U.S. patent application number 15/597305 was filed with the patent office on 2017-11-16 for compositions, methods, systems and kits for nucleic acid amplification and analysis.
The applicant listed for this patent is Ampliwise Inc.. Invention is credited to Mindy Su, Xing Su, Kai Wu.
Application Number | 20170327879 15/597305 |
Document ID | / |
Family ID | 56014486 |
Filed Date | 2017-11-16 |
United States Patent
Application |
20170327879 |
Kind Code |
A1 |
Wu; Kai ; et al. |
November 16, 2017 |
COMPOSITIONS, METHODS, SYSTEMS AND KITS FOR NUCLEIC ACID
AMPLIFICATION AND ANALYSIS
Abstract
The disclosure provides compositions, methods, systems and kits
for amplifying and assaying nucleic acids.
Inventors: |
Wu; Kai; (Mountain View,
CA) ; Su; Mindy; (Cupertino, CA) ; Su;
Xing; (Cupertino, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Ampliwise Inc. |
Santa Clara |
CA |
US |
|
|
Family ID: |
56014486 |
Appl. No.: |
15/597305 |
Filed: |
May 17, 2017 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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PCT/US2015/061252 |
Nov 18, 2015 |
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15597305 |
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62082534 |
Nov 20, 2014 |
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62082538 |
Nov 20, 2014 |
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62082541 |
Nov 20, 2014 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12P 19/34 20130101;
C12Q 1/6853 20130101; C12Q 1/6827 20130101; C12Q 1/686
20130101 |
International
Class: |
C12Q 1/68 20060101
C12Q001/68 |
Claims
1.-16. (canceled)
17. A set of particles for nucleic acid amplification, comprising:
a first particle comprising a first primer having a 5' end and a 3'
end, wherein said first primer is linked to said first particle via
said 5' end of said first primer, and wherein said first primer
exhibits sequence homology to a target nucleic acid strand at a 5'
end of said target nucleic acid strand; and a second particle
comprising a second primer having a 5' end and a 3' end, wherein
said second primer is linked to said second particle via said 5'
end of said second primer, and wherein said second primer exhibits
sequence homology to a complement nucleic acid strand of said
target nucleic acid strand at a 5' end of said complement nucleic
acid strand, wherein a sequence of said target nucleic acid strand
to which said first primer exhibits homology is separated from a
sequence of said target nucleic acid strand to which said second
primer exhibits complementarity by at least about 10
nucleotides.
18.-20. (canceled)
21. The set of particles of claim 17, wherein said first particle
and/or said second particle are contained in a partition.
22. The set of particles of claim 21, wherein said partition is a
droplet, a well or a vessel.
23.-25. (canceled)
26. The set of particles of claim 17, wherein said first particle
comprises at least two of said first primer or two of said second
primer.
27. (canceled)
28. (canceled)
29. The set of particles of claim 17, wherein said first and/or
second particle comprise a material selected from the group
consisting of gold, silver, copper, platinum, palladium, a metal
oxide, a polymer, carbon and combinations thereof.
30. An isolated nucleic acid complex comprising a double-stranded
nucleic acid molecule having at least a first strand and a second
strand that is at least partially complementary to said first
strand, wherein said first strand is coupled to a first particle at
a 5' end of said first strand, and wherein said second strand is
coupled to a separate second particle at a 5' end of said second
strand.
31. The isolated nucleic acid complex of claim 30, wherein said
double-stranded nucleic acid molecule comprises a first end
sequence and a second end sequence, wherein said first end sequence
is complexed with said first particle via a first capture sequence
linked to said first particle, wherein said second end sequence is
complexed with said second particle via a second capture sequence
linked to said second particle, wherein said first capture sequence
is at least partially complementary to said first end sequence and
said second capture sequence is at least partially complementary to
said second end sequence.
32. The isolated nucleic acid complex of claim 31, wherein said
first capture sequence is linked to said first particle at a 3' end
of said first capture sequence, or wherein said second capture
sequence is linked to said second particle at a 3' end of said
second capture sequence.
33. (canceled)
34. The isolated nucleic acid complex of claim 30, wherein said
first strand is covalently linked to said first particle at said 5'
end of said first strand, or wherein said second strand is
covalently linked to said second particle at said 5' end of said
second strand.
35. (canceled)
36. The isolated nucleic acid complex of claim 30, wherein said
nucleic acid complex is not immobilized to a support.
37.-39. (canceled)
40. The isolated nucleic acid complex of claim 30, wherein said
first particle and said second particle comprise a dimension of
about 0.5 nanometers (nm) to about 100 nanometers.
41. (canceled)
42. The isolated nucleic acid complex of claim 30, wherein said
nucleic acid complex is contained in a partition.
43. The isolated nucleic acid complex of claim 42, wherein said
partition is a droplet, a well or a vessel.
44.-47. (canceled)
48. The isolated nucleic acid complex of claim 30, wherein said
first and/or second particle comprise a material selected from the
group consisting of gold, silver, copper, platinum, palladium, a
metal oxide, a polymer, carbon and combinations thereof.
49. A kit for assaying the presence or absence of a target nucleic
acid strand in a sample having or suspected of having said target
nucleic acid strand, comprising: a first particle and a second
particle, wherein said first particle comprises a first primer
having a first nucleic acid sequence that exhibits sequence
homology to a portion of a target nucleic acid strand, wherein said
second particle comprises a second primer having a second nucleic
acid sequence that exhibits sequence homology to a portion of a
complement nucleic acid strand of said target nucleic acid strand,
wherein said first nucleic acid sequence is different than said
second nucleic acid sequence; instructions for using said first and
second particles to identify the presence or absence of said target
nucleic acid strand in the sample via a primer extension
reaction.
50. The kit of claim 49, wherein said first and second particles
are contained in a vessel.
51. (canceled)
52. The kit of claim 49, wherein said first and/or second particle
comprise a material selected from the group consisting of gold,
silver, copper, platinum, palladium, a metal oxide, a polymer,
carbon and combinations thereof.
53. The kit of claim 49, further comprising one or more reagents
suitable for generating a water-in-oil emulsion.
54. (canceled)
55. The kit of claim 49, further comprising a detectable species
that permits the identification of said target nucleic acid
strand.
56. (canceled)
57. The kit of claim 49, further comprising reagents necessary for
performing said primer extension reaction.
58. The kit of claim 57, wherein said reagents comprise a
polymerase and nucleotides.
59.-131. (canceled)
Description
CROSS-REFERENCE
[0001] This application is a continuation of PCT International
Application No. PCT/US2015/061252, filed Nov. 18, 2015, which
claims priority to U.S. Provisional Patent Application No.
62/082,534 filed on Nov. 20, 2014, U.S. Provisional Patent
Application No. 62/082,538 filed on Nov. 20, 2014 and U.S.
Provisional Patent Application No. 62/082,541 filed on Nov. 20,
2014, which applications are herein incorporated by reference in
their entireties for all purposes.
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 Jun. 30, 2017, is named 46846701301SL.txt and is 757 bytes in
size.
BACKGROUND
[0003] Nucleic acid amplification methods permit amplification of
nucleic acid molecules in a sample, such as a biological sample. A
nucleic acid molecule can be amplified, via, for example, thermal
cycling based approaches (e.g., polymerase chain reaction (PCR)) or
via isothermal approaches. Following amplification of a nucleic
acid molecule, amplification products can be detected and the
results of detection interpreted by an end-user. Nucleic acid
amplification may also be useful in preparing a nucleic acid
molecule for subsequent analysis in numerous applications related
to nucleic acid analysis such as, for example in detecting target
nucleic acid sequences, single nucleotide polymorphisms (SNPs),
sequence mutations (e.g., deletions, insertions), detecting rare
nucleic acid molecules/sequences in a sample and/or preparing a
nucleic acid molecule for a sequencing reaction. Thus, due to the
applicability of nucleic acid amplification to a wide range of
applications, there exists a need for compositions, kits, methods
and systems useful for amplifying nucleic acid molecules and/or for
analyzing amplified nucleic acid molecules that are generated from
nucleic acid amplification.
SUMMARY
[0004] The disclosure provides compositions, kits, methods and
systems for the amplification and analysis of nucleic acids. In
addition, the disclosure provides compositions, kits, methods and
system for the generation and analysis of complexes that comprise
particles and nucleic acids. The methods and compositions disclosed
herein find a wide array of utilities in, for example, research,
environmental testing, forensic identification and clinical
diagnostics.
[0005] An aspect of the disclosure provides a set of particles for
nucleic acid amplification. The set of particles can comprise a
first particle that comprises a first primer having a 5' end and a
3' end, where the first primer is linked to the first particle via
the 5' end of the first primer, and where the first primer exhibits
sequence homology to a target nucleic acid strand at a 5' end of
the target nucleic acid strand. The set of particles can also
comprise a second particle comprising a second primer having a 5'
end and a 3' end, where the second primer is linked to the second
particle via the 5' end of the second primer, and where the second
primer exhibits sequence homology to a complement nucleic acid
strand of the target nucleic acid strand at a 5' end of the
complement nucleic acid strand. Moreover, a sequence on the target
nucleic acid strand to which the first primer exhibits homology can
be different than a sequence on the complement nucleic acid strand
to which the second primer exhibits homology.
[0006] In some embodiments, the first particle and/or the second
particle can be contained in a partition that can be, for example,
a droplet in an emulsion, a well or a vessel. In cases where the
partition is a well, the well can be well among an array of wells.
In some embodiments, the 3' end of the first primer may be adapted
to be extended in a primer extension reaction to form a copy of the
target nucleic acid strand or a portion thereof. In some
embodiments, the 3' end of the second primer may be adapted to be
extended in a primer extension reaction to form the complement
nucleic acid strand or a portion thereof. In some embodiments, the
first particle may comprise at least two of the first primer and/or
the second particle may comprise at least two of the second primer.
In some embodiments, the set of particles can further comprise a
third particle that may comprise the first primer and/or second
primer.
[0007] In some embodiments, the first and/or second particle may be
selected from the group consisting of solid particles, porous
particles, nanoparticles, beads, microparticles, metal particles,
magnetic particles, semiconductor particles, polymeric particles,
and nucleic acid particles. In some embodiments, the first or
second primer may be directly linked to a respective one of the
first and second particles. In some embodiments, the first or
second primer may be linked to a respective one of the first and
second particles through at least one linker. The linker can be
selected from the group consisting of a nucleic acid, a phosphate
moiety, an amino acid, a peptide, a hydrocarbon chain, a
polyethylene glycol (PEG), a polysaccharide and combinations
thereof. In some embodiments, the first and/or second particle may
comprise a material selected from the group consisting of gold,
silver, copper, platinum, palladium, a metal oxide, a polymer,
carbon and combinations thereof. In some embodiments, the first
particle and/or second particle may have a dimension of about 0.5
nm to about 100 nm. In some embodiments, the first particle and/or
second particle may have a dimension of about 1 nm to about 20
nm.
[0008] An additional aspect of the disclosure provides a set of
particles for nucleic acid amplification. The set of particles can
comprise a first particle that comprises a first primer having a 5'
end and a 3' end, where the first primer is linked to the first
particle via the 5' end of the first primer, and where the first
primer exhibits sequence homology to a target nucleic acid strand
at a 5' end of the target nucleic acid strand. The set of particles
can also comprise a second particle that comprises a second primer
having a 5' end and a 3' end, where the second primer is linked to
the second particle via the 5' end of the second primer, and where
the second primer exhibits sequence homology to a complement
nucleic acid strand of the target nucleic acid strand at a 5' end
of the complement nucleic acid strand. Moreover, a sequence of the
target nucleic acid strand to which the first primer exhibits
homology may be separated from a sequence of the target nucleic
acid strand to which the second primer exhibits complementarity by
at least about 10 nucleotides.
[0009] In some embodiments, the sequence on the target nucleic acid
strand to which the first primer exhibits homology may be separated
from the sequence on the target nucleic acid strand to which the
second primer exhibits complementarity by at least about 25
nucleotides, at least about 50 nucleotides, or at least about 100
nucleotides. In some embodiments, the first particle and/or the
second particle may be contained in a partition such as, for
example, a droplet in an emulsion, a well or a vessel. In cases
where the partition is a well, the well may be among an array of
wells.
[0010] In some embodiments, the first particle may comprise at
least two of the first primer and/or the second particle may
comprise at least two of the second primer. In some embodiments,
the first and/or second particle may be selected from the group
consisting of solid particles, porous particles, nanoparticles,
beads, microparticles, metal particles, magnetic particles,
semiconductor particles, polymeric particles, and nucleic acid
particles. In some embodiments, the first and/or second particle
may comprise a material selected from the group consisting of gold,
silver, copper, platinum, palladium, a metal oxide, a polymer,
carbon and combinations thereof. In some embodiments, the first
particle and/or second particle may have a dimension of about 0.5
nm to about 100 nm. In some embodiments, the first particle and/or
second particle may have a dimension of about 1 nm to about 20
nm.
[0011] An additional aspect of the disclosure provides an isolated
nucleic acid complex. The isolated nucleic acid complex can
comprise a double-stranded nucleic acid molecule having at least a
first strand and a second strand that is at least partially
complementary to the first strand. The first strand can be coupled
to a first particle at a 5' end of the first strand and the second
strand can be coupled to a separate second particle at a 5' end of
the second strand.
[0012] In some embodiments, the double-stranded nucleic acid
molecule can comprise a first end sequence and a second end
sequence, wherein the first end sequence is complexed with the
first particle via a first capture sequence linked to the first
particle. The second end sequence can be complexed with the second
particle via a second capture sequence linked to the second
particle. Moreover, the first capture sequence can be at least
partially complementary to the first end sequence and the second
capture sequence can be at least partially complementary to the
second end sequence.
[0013] In some embodiments, the first capture sequence may be
linked to the first particle at a 3' end of the first capture
sequence and/or the second capture sequence may be linked to the
second particle at a 3' end of the second capture sequence. In some
embodiments, the first strand may be covalently linked to the first
particle at the 5' end of the first strand and/or the second strand
may be covalently linked to the second particle at the 5' end of
the second strand. In some embodiments, the nucleic acid complex
may not be immobilized to a support.
[0014] In some embodiments, the isolated nucleic acid complex may
further comprise at least three particles, at least five particles
or at least ten particles each of which particles can be linked to
another particle through an additional double-stranded nucleic acid
molecule that is substantially the same as the double-stranded
nucleic acid molecule. In some embodiments, the first particle and
the second particle may comprise a dimension of about 0.5
nanometers (nm) to about 100 nanometers. In some embodiments, the
first particle and the second particle may comprise a dimension of
about 1 nm to about 20 nm.
[0015] In some embodiments, the nucleic acid complex may be
contained in a partition such as, for example, a droplet in an
emulsion, a well or a vessel. In cases where the partition is a
well, the well may be a well among an array of wells. In some
embodiments, the first and/or second particle may be selected from
the group consisting of solid particles, porous particles,
nanoparticles, beads, microparticles, metal particles, magnetic
particles, semiconductor particles, polymeric particles, and
nucleic acid particles. In some embodiments, the first and/or
second particle may comprise a material selected from the group
consisting of gold, silver, copper, platinum, palladium, a metal
oxide, a polymer, carbon and combinations thereof.
[0016] An additional aspect of the disclosure provides a kit for
assaying the presence or absence of a target nucleic acid strand in
a sample having or suspected of having the target nucleic acid
strand. The kit can comprise a first particle and a second
particle. The first particle may comprise a first primer having a
first nucleic acid sequence that exhibits sequence homology to a
portion of a target nucleic acid strand and the second particle may
comprise a second primer having a second nucleic acid sequence that
exhibits sequence homology to a portion of a complement nucleic
acid strand of the target nucleic acid strand. Moreover, the first
nucleic acid sequence may be different than the second nucleic acid
sequence. The kit can also comprise instructions for using the
first and second particles to identify the presence or absence of
the target nucleic acid strand in the sample via a primer extension
reaction.
[0017] In some embodiments, the first and/or second particles may
be contained in a vessel. In some embodiments, the first and/or
second particle may be selected from the group consisting of solid
particles, porous particles, nanoparticles, beads, microparticles,
metal particles, magnetic particles, semiconductor particles,
polymeric particles, and nucleic acid particles. In some
embodiments, the first and/or second particle may comprise a
material selected from the group consisting of gold, silver,
copper, platinum, palladium, a metal oxide, a polymer, carbon and
combinations thereof.
[0018] In some embodiments, the kit may further comprise one or
more reagents suitable for generating a water-in-oil emulsion. Such
reagents include, for example, a buffer, oil, and a surfactant. In
some embodiments, the kit may further comprise a detectable species
that permits the identification of the target nucleic acid strand.
The detectable species may be, for example, an optically-responsive
species. In some embodiments, the kit may further comprise reagents
necessary for performing the primer extension reaction. Such
reagents include, for example, a polymerase and nucleotides.
[0019] An additional aspect of the disclosure provides a method for
assaying the presence of a target nucleic acid molecule in a sample
having or suspected of having the target nucleic acid molecule. The
method can comprise subjecting the sample to a nucleic acid
amplification reaction in a reaction mixture under conditions to
yield an amplified target nucleic acid molecule linked to at least
a first particle and a second particle, which amplified target
nucleic acid molecule comprises at least a portion of a sequence of
the target nucleic acid molecule. The reaction mixture can comprise
the first particle having a first primer with a 5' end and a 3'
end. The first primer can be linked to the first particle via the
5' end of the first primer and the first primer can exhibit
sequence homology to a strand of the target nucleic acid molecule
at a 5' end of the strand of the target nucleic acid molecule. The
reaction mixture can also comprise the second particle having a
second primer with a 5' end and a 3' end. The second primer can be
linked to the second particle via the 5' end of the second primer
and the second primer can exhibit sequence homology to a complement
strand of the strand of the target nucleic acid molecule at a 5'
end of the complement strand. A sequence on the strand of the
target nucleic acid molecule to which the first primer exhibits
homology may be different than a sequence of the complement strand
to which the second primer exhibits homology. Moreover, the first
primer and the second primer can be adapted to be extended in a
primer extension reaction to form a copy of the target nucleic acid
molecule or a portion thereof.
[0020] In some embodiments, the reaction mixture may further
comprise a polymerase. Such a polymerase may extend the 3' end of
the first primer in a primer extension reaction to form a copy of
the strand of the target nucleic acid or a portion thereof.
Moreover, such a polymerase may extend the 3' end of the second
primer in a primer extension reaction to form the complement strand
or a portion thereof. In some embodiments, the reaction mixture may
further comprise an optically-responsive species such as, for
example, a dye or fluorescent protein. In some embodiments, the
reaction mixture may be contained in a partition such as, for
example, a droplet in an emulsion, a well or a vessel. In cases
where the partition is a well, the well may be a well in an array
of wells.
[0021] In some embodiments, the method can further comprise
detecting the amplified target nucleic acid molecule linked to the
first particle and second particle. The amplified target nucleic
acid molecule can detected, for example, optically, electrically,
physically, spectroscopically, electrochemically or
electrostatically. In some embodiments, a plurality of amplified
target nucleic acid molecules is detected. In some embodiments, the
target nucleic acid molecule may be single-stranded.
[0022] An additional aspect of the disclosure provides a method for
generating a nucleic acid complex comprising a target nucleic acid
molecule. The method can comprise, in a reaction mixture,
amplifying the target nucleic acid molecule with a forward primer
and a reverse primer, under conditions that yield the nucleic acid
complex. The nucleic acid complex can comprise an amplified target
nucleic acid molecule, where the amplified target nucleic acid
molecule comprises a first strand and a second strand that is at
least partially complementary to the first strand. The first strand
can be coupled to a first particle at a 5' end of the first strand
and the second strand can be coupled to the second particle at a 5'
end of the second strand.
[0023] In some embodiments, the forward primer may be linked to the
first particle and the reverse primer may be linked to the second
particle. In some embodiments, the reaction mixture may comprise
the first particle and the second particle. In some embodiments,
the method may further comprise amplifying the target nucleic acid
molecule with a third particle linked thereto the forward primer or
the reverse primer to yield the nucleic acid complex. The nucleic
acid complex can further comprise the third particle coupled to the
first particle and/or the second particle via an additional
amplified target nucleic acid molecule.
[0024] In some embodiments, the method may further comprise
detecting the nucleic acid complex. The nucleic acid complex may be
detected, for example, optically, spectroscopically, physically,
electrically, electrochemically or electrostatically. In some
embodiments, the first particle and the second particle may be
metallic particles and detection of the nucleic acid complex may be
effected by visual examination. In some embodiments, the method may
further comprise detecting the nucleic acid complex with the aid of
an optically-responsive species.
[0025] In some embodiments, the method may further comprise
isolating the nucleic acid complex by centrifugation, magnetic
separation, sedimentation, filtration, chromatography, capillary
action or affinity capture of the nucleic acid complex. In some
embodiments, the method may further comprise isolating the nucleic
acid complex by affinity capture of the nucleic acid complex on a
support. In some embodiments, the reaction mixture may be contained
in a partition such as, for example, a droplet in an emulsion or a
well. In some embodiments, the method may further comprise
releasing the nucleic acid complex from the partition and detecting
the released nucleic acid complex.
[0026] Another aspect of the disclosure provides a method for
identifying the presence or absence of a target nucleic acid
molecule in a sample. The method can comprise providing a solution
that is suspected of containing the target nucleic acid molecule.
The target nucleic acid molecule can comprise a first nucleic acid
strand linked to a first particle, and a second nucleic acid strand
linked to a second particle. The second strand can be hybridized to
the first strand via sequence complementarity to yield a nucleic
acid complex. The method can further comprise detecting a signal
indicative of the presence or absence of the nucleic acid complex
in the solution, thereby identifying the presence of the target
nucleic acid molecule.
[0027] In some embodiments, providing the solution can comprise
providing the solution to a detector and the detector can detect
the signal indicative of the presence or absence of the nucleic
acid complex in the solution. In some embodiments, the signal can
be indicative of an optical property, physical property, electrical
property, electrostatic property, or electrochemical property of
the nucleic acid complex. In some embodiments, the first particle
and the second particle may be metallic particles and the signals
can be detected by visual examination. In some embodiments, the
signal may be generated from the activity of an
optically-responsive species such as, for example, a dye or
fluorescent protein. Examples of a dye include SYBR green I, SYBR
green II, SYBR gold, ethidium bromide, methylene blue, Pyronin Y,
DAPI, acridine orange, Blue View or phycoerythrin.
[0028] In some embodiments, the solution may be contained within a
partition such as, for example, a droplet in an emulsion or a well
(e.g., a well in an array of wells). In some embodiments, the
solution may comprise a plurality of nucleic acid complexes that
comprise two or more particles. In some embodiments, the nucleic
acid complex may not be affixed to a support.
[0029] Another aspect of the disclosure provides a method for
assaying a sample for the presence or absence of a target nucleic
acid sequence. The method can comprise receiving a request to assay
the sample for the presence or absence of the target nucleic acid
sequence. The method can further comprise assaying the sample for
the presence or absence of the target nucleic acid sequence by
detecting at least one nucleic acid complex that comprises a
double-stranded nucleic acid molecule linked to at least a first
particle and a second particle. The double-stranded nucleic
molecule can comprise a first single-stranded nucleic acid molecule
and a second single-stranded nucleic acid molecule that is
complementary to the first single-stranded nucleic acid molecule.
At least one of the first single-stranded nucleic acid molecule and
the second single-stranded nucleic acid molecule can comprise the
target nucleic acid sequence. Moreover, the method can further
comprise generating a report that is indicative of the presence or
absence of the target nucleic acid sequence in the sample.
[0030] In some embodiments, assaying the sample for the presence or
absence of the target nucleic acid sequence can comprise detecting
a signal that is indicative of the presence or absence of the
nucleic acid complex. The signal can be, for example, indicative of
an optical property, physical property, electrical property,
spectroscopic property, electrostatic property, or electrochemical
property of the nucleic acid complex. In some embodiments, the
signal may be generated from the activity of an
optically-responsive species such as, for example, a dye. Examples
of dyes include SYBR green I, SYBR green II, SYBR gold, ethidium
bromide, methylene blue, Pyronin Y, DAPI, acridine orange, Blue
View or phycoerythrin.
[0031] In some embodiments, the nucleic acid complex may be
detected in a partition such as, for example, a droplet in an
emulsion or a well (e.g., a well in an array of wells). In some
embodiments, the nucleic acid complex may comprise a plurality of
double-stranded nucleic acid molecules linked to greater than two
particles. In some embodiments, the report is an electronic report
that can be presented on a user interface of an electronic display
of an electronic device.
[0032] An additional aspect of the disclosure provides a method for
generating a nucleic acid complex comprising a target nucleic acid
molecule. The method can comprise, in a reaction mixture,
amplifying the target nucleic acid molecule with a forward primer
and a reverse primer to yield an amplified target nucleic acid
molecule. The forward primer and the reverse primer can each
comprise a 3' end capable of being extended in a primer extension
reaction and a hairpin structure. The amplified target nucleic acid
molecule can comprise a first strand comprising a first overhang
sequence at one end of the amplified target nucleic acid molecule
and a second strand comprising a second overhang sequence at the
other end of the amplified target nucleic acid molecule. Moreover,
the method can further comprise contacting the amplified target
nucleic acid molecule with a first particle and a second particle
to yield a nucleic acid complex. The nucleic acid complex can
comprise the amplified target nucleic acid molecule that is
complexed with the first particle via sequence complementarity
between the first overhang sequence and a first capture sequence
linked to the first particle; and the second particle via sequence
complementarity between the second overhang sequence and a second
capture sequence linked to the second particle.
[0033] In some embodiments, the forward primer and the reverse
primer further may comprise a spacer region which cannot be copied
via a primer extension reaction. Such a spacer region may be
selected from the group consisting of C3 spacer, an abasic site, a
carbonaceous linker, polyethylene glycol (PEG) and combinations
thereof. In some embodiments, the method may further comprise
ligating the first strand to the second capture sequence and/or
ligating the second strand to the first capture sequence.
[0034] Another aspect of the disclosure provides a method for
nucleic acid amplification. The method can comprise annealing a
forward primer linked to a first particle to a nucleic acid strand
and a reverse primer linked to a second particle to a complement
strand of the nucleic acid strand. The method can further comprise
extending the forward primer and the reverse primer in a
template-directed manner to yield a first double-stranded nucleic
acid molecule linked to the first particle and a second
double-stranded nucleic acid molecule linked to the second
particle. The method may further comprise denaturing the first
double-stranded nucleic acid molecule and the second
double-stranded nucleic acid molecule to generate a first
single-stranded molecule linked to the first particle and a second
single-stranded molecule linked to the second particle. The method
can further comprise repeating annealing, extending and denaturing
as described above can be repeated by annealing the forward primer
to the second single-stranded molecule and annealing the reverse
primer to the first single-stranded molecule to yield a nucleic
acid complex comprising an amplified double-stranded nucleic acid
molecule. The amplified double-stranded nucleic acid molecule can
linked at one end to the first particle and linked at its other end
to the second particle.
[0035] In some embodiments, the method is performed in a partition.
In some embodiments, the method may further comprise amplifying the
nucleic acid strand with a third particle linked thereto the
forward primer or the reverse primer to yield the nucleic acid
complex that further comprises the third particle coupled to the
first particle and/or the second particle via an additional
amplified double-stranded nucleic acid molecule. In some
embodiments, the method can further comprise detecting the nucleic
acid complex optically, spectroscopically physically, electrically,
electrochemically, or electrostatically.
[0036] An additional aspect of the disclosure provides a system for
analyzing the content(s) of a solution. The system can comprise a
detection cell that is adapted to contain or direct a solution
containing a nucleic acid complex that comprises a double-stranded
nucleic acid molecule that is linked to at least a first particle
and a second particle. The double-stranded nucleic molecule can
comprise a first single-stranded nucleic acid molecule and a second
single-stranded nucleic acid molecule that is complementary to at
least a portion of the first single-stranded nucleic acid molecule.
The system can further comprise a detector that can be linked to
the detection cell and detects signals indicative of the presence
or absence of a nucleic acid complex in the solution. Moreover, the
system can further comprise a computer processor that can be linked
to the detector and programmed to receive signals from the
detector, which signals are indicative of the presence or absence
of the nucleic acid complex. The computer processor can also be
programmed to determine if the nucleic acid complex is present or
absent in the solution based on the detected signals.
[0037] In some embodiments, the detection cell may comprise a
vessel or an array of wells. In some embodiments, the detection
cell may comprise a support. In some embodiments, the detection
cell may comprise a fluid flow path that may be, for example, a
microfluidic channel. In some embodiments, the detector may be
selected from the group consisting of an optical detector, a
spectroscopic detector and an electrochemical detector.
[0038] In some embodiments, the detection cell may further comprise
the solution. In some embodiments, the solution may be contained
within a partition such as, for example, a well (e.g., a well in an
array of wells) or a droplet in an emulsion. In some embodiments,
the nucleic acid complex may comprise a plurality of
double-stranded nucleic acid molecules linked to greater than two
particles.
[0039] Additional aspects and advantages of the present disclosure
will become readily apparent to those skilled in this art from the
following detailed description, wherein only illustrative
embodiments of the present disclosure are shown and described. As
will be realized, the present disclosure is capable of other and
different embodiments, and its several details are capable of
modifications in various obvious respects, all without departing
from the disclosure. Accordingly, the drawings and description are
to be regarded as illustrative in nature, and not as
restrictive.
INCORPORATION BY REFERENCE
[0040] All publications, patents, and patent applications mentioned
in this specification are herein incorporated by reference to the
same extent as if each individual publication, patent, or patent
application was specifically and individually indicated to be
incorporated by reference.
BRIEF DESCRIPTION OF THE DRAWINGS
[0041] The novel features of the invention are set forth with
particularity in the appended claims. A better understanding of the
features and advantages of the present invention will be obtained
by reference to the following detailed description that sets forth
illustrative embodiments, in which the principles of the invention
are utilized, and the accompanying drawings (also "Figure" and
"Fig." herein), of which:
[0042] FIG. 1 is a schematic depiction of an example method of
isolating and detecting a nucleic acid complex and/or a nucleic
acid molecule of a nucleic acid complex;
[0043] FIG. 2 is a schematic depiction of an example method of
isolating and detecting a nucleic acid complex and/or nucleic acid
molecule of a nucleic acid complex;
[0044] FIG. 3 (panel A) and FIG. 3 (panel B) are schematic
depictions of an example primer (SEQ ID NO: 1) used in nucleic acid
amplification;
[0045] FIG. 3 (panel C) is a schematic depiction of an example
method of using an example primer in nucleic acid
amplification;
[0046] FIG. 4 is a schematic depiction of an example method for
nucleic acid amplification to yield a nucleic acid complex;
[0047] FIG. 5 is a schematic depiction of an example method for
nucleic acid amplification to yield a nucleic acid complex;
[0048] FIG. 6 is a schematic depiction of an example method for
nucleic acid amplification to yield a nucleic acid complex and
isolation of the nucleic acid complex;
[0049] FIG. 7 is a schematic depiction of an example method for
multiplex nucleic acid amplification;
[0050] FIG. 8 is a schematic depiction of an example method for
multiplex nucleic acid amplification;
[0051] FIG. 9 is a schematic depiction of an example method for
processing a nucleic acid complex for completing a sequencing
reaction; and
[0052] FIG. 10 is a schematic depiction of an example computer
system that comprises a computer processor.
DETAILED DESCRIPTION
[0053] While various embodiments of the invention have been shown
and described herein, it will be obvious to those skilled in the
art that such embodiments are provided by way of example only.
Numerous variations, changes, and substitutions may occur to those
skilled in the art without departing from the invention. It should
be understood that various alternatives to the embodiments of the
invention described herein may be employed.
[0054] As used herein, the singular form "a", "an" and "the"
include plural references unless the context clearly dictates
otherwise. For example, the term "a nucleic acid molecule" includes
a plurality of nucleic acid molecules, including mixtures
thereof.
[0055] As used herein, the terms "amplifying" and "amplification"
are used interchangeably and generally refer to producing one or
more copies of a nucleic acid.
[0056] As used herein, the terms "anneal" and "annealing" generally
refer to the binding of one nucleic acid molecule (e.g., a primer)
with another nucleic acid molecule (e.g., a template nucleic acid
molecule) via complementarity between the nucleic acid
molecules.
[0057] As used herein, the term "capture sequence" generally refers
to a nucleic acid sequence that is associated with a support and is
capable of coupling (e.g., hybridizing) with a nucleic acid
molecule via sequence complementarity such that the coupling of the
nucleic acid molecule with the capture sequence immobilizes the
nucleic acid molecule to the support.
[0058] "Complementarity" or "complementary" generally refer to the
ability of a nucleic acid molecule to form hydrogen bond(s) with
another nucleic acid molecule by either Watson-Crick or other types
of base-pairing. "Partially complementary" generally means that a
portion of a first nucleic acid sequence will hydrogen bond with a
portion of a second nucleic acid sequence.
[0059] As used herein, the terms "denaturing" and "denaturation"
are used interchangeably and generally refer to the full or partial
unwinding of the helical structure of a double-stranded nucleic
acid, and in some embodiments the unwinding of the secondary
structure of a single stranded nucleic acid.
[0060] As used herein, a "detectable species" generally refers to a
composition that yields a detectable signal, the presence or
absence of which can be used to detect the presence of a nucleic
acid and/or copies of a nucleic acid. In some embodiments, a
detectable species may be an optically-responsive species. As used
herein, the term "optically-responsive species" generally refers to
a detectable species that yields a detectable signal in the
presence (or absence) of electromagnetic radiation, such as, for
example, light. Examples of detectable moieties are provided
elsewhere herein.
[0061] As used herein, the term "linked" and "coupled" are used
interchangeably and generally refer to the association of two
species. Two species may be linked or coupled in any suitable way
including direct linkage or coupling (e.g., direct attachment
between species), indirect linkage or coupling (e.g., attachment
via a linker coupled to both species), covalent attachment, or
non-covalent attachment (e.g., hybridization between nucleic acid
molecules, binding of members of an affinity binding pair, ionic
interactions, hydrophobic interactions, Van der Waals forces, etc.)
and combinations thereof.
[0062] As used herein, the term "melting temperature" (T.sub.m)
generally refers to the temperature at which two single-stranded
nucleic acid molecules that are hybridized and form a
double-stranded molecule dissociate from each other. In some
embodiments, a melting temperature can refer to a temperature at
which about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%,
60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or more identical
nucleic acid strands of a population of identical double-stranded
nucleic acid molecules dissociate from their complement strands.
For example, the melting temperature of a primer may refer to the
temperature at which about half of the molecules of the primer in a
population of identical primers hybridized to a nucleic acid
molecule dissociate from their complementary sequence on their
respective nucleic acid molecules. In some embodiments, the melting
temperature of a primer may be from about 20.degree. C. to about
80.degree. C. In some embodiments, the melting temperature of a
primer may be from about 25.degree. C. to about 75.degree. C. In
some embodiments, the melting temperature of a primer may be from
about 25.degree. C. to about 60.degree. C. In some embodiments, the
melting temperature of a primer may be about 20.degree. C.,
21.degree. C., 22.degree. C., 23.degree. C., 24.degree. C.,
25.degree. C., 26.degree. C., 27.degree. C., 28.degree. C.,
29.degree. C., 30.degree. C., 31.degree. C., 32.degree. C.,
33.degree. C., 34.degree. C., 35.degree. C., 36.degree. C.,
37.degree. C., 38.degree. C., 39.degree. C., 40.degree. C.,
41.degree. C., 42.degree. C., 43.degree. C., 44.degree. C.,
45.degree. C., 46.degree. C., 47.degree. C., 48.degree. C.,
49.degree. C., 50.degree. C., 51.degree. C., 52.degree. C.,
53.degree. C., 54.degree. C., 55.degree. C., 56.degree. C.,
57.degree. C., 58.degree. C., 59.degree. C., 60.degree. C.,
61.degree. C., 62.degree. C., 63.degree. C., 64.degree. C.,
65.degree. C., 66.degree. C., 67.degree. C., 68.degree. C.,
69.degree. C., 70.degree. C., or higher.
[0063] As used herein, the terms "nucleic acid" and "nucleic acid
molecule" are used interchangeably and generally refer to a
polymeric form of nucleotides of any length, either
deoxyribonucleotides (dNTPs) or ribonucleotides (rNTPs), or analogs
thereof. Nucleic acids may have any three dimensional structure,
and may perform any function, known or unknown. Non-limiting
examples of nucleic acids include deoxyribonucleic acid (DNA),
ribonucleic acid (RNA), a peptide nucleic acid (PNA), coding or
non-coding regions of a gene or gene fragment, loci (locus) defined
from linkage analysis, exons, introns, messenger RNA (mRNA),
transfer RNA, ribosomal RNA, short interfering RNA (siRNA),
short-hairpin RNA (shRNA), micro-RNA (miRNA), ribozymes, cDNA,
recombinant nucleic acids, branched nucleic acids, plasmids,
vectors, isolated DNA of any sequence, isolated RNA of any
sequence, nucleic acid probes, and primers. A nucleic acid may
comprise one or more modified nucleotides, such as methylated
nucleotides and nucleotide analogs such as, for example, locked
nucleic acids (LNA), fluorinated nucleic acids (FNA), bridged
nucleic acids and thio-nucleotides. If present, modifications to
the nucleotide structure may be made before or after assembly of
the nucleic acid. The sequence of nucleotides of a nucleic acid may
be interrupted by non-nucleotide components, such as, for example a
linker or other type of spacer. A nucleic acid may be further
modified after polymerization, such as by conjugation or binding
with a detectable species. In some embodiments, a nucleic acid may
be a primer that, in some embodiments, can be used to amplify
another nucleic acid molecule.
[0064] As used herein, the term "nucleic acid complex" generally
refers to a complex comprising a plurality of particles linked
together via one or more double-stranded nucleic acid molecules. A
nucleic acid complex may comprise at least 2, 3, 4, 5, 6, 7, 8, 9,
10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,
27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43,
44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100
or more particles associated with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,
11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,
28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44,
45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 or
more double-stranded nucleic acid molecules.
[0065] As used herein, the term "primer" generally refers to a
nucleic acid molecule that is capable of hybridizing with a
template nucleic acid molecule and capable of being extended in a
template-directed manner via the template nucleic acid
molecule.
[0066] A "primer extension reaction" generally refers to the
binding (e.g., "annealing") of a primer to a strand of nucleic
acid, followed by incorporation of nucleotides to the primer (e.g.,
"extension" of or "extending" the primer), using the strand of
nucleic acid as a template. A primer extension reaction may be
completed with the aid of an enzyme, such as, for example a
polymerase.
[0067] As used herein, the term "reaction mixture" generally refers
to a composition comprising one or more reagents necessary to
complete denaturation of a double-stranded nucleic acid molecule,
annealing of a primer to a strand of nucleic acid, extension of the
primer in a primer extension reaction and/or nucleic acid
amplification, with non-limiting examples of such reagents that
include one or more primers having specificity for a target nucleic
acid, a polymerase, suitable buffers, co-factors (e.g., divalent
and monovalent cations), nucleotides (e.g., deoxyribonucleotides
(dNTPs)), any other enzymes, surfactants and additives that can
modulate nucleic acid hybridization. In some embodiments, a
reaction mixture can also comprise one or more detectable
species.
[0068] As used herein, the term "sequence homology" generally
refers to a degree of sequence congruence between two or more
nucleic acid molecules. For example, a nucleic acid molecule can
exhibit at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%,
98%, 99% or 100% sequence homology with one or more other nucleic
acid molecules when optimally aligned with the one or more nucleic
acid molecules. Sequence homology may occur at the 5' ends of a
plurality of nucleic acid molecules (e.g., two nucleic acid
molecules may have a common nucleic acid sequence each at the 5'
end of each nucleic acid molecule), at the 3' ends of a plurality
of nucleic acid molecules (e.g., two nucleic acid molecules may
have a common nucleic acid sequence at the 3' end of each nucleic
acid molecule); or may occur at different locations of different
nucleic acid molecules (e.g., one nucleic acid molecule may have a
common nucleic acid sequence at its 5' end and another nucleic acid
molecule may have the common nucleic acid sequence between its 5'
end and 3' end).
[0069] As used herein, the term "support" generally refers to a
species on which another species can be immobilized. Non-limiting
examples of supports include a particle, a surface of a well, a
surface of a vessel, a solid surface, a planar surface, a surface
of an array, a porous surface (e.g., a micro-cavity of a porous
surface), a resin (e.g., a resin in a column) and a fiber (e.g., a
fiber in a membrane or support). Moreover, a support can comprise
any suitable material with non-limiting examples that include a
metal, a metal oxide, carbonaceous materials and polymeric species.
A support may be used, for example, to immobilize a nucleic acid
molecule and/or may be used to immobilize a nucleic acid
complex.
[0070] As used herein, the terms "target nucleic acid" and "target
nucleic acid molecule" are used interchangeably and generally refer
to a nucleic acid molecule in a starting population of nucleic acid
molecules having a target sequence whose presence, amount, and/or
nucleotide sequence, or changes in one or more of these, are
desired to be determined. In some embodiments, a target nucleic
acid molecule may be double-stranded. In some embodiments, a target
nucleic acid molecule may be single-stranded. In general, the term
"target nucleic acid strand" refers to a single-stranded target
nucleic acid molecule. In general, the term "target nucleic acid
sequence" refers to a nucleic acid sequence on a strand of target
nucleic acid. A target nucleic acid molecule or target nucleic acid
sequence may be a portion of a gene, a regulatory sequence, genomic
DNA, cDNA, RNA including mRNA, miRNA, rRNA, or others. The target
nucleic acid sequence or target nucleic acid molecule may be a
target nucleic acid sequence or target nucleic acid molecule from a
sample or a secondary target such as a product of an amplification
reaction.
[0071] Various aspects of the disclosure provide sets of particles
for nucleic acid amplification and isolated nucleic acid complexes
that comprise nucleic acid molecules linked to a plurality of
particles. Sets of particles can include a first and second
particle each associated with a nucleic acid molecule such as
primer and/or capture sequence. Isolated nucleic acid complexes can
include a first particle and a second particle, which first and
second particles are linked via a double-stranded nucleic acid
molecule linked to both the first particle and the second
particle.
[0072] In one aspect, the disclosure provides a set of particles
for nucleic acid amplification that includes a first particle and a
second particle. The first particle may comprise a first primer,
having a 5' end and a 3' end and linked to the first particle via
the 5' end of the first primer. The first primer can exhibit
sequence homology to a target nucleic acid strand at a 5' end of
the target nucleic acid strand. The second particle may comprise a
second primer, having a 5' end and a 3' end and linked to the
second particle via the 5' end of the second primer. The second
primer can exhibit sequence homology to a complement nucleic acid
strand of the target nucleic acid strand at a 5' end of the
complement nucleic acid strand. In addition, a sequence on the
target nucleic acid strand to which the first primer exhibits
homology can be different than a sequence on the complement nucleic
acid strand to which the second primer exhibits homology.
[0073] In another aspect, the disclosure provides a set of
particles for nucleic acid amplification that includes a first
particle and a second particle. The first particle may comprise a
first primer, having a 5' end and a 3' end and linked to the first
particle via the 5' end of the first primer. The first primer can
exhibit sequence homology to a target nucleic acid strand at a 5'
end of the target nucleic acid strand. The second particle may
comprise a second primer, having a 5' end and a 3' end and linked
to the second particle via the 5' end of the second primer. The
second primer can exhibit sequence homology to a complement nucleic
acid strand of the target nucleic acid strand at a 5' end of the
complement nucleic acid strand. In addition, a sequence of the
target nucleic acid strand to which the first primer exhibits
homology may be separated from a sequence of the target nucleic
acid strand to which the second primer exhibits complementarity by
at least about 10 nucleotides.
[0074] In any set of particles described herein, a sequence on the
target nucleic acid strand to which the first primer exhibits
homology may be separated from a sequence on the target nucleic
acid strand to which the second primer exhibits complementarity by
at least about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,
23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39,
40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56,
57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73,
74 75 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90,
91, 92, 93, 94, 95, 96, 97, 98, 99, 100 or more nucleotides.
Additionally, in any set of particles described herein, the first
primer can exhibit at least about 60%, 65%, 70%, 75%, 80%, 85%,
90%, 95%, 96%, 97%, 98%, 99% or 100% sequence homology to the
target nucleic acid strand at a 5' end of the target nucleic acid
strand when optimally aligned with the target nucleic acid strand.
Similarly, in any set of particles described herein, the second
primer can exhibit at least about 60%, 65%, 70%, 75%, 80%, 85%,
90%, 95%, 96%, 97%, 98%, 99% or 100% sequence homology to the
complement nucleic acid strand of the target nucleic acid strand at
a 5' end of the complement nucleic acid strand when optimally
aligned with the complement nucleic acid strand.
[0075] In any set of particles described herein, the first particle
may comprise at least about 1, 2, 10, 50 100, 500 1000, 5000,
10000, 50000, 100000, 500000, 1000000, 5000000, 10000000 or more
molecules of the first primer. Moreover, the second particle may
comprise at least about 1, 2, 10, 50 100, 500 1000, 5000, 10000,
50000, 100000, 500000, 1000000, 5000000, 10000000 or more molecules
of the second primer. In any set of particles described herein, the
first particle and second particle may also comprise the second
primer and first primer, respectively. Moreover, the length of the
first primer and the second primer linked to the first and second
particles, respectively, may vary depending upon the particular
application. For example, the length of the first primer or second
primer may be about 1 nucleotide to about 100 nucleotides in
length; about 5 to about 50 nucleotides in length; about 5 to about
30 nucleotides in length; or about 15 to about 30 nucleotides in
length. In some embodiments, the length of the first primer or the
second primer may be about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,
14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,
31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47,
48, 49, 50 or more nucleotides in length.
[0076] Additionally, the first primer may be linked to the first
particle and the second primer may be linked to the second
particle, including via direct attachment or indirect attachment
via, for example, at least one linker. Non-limiting examples of
such linkers include a polymeric species, a nucleic acid, a
phosphate moiety, an amino acid, a peptide, a hydrocarbon chain, a
polysaccharide, a polyethylene glycol (PEG) and combinations
thereof. Direct and indirect attachments between primers and
particles may be via covalent bonds (e.g., covalent bonds between
primer and particle, covalent bonds between linker and particle
and/or primer), or non-covalent interactions (e.g., hybridization
between nucleic acid molecules, binding of members of an affinity
binding pair (e.g., streptavidin/biotin), ionic interactions,
hydrophobic interactions, Van der Waals forces, etc.) and
combinations thereof.
[0077] Moreover, the particles in any set of particles described
herein may be linked to respective primers via any suitable
chemistry or combination of chemistries (e.g., for functionalizing
particles with groups that may be used for linking primers and
particles). Non-limiting examples of such chemistries include thiol
chemistry, phosphonic acid chemistry (e.g., in cases where a
particle comprises a metal oxide),
1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) chemistry,
aldehyde chemistry, epoxy chemistry, homobifunctional cross-linker
chemistry (e.g., via amine functional groups, via thiol functional
groups), heterobifunctional cross-linker chemistry (e.g., via amine
functional groups, via thiol functional groups),
1,4-phenylenedisiothiocyanate (PDC) chemistry, organosilanization,
ionized gas treatments, UV irradiation, click chemistry, diacetone
acrylamide crosslinking and combinations thereof.
[0078] In any set of particles described herein, the 3' end of the
first primer may be adapted to be extended in a primer extension
reaction to form a copy of the target nucleic acid strand or a
portion thereof. Moreover, the 3' end of the second primer may be
adapted to be extended in a primer extension reaction to form the
complement nucleic acid strand or a portion thereof. In addition,
any set of particles described herein may also comprise more
particles than a first particle and a second particle, where each
additional particle comprises the first and/or second primer. For
example, a set of particles described herein may comprise about 2,
3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 100, 500,
1000, 5000, 10000, 50000, 100000, 500000, 1000000, 5000000,
10000000, 50000000, 100000000, 500000000, 1000000000 or more
particles each comprising the first and/or second primer.
[0079] In another aspect, the disclosure provides an isolated
nucleic acid complex. The isolated nucleic acid complex may
comprise a double-stranded nucleic acid molecule having at least a
first strand and a second strand that is at least partially
complementary to the first strand. The first strand can be coupled
to a first particle at a 5' end of the first strand and the second
strand can be coupled to a separate second particle at a 5' end of
the second strand.
[0080] In some embodiments, the first strand of the double-stranded
nucleic acid molecule may be coupled to the first particle and the
second strand of the double-stranded nucleic acid molecule may be
coupled to the second particle, including via direct attachment or
indirect attachment as described elsewhere herein for linking of
primers and particles. Direct and indirect attachments between the
first and second strands and respective particles may be via
covalent bonds, non-covalent interactions and combinations thereof
also as described elsewhere herein. For example, the first strand
may be covalently linked to the first particle at a 5' end of the
first strand and/or the second strand may be covalently linked to
the second particle at a 5' end of the second strand. Furthermore,
in some embodiments, the isolated nucleic acid complex may not be
immobilized to a support and instead, for example, suspended or
free-floating in a liquid medium.
[0081] In some embodiments, the double-stranded nucleic acid
molecule may include a first end sequence and a second end
sequence. Each of the first and second end sequences can be a
single-stranded nucleic acid sequence linked to the 5' or 3' ends
of the first and second strands, respectively, of the
double-stranded nucleic acid molecule. The first end sequence can
be complexed with the first particle via a first capture sequence
linked to the first particle and the second end sequence can be
complexed with the second particle via a second capture sequence
linked to the second particle. The first capture sequence can be an
oligonucleotide linked to the first particle and configured to
hybridize with the first end sequence of the double-stranded
nucleic acid molecule via sequence complementarity. The first
capture sequence may be fully or at least partially complementary
to the first end sequence. Additionally, the second capture
sequence can be an oligonucleotide linked to the second particle
and configured to hybridize with the second end sequence of the
double-stranded nucleic acid molecule via sequence complementarity.
The second capture sequence may be fully or at least partially
complementary to the second end sequence. In some embodiments, the
first and/or second capture sequences may comprise a locked nucleic
acid (LNA).
[0082] In some embodiments, the first capture sequence may be
linked to the first particle at a 3' end of the first capture
sequence and/or the second capture sequence may be linked to the
second particle at a 3' end of the second capture sequence.
Additionally, the first capture sequence may be linked to the first
particle and the second capture sequence may be linked to the
second particle, including via direct attachment or indirect
attachment as described elsewhere herein for linking of primers and
particles. Direct and indirect attachments between capture
sequences and particles may be via covalent bonds, non-covalent
interactions and combinations thereof also as described elsewhere
herein.
[0083] In some embodiments, the first particle may comprise at
least about 1, 2, 10, 50 100, 500 1000, 5000, 10000, 50000, 100000,
500000, 1000000, 5000000, 10000000 or more molecules of the first
capture sequence. Moreover, the second particle may comprise at
least about 1, 2, 10, 50 100, 500 1000, 5000, 10000, 50000, 100000,
500000, 1000000, 5000000, 10000000 or more molecules of the second
capture sequence. In some embodiments, the first particle and
second particle may also comprise the second capture sequence and
first capture sequence, respectively.
[0084] Moreover, the length of the first and second capture
sequences may vary depending upon the particular application. For
example, the length of the first or second capture sequence may be
about 1 nucleotide to about 100 nucleotides in length; about 5 to
about 50 nucleotides in length; about 5 to about 30 nucleotides in
length; or about 15 to about 30 nucleotides in length. In some
embodiments, the length of the first or second capture sequence may
be about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,
18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,
35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or
more nucleotides in length.
[0085] The length of the first and second end sequences may vary
depending upon the particular application. For example, the length
of the first or second end sequence may be about 1 nucleotide to
about 100 nucleotides in length; about 5 to about 50 nucleotides in
length; about 5 to about 30 nucleotides in length; or about 15 to
about 30 nucleotides in length. In some embodiments, the length of
the first or second end sequence may be about 2, 3, 4, 5, 6, 7, 8,
9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,
26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42,
43, 44, 45, 46, 47, 48, 49, 50 or more nucleotides in length.
[0086] In some embodiments, the isolated nucleic acid complex may
comprise at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,
15, 16, 17, 18, 19, 20, 21, 22, 22, 23, 24, 25, 26, 27, 28, 29, 30,
31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47,
48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 or more
particles associated with about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,
12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,
29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45,
46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 or more
double-stranded nucleic acid molecules. Each particle of the
isolated nucleic acid complex can be linked to another particle in
the isolated nucleic acid complex through an additional
double-stranded nucleic acid molecule that is substantially the
same as the double-stranded nucleic acid. The term "substantially
the same" as used herein refers to a degree of sequence homology
between the double-stranded nucleic acid molecule and the
additional double-stranded nucleic acid molecule of at least about
60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100%.
[0087] Any particle, set of particles, or nucleic acid complex
described herein may comprise any suitable type of particle.
Non-limiting examples of particle types include solid particles,
porous particles, nanoparticles (e.g., nanotubes, nanorods,
nanoshells, spherical nanoparticles), microparticles, beads, metal
particles, magnetic particles (e.g., magnetic nanoparticles),
semiconductor particles (e.g., quantum dots), polymeric particles,
nucleic acid particles (e.g., DNA-containing particles,
RNA-containing particles, etc), fluorescent particles, colorimetric
particles, composites thereof and combinations thereof. Moreover,
any particle, particle of a set of particles, or particle of an
isolated nucleic acid complex described herein may comprise any
suitable material or plurality of materials (e.g., particles of one
material coated with another material, particles made of composite
materials, etc.). Non-limiting examples of materials that a
particle can comprise include metals (e.g., silver, gold, platinum,
copper, palladium), metal oxides (e.g., Al.sub.2O.sub.3, NiO,
Fe.sub.2O.sub.3, ZrO.sub.2, MoO.sub.3, CeO.sub.2, Y.sub.2O.sub.3,
TiO.sub.2, ZnO, SnO, ITO, Co.sub.3O.sub.4), polymers (e.g.,
polyacrylamide, polystyrene, dextrose, polyaniline, polypyrrole,
polyacetylene, a fluorescent protein, carbon, composites thereof
and combinations thereof. In some embodiments, a first particle and
a second particle in a set of particles and/or a nucleic acid
complex may comprise the same materials. In some embodiments, a
first particle and a second particle in a set of particles and/or a
nucleic acid complex may comprise different materials. For example,
a set of particles or nucleic acid complex may comprise a first
particle that comprises gold and a second particle that comprises
silver.
[0088] Any particle, particle of a set of particles, or particle of
an isolated nucleic acid complex described herein may have varied
particle size depending upon, for example, the particular
application envisioned. In general, "particle size" as used herein
generally refers to the size of a dimension of a particle. Such a
dimension, for example, may be a diameter, a circumference, a
perimeter, a hydrodynamic diameter, a radius, a length, a width, or
a depth of a particle. In some embodiments, a particle may have a
dimension of about 0.1 nanometers (nm) to about 100 nm; about 0.5
nm to about 100 nm; about 1 nm to about 20 nm; about 1 nm to about
10 nm; or about 1 nm to 5 nm. In some embodiments a particle may
have a dimension of at most 100 nm, at most 50 nm, at most 20 nm,
at most 10 nm or at most 5 nm. In some embodiments, a particle may
have a dimension of about 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,
11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55,
60, 65, 70, 75, 80, 85, 90, 100 nm or the dimension may be larger
or smaller depending on the particular particle.
[0089] Any particle, particle of a set of particles, or particle of
an isolated nucleic acid complex described herein may have any
suitable shape/configuration that may be a regular
shape/configuration or may be an irregular shape/configuration.
Non-limiting examples of particle shapes include spheres, rods,
hollow shells, tubes, elliptical particles, core-shell particles
(e.g., coated particles), agglomerations of smaller particles and
combinations thereof. Moreover, any particle, particle of a set of
particles, or particle of an isolated nucleic acid complex
described herein may or may not be linked with a support. In
embodiments where a particle, particle of a set of particles or
particle of an isolated nucleic acid complex is not linked with a
support, the particle(s) may be, for example, free-floating or
suspended in a liquid medium (e.g., aqueous medium).
[0090] In some embodiments, a particle, particle of a set of
particles, or particle of an isolated nucleic acid complex
described herein may comprise an affinity capture agent that is
configured to bind to a binding partner. A suitable affinity of the
affinity capture agents for its binding partner generally can allow
the two species to bind. Affinity capture agents may be useful in
isolating a particle, particle of a set of particles or a nucleic
acid complex as described elsewhere herein. Non-limiting examples
of affinity capture agents include a non-nucleic acid member of a
binding pair (e.g., streptavidin or biotin from a
streptavidin-biotin binding pair), a capture sequence (e.g., a
capture sequence comprising a locked nucleic acid (LNA)),
antibody-antigen pairs (e.g., anti-fluorescein antibody and
fluorescein, anti digoxigenin antibody and digoxigenin),
leptin-sugar pairs, aptamers and binding pairs, polypeptides with
their binding pairs and or other types of polymers with their
binding pairs.
[0091] For example, a particle, a particle of a set of particles,
or a particle of an isolated nucleic acid complex described herein
may comprise a free primer or a capture sequence that has not
coupled with another nucleic acid molecule. In another example, a
particle, a particle of a set of particles, or a particle of an
isolated nucleic acid complex described herein may comprise a
member of a binding pair such as, for example, one or both of
streptavidin or biotin. Additionally, an affinity capture agent may
be linked to a particle, including via direct attachment or
indirect attachment as described elsewhere herein for linking of
primers and particles. Direct and indirect attachments between
affinity capture sequences and particles may be via covalent bonds,
non-covalent interactions and combinations thereof also as
described elsewhere herein.
[0092] Furthermore, any particle, set of particles, or isolated
nucleic acid complex described herein may be contained in a
partition. The partition may be any suitable type of partition with
non-limiting examples of partitions that include a droplet (e.g., a
droplet in an emulsion, such as, for example, a water-in-oil
emulsion or oil-in-water emulsion), a vessel (e.g., any suitable
type of tube, a capillary tube, a centrifuge tube, a cuvette, a
pipette tip, a bag, a box, a container) and combinations
thereof.
[0093] In some embodiments, a partition may be a well (e.g., a well
among an array of wells such as a well in a microwell plate, a
microwell, a nanowell). A well can be microfabricated in (e.g.,
etched into a silicon substrate) or on (e.g., depositing well
materials onto a substrate) a substrate such as, for example a
silicon substrate. Moreover, the surface(s) within a well can have
different properties than a bulk surface substrate in or on which
the well is included. For example, the well surface may be
hydrophilic and the bulk substrate surface may be hydrophobic or
vice versa. Additionally, in some embodiments, the surface of a
well and a bulk substrate can be differentially modified with
various functional groups such that they each selectively capture
particular chemical agents (e.g., chemical agents having affinity
for particles, a set of particles, nucleic acid complexes,
etc.).
[0094] A substrate comprising wells can be placed in or be included
as part of a flow-cell chamber that has an inlet and an outlet for
liquid transferred. In some embodiments, such a flow-cell may also
include a flat or curved inner surface where wells are patterned. A
liquid phase solution can be passed through the flow-cell such that
the wells are exposed to a solution. Depending on the physical
and/or chemical properties of the solution and surface properties
of the wells and/or other flow-cell surfaces, the solution can be
partitioned into the wells or selectively prohibited from entering
the wells. For example, an oil solution flowed over wells can avoid
partitioning when well surfaces contain a hydrophilic surface,
whereas an aqueous solution flowed over the wells can be
partitioned into the wells. A dimension of a well can be any
suitable dimension. For example, a dimension of a well can be about
1 nanometer ("nm") to about 1 millimeter ("mm"), 10 nm to 1 mm, 100
nm to 1 mm, 1 micrometer (".mu.m") to 1 mm, 10 .mu.m to 1 mm or 100
.mu.m to 1 mm.
[0095] In cases where a partition is a droplet in an emulsion, the
droplet may be generated in any suitable way including bulk
emulsification methods and/or with the aid of a microfluidic
device. Bulk emulsification and/or microfluidic devices may also be
useful in partitioning particles into droplets. Moreover, the
volume of a droplet can be any suitable volume. For example, the
volume of a droplet can be about 1 femtoliter ("fL") to 100 .mu.L
microliter (".mu.L"), 10 fL to 10 .mu.L, 100 fL to 10 .mu.L, 1
picoliter ("pL") to 1 .mu.L, 1 nanoliter ("nL") to 10 .mu.L, 10 nL
to 10 .mu.L, 100 nL to 10 .mu.L or 1 .mu.L to 10 .mu.L.
[0096] Additional aspects of the disclosure provide methods for
assaying or identifying the presence of a target nucleic acid
molecule/target nucleic acid sequence in a sample, methods for
generating a nucleic acid complex comprising a target nucleic acid
molecule and methods for nucleic acid amplification.
[0097] In another aspect, the disclosure provides a method for
assaying the presence of a target nucleic acid molecule in a sample
having or suspected of having the target nucleic acid molecule. The
method comprises subjecting the sample to a nucleic acid
amplification reaction in a reaction mixture under conditions to
yield an amplified target nucleic acid molecule linked to at least
a first particle and a second particle, where the amplified target
nucleic acid molecule comprises at least a portion of a sequence of
the target nucleic acid molecule. The reaction mixture may comprise
the first particle and the second particle. The first particle can
have a first primer, with a 5' end and a 3' end, linked to the
first particle via the 5' end of the first primer. The first primer
can exhibit sequence homology to a strand of the target nucleic
acid molecule at a 5' end of the strand of the target nucleic acid
molecule. The second particle can have a second primer, with a 5'
end and a 3' end, linked to the second particle via the 5' end of
the second primer. The second primer can exhibit sequence homology
to a complement strand of the strand of the target nucleic acid
molecule at a 5' end of the complement strand. In addition, a
sequence on the strand of the target nucleic acid molecule to which
the first primer exhibits homology may be different than a sequence
of the complement strand to which the second primer exhibits
homology. Moreover, the first primer and the second primer can be
adapted to be extended in a primer extension reaction to form a
copy of the target nucleic acid molecule or a portion thereof.
[0098] In some embodiments, the first primer can exhibit at least
about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or
100% sequence homology to the 5' end of the strand of the target
nucleic acid molecule when optimally aligned with the strand.
Similarly, the second primer can exhibit at least about 60%, 65%,
70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence
homology to the 5' end of the complement strand when optimally
aligned with the complement strand. Furthermore, in some
embodiments, the target nucleic acid molecule may be
single-stranded or, in some embodiments, the target nucleic acid
molecule may be double-stranded. Additionally, the first primer may
be linked to the first particle and the second primer may be linked
to the second particle, including via direct attachment or indirect
attachment as described elsewhere herein. Direct and indirect
attachments between primers and particles may be via covalent
bonds, non-covalent interactions and combinations thereof also as
described elsewhere herein.
[0099] The reaction mixture may also comprise reagents necessary
for the amplification of the target nucleic acid molecule,
including a polymerase and other such reagents necessary for
amplification of a nucleic acid molecule described elsewhere
herein. The polymerase may extend the 3' end of the first primer in
a primer extension reaction to form a copy of the strand of the
target nucleic acid or a portion thereof and/or may extend the 3'
end of the second primer in a second primer extension reaction to
form the complement strand or a portion thereof. Moreover, in some
embodiments, the reaction mixture may be contained in a partition.
Any suitable partition may be used to contain the reaction mixture
including example types of partitions described elsewhere
herein.
[0100] In some embodiments, the method may further comprise
detecting the amplified target nucleic acid molecule linked to the
first particle and the second particle. The amplified target
nucleic acid molecule may be detected, including via modes of
detection described elsewhere herein, such as, for example, optical
detection. For example, the reaction mixture may further comprise
an optically-responsive species and detection may be achieved via
the optically-responsive species (e.g., a dye or other
optically-responsive species described elsewhere herein). The
optically-responsive species may interact with the amplified target
nucleic acid molecule, such that upon successful amplification of
the target nucleic acid, a detectable signal (or lack thereof) can
be detected from the optically-responsive species. In some
embodiments, the amplified target nucleic acid molecule may be
detected by detecting either or both of the first or second
particles or the assembly of the amplified target nucleic acid
molecule and first and second particles (e.g., detection of a
nucleic acid complex as described elsewhere herein). In some
embodiments, the assembly of the amplified target nucleic acid
molecule and first and second particles may be isolated prior to
detection, using, for example, a mode of isolation described
elsewhere herein.
[0101] In some embodiments, the method may comprise producing a
plurality of amplified target nucleic acid molecules and, in some
embodiments, detecting the plurality of amplified target nucleic
acid molecules. In such cases, the reaction mixture may comprise
more than two particles to which the plurality of amplified nucleic
acid molecules can be linked. Each additional particle may comprise
either or both of the first primer and the second primer.
[0102] In another aspect, the disclosure provides a method for
identifying the presence or absence of a target nucleic acid
molecule in a sample. The method comprises providing a solution
that is suspected of containing the target nucleic acid molecule,
where the target nucleic acid molecule comprises a first nucleic
acid strand linked to a first particle and a second nucleic acid
strand linked to a second particle. The second nucleic acid strand
can be hybridized to the first nucleic acid strand via sequence
complementarity to yield a nucleic acid complex. In addition, the
method also comprises detecting a signal indicative of the presence
or absence of the nucleic acid complex in the solution, thereby
identifying the presence of the target nucleic acid molecule.
[0103] In some embodiments, the solution may be provided to a
detector and the detector may detect the signal indicative of the
presence or absence of the nucleic acid complex in the solution.
Detection of the signal indicative of the presence or absence of
the nucleic acid complex in the solution may be completed via any
suitable detection method and, in some embodiments, detector.
Example modes of detection and detection are described elsewhere
herein. In some embodiments, the signal indicative of the presence
or absence of the nucleic acid complex can be indicative of an
optical property (e.g., an optically-responsive species associated
with the nucleic acid complex, such as, for example, an example dye
described elsewhere herein), physical property (e.g., density, size
of the nucleic acid complex), an electrical property (e.g.,
conductance, impedance), an electrostatic property, and/or an
electrochemical property of the nucleic acid complex. In some
embodiments, the first and second particles may be metallic
particles and detection of the signals may be detected by visual
examination as described elsewhere herein.
[0104] The solution comprising the nucleic acid complex may be any
suitable solution, such as, for example, a nucleic acid
amplification reaction mixture as described elsewhere herein or a
reaction mixture that has been processed (e.g., a reaction mixture
diluted with a diluent such as water, buffer other liquid medium,
or combination thereof). In some embodiments, the solution may be
an aqueous solution comprising the nucleic acid complex. In some
embodiments, the solution may comprise a nucleic acid complex that
has been isolated from a reaction mixture, using any suitable mode
of isolation including examples of isolating a nucleic acid complex
described elsewhere herein. In some embodiments, the solution may
be contained within a partition. Any suitable type of partition may
be used to contain the solution including example types of
partitions described elsewhere herein. In some embodiments, the
solution may comprise nucleic acid complexes that comprise two or
more particles (e.g., nucleic acid complexes that comprise 2, 3, 4,
5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more
particles). In some embodiments, the solution may comprise a
plurality of nucleic acid complexes that comprise two or more
particles, where, in some embodiments, the detected signal is
indicative of the presence or absence of a plurality of nucleic
acid complexes. In some embodiments, the nucleic acid complex may
or may not be immobilized or affixed (e.g., linked) to a support in
the solution. In embodiments where the nucleic acid complex is not
immobilized or affixed to a support in the solution, the nucleic
acid complex may be suspended and/or free-floating within the
solution.
[0105] Additionally, the first nucleic acid strand may be linked to
the first particle and the second nucleic acid strand may be linked
to the second particle, including via direct attachment or indirect
attachment as described elsewhere herein. Direct and indirect
attachments between target nucleic acid strands and particles may
be via covalent bonds, non-covalent interactions and combinations
thereof also as described elsewhere herein.
[0106] In another aspect, the disclosure provides a method for
assaying a sample for the presence or absence of a target nucleic
acid sequence. The method comprises receiving a request to assay
the sample for the presence or absence of the target nucleic acid
sequence and assaying the sample for the presence or absence of the
target nucleic acid sequence. Assaying can be completed by
detecting at least one nucleic acid complex that comprises a
double-stranded nucleic acid molecule linked to at least a first
particle and a second particle. The double-stranded nucleic
molecule can comprise a first single-stranded nucleic acid molecule
and a second single-stranded nucleic acid molecule that is
complementary to the first single-stranded nucleic acid molecule.
Moreover, at least one of the first single-stranded nucleic acid
molecule and the second single-stranded nucleic acid molecule may
comprise the target nucleic acid sequence. In addition, the method
can further comprise generating a report that is indicative of the
presence or absence of the target nucleic acid sequence in the
sample.
[0107] In some embodiments, assaying may further comprise detecting
a signal that is indicative of the presence or absence of the
nucleic acid complex. Detection of the signal indicative of the
presence or absence of the nucleic acid complex in the solution may
be completed via any suitable detection method such as, for
example, the example modes of detection described elsewhere herein.
The detected signal may be indicative of an optical property (e.g.,
a signal generated from the activity of an optically-responsive
species (such as, for example, a dye or other optically-responsive
species described elsewhere herein) associated with the nucleic
acid complex), a physical property (e.g., density or size), an
electrical property (e.g., conductance, impedance), an
electrostatic property, and/or an electrochemical property of the
nucleic acid complex. In some embodiments, the nucleic acid complex
may be detected in a partition. The nucleic acid complex may be
detected in any suitable type of partition including example types
of partitions described elsewhere herein. In some embodiments, the
nucleic acid may be isolated prior to detection (e.g., isolated
from a reaction mixture), as described elsewhere herein. In some
embodiments, the nucleic acid complex may comprise a plurality of
double-stranded nucleic acid molecules linked to greater than two
particles as described elsewhere herein.
[0108] Moreover, the report that is generated may be any suitable
type of report. A report may include any number of desired
elements, with non-limiting examples that include information
regarding the presence or absence of the target nucleic acid
sequence in the sample, the presence or absence of the nucleic acid
complex in the sample, the target nucleic acid sequence, the number
of nucleic acid molecules detected in the sample, the number of
copies of the target nucleic acid sequence detected in the sample,
etc. The report may be provided as a printed report (e.g., a hard
copy) and/or may be provided as an electronic report. An electronic
report may be presented on a user interface (UI) such as a UI on an
electronic display of an electronic device. In some examples, an
electronic display may include a resistive or capacitive touch
screen. Non-limiting examples of electronic displays include a
monitor or television, a screen operatively linked with a detector,
a tablet computer screen, a mobile device screen, a portable
computer screen, and the like. In some embodiments a UI may be a
graphical user interface (GUI) that is configured to provide a
report to a user. A GUI can include textual, graphical and/or audio
components. Both a printed and an electronic report may be stored
in files or in databases, respectively, such that they are
accessible for future use. Moreover, a report may be transmitted to
a local or remote location using any suitable communication medium
including, for example, a network connection, a wireless
connection, and/or an internet connection.
[0109] In some embodiments, the request to assay the sample may be
received from any type of requestor, with non-limiting types of
requestors that include a research professional (e.g., scientist,
lab technician, professor, etc.), a health-care professional (e.g.,
a nurse, a doctor, a physician's assistant, a medical technician,
etc.), a research organization (e.g., a laboratory, a research
hospital, a research center, a research institute, and academic
institution, a university), a health-care organization (e.g., a
hospital, a nursing home, a hospice, a medical center, a clinic), a
public health organization (e.g., government agencies such as, for
example, the U.S. Centers for Disease Control (CDC)) and
combinations thereof. In some embodiments, the generated report may
be provided to the requestor and/or any other desired recipient.
Example types of requestors described above may also be recipients
of a report.
[0110] In another aspect, the disclosure provides a method for
generating a nucleic acid complex comprising a target nucleic acid
molecule. The method comprises, in a reaction mixture, amplifying
the target nucleic acid molecule with a forward primer and a
reverse primer, under conditions that yield the nucleic acid
complex. The nucleic acid complex can comprise an amplified target
nucleic acid molecule, where the amplified target nucleic acid
molecule comprises a first strand and a second strand that is at
least partially complementary to the first strand. The first strand
can be coupled to a first particle at a 5' end of the first strand
and the second strand can be coupled to the second particle at a 5'
end of the second strand.
[0111] In some embodiments, the forward primer may be linked to the
first particle and the reverse primer may be linked to the second
particle. The forward and reverse primers may be linked to the
first particle and second particle, respectively, including via
direct attachment or indirect attachment as described elsewhere
herein. Direct and indirect attachments between primers and
particles may be via covalent bonds, non-covalent interactions and
combinations thereof also as described elsewhere herein.
[0112] In some embodiments, the reaction mixture may also comprise
the first particle and the second particle and any other reagents
necessary for amplification of the target nucleic acid molecule as
described elsewhere herein. Moreover, the reaction mixture can
comprise additional particles coupled to additional forward and/or
reverse primers to yield nucleic acid complexes comprising more
than two particles coupled to additional amplified target nucleic
acid molecules as described elsewhere herein. For example, in some
embodiments, the method can further comprise amplifying the target
nucleic acid molecule with a third particle linked thereto the
forward primer or the reverse primer to yield the nucleic acid
complex. In such embodiments, the nucleic acid complex can further
comprise the third particle coupled to the first particle and/or
the second particle via an additional amplified target nucleic acid
molecule.
[0113] Additionally, in some embodiments, the method further
comprises detecting the nucleic acid complex. Detection of the
nucleic acid complex may be achieved via any suitable mode,
including example modes of detection (e.g., optical (e.g., with the
aid of an optically-responsive species), spectroscopic,
electrochemical, electrostatic, physical, electrical, etc.)
described elsewhere herein. In some embodiments, the first and
second particle may be metallic particles and detection of the
nucleic acid complex may be effected by visual examination.
[0114] In some embodiments, the reaction mixture may be contained
in a partition. Any suitable type of partition may be used to
contain the reaction mixture including example type of partitions
described elsewhere herein. In embodiments where the reaction
mixture is contained in a partition, the method may further
comprise releasing the nucleic acid complex from the partition and
detecting the released nucleic acid complex. In some embodiments,
the method further comprises isolating the nucleic acid complex,
which isolation may occur before, during or following any detection
of the nucleic acid complex. Any suitable mode of isolation of the
nucleic acid complex may be used including example modes of
isolation (e.g., centrifugation, magnetic separation,
chromatography, capillary action, chromatography, filtration,
sedimentation, affinity capture (e.g., affinity capture on a
support), etc.) described elsewhere herein.
[0115] In another aspect, the disclosure provides a method for
generating a nucleic acid complex comprising a target nucleic acid
molecule. The method comprises, in a reaction mixture, amplifying
the target nucleic acid molecule with a forward primer and a
reverse primer to yield an amplified target nucleic acid molecule.
The forward primer and the reverse primer can each comprise a 3'
end capable of being extended in a primer extension reaction and a
hairpin structure. Additionally, the amplified target nucleic acid
molecule can comprise a first strand comprising a first overhang
sequence at one end of the amplified target nucleic acid molecule
and a second strand comprising a second overhang sequence at the
other end of the amplified target nucleic acid molecule. Moreover,
the method further comprises contacting the amplified target
nucleic acid molecule with a first particle and a second particle
to yield a nucleic acid complex. The nucleic acid complex can
comprise the amplified target nucleic acid molecule complexed with
the first particle and the second particle. The amplified target
nucleic acid molecule may be complexed with the first particle via
sequence complementarity between the first overhang sequence and a
first capture sequence linked to the first particle and complexed
with the second particle via sequence complementarity between the
second overhang sequence and a second capture sequence linked to
the second particle.
[0116] In some embodiments, the method further comprises ligating
the first strand to the second capture sequence and/or ligating the
second strand to the first capture sequence. Ligation may be
achieved via any suitable method including via the action of a
ligase enzyme present in the reaction mixture such as, for example,
a DNA ligase. In addition, the reaction mixture may comprise one or
more reagents necessary for amplification of the target nucleic
acid molecule, such reagents necessary for nucleic acid
amplification described elsewhere herein.
[0117] In some embodiments, the method further comprises isolating
and/or detecting the nucleic acid complex. Isolation and/or
detection of the nucleic acid complex may be completed using any
suitable modes of isolation and detection, including example modes
of isolation and detection described elsewhere herein.
[0118] As used herein, an "overhang sequence" generally refers to a
single-stranded sequence associated with the 5' or 3' end of a
strand of a double-stranded nucleic acid molecule. An overhang
sequence may be useful for linking (e.g., via hybridization via
sequence complementarity) a double-stranded nucleic acid molecule
with another nucleic acid molecule.
[0119] Moreover, in some embodiments, the forward primer and/or the
reverse primer may comprise a spacer region which cannot be copied
via a primer extension reaction. The spacer region of a primer may
be useful in generating an overhang sequence on an amplified target
nucleic molecule that is derived from the primer. The overhang
sequence can be generated because any primer sequence 5' of a
spacer region may also not be copied due to the presence of the
spacer region preventing further action of a polymerase during a
primer extension reaction. A spacer region may comprise a single
nucleotide, a series of nucleotides, and/or a non-nucleic acid
species. Non-limiting examples of species that can be included in a
spacer region include a polyethylene glycol (PEG),
1',2'-Dideoxyribose (an abasic site), a carbonaceous linker (e.g.,
a multi-carbon linker, a C3 spacer, Spacer, Spacer 9, Spacer 18
available from Integrated DNA Technologies) and combinations
thereof.
[0120] An example of a forward or reverse primer having a 3' end
capable of being extended in a primer extension reaction, having a
hairpin structure and a spacer region is schematically depicted in
FIG. 3A. As shown in FIG. 3A, a primer 300 is configured to
comprise a hairpin structure and comprises a 3' end that is capable
of being extended in a primer extension reaction. In the loop
region 301 of the hairpin structure, the sequence of the primer 300
comprises a spacer region 302 that cannot be copied via a primer
extension reaction. The primer also comprises a stem region 303.
Moreover, a linear configuration of the primer shown in FIG. 3A is
schematically depicted in FIG. 3B. As shown in FIG. 3B, the primer
comprise an overhang sequence region 304 that is 5' of the spacer
region and a priming sequence region 305 that is 3' of the spacer
region 302. In the hairpin configuration shown in FIG. 3A, a
portion of the overhang sequence region 304 is hybridized with a
portion of the priming sequence region 305, which forms the stem
region 303.
[0121] The melting temperature (T.sub.m,1) of the overhang sequence
304 when hybridized with a target nucleic acid sequence on a
nucleic acid molecule may be less than the melting temperature
(T.sub.m,2) of the portion of the overhang sequence region 304
hybridized with the portion of the priming sequence region 305 in
the stem region 303, which melting temperatures may both be less
than the melting temperature (T.sub.m,3) of the priming sequence
305 when hybridized with its target nucleic acid sequence on a
template nucleic acid molecule. When exposed to a temperature that
is greater than T.sub.m,2, the primer 300 shown in FIG. 3A can
adopt the linear configuration shown in FIG. 3B. Moreover, where
such a temperature is also less than T.sub.m,3, the priming
sequence 305 can prime a target nucleic acid sequence because the
priming sequence 305 does not melt from the target nucleic acid
sequence at temperatures less than T.sub.m,3. For the example
primer 300 shown in FIG. 3A and FIG. 3B, T.sub.m,1 is 28.degree.
C., T.sub.m,2 is 55.degree. C. and T.sub.m,3 is 59.degree. C.
[0122] An example of the functionality of primer 300 shown in FIG.
3A and FIG. 3B is schematically depicted in FIG. 3C. As shown in
FIG. 3C, primer 300 is provided in a reaction mixture and upon
exposure 310 to the appropriate temperature (T) (e.g., T.sub.m,1
and T.sub.m,2<T<T.sub.m,3), primer 300 linearizes. The
linearized primer 300 can then prime 320 a nucleic acid molecule
306 via priming sequence region 305 and the nucleic acid sequence
for the priming region 305 on nucleic acid molecule 306 that is
also included in the reaction mixture. The priming sequence 305 can
be extended 307 at its 3' end in a primer extension reaction (e.g.,
via the action of a polymerase). Moreover, nucleic acid molecule
306 is not extended to include a sequence complementary to overhang
sequence 304 in the primer extension reaction (or a subsequent
primer extension reaction) due to the presence of the spacer region
302. The primer extension reaction yields a double-stranded nucleic
acid molecule comprising primer 300, nucleic acid molecule 306
linked to single-stranded overhang sequence 304.
[0123] The reaction mixture may also include a particle 308 that is
coupled to a capture sequence 309 at the 3' end of the capture
sequence. The capture sequence 309 may comprise a sequence that is
at least partially complementary to the overhang sequence 304. In
such cases, overhang sequence 304 can hybridize 330 with capture
sequence 309 such that the double-stranded molecule linked to
overhang sequence 304 is coupled to particle 308. Following
hybridization of overhang sequence 304 and capture sequence 309,
the 3' end of nucleic acid molecule 306 of the double-stranded
nucleic acid molecule can be ligated to the capture sequence 309 at
its 5' end such that the double-stranded molecule is covalently
immobilized to the particle 308.
[0124] An example method of amplifying a double-stranded nucleic
acid molecule (e.g., a target nucleic acid molecule) with a primer
similar to primer 300 shown in FIG. 3A/FIG. 3B and generating a
nucleic acid complex is schematically depicted in FIG. 4. As shown
in FIG. 4, a double stranded nucleic-acid molecule 401, a forward
primer 402, a reverse primer 403 and other reagents (e.g., a
polymerase, dNTPs, co-factors, etc.) necessary for amplification of
double-stranded nucleic acid molecule 401 may be provided in a
reaction mixture. The forward primer 402 and the reverse primer 403
are configured to comprise a hairpin structure and a 3' end that
can be extended in a primer extension reaction, similar to the
example primer 300 depicted in FIG. 3A and FIG. 3B.
[0125] The reaction mixture may then be subject to conditions 410
suitable for amplification of the double-stranded nucleic acid
molecule 401 via forward primer 402 and reverse primer 403 to
generate a plurality of amplified double-stranded nucleic acid
molecules 404. For example, the temperature of the reaction mixture
may be cycled such that the double-stranded nucleic acid molecule
401 is amplified via forward primer 402 and reverse primer 403. One
strand of an individual amplified double-stranded nucleic acid
molecule may comprise a first overhang sequence 405 at its 5' end
and the other strand of the individual amplified double-stranded
nucleic acid molecule may comprise a second overhang sequence 406
at its 5' end.
[0126] The amplified double-stranded nucleic acid molecules 404 may
then be contacted with one or more first particles 406 and one or
more second particles 407. In some cases, the reaction mixture may
initially comprise the first particles 406 and the second particles
407 prior to amplification of the double-stranded nucleic acid
molecule 401. In other cases, the first particles 406 and the
second particles 407 may be added to the reaction during or after
amplification of the double-stranded nucleic acid molecule 401.
Additionally, an individual first particle of first particles 406
comprises one or more copies of a first capture sequence 408 that
is coupled to the individual first particle at its 3' end. The
first capture sequence 408 is at least partially complementary to
the overhang sequences 405 of the amplified double-stranded nucleic
acid molecules 404. Moreover, an individual second particle of
second particles 407 comprises one or more copies of a second
capture sequence 409 that is coupled to the individual second
particle at its 3' end. The second capture sequence 409 is at least
partially complementary to the overhang sequences 406 of the
amplified double-stranded nucleic acid molecules 404.
[0127] Upon contact with the first particles 406 and the second
particles 407, overhang sequences 405 and 406 can hybridize with
capture sequences 408 and 409, respectively via sequence
complementarity such that an individual amplified double-stranded
nucleic acid molecule 404 is coupled to an individual first
particle 406 and/or an individual second particle 407. As the
overhang sequences are positioned at the 5' ends of each strand of
an individual amplified double-stranded nucleic acid molecule 404,
each amplified double-stranded nucleic acid molecule 404 is coupled
to an individual first particle 406 and individual second particle
407 at the 5' end of one of its component strands. In cases where
one or both of the first particles 406 and second particles 407
comprise a plurality of capture sequences 408 and 409,
respectively, multiple amplified double-stranded nucleic acid
molecules 404 can couple to each particle such that a nucleic acid
complex 411 can be generated. Following hybridization of capture
sequences and overhang sequences, the 3' ends of each strand of
each amplified double-stranded nucleic acid molecule can be ligated
(e.g., via the action of a ligase) to its adjacent capture sequence
408 or 409 such that the double-stranded molecule is covalently
attached to the respective capture sequence. Where appropriate, the
nucleic acid complex 411 and/or the amplified double-stranded
nucleic acid molecules of the nucleic acid complex 411 can then be
isolated and/or detected as described elsewhere herein.
[0128] In another aspect, the disclosure provides a method for
nucleic acid amplification. The method comprises annealing a
forward primer linked to a first particle to a nucleic acid strand
and annealing a reverse primer linked to a second particle to a
complement strand of the nucleic acid strand. Next, the method
further comprises extending the forward primer and the reverse
primer in a template-directed manner to yield a first
double-stranded nucleic acid molecule linked to the first particle
and a second double-stranded nucleic acid molecule linked to the
second particle. Additionally, the method further comprises
denaturing the first double-stranded nucleic acid molecule and the
second double-stranded nucleic acid molecule to generate a first
single-stranded molecule linked to the first particle and a second
single-stranded molecule linked to the second particle.
Furthermore, the method further comprises annealing the forward
primer to the second single-stranded molecule and annealing the
reverse primer to the first single-stranded molecule to yield a
nucleic acid complex comprising an amplified double-stranded
nucleic acid molecule. The amplified double-stranded nucleic acid
molecule can be linked at one end to the first particle and linked
at its other end to the second particle.
[0129] The steps of the method of may be performed in a reaction
mixture that comprises the first particle linked to the forward
primer, the second particle linked to the second primer, the
nucleic acid strand and one or more reagents necessary for nucleic
acid amplification. Moreover, such a reaction mixture may also
comprise additional particles (e.g., a third, fourth, fifth, sixth,
seventh, eighth, ninth, tenth, eleventh, twelfth, thirteenth,
fourteenth, fifteenth, sixteenth, seventeenth, eighteenth,
nineteenth, twentieth or greater particle) linked to the forward
and/or reverse primers such that nucleic acid complexes comprising
additional particles linked to additional amplified double-stranded
nucleic acid molecules are generated. For example, in some
embodiments, the method can further comprise amplifying the nucleic
acid strand with a third particle linked thereto the forward primer
or the reverse primer to yield the nucleic acid complex, where the
nucleic acid further comprises the third particle coupled to the
first particle and/or the second particle via an additional
amplified double-stranded nucleic acid molecule.
[0130] In some embodiments, one or more steps of the method may be
performed in a partition. In some embodiments, the entire method
can be performed in a partition. One or more steps of the method or
the entire method may be performed in any suitable type of
partition including example types of partitions described elsewhere
herein. Additionally, one or more steps of the method (up to all
steps of the method) may be repeated for one or more cycles,
whereby nucleic acid complexes comprising greater than two
particles and/or a plurality of nucleic acid complexes can be
generated.
[0131] In some embodiments, the method further comprises isolating
and/or detecting the nucleic acid complex. Isolation and/or
detection of the nucleic acid complex may be completed using any
suitable modes of isolation and/or detection including example
modes of isolation and detection (e.g., optical detection,
spectroscopic detection, physical detection, electrical detection,
electrochemical detection, electrostatic detection) described
elsewhere herein.
[0132] An example method of amplifying a double-stranded nucleic
acid molecule with forward and reverse primers coupled to particles
to yield a nucleic acid complex is schematically depicted in FIG.
5. As shown in FIG. 5, a double stranded nucleic-acid molecule 501;
a plurality of first particles 502 that are each coupled (e.g.,
covalently coupled) to one or more copies of a forward primer 503
at its 5' end; a plurality of second particles 504 that are each
coupled (e.g., covalently coupled) to one or more copies of a
reverse primer 505 at its 5' end; and other reagents (e.g., a
polymerase, dNTPs, co-factors, etc.) necessary for amplification of
double-stranded nucleic acid molecule 501 may be provided in a
reaction mixture. Forward primer 503 exhibits sequence homology
with the 5' end of strand 501a of double-stranded nucleic acid
molecule 501 and is also at least partially complementary to at
least a portion of the 3' end of strand 501b of double-stranded
nucleic acid molecule 501. Reverse primer 504 exhibits sequence
homology with the 5' end of strand 501b of double-stranded nucleic
acid molecule 501 and is also at least partially complementary to
at least a portion of the 3' end of strand 501a of double-stranded
nucleic acid molecule 501. The reaction mixture can be subjected to
conditions 510 suitable to denature the double-stranded nucleic
acid molecule 501 into its component strands 501a and 501b. For
example, the reaction mixture may be brought up to a denaturing
temperature suitable to denature double-stranded nucleic acid
molecule 501.
[0133] Following denaturation of double-stranded nucleic acid
molecule 501, strands 501a and 501b can be contacted with an
individual second particle 504 and an individual first particle
502, respectively. The reaction mixture can then be subject to
conditions suitable such that an individual copy of forward primer
503 can anneal to its complementary sequence at the 3' end of
strand 501b and such that an individual copy of reverse primer 505
can anneal with its complementary sequence at the 3' end of strand
501a. For example, the temperature of the reaction mixture may be
brought to an annealing temperature such that forward primer 503
and reverse primer 504 anneal to their respective sequences on
strands 501b and 501a.
[0134] Following annealing of forward primer 503 and reverse primer
505, the reaction mixture may be brought to conditions 520 such
that the 3' ends of forward primer 503 and reverse primer 505 are
extended in a template-directed manner in a primer extension
reaction (e.g., via the action of a polymerase). For example, the
temperature of the reaction mixture may be brought to an extension
temperature at which the primer extension reaction can take place.
After extension of forward primer 503 and extension of reverse
primer 505, the individual first particle 502 and individual second
particle 504 are coupled to amplified double-stranded nucleic acid
molecules that comprise component strands that are substantially
the same or are the same as strands 501a and 501b of
double-stranded nucleic acid molecule 501. Individual first
particle 502 is coupled to its amplified double-stranded nucleic
acid molecule via the 5' end of a nucleic acid strand that is
substantially the same or the same as nucleic acid strand 501a of
double-stranded nucleic acid molecule 501. Individual second
particle 504 is coupled to its amplified double-stranded nucleic
acid molecule via the 5' end of a nucleic acid strand that is
substantially the same or the same as nucleic acid strand 501b of
double-stranded nucleic acid molecule 501.
[0135] The cycle of denaturing, annealing of additional copies of
forward primer 503 and reverse primer 505 can be repeated for each
amplified double-stranded nucleic acid molecules to generate
additional amplified double-stranded nucleic acid molecules by
subjecting the reaction mixture to appropriate conditions 530. For
example, the temperature of the reaction mixture may be cycled
through suitable denaturation, annealing and extension
temperatures, as described above. The additional copies of forward
primer 503 and reverse primer 505 can be provided by additional
individual first particles 502 and individual second particles 504
in the reaction mixture. Copies of forward primer 503 coupled to
additional individual first particles 502 and copies of reverse
primer 505 coupled to additional individual second particles 504
can anneal to complementary sequences on nucleic acid strands
coupled to individual second particles 504 and individual first
particles 502, respectively. The primers can then be extended in a
subsequent primer extension reaction such that an additional
amplified double-stranded nucleic acid molecule is generated that
is coupled to both an individual first particle 502 and an
individual second particle 504. The process can repeat over a
desired number of cycles such that a nucleic acid complex 506 is
generated via additional copies of forward primer 503 and reverse
primer 504 coupled to first particles 502 and second particles 504,
respectively, in the reaction mixture. Where appropriate, the
nucleic acid complex 506 and/or the double-stranded nucleic acid
molecules of the nucleic acid complex 506 can then be detected as
described elsewhere herein.
[0136] In various aspects described herein, a method may further
comprise isolating a nucleic acid complex. For example, a nucleic
acid complex may be isolated from a reaction mixture after an
amplification reaction. In some embodiments, a nucleic acid complex
may be isolated from a reaction mixture prior to, during, or after
detecting the nucleic acid complex. Any suitable mode of isolating
a nucleic acid complex may be used. Non-limiting examples of modes
of isolating a nucleic acid complex include centrifugation,
magnetic separation (e.g., via magnetic properties of a particle or
a particle of a nucleic acid complex), chromatography (e.g., size
chromatography, affinity chromatography), electrophoresis,
capillary action (e.g., capillary action through a solid matrix
such as filter paper or a pregnancy test strip), filtration,
sedimentation, affinity capture and combinations thereof.
[0137] Where affinity capture is used to isolate a nucleic acid
complex, one or more particles of a nucleic acid complex may
comprise an affinity capture agent that binds to its binding
partner, which may, for example, be immobilized on a support.
Binding of the affinity capture agent of the nucleic acid complex
with its binding pair immobilized to the support can immobilize the
nucleic acid complex to the support. For example, one or more
particles of a nucleic acid complex may comprise a primer that has
not been extended or a capture sequence that has not coupled with
another nucleic acid molecule. Such a primer or capture sequence
may hybridize with a complementary sequence that may be immobilized
to a support, such that hybridization of the primer or capture
sequence with the complementary sequence isolates the associated
nucleic acid complex by immobilizing the nucleic acid complex to
the support. In another example, one or more particles of a nucleic
acid complex may comprise one or more streptavidin moieties that
bind with respective biotin moieties immobilized to a support.
Alternatively, for example, the one or more particles may comprise
the biotin and the support may comprise the streptavidin. In either
case, binding of the streptavidin and biotin can immobilize the
nucleic acid complex to the support.
[0138] An example of generating a nucleic acid complex and
isolating a nucleic acid complex is schematically depicted in FIG.
6. As shown in FIG. 6, a water-in-oil emulsion 601 is provided in a
vessel 602. The water-in-oil emulsion comprises a plurality of
droplets in a continuous oil phase 603, where each droplet
comprises an aqueous reaction mixture 604 that comprises a
plurality of two types of particles 605 and 606. The first type of
particle 605 can comprise a forward primer the second type of
particle 606 may comprise a reverse primer. Alternatively, the
first type of particle 605 and second type of particle 606 may
comprise a first and second capture sequence, as described
elsewhere herein, capable of hybridizing with an overhang sequence
of an amplified nucleic acid molecule. The particles in each
droplet are free in the droplet reaction mixtures and not initially
associated with a nucleic acid complex. Moreover, positive droplets
607 comprise a template nucleic acid molecule 608, whereas negative
droplets 609 do not comprise the template nucleic acid molecule
608. The template nucleic acid molecule may be single-stranded or
may be double-stranded.
[0139] Positive droplets 607 and negative droplets 609 are
subjected to conditions 610 suitable to amplify the template
nucleic acid molecule 608 to generate amplified nucleic acid
molecules associated with a nucleic acid complex 611. As positive
droplets 607 comprise the template nucleic acid molecule, the
nucleic acid complex 611 is generated positive droplets 607
following amplification of the template nucleic acid molecule 608.
Moreover, no nucleic acid complex 611 is generated in negative
droplets 609 because negative droplets 609 do not comprise the
template nucleic acid molecule 608 and, thus, no amplification
occurs.
[0140] The water-in-oil emulsion 601 can then be broken 620 into
the continuous oil phase 603 and an aqueous phase 612 that
comprises a pool of the aqueous reaction mixtures 604 from the
positive droplets 607 and negative droplets 609 of the water-in-oil
emulsion 601. The aqueous phase 612 can be separated from the
continuous oil phase 603 such as, for example, by pouring the oil
off from the aqueous phase via separation funnel or transfer out of
the vessel 602 via pipetting. The isolated aqueous phase 612 can
then be applied 630 to a surface (e.g., an array, such as a nucleic
acid array) 613 immobilized to an affinity capture agent 614 such
that the nucleic acid complex 611 in aqueous phase 612 are
immobilized to the surface. For example, the affinity capture agent
614 may be a capture sequence that is complementary to a primer or
capture sequence associated with one or more particles of the
nucleic acid complex 611. In another example, the affinity capture
agent 614 may be a member of a binding pair that binds with the
other member of the binding pair that is coupled to one or more
particles of the nucleic acid complex 611. The isolated complex 611
may then be detected, including via example modes of detection
described elsewhere herein.
[0141] In various aspects described herein, a method may comprise
detecting one or more nucleic acid molecules (e.g., amplified
nucleic acid molecules, double-stranded nucleic acid molecules,
single-stranded nucleic acid molecules, target nucleic acid
molecules, nucleic acid molecules associated with a nucleic acid
complex), nucleic acid sequences (e.g., target nucleic acid
sequences) and/or nucleic acid complexes. Detection of any of these
species may be qualitative and, in some embodiments, quantitative.
In some embodiments, the detection of a nucleic acid complex may be
used to determine or assay the absence or presence of a nucleic
acid molecule (e.g., a target nucleic acid molecule) and/or nucleic
acid sequence (e.g., a target nucleic acid sequence) that is
component of the nucleic acid complex. For example, the number of
nucleic acid complexes detected can be indicative of the copy
number of target nucleic acid molecule in a sample that is
amplified to generate the nucleic complexes. Detection of a nucleic
acid molecule, nucleic acid sequence or nucleic acid complex may be
accomplished with any suitable detection method or modality. In
some embodiments, a nucleic acid complex may be isolated prior to,
during, or after detection. The particular type of detection method
used may depend, for example, on the particular species being
detected, other species present during detection, whether or not a
detectable species is present, the particular type of detectable
species to-be-used and/or the particular application.
[0142] Non-limiting examples of detection methods include optical
detection, electrical detection, physical detection, spectroscopic
detection, electrostatic detection and electrochemical detection.
Accordingly, a nucleic acid complex, nucleic acid molecule or
nucleic acid sequence may be detected by detecting signals (e.g.,
signals indicative of an optical property, a spectroscopic
property, an electrostatic property or an electrochemical property
of the nucleic acid molecule, nucleic acid sequence, and/or nucleic
acid complex or an associated detectable species) that are
indicative of the presence or absence of the nucleic acid molecule,
nucleic acid sequence and/or nucleic acid complex. Optical
detection methods include, but are not limited to, visual
inspection (e.g., detection via the eye, observing an optical
property or optical event without the aid of an optical detector),
fluorimetry (e.g., fluorescence, fluorescent energy transfer,
quenched fluorescence), UV-vis light absorbance and colorimetric
detection. Imaging (e.g., microscopy) equipped with an optical mode
of detection (e.g., fluorescence microscopy, light microscopy,
etc.) can also be used for optical detection. Spectroscopic
detection methods include, but are not limited to, mass
spectrometry, nuclear magnetic resonance (NMR) spectroscopy, and
infrared spectroscopy. Electrostatic detection methods include, but
are not limited to, gel based techniques, such as, for example, gel
electrophoresis. Such gel techniques may also be useful for
isolating a nucleic acid complex as described elsewhere herein.
Electrochemical detection methods include, but are not limited to,
amperometry. Electrical detection methods include, but are not
limited to, detection of conductance or impedance. Physical
detection methods include, but are not limited to, detection of
particle size, density, sedimentation rate and/or viscosity.
[0143] In some embodiments, detection of a nucleic acid molecule, a
particle, a set of particles or a nucleic complex may be achieved
with fluid flow. A fluid (e.g., solution, reaction mixture)
comprising the species to be detected can be flowed past a suitable
detector such that the detector detects the species as it flows
past the detector. Alternatively, detection may be completed
statically in that a fluid comprising the species to be detected is
provided to a detector and detection occurs without fluid flow. For
example, a species to be detected may be applied to a slide or
other surface and imaged using a camera equipped with an
appropriate detector.
[0144] Detection of a particle, set of particles or a nucleic acid
comprising particles may be achieved, at least in part, based on or
more physical properties of the particle(s) or nucleic acid
complex. In some embodiments detection of one or more particles or
a nucleic acid complex comprising particles may be based, at least
in part, by the size of a particle, the size particles in a nucleic
acid complex or the size of a nucleic acid complex. In some
embodiments, detection of one or more particles or a nucleic acid
complex comprising particles may be based, at least in part, by the
density of a particle, the densities of particles in a nucleic acid
complex or the density of a nucleic acid complex. In some
embodiments, detection of one or more particles or a nucleic acid
complex may be based on sedimentation rate and/or viscosity of the
particle(s) or nucleic acid complex. Moreover, detection of one or
more particles or a nucleic acid comprising particles may be
achieved, at least in part, based on or more electrical properties
of the particle(s) or nucleic acid complex.
[0145] In some embodiments, detecting a nucleic acid molecule,
nucleic acid sequence or nucleic acid complex may be achieved with
the aid of a detectable species. A detectable species may be linked
or coupled with a nucleic acid molecule, a nucleic acid molecule
associated with a nucleic acid complex and/or any other component
of a nucleic acid complex, including covalently and non-covalently
(e.g., including intercalation of a double-stranded nucleic acid
molecule). Moreover, a detectable species may be included in a
reaction mixture that is used to generate a nucleic acid complex
and/or for nucleic acid amplification, as described elsewhere
herein. Non-limiting examples of detectable species include
optically-responsive species (e.g., optically-responsive dyes,
optically-responsive oligonucleotide probes (e.g., TaqMan probes,
TaqMan Tamara probes, TaqMan MGB probes, Lion probes, molecular
beacons)) and radiolabels (e.g., .sup.14C, .sup.123I, .sup.124I,
.sup.125I, .sup.131I, .sup.99mTc, .sup.35S, or .sup.3H).
[0146] In some embodiments, a detectable species may be an
optically-responsive dye. (e.g., a fluorescent dye) that generates
(or fails to generate a signal) when subjected to the appropriate
conditions. Non-limiting examples of dyes include SYBR green, SYBR
blue, DAPI, propidium iodine, Hoeste, SYBR gold, ethidium bromide,
acridines, proflavine, acridine orange, acriflavine, fluorcoumanin,
ellipticine, daunomycin, chloroquine, distamycin D, chromomycin,
homidium, mithramycin, ruthenium polyp yridyls, anthramycin,
methylene blue, phenanthridines and acridines, propidium iodide,
hexidium iodide, dihydroethidium, ethidium homodimer-1 and -2,
ethidium monoazide, and ACMA, Hoechst 33258, Hoechst 33342, Hoechst
34580, DAPI, Pyronin Y, Blue View, acridine orange, 7-AAD,
actinomycin D, LDS751, phycoerythrin, hydroxystilbamidine, SYTOX
Blue, SYTOX Green, SYTOX Orange, POPO-1, POPO-3, YOYO-1, YOYO-3,
TOTO-1, TOTO-3, JOJO-1, LOLO-1, BOBO-1, BOBO-3, PO-PRO-1, PO-PRO-3,
BO-PRO-1, BO-PRO-3, TO-PRO-1, TO-PRO-3, TO-PRO-5, JO-PRO-1,
LO-PRO-1, YO-PRO-1, YO-PRO-3, PicoGreen, OliGreen, RiboGreen, SYBR
Gold, SYBR Green I, SYBR Green II, SYBR DX, SYTO-40, -41, -42, -43,
-44, -45 (blue), SYTO-13, -16, -24, -21, -23, -12, -11, -20, -22,
-15, -14, -25 (green), SYTO-81, -80, -82, -83, -84, -85 (orange),
SYTO-64, -17, -59, -61, -62, -60, -63 (red), fluorescein,
fluorescein isothiocyanate (FITC), tetramethyl rhodamine
isothiocyanate (TRITC), rhodamine, tetramethyl rhodamine,
R-phycoerythrin, Cy-2, Cy-3, Cy-3.5, Cy-5, Cy5.5, Cy-7, Texas Red,
Phar-Red, allophycocyanin (APC), CellTracker Green, 7-AAD, ethidium
homodimer I, ethidium homodimer II, ethidium homodimer III,
umbelliferone, eosin, a fluorescent protein (e.g., green
fluorescent protein, red fluorescent protein), erythrosin,
coumarin, methyl coumarin, pyrene, malachite green, stilbene,
lucifer yellow, cascade blue, dichlorotriazinylamine fluorescein,
dansyl chloride, fluorescent lanthanide complexes such as those
including europium and terbium, carboxy tetrachloro fluorescein, 5
and/or 6-carboxy fluorescein (FAM), 5- (or 6-)
iodoacetamidofluorescein, 5-{[2(and
3)-5-(Acetylmercapto)-succinyl]amino} fluorescein
(SAMSA-fluorescein), lissamine rhodamine B sulfonyl chloride, 5
and/or 6 carboxy rhodamine (ROX), 7-amino-methyl-coumarin,
7-Amino-4-methylcoumarin-3-acetic acid (AMCA), BODIPY fluorophores,
8-methoxypyrene-1,3,6-trisulfonic acid trisodium salt,
3,6-Disulfonate-4-amino-naphthalimide, phycobiliproteins,
AlexaFluor 350, 405, 430, 488, 532, 546, 555, 568, 594, 610, 633,
635, 647, 660, 680, 700, 750, and 790 dyes, DyLight 350, 405, 488,
550, 594, 633, 650, 680, 755, and 800 dyes, or other
fluorophores.
[0147] In some embodiments, a nucleic acid complex may be detected
via an optical change observed upon formation of a nucleic acid
complex. For example, as described elsewhere herein, a nucleic acid
complex may be generated from a plurality of particles. The
particles may be, for example, metallic particles (e.g., metallic
nanoparticles) such as, for example, gold nanoparticles or silver
nanoparticles. Free particles, prior to the formation of a nucleic
acid complex, may exhibit a first color and the nucleic acid
complex comprising the same particles may exhibit a second color.
The second color may be detected (e.g., with visual examination
and/or with a detector) to identify/quantify the nucleic acid
complex and a color change (or absence thereof) from the first
color to the second color may be used to assay for the presence or
absence of the nucleic acid complex. In some embodiments, the
nucleic acid complex may be isolated from other species in order to
observe any color change. Moreover, other suitable optical
properties of a particle may be used for detection of a nucleic
acid complex such as, for example, optical signals received from a
semiconductor particle.
[0148] In some embodiments, a nucleic acid complex may comprise at
least two different types of particles (e.g., particles having two
different material compositions, such as, for example gold
particles and silver particles). Optical detection of the nucleic
acid complex can occur via energy transfer between the two types of
particles. For example, a first type of particle can have a
wavelength 1a and emission wavelength 1e. A second type of particle
can have absorption wavelength 2a and emission wavelength 2e.
Emission wavelength 2e can be detected when a solution comprising
the nucleic acid complex is excited with wavelength 1a when a
nucleic acid complex is present. In the absence of a nucleic acid
complex, the first particle and second particle are separate and
emission wavelength 2e is not detected when the particles are
excited with wavelength 1a.
[0149] An example of generating a nucleic acid complex via nucleic
acid amplification, isolating the generated nucleic acid complex
and detecting the nucleic acid complex is schematically depicted in
FIG. 1. Two reaction mixtures 101 and 102 are provided. Both
reaction mixtures comprise a first type of particle 103 that
comprises a forward primer and a second type of particle 104 that
comprises a reverse primer. Alternatively, particle 103 and
particle 104 may comprise a first and second capture sequence, as
described elsewhere herein, capable of hybridizing with an overhang
sequence of an amplified nucleic acid molecule. Particles 103 and
104 are free in the reaction mixtures and not initially associated
with a nucleic acid complex. In some embodiments, particles 103 and
104 are both metallic particles, such as, for example gold or
silver nanoparticles. Reaction mixture 101 comprises a template
nucleic acid molecule 105 that can be amplified in the presence of
particles 104 and 105 such that a nucleic acid complex is generated
comprising one or more amplified nucleic acid molecules and
particles 104 and 105. Reaction mixture 102 does not comprise the
template nucleic acid molecule 105. The template nucleic acid
molecule may be single-stranded or may be double-stranded.
Moreover, the color of both reaction mixtures is red due to the
color of particles 103 and 104.
[0150] Reaction mixtures 101 and 102 are subjected to conditions
106 suitable to amplify the template nucleic acid molecule 105 to
generate amplified nucleic acid molecules associated with a nucleic
acid complex 107. As reaction mixture 101 comprises the template
nucleic acid molecule, the nucleic acid complex 107 is generated in
reaction mixture 101 following amplification of the template
nucleic acid molecule 105. Moreover, no nucleic acid complex 107 is
generated in reaction mixture 102 because reaction mixture 102 does
not comprise the template nucleic acid molecule 105 and, thus, no
amplification occurs. Upon formation of the nucleic acid complex
107, the color of reaction mixture 101 shifts from a red color to a
blue color due to the coupling of particles 104 and 105 in nucleic
acid complex 107. The color of reaction mixture 102 remains red,
since no nucleic acid complex is generated.
[0151] The blue color of reaction mixture 101 can be detected with
visual examination to determine that reaction mixture 101 comprises
nucleic acid complex 107 (and, thus, amplified nucleic acid
molecules). The red color of reaction mixture 102 can be detected
with visual examination to determined that reaction mixture 102
does not comprise nucleic acid complex 107 (and, thus, does not
comprise an amplified nucleic acid molecule). In cases where
isolation of the nucleic acid complex 107 is helpful to observe its
blue color (e.g., due to, for example, possible interference from
other species in reaction mixture 101), the nucleic acid 107 may be
isolated 108 (e.g., via chromatography or filtration) from reaction
mixture 101.
[0152] The presence 109 of a blue band is indicative of the blue
color of the nucleic acid complex 107 in the reaction mixture (and,
thus, template nucleic acid molecule 105 in the reaction mixture).
The absence 110 of a blue band is indicative that reaction mixture
102 does not comprise nucleic acid complex 107 (and, thus, does not
comprise nucleic acid molecule 105). The blue color of the nucleic
acid complex 107 is a result of particles 104 and 105 aggregating
(via amplification) to generate nucleic acid complex 107.
[0153] An example of isolating and detecting a nucleic acid complex
is schematically depicted in FIG. 2. As shown, a reaction mixture
201 is contained in a vessel 202 and comprises a nucleic acid
complex. The vessel 202 is enclosed in a sealed vessel 203. A
sealed vessel 203 can be used to prevent contamination of the
reaction mixture 201. Vessel 203 also comprises a solid matrix 204
(e.g., a piece of filter paper or pregnancy test strip) and a
mechanism 205 (e.g., a puncture mechanism) capable of generating an
outlet in the bottom of vessel 202. The outlet is generated 206
such that reaction mixture 201 is applied 207 to solid matrix 204.
Via flow due to capillary action 210, the contents of reaction
mixture 201 are separated. Due to its larger size compared to other
reagents in reaction mixture 201 (e.g., free particles, reagents
for primer extension reactions, primers, etc.), the movement of the
nucleic acid complex in reaction mixture 201 is retarded compared
to the other reagents. Thus, a band 208 can be generated on the
solid matrix 204 that corresponds to the nucleic acid complex and
is separate from a band 209 that is generated further down the
solid matrix and represents other reagents in the reaction mixture
201. The separation of the bands 208 and 209 can be detected upon
visual examination (where possible) or may be detected with a
suitable type of detector (e.g., an optical detector). Moreover,
the absence of band 208 indicates that reaction mixture 201 does
not comprise a nucleic acid complex.
[0154] Various aspects described herein may be useful in conducting
multiplex nucleic acid amplification reactions. Multiplex nucleic
acid amplifications may be useful in a variety of different
applications with non-limiting examples that include detection and
quantification of small percentage gene copy number differences
with precision; detection and quantification of sequence mutations
(e.g., insertions, deletions) and rare-mutations (e.g., rare
mutations in low-prevalence targets associated with cancer);
detection and quantification of pathogens (e.g., detection and
absolute quantification of bacterial and viral loads, detection and
quantification of low-level pathogens that contaminate food and
water supplies); generation of references and standards (e.g.,
generation of absolute reference standards for genetic
measurements, metrology, and cross-laboratory measurements);
preparation of sequencing libraries; quantification of sequencing
libraries with or without reference standards (e.g., absolute
quantification of sequencing libraries and validation of sequencing
results without reference standards); detection and quantification
of gene expression changes (e.g., detection of gene express changes
for absolute transcript quantification without a reference gene);
detection and quantification of genetically modified organisms
(GMOs) (e.g., detection and absolute quantification of a foreign
gene(s) in plants); detection and quantification of circulating
tumor cells (CTCs) or other rare nucleic acids that may be present
in the blood; analysis of gene copy number in a sample; analysis of
pathogen loads and pathogen load changes in a biological sample;
analysis of transcription level in response to drug treatment;
environmental testing; forensic identification; clinical
diagnostics and detection of a nucleotide polymorphism or genotype,
such as HLA or HMC typing.
[0155] In some embodiments, compositions and methods described
herein may be useful for RNA analysis. In such analysis, RNA can be
reverse transcribed via the action of a reverse transcriptase to
generate cDNA. The cDNA can then be amplified to generate a nucleic
acid complex using particles or a set of particles described
elsewhere herein. The nucleic acid complex, once formed, can then
be isolated and/or detected using any suitable methods, including
those described elsewhere herein.
[0156] An example method of multiplex nucleic acid amplification is
schematically depicted in FIG. 7. As shown in FIG. 7, a nucleic
acid sample 701 (e.g., a sample comprising DNA) that is suspected
to comprise or comprises one or more target nucleic acid sequences
is provided in a vessel 702. An aliquot of the nucleic acid sample
701 can be provided to each of a plurality of vessels 703. In the
example shown in FIG. 7, the nucleic acid sample is aliquoted into
four vessels, however aliquots of the nucleic acid sample 701 may
be provided to any suitable number of vessels. In each vessel, a
primer set (e.g., a primer set comprising at least a forward primer
and a reverse primer) is provided that is configured to amplify a
nucleic acid molecule that comprises a unique target nucleic acid
sequence. For example, a forward primer may be provided that is at
least partially complementary to a target nucleic acid sequence and
a reverse primer may be provided that is at least partially
complementary to a complement of the target nucleic acid
sequence.
[0157] In the example shown in FIG. 7, each primer set is indicated
by "A", "B", "C" and "D" and each set is configured to amplify a
nucleic acid molecule that comprises one of four unique target
nucleic acid sequences. Moreover, the primers in each primer set
may be provided as coupled to a particle (e.g., coupling via the 5'
end of a primer) as described elsewhere herein or may be provided
free from a particle as described elsewhere herein. In cases where
the primer is provided free from a particle, the primer may be
configured in a hairpin structure and the hairpin structure may
also include an overhang sequence that is unique to the particular
priming sequence of the particular primer (and, thus, the
particular target nucleic acid sequence). In addition to the primer
sets, additional reagents necessary for amplifying a nucleic acid
molecule (e.g., a polymerase, dNTPs, suitable buffers, co-factors)
and generating a nucleic acid complex (e.g., particles coupled to
capture sequences complementary to any overhang sequences, etc.)
are also provided to the vessels 703 to generate a pre-reaction
mixture 704 in each vessel.
[0158] Reagents suitable for generating a water-in-oil emulsion
(e.g., oil, a surfactant, etc.) can then be added 710 to each
pre-reaction mixture 704 such that a water-in-oil emulsion 705
comprising a plurality of aqueous droplets in a continuous oil
phase is generated in each vessel. The aqueous droplets in each
water-in-oil emulsion 705 comprise an aqueous reaction mixture that
includes the contents of the corresponding pre-reaction mixture
704. In some embodiments, a water-in-oil emulsion 705 may be
generated such that some of the aqueous droplets in the
water-in-oil emulsion 705 do not comprise a nucleic acid molecule
to be amplified. Such control may be exerted, for example, by
controlling the level of dilution of the sample in the pre-reaction
mixtures 704 and/or the amount of reagents added 710 to generate
the water-in-oil emulsion 705.
[0159] The droplets from each water-in-oil emulsion 705 can then be
pooled 720 into a common water-in-oil emulsion 706 provided to
common vessel 707. The common water-in-oil emulsion 706 (or
water-in-oil emulsions 703) may then be subject to conditions 730
suitable for amplifying nucleic acid molecules in each droplet
(e.g., via the primer set present in each droplet) and generating a
nucleic acid complex during or after amplification as described
elsewhere herein. For example, the temperature of the common
water-in-oil emulsion 706 may be cycled as described elsewhere
herein. As a result of amplification, nucleic acid complexes may be
generated 740 (e.g., corresponding to the closed circles in 740) in
droplets that comprise a particular primer set and one or more
nucleic acid molecules that comprise the corresponding target
nucleic acid sequence. In a droplet where no nucleic acid molecule
is present or no nucleic acid molecule that comprises the target
nucleic acid sequence corresponding to the primer set in the
droplet is present, a nucleic acid complex may not be generated
(e.g., corresponding to the open circles in 740) in the droplet due
to the lack of the target nucleic acid sequence.
[0160] The common water-in-oil emulsion 706 can then be broken 750
such that a two-phase mixture comprising the oil 708 from the
continuous oil phase of the common water-in-oil emulsion 706 and a
pooled aqueous mixture 709 that comprises the aqueous reaction
mixtures of the droplets of the common water-in-oil emulsion 706.
The pooled aqueous mixture 709 can be separated from the oil 708
and provided 760 to an array 711. Each position of the array 711
can comprise a surface immobilized thereto one or more capture
sequences that are at least partially complementary to a free
primer or capture sequence associated with a particular target
nucleic acid sequence (and, thus, nucleic acid complex). The
positions of the array can be arranged such that a particular
column, row or other configuration of positions corresponds to a
particular capture sequence or set of capture sequences and, thus,
a particular target nucleic acid sequence.
[0161] In the example shown in FIG. 7, each column of the array 711
corresponds to a particular primer set (e.g., "A", "B", "C" and "D"
described above) corresponding to a particular target nucleic acid
sequence and is labeled "A'", "B'", "C'" and "D'". Via the capture
sequences immobilized to the array 711, the corresponding free
primer(s) or capture sequence(s) of the nucleic acid complexes that
are generated can bind to their appropriate positions on the array
711. As each free primer or capture sequence of a particular
nucleic acid complex is unique to a particular target nucleic acid
sequence, the nucleic acid complex can be identified as
corresponding to the particular target nucleic acid sequence based
on which site(s) it binds to on the array 711. In the example shown
in FIG. 7, a closed circle at an array position represents binding
of a nucleic acid complex and an open circle at an array position
represents no binding of a nucleic acid complex at the array
position. Binding of a nucleic acid complex can be detected via any
suitable mode, including modes of detection described elsewhere
herein. The positions that comprise a particular bound nucleic acid
complex (and, thus, a particular target nucleic acid sequence) can
be counted 770 (e.g., digitally counted) and the counting data used
for a downstream application.
[0162] An additional example of multiplex nucleic acid
amplification and its possible use in nucleic acid sequencing is
schematically depicted in FIG. 8. As shown in FIG. 8, a nucleic
acid sample 801 (e.g., a sample comprising DNA) that is suspected
to comprise or comprises one or more target nucleic acid sequences
is provided in a vessel 802. An aliquot of the nucleic acid sample
801 can be provided to each of a plurality of vessels 803. In the
example shown in FIG. 8, the nucleic acid sample is aliquoted into
four vessels, however aliquots of the nucleic acid sample 801 may
be provided to any suitable number of vessels. In each vessel, a
primer set (e.g., a primer set comprising at least a forward primer
and a reverse primer) is provided that is configured to amplify a
nucleic acid molecule that comprises a unique target nucleic acid
sequence. For example, a forward primer may be provided that is at
least partially complementary to a target nucleic acid sequence and
a reverse primer may be provided that is at least partially
complementary to a complement of the target nucleic acid
sequence.
[0163] In the example shown in FIG. 8, each primer set is indicated
by "A", "B", "C" and "D" and each set is configured to amplify a
nucleic acid molecule that comprises one of four unique target
nucleic acid sequences. Moreover, the primers in each primer set
may be provided free from a particle as described elsewhere herein.
In cases where the primer is provided free from a particle, the
primer may be configured in a hairpin structure (e.g., as in the
example shown in FIG. 3 and FIG. 4) and the hairpin structure may
also include an overhang sequence that is unique to the particular
priming sequence of the particular primer (and, thus, the
particular target nucleic acid sequence). In addition to the primer
sets, additional reagents necessary for amplifying a nucleic acid
molecule (e.g., a polymerase, dNTPs, suitable buffers, co-factors)
and generating a nucleic acid complex (e.g., particles coupled to
capture sequences complementary to any overhang sequences) are also
provided to the vessels 803 to generate a pre-reaction mixture 804
in each vessel. In some embodiments, particles provided with
capture sequences may also comprise a common affinity capture
agent, such as a member of binding-pair (e.g., biotin).
[0164] Reagents suitable for generating a water-in-oil emulsion
(e.g., oil, a surfactant, etc.) can then be added 810 to each
pre-reaction mixture 804 such that a water-in-oil emulsion 805
comprising a plurality of aqueous droplets in a continuous oil
phase is generated in each vessel. The aqueous droplets in each
water-in-oil emulsion 805 comprise an aqueous reaction mixture that
includes the contents of the corresponding pre-reaction mixture
804. In some embodiments, a water-in-oil emulsion 805 may be
generated such that some of the aqueous droplets in the
water-in-oil emulsion 805 do not comprise a nucleic acid molecule
to be amplified. Such control may be exerted, for example, by
controlling the level of dilution of the sample in the pre-reaction
mixtures 804 and/or the amount of reagents added 810 to generate
the water-in-oil emulsion 805.
[0165] The droplets from each water-in-oil emulsion 805 can then be
pooled 820 into a common water-in-oil emulsion 806 provided to
common vessel 807. The common water-in-oil emulsion 806 (or
water-in-oil emulsions 803) may then be subject to conditions 830
suitable for amplifying nucleic acid molecules in each droplet
(e.g., via the primer set present in each droplet) and generating a
nucleic acid complex during or after amplification as described
elsewhere herein. For example, the temperature of the common
water-in-oil emulsion 806 may be cycled as described elsewhere
herein. As a result of amplification, nucleic acid complexes may be
generated 840 (e.g., corresponding to the closed circles in 840) in
droplets that comprise a particular primer set and one or more
nucleic acid molecules that comprise the corresponding target
nucleic acid sequence. In a droplet where no nucleic acid molecule
is present or no nucleic acid molecule that comprises the target
nucleic acid sequence corresponding to the primer set in the
droplet is present, a nucleic acid complex may not be generated
(e.g., corresponding to the open circles in 840) in the droplet due
to the lack of the target nucleic acid sequence. The nucleic acid
complexes that are generated may be structurally similar to the
example nucleic acid complex 411 shown in FIG. 4, in that the
amplified double-stranded nucleic acid molecules of the generated
nucleic acid complex can be coupled to the particles of the
generated nucleic acid complex via hybridization between capture
sequences of the particles and overhang sequences of the amplified
double-stranded nucleic acid molecules.
[0166] The common water-in-oil emulsion 806 can then be broken 850
such that a two-phase mixture comprising the oil 808 from the
continuous oil phase of the common water-in-oil emulsion 806 and a
pooled aqueous mixture 809 that comprises the aqueous reaction
mixtures of the droplets of the common water-in-oil emulsion 806.
The pooled aqueous mixture 809 can be separated from the oil 808,
if desired. Additionally, the pooled aqueous mixture 809 can be
subject to conditions suitable to ligate (e.g., via the addition of
a ligase to the pooled aqueous mixture 809 or a ligase already
present in the pooled aqueous mixture 809) 860 amplified
double-stranded nucleic acid molecules associated with nucleic acid
complexes to respective capture sequences associated with the
amplified double-stranded nucleic acid molecules. Following
ligation of amplified double-stranded nucleic acid molecules to
capture sequences, nucleic acid complexes can be denatured 870 into
component particles comprising single strands of amplified
double-stranded nucleic acid molecules, such that particles and
single-strands are no longer associated in a nucleic acid
complex.
[0167] Component particles and strands can then be provided 880 to
a sequencing array 811. Each position of the sequencing array 811
can comprise a surface immobilized thereto one or more affinity
capture agents that can bind with a nucleic acid complex. In some
embodiments, for example, the one or more affinity capture agents
may comprise capture sequences that are at least partially
complementary to a free primer or capture sequence associated with
a particular target nucleic acid sequence or even the target
nucleic acid sequence itself. The positions of the array can be
arranged such that a particular column, row or other configuration
of positions corresponds to a particular capture sequence or set of
capture sequences and, thus, a particular target nucleic acid
sequence. In cases where the component particles comprise a common
affinity capture agent (e.g., biotin, an affinity capture agent may
comprise an agent (e.g., streptavidin) capable of binding the
common affinity capture agent (e.g., biotin).
[0168] Via the affinity capture agents immobilized to the
sequencing array 811, the corresponding binding species (e.g., free
primer(s), capture sequence(s), a common affinity capture agent or
a sequence of the single-stranded nucleic acid molecule coupled to
a particle) of the component particles can bind to positions on the
sequencing array 811. In the example shown in FIG. 8, a closed
circle at an array position represents binding of particles and an
open circle at an array position represents no binding of particles
at the array position. The single-stranded nucleic acid molecules
coupled to the component particles can then serve as templates in a
nucleic acid sequencing reaction 890, such as, for example, a
sequencing-by-synthesis sequencing reaction. Sequences obtained
from the sequence reaction can be detected via any suitable mode,
including example modes of detection described elsewhere
herein.
[0169] An example of processing a nucleic acid complex for a
sequencing reaction is shown in FIG. 9. As shown in FIG. 9, a
nucleic acid complex 901 generated during nucleic acid
amplification may comprise a plurality of particles 902 coupled to
amplified double-stranded nucleic acid molecules 903 via
hybridization of capture sequences coupled to the particles (e.g.,
at the 3' ends of the capture sequences) with overhang sequences
coupled to the amplified double-stranded nucleic acid molecules 903
(e.g., at the 5'ends of the component strands of the amplified
double-stranded nucleic acid molecules). The nucleic acid complex
901 may be then subject to conditions suitable to ligate 910 the
amplified double-stranded nucleic acid molecules 903 to respective
capture sequences.
[0170] Following ligation, the nucleic acid complex may be
denatured 920 (e.g., at a denaturing temperature and/or with the
addition of a denaturing agent (e.g., an alkaline agent)) into
component particles 904. Denaturation can separate amplified
double-stranded nucleic acid molecules 903 into component particles
904 by separating the strands of amplified double-stranded nucleic
acid molecules 903 into component single-stranded nucleic acid
molecules 905. As shown in FIG. 9, example component particles 904
are separated and coupled to single-stranded nucleic acid molecules
905 at a 3' end of the single-stranded molecules 905.
[0171] The component particles 904 can then be provided to a
surface 906 that is associated with one or more affinity capture
agents 907 and 908. In the example shown in FIG. 9, surface 906
comprises capture sequences 907 and 908 that are at least partially
complementary to a free capture sequence on the component particles
904 or other sequences on the component particles 904. The capture
sequences 907 and 908 can bind the component particles 904 via
hybridization with the appropriate species coupled to the component
particles 904. In some embodiments, the component particles 904 may
comprise a common member of a binding pair (e.g., biotin) and the
affinity capture agent of the surface 906 may comprise the other
member of the binding pair (e.g., streptavidin). The two members of
the binding pair can bind, such that the component particles 904
bind with the surface 906.
[0172] As shown in FIG. 9, binding immobilizes the single-stranded
nucleic acid molecules 905 that are associated with the component
particles to the surface 906. The single-stranded molecules 905 can
serve as templates in a sequencing reaction, such as, for example a
sequencing-by-synthesis reaction. Detection of the sequencing
reaction can be completed via any suitable mode of detection,
including example modes of detection described elsewhere
herein.
[0173] In various aspects described herein, a method may comprise
amplification of a nucleic acid molecule. In general, nucleic acid
amplification may occur in a reaction mixture in which the nucleic
acid molecule to be amplified is provided along with additional
reagents (e.g., forward primers, reverse primers, polymerases,
dNTPs, co-factors, suitable buffers, etc.) necessary for
amplification of the nucleic acid molecule. The reaction mixture
may then be subjected to conditions (e.g., appropriate
temperatures, addition/removal of heat, buffer concentrations,
etc.) suitable for amplifying the nucleic acid molecule.
[0174] For example, a single or double-stranded nucleic acid
molecule may be provided in a reaction mixture that also comprises
additional reagents (e.g., a forward primers and reverse primers
described elsewhere herein, a polymerase, dNTPs, co-factors,
buffers, other enzymes (e.g., a reverse transcriptase to generate
cDNA from RNA, a ligase, etc.) necessary for amplification of the
single or double-stranded nucleic acid molecule. In some
embodiments, the temperature of the reaction mixture may be cycled
repeatedly through a denaturation temperature (e.g., to denature,
separate or melt double-stranded nucleic acid molecules into
component nucleic acid strands), an annealing temperature (e.g., to
anneal or hybridize a primer to each of the component nucleic acid
strands) and an extension temperature (e.g., to extend or add
nucleotides to the annealed primers in a primer extension reaction
via the action of a polymerase) in order to amplify the
single-stranded or double-stranded nucleic acid molecule. The
cycling of the temperature of a reaction mixture may be achieved,
for example, with the aid of any suitable thermocycler instrument
or other type of heating device. In some embodiments, denaturation
of a double-stranded nucleic acid molecule may be achieved via a
denaturing agent, such as, for example an alkaline agent (e.g.
sodium hydroxide (NaOH)). In some cases, amplification of a nucleic
acid may be achieved isothermally such as, for example, without a
change in temperature of a reaction mixture.
[0175] A nucleic acid amplification reaction can include the use
and action of a polymerase. During a primer extension reaction, a
polymerase can generally add, in template-directed fashion,
nucleotides to the 3' end of a primer annealed to a single-stranded
nucleic acid molecule. Any suitable polymerase may be used for a
primer extension reaction, including commercially available
polymerases. Non-limiting examples of polymerases include Taq
polymerase, Tth polymerase, Tli polymerase, Pfu polymerase, VENT
polymerase, DEEPVENT polymerase, EX-Taq polymerase, LA-Taq
polymerase, Expand polymerases, Sso polymerase, Poc polymerase, Pab
polymerase, Mth polymerase, Pho polymerase, Phusion DNA polymerase,
ES4 polymerase, Tru polymerase, Tac polymerase, Tne polymerase, Tma
polymerase, Tih polymerase, Tfi polymerase, Platinum Taq
polymerases, Hi-Fi polymerase, Tbr polymerase, Tfl polymerase,
Pfutubo polymerase, Pyrobest polymerase, Pwo polymerase, KOD
polymerase, Bst polymerase, Sac polymerase, Klenow fragment,
polymerase with 3' to 5' exonuclease activity, KleTaq DNA
polymerase, and variants, modified products and derivatives
thereof.
[0176] In some embodiments, a suitable denaturation temperature may
be, for example, about 60.degree. C., 61.degree. C., 62.degree. C.,
63.degree. C., 64.degree. C., 65.degree. C., 66.degree. C.,
67.degree. C., 68.degree. C., 69.degree. C., 70.degree. C.,
71.degree. C., 72.degree. C., 73.degree. C., 74.degree. C.,
75.degree. C., 76.degree. C., 77.degree. C., 78.degree. C.,
79.degree. C., 80.degree. C., 81.degree. C., 82.degree. C.,
83.degree. C., 84.degree. C., 85.degree. C., 86.degree. C.,
87.degree. C., 88.degree. C., 89.degree. C., 90.degree. C.,
91.degree. C., 93.degree. C., 94.degree. C., 95.degree. C.,
96.degree. C., 97.degree. C., 98.degree. C., 99.degree. C.,
100.degree. C., 101.degree. C., 102.degree. C., 103.degree. C.,
104.degree. C., 105.degree. C. or higher. In some embodiments, a
suitable annealing temperature may be, for example, about
45.degree. C., 46.degree. C., 47.degree. C., 48.degree. C.,
49.degree. C., 50.degree. C., 51.degree. C., 52.degree. C.,
53.degree. C., 54.degree. C., 55.degree. C., 56.degree. C.,
57.degree. C., 58.degree. C., 59.degree. C., 60.degree. C.,
61.degree. C., 62.degree. C., 63.degree. C., 64.degree. C.,
65.degree. C., 66.degree. C., 67.degree. C., 68.degree. C.,
69.degree. C., 70.degree. C., 71.degree. C., 72.degree. C.,
73.degree. C., 74.degree. C., 75.degree. C., 76.degree. C.,
77.degree. C., 78.degree. C., 79.degree. C., 80.degree. C., or
higher. In some embodiments, a suitable extension temperature may
be, for example, about 45.degree. C., 46.degree. C., 47.degree. C.,
48.degree. C., 49.degree. C., 50.degree. C., 51.degree. C.,
52.degree. C., 53.degree. C., 54.degree. C., 55.degree. C.,
56.degree. C., 57.degree. C., 58.degree. C., 59.degree. C.,
60.degree. C., 61.degree. C., 62.degree. C., 63.degree. C.,
64.degree. C., 65.degree. C., 66.degree. C., 67.degree. C.,
68.degree. C., 69.degree. C., 70.degree. C., 71.degree. C.,
72.degree. C., 73.degree. C., 74.degree. C., 75.degree. C.,
76.degree. C., 77.degree. C., 78.degree. C., 79.degree. C.,
80.degree. C., or higher. In some embodiments, annealing and
denaturation steps may be combined such that they occur at the same
temperature.
[0177] Any suitable type of nucleic acid amplification reaction may
be used to amplify a nucleic acid molecule. One example of a
nucleic acid amplification reaction is a polymerase chain reaction
(PCR) that relies on repeated cycles of primer annealing, primer
extension and denaturing of amplified nucleic acid molecules as
described above. Additional non-limiting examples of types of
nucleic acid amplification reactions include reverse transcription,
ligase chain reaction, nested amplification, multiplex
amplification, helicase-dependent amplification, asymmetric
amplification, rolling circle amplification, multiple displacement
amplification (MDA); and variants of PCR that include real-time
PCR, hot start PCR, inverse PCR, methylation-specific PCR,
allele-specific PCR, assembly PCR, asymmetric PCR, miniprimer PCR,
multiplex PCR, nested PCR, overlap-extension PCR, digital PCR,
emulsion PCR, dial-out PCR, helicase-dependent PCR, nested PCR,
thermal asymmetric interlaced PCR, and touchdown PCR. In some
embodiments, digital PCR and other amplification processes can be
used in conjunction with any of the amplification methods,
particles, primers and/or capture sequences described herein.
[0178] Additional aspects of the disclosure provide systems for
assaying or identifying the presence of a target nucleic acid
molecule/target nucleic acid sequence in a sample and/or executing
methods of the disclosure. In one aspect, the disclosure provides a
system for analyzing the content(s) of a solution. The system can
comprise a detection cell that is adapted to contain or direct a
solution containing a nucleic acid complex that comprises a
double-stranded nucleic acid molecule linked to at least a first
particle and a second particle. The double-stranded nucleic
molecule may comprise a first single-stranded nucleic acid molecule
and a second single-stranded nucleic acid molecule that is
complementary to at least a portion of the first single-stranded
nucleic acid molecule. In addition, the system may also comprise a
detector that is linked to the detection cell and detects signals
indicative of the presence or absence of the nucleic acid complex
in the solution. Moreover, the system may also comprise a computer
processor that is linked to the detector and programmed to receive
signals (e.g., signals are indicative of the presence of absence of
the nucleic acid complex) from the detector and determine if the
nucleic acid complex is present or absent in the solution based on
the detected signals.
[0179] In some embodiments the detection cell may comprise a vessel
(e.g., an example type of vessel described elsewhere herein), a
well (e.g., a microwell, a machine microwell, micro-fabricated
micro-wells or cavities), an array of wells (e.g., a microwell
plate, a molded microwell), and/or a support (e.g., an example type
of support described elsewhere herein). In some embodiments, the
detection cell may comprise a fluid flow path, such as one or more
channels of a microfluidic device. In such embodiments, the
solution may be provided to and/or directed within the detection
cell and/or other system component via fluid flow. In some
embodiments, a system may also comprise one or more pumps that can
be configured to flow the solution to and/or through the detection
cell and any other system components. In some embodiments, the one
or more pumps may be fluidically connected to the detection cell
and/or the detector in order to provide and/or the solution to the
detection cell. Detection of nucleic acid complexes can occur as
nucleic acid complexes flow past the detector in the fluid flow
path. In some embodiments, the system may comprise one or more
reagent reservoirs, where and individual reagent reservoir can
contain the solution and/or any other reagents for the detection of
the nucleic acid complex (e.g., a detectable species) prior to its
containment in the solution and/or reagents. The reagent reservoirs
may be fluidically connected to the detection cell via a fluid flow
path, such as one or more channels of a microfluidic device.
[0180] In some embodiments, the detection cell may comprise the
solution. Moreover, the solution may or may not be contained within
a partition. Where the solution is contained in a partition, the
partition may be any suitable type of partition, including example
types of partitions (e.g., droplets of an emulsion, wells,
cavities) described elsewhere herein. In some embodiments, the
nucleic acid complex may comprise a plurality of double-stranded
nucleic acid molecules linked to greater than two particles as
described elsewhere herein. In some embodiments, the solution may
comprise a plurality of nucleic acid complexes. The detector can be
configured to detect the plurality of nucleic acid complexes and
the computer processor can be programmed to determine the presence
of the plurality of the nucleic acid complexes (and/or any
associated nucleic acid molecules associated with the nucleic acid
complexes).
[0181] Moreover, the detector may be any suitable type of detector,
the type of which may depend upon the particular mode of detection
utilized. For example, where a system is configured for optical
detection, the detector can be an optical detector (e.g., a
spectrophotometer, a UV-vis light absorbance spectrophotometer, a
fluorimeter, a colorimeter, a camera (e.g., a charge-coupled device
(CCD) camera, an electron charge coupled device (EMCCD) camera),
photodiode, a photodiode array an etc.). Additional non-limiting
examples of detectors include a spectroscopic detector (e.g., a
mass spectrometer, a nucleic magnetic resonance (NMR) spectrometer,
a particle sizer, an electrical detector, an infrared spectrometer,
an electron paramagnetic resonance (EPR) spectrometer) and an
electrochemical detector.
[0182] Linkages between various system components can depend on the
particular system components being linked. The detection cell and
the detector may be electronically linked such that the detection
cell electronically communicates with the detector. Moreover, a
linkage between the detector and a detection cell may depend, for
example, on the particular mode of detection. For example, a
detector and a detection cell may be optically linked such that
light passes through, from and/or to the detection cell to/from the
detector. Moreover, the computer processor may be electronically
linked to the detector such that the signals detected by the
detector are transmitted by the detector and received by the
computer processor electronically. In some embodiments, the
detection cell may also be linked (e.g., electronically linked) to
the computer processor. In addition, the computer processor may
also be programmed to control detector operation (e.g., such as
timing of detection, detector configuration for particular
detection modes or solution, etc.), detection cell operation, or
operation of any other component (e.g., pumps). Furthermore,
various components of the system may be contained in a housing. In
some embodiments, the detector and detection cell may be contained
in the same housing or may be contained in separate housings. In
some embodiments, the computer processor may be contained in the
same housing as the detector and/or detection cell or may be
contained in a separate housing. In some embodiments, all
components of the system may be included in a single housing.
[0183] In some embodiments, the computer processor may be included
as part of a computer system. The computer system may be housed
separately from the detector and/or detection cell or may be housed
together with one or both components. For example, as shown in FIG.
10, the computer processor 1005 (e.g., a central processing unit
(CPU)) may be included as part of computer system 1001 and can be a
single core or multi core processor, or a plurality of processors
for parallel processing. The computer system 1001 can also include
memory or memory location 1010 (e.g., random-access memory,
read-only memory, flash memory), electronic storage unit 1015
(e.g., hard disk), 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 (e.g., keyboards,
mice, sounds systems, microphones, printers, or other input or
output devices) can be in communication with the computer processor
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 embodiments, may be 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 embodiments 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.
[0184] The computer processor 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
computer processor 1005 can include fetch, decode, execute, and
writeback. The storage unit 1015 can store files, such as drivers,
libraries and saved programs. The storage unit 1015 can store
programs generated by users and recorded sessions, as well as
output(s) associated with the programs. The storage unit 1015 can
store user data, e.g., user preferences and user programs. The
computer system 1001, in some embodiments, can include one or more
additional data storage units that are external to the computer
system 1001, such as an additional storage unit that is located on
a remote server that is in communication with the computer system
1001 through an intranet or the Internet.
[0185] 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. 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.
[0186] Methods described herein and instructions for operating the
detection cell, detector and any other component of the system
(e.g., pumps) 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
embodiments, 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. The code can be pre-compiled and configured for use with a
machine have 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.
[0187] 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 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.
[0188] 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 dynamic memory, such as main memory of such a
computer platform. Tangible transmission media include 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, 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.
[0189] Additional aspects of the disclosure provide kits that may
include particle sets, primers, other reagents (e.g., one or more
reagents necessary for amplification of a nucleic acid molecule),
and instructions suitable for amplifying a nucleic acid molecule,
conducting primer extension reactions, assaying for a target
nucleic acid molecule and/or generating (and, in some cases,
detecting) a nucleic acid complex. In one aspect, the disclosure
provides a kit for assaying the presence or absence of a target
nucleic acid strand in a sample having or suspected of having the
target nucleic acid strand. The kit may comprise a first particle,
a second particle, and instructions for using the first and second
particles to identify the presence or absence of the target nucleic
acid strand in the sample via a primer extension reaction. The
first particle may comprise a first primer that has a first nucleic
acid sequence that exhibits sequence homology to a portion of the
target nucleic acid strand. The second particle may comprise a
second primer that has a second nucleic acid sequence that exhibits
sequence homology to a portion of a complement nucleic acid strand
of the target nucleic acid strand. In addition, the first nucleic
acid sequence may be different than the second nucleic acid
sequence.
[0190] In some embodiments, the first particle and/or the second
particle may be contained in a vessel. The first and/or the second
particle may be contained in any suitable type of vessel including
example types of vessels described elsewhere herein. Moreover, the
first particle and/or the second particle may be any suitable type
of particle (including example types of particles described
elsewhere herein), may comprise any suitable type of material
(including example types of particle materials described elsewhere
herein), and/or may be of any suitable particle size (including
example particle sizes described elsewhere herein). In some
embodiments, the first particle and second particle comprise the
same materials. In some embodiments, the first particle and second
particle may comprise different materials.
[0191] In some embodiments, the kit may further comprise one or
more reagents suitable for generating a water-in-oil emulsion or
oil-in water emulsion. Non-limiting examples of such reagents
include an aqueous media (e.g., water, a buffer, etc), an oil
(e.g., mineral oil) and a surfactant (e.g., ABIL EM90 (from Evonik
Industries)), ABIL WE 90 (from Evonik Industries), Triton-X100,
Tween 80, Span 80). In some embodiments, the kit may further
comprise a detectable species (e.g., an optically-responsive
species) that can permit identification of the target nucleic acid
strand and/or an amplified target nucleic acid strand. In some
embodiments, the kit may further comprise one or more reagents
included in a reaction mixture, such as reagents (e.g., polymerase,
nucleotides (e.g., dNTPs), other enzymes, buffers, co-factors,
etc.) necessary for performing the primer extension reaction.
EXAMPLES
Example 1: Preparation of Polymeric Particles
[0192] Provided herein is an example method of generating polymeric
particles that may be used to amplify nucleic acid molecules and/or
generate nucleic acid complexes as described elsewhere herein.
[0193] An aqueous solution containing 8% acrylamide and 5%
bis-acrylamide is prepared and degassed with nitrogen gas (N.sub.2)
for 10 minutes. Separately, an oil solution containing 10%
surfactant Abil We 90 (from Evonik Industries) in mineral oil is
prepared and degassed under high vacuum for 10 minutes. 200 .mu.L
of the aqueous solution is then mixed with 10 .mu.L of 6% ammonium
persulfate (NH.sub.4).sub.2S.sub.2O.sub.8 and 20 .mu.L of
acrylamide-modified oligonucleotides (e.g., DNA oligonucleotides)
or acrydite-modified oligonucleotides to generate a modified
aqueous solution. Separately, 400 .mu.L of the oil solution is
mixed with 4 .mu.L of tetramethylethylenediamine (TEMED) to
generate a modified oil solution.
[0194] The modified aqueous solution and modified oil solution is
then mixed together and immediately vortexed at about 2500 rpm for
2 minutes to emulsify the mixture. The emulsified mixture is then
allowed to stand at ambient temperature for about three hours such
that particles are generated in the solution.
[0195] Next, 10 mL of ethanol is added to the emulsion and the
particles vortexed and then centrifuged at 5000 rpm for 6 minutes
(min). Following centrifugation, the supernatant is removed from
the mixture via, for example, pipetting. Washing of the particles
with 10 mL ethanol followed by vortexing and centrifugation at 5000
rpm for 6 min is then repeated for two additional cycles. The
particles are then washed with two cycles of 10 mL water followed
by vortexing and centrifugation at 5000 rpm for 6 min. The washed
beads are then suspended in 20 mL water and larger particles are
allowed to settle to the bottom of the mixture via gravity. Smaller
particles remain at the top of the mixture. The smaller particles
at the top of the solution can then be removed, diluted with water
and filtered through a filter or membrane that selects particles of
the desired particle size. The selected particles can then be
quantified by SYBR Green staining.
[0196] While preferred embodiments of the present invention have
been shown and described herein, it will be obvious to those
skilled in the art that such embodiments are provided by way of
example only. Numerous variations, changes, and substitutions will
now occur to those skilled in the art without departing from the
invention. It should be understood that various alternatives to the
embodiments of the invention described herein may be employed in
practicing the invention. It is intended that the following claims
define the scope of the invention and that methods and structures
within the scope of these claims and their equivalents be covered
thereby.
Sequence CWU 1
1
1136DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primermodified_base(13)..(13)Abasic nucleotide or not
present 1tttggtgttt atngtgctat aaacaccagc ctccca 36
* * * * *