In
some instances, an amount of the organic solvent is at least about
5% by volume based on the total volume of the hybridizing
composition. In some instances, an amount of the organic solvent is
in the range of about 5% to 95% by volume based on the total volume
of the hybridizing composition. In some instances, an amount of the
pH buffer is no greater than 90% by volume based on the total
volume of the hybridizing composition. In some instances, the
hybridizing composition further comprises a molecular crowding
agent. In some instances, the molecular crowding agent is selected
from the group consisting of polyethylene glycol (PEG), dextran,
hydroxypropyl methyl cellulose (HPMC), hydroxyethyl methyl
cellulose (HEMC), hydroxybutyl methyl cellulose, hydroxypropyl
cellulose, methylcellulose, and hydroxyl methyl cellulose, and any
combination thereof. In some instances, the molecular crowding
agent is polyethylene glycol (PEG). In some instances, the
molecular crowding agent has a molecular weight in the range of
about 5,000 to 40,000 Daltons. In some instances, an amount of the
molecular crowding agent is at least about 5% by volume based on
the total volume of the hybridizing composition. In some instances,
an amount of the molecular crowding agent is less than 50% by
volume based on the total volume of the hybridizing composition. In
some instances, methods described herein further comprise an
additive for controlling a melting temperature of the target
nucleic acid. In some instances, an amount of the additive for
controlling melting temperature of the target nucleic acid is at
least about 2% by volume based on the total volume of the
hybridizing composition. In some instances, an amount of the
additive for controlling melting temperature of the nucleic acid is
in the range of about 2% to 50% by volume based on the total volume
of the hybridizing composition. In some instances, the pH buffer
comprises at least one buffering agent selected from the group
consisting of Tris, HEPES, TAPS, Tricine, Bicine, Bis-Tris, sodium
hydroxide (NaOH), potassium hydroxide (KOH), TES, EPPS, and MOPS.
In some instances, the pH buffer further comprises a second organic
solvent. In some instances, the pH buffer comprises MOPS and
methanol. In some instances, an amount of the pH buffer is
effective to maintain the pH of the hybridizing composition to be
in the range of about 3 to about 10. In some instances, the surface
bound nucleic acid is coupled to the surface through covalent or
noncovalent bonding. In some instances, the polymer surface
comprises one or more hydrophilic polymer layers, and the surface
bound nucleic acid is coupled to the one or more hydrophilic
polymer layers. In some instances, no more than 10% of the target
nucleic acid is associated with the surface without hybridizing to
the polymer surface bound nucleic acid. In some instances, the one
or more hydrophilic polymer layers comprises a molecule selected
from the group consisting of polyethylene glycol (PEG), poly(vinyl
alcohol) (PVA), poly(vinyl pyridine), poly(vinyl pyrrolidone)
(PVP), poly(acrylic acid) (PAA), polyacrylamide,
poly(N-isopropylacrylamide) (PNIPAM), poly(methyl methacrylate)
(PMA), poly(2-hydroxylethyl methacrylate) (PHEMA),
poly(oligo(ethylene glycol) methyl ether methacrylate) (POEGMA),
polyglutamic acid (PGA), poly-lysine, poly-glucoside, streptavidin,
and dextran. In some instances, the one or more hydrophilic polymer
layers comprises at least one dendrimer.
[0282] In some instances, bringing the surface bound nucleic acid
molecule into contact with the hybridizing composition is performed
for a period of no more than 25 minutes. In some instances,
bringing the surface bound nucleic acid molecule into contact with
the hybridizing composition is performed for a period of no more
than 15 minutes. In some instances, bringing the surface bound
nucleic acid molecule into contact with the hybridizing composition
is performed for a period of between 2-25 minutes. In some
instances, the isothermal conditions are at a temperature in the
range of about 30 to 70 degrees Celsius. In some instances,
attaching the target nucleic acid molecule to the surface comprises
hybridizing the target nucleic acid molecule to the surface bound
nucleic acid molecule with a hybridization stringency of at least
80%. In some instances, attaching the target nucleic acid molecule
to the surface comprises hybridizing the target nucleic acid
molecule to the surface bound nucleic acid molecule with an
increased hybridization efficiency, as compared to a comparable
hybridization reaction wherein the organic buffer is a
saline-sodium citrate and hybridizing is performed for 120 minutes
at 90 degrees Celsius for 5 minutes followed by cooling for 120
minutes to reach a final temperature of 37 degrees Celsius. In some
instances, the target nucleic acid molecule is present in the
hybridizing composition at a 1 nanomolar concentration or less. In
some instances, the target nucleic acid is present in the
hybridizing composition at a 250 picomolar concentration or less.
In some instances, the target nucleic acid molecule is present in
the hybridizing composition at a 100 picomolar concentration or
less. In some instances, the target nucleic acid molecule is
present in the hybridizing composition at a 50 picomolar
concentration or less. In some instances, methods further comprise
hybridizing at least a portion of the surface bound nucleic acid
molecule to at least a portion of the target nucleic acid molecule
in the hybridizing composition, wherein hybridizing does not
consist of cooling. In some instances, bringing the surface bound
nucleic acid into contact with the hybridizing composition
comprising the target nucleic acid is performed under conditions of
stringency that prevent the target nucleic acid molecule from
hybridizing to a non-complementary nucleic acid molecule. In some
instances, the stringency is at least or about 70%, 80%, or 90%. In
some instances, the stringency is at least 80%. Provided herein are
methods of attaching a target nucleic acid molecule to a surface,
the method comprising: (a) providing at least one surface bound
nucleic acid molecule, wherein the at least one surface bound
nucleic acid molecule is coupled to a surface; and (b) bringing a
hybridizing composition comprising a target nucleic acid molecule
into contact with the at least one surface bound nucleic acid
molecule, wherein the hybridizing composition comprises: (i) at
least one organic solvent; and (ii) a pH buffer. In some instances,
the surface exhibits a level of non-specific Cy3 dye absorption of
less than about 0.25 molecules/.mu.m.sup.2. In some instances, no
more than 5% of a total number of the target nucleic acid molecule
is associated with the surface without hybridizing to the surface
bound nucleic acid molecule. In some instances, bringing the
hybridizing composition into contact with the at least one surface
bound nucleic acid molecule is performed under isothermal
conditions. In some instances, the surface bound nucleic acid
molecule is coupled to the surface by being tethered to the
surface. In some instances, the surface is a hydrophilic polymer
surface. In some instances, the surface has a water contact angle
of less than 45 degrees.
[0283] In some instances, the at least one organic solvent has a
dielectric constant of no greater than about 115 when measured at
68 degrees Fahrenheit. In some instances, the organic solvent is a
polar aprotic solvent. In some instances, the organic solvent is an
organic solvent having a dielectric constant of no greater than 40
when measured at 70 degrees Fahrenheit. In some instances, the
organic solvent is acetonitrile, alcohol, or formamide. In some
instances, the organic solvent comprises at least one functionality
selected from hydroxy, nitrile, lactone, sulfone, sulfite, and
carbonate. In some instances, the organic solvent is miscible with
water. In some instances, the organic solvent is present in an
amount effective to denature a double stranded nucleic acid. In
some instances, an amount of the organic solvent is at least about
5% by volume based on the total volume of the hybridizing
composition. In some instances, an amount of the organic solvent is
in the range of about 5% to 95% by volume based on the total volume
of the hybridizing composition. In some instances, an amount of the
pH buffer is no greater than 90% by volume based on the total
volume of the hybridizing composition. In some instances, the
hybridizing composition further comprises a molecular crowding
agent. In some instances, the molecular crowding agent is selected
from the group consisting of polyethylene glycol (PEG), dextran,
hydroxypropyl methyl cellulose (HPMC), hydroxyethyl methyl
cellulose (HEMC), hydroxybutyl methyl cellulose, hydroxypropyl
cellulose, methylcellulose, and hydroxyl methyl cellulose, and any
combination thereof. In some instances, the molecular crowding
agent is polyethylene glycol (PEG). In some instances, the
molecular crowding agent has a molecular weight in the range of
about 5,000 to 40,000 Daltons. In some instances, an amount of the
molecular crowding agent is at least about 5% by volume based on
the total volume of the hybridizing composition. In some instances,
an amount of the molecular crowding agent is less than 50% by
volume based on the total volume of the hybridizing composition. In
some instances, methods further comprise an additive for
controlling a melting temperature of the target nucleic acid. In
some instances, an amount of the additive for controlling the
melting temperature of the target nucleic acid molecule is at least
about 2% by volume based on the total volume of the hybridizing
composition. In some instances, an amount of the additive for
controlling the melting temperature of the nucleic acid is in the
range of about 2% to 50% by volume based on the total volume of the
hybridizing composition. In some instances, the pH buffer comprises
at least one buffering agent selected from the group consisting of
Tris, HEPES, TAPS, Tricine, Bicine, Bis-Tris, sodium hydroxide
(NaOH), potassium hydroxide (KOH), TES, EPPS, and MOPS. In some
instances, the pH buffer further comprises a second organic
solvent. In some instances, the pH buffer comprises MOPS and
methanol. In some instances, an amount of the pH buffer is
effective to maintain the pH of the hybridizing composition to be
in the range of about 3 to about 10.
[0284] In some instances, the surface bound nucleic acid molecule
is coupled to the surface through covalent or noncovalent bonding.
In some instances, the polymer surface comprises one or more
hydrophilic polymer layers, wherein the surface bound nucleic acid
is coupled to the one or more hydrophilic polymer layers. In some
instances, no more than 10% of the total number of the target
nucleic acid molecule is associated with the surface without
hybridizing to the polymer surface bound nucleic acid molecule. In
some instances, the one or more hydrophilic polymer layers
comprises a molecule selected from the group consisting of
polyethylene glycol (PEG), poly(vinyl alcohol) (PVA), poly(vinyl
pyridine), poly(vinyl pyrrolidone) (PVP), poly(acrylic acid) (PAA),
polyacrylamide, poly(N-isopropylacrylamide) (PNIPAM), poly(methyl
methacrylate) (PMA), poly(2-hydroxylethyl methacrylate) (PHEMA),
poly(oligo(ethylene glycol) methyl ether methacrylate) (POEGMA),
polyglutamic acid (PGA), poly-lysine, poly-glucoside, streptavidin,
and dextran. In some instances, the one or more hydrophilic polymer
layers comprises at least one dendrimer. In some instances,
bringing the surface bound nucleic acid molecule into contact with
the hybridizing composition is performed for a period of no more
than 25 minutes. In some instances, bringing the surface bound
nucleic acid molecule into contact with the hybridizing composition
is performed for a period of no more than 15 minutes. In some
instances, bringing the surface bound nucleic acid molecule into
contact with the hybridizing composition is performed for a period
between 2-25 minutes. In some instances, the isothermal conditions
are at a temperature in the range of about 30 to 70 degrees
Celsius. In some instances, attaching the target nucleic acid
molecule to the surface comprises hybridizing the target nucleic
acid molecule to the surface bound nucleic molecule with a
hybridization stringency of at least 80%. In some instances,
attaching the target nucleic acid molecule to the surface comprises
hybridizing the target nucleic acid molecule to the surface bound
nucleic acid molecule with an increased hybridization efficiency,
as compared to a comparable hybridization reaction wherein the
organic buffer is a saline-sodium citrate and hybridizing is
performed for 120 minutes at 90 degrees Celsius for 5 minutes
followed by cooling for 120 minutes to reach a final temperature of
37 degrees Celsius. In some instances, the target nucleic acid
molecule is present in the hybridizing composition at a 1 nanomolar
concentration or less. In some instances, the target nucleic acid
molecule is present in the hybridizing composition at a 250
picomolar concentration or less. In some instances, the target
nucleic acid molecule is present in the hybridizing composition at
a 100 picomolar concentration or less. In some instances, the
target nucleic acid molecule is present in the hybridizing
composition at a 50 picomolar concentration or less. In some
instances, methods further comprise hybridizing at least a portion
of the surface bound nucleic acid molecule to at least a portion of
the target nucleic acid molecule in the hybridizing composition,
wherein hybridizing does not consist of cooling.
[0285] Provided herein are methods of sequencing a target nucleic
acid molecule, the methods comprising: (a) bringing a surface bound
nucleic acid molecule coupled to a surface into contact with a
hybridizing composition comprising a target nucleic acid molecule,
wherein the hybridizing composition comprises: (i) at least one
organic solvent; and (ii) a pH buffer; (b) amplifying the target
nucleic acid molecule to form a plurality of clonally-amplified
clusters of the target nucleic acid; and (c) determining the
identity of the target nucleic acid molecule, wherein a
fluorescence image of the surface comprising the plurality of
clonally-amplified clusters of the target nucleic acid molecule
exhibits a contrast-to-noise ratio (CNR) of at least 20 when the
fluorescence image is captured using a fluorescence imaging system
under non-signal saturating conditions. In some instances, methods
described herein further comprise hybridizing the target nucleic
acid molecule to the at least one surface bound nucleic acid
coupled to the surface. In some instances, the CNR is at least 50.
In some instances, the organic solvent is a polar aprotic solvent.
In some instances, the organic solvent is an organic solvent having
a dielectric constant of no greater than 40 as measured at 70
degrees Fahrenheit. In some instances, the organic solvent is
acetonitrile, alcohol, or formamide. In some instances, the organic
solvent comprises at least one functionality selected from hydroxy,
nitrile, lactone, sulfone, sulfite, and carbonate. In some
instances, the organic solvent is miscible with water. In some
instances, the organic solvent is present in an amount effective to
denature a double stranded nucleic acid. In some instances, an
amount of the organic solvent is at least about 5% by volume based
on the total volume of the hybridizing composition. In some
instances, an amount of the organic solvent is in the range of
about 5% to 95% by volume based on the total volume of the
hybridizing composition. In some instances, an amount of the pH
buffer is no greater than 90% by volume based on the total volume
of the hybridizing composition. In some instances, the hybridizing
composition further comprises a molecular crowding agent. In some
instances, the molecular crowding agent is selected from the group
consisting of polyethylene glycol (PEG), dextran, hydroxypropyl
methyl cellulose (HPMC), hydroxyethyl methyl cellulose (HEMC),
hydroxybutyl methyl cellulose, hydroxypropyl cellulose,
methylcellulose, and hydroxyl methyl cellulose, and any combination
thereof. In some instances, the molecular crowding agent is
polyethylene glycol (PEG). In some instances, the molecular
crowding agent has a molecular weight in the range of about 5,000
to 40,000 Daltons. In some instances, an amount of the molecular
crowding agent is at least about 5% by volume based on the total
volume of the hybridizing composition. In some instances, an amount
of the molecular crowding agent is less than 50% by volume based on
the total volume of the hybridizing composition. In some instances,
methods described herein further comprise an additive for
controlling a melting temperature of the target nucleic acid
molecule. In some instances, an amount of the additive for
controlling melting temperature of the target nucleic acid is at
least about 2% by volume based on the total volume of the
hybridizing composition. In some instances, an amount of the
additive for controlling melting temperature of the nucleic acid
molecule is in the range of about 2% to 50% by volume based on the
total volume of the hybridizing composition. In some instances, the
pH buffer comprises at least one buffering agent selected from the
group consisting of Tris, HEPES, TAPS, Tricine, Bicine, Bis-Tris,
sodium hydroxide (NaOH), potassium hydroxide (KOH), TES, EPPS, and
MOPS. In some instances, the pH buffer further comprises a second
organic solvent. In some instances, the pH buffer comprises MOPS
and methanol. In some instances, an amount of the pH buffer is
effective to maintain the pH of the hybridizing composition to be
in the range of about 3 to about 10.
[0286] In some instances, the surface bound nucleic acid molecule
is coupled to the surface through covalent or noncovalent bonding.
In some instances, the polymer surface comprises one or more
hydrophilic polymer layers, wherein the surface bound nucleic acid
molecule is coupled to the one or more hydrophilic polymer layers.
In some instances, the polymer surface exhibits a level of
non-specific Cyanine3 (Cy3) dye absorption of less than about 0.25
molecules per micrometer squared (.mu.m.sup.2). In some instances,
no more than 5% of a total number of the target nucleic acid
molecule is associated with the surface without hybridizing to the
surface bound nucleic acid molecule. In some instances, no more
than 10% of the total number of the target nucleic acid molecule is
associated with the surface without hybridizing to the surface
bound nucleic acid molecule. In some instances, the one or more
hydrophilic polymer layers comprises a molecule selected from the
group consisting of polyethylene glycol (PEG), poly(vinyl alcohol)
(PVA), poly(vinyl pyridine), poly(vinyl pyrrolidone) (PVP),
poly(acrylic acid) (PAA), polyacrylamide,
poly(N-isopropylacrylamide) (PNIPAM), poly(methyl methacrylate)
(PMA), poly(2-hydroxylethyl methacrylate) (PHEMA),
poly(oligo(ethylene glycol) methyl ether methacrylate) (POEGMA),
polyglutamic acid (PGA), poly-lysine, poly-glucoside, streptavidin,
and dextran. In some instances, the one or more hydrophilic polymer
layers comprise at least one dendrimer. In some instances, bringing
the surface bound nucleic acid molecule into contact with the
hybridizing composition is performed under isothermal conditions.
In some instances, bringing the surface bound nucleic acid molecule
into contact with the hybridizing composition is performed at a
temperature in the range of about 30 to 70 degrees Celsius. In some
instances, bringing the surface bound nucleic acid molecule into
contact with the hybridizing composition is performed for a period
of no more than 25 minutes. In some instances, methods described
herein further comprise removing the hybridizing composition from
the surface after the period of no more than 25 minutes. In some
instances, bringing the surface bound nucleic acid molecule into
contact with the hybridizing composition is performed for a period
between 2-25 minutes. In some instances, bringing the surface bound
nucleic acid molecule into contact with the hybridizing composition
is performed for a period between 2-4 minutes. In some instances,
bringing the surface bound nucleic acid molecule into contact with
the hybridizing composition is performed for a period of 2 minutes.
In some instances, the at least one surface bound nucleic acid
molecule is circular. In some instances, methods further comprise
hybridizing at least a portion of the surface bound nucleic acid
molecule to at least a portion of the target nucleic acid in the
hybridizing composition, which hybridizing does not consist of
cooling. In some instances, bringing the surface bound nucleic acid
into contact with the hybridizing composition comprising the target
nucleic acid is performed under conditions of stringency that
prevent the target nucleic acid from hybridizing to a
non-complementary nucleic acid. In some instances, the stringency
is at least or about 70%, 80%, or 90%. In some instances, the
stringency is at least 80%.
[0287] Provided herein are compositions for hybridizing a target
nucleic acid molecule to a surface bound nucleic acid molecule, the
compositions comprising: (a) a target nucleic acid molecule; (b) at
least one organic solvent; and (c) a pH buffer. In some instances,
no more than 10% of a total number of the target nucleic acid
molecule is associated with the surface without hybridizing to the
surface bound nucleic acid molecule. In some instances, no more
than 5% of the total number of the target nucleic acid molecule is
bound to the surface without hybridizing to the surface bound
nucleic acid molecule.
[0288] In some instances, the organic solvent is a polar aprotic
solvent. In some instances, the organic solvent is an organic
solvent having a dielectric constant of no greater than 40 when
measured at 70 degrees Fahrenheit. In some instances, the organic
solvent is acetonitrile, alcohol, or formamide. In some instances,
the organic solvent comprises at least one functionality selected
from hydroxy, nitrile, lactone, sulfone, sulfite, and carbonate. In
some instances, the organic solvent is miscible with water. In some
instances, the organic solvent is present in an amount effective to
denature a double stranded nucleic acid. In some instances, an
amount of the organic solvent is at least about 5% by volume based
on the total volume of the composition. In some instances, an
amount of the organic solvent is in the range of about 5% to 95% by
volume based on the total volume of the composition. In some
instances, the pH buffer system comprises a pH buffer. In some
instances, an amount of the pH buffer is no greater than 90% by
volume based on the total volume of the composition. In some
instances, the composition further comprises a molecular crowding
agent. In some instances, the molecular crowding agent is selected
from the group consisting of polyethylene glycol (PEG), dextran,
hydroxypropyl methyl cellulose (HPMC), hydroxyethyl methyl
cellulose (HEMC), hydroxybutyl methyl cellulose, hydroxypropyl
cellulose, methylcellulose, and hydroxyl methyl cellulose, and any
combination thereof. In some instances, the molecular crowding
agent is polyethylene glycol (PEG). In some instances, the
molecular crowding agent has a molecular weight in the range of
about 5,000 to 40,000 Daltons. In some instances, an amount of the
molecular crowding agent is at least about 5% by volume based on
the total volume of the composition. In some instances, an amount
of the molecular crowding agent is less than 50% by volume based on
the total volume of the composition. In some instances, the
compositions for hybridizing a target nucleic acid molecule to a
surface bound nucleic acid molecule further comprise an additive
for controlling a melting temperature of the target nucleic acid
molecule. In some instances, an amount of the additive for
controlling the melting temperature of the target nucleic acid
molecule is at least about 2% by volume based on the total volume
of the composition. In some instances, an amount of the additive
for controlling the melting temperature of the nucleic acid
molecule is in the range of about 2% to 50% by volume based on the
total volume of the composition. In some instances, the pH buffer
comprises at least one buffering agent selected from the group
consisting of Tris, HEPES, TAPS, Tricine, Bicine, Bis-Tris, sodium
hydroxide (NaOH), potassium hydroxide (KOH), TES, EPPS, and MOPS.
In some instances, the pH buffer further comprises a second organic
solvent. In some instances, the pH buffer comprises MOPS and
methanol. In some instances, an amount of the pH buffer is
effective to maintain the pH of the composition to be in the range
of about 3 to about 10.
[0289] In some instances, the surface bound nucleic acid molecule
is coupled to a surface through covalent or noncovalent bonding. In
some instances, the surface is a hydrophilic polymer surface. In
some instances, the polymer surface comprises one or more
hydrophilic polymer layers, wherein the surface bound nucleic acid
molecule is coupled to the one or more hydrophilic polymer layers.
In some instances, the one or more hydrophilic polymer layers
comprises a molecule selected from the group consisting of
polyethylene glycol (PEG), poly(vinyl alcohol) (PVA), poly(vinyl
pyridine), poly(vinyl pyrrolidone) (PVP), poly(acrylic acid) (PAA),
polyacrylamide, poly(N-isopropylacrylamide) (PNIPAM), poly(methyl
methacrylate) (PMA), poly(2-hydroxylethyl methacrylate) (PHEMA),
poly(oligo(ethylene glycol) methyl ether methacrylate) (POEGMA),
polyglutamic acid (PGA), poly-lysine, poly-glucoside, streptavidin,
and dextran. In some instances, the one or more hydrophilic polymer
layers comprises at least one dendrimer. In some instances, the
target nucleic acid molecule is present in the composition at a 1
nanomolar concentration or less. In some instances, the target
nucleic acid molecule is present in the composition at a 250
picomolar concentration or less. In some instances, the target
nucleic acid molecule is present in the composition at a 100
picomolar concentration or less. In some instances, the target
nucleic acid molecule is present in the composition at a 50
picomolar concentration or less.
[0290] Provided herein, in some instances, are microfluidic
systems, comprising the compositions described herein. In some
instances, the microfluidic systems comprise a flow cell device. In
some instances, the flow cell device is a microfluidic chip flow
cell. In some instances, the flow cell device is a capillary flow
cell device. In some instances, at least one surface of the flow
cell device comprises one or more hydrophilic polymer layers
comprising a molecule selected from the group consisting of
polyethylene glycol (PEG), poly(vinyl alcohol) (PVA), poly(vinyl
pyridine), poly(vinyl pyrrolidone) (PVP), poly(acrylic acid) (PAA),
polyacrylamide, poly(N-isopropylacrylamide) (PNIPAM), poly(methyl
methacrylate) (PMA), poly(2-hydroxylethyl methacrylate) (PHEMA),
poly(oligo(ethylene glycol) methyl ether methacrylate) (POEGMA),
polyglutamic acid (PGA), poly-lysine, poly-glucoside, streptavidin,
and dextran. In some instances, the flow cell device comprises a
composition described herein formulated as a fluid. In some
instances, the flow cell device comprises one or more surface bound
nucleic acid molecules coupled to the at least one surface of the
flow cell. In some instances, a target nucleic acid molecule in the
composition is hybridized to the one or more surface bound nucleic
acid molecules coupled to the at least one surface of the flow
cell. In some instances, the flow cell device is operatively
coupled to an imaging system configured to capture an image of the
at least one surface of the flow cell comprising the hybridized
target nucleic acid molecule and the one or more surface bound
nucleic acid molecules. Methods described herein comprise
determining an identity of the target nucleic acid molecule using
the microfluidic systems described herein.
[0291] Provided herein are kits comprising: (a) a surface; and (b)
a composition comprising: (i) at least one organic solvent; and
(ii) a pH buffer. In some instances, the surface comprises one or
more surface bound nucleic acid molecules coupled to the surface.
In some instances, the surface is a hydrophilic polymer surface. In
some instances, the surface has a water contact angle of less than
45 degrees. In some instances, the hydrophilic polymer surface
comprises one or more hydrophilic polymer layers, and wherein the
surface bound nucleic acid is coupled to the one or more
hydrophilic polymer layers. In some instances, the one or more
hydrophilic polymer layers comprises a molecule selected from the
group consisting of polyethylene glycol (PEG), poly(vinyl alcohol)
(PVA), poly(vinyl pyridine), poly(vinyl pyrrolidone) (PVP),
poly(acrylic acid) (PAA), polyacrylamide,
poly(N-isopropylacrylamide) (PNIPAM), poly(methyl methacrylate)
(PMA), poly(2-hydroxylethyl methacrylate) (PHEMA),
poly(oligo(ethylene glycol) methyl ether methacrylate) (POEGMA),
polyglutamic acid (PGA), poly-lysine, poly-glucoside, streptavidin,
and dextran. In some instances, the kit further comprises
instructions for hybridizing the one or more surface bound nucleic
acid molecules to one or more target nucleic acid molecules. In
some instances, the kit further comprises instructions for
determining the identity of the one or more target nucleic acid
molecules.
[0292] In some instances, the organic solvent is a polar aprotic
solvent. In some instances, the organic solvent is an organic
solvent having a dielectric constant of no greater than 40 when
measured at 70 degrees Fahrenheit. In some instances, the organic
solvent is acetonitrile, alcohol, or formamide. In some instances,
the organic solvent comprises at least one functionality selected
from hydroxy, nitrile, lactone, sulfone, sulfite, and carbonate. In
some instances, the organic solvent is miscible with water. In some
instances, the organic solvent is present in an amount effective to
denature a double stranded nucleic acid. In some instances, an
amount of the organic solvent is at least about 5% by volume based
on the total volume of the composition. In some instances, an
amount of the organic solvent is in the range of about 5% to 95% by
volume based on the total volume of the composition. In some
instances, the pH buffer system comprises a pH buffer. In some
instances, an amount of the pH buffer is no greater than 90% by
volume based on the total volume of the composition. In some
instances, the composition further comprises a molecular crowding
agent. In some instances, the molecular crowding agent is selected
from the group consisting of polyethylene glycol (PEG), dextran,
hydroxypropyl methyl cellulose (HPMC), hydroxyethyl methyl
cellulose (HEMC), hydroxybutyl methyl cellulose, hydroxypropyl
cellulose, methylcellulose, and hydroxyl methyl cellulose, and any
combination thereof. In some instances, the molecular crowding
agent is polyethylene glycol (PEG). In some instances, the
molecular crowding agent has a molecular weight in the range of
about 5,000 to 40,000 Daltons. In some instances, an amount of the
molecular crowding agent is at least about 5% by volume based on
the total volume of the composition. In some instances, an amount
of the molecular crowding agent is less than 50% by volume based on
the total volume of the composition. In some instances, the
compositions for hybridizing a target nucleic acid molecule to a
surface bound nucleic acid molecule further comprise an additive
for controlling a melting temperature of the one or more target
nucleic acid molecules. In some instances, an amount of the
additive for controlling melting temperature of the one or more
target nucleic molecules acid is at least about 2% by volume based
on the total volume of the composition. In some instances, an
amount of the additive for controlling melting temperature of the
nucleic acid is in the range of about 2% to 50% by volume based on
the total volume of the composition. In some instances, the pH
buffer comprises at least one buffering agent selected from the
group consisting of Tris, HEPES, TAPS, Tricine, Bicine, Bis-Tris,
sodium hydroxide (NaOH), potassium hydroxide (KOH), TES, EPPS, and
MOPS. In some instances, the pH buffer further comprises a second
organic solvent. In some instances, the pH buffer comprises MOPS
and methanol. In some instances, an amount of the pH buffer is
effective to maintain the pH of the composition to be in the range
of about 3 to about 10.
[0293] Provided herein are methods of using the kits described
herein. In some instances, the surface bound nucleic acid molecules
is coupled to the surface by a covalent or a noncovalent bond. In
some instances, the methods comprise: (a) combining the one or more
target nucleic acid molecules and the composition of the kit to
form a master mix; and (b) bringing the master mix into contact
with the one or more surface bound nucleic acid molecules coupled
to the surface provided in the kit. In some instances, the methods
further comprise (c) hybridizing the one or more target nucleic
acid molecules with the one or more surface bound nucleic acid
molecules coupled to the surface. In some instances, the surface
exhibits a level of non-specific Cy3 dye absorption of less than
about 0.25 molecules/.mu.m.sup.2. In some instances, no more than
10% of a total number of the one or more target nucleic acid
molecules is associated with the surface without hybridizing to the
surface bound nucleic acid molecule. In some instances, no more
than 5% of the total number of the one or more target nucleic acid
molecules is associated with the surface without hybridizing to the
one or more surface bound nucleic acid molecules. In some
instances, hybridizing the one or more target nucleic acid
molecules with the one or more surface bound nucleic acid molecules
coupled to the surface is performed under isothermal conditions. In
some instances, the isothermal conditions are performed at a
temperature in a range of 30 to 70 degrees Celsius. In some
instances, the methods further comprise (d) amplifying the target
nucleic acid hybridized to the surface bound nucleic acid to form a
plurality of clonally-amplified clusters of the one or more target
nucleic acid molecules coupled to the surface; and (c) determining
the identity of the one or more target nucleic acid molecules. In
some instances, a fluorescence image of the surface comprising the
plurality of clonally-amplified clusters of the one or more target
nucleic acid molecules exhibits a contrast-to-noise ratio (CNR) of
at least 20 when the fluorescence image is captured using a
fluorescence imaging system under non-signal saturating conditions.
In some instances, the CNR is at least 50.
[0294] In some instances, hybridizing the surface bound nucleic
acid and the target nucleic acid is performed for a period of no
more than 25 minutes. In some instances, methods of using the kits
described herein further comprise removing the composition from the
surface after the period of no more than 25 minutes. In some
instances, hybridizing the surface bound nucleic acid and the
target nucleic acid is performed for a period between 2-25 minutes.
In some instances, hybridizing the one or more surface bound
nucleic acid molecules and the one or more target nucleic acid
molecules is performed for a period of between 2-4 minutes. In some
instances, hybridizing the one or more surface bound nucleic acid
molecules and the one or more target nucleic acid molecules is
performed for a period of 2 minutes. In some instances, the at
least one surface bound nucleic acid is circular. In some
instances, hybridizing does not consist of cooling. In some
instances, bringing the master mix into contact with the one or
more surface bound nucleic acid molecules is performed under
conditions of stringency that prevent the one or more target
nucleic acid molecules from hybridizing to a non-complementary
nucleic acid. In some instances, the stringency is at least or
about 70%, 80%, or 90%. In some instances, the stringency is at
least 80%.
[0295] Provided herein are systems comprising: (a) a surface
comprising one or more surface bound nucleic acids molecules
coupled to the surface; (b) one or more target nucleic acid
molecules; and (c) a composition comprising: (i) at least one
organic solvent; and (ii) a pH buffer. In some instances, the
systems further comprise a fluorescence imaging device. In some
instances, the surface is a hydrophilic polymer surface. In some
instances, the surface has a water contact angle of less than 45
degrees. In some instances, the hydrophilic polymer surface
comprises one or more hydrophilic polymer layers, wherein the one
or more surface bound nucleic acid molecules is coupled to the one
or more hydrophilic polymer layers. In some instances, the one or
more hydrophilic polymer layers comprises a molecule selected from
the group consisting of polyethylene glycol (PEG), poly(vinyl
alcohol) (PVA), poly(vinyl pyridine), poly(vinyl pyrrolidone)
(PVP), poly(acrylic acid) (PAA), polyacrylamide,
poly(N-isopropylacrylamide) (PNIPAM), poly(methyl methacrylate)
(PMA), poly(2-hydroxylethyl methacrylate) (PHEMA),
poly(oligo(ethylene glycol) methyl ether methacrylate) (POEGMA),
polyglutamic acid (PGA), poly-lysine, poly-glucoside, streptavidin,
and dextran.
[0296] In some instances, the organic solvent is an organic solvent
having a dielectric constant of no greater than 40 when measured at
70 degrees Fahrenheit. In some instances, the organic solvent is
acetonitrile, alcohol, or formamide. In some instances, the organic
solvent comprises at least one functionality selected from hydroxy,
nitrile, lactone, sulfone, sulfite, and carbonate. In some
instances, the organic solvent is miscible with water. In some
instances, the organic solvent is present in an amount effective to
denature a double stranded nucleic acid. In some instances, an
amount of the organic solvent is at least about 5% by volume based
on the total volume of the composition. In some instances, an
amount of the organic solvent is in the range of about 5% to 95% by
volume based on the total volume of the composition. In some
instances, the pH buffer system comprises a pH buffer. In some
instances, an amount of the pH buffer is no greater than 90% by
volume based on the total volume of the composition. In some
instances, the composition further comprises a molecular crowding
agent. In some instances, the molecular crowding agent is selected
from the group consisting of polyethylene glycol (PEG), dextran,
hydroxypropyl methyl cellulose (HPMC), hydroxyethyl methyl
cellulose (HEMC), hydroxybutyl methyl cellulose, hydroxypropyl
cellulose, methyl cellulose, and hydroxyl methyl cellulose, and any
combination thereof. In some instances, the molecular crowding
agent is polyethylene glycol (PEG). In some instances, the
molecular crowding agent has a molecular weight in the range of
about 5,000 to 40,000 Daltons. In some instances, an amount of the
molecular crowding agent is at least about 5% by volume based on
the total volume of the composition. In some instances, an amount
of the molecular crowding agent is less than 50% by volume based on
the total volume of the composition. In some instances, the systems
described herein further comprise an additive for controlling a
melting temperature of the target nucleic acid. In some instances,
an amount of the additive for controlling melting temperature of
the one or more target nucleic acid molecules is at least about 2%
by volume based on the total volume of the composition. In some
instances, an amount of the additive for controlling melting
temperature of the one or more nucleic acid molecules is in the
range of about 2% to 50% by volume based on the total volume of the
composition. In some instances, the pH buffer comprises at least
one buffering agent selected from the group consisting of Tris,
HEPES, TAPS, Tricine, Bicine, Bis-Tris, sodium hydroxide (NaOH),
potassium hydroxide (KOH), TES, EPPS, and MOPS. In some instances,
the pH buffer further comprises a second organic solvent. In some
instances, the pH buffer comprises MOPS and methanol. In some
instances, an amount of the pH buffer is effective to maintain the
pH of the composition to be in the range of about 3 to about
10.
[0297] Provided herein are methods of using the systems described
herein. In some instances, the one or more surface bound nucleic
acid molecules is coupled to the surface by a covalent or a
noncovalent bond. In some instances, the methods comprise: (a)
combining the one or more target nucleic acid molecules and the
composition of the system to form a master mix; (b) bringing the
master mix into contact with the one or more surface bound nucleic
acid molecules coupled to the surface provided in the system; (c)
hybridizing the one or more target nucleic acid molecules with the
one or more surface bound nucleic acid molecules coupled to the
surface; (d) amplifying the one or more target nucleic acid
molecules hybridized to the one or more surface bound nucleic acid
molecules to form a plurality of clonally-amplified clusters of the
one or more target nucleic acid molecules coupled to the surface;
and (e) determining the identity of the one or more target nucleic
acid molecules by capturing an image of the surface with the
fluorescence imaging device. In some instances, the surface
exhibits a level of non-specific Cy3 dye absorption of less than
about 0.25 molecules/.mu.m.sup.2. In some instances, hybridizing
the one or more target nucleic acid molecules with the one or more
surface bound nucleic acid molecules coupled to the surface is
performed under isothermal conditions. In some instances, the
isothermal conditions are performed at a temperature in a range of
30 to 70 degrees Celsius. In some instances, no more than 10% of a
total number of the one or more target nucleic acid molecules is
associated with the surface without hybridizing to the one or more
surface bound nucleic acid molecules. In some instances, no more
than 5% of the total number of the one or more target nucleic acid
molecules is associated with the surface without hybridizing to the
one or more surface bound nucleic acid molecules. In some
instances, a fluorescence image of the surface comprising the
amplified one or more target nucleic acid molecules exhibits a
contrast-to-noise ratio (CNR) of at least 20 when the fluorescence
image is captured using the fluorescence imaging device under
non-signal saturating conditions. In some instances, the CNR is at
least 50.
[0298] In some instances, hybridizing the one or more surface bound
nucleic acid molecules and the one or more target nucleic acid
molecules is performed for a period of no more than 25 minutes. In
some instances, the methods disclosed herein further comprise
removing the composition from the surface after the period of no
more than 25 minutes. In some instances, hybridizing the one or
more surface bound nucleic acid molecules and the one or more
target nucleic acid molecules is performed for a period between
2-25 minutes. In some instances, hybridizing the one or more
surface bound nucleic acid molecules and the one or more target
nucleic acid molecules is performed for a period between 2-4
minutes. In some instances, hybridizing the one or more surface
bound nucleic acid molecules and the one or more target nucleic
acid molecules is performed for a period of 2 minutes. In some
instances, the at least one surface bound nucleic acid is circular.
In some instances, hybridizing does not consist of cooling. In some
instances, bringing the one or more surface bound nucleic acid
molecules into contact with the hybridizing composition comprising
the one or more target nucleic acid molecules is performed under
conditions of stringency that prevent the one or more target
nucleic acid molecules from hybridizing to a non-complementary
nucleic acid molecule. In some instances, the stringency is at
least or about 70%, 80%, or 90%. In some instances, the stringency
is at least 80%.
Illustrative Embodiment 3
[0299] Disclosed herein are methods of determining an identity of a
nucleotide in a target nucleic acid sequence comprising: (a)
providing a composition comprising: (i) two or more copies of said
target nucleic acid sequence; (ii) two or more primer nucleic acid
molecules that are complementary to one or more regions of said
target nucleic acid sequence; and (iii) two or more polymerase
molecules; (b) contacting said composition with a polymer
nucleotide conjugate under conditions sufficient to allow a
multivalent binding complex to be formed between said
polymer-nucleotide conjugate and said two or more copies of said
target nucleic acid sequence in said composition of (a), wherein
the polymer-nucleotide conjugate comprises two or more copies of a
nucleotide moiety and optionally one or more detectable labels; and
(c) detecting said multivalent binding complex, thereby determining
the identity of said nucleotide in the target nucleic acid
sequence. In some instances, the target nucleic acid sequence is
DNA. In some instances, the detection of the multivalent binding
complex is performed in the absence of unbound or solution-borne
polymer nucleotide conjugates. In some instances, the target
nucleic acid sequence has been replicated or amplified or has been
produced by replication or amplification. In some instances, the
one or more detectable labels are fluorescent labels. In some
instances, detecting the multivalent complex comprises a
fluorescence measurement. In some instances, the contacting
comprises use of one type of polymer-nucleotide conjugate. In some
instances, the contacting comprises use of two or more types of
polymer-nucleotide conjugates. In some instances, each type of the
two or more types of polymer-nucleotide conjugate comprises a
different type of nucleotide moiety. In some instances, the
contacting comprises use of three types of polymer-nucleotide
conjugates, wherein each type of the three types of
polymer-nucleotide conjugates comprises a different type of
nucleotide moiety. In some instances, the polymer-nucleotide
conjugate comprises a blocked nucleotide moiety. In some instances,
the blocked nucleotide is a 3'-O-azidomethyl nucleotide, a
3'-O-methyl nucleotide, or a 3'-O-alkyl hydroxylamine nucleotide.
In some instances, said contacting occurs in the presence of an ion
that stabilizes said multivalent binding complex. In some
instances, the contacting is done in the presence of strontium
ions, magnesium ions, calcium ions, or any combination thereof. In
some instances, the polymerase molecules are catalytically
inactive. In some instances, the polymerase molecules have been
rendered catalytically inactive by mutation or chemical
modification. In some instances, the polymerase molecules have been
rendered catalytically inactive by the absence of a necessary ion
or cofactor. In some instances, the polymerase molecules are
catalytically active. In some instances, the polymer-nucleotide
conjugate does not comprise a blocked nucleotide moiety. In some
instances, the multivalent binding complex has a persistence time
of greater than 2 seconds. In some instances, the method can be
carried out at a temperature within a range of 25.degree. C. to
62.degree. C. In some instances, the polymer-nucleotide conjugate
further comprises one or more fluorescent labels and the two or
more copies of the target nucleic acid sequence are deposited on,
attached to, or hybridized to a surface, wherein a fluorescence
image of the multivalent binding complex on the surface has a
contrast to noise ratio in the detecting step of greater than 20.
In some instances, the composition of (a) is deposited on a surface
using a buffer that incorporates a polar aprotic solvent. In some
instances, the contacting is performed under a condition that
stabilizes said multivalent binding complex when said nucleotide
moiety is complementary to a next base of said target nucleic acid
sequence and destabilizes said multivalent binding complex when
said nucleotide moiety is not complementary to said next base of
said target nucleic acid sequence. In some instances, said
polymer-nucleotide conjugate comprises a polymer having a plurality
of branches and said two or more nucleotide moieties are attached
to said branches. In some instances, said polymer has a star, comb,
cross-linked, bottle brush, or dendrimer configuration. In some
instances, said polymer-nucleotide conjugate comprises one or more
binding groups selected from the group consisting of an avidin, a
biotin, an affinity tag, and combinations thereof. In some
instances, the method further comprises a dissociation step that
destabilizes said multivalent binding complex formed between the
composition of (a) and the polymer-nucleotide conjugate, said
dissociation step enabling removal of said polymer-nucleotide
conjugate. In some instances, the method further comprises an
extension step to incorporate a nucleotide that is complementary to
a next base of the target nucleic acid sequence into said two or
more primer nucleic acid molecules. In some instances, the
extension step occurs concurrently with or after said dissociation
step.
[0300] Disclosed herein are methods of determining an identity of a
nucleotide in a target nucleic acid sequence comprising: (a)
providing a composition comprising: (i) two or more copies of said
target nucleic acid sequence; (ii) two or more primer nucleic acid
molecules that are complementary to one or more regions of said
target nucleic acid sequence; and (ii). two or more polymerase
molecules; (b) contacting said composition with a polymer
nucleotide conjugate under conditions sufficient to allow a
multivalent complex to be formed between said polymer-nucleotide
conjugate and said two or more copies of said target nucleic acid
sequence in said composition of (a), wherein the polymer-nucleotide
conjugate comprises two or more copies of a reversibly terminated
nucleotide moiety and optionally one or more cleavable detectable
labels; and (c) detecting said multivalent complex, thereby
determining the identity of said nucleotide in the target nucleic
acid sequence. In some instances, the target nucleic acid sequence
is DNA. In some instances, the method further comprises contacting
the composition of (a) with reversibly terminated nucleotides or
polymer-nucleotide conjugates comprising two or more copies of a
reversibly terminated nucleotide following the detection of said
multivalent binding complex. In some instances, the target nucleic
acid sequence has been replicated or amplified or has been produced
by replication or amplification. In some instances, the one or more
detectable labels are fluorescent labels. In some instances,
detecting the multivalent complex comprises a fluorescence
measurement. In some instances, the contacting comprises use of one
type of polymer-nucleotide conjugate. In some instances, the
contacting comprises use of two or more types of polymer-nucleotide
conjugates. In some instances, each type of the two or more types
of polymer-nucleotide conjugate comprises a different type of
nucleotide moiety. In some instances, the contacting comprises use
of three types of polymer-nucleotide conjugate, wherein each type
of the three types of polymer-nucleotide conjugate comprises a
different type of nucleotide moiety. In some instances, the
polymer-nucleotide conjugate comprises a blocked nucleotide moiety.
In some instances, the blocked nucleotide is a 3'-O-azidomethyl,
3'-O-methyl, or 3'-O-alkyl hydroxylamine. In some instances, said
contacting occurs in the presence of an ion that stabilizes said
multivalent binding complex. In some instances, the polymerase
molecules are catalytically inactive. In some instances, the
polymerase molecules have been rendered catalytically inactive by
mutation or chemical modification. In some instances, the
polymerase molecules are catalytically active. In some instances,
the polymer-nucleotide conjugate does not comprise a blocked
nucleotide moiety. In some instances, the method can be carried out
at a temperature within a range of 25.degree. C. to 80.degree. C.
In some instances, the polymer-nucleotide conjugate further
comprises one or more fluorescent labels and the two or more copies
of the target nucleic acid sequence are deposited on, attached to,
or hybridized to a surface, wherein a fluorescence image of the
multivalent binding complex on the surface has a contrast to noise
ratio in the detecting step of greater than 20.
[0301] Also disclosed herein are systems comprising: (a) one or
more computer processors individually or collectively programmed to
implement a method comprising: (i) contacting a substrate
comprising multiple copies of a target nucleic acid sequence
tethered to a surface of the substrate with a reagent comprising a
polymerase and one or more primer nucleic acid sequences that are
complementary to one or more regions of said target nucleic acid
sequence to form a primed target nucleic acid sequence; (ii)
contacting the substrate surface with a reagent comprising a
polymer nucleotide conjugate under conditions sufficient to allow a
multivalent binding complex to be formed between said
polymer-nucleotide conjugate and two or more copies of said primed
target nucleic acid sequence, wherein the polymer-nucleotide
conjugate comprises two or more copies of a known nucleotide moiety
and a detectable label; (iii) acquiring and processing an image of
the substrate surface to detect said multivalent binding complex,
thereby determining the identity of a nucleotide in the target
nucleic acid sequence. In some instances, the system further
comprises a fluidics module configured to deliver a series of
reagents to the substrate surface in a specified sequence and for
specified time intervals. In some instances, the system further
comprises an imaging module configured to acquire images of the
substrate surface. In some instances, (ii) and (iii) are repeated
two or more times thereby determining the identity of a series of
two or more nucleotides in the target nucleic acid sequence. In
some instances, the series of steps further comprise a dissociation
step that destabilizes said multivalent binding complex, said
dissociation step enabling removal of said polymer-nucleotide
conjugate. In some instances, the series of steps further comprises
an extension step to incorporate a nucleotide that is complementary
to a next base of the target nucleic acid sequence into said two or
more primer nucleic acid molecules. In some instances, the
extension step occurs concurrently with or after said dissociation
step. In some instances, the detectable label comprises a
fluorophore and the images comprise fluorescence images. In some
instances, the fluorescence images of the multivalent binding
complex on the substrate surface has a contrast-to-noise ratio of
greater than 20 when the fluorophore is cyanine dye 3 (Cy3) and the
image is acquired using an inverted fluorescence microscope
equipped with a 20.times. objective, NA=0.75, dichroic mirror
optimized for 532 nm light, a bandpass filter optimized for Cyanine
dye-3 emission, and a camera, under non-signal saturating
conditions while the surface is immersed in 25 mM ACES, pH 7.4
buffer. In some instances, the series of steps is completed in less
than 60 minutes. In some instances, the series of steps is
completed in less than 30 minutes. In some instances, the series of
steps is completed in less than 10 minutes. In some instances, an
accuracy of base-calling is characterized by a Q-score of greater
than 25 for at least 80% of the nucleotide identities determined.
In some instances, an accuracy of base-calling is characterized by
a Q-score of greater than 30 for at least 80% of the nucleotide
identities determined. In some instances, an accuracy of
base-calling is characterized by a Q-score of greater than 40 for
at least 80% of the nucleotide identities determined.
[0302] Disclosed herein are compositions comprising: a) a polymer
core; and b) two or more nucleotide, nucleotide analog, nucleoside,
or nucleoside analog moieties attached to the polymer core; wherein
the length of the linker is dependent on the nucleotide, nucleotide
analog, nucleoside, or nucleoside analog moiety that is attached to
the polymer core. Also disclosed herein are compositions
comprising: a) a mixture of polymer-nucleotide conjugates, wherein
each polymer-nucleotide conjugate comprises: i) a polymer core; and
ii) two or more nucleotide, nucleotide analog, nucleoside, or
nucleoside analog moieties attached to the polymer core, wherein
the length of the linker is dependent on the nucleotide, nucleotide
analog, nucleoside, or nucleoside analog moiety that is attached to
the polymer core; and wherein the mixture comprises
polymer-nucleotide conjugates having at least two different types
of attached nucleotides, nucleotide analogs, nucleosides, or
nucleoside analog moieties. In some instances, the polymer core
comprises a polymer having a plurality of branches and the two or
more nucleotide, nucleotide analog, nucleoside, or nucleoside
analog moieties are attached to said branches. In some instances,
polymer has a star, comb, cross-linked, bottle brush, or dendrimer
configuration. In some instances, the polymer-nucleotide conjugate
comprises one or more binding groups selected from the group
consisting of an avidin, a biotin, an affinity tag, and
combinations thereof. In some instances, the polymer core comprises
a branched polyethylene glycol (PEG) molecule. In some instances,
the polymer-nucleotide conjugate comprises a blocked nucleotide
moiety. In some instances, the blocked nucleotide is a
3'-O-azidomethyl nucleotide, a 3'-O-methyl nucleotide, or a
3'-O-alkyl hydroxylamine nucleotide. In some instances, the
polymer-nucleotide conjugate further comprises one or more
fluorescent labels.
[0303] In some instances, the present disclosure provides methods
of determining the identity of a nucleotide in a target nucleic
acid comprising the steps, without regard to any particular order
of operations, 1) providing a composition comprising: a target
nucleic acid comprising two or more repeats of an identical
sequence; two or more primer nucleic acids complementary to one or
more regions of said target nucleic acid; and two or more
polymerase molecules; 2) contacting said composition with a
multivalent binding or incorporation composition comprising a
polymer-nucleotide conjugate under conditions sufficient to allow a
binding or incorporated complex to be formed between said
polymer-nucleotide conjugate and the composition of step (a),
wherein the polymer-nucleotide conjugate comprises two or more
copies of a nucleotide and optionally one or more detectable
labels; and 3) detecting said binding or incorporated complex,
thereby establishing the identity of said nucleotide in the target
nucleic acid polymer. In some further instances, the present
disclosure provides said method, wherein the target nucleic acid is
DNA, and/or wherein the target nucleic acid has been replicated,
such as by any commonly practiced method of DNA replication or
amplification, such as rolling circle amplification, bridge
amplification, helicase dependent amplification, isothermal bridge
amplification, rolling circle multiple displacement amplification
(RCA/MDA), and/or recombinase based methods of replication or
amplification. In some further instances, the present disclosure
provides said method, wherein the detectable label is a fluorescent
label and/or wherein detecting the complex comprises a fluorescence
measurement. In some further instances, the present disclosure
provides said method wherein the multivalent binding composition
comprises one type of polymer-nucleotide conjugate, wherein the
multivalent binding composition comprises two or more types of
polymer-nucleotide conjugates, and/or wherein each type of the two
or more types of polymer-nucleotide conjugates comprises a
different type of nucleotide. In some instances, the present
disclosure provides said method wherein the binding complex or
incorporated complex further comprises a blocked nucleotide,
especially wherein the blocked nucleotide is a 3'-O-azidomethyl
nucleotide, a 3'-O-alkyl hydroxylamino nucleotide, or a 3'-O-methyl
nucleotide. In some further instances, the present disclosure
provides said method wherein the contacting is done in the presence
of strontium ions, barium, magnesium ions, and/or calcium ions. In
some instances, the present disclosure provides said method wherein
the polymerase molecule is catalytically inactive, such as where
the polymerase molecule been rendered catalytically inactive by
mutation, by chemical modification, or by the absence of a
necessary ion or cofactor. In some instances, the present
disclosure also provides said method wherein the polymerase
molecule is catalytically active, and/or wherein the binding
complex does not comprise a blocked nucleotide. In some instances,
the present disclosure provides said method wherein the binding
complex has a persistence time of greater than 2 seconds and/or
wherein the method is or may be carried out at a temperature of at
or above 15.degree. C., at or above 20.degree. C., at or above
25.degree. C., at or above 35.degree. C., at or above 37.degree.
C., at or above 42.degree. C., at or above 55.degree. C., at or
above 60.degree. C., or at or above 72.degree. C., or within a
range defined by any of the foregoing. In some instances, the
present disclosure provides said method wherein the binding complex
is deposited on, attached to, or hybridized to, a surface showing a
contrast to noise ratio in the detecting step of greater than 20.
In some instances, the present disclosure provides said method
wherein the composition is deposited under buffer conditions
incorporating a polar aprotic solvent. In some instances, the
present disclosure provides said method wherein the contacting is
performed under a condition that stabilizes said binding complex
when said nucleotide is complementary to a next base of said target
nucleic acid and destabilizes said binding complex when said
nucleotide is not complementary to said next base of said target
nucleic acid. In some instances, the present disclosure provides
said method wherein said polymer-nucleotide conjugate comprises a
polymer having a plurality of branches and said plurality of copies
of said first nucleotide are attached to said branches, especially
wherein said first polymer has a star, comb, cross-linked, bottle
brush, or dendrimer configuration. In some instances, the present
disclosure provides said method wherein said polymer-nucleotide
conjugate comprises one or more binding groups selected from the
group consisting of avidin, biotin, affinity tag, and combinations
thereof. In some instances, the present disclosure provides said
method further comprising a dissociation step that destabilizes
said binding complex formed between the composition of (a) and the
polymer-nucleotide conjugate to remove said polymer-nucleotide
conjugate. In some instances, the present disclosure provides said
method further comprising an extension step to incorporate into
said primer nucleic acid a nucleotide that is complementary to said
next base of the target nucleic acid, and optionally wherein the
extension step occurs currently as or after said dissociation
step.
[0304] In some instances, the present disclosure provides a
composition comprising a branched polymer having two or more
branches and two or more copies of a nucleotide, wherein said
nucleotide is attached to a first plurality of said branches or
arms, and optionally, wherein one or more interaction moieties are
attached to a second plurality of said branches or arms. In some
instances, said composition may further comprise one or more labels
on the polymer. In some instances, the present disclosure provides
said composition wherein the nucleoside has a surface density of at
least 4 nucleotides per polymer. In some instances, the present
disclosure provides said composition comprising or incorporating a
nucleotide or nucleotide analog that is modified so as to prevent
its incorporation into an extending nucleic acid chain during a
polymerase reaction. In some instances, said composition may
comprise or incorporate a nucleotide or nucleotide analog that is
reversibly modified so as to prevent its incorporation into an
extending nucleic acid chain during a polymerase reaction. In some
instances, the present disclosure provides said composition wherein
one or more labels comprise a fluorescent label, a FRET donor,
and/or a FRET acceptor. In some instances, said composition may
comprise 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or more
branches or arms, or 2, 4, 8, 16, 32, 64, or more, branches or
arms. In some instances, the branches or arms may radiate from a
central moiety. In some instances, said composition may comprise
one or more interaction moieties, which interaction moieties may
comprise avidin or streptavidin; a biotin moiety; an affinity tag;
an enzyme, antibody, minibody, receptor, or other protein; a
non-protein tag; a metal affinity tag, or any combination thereof.
In some instances, the present disclosure provides said composition
wherein the polymer comprises polyethylene glycol, polypropylene
glycol, polyvinyl acetate, polylactic acid, or polyglycolic acid.
In some instances, the present disclosure provides said composition
wherein the nucleotide or nucleotide analog is attached to the
branch or arm through a linker; and especially wherein the linker
comprises PEG, and wherein the PEG linker moiety has an average
molecular weight of about 1K Da, about 2K Da, about 3K Da, about 4K
Da, about 5K Da, about 10K Da, about 15K Da, about 20K Da, about
50K Da, about 100K Da, about 150K Da, or about 200K Da, or greater
than about 200K Da. In some instances, the present disclosure
provides said composition wherein the linker comprises PEG, and
wherein the PEG linker moiety has an average molecular weight of
between about 5K Da and about 20K Da. In some instances, the
present disclosure provides said composition wherein at least one
nucleotide or nucleotide analog comprises a deoxyribonucleotide, a
ribonucleotide, a deoxyribonucleoside, or a ribonucleoside; and/or
wherein the nucleotide or nucleotide analog is conjugated to the
linker through the 5' end of the nucleotide or nucleotide analog.
In some instances, the present disclosure provides said composition
wherein one of the nucleotides or nucleotide analogs comprises
deoxyadenosine, deoxyguanosine, thymidine, deoxyuridine,
deoxycytidine, adenosine, guanosine, 5-methyl-uridine, and/or
cytidine; and wherein the length of the linker is between 1 nm and
1,000 nm. In some instances, the present disclosure provides said
composition wherein at least one nucleotide or nucleotide analog is
a nucleotide that has been modified to inhibit elongation during a
polymerase reaction or a sequencing reaction, such as wherein the
at least one nucleotide or nucleotide analog is a nucleotide that
lacks a 3' hydroxyl group; a nucleotide that has been modified to
contain a blocking group at the 3' position; and/or a nucleotide
that has been modified with a 3'-O-azido group, a 3'-O-azidomethyl
group, a 3'-O-alkyl hydroxylamino group, a 3'-phosphorothioate
group, a 3'-O-malonyl group, or a 3'-O-benzyl group. In some
instances, the present disclosure provides said composition wherein
at least one nucleotide or nucleotide analog is a nucleotide that
has not been modified at the 3' position.
[0305] In some instances, the present disclosure provides a method
of determining the sequence of a nucleic acid molecule comprising
the steps, without regard to any particular order, of 1) providing
a nucleic acid molecule comprising a template strand and a
complementary strand that is at least partially complementary to
the template strand; 2) contacting the nucleic acid molecule with
the one or more nucleic acid binding composition according to any
of the examples disclosed herein; 3) detecting binding of the
nucleic acid binding composition to the nucleic acid molecule, and
4) determining an identity of a terminal nucleotide to be
incorporated into said complementary strand of said nucleic acid
molecule. In some instances, the present disclosure provides a
method of determining the sequence of a nucleic acid molecule
comprising the steps, without regard to any particular order, of 1)
providing a nucleic acid molecule comprising a template strand and
a complementary strand that is at least partially complementary to
the template strand; 2) contacting the nucleic acid molecule with
the one or more nucleic acid binding compositions according to any
of the examples disclosed herein; 3) detecting partial or complete
incorporation of the nucleic acid binding composition to the
nucleic acid molecule, and 4) determining an identity of a terminal
nucleotide to be incorporated into said complementary strand of
said nucleic acid molecule from the partial or complete
incorporation of the examples described herein. In some instances,
the present disclosure provides said method further comprising
incorporating said terminal nucleotide into said complementary
strand, and repeating said contacting, detecting, and incorporating
steps for one or more additional iterations, thereby determining
the sequence of said template strand of said nucleic acid molecule.
In some instances, the present disclosure provides said method,
wherein said nucleic acid molecule is tethered to a solid support;
and, in some examples, wherein the solid support comprises a glass
or polymer substrate, at least one hydrophilic polymer coating
layer, and a plurality of oligonucleotide molecules attached to at
least one hydrophilic polymer coating layer. In some instances, the
present disclosure provides said method, further comprising
examples wherein at least one hydrophilic polymer coating layer
comprises PEG; and/or wherein at least one hydrophilic polymer
layer comprises a branched hydrophilic polymer having at least 8
branches. In some instances, the present disclosure provides said
method, wherein the plurality of oligonucleotide molecules is
present at a surface density of at least 500 molecules/mm.sup.2, at
least 1,000 molecules/mm.sup.2, at least 5,000 molecules/mm.sup.2,
at least 10,000 molecules/mm.sup.2, at least 20,000
molecules/mm.sup.2, at least 50,000 molecules/mm.sup.2, at least
100,000 molecules/mm.sup.2, or at least 500,000 molecules/mm.sup.2.
In some instances, the present disclosure provides said method,
wherein said nucleic acid molecule has been clonally-amplified on a
solid support. In some instances, the present disclosure provides
said method, wherein the clonal amplification comprises the use of
a polymerase chain reaction (PCR), multiple displacement
amplification (MDA), transcription-mediated amplification (TMA),
nucleic acid sequence-based amplification (NASBA), strand
displacement amplification (SDA), real-time SDA, bridge
amplification, isothermal bridge amplification, rolling circle
amplification (RCA), circle-to-circle amplification,
helicase-dependent amplification, recombinase-dependent
amplification, single-stranded binding (SSB) protein-dependent
amplification, or any combination thereof. In some instances, the
present disclosure provides said method, wherein the one or more
nucleic acid binding compositions are labeled with fluorophores and
the detecting step comprises use of fluorescence imaging; and
especially wherein the fluorescence imaging comprises dual
wavelength excitation/four wavelength emission fluorescence
imaging. In some instances, the present disclosure provides said
method, wherein four different nucleic acid binding compositions,
each comprising a different nucleotide or nucleotide analog, are
used to determine the identity of the terminal nucleotide, wherein
the four different nucleic acid binding compositions are labeled
with separate respective fluorophores, and wherein the detecting
step comprises simultaneous excitation at a wavelength sufficient
to excite all four fluorophores and imaging of fluorescence
emission at wavelengths sufficient to detect each respective
fluorophore. In some instances, the present disclosure provides
said method, wherein four different nucleic acid binding
compositions, each comprising a different nucleotide or nucleotide
analog, are used to determine the identity of the terminal
nucleotide, wherein the four different nucleic acid binding
compositions are labeled with cyanine dye 3 (Cy3), cyanine dye 3.5
(Cy3.5), cyanine dye 5 (Cy5), and cyanine dye 5.5. (Cy5.5)
respectively, and wherein the detecting step comprises simultaneous
excitation at any two of 532 nm, 568 nm and 633 nm, and imaging of
fluorescence emission at about 570 nm, 592 nm, 670 nm, and 702 nm
respectively; and/or wherein the fluorescence imaging comprises
dual wavelength excitation/dual wavelength emission fluorescence
imaging. In some instances, the present disclosure provides said
method, wherein four different nucleic acid binding compositions,
each comprising a different nucleotide or nucleotide analog, are
used to determine the identity of the terminal nucleotide, wherein
one, two, three, or four different nucleic acid binding
compositions are respectively labeled, each with a with distinct
fluorophore or set of fluorophores, and wherein the detecting step
comprises simultaneous excitation at a wavelength sufficient to
excite one, two, three, or four fluorophores or sets of
fluorophores, and imaging of fluorescence emission at wavelengths
sufficient to detect each respective fluorophore. In some
instances, the present disclosure provides said method, wherein
three different nucleic acid binding or incorporation compositions,
each comprising a different nucleotide or nucleotide analog, are
used to determine the identity of the terminal nucleotide, wherein
one, two, or three different nucleic acid binding or incorporation
compositions are respectively labeled, each with a with distinct
fluorophore or set of fluorophores, and wherein the detecting step
comprises simultaneous excitation at a wavelength sufficient to
excite one, two, or three, fluorophores or sets of fluorophores,
and imaging of fluorescence emission at wavelengths sufficient to
detect each respective fluorophore, and wherein detection of the
fourth nucleotide is determined or determinable with reference to
the location of "dark" or unlabeled spots or target nucleotides. In
some instances, the present disclosure provides said method,
wherein the multivalent binding or incorporation composition may
comprise three types of polymer-nucleotide conjugates and wherein
each type of the three types of polymer-nucleotide conjugates
comprises a different type of nucleotide. In some instances, the
present disclosure provides said method, wherein the detection of
the binding or incorporation complex is performed in the absence of
unbound or solution-borne polymer nucleotide conjugates.
[0306] In some instances, the present disclosure provides said
method, wherein four different nucleic acid binding compositions,
or three different nucleic acid binding or incorporation
compositions, each comprising a different nucleotide or nucleotide
analog, are used to determine the identity of the terminal
nucleotide, wherein one of the four or three different nucleic acid
binding or incorporation compositions is labeled with a first
fluorophore, one is labeled with a second fluorophore, one is
labeled with both the first and second fluorophore, and one is not
labeled or is absent, and wherein the detecting step comprises
simultaneous excitation at a first excitation wavelength and a
second excitation wavelength and images are acquired at a first
fluorescence emission wavelength and a second fluorescence emission
wavelength. In some instances, the present disclosure provides said
method, wherein the first fluorophore is Cy3, the second
fluorophore is Cy5, the first excitation wavelength is 532 nm or
568 nm, the second excitation wavelength is 633 nm, the first
fluorescence emission wavelength is about 570 nm, and the second
fluorescence emission wavelength is about 670 nm. In some
instances, the present disclosure provides said method, wherein the
detection label can comprise one or more portions of a fluorescence
resonance energy transfer (FRET) pair, such that multiple
classifications can be performed under a single excitation and
imaging step. In some instances, the present disclosure provides
said method, wherein a sequencing reaction cycle comprising the
contacting, detecting, and incorporating/extending steps is
performed in less than 30 minutes in less than 20 minutes, or in
less than 10 minutes. In some instances, the present disclosure
provides said method, wherein an average Q-score for base calling
accuracy over a sequencing run is greater than or equal to 30,
and/or greater than or equal to 40. In some instances, the present
disclosure provides said method, wherein at least 50%, at least
60%, at least 70%, at least 80%, or at least 90% of the terminal
nucleotides identified have a Q-score of greater than 30 and/or
greater than or equal to 40. In some instances, the present
disclosure provides said method, herein at least 95% of the
terminal nucleotides identified have a Q-score of greater than
30.
[0307] In some instances, the present disclosure provides a reagent
comprising one or more nucleic acid binding compositions as
disclosed herein and a buffer. For example, in some instances, the
present disclosure provides a reagent, wherein said reagent
comprises 1, 2, 3, 4, or more nucleic acid binding or incorporation
compositions, wherein each nucleic acid binding or incorporation
composition comprises a single type of nucleotide. In some
instances, a reagent of the present disclosure comprises 1, 2, 3,
4, or more nucleic acid binding or incorporation compositions,
wherein each nucleic acid binding or incorporation composition
comprises a single type of nucleotide or nucleotide analog, and
wherein said nucleotide or nucleotide analog may respectively
correspond to one or more from the group consisting of adenosine
triphosphate (ATP), adenosine diphosphate (ADP), adenosine
monophosphate (AMP), deoxyadenosine triphosphate (dATP),
deoxyadenosine diphosphate (dADP), and deoxyadenosine monophosphate
(dAMP); one or more from the group consisting of thymidine
triphosphate (TTP), thymidine diphosphate (TDP), thymidine
monophosphate (TMP), deoxythymidine triphosphate (dTTP),
deoxythymidine diphosphate (dTDP), deoxythymidine monophosphate
(dTMP), uridine triphosphate (UTP), uridine diphosphate (UDP),
uridine monophosphate (UMP), deoxyuridine triphosphate (dUTP),
deoxyuridine diphosphate (dUDP), and deoxyuridine monophosphate
(dUMP); one or more from the group consisting of cytidine
triphosphate (CTP), cytidine diphosphate (CDP), cytidine
monophosphate (CMP), deoxycytidine triphosphate (dCTP),
deoxycytidine diphosphate (dCDP), and deoxycytidine monophosphate
(dCMP); and one or more from the group consisting of guanosine
triphosphate (GTP), guanosine diphosphate (GDP), guanosine
monophosphate (GMP), deoxyguanosine triphosphate (dGTP),
deoxyguanosine diphosphate (dGDP), and deoxyguanosine monophosphate
(dGMP). In some other examples or some further examples, the
present disclosure provides a reagent comprising or further
comprising 1, 2, 3, 4, or more nucleic acid binding or
incorporation compositions, wherein each nucleic acid binding or
incorporation composition comprises a single type of nucleotide or
nucleotide analog, and wherein said nucleotide or nucleotide analog
may respectively correspond to one or more from the group
consisting of ATP, ADP, AMP, dATP, dADP, dAMP, TTP, TDP, TMP, dTTP,
dTDP, dTMP, UTP, UDP, UMP, dUTP, dUDP, dUMP, CTP, CDP, CMP, dCTP,
dCDP, dCMP, GTP, GDP, GMP, dGTP, dGDP, and dGMP.
[0308] Disclosed herein are kits comprising any of the nucleic acid
binding or incorporation compositions disclosed herein and/or any
of the reagents disclosed herein, and/or one or more buffers; and
instructions for the use thereof.
[0309] Disclosed herein are systems for performing any of the
methods disclosed herein, comprising any of the nucleic acid
binding or incorporation compositions disclosed herein, and/or any
of the reagents disclosed herein. In some instances, a system is
configured to iteratively perform the sequential contacting of
tethered, primed nucleic acid molecules with said nucleic acid
binding or incorporation compositions and/or said reagents; and for
the detection of binding or incorporation of the disclosed nucleic
acid binding or incorporation compositions to the one or more
primed nucleic acid molecules.
[0310] In some instances, the present disclosure provides a
composition comprising a particle (e.g., a nanoparticle or polymer
core), said particle comprising a plurality of enzyme or protein
binding or incorporation substrates, wherein the enzyme or protein
binding or incorporation substrates bind with one or more enzymes
or proteins to form one or more binding or incorporation complexes
(e.g., a multivalent binding or incorporation complex), and wherein
said binding or incorporation may be monitored or identified by
observation of the location, presence, or persistence of the one or
more binding or incorporation complexes. In some instances, said
particle may comprise a polymer, branched polymer, dendrimer,
liposome, micelle, nanoparticle, or quantum dot. In some instances,
said substrate may comprise a nucleotide, a nucleoside, a
nucleotide analog, or a nucleoside analog. In some instances, the
enzyme or protein binding or incorporation substrate may comprise
an agent that can bind with a polymerase. In some instances, the
enzyme or protein may comprise a polymerase. In some instances,
said observation of the location, presence, or persistence of one
or more binding or incorporation complexes may comprise
fluorescence detection. In some instances, the present disclosure
provides a composition comprising multiple distinct particles as
disclosed herein, wherein each particle comprises a single type of
nucleoside or nucleoside analog, and wherein each nucleoside or
nucleoside analog is associated with a fluorescent label of a
detectably different emission or excitation wavelength. In some
instances, the present disclosure provides said composition further
comprising one or more labels, e.g., fluorescence labels, on the
particle. In some instances, the present disclosure provides said
composition wherein the composition comprises at least 2, 4, 6, 8,
10, 12, 14, 16, 18, 20, or more than 20 tethered nucleotides,
nucleotide analogs, nucleosides, or nucleoside analogs tethered to
the particle. In some instances, the present disclosure provides
said composition wherein the nucleoside or nucleoside analog is
present at a surface density of between 0.001 and 1,000,000 per
.mu.m.sup.2, between 0.01 and 1,000,000 per .mu.m.sup.2, between
0.1 and 1,000,000 per .mu.m.sup.2, between 1 and 1,000,000 per
.mu.m.sup.2, between 10 and 1,000,000 per .mu.m.sup.2, between 100
and 1,000,000 per .mu.m.sup.2, between 1,000 and 1,000,000 per
.mu.m.sup.2, between 1,000 and 100,000 per .mu.m.sup.2, between
10,000 and 100,000 per .mu.m.sup.2, or between 50,000 and 100,000
per .mu.m.sup.2, or within a range defined by any two of the
foregoing values. In some instances, the present disclosure
provides said composition wherein the nucleoside or nucleoside
analog is present within a nucleotide or nucleotide analog. In some
instances, the present disclosure provides said composition wherein
the composition comprises or incorporates a nucleotide or
nucleotide analog that is modified so as to prevent its
incorporation into an extending nucleic acid chain during a
polymerase reaction. In some instances, the present disclosure
provides said composition wherein the composition comprises or
incorporates a nucleotide or nucleotide analog that is reversibly
modified so as to prevent its incorporation into an extending
nucleic acid chain during a polymerase reaction. In some instances,
the present disclosure provides said composition wherein one or
more labels comprise a fluorescent label, a FRET donor, and/or a
FRET acceptor. In some instances, the present disclosure provides
said composition wherein the substrate (e.g., nucleotide,
nucleotide analog, nucleoside, or nucleoside analog) is attached to
the particle through a linker. In some instances, the present
disclosure provides said composition wherein at least one
nucleotide or nucleotide analog is a nucleotide that has been
modified to inhibit elongation during a polymerase reaction or a
sequencing reaction, such as, for example, a nucleotide that lacks
a 3' hydroxyl group; a nucleotide that has been modified to contain
a blocking group at the 3' position; a nucleotide that has been
modified with a 3'-O-azido group, a 3'-O-azidomethyl group, a
3'-O-alkyl hydroxylamino group, a 3'-phosphorothioate group, a
3'-O-malonyl group, or a 3'-O-benzyl group; and/or a nucleotide
that has not been modified at the 3' position.
[0311] In some instances, the present disclosure provides a method
of determining the sequence of a nucleic acid molecule comprising
the steps, without regard to order, of 1) providing a nucleic acid
molecule comprising a template strand and a complementary strand
that is at least partially complementary to the template strand; 2)
contacting the nucleic acid molecule with the one or more nucleic
acid binding or incorporation composition according to any of the
instances disclosed herein; 3) detecting binding or incorporation
of the nucleic acid binding or incorporation composition to the
nucleic acid molecule, and 4) determining an identity of a terminal
nucleotide to be incorporated into said complementary strand of
said nucleic acid molecule. In some instances, said method may
further comprise incorporating said terminal nucleotide into said
complementary strand, and repeating said contacting, detecting, and
incorporating steps for one or more additional iterations, thereby
determining the sequence of said template strand of said nucleic
acid molecule. In some instances, the present disclosure provides
said method wherein said nucleic acid molecule has been
clonally-amplified on a solid support. In some instances, the
present disclosure provides said method wherein the clonal
amplification comprises the use of a polymerase chain reaction
(PCR), multiple displacement amplification (MDA),
transcription-mediated amplification (TMA), nucleic acid
sequence-based amplification (NASBA), strand displacement
amplification (SDA), real-time SDA, bridge amplification,
isothermal bridge amplification, rolling circle amplification,
circle-to-circle amplification, helicase-dependent amplification,
recombinase-dependent amplification, single-stranded binding (SSB)
protein-dependent amplification, or any combination thereof. In
some instances, the present disclosure provides said method wherein
a sequencing reaction cycle comprising the contacting, detecting,
and incorporating steps is performed in less than 30 minutes, less
than 20 minutes, or in less than 10 minutes. In some instances, the
present disclosure provides said method wherein an average Q-score
for base calling accuracy over a sequencing run is greater than or
equal to 30, or greater than or equal to 40. In some instances, the
present disclosure provides said method wherein at least 50%, at
least 60%, at least 70%, at least 80%, or at least 90% of the
terminal nucleotides identified have a Q-score of greater than 30;
or greater than 40. In some instances, the present disclosure
provides said method wherein at least 95% of the terminal
nucleotides identified have a Q-score of greater than 30.
[0312] In some instances, the present disclosure provides a reagent
comprising one or more nucleic acid binding or incorporation
compositions as disclosed herein, and a buffer. In some instances,
the present disclosure provides said reagent, wherein said reagent
comprises 1, 2, 3, 4, or more nucleic acid binding or incorporation
compositions, wherein each nucleic acid binding or incorporation
composition comprises a single type of nucleotide or nucleotide
analog, and wherein said nucleotide or nucleotide analog comprises
a nucleotide, nucleotide analog, nucleoside, or nucleoside analog.
In some instances, the present disclosure provides said method
wherein said reagent comprises 1, 2, 3, 4, or more nucleic acid
binding or incorporation compositions, wherein each nucleic acid
binding or incorporation composition comprises a single type of
nucleotide or nucleotide analog, and wherein said nucleotide or
nucleotide analog may respectively correspond to one or more from
the group consisting of ATP, ADP, AMP, dATP, dADP, and dAMP; one or
more from the group consisting of TTP, TDP, TMP, dTTP, dTDP, dTMP,
UTP, UDP, UMP, dUTP, dUDP, and dUMP; one or more from the group
consisting of CTP, CDP, CMP, dCTP, dCDP, and dCMP; and one or more
from the group consisting of GTP, GDP, GMP, dGTP, dGDP, and dGMP.
In some instances, the present disclosure provides said method
wherein said reagent comprises 1, 2, 3, 4, or more nucleic acid
binding or incorporation compositions, wherein each nucleic acid
binding or incorporation composition comprises a single type of
nucleotide or nucleotide analog, and wherein said nucleotide or
nucleotide analog may respectively correspond to one or more from
the group consisting of ATP, ADP, AMP, dATP, dADP, dAMP TTP, TDP,
TMP, dTTP, dTDP, dTMP, UTP, UDP, UMP, dUTP, dUDP, dUMP, CTP, CDP,
CMP, dCTP, dCDP, dCMP, GTP, GDP, GMP, dGTP, dGDP, and dGMP.
[0313] In some instances, the present disclosure provides a kit
comprising any of the compositions disclosed herein; and/or any of
the reagents disclosed herein; one or more buffers; and
instructions for the use thereof.
[0314] In some instances, the present disclosure provides a system
for performing any of the methods disclosed herein; wherein said
methods may comprise use of any of the compositions as disclosed
herein; and/or any of the reagents as disclosed herein; one or more
buffers, and one or more nucleic acid molecules optionally tethered
or attached to a solid support, wherein said system is configured
to iteratively perform for the sequential contacting of said
nucleic acid molecules with said composition and/or said reagent;
and for the detection of binding or incorporation of the nucleic
acid binding or incorporation compositions to the one or more
nucleic acid molecules.
[0315] In some instances, the present disclosure provides a
composition as disclosed herein for use in increasing the contrast
to noise ratio (CNR) of a labeled nucleic acid complex bound to or
associated with a surface.
[0316] In some instances, the present disclosure provides a
composition as disclosed herein for use in establishing or
maintaining control over the persistence time of a signal from a
labeled nucleic acid complex bound to or associated with a
surface.
[0317] In some instances, the present disclosure provides a
composition as disclosed herein for use in establishing or
maintaining control over the persistence time of a fluorescence,
luminescence, electrical, electrochemical, colorimetric,
radioactive, magnetic, or electromagnetic signal from a labeled
nucleic acid complex bound to or associated with a surface.
[0318] In some instances, the present disclosure provides a
composition as disclosed herein for use in increasing the
specificity, accuracy, or read length of a nucleic acid sequencing
and/or genotyping application.
[0319] In some instances, the present disclosure provides a
composition as disclosed herein for use in increasing the
specificity, accuracy, or read length in a sequencing by binding or
incorporation, sequencing by synthesis, single molecule sequencing,
or ensemble sequencing method.
[0320] In some instances, the present disclosure provides a reagent
as disclosed herein for use in increasing the contrast to noise
ratio (CNR) of a labeled nucleic acid complex bound to or
associated with a surface.
[0321] In some instances, the present disclosure provides a reagent
as disclosed herein for use in establishing or maintaining control
over the persistence time of a signal from a labeled nucleic acid
complex bound to or associated with a surface.
[0322] In some instances, the present disclosure provides a reagent
as disclosed herein for use in establishing or maintaining control
over the persistence time of a fluorescence, luminescence,
electrical, electrochemical, colorimetric, radioactive, magnetic,
or electromagnetic signal from a labeled nucleic acid complex bound
to or associated with a surface.
[0323] In some instances, the present disclosure provides a reagent
as disclosed herein for use in increasing the specificity,
accuracy, or read length of a nucleic acid sequencing and/or
genotyping application.
[0324] In some instances, the present disclosure provides a reagent
as disclosed herein for use in increasing the specificity,
accuracy, or read length in a sequencing by binding or
incorporation, sequencing by synthesis, single molecule sequencing,
or ensemble sequencing method.
Definitions
[0325] Unless otherwise defined, all of the technical terms used
herein have the same meaning as commonly understood by one of
ordinary skill in the art in the field to which this disclosure
belongs.
[0326] As used in this specification and the appended enumerated
embodiments, the singular forms "a", "an", and "the" include plural
references unless the context clearly dictates otherwise. Any
reference to "or" herein is intended to encompass "and/or" unless
otherwise stated.
[0327] As used herein, the term "about" a number refers to that
number plus or minus 10% of that number. The term "about" when used
in the context of a range refers to that range minus 10% of its
lowest value and plus 10% of its greatest value.
[0328] As used herein, the terms "DNA hybridization" and "nucleic
acid hybridization" are used interchangeably and are intended to
cover any type of nucleic acid hybridization, e.g., DNA
hybridization or RNA hybridization, unless otherwise specified.
[0329] As used herein, the term "isothermal" refers to a condition
in which the temperature remains substantially constant. A
temperature that is "substantially constant" may deviate (e.g.,
increase or decrease) over a period of time by no more than 0.25
degrees, 0.50 degrees, 0.75 degrees, or 1.0 degrees.
[0330] The terms "anneal" or "hybridize," are used herein
interchangeably to refer to the ability of two nucleic acid
molecules to combine together. In some cases, the "combining"
refers to Watson-Crick base pairing between the bases in each of
the two nucleic acid molecules.
[0331] As used herein, "hybridization specificity" refers to a
measure of the ability of nucleic acid molecules (e.g., adapter
sequences, primer sequences, or oligonucleotide sequences) to
correctly hybridize to a region of a target nucleic acid molecule
with a nucleic acid sequence that is completely complementary to
the nucleic acid molecule.
[0332] As used herein, "hybridization sensitivity" refers to a
concentration range of sample (or target) nucleic molecules in
which hybridization occurs with high specificity. In some cases, as
little as 50 picomolar concentration of sample nucleic acid
molecules in which hybridization with high specify is achieved with
the methods, compositions, systems and kits described herein. In
some instances, the range is between about 1 nanomolar to about 50
picomolar concentrations of sample nucleic acid molecules.
[0333] As used herein, "hybridization efficiency" refers to a
measure of the percentage of total available nucleic acid molecules
(e.g., adapter sequences, primer sequences, or oligonucleotide
sequences) that are hybridized to the region of the target nucleic
acid molecule with the nucleic acid sequence that is completely
complementary to the nucleic acid molecule.
[0334] As used herein, the term "hybridization stringency" refer to
a percentage of nucleotide bases within at least a portion of a
nucleic acid sequence undergoing a hybridization (e.g., a
hybridization region) reaction that is complementary through
standard Watson-Crick base pairing. In a non-limiting example, a
hybridization stringency of 80% means that a stable duplex can be
formed in which 80% of the hybridization region undergoes
Watson-Crick base pairing. A higher hybridization stringency means
a higher degree of Watson-Crick base pairing is required in a given
hybridization reaction in order to form a stable duplex.
[0335] As used herein, the terms, "isolate" and "purify," are used
interchangeably herein unless specified otherwise.
[0336] As used herein, "nucleic acid" (also referred to as a
"polynucleotide", "oligonucleotide", ribonucleic acid (RNA), or
deoxyribonucleic acid (DNA)) is a linear polymer of two or more
nucleotides joined by covalent internucleosidic linkages, or
variants or functional fragments thereof. In naturally occurring
examples of nucleic acids, the internucleoside linkage is a
phosphodiester bond. However, other examples optionally comprise
other internucleoside linkages, such as phosphorothiolate linkages
and may or may not comprise a phosphate group. Nucleic acids
include double- and single-stranded DNA, as well as double- and
single-stranded RNA, DNA/RNA hybrids, peptide-nucleic acids (PNAs),
hybrids between PNAs and DNA or RNA, and may also include other
types of nucleic acid modifications.
[0337] As used herein, a "nucleotide" refers to a nucleotide,
nucleoside, or analog thereof. The nucleotide refers to both
naturally occurring and chemically modified nucleotides and can
include but are not limited to a nucleoside, a ribonucleotide, a
deoxyribonucleotide, a protein-nucleic acid residue, or
derivatives. Examples of the nucleotide includes an adenine, a
thymine, a uracil, a cytosine, a guanine, or residue thereof; a
deoxyadenine, a deoxythymine, a deoxyuracil, a deoxycytosine, a
deoxyguanine, or residue thereof; a adenine PNA, a thymine PNA, a
uracil PNA, a cytosine PNA, a guanine PNA, or residue or
equivalents thereof, an N- or C-glycoside of a purine or pyrimidine
base (e.g., a deoxyribonucleoside containing 2-deoxy-D-ribose or
ribonucleoside containing D-ribose).
[0338] "Complementary," as used herein, refers to the topological
compatibility or matching together of interacting surfaces of a
ligand molecule and its receptor. Thus, the receptor and its ligand
can be described as complementary, and furthermore, the contact
surface characteristics are complementary to each other.
[0339] "Branched polymer", as used herein, refers to a polymer
having a plurality of functional groups that help conjugate a
biologically active molecule such as a nucleotide, and the
functional group can be either on the side chain of the polymer or
directly attached to a central core or central backbone of the
polymer. The branched polymer can have a linear backbone with one
or more functional groups coming off the backbone for conjugation.
The branched polymer can also be a polymer having one or more
sidechains, wherein the one or more side chains has a site suitable
for conjugation. Examples of the functional group include but are
limited to hydroxyl, ester, amine, carbonate, acetal, aldehyde,
aldehyde hydrate, alkenyl, acrylate, methacrylate, acrylamide,
active sulfone, hydrazide, thiol, alkanoic acid, acid halide,
isocyanate, isothiocyanate, maleimide, vinylsulfone,
dithiopyridine, vinylpyridine, iodoacetamide, epoxide, glyoxal,
dione, mesylate, tosylate, and tresylate.
[0340] "Polymerase," as used herein, refers to an enzyme that
contains a nucleotide binding moiety and helps formation of a
binding complex between a target nucleic acid and a complementary
nucleotide. The polymerase can have one or more activities
including, but not limited to, base analog detection activities,
DNA polymerization activity, reverse transcriptase activity, DNA
binding or incorporation, strand displacement activity, and
nucleotide binding or incorporation and recognition. The polymerase
can include catalytically inactive polymerase, catalytically active
polymerase, reverse transcriptase, and other enzymes containing a
nucleotide binding or incorporation moiety.
[0341] "Persistence time," as used herein, refers to the length of
time that a binding complex, which is formed between the target
nucleic acid, a polymerase, and a conjugated or unconjugated
nucleotide, remains stable without any binding component
dissociating from the binding complex. The persistence time is
indicative of the stability of the binding complex and strength of
the binding interactions. Persistence time can be measured by
observing the onset and/or duration of a binding complex, such as
by observing a signal from a labeled component of the binding
complex. For example, a labeled nucleotide or a labeled reagent
comprising one or more nucleotides may be present in a binding
complex, thus allowing the signal from the label to be detected
during the persistence time of the binding complex. One
non-limiting example of label is a fluorescent label.
[0342] Abbreviations
[0343] Dimethyl sulfoxide (DMSO),
[0344] Dimethylformamide (DMF),
[0345] 3-(N-morpholino)propanesulfonic acid (MOPS),
[0346] Acetonitrile (ACN)
[0347] 2-(N-morpholino)ethanesulfonic acid (MES)
[0348] saline-sodium citrate (SSC)
[0349] Formamide (Form.)
[0350] Tris(hydroxymethyl)aminomethane (Tris)
Examples
[0351] These examples are provided for illustrative purposes only
and not to limit the scope of the examples and enumerated
embodiments provided herein.
Example 1--DNA Hybridization on Low Non-Specific Binding
Surface
[0352] FIGS. 1A-B provide examples of the optimized hybridization
achieved on low binding surface using the disclosed hybridization
method (FIG. 1A) with reduced concentrations of hybridization
reporter probe and shortened hybridization times, as compared to
the results achieved using a traditional hybridization protocol on
the same low binding surface (FIG. 1B).
[0353] FIG. 1A shows hybridization reactions on the low binding
surface according to the embodiments described herein. The rows
provide two test hybridization conditions, hybridization condition
1 ("Hyb 1") and hybridization condition 2 ("Hyb 2"). Hyb 1 refers
to the hybridization buffer composition C10 from Table 1. Hyb 2
refers to the hybridization buffer composition D18 from Table 1. A
hybridization reporter probe (complementary oligonucleotide
sequences labeled with a Cy.TM.3 fluorophore at the 5' end) at
concentrations reported in FIG. 1A (10 nM, 1 nM, 250 pM, 100 pM,
and 50 pM) were hybridized in the buffer compositions at 60 degrees
Celsius for 2 minutes.
[0354] FIG. 1B shows hybridization reactions on the low binding
surface according to a standard hybridization protocol with
standard hybridization conditions ("Standard Hyb Conditions"). A
standard hybridization buffer of 2.times.-5.times. saline-sodium
citrate (SSC) was used with same hybridization reporter probe above
at the same concentrations above, as shown in FIG. 1A. The standard
hybridization reaction was performed at 90 degrees Celsius with a
slow cool process (2 hours) to reach 37 degrees Celsius.
[0355] For each hybridization reaction provided in FIG. 1A and FIG.
1B, the top row for each hybridization reaction is test ("T"),
which is the complementary oligos (e.g.,
CY3.TM.-5'-ACCCTGAAAGTACGTGCATTACATG-3'), and the bottom row for
each hybridization reach is a control ("C"), which is a
noncomplementary oligos (e.g.,
CY3.TM.-5'-ATGTCTATTACGTCACACTATTATG-3').
[0356] The surfaces used for all testing conditions were ultra-low
non-specific binding surfaces having a level of non-specific Cy3
dye absorption corresponding to less than or equal to about 0.25
molecules/.mu.m.sup.2. In this example, the low non-specific
binding surfaces used were a glass substrates that were
functionalized with Silane-PEG-5K-COOH (Nanocs Inc.).
[0357] Following completion of the hybridization reactions, wells
were washed with 50 mM Tris pH 8.0; 50 mM NaCl.
[0358] Images were obtained using an inverted microscope (Olympus
IX83) equipped with 100.times.TIRF objective, NA=1.4 (Olympus),
dichroic mirror optimized for 532 nm light (Semrock,
Di03-R532-t1-25.times.36), a bandpass filter optimized for Cy3
emission, (Semrock, FF01-562/40-25), and a camera (sCMOS, Andor
Zyla) under non-signal saturating conditions for 1 s, (Laser
Quantum, Gem 532, <1 W/cm.sup.2 at the sample) while the sample
was immersed in a buffer (25 mM ACES, pH 7.4 buffer). Images were
collected as described above, and the results are shown in FIG. 1A
(optimized) and FIG. 1B (standard).
[0359] A significant signal was observed from the reaction with 250
picomolar (pM) in both Hyb 1 and Hyb 2 hybridization reactions
(FIG. 1A), as compared with the negative control. In contrast, no
signal was observed from the reaction with 250 pM in the Standard
Hyb conditions, as compared with the negative control. The same
result was observed for lower input concentrations (e.g., 100 pM,
50 pM) of the hybridization reporter probe. FIG. 1A shows more than
200-fold decrease in input DNA (labeled oligo) required for
specific DNA capture on low non-specific binding surfaces tested, a
50.times. decrease in hybridization times, and a reduction in the
hybridization temperatures by half, as compared with standard
hybridization methods and reagents on the same low non-specific
binding substrates (FIG. 1B). The buffer compositions and methods
described herein boast improved hybridization specificity,
decreased workflow times and increased hybridization
sensitivity.
Example 2
[0360] Buffer compositions according to various embodiments
described herein were optimized to facilitate hybridization of
monotemplate oligonucleotide fragments to the low non-specific
binding surface described herein.
[0361] Preparing the low non-specific binding surfaces. Glass
substrates (175 um 22.times.60 mm.sup.2, Corning Glass) were
cleaned with KOH and ethanol. Low binding glass surfaces were
prepared by incubating Silane-PEG5K-NHS (Nanocs) in ethanol at 65
degrees for 30 minutes. Oligonucleotides with 5' modified NH.sub.2
were grafted to these surfaces in a mixture of 1 micromolar (uM),
5.1 .mu.M, and 46 uM oligonucleotides in methanol/phosphate buffer
for 20 minutes, to form immobilized oligonucleotides coupled to the
glass substrates.
[0362] Circularizing monotemplate oligonucleotide fragments into
library. Monotemplate oligonucleotide fragments (approximately 100
base pairs in length) were circularized using splint ligation
protocol that contained complementary fragments to surface grafted
primers.
[0363] Hybridizing the circularized library to immobilized
oligonucleotides. Following circularization of the library,
circular library fragments were added at a concentration of 100
picomolar (pM) in various test hybridization test mixtures
indicated by rows B-F. Individual buffer/library hybridization
mixtures were added to 384 well plate with the functionalized
surface affixed at 50 degrees Celsius for 4 minutes.
[0364] Visualizing hybridization using test buffer compositions.
Intercalating DNA stain was added to the buffer/library
hybridization mixtures following the hybridization reaction to
visualize the hybridization of the circularized libraries. The 384
well plate was imaged using a fluorescence microscope and 488
nanometer (nm) excitation with a 60.times. water immersion
objective (1.2 NA, Olympus) (See FIG. 3). A number of buffer
compositions were tested for the hybridization of target nucleic
acid (e.g., circularized library) with surface bound nucleic acid
(e.g., immobilized oligonucleotides). Table 1 provides the buffer
compositions and immobilized oligonucleotide concentrations for
each reaction seen in FIG. 3, with columns 10-21 in Table 1
corresponding with columns 10-21 of FIG. 3, and rows B-F
corresponding to row B-F of FIG. 3. F10 and F11 are negative
controls using standard hybridization conditions, where no
background signal was detected signifying both the validity of the
negative control and the low non-specific binding nature of
surfaces tested.
TABLE-US-00001 TABLE 1 Buffer compositions tested for hybridizing
target nucleic acid with surface bound nucleic acid Graft
concentration 1 uM 5.1 uM 46 uM 9 10 11 12 13 14 15 16 17 18 19 20
21 B Cracked 75% 75% 2x 25% Std 30% Std 50% Std Std Std Std ACN +
ACN + SSC ACN + buf. + PEG ACN + MES Phos 2xSSC + 5% PEG + 50% 10%
30% Std PEG Form. buf. C 1 uM 50% 50% 4x 25% Std 20% Std + Std +
Tris + Tris + Std Std 31- ACN + ACN + SSC ACN + buf. + PEG + 2 2
1xSSC 1xSSC buff + buff + NH2- Mes Tris MES + 10% 2x 5% 5% Cy3 20%
PEG + SSC PEG + PEG + PEG + 5 % 30% 30% 10% Form. Form. Form. Form.
D 1 uM 25% 25% 10x 50% Std 10% Std + Std + 25% 25% Std Std 31- ACN
+ ACN + SSC EtOH + buf. + PEG + 4 4 ACN + ACN + buff + buff + NH2-
MES + Tris + 2x 10% 2x MES + MES + 10% 10% Cy3 2xSSC 2xSSC SSC PEG
+ SSC + 20% 20% PEG + PEG + 10% 5% PEG + PEG + 5% 5% Form. Form.
10% 10% Form. Form. Form. Form. E 1 uM MES + Tris + 20x 50% Std 5%
Std + Std + Std Std 10% 10% 31- 1xSSC 1xSSC SSC EtOH + buf. + Form.
+ 6 6 buf. + buf. + PEG + PEG + NH2- 2x 20% 2xSSC 20% 20% 2x 2x Cy3
SSC + PEG + PEG + PEG + SSC + SSC + 10% 10% 10% 10% 5% 5% PEG Form.
Form. Form. Form. Form. F 10 nM 10 nM 10nM 10x Std Std 10% Std +
Std + Std Std 10% 10% 31- 31- 31- SSC + buf. + Form. + 8 8 buf. +
buf. + Form. + Form. + NH2- NH2- NH2- 10% 10% 2xSSC 10% 10% 2xSSC
2xSSC Cy3 Cy3 Cy3 Form. Form. Form. Form.
[0365] "Graft" concentration refers to the concentration of surface
bound oligos. Spot counts for each of the hybridization conditions
were tabulated, whereby higher counts indicated more effective
hybridization buffer formulations as shown in FIG. 4. Table 1
provides the buffer compositions and immobilized oligonucleotide
concentrations for each reaction seen in FIG. 4, with columns 10-21
in Table 1 corresponding with columns 10-21 of FIG. 4, and rows B-F
corresponding to row B-F of FIG. 4.
[0366] Amplifying the hybridized target nucleic acid with surface
bound nucleic acid. Following hybridization, the target nucleic
acids were amplified to quantify hybridization effectiveness.
Rolling circle amplification (RCA) was performed using
amplification mixes with Bst according to manufacturer's
instructions (New England Biolabs.RTM.). The amplified colonies of
target nucleic acids were further amplified using a RCA/PCR
amplification strategy, whereby PCR cycles were performed on the
RCA multimer nanoball to improve the detection sensitivity of the
assay and more stringently quantify hybridized library.
[0367] The resulting surface amplified products were again stained
with intercalating DNA stains and imaged to verify hybridization
specificity and effectiveness (See FIG. 5). Table 1 provides the
buffer compositions and immobilized oligonucleotide concentrations
for each reaction seen in FIG. 5, with columns 10-21 in Table 1
corresponding with columns 10-21 of FIG. 5, and rows B-F
corresponding to row B-F of FIG. 5.
[0368] Analysis of Hybridization Buffers and conditions.
Hybridization conditions were evaluated based on the correlation of
maximum spot counts from FIG. 3, FIG. 4, and FIG. 5. Hybridization
buffer C10, D18, and E21 showed the highest spot count, as compared
to the negative controls provided in F10 and F11 in which water,
instead of hybridization buffer, was used. in FIG. 4. This result
was validated in FIG. 5 after amplification.
Example 3
[0369] In this example, the non-specific binding of cyanine 3 dye
(Cy3)-labeled molecules was measured on the low non-specific
binding surfaces disclosed herein. In independent non-specific
binding assays, 1 uM labeled Cy3 dCTP (GE Amersham), 1 uM Cy5 dGTP
dye (Jena Biosciences), 10 uM Aminoallyl-dUTP--ATTO-647N (Jena
Biosciences), 10 uM Aminoallyl-dUTP--ATTO-Rho11 (Jena Biosciences),
10 .mu.M Aminoallyl-dUTP--ATTO-Rho11 (Jena Biosciences), 10 .mu.M
cCTP--Cy3.5 (GE Amersham), and 10 .mu.M
7-Propargylamino-7-deaza-dGTP--Cy3 (Jena Biosciences) were
incubated individually on the low non-specific binding surfaces
described in Example 2 (Glass substrates treated with Silane-PEG5K,
Nanocs) at 37.degree. C. for 15 minutes in a 384 well plate format.
Each well was rinsed 2-3.times. with 50 .mu.l deionized RNase/DNase
Free water and 2-3.times. with 25 mM ACES buffer pH 7.4. The 384
well plates were imaged at single molecule resolution on an Olympus
IX83 microscope (Olympus Corp., Center Valley, Pa.) with TIRF
objective (100.times., 1.4 NA, Olympus), a sCMOS camera (Zyla 4.2,
Andor), an illumination source with excitation wavelengths of 532
nm or 635 nm. Dichroic mirrors were purchased from Semrock (IDEX
Health & Science, LLC, Rochester, N.Y.), e.g., 405, 488, 532,
or 633 nm dichroic reflectors/beamsplitters, and band pass filters
were chosen as 532 LP or 645 LP concordant with the appropriate
excitation wavelength. 5.
[0370] The imaging set-up enabled the visualization of single dye
molecules bound to the substrates. Individual fluorescent spots
were counted and the total spot numbers were divided by the
respective area of the ROI. For example, with a 100.times.
objective and Andor sCMOS camera, which has a pixel size of 6.5
microns, it is possible to calculate the area of a region of
interest (ROI).
[0371] A low non-specific binding of the dye molecules above of
less than or equal to about 0.50 molecules per .mu.m.sup.2 was
observed. Some non-specific binding of the dye molecules of less
than or equal to 0.25 molecules per .mu.m.sup.2 was observed.
Example 4
[0372] A nucleic acid sequencing reaction is performed using the
workflow provided in FIG. 2 using the disclosed hybridization
compositions and methods from Example 1 and Example 2 on the
surfaces used in Examples 1-3. In this non-limiting example, the
processing times that are achieved are also provided in FIG. 2.
Example 5: Preparation of Multivalent Binding Composition
[0373] One type of multi-armed substrate, as shown in FIG. 16A was
made by reacting propargylamine dNTPs with Biotin-PEG-NHS. This
aqueous reaction was driven to completion and purified; resulting
in a pure Biotin-PEG-dNTP species. In separate reactions, several
different PEG lengths were used, corresponding to average molecular
weights varying from 1K Da to 20K Da. The Biotin-PEG-dNTP species
were mixed with either freshly prepared or commercially-sourced
dye-labeled streptavidin (SA) using a Dye:SA ratio of 3-5:1. Mixing
of Biotin-PEG-dNTP with dye-labeled streptavidin was done in the
presence of excess biotin-PEG-dNTP to ensure saturation of the
biotin binding sites on each streptavidin tetramer. Complete
complexes were purified away from excess biotin-PEG-dNTP by size
exclusion chromatography. Each nucleotide type was conjugated and
purified separately, then mixed together to create a four-base mix
for sequencing.
[0374] Another type of multi-armed substrate as shown in FIG. 16A
was made in a single pot by reacting multi-arm PEG NHS with excess
Dye-NH2 and propargylamine dNTPs. Various multi-arm PEG NHS
variants were used ranging from 4-16 arms and ranging in molecular
weight from 5K Da to 40K Da. After reacting, excess small molecule
dye and dNTP were removed by size exclusion chromatography. Each
nucleotide type was conjugated and purified independently then
mixed together to create a four-base mix for sequencing.
[0375] Class II substrates as shown in FIG. 16B were made using one
pot reactions to simultaneously conjugate dye and dNTP.
Alkyne-PEG-NHS was reacted with excess propargylamine dNTP. This
product (Alkyne-PEG-dNTP) was then purified to homogeneity by
chromatography. Multiple PEG lengths were used, with average
molecular weights varying between 1K Da and 20K Da. Dendrimer cores
containing a variable, discrete number (12, 24, 48, 96) of azide
conjugation sites were used. Conjugation of Alkyne-Dye and
Alkyne-PEG-dNTP to the dendrimer core occurred in a one pot
reaction containing excess dye and dNTP species via copper-mediated
click chemistry. After reacting, excess small molecule dye and dNTP
were removed by size exclusion chromatography. Each nucleotide type
was conjugated and purified independently then mixed together to
create a four-base mix for sequencing. We note that this scheme
allows the ready substitution of alternative cores, such as
dextrans, other polymers, proteins, etc.
[0376] Class III polymer-nucleotide conjugates as shown in FIG. 16C
were constructed by reacting 4- or 8-arm PEG NHS with a saturating
mixture of biotin and propargylamine dNTP. This reaction was then
purified by size exclusion chromatography. The result of this
reaction was a multi-arm PEG containing a discrete distribution of
biotin and nucleotides. This heterogeneous population was then
reacted with dye-labeled streptavidin and purified by size
exclusion chromatography. Each nucleotide type was conjugated and
purified independently then mixed together to create a four-base
mix for sequencing. We note that the distribution of biotin and
nucleotide is tunable by the input ratio of Biotin-NH2 to
propargylamine dNTP.
Example 6: Detection of Ternary Complex
[0377] Binding reactions using the multivalent binding composition
having PEG polymer-nucleotide conjugates were analyzed to detect
possible formation of ternary binding complex, and the fluorescence
images of the various steps are illustrated in FIGS. 18A-18J. In
FIG. 18A, red and green fluorescent images post exposure of DNA
rolling circle application (RCA) templates (G and A first base) to
500 nM base labeled nucleotides (A-Cy3 and G-Cy5) in exposure
buffer containing 20 nM Klenow polymerase and 2.5 mM Sr+2.
Multivalent PEG-substrate compositions were prepared using varying
ratios of 4-armed PEG-amine (4ArmPEG-NH), biotin-PEG-amine
(Biotin-PEG-NH), and nucleotide (Nuc) as follows: Samples PB1 and
PB5, 4ArmPEG-NH: Biotin-PEG-NH:Nuc=0.25:1:0.5; Sample PB2,
4ArmPEG-NH: Biotin-PEG-NH:Nuc=0.125:0.5:0.25; Sample PB3,
4ArmPEG-NH: Biotin-PEG-NH:Nuc=0.25:1:0.5. Images were collected
after washing with imaging buffer with the same composition as the
exposure buffer but containing no nucleotides or polymerase.
[0378] Contrast was scaled to maximize visualization of the dimmest
signals, but no signals persisted following washing with imaging
buffer (FIG. 18A, inset). In FIGS. 18B-18E, the fluorescence images
showing multivalent PEG-nucleotide (base-labeled) ligands at 500 nM
after mixing in the exposure buffer and imaging in the imaging
buffer as above (FIG. 18B: PB1; FIG. 18C: PB2; FIG. 18D: PB3; FIG.
18E: PB5). FIG. 18F: fluorescence image showing multivalent
PEG-nucleotide (base-labeled) ligand PB5 at 2.5 uM after mixing in
the exposure buffer and imaging in the imaging buffer as above. In
FIGS. 18G-18I, the fluorescence images showing further base
discrimination by exposure of multivalent ligands to inactive
mutants of Klenow polymerase (FIG. 18G: D882H; FIG. 18H: D882E;
FIG. 18I: D882A, and the wild type Klenow (control) enzyme is shown
in FIG. 18J).
[0379] Using multivalent ligands formulations, the base
discrimination can be enabled by providing polymerase-ligand
interactions having increased avidity. In addition, it is shown
that increased concentration of multivalent ligands can generate
higher signals, as well as various Klenow mutations that knock out
catalytic activity, and can be used for avidity-based
sequencing.
Example 7: Sequencing of Target Nucleic Acid Molecules Using
Ternary Complexes
[0380] In order to demonstrate sequencing based on multivalent
ligand reporters, 4 known templates were amplified using RCA
methods on a low binding substrate. Successive cycles were exposed
to exposure buffer containing 20 nM Klenow polymerase and 2.5 mM
Sr+2 and washed with imaging buffer and imaged. After imaging, the
substrates were washed with wash buffer (EDTA and high salt) and
blocked nucleotides were added to proceed to the next base. The
cycle was repeated for 5 cycles. Spots were detected using standard
imaging processing and spot detection and the sequences were called
using a two-color green and red scheme (G-Cy3 and A-Cy5) to
identify the templates being cycled. As shown in FIG. 19A and FIG.
19B, multivalent ligands are able to provide base discrimination
through all 5 sequencing cycles.
Example 8: Control of Nucleotide Dissociation from Ternary
Complex
[0381] Ternary complexes are prepared and imaged as in Example 6.
The complexes are imaged over varying lengths of time to
demonstrate the persistence of the ternary complex, e.g., as long
as 60 seconds. After a length of time, the complexes are washed
with a buffer identical to the buffer used for the formation of the
complexes, only lacking any divalent cation, e.g., 10 mM Tris pH
8.0, 0.5 mM EDTA, 50 mM NaCl, 0.016% Triton X100 (without SrOAc),
or, in another example, the complexes are washed with a buffer
identical to the buffer used for the formation of the complexes,
which contains a chelating agent but otherwise lacks any divalent
cation, e.g., 10 mM Tris pH 8.0, 0.5 mM EDTA, 50 mM NaCl, 0.016%
Triton X100 (without SrOAc), with 100 nm-100 mM EDTA. The
fluorescence from the complexes is observed over time allowing
observation and quantitation of the dissociation of the ternary
complexes. A representative time course of this dissolution is
shown in FIG. 17.
Example 9: Extension of Target Nucleic Acid Complementary
Sequence
[0382] After preparing, imaging, and dissociating ternary complexes
as in Example 8, a deblocking solution is flowed into the chamber
containing the bound DNA molecules, sufficient to remove the
blocking moiety, such as an O-azidomethyl group, an O-alkyl
hydroxylamino group, or an O-amino group, from the 3' end of the
elongating DNA strand. Either following or concurrently with this,
an extension solution is flowed into the chamber containing the
bound DNA molecules. The extension solution contains a buffer, a
divalent cation sufficient to support polymerase activity, an
active polymerase, and an appropriate amount of all four
nucleotides, where the nucleotides are blocked such that they are
incapable of supporting further elongation after the addition of a
single nucleotide to the elongating DNA strand, such as by
incorporation of a 3'-O-azidomethyl group, a 3'-O-alkyl
hydroxylamino group, or a 3'-O-amino group. The elongating strand
is thus extended by one and only one base, and the binding of
catalytically inactive polymerase and multivalent binding substrate
can be used to call the next base in the cycle.
[0383] In another example, the nucleotides attached to the
multivalent substrate may be attached through a labile bond, such
that a buffer may be flowed into the chamber containing the bound
DNA molecules containing a divalent cation or other cofactor
sufficient to render the polymerase catalytically active. Prior to,
after, or concurrently with this, conditions may be provided that
are sufficient to cleave the base from the multivalent substrate
such that it may be incorporated into the elongating strand. This
cleavage and incorporation results in the dissociation of the label
and the polymer backbone of the multivalent substrate while
extending the elongating DNA strand by exactly one base. Washing to
remove used polymer backbone is carried out, and new multivalent
substrate is flowed into the chamber containing the bound DNA
molecules, allowing the new base to be called as in Example 5.
Example 10. Use of Polymer-Nucleotide Conjugates with Various
Lengths of PEG Branch
[0384] The polymer-nucleotide conjugates having varying PEG arm
lengths described in Example 7 were subjected to a single
sequencing cycle and imaged as described in Example 5. As shown in
FIGS. 20A-20G, increasing the length of the PEG branches led to
increased signal up to a length corresponding to an apparent
average PEG MW of 5K Da (FIGS. 20A-20D). The use of longer PEG arms
than this led to decreases in the fluorescence signal for both
Cy3-A and Cy5-G (FIG. 20E-20G). Quantitative measurements of signal
intensity are shown graphically in FIG. 21.
Example 11: Enhancement of Multivalent Substrate Binding by
Addition of Detergent
[0385] Multivalent substrates were prepared and assembled into
binding complexes in the presence and absence of detergent: one set
using 10 mM Tris pH 8.0, 0.5 mM EDTA, 50 mM NaCl, 5 mM SroAc, 0%
TritonX100 (Condition A), and one set using 10 mM Tris pH 8.0, 0.5
mM EDTA, 50 mM NaCl, 5 mM SroAc, 0.016% Triton X100. FIG. 22 shows
normalized fluorescence from these multivalent substrates bound to
DNA clusters, with the substrate complexes formed in the presence
(condition B) of Triton-X100 (0.016%) showing clearly enhanced
fluorescence intensity.
Example 12. Evaluation of Multivalent Substrate Binding Time
Courses
[0386] Multivalent substrates were prepared and assembled into
binding complexes as in Example 6. Complexes were also formed under
identical buffer conditions using free labeled nucleotides.
Complexes were imaged over the course of 60 min. to characterize
the persistence time of the complexes. FIGS. 23A-23B shows
representative results. Multivalent binding complexes are stable
over timescales of >60 minutes (FIG. 23B) while labeled free
nucleotides dissociate in less than one minute (FIG. 23A).
[0387] While preferred embodiments of the compositions and methods
disclosed herein 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 present disclosure. It should be understood that
various alternatives to the embodiments of the methods and
compositions described herein may be employed in any combination in
practicing the methods and compositions of the present disclosure.
Sequence CWU 1
1
15120DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotidemisc_feature(1)..(20)This sequence may
encompass 1-20 nucleotidesSee specification as filed for detailed
description of substitutions and preferred embodiments 1tttttttttt
tttttttttt 20220DNAArtificial SequenceDescription of Artificial
Sequence Synthetic oligonucleotidemisc_feature(1)..(20)This
sequence may encompass 1-20 nucleotidesSee specification as filed
for detailed description of substitutions and preferred embodiments
2aaaaaaaaaa aaaaaaaaaa 20325DNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotide 3accctgaaag
tacgtgcatt acatg 25425DNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotide 4atgtctatta
cgtcacacta ttatg 25537DNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotide 5cgcacttttt
accgcttttt cagcgttttt tgcacac 37637DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 6cgcacttttt accccttttt cagcgttttt tgcacac
37737DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 7cgcccttttt accgcttttt cagagttttt tgcacac
37837DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 8cgcccttttt accccttttt cagcgttttt tgcacac
37937DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 9cgcacttttt accgcttttt cagcgttttt tgcccac
371037DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 10cgcacttttt accccttttt ccgcgttttt
tgcacac 371137DNAArtificial SequenceDescription of Artificial
Sequence Synthetic oligonucleotide 11cgcacttttt accgcttttt
cagcgttttt tacacac 371237DNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotide 12cgcccttttt
accccttttt cagcgttttt tgcccac 371337DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 13cgcccttttt accgcttttt cagcgttttt tgcacac
371437DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 14cgaacttttt accgcttttt cagcgttttt
tgcacac 371537DNAArtificial SequenceDescription of Artificial
Sequence Synthetic oligonucleotide 15cgcccttttt accgcttttt
cagcgttttt tgcagac 37
* * * * *