U.S. patent application number 16/953547 was filed with the patent office on 2021-08-19 for determination of cells using amplification.
This patent application is currently assigned to President and Fellows of Harvard College. The applicant listed for this patent is President and Fellows of Harvard College. Invention is credited to David A. Weitz, Huidan Zhang.
Application Number | 20210254129 16/953547 |
Document ID | / |
Family ID | 1000005564610 |
Filed Date | 2021-08-19 |
United States Patent
Application |
20210254129 |
Kind Code |
A1 |
Weitz; David A. ; et
al. |
August 19, 2021 |
DETERMINATION OF CELLS USING AMPLIFICATION
Abstract
The present invention generally relates to microfluidics and, in
particular, to systems and methods for determining cells using
amplification. In one set of embodiments, cells are encapsulated
within droplets and nucleic acids from the cells amplified within
the droplets. The droplets may then be pooled together and the
amplified nucleic acids can be determined using PCR or other
suitable techniques. In some embodiments, techniques such as these
can be used to detect relatively rare cells that may be present,
e.g., if the droplets are amplified using conditions able to
selectively amplify nucleic acids arising from the relatively rare
cells.
Inventors: |
Weitz; David A.; (Cambridge,
MA) ; Zhang; Huidan; (Cambridge, MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
President and Fellows of Harvard College |
Cambridge |
MA |
US |
|
|
Assignee: |
President and Fellows of Harvard
College
Cambridge
MA
|
Family ID: |
1000005564610 |
Appl. No.: |
16/953547 |
Filed: |
November 20, 2020 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15557622 |
Jan 18, 2018 |
10876156 |
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PCT/US2016/022021 |
Mar 11, 2016 |
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16953547 |
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62133140 |
Mar 13, 2015 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12Q 2600/158 20130101;
C12N 15/1075 20130101; C12Q 1/689 20130101; C12Q 1/6886 20130101;
C12Q 1/686 20130101; C12Q 2600/106 20130101; C12Q 2600/156
20130101; C12Q 1/6806 20130101 |
International
Class: |
C12Q 1/686 20060101
C12Q001/686; C12Q 1/689 20060101 C12Q001/689; C12N 15/10 20060101
C12N015/10; C12Q 1/6806 20060101 C12Q001/6806; C12Q 1/6886 20060101
C12Q001/6886 |
Claims
1-50. (canceled)
51. A method, comprising: within a plurality of droplets
encapsulating nucleic acids arising from a plurality of cells,
applying conditions able to selectively amplify a target nucleic
acid sequence suspected of being present within the nucleic acids;
combining the interiors of the droplets together to form a combined
fluid; and determining amplified nucleic acids contained within the
combined fluid.
52. A method, comprising: encapsulating cells within a plurality of
droplets; lysing at least some of the cells within the plurality of
droplets to release nucleic acids from the cells into the interior
of the droplets; within the interior of the droplets, applying
conditions able to selectively amplify a target nucleic acid
sequence suspected of being present within the nucleic acids
released from the cells; and determining amplified nucleic acids
contained within the droplets.
53. The method of claim 51, wherein the target nucleic acid
sequence is a DNA sequence.
54. The method of claim 51, wherein the target nucleic acid
sequence is an RNA sequence.
55. The method of claim 51, wherein the target nucleic acid
sequence is an mRNA sequence.
56. The method of claim 51, wherein applying conditions able to
selectively amplify a target nucleic acid sequence comprises
applying conditions able to cause amplification via PCR.
57. The method of claim 56, wherein applying conditions able to
selectively amplify a target nucleic acid sequence comprises
applying conditions able to cause amplification via RT-PCR.
58. The method of claim 51, wherein applying conditions able to
selectively amplify a target nucleic acid sequence comprises using
PCR to amplify the target nucleic acid sequence.
59. The method of claim 51, wherein applying conditions able to
selectively amplify a target nucleic acid sequence comprises adding
a polymerase to at least some of the interior of the droplets.
60. The method of claim 59, comprising adding a polymerase to at
least some of the interior of the droplets via injection.
61. The method of claim 52, wherein the target nucleic acid
sequence is a DNA sequence.
62. The method of claim 52, wherein the target nucleic acid
sequence is an RNA sequence.
63. The method of claim 52, wherein the target nucleic acid
sequence is an mRNA sequence.
64. The method of claim 52, wherein applying conditions able to
selectively amplify a target nucleic acid sequence comprises
applying conditions able to cause amplification via PCR.
65. The method of claim 64, wherein applying conditions able to
selectively amplify a target nucleic acid sequence comprises
applying conditions able to cause amplification via RT-PCR.
66. The method of claim 52, wherein applying conditions able to
selectively amplify a target nucleic acid sequence comprises using
PCR to amplify the target nucleic acid sequence.
67. The method of claim 52, wherein applying conditions able to
selectively amplify a target nucleic acid sequence comprises adding
a polymerase to at least some of the interior of the droplets.
68. The method of claim 67, comprising adding a polymerase to at
least some of the interior of the droplets via injection.
Description
RELATED APPLICATIONS
[0001] This application claims the benefit of U.S. Provisional
Patent Application Ser. No. 62/133,140, filed Mar. 13, 2015,
entitled "Determination of Cells Using Amplification," by Weitz, et
al., incorporated herein by reference in its entirety.
FIELD
[0002] The present invention generally relates to microfluidics
and, in particular, to systems and methods for determining cells
using amplification.
BACKGROUND
[0003] The determination of rare cells in samples, such as clinical
samples, represents an unresolved and poorly addressed medical
issue. These rare cells usually exist in the early stage of
diseases and may result in serious consequences if not detected in
a timely manner. Most traditional molecular biology techniques,
such as real-time quantitative PCR, have limited sensitivity due to
the presence of a large amount of background cells, inhibitors,
noise, or the like. In addition, the cells in samples may be highly
heterogeneous, such that bulk methods used for the detection of
target cells at the population level are usually incapable of
providing single-cell resolution or detection. Therefore,
determining these significant rare cells individually, with
sufficiently high sensitivity and specificity, remains
challenging.
SUMMARY
[0004] The present invention generally relates to microfluidics
and, in particular, to systems and methods for determining cells
using amplification. The subject matter of the present invention
involves, in some cases, interrelated products, alternative
solutions to a particular problem, and/or a plurality of different
uses of one or more systems and/or articles.
[0005] In one set aspect, the present invention is generally
directed to a method. In one set of embodiments, the method
includes acts of encapsulating cells within a plurality of
microfluidic droplets; lysing at least some of the cells within the
plurality of droplets to release nucleic acids from the cells into
the interior of the droplets; within the interior of the droplets,
applying conditions able to selectively amplify a target nucleic
acid sequence suspected of being present within the nucleic acids
released from the cells; combining the interiors of the droplets
together to form a combined fluid; and determining amplified
nucleic acids contained within the combined fluid.
[0006] In another set of embodiments, the method comprises, within
a plurality of droplets encapsulating nucleic acids arising from a
plurality of cells, applying conditions able to selectively amplify
a target nucleic acid sequence suspected of being present within
the nucleic acids; combining the interiors of the droplets together
to form a combined fluid; and determining amplified nucleic acids
contained within the combined fluid.
[0007] The method, in yet another set of embodiments, includes
encapsulating cells within a plurality of droplets; lysing at least
some of the cells within the plurality of droplets to release
nucleic acids from the cells into the interior of the droplets;
within the interior of the droplets, applying conditions able to
selectively amplify a target nucleic acid sequence suspected of
being present within the nucleic acids released from the cells; and
determining amplified nucleic acids contained within the
droplets.
[0008] In another aspect, the present invention encompasses methods
of making one or more of the embodiments described herein. In still
another aspect, the present invention encompasses methods of using
one or more of the embodiments described herein.
[0009] Other advantages and novel features of the present invention
will become apparent from the following detailed description of
various non-limiting embodiments of the invention when considered
in conjunction with the accompanying figures. In cases where the
present specification and a document incorporated by reference
include conflicting and/or inconsistent disclosure, the present
specification shall control. If two or more documents incorporated
by reference include conflicting and/or inconsistent disclosure
with respect to each other, then the document having the later
effective date shall control.
BRIEF DESCRIPTION OF THE DRAWINGS
[0010] Non-limiting embodiments of the present invention will be
described by way of example with reference to the accompanying
figures, which are schematic and are not intended to be drawn to
scale. In the figures, each identical or nearly identical component
illustrated is typically represented by a single numeral. For
purposes of clarity, not every component is labeled in every
figure, nor is every component of each embodiment of the invention
shown where illustration is not necessary to allow those of
ordinary skill in the art to understand the invention. In the
figures:
[0011] FIG. 1 illustrates cell amplification in accordance with one
embodiment of the invention;
[0012] FIG. 2 illustrates a technique for PCR in accordance with
certain embodiments of the invention; and
[0013] FIGS. 3A-3B illustrate data illustrate detection of mutants
using an embodiment of the invention.
DETAILED DESCRIPTION
[0014] The present invention generally relates to microfluidics
and, in particular, to systems and methods for determining cells
using amplification. In one set of embodiments, cells are
encapsulated within droplets and nucleic acids from the cells
amplified within the droplets. The droplets may then be pooled
together and the amplified nucleic acids can be determined using
PCR or other suitable techniques. In some embodiments, techniques
such as these can be used to detect relatively rare cells that may
be present, e.g., if the droplets are amplified using conditions
able to selectively amplify nucleic acids arising from the
relatively rare cells.
[0015] Referring to FIG. 1, one aspect of the invention is now
discussed. However, in other embodiments, other systems and methods
may be used as well, e.g., as described below. In FIG. 1, a
population of cells 10 is shown. Most of the cells are of a first
cell type 11 (or more than one cell type), while there are rare
cells of a second type 12 that are contained within the population.
It is these rare cells that are of primary interest, e.g., as
target cells. As non-limiting examples, the first cells may be
normal cells while the second cells may be cancerous or diseased
cells, the first cells may be human cells while the second cells
may be non-human cells (for example, pathogenic cells such as
bacteria or the like), etc.
[0016] In some cases, the second (or target) cells may be quite
rare within the overall population of cells. For example, only 1
out of 1,000, 10,000, 100,000, or 1,000,000 of the cells may be the
second cell type, while the other cells may be of the first cell
type. This rarity can cause problems in determining the second cell
type under some circumstances. As an illustrative non-limiting
example, many existing techniques for determining cells or nucleic
acids in a population of cells have a false positive error rate. As
an example, the error rate may be 1%, i.e., out of every 100 cells
studied, one is erroneously positively identified (i.e., as a
second cell type) when it should have been negatively identified
(i.e., as a first cell type). However, even relatively low error
rates (such as 1%) may still nonetheless be quite substantial
compared to the rarity of the second cell type within the
population. For instance, if there is only 1 second cell present
for every 1,000 first cells in a population, then even a false
positive error rate of 1% would still result in about 90% of all
cells positively identified being incorrectly identified.
[0017] However, various embodiments as discussed herein can
increase the ability to correctly determine rare cells within a
population of cells. As a non-limiting illustrative example, as
shown in FIG. 1, a population of cells 10 contains a first cell
type 11 (or more than one type) and a second cell type 12. (As
noted above, second cell type 12 may be exceedingly rare compared
to first cell type 11 in certain embodiments, although such extreme
ratios are not accurately depicted in FIG. 1 in the interests of
clarity.) The population of cells may be encapsulated within a
plurality of droplets 20, e.g., microfluidic droplets. The ratio of
encapsulation may be 1:1 (i.e., one cell to one droplet), or any
other suitable encapsulation ratio. (1:1 is also used here in the
interests of clarity.)
[0018] Within the droplets, the cells may then be lysed in some
fashion to release nucleic acids within the droplets, e.g., DNA
and/or RNA from the cells may be released from the cells within the
droplets. The lysing can be performed using any suitable technique
for lysing cells. Non-limiting examples include ultrasound or
exposure to suitable agents such as surfactants. In some cases, the
exact technique chosen may depend on the type of cell being lysed;
many such cell lysing techniques will be known by those of ordinary
skill in the art.
[0019] Next, nucleic acids can be amplified within the droplets.
Various techniques may be used to amplify the nucleic acid with the
droplets, for example, PCR (polymerase chain reaction) techniques
such as RT-PCR (reverse transcription polymerase chain reaction),
or other techniques including those discussed herein. In certain
embodiments, various PCR reagents may be added to the droplets
(e.g., deoxyribonucleotides, primers, polymerases, reverse
transcriptases, etc.), e.g., via injection or merging with other
droplets, and/or the droplets may be subjected to conditions (such
as temperature changes) to facilitate PCR amplification of nucleic
acid with the droplets.
[0020] In some cases, the amplification conditions may be selected
to amplify certain nucleic acids, relative to other nucleic acids,
e.g., nucleic acids arising from the second cell types relative to
the first cell types. For instance, as is shown in FIG. 1, nucleic
acids 22 from cells 12 may be substantially amplified, relative to
nucleic acids 21 from cells 11. Thus, as a non-limiting example,
primers for PCR amplification may be selected so as to selectively
amplify bacterial nucleic acids (or specific species of bacteria),
relative to nucleic acids from human cells, e.g., to assist in the
determination of bacteria within a sample. In some cases,
relatively large amplifications may be achieved. For example, a
nucleic acid may be amplified by at least about 10.sup.3, at least
about 10.sup.4, at least about 10.sup.5, at least about 10.sup.6,
or at least about 10.sup.7-fold within a droplet.
[0021] Containing individual cells in droplets may facilitate the
amplification of desired or target nucleic acids (e.g., arising
from the second cell types), relative to other nucleic acids (e.g.,
arising from the first cell types), at least in some embodiments.
Since the droplets contain relatively few numbers of cells (for
instance, zero or only one cell per droplet), there are fewer
chances for competition reactions or other reactions, where
undesired nucleic acids (e.g., from the first cell types) are
amplified relative to the target nucleic acids (e.g., from the
second cell types). Thus, the amplification of the second cell
types may be enhanced by performing amplification within droplets,
allowing for relatively rare nucleic acids within the population of
nucleic acids to be substantially amplified relative to other
nucleic acids within the population. In contrast, amplification of
a population of different nucleic acids arising from different
cells, i.e., without droplets, may result in competitive reactions,
background cells, inhibitors, etc. that often can impede the
amplification of the target nucleic acids.
[0022] After amplification, the interiors of the droplets may be
merged or pooled together in some fashion. For example, the
droplets may be "burst" or disrupted, for example, mechanically or
by applying ultrasound, to release their contents, or the droplets
may be coalesced together, for example, using suitable dipole
moments or electric fields. The nucleic acids may thus be mixed
together, e.g., into a common "pool," as is shown in FIG. 1. The
population of nucleic acids (e.g., which may contain a greater
population of target nucleic acids arising from the second cell
types) may then be analyzed or determined in some fashion,
qualitatively or quantitatively, for example, using techniques
known to those of ordinary skill in the art. Examples of such
techniques include digital PCR, qPCR, or other techniques as
discussed herein. As nucleic acids arising from the second cell
types may be substantially amplified, relative to the original
population of cells, they may be easier to determine. Thus, for
example, it could be concluded that the original population of
cells contains (or does not contain) cancer cells, bacterial cells,
or other target cells of interest.
[0023] The above discussion is a non-limiting example of one
embodiment of the present invention that can be used to determine
cells using amplification, e.g., within droplets. However, other
embodiments are also possible. Accordingly, more generally, various
aspects of the invention are directed to various systems and
methods for determining cells using amplification.
[0024] As mentioned, certain aspects of the invention are generally
directed to systems and methods of determining relatively rare
cells within a population of cells. The population of cells may
comprise one, or more than one, type of cell. The rare cells may,
for example, be only 1 out of about 1,000, about 3,000, about
5,000, about 10,000, about 30,000, about 50,000 about 100,000,
about 300,000, about 500,000, or about 1,000,000 of the cells
contained within the population of cells. As non-limiting examples,
the population of cells may be taken from a blood sample, a biopsy,
a tissue sample, a tissue culture, a sample from another biological
fluid (such as urine or sweat), or the like. As addition examples,
the cells may also be taken from a certain organ or tissue (e.g.,
cardiac cells, immune cells, muscle cells, cancer cells, etc.),
cells from a specific individual or species (e.g., human cells,
mouse cells, bacteria, etc.), cells from different organisms, cells
from a naturally-occurring sample (e.g., pond water, soil, etc.),
or the like. The rare cells may be, for example, cancer cells, or
pathogens such as bacteria that are present in the sample (e.g.,
which may cause a disease in a subject), or the like. Thus, as a
non-limiting example, in one embodiment, blood may be taken from a
subject (such as a human subject), and analyzed to determine tumor
cells (e.g., leukemia or other cancer cells) contained within the
blood. As another non-limiting example, blood may be taken from a
subject (such as a human subject) may be analyzed to determine
pathogens such as bacteria that may be present.
[0025] In some cases, the cells may be encapsulated within a
plurality of droplets, such as microfluidic droplets. Various
techniques for creating or manipulating droplets are known;
non-limiting examples include those in Int. Pat. Apl. Pub. No. WO
2004/091763, entitled "Formation and Control of Fluidic Species,"
by Link et al.; Int. Pat. Apl. Pub. No. WO 2004/002627, entitled
"Method and Apparatus for Fluid Dispersion," by Stone et al.; Int.
Pat. Apl. Pub. No. WO 2006/096571, entitled "Method and Apparatus
for Forming Multiple Emulsions," by Weitz et al.; Int. Pat. Apl.
Pub. No. WO 2005/021151, entitled "Electronic Control of Fluidic
Species," by Link et al.; Int. Pat. Apl. Pub. No. WO 2011/056546,
entitled "Droplet Creation Techniques," by Weitz, et al., each
incorporated herein by reference in its entirety.
[0026] The cells may be encapsulated in the droplets at any
suitable average ratio. For example, in one set of embodiments, the
cells are encapsulated at about a 1:1 ratio, e.g., at an average of
about 1 cell per droplet. However, in some cases, lower ratios of
cells per droplet may be desired. For instance, the ratio may be
less than about 1:10, less than about 1:30, less than about 1:50,
less than about 1:100, less than about 1:300, less than about
1:500, less than about 1:1,000, less than about 1:3,000, less than
about 1:500, less than about 1:10,000, less than about 1:30,000,
less than about 1:50,000, less than about 1:100,000, less than
about 1:300,000, less than about 1:500,000, less than about
1:000,000, or any other suitable average ratio of cells to
droplets. Such ratios may be useful, for example, to ensure that
most of the droplets encapsulate either zero or one cell, and
thereby limit the number of droplets containing two or more cells.
However, in some embodiments, average ratios higher than 1:1 may
also be used, e.g., at least about 2:1, at least about 3:1, at
least about 5:1, at least about 10:1, at least about 50:1, at least
about 100:1 of cells to droplets, etc.
[0027] After encapsulation, the cells may be lysed within the
droplets, e.g., to release nucleic acids into the interiors of the
droplets. As non-limiting examples, the cells within the droplets
may be exposed to a lysing chemical (e.g., pure water, a surfactant
such as Triton-X or SDS, an enzyme such as lysozyme, lysostaphin,
zymolase, cellulase, mutanolysin, glycanases, proteases, mannase,
etc.), or a physical condition (e.g., ultrasound, ultraviolet
light, mechanical agitation, etc.). Lysing chemicals may be added,
for example, by merging the droplets with other droplets containing
lysing chemicals, through injection techniques, or the like, for
instance as discussed herein. See also Int. Pat. Apl. Pub. No. WO
2010/151776, incorporated herein by reference in its entirety.
[0028] In some embodiments, nucleic acids within the droplets may
be tagged or "barcoded," e.g., joined to other nucleic acids that
can be used to uniquely identify the nucleic acids. However, this
is optional and in other embodiments, no barcoding step is
required. In some embodiments, one or more "tags" may be present
within or added to a droplet, which can be analyzed or used, for
instance, to determine the identity and/or history of the droplet,
to determine cells within the droplets, to determine nucleic acids
within the droplet, or the like. In some cases, the tags may be
chosen to be relatively inert relative to other components of the
droplet. The tags may be present initially in the droplet, and/or
subsequently added. For instance, tags may be added when the
droplet is exposed to one or more conditions (or proximate in time
to such exposure). In some cases, more than one tag may be present
in a droplet. Non-limiting examples of tagging or barcoding are
discussed in U.S. Pat. Apl. Ser. No. 61/981,123, entitled "Systems
and Methods for Droplet Tagging," by Bernstein, et al., filed Apr.
17, 2014; U.S. Pat. Apl. Ser. No. 61/981,108, entitled "Methods and
Systems for Droplet Tagging and Amplification," by Weitz, et al.,
filed Apr. 17, 2014; or U.S. Pat. Apl. Ser. No. 62/072,950,
entitled "Methods and Systems for Barcoding Nucleic Acids using
Transposons," each incorporated herein by reference in its
entirety.
[0029] After release, the nucleic acids may be amplified within the
droplets in any suitable fashion. Non-limiting examples of suitable
techniques include PCR (polymerase chain reaction) or other
amplification techniques. Typically, in PCR, the nucleic acids are
heated to cause dissociation of the nucleic acids into single
strands, and a heat-stable DNA polymerase (such as Taq polymerase)
is used to amplify the nucleic acid. This process is often repeated
multiple times to amplify the nucleic acids.
[0030] Thus, in one set of embodiments, PCR may be performed within
the droplets. For example, the droplets may contain a polymerase
(such as Taq polymerase), and DNA nucleotides, and the droplets may
be processed (e.g., via repeated heated and cooling) to amplify the
nucleic acid within the droplets. The polymerase and nucleotides
may be added at any suitable point, e.g., before, during, or after
release of nucleic acids from cells within the droplets. For
instance, as a non-limiting example, a droplet containing
polymerase or DNA nucleotides may be fused to a droplet containing
nucleic acids (e.g., arising from a cell) to allow amplification of
the nucleic acids to occur, and/or injection techniques may be used
to introduce polymerase or DNA nucleotides. Those of ordinary skill
in the art will be aware of suitable PCR techniques and variations,
such as assembly PCR or polymerase cycling assembly, which may be
used in some embodiments to produce an amplified nucleic acid.
[0031] The nucleic acids may be amplified to any suitable extent.
The degree of amplification may be controlled, for example, by
controlling factors such as the temperature, cycle time, or amount
of enzyme and/or deoxyribonucleotides contained within the
droplets. For instance, in some embodiments, a population of
droplets may have at least about 10,000, at least about 30,000, at
least about 50,000, at least about 100,000, at least about 150,000,
at least about 200,000, at least about 250,000, at least about
300,000, at least about 400,000, at least about 500,000, at least
about 750,000, at least about 1,000,000 or more molecules of the
amplified nucleic acid per droplet. In some embodiments, the
nucleic acids may be amplified by at least about 10.sup.3-, at
least about 10.sup.4-, at least about 10.sup.5-, at least about
10.sup.6-, or at least about 10.sup.7-fold within a droplet.
[0032] In addition, suitable primers may be used to initiate
polymerization, e.g., P5 and P7, or other primers known to those of
ordinary skill in the art. In some embodiments, primers may be
added to the droplets, or the primers may be present on one or more
of the nucleic acids within the droplets. Those of ordinary skill
in the art will be aware of suitable primers, many of which can be
readily obtained commercially.
[0033] In one set of embodiments, the primers are chosen on the
basis of their ability to selectively amplify certain types of
nucleic acids, relative to other types of nucleic acids. For
example, the primer may selectively bind to and allow amplification
of nucleic acids arising from a target cell, relative to other
nucleic acids from other cells that may be present in a population
of cells. The primers can be chosen on their ability to bind to DNA
or RNA (for example, mRNA) that may be present within the cells.
The primers may, for example, arise from human cells or non-human
cells, e.g., bacterial cells, depending on the application. The
primers may also be specific to one cell or one type of cell, or
the primers may be non-specific, or specific to a class or type of
cell. For instance, the primer may be include non-specific
bacterial primers that are generally able to recognize nucleic
acids from more than one species of bacteria. In addition, more
than one type of primer may be used in certain embodiments. For
example, in one set of embodiments, a plurality of primers able to
bind bacteria, cancer cells, or the like may be used, e.g., to
facilitate amplification of nucleic acids arising from those cells,
relative to other cells in the population. Many such primer "kits"
can be obtained commercially.
[0034] In some embodiments, various PCR reagents may be added to
the droplets (e.g., deoxyribonucleotides, primers, polymerases,
reverse transcriptases, etc.), e.g., via injection or merging with
other droplets, e.g., as discussed herein. In some cases, at least
some of the PCR reagents may be added using other techniques, or at
least some of the PCR reagents may be present within the droplets
at formation (e.g., when the cells are encapsulated within the
droplets. Techniques for merging or coalescing droplets or
injecting a fluid into a droplet are known to those of ordinary
skill in the art, and include those techniques discussed herein.
See, for example, U.S. Pat. Apl. Pub. No. 2012-0132288, entitled
"Fluid Injection," by Weitz, et al., Int. Pat. Apl. Pub. No. WO
2004/091763, entitled "Formation and Control of Fluidic Species,"
by Link et al.; Int. Pat. Apl. Pub. No. WO 2004/002627, entitled
"Method and Apparatus for Fluid Dispersion," by Stone et al., Int.
Pat. Apl. Pub. No. WO 2005/021151, entitled "Electronic Control of
Fluidic Species," by Link et al.; Int. Pat. Apl. Pub. No. WO
2011/056546, entitled "Droplet Creation Techniques," by Weitz, et
al., each incorporated herein by reference in its entirety.
[0035] In addition, in some embodiments, the droplets may be
subjected to temperature changes and/or other conditions in order
to facilitate PCR amplification of nucleic acid with the droplets.
For example, in PCR reactions, the nucleic acids may be heated
(e.g., to a temperature of at least about 50.degree. C., at least
about 70.degree. C., or least about 90.degree. C. in some cases) to
cause dissociation of the nucleic acids into single strands, and a
heat-stable DNA polymerase (such as Taq polymerase) is used to
amplify the nucleic acid. This process is often repeated multiple
times to amplify the nucleic acids. Those of ordinary skill in the
art will be aware of a variety of different PCR techniques that may
be used, such as RT-PCR.
[0036] After amplification, the interiors of the droplets may be
merged or pooled together in some fashion. For example, in some
embodiments, the droplets may be disrupted (or "broken") using
techniques such as mechanical disruption, chemical disruption (for
example, suitable surfactants), and/or ultrasound, e.g., to release
their contents. In another set of embodiments, the droplets may be
merged or coalesced together, for example, using dipole moments or
electric fields. See, e.g., U.S. patent application Ser. No.
11/698,298, filed Jan. 24, 2007, entitled "Fluidic Droplet
Coalescence," by Ahn, et al., published as U.S. Patent Application
Publication No. 2007/0195127 on Aug. 23, 2007, incorporated herein
by reference in its entirety. For example, two droplets can be
given opposite electric charges (i.e., positive and negative
charges, not necessarily of the same magnitude), which can increase
the electrical interaction of the two droplets such that fusion or
coalescence of the droplets can occur due to their opposite
electric charges. For instance, an electric field may be applied to
the droplets, the droplets may be passed through a capacitor, a
chemical reaction may cause the droplets to become charged, etc. In
some embodiments, if the droplets are electrically charged with
opposite charges (which can be, but are not necessarily of, the
same magnitude), the droplets may be able to fuse or coalesce. As
another example, the droplets may not necessarily be given opposite
electric charges (and, in some cases, may not be given any electric
charge), and are fused through the use of dipoles induced in the
droplets that causes the droplets to coalesce.
[0037] After combination, the nucleic acids that are collected
together may be analyzed or determined using any methods known to
those of ordinary skill in the art. For instance, in some
embodiments, the presence (or absence) of nucleic acids arising
from relatively rare target cells may be determined. In some cases,
the concentration may be determined, e.g., to determine the
relative amount or percentage of the target cells in the initial
population. The nucleic acids may also be sequenced in some
embodiments. A variety of techniques may be used, for example,
sequencing techniques such as chain-termination sequencing,
sequencing-by-hybridization, Maxam-Gilbert sequencing,
dye-terminator sequencing, chain-termination methods, Massively
Parallel Signature Sequencing (Lynx Therapeutics), polony
sequencing, pyrosequencing, sequencing by ligation, ion
semiconductor sequencing, DNA nanoball sequencing, single-molecule
real-time sequencing, nanopore sequencing, microfluidic Sanger
sequencing, digital RNA sequencing ("digital RNA-seq"), etc.
[0038] Additional details regarding systems and methods for
manipulating droplets in a microfluidic system follow, in
accordance with certain aspects of the invention. For example,
various systems and methods for screening and/or sorting droplets
are described in U.S. patent application Ser. No. 11/360,845, filed
Feb. 23, 2006, entitled "Electronic Control of Fluidic Species," by
Link, et al., published as U.S. Patent Application Publication No.
2007/000342 on Jan. 4, 2007, incorporated herein by reference. As a
non-limiting example, in some aspects, by applying (or removing) a
first electric field (or a portion thereof), a droplet may be
directed to a first region or channel; by applying (or removing) a
second electric field to the device (or a portion thereof), the
droplet may be directed to a second region or channel; by applying
a third electric field to the device (or a portion thereof), the
droplet may be directed to a third region or channel; etc., where
the electric fields may differ in some way, for example, in
intensity, direction, frequency, duration, etc.
[0039] As mentioned, certain embodiments comprise a droplet
contained within a carrying fluid. For example, there may be a
first phase forming droplets contained within a second phase, where
the surface between the phases comprises one or more proteins. For
example, the second phase may comprise oil or a hydrophobic fluid,
while the first phase may comprise water or another hydrophilic
fluid (or vice versa). It should be understood that a hydrophilic
fluid is a fluid that is substantially miscible in water and does
not show phase separation with water at equilibrium under ambient
conditions (typically 25.degree. C. and 1 atm). Examples of
hydrophilic fluids include, but are not limited to, water and other
aqueous solutions comprising water, such as cell or biological
media, ethanol, salt solutions, saline, blood, etc. In some cases,
the fluid is biocompatible.
[0040] Similarly, a hydrophobic fluid is one that is substantially
immiscible in water and will show phase separation with water at
equilibrium under ambient conditions. As previously discussed, the
hydrophobic fluid is sometimes referred to by those of ordinary
skill in the art as the "oil phase" or simply as an oil.
Non-limiting examples of hydrophobic fluids include oils such as
hydrocarbons oils, silicon oils, fluorocarbon oils, organic
solvents, perfluorinated oils, perfluorocarbons such as
perfluoropolyether, etc. Additional examples of potentially
suitable hydrocarbons include, but are not limited to, light
mineral oil (Sigma), kerosene (Fluka), hexadecane (Sigma), decane
(Sigma), undecane (Sigma), dodecane (Sigma), octane (Sigma),
cyclohexane (Sigma), hexane (Sigma), or the like. Non-limiting
examples of potentially suitable silicone oils include 2 cst
polydimethylsiloxane oil (Sigma). Non-limiting examples of
fluorocarbon oils include FC3283 (3M), FC40 (3M), Krytox GPL
(Dupont), etc. In addition, other hydrophobic entities may be
contained within the hydrophobic fluid in some embodiments.
Non-limiting examples of other hydrophobic entities include drugs,
immunologic adjuvants, or the like.
[0041] Thus, the hydrophobic fluid may be present as a separate
phase from the hydrophilic fluid. In some embodiments, the
hydrophobic fluid may be present as a separate layer, although in
other embodiments, the hydrophobic fluid may be present as
individual fluidic droplets contained within a continuous
hydrophilic fluid, e.g. suspended or dispersed within the
hydrophilic fluid. This is often referred to as an oil/water
emulsion. The droplets may be relatively monodisperse, or be
present in a variety of different sizes, volumes, or average
diameters. In some cases, the droplets may have an overall average
diameter of less than about 1 mm, or other dimensions as discussed
herein. In some cases, a surfactant may be used to stabilize the
hydrophobic droplets within the hydrophilic liquid, for example, to
prevent spontaneous coalescence of the droplets. Non-limiting
examples of surfactants include those discussed in U.S. Pat. Apl.
Pub. No. 2010/0105112, incorporated herein by reference. Other
non-limiting examples of surfactants include Span80 (Sigma),
Span80/Tween-20 (Sigma), Span80/Triton X-100 (Sigma), Abil EM90
(Degussa), Abil we09 (Degussa), polyglycerol polyricinoleate
"PGPR90" (Danisco), Tween-85, 749 Fluid (Dow Corning), the ammonium
carboxylate salt of Krytox 157 FSL (Dupont), the ammonium
carboxylate salt of Krytox 157 FSM (Dupont), or the ammonium
carboxylate salt of Krytox 157 FSH (Dupont). In addition, the
surfactant may be, for example, a peptide surfactant, bovine serum
albumin (BSA), or human serum albumin.
[0042] The droplets may have any suitable shape and/or size. In
some cases, the droplets may be microfluidic, and/or have an
average diameter of less than about 1 mm. For instance, the droplet
may have an average diameter of less than about 1 mm, less than
about 700 micrometers, less than about 500 micrometers, less than
about 300 micrometers, less than about 100 micrometers, less than
about 70 micrometers, less than about 50 micrometers, less than
about 30 micrometers, less than about 10 micrometers, less than
about 5 micrometers, less than about 3 micrometers, less than about
1 micrometer, etc. The average diameter may also be greater than
about 1 micrometer, greater than about 3 micrometers, greater than
about 5 micrometers, greater than about 7 micrometers, greater than
about 10 micrometers, greater than about 30 micrometers, greater
than about 50 micrometers, greater than about 70 micrometers,
greater than about 100 micrometers, greater than about 300
micrometers, greater than about 500 micrometers, greater than about
700 micrometers, or greater than about 1 mm in some cases.
Combinations of any of these are also possible; for example, the
diameter of the droplet may be between about 1 mm and about 100
micrometers. The diameter of a droplet, in a non-spherical droplet,
may be taken as the diameter of a perfect mathematical sphere
having the same volume as the non-spherical droplet.
[0043] In some embodiments, the droplets may be of substantially
the same shape and/or size (i.e., "monodisperse"), or of different
shapes and/or sizes, depending on the particular application. In
some cases, the droplets may have a homogenous distribution of
cross-sectional diameters, i.e., in some embodiments, the droplets
may have a distribution of average diameters such that no more than
about 20%, no more than about 10%, or no more than about 5% of the
droplets may have an average diameter greater than about 120% or
less than about 80%, greater than about 115% or less than about
85%, greater than about 110% or less than about 90%, greater than
about 105% or less than about 95%, greater than about 103% or less
than about 97%, or greater than about 101% or less than about 99%
of the average diameter of the microfluidic droplets. Some
techniques for producing homogenous distributions of
cross-sectional diameters of droplets are disclosed in
International Patent Application No. PCT/US2004/010903, filed Apr.
9, 2004, entitled "Formation and Control of Fluidic Species," by
Link, et al., published as WO 2004/091763 on Oct. 28, 2004,
incorporated herein by reference. In addition, in some instances,
the coefficient of variation of the average diameter of the
droplets may be less than or equal to about 20%, less than or equal
to about 15%, less than or equal to about 10%, less than or equal
to about 5%, less than or equal to about 3%, or less than or equal
to about 1%. However, in other embodiments, the droplets may not
necessarily be substantially monodisperse, and may instead exhibit
a range of different diameters.
[0044] Those of ordinary skill in the art will be able to determine
the average diameter of a population of droplets, for example,
using laser light scattering or other known techniques. The
droplets so formed can be spherical, or non-spherical in certain
cases. The diameter of a droplet, in a non-spherical droplet, may
be taken as the diameter of a perfect mathematical sphere having
the same volume as the non-spherical droplet.
[0045] In some embodiments, one or more droplets may be created
within a channel by creating an electric charge on a fluid
surrounded by a liquid, which may cause the fluid to separate into
individual droplets within the liquid. In some embodiments, an
electric field may be applied to the fluid to cause droplet
formation to occur. The fluid can be present as a series of
individual charged and/or electrically inducible droplets within
the liquid. Electric charge may be created in the fluid within the
liquid using any suitable technique, for example, by placing the
fluid within an electric field (which may be AC, DC, etc.), and/or
causing a reaction to occur that causes the fluid to have an
electric charge.
[0046] The electric field, in some embodiments, is generated from
an electric field generator, i.e., a device or system able to
create an electric field that can be applied to the fluid. The
electric field generator may produce an AC field (i.e., one that
varies periodically with respect to time, for example,
sinusoidally, sawtooth, square, etc.), a DC field (i.e., one that
is constant with respect to time), a pulsed field, etc. Techniques
for producing a suitable electric field (which may be AC, DC, etc.)
are known to those of ordinary skill in the art. For example, in
one embodiment, an electric field is produced by applying voltage
across a pair of electrodes, which may be positioned proximate a
channel such that at least a portion of the electric field
interacts with the channel. The electrodes can be fashioned from
any suitable electrode material or materials known to those of
ordinary skill in the art, including, but not limited to, silver,
gold, copper, carbon, platinum, copper, tungsten, tin, cadmium,
nickel, indium tin oxide ("ITO"), etc., as well as combinations
thereof.
[0047] In another set of embodiments, droplets of fluid can be
created from a fluid surrounded by a liquid within a channel by
altering the channel dimensions in a manner that is able to induce
the fluid to form individual droplets. The channel may, for
example, be a channel that expands relative to the direction of
flow, e.g., such that the fluid does not adhere to the channel
walls and forms individual droplets instead, or a channel that
narrows relative to the direction of flow, e.g., such that the
fluid is forced to coalesce into individual droplets. In some
cases, the channel dimensions may be altered with respect to time
(for example, mechanically or electromechanically, pneumatically,
etc.) in such a manner as to cause the formation of individual
droplets to occur. For example, the channel may be mechanically
contracted ("squeezed") to cause droplet formation, or a fluid
stream may be mechanically disrupted to cause droplet formation,
for example, through the use of moving baffles, rotating blades, or
the like.
[0048] Some embodiments of the invention generally relate to
systems and methods for fusing or coalescing two or more droplets
into one droplet, e.g., where the two or more droplets ordinarily
are unable to fuse or coalesce, for example, due to composition,
surface tension, droplet size, the presence or absence of
surfactants, etc. In certain cases, the surface tension of the
droplets, relative to the size of the droplets, may also prevent
fusion or coalescence of the droplets from occurring.
[0049] As a non-limiting example, two droplets can be given
opposite electric charges (i.e., positive and negative charges, not
necessarily of the same magnitude), which can increase the
electrical interaction of the two droplets such that fusion or
coalescence of the droplets can occur due to their opposite
electric charges. For instance, an electric field may be applied to
the droplets, the droplets may be passed through a capacitor, a
chemical reaction may cause the droplets to become charged, etc.
The droplets, in some cases, may not be able to fuse even if a
surfactant is applied to lower the surface tension of the droplets.
However, if the droplets are electrically charged with opposite
charges (which can be, but are not necessarily of, the same
magnitude), the droplets may be able to fuse or coalesce. As
another example, the droplets may not necessarily be given opposite
electric charges (and, in some cases, may not be given any electric
charge), and are fused through the use of dipoles induced in the
droplets that causes the droplets to coalesce. Also, the two or
more droplets allowed to coalesce are not necessarily required to
meet "head-on." Any angle of contact, so long as at least some
fusion of the droplets initially occurs, is sufficient. See also,
e.g., U.S. patent application Ser. No. 11/698,298, filed Jan. 24,
2007, entitled "Fluidic Droplet Coalescence," by Ahn, et al.,
published as U.S. Patent Application Publication No. 2007/0195127
on Aug. 23, 2007, incorporated herein by reference in its
entirety.
[0050] In one set of embodiments, a fluid may be injected into a
droplet. The fluid may be microinjected into the droplet in some
cases, e.g., using a microneedle or other such device. In other
cases, the fluid may be injected directly into a droplet using a
fluidic channel as the droplet comes into contact with the fluidic
channel. Other techniques of fluid injection are disclosed in,
e.g., International Patent Application No. PCT/US2010/040006, filed
Jun. 25, 2010, entitled "Fluid Injection," by Weitz, et al.,
published as WO 2010/151776 on Dec. 29, 2010; or International
Patent Application No. PCT/US2009/006649, filed Dec. 18, 2009,
entitled "Particle-Assisted Nucleic Acid Sequencing," by Weitz, et
al., published as WO 2010/080134 on Jul. 15, 2010, each
incorporated herein by reference in its entirety.
[0051] The following documents are incorporated herein by reference
in their entirety for all purposes: U.S. Pat. Apl. Ser. No.
62/106,981, entitled "Systems, Methods, and Kits for Amplifying or
Cloning Within Droplets," by Weitz, et al.; U.S. Pat. Apl. Pub. No.
2010-0136544, entitled "Assay and Other Reactions Involving
Droplets," by Agresti, et al.; Int. Pat. Apl. Pub. No. WO
2004/091763, entitled "Formation and Control of Fluidic Species,"
by Link et al.; Int. Pat. Apl. Pub. No. WO 2004/002627, entitled
"Method and Apparatus for Fluid Dispersion," by Stone et al.; Int.
Pat. Apl. Pub. No. WO 2006/096571, entitled "Method and Apparatus
for Forming Multiple Emulsions," by Weitz et al.; Int. Pat. Apl.
Pub. No. WO 2005/021151, entitled "Electronic Control of Fluidic
Species," by Link et al.; Int. Pat. Apl. Pub. No. WO 2011/056546,
entitled "Droplet Creation Techniques," by Weitz, et al.; Int. Pat.
Apl. Pub. No. WO 2010/033200, entitled "Creation of Libraries of
Droplets and Related Species," by Weitz, et al.; U.S. Pat. Apl.
Pub. No. 2012-0132288, entitled "Fluid Injection," by Weitz, et
al.; Int. Pat. Apl. Pub. No. WO 2008/109176, entitled "Assay And
Other Reactions Involving Droplets," by Agresti, et al.; Int. Pat.
Apl. Pub. No. WO 2010/151776, entitled "Fluid Injection," by Weitz,
et al.; U.S. Pat. Apl. Ser. No. 61/981,123, entitled "Systems and
Methods for Droplet Tagging," by Bernstein, et al.; U.S. Pat. Apl.
Ser. No. 61/981,108, entitled "Methods and Systems for Droplet
Tagging and Amplification," by Weitz, et al.; and Int. Pat. Apl.
Pub. No. PCT/US2014/037962, filed May 14, 2014, entitled "Rapid
Production of Droplets," by Weitz, et al.
[0052] Also incorporated herein by reference in its entirety is
U.S. Provisional Patent Application Ser. No. 62/133,140, filed Mar.
13, 2015, entitled "Determination of Cells Using Amplification," by
Weitz, et al.
[0053] The following examples are intended to illustrate certain
embodiments of the present invention, but do not exemplify the full
scope of the invention.
Example 1
[0054] This example demonstrates a two-step drop-based
amplification strategy to overcome detection errors often seen in
other amplification methods, in accordance with some embodiments of
the invention. In the first-step, single cells are encapsulated in
droplets (e.g., microfluidic droplets), and amplification of
nucleic acids is performed within the droplets. This amplification
procedure is similar to other amplification techniques, but the
goal is to amplify the targets from a single cell rather than
actual detection. The resulting PCR amplicons or amplified nucleic
acid are then pooled together, and used as a template for the
second-step, which includes determination of the amplified nucleic
acids, e.g., by digital PCR, counting, etc. Expanding the original
single cell by a high degree (for example, about 10.sup.3 to
10.sup.7 amplicons per target gene) results in several potential
advantages, including statistically-valid results, noise reduction,
or the ability to divide the sample into different portions (e.g.,
for different subsequent analyses), e.g., without losing the
coverage.
[0055] This example demonstrates an application of this two-step
drop-based amplification strategy to detect and quantify very rare
drug-resistance cells carrying PLCG1 mutations from a leukemia
patient's blood. As estimated by a mathematic model, the mutation
rate is around one in a million. Mutation-specific primers were
designed and used to first amplify PLCG1 genes in both mutant and
wild-type cells in bulk. By running agarose gel electrophoresis, it
was observed that only PLCG1 gene in mutant cells could be
amplified. The cells were then encapsualted into drops with lysis
buffer, followed by injecting RT-PCR reagent and RT-PCR. In the
drop detection results of the mutant cells, two populations were
observed: one with low fluorescence intensity indicates the empty
drops, and the other with high fluorescence intensity indicates the
drops containing PLCG1 mutant cells. In the wild-type cell sample,
0.1% bright drops were observed, even though in theory, no bright
drops should have been observed Therefore, after the first-step of
amplification, it would not be possible to accurately quantify
mutant cells if the mutation rate is one in a million.
[0056] All of the amplicons were polled together, and a second-step
gene-specific digital PCR was performed. The drops in which
amplification occur contained about 10.sup.7 amplicons, while the
"noise" drops contained unspecific amplicons or nothing, so the
difference between mutant and wild-type cells has been enlarged
from 1:0 to 10.sup.7:0. Thus, these results are more statistically
significant, rather than stochastic. To be able to accurately
quantify the number of mutant cells, a serial dilution was prepared
by mixing the mutant and wild-type cells at different ratios down
to 1:10.sup.6. After two-step amplification, a standard curve was
established and used to observe a linear correlation between the
dilution factor and cell number. Using this standard curve, mutant
cells in real clinical samples could be quantified.
Example 2
[0057] This example evaluates antimicrobial drug efficacy in
patients with infectious diseases. One of the most important
markers for studying their efficacy is gene up-regulation or
down-regulation. By comparing the expression level (mRNA) of
certain genes in the presence or absence of drugs, the
effectiveness of the drug on a microbe can be determined.
[0058] However, in the detection of certain gene expression, early
stage gene expression of infection pathogen concentration is
usually very low, and mRNA in bacteria is also very low, about 10
mRNAs per gene. In addition, the primers that should be used for
amplification often aren't known because it is not known which
pathogen exists in a sample, out of many possible pathogen
candidates. However, in this example, all pathogen candidate
primers are first used to amplify all candidate bacteria mRNA
fragments in the sample, and then the samples are divided and
digital PCR mixtures prepared for individual candidates for
detection. In some cases, each digital PCR mixture can be barcoded,
e.g., with fluorescence dye, for simultaneous detection.
Example 3
[0059] Following are example material and methods useful for
certain embodiments of the invention.
[0060] Detection of rare mutant cells with the presence of host
cells. Preparation of cells expressing mutant and wild-type target
gene. A stable mouse leukemia cell line, 30019, that expresses
wild-type and mutant Phospholipase C (PLCG-1) gene were maintained
in RPMI-1640 supplemented with 10% low-endotoxin fetal calf serum,
100 U penicillin/ml, 100 micrograms/ml streptomycin, 15
micrograms/ml gentamycin, 1% glutamine, 50.times.10.sup.-6 M
2-mercaptoethanol, and 1000 ng/ml G418. These cells were harvested
and counted before they were co-flowed into the microfluidic device
for single-cell analysis. The 30019 cells were mixed with white
blood cells at 1:1,000, 1:10,000, 1:100,000, and 1:1,000,000
ratios, respectively.
[0061] Preparation of microfluidic devices. Polydimethylsiloxane
(PDMS) microfluidic devices were fabricated using standard soft
lithographic methods. The microfluidic channel walls are rendered
hydrophobic by treating them with Aquapel (PPG, Pittsburgh,
Pa.).
[0062] Preparation of a 2.times.cell lysis buffer and a
2.times.RT-PCR cocktail. The cell lysis buffer contained 1 M
Tris-HCl pH 8.0, 10% Tween 20, 100 mg/ml proteinase K and 2,000
U/ml DNase I. Primers for amplifying Phospholipase C (PLCG-1) gene
were bought from IDT, which were
F-5'-GGGTAAGTGGCATGAGCAAGAAAGAACC-3' and R-5'-TTTCTGCGCTTTGTGG
TTTATGAA-3'. The Taqman probe was purchased from Life Tech, and its
sequence was 5'-FAM-ACACAGGAGAAGGTGACATTTGAA-3'-MGB. The 50
microliter 2.times.RT-PCR cocktails contained 4 microliters of
OneStep RT-PCR enzyme mixed with 2.times.OneStep RT-PCR buffer from
Qiagen, 800 micromolar dNTPs, 0.6 micromolar forward and reverse
primers, 0.5 micromolar Taqman probe, 0.4 microgram/microliter BSA,
and 0.4% Tween 20.
[0063] Generation of monodisperse aqueous drops containing cells
and lysis of cells. A microfluidic chip was used that contained a
co-flow drop maker with a cross section of 35 micrometer.sup.2 to
generate 50 micrometer (diameter) monodisperse aqueous drops in
fluorinated oil, HFE-7500 (3M, Saint Paul, Minn., U.S.A),
containing 2% (w/w) Krytox-PEG diblock co-polymer surfactant (RAN
Biotech, Beverly, Mass.). The cell lysis buffer and different cell
mixtures were encapsulated in drops via co-flow in different
channels at a 1:1 ratio. The drops were collected in a PCR tube and
covered with mineral oil. To lyse the cells within these drops, the
following protocol was used: 37.degree. C. for 10 min, 50.degree.
C. for 20 min, 70.degree. C. for 10 min, then the drops containing
lysed cells were kept on ice.
[0064] Picoinjection of 2.times.RT-PCR reagent and RT-PCR. The
drops containing lysed cells were flowed into a microfluidic
pico-injection device and a 2.times.RT-PCR cocktail was injected
into the drop by electro-coalescence. See, e.g., U.S. Pat. Apl.
Pub. No. 2012/0132288, incorporated herein by reference in its
entirety. The drops were spaced on chip by oil with 2% w/w
surfactant. The device electrodes were connected to a high voltage
TREK 2210 amplifier (TREK, Lockport, NY) which supplied a 100 V
sine wave at a frequency of 25 kHz. The flow rate of the PCR
cocktail was chosen to ensure that the buffer was added at -1:1
ratio upon coalescence. Typical flow rates fulfilling these
requirements were 300 microliters/hr for the oil, 60 microliters/hr
for the barcode primer-drops, and 30 microliters/hr for the PCR
cocktail. The drops were collected in PCR tubes and covered with
mineral oil to prevent evaporation. The following RT-PCR protocol
was used: 50.degree. C. for 30 min, 95.degree. C. for 10 min, 2
cycles of 94.degree. C. for 15 s, 64.degree. C. for 8 min, and 38
cycles of 95.degree. C. for 15 s, 62.degree. C. for 1 min.
[0065] Second-round digital PCR. To obtain the templates for the
second-round digital PCR, 25 microliters of
1H,1H,2H,2H-perfluoro-1-octanol (PFO; Sigma-Aldrich, St. Louis,
Mo.) was added to the emulsion and gently centrifuged to separate
the phases. PCR products from the first-round RT-PCR were all in
the liquid phase. The PCR products were diluted by 1,000 fold and
used for the second-round digital PCR. The 25 microliter PCR
cocktail contained 1 microliter of OneStep RT-PCR enzyme mix with
1.times.OneStep RT-PCR buffer from Qiagen, 400 micromolar dNTPs,
0.25 micromolar forward and reverse primers, 0.24 micromolar Taqman
probe, 0.2 micrograms/microliter BSA, 0.2% Tween 20, and 1
microliter of diluted PCR products. A microfluidic device
containing a flow-focusing drop maker with a cross section of 15
micrometers.times.25 micrometers was used to generate 25 micrometer
monodisperse aqueous drops in HFE-7500 containing 2% (w/w)
surfactant. The flow was driven by applying a -0.4 psi vacuum at
the outlet (1 psi=6895 Pa). The drops were collected in a PCR tube
and covered with mineral oil to prevent evaporation. The following
RT-PCR protocol was used: 95.degree. C. for 10 min, 40 cycles of 2
cycles of 94.degree. C. for 15 s, 64.degree. C. for 8 min, and 38
cycles of 95.degree. C. for 15 s, 62.degree. C. for 1 min.
[0066] Fluorescence detection of drops after second-round digital
PCR. After amplification the drops were re-injected into the
microfluidic reading and sorting device. To achieve a stream of
evenly spaced drops for detection, flows were combined from the
drops and HFE-7500 oil with 1% surfactant in a "T" junction, where
the flow rate of the drops was 15 microliters/h and that of the oil
was 180 microliters/h. This stream flowed through a 25
micrometer.times.25 micrometer channel, and as the drops passed the
focal point of an excitation laser (488 nm), their fluorescence was
collected by a microscope objective and focused onto a
photomultiplier tube (PMT) from Hammamatsu. The pulses were
acquired by a real-time field-programmable gate array card from
National Instruments and recorded by a LabView program and analyzed
using a MATLAB code. The pulse height was used as a measure of drop
fluorescence. The pulse width, which was the duration of time for a
drop to pass through the laser, was used as a measure of drop size.
The sensitivity of the PMT was sufficiently high to detect drops
not containing target templates, due to the intrinsic fluorescence
of the Taqman probe.
[0067] Quantification of mutant cells in the sample. To quantify
the mutant cells in the original sample, the fluorescence detection
results from these cell mixture samples were used to establish a
standard curve. The X axis was the number of the mutant cells in
the cell mixture, and the Y axis was the number of bright drops
after second-round digital PCR. When the mutant cells in an unknown
sample were quantified, first-round RT-PCR and second-round PCR
were performed to get the number of bright drops, thereby obtaining
the corresponding number of the mutant cells.
[0068] Evaluation of antimicrobial drug efficacy in patients with
infectious diseases. Purify bacteria RNA from blood. Bacteria RNAs
were purified from blood with and without antibiotic treatment
using a Qiagen blood RNA kit, with some modification. Briefly,
protection buffer was added to prevent the bacteria expression
profile from being changed during the purification procedure. Then,
the red blood cells were lysed and centrifuged to collect the
bacteria and white blood cells. To lyse the bacteria and white
blood cells, proteinase K was added to the cell pellet and
incubated at room temperature for 10 min, followed by adding RLT
lysis buffer in the kit. The addition of proteinase K also
prevented the bacteria RNA from being digested by cellular RNase.
The cell lysate was loaded onto a Qiagen Column, so that the RNA
could bind to the silicon membrane. After two steps of washing, the
RNA was eluted into DEPC water and frozen at -80.degree. C.
[0069] First-round digital RT-PCR. A 25 microliter PCR cocktail
containing 1 microliter of OneStep RT-PCR enzyme was mixed with
1.times.OneStep RT-PCR buffer from Qiagen, 400 micromolar dNTPs,
0.01 micromolar each forward and reverse primers for amplifying
total 48 different bacteria, 0.24 micromolar Taqman probe, 0.2
microgram/microliter BSA, 0.2% Tween 20, and 5 microliter of
purified bacteria RNA. A microfluidic device containing a
flow-focusing drop maker with a cross section of 25
micrometer.times.25 micrometer was used to generate 35 micrometer
monodisperse aqueous drops in HFE-7500 containing 2% (w/w)
surfactant. The flow was driven by applying a -0.4 psi vacuum at
the outlet. The drops were collected in a PCR tube and covered with
mineral oil to prevent evaporation. The following RT-PCR protocol
was used: 50.degree. C. for 20 min, 95.degree. C. for 10 min, 40
cycles of 95.degree. C. for 30 s, 60.degree. C. for 5 min and
72.degree. C. 1 min.
[0070] Second-round multiplex digital PCR to quantify the bacteria
mRNA. To obtain the templates for the second-round digital PCR, 25
microliters of 1H,1H,2H,2H-perfluoro-1-octanol (PFO; Sigma-Aldrich,
St. Louis, Mo.) was added to the emulsion and gently centrifuged to
separate the phases. PCR products from the first-round RT-PCR were
all in the liquid phase. In total, there were 48 PCR cocktails for
48 different genes which belong to 48 different bacteria. Each 25
microliter PCR cocktail contained 1 microliter of OneStep RT-PCR
enzyme mixed with 1.times.OneStep RT-PCR buffer from Qiagen, 400
micromolar dNTPs, 0.25 micromolar each forward and reverse primers,
0.24 micromolar Taqman probe, 0.2 microgram/microliter BSA, 0.2%
Tween 20, and 1 microliter of purified bacteria RNA. To be able to
distinguish every reaction, a combination of two fluorescence dyes,
Texas red and Alexa 680, was added at different concentrations to
each cocktail as its unique barcode. A 48-parallel drop maker was
then used to generate drops from all PCR cocktails simultaneously
in a pressure-driven chamber. See, e.g., Int. Pat. Apl. Pub. No. WO
2014/186440, published on Nov. 20, 2014, incorporated herein by
reference in its entirety. The resulting drops were collected in a
PCR tube and covered with mineral oil to prevent evaporation. The
following RT-PCR protocol was used: 50.degree. C. for 20 min,
95.degree. C. for 10 min, 40 cycles of 95.degree. C. for 30 s,
60.degree. C. for 5 min and 72.degree. C. 1 min.
[0071] Fluorescence detection of drops after second-round digital
PCR. After amplification, the drops were re-injected into the
microfluidic reading and sorting device. To achieve a stream of
evenly spaced drops for detection, flows from the drops and
HFE-7500 oil with 1% surfactant were combined in a "T" junction,
where the flow rate of the drops was 15 microliters/h and that of
the oil was 180 microliters/h. This stream flowed through a 25
micrometer.times.25 micrometer channel, and the drops passed the
focal point of the excitation laser (488 nm). The fluorescence of
each drop was collected by a microscope objective and focused onto
a photomultiplier tube (PMT) from Hammamatsu. The pulses were
acquired by a real-time field-programmable gate array card from
National Instruments and recorded by a LabView program and analyzed
using MATLAB code. From each drop, three colors were detected:
green, Taqman probe signal; red, Texas red; far red, Alexa 680,
where red and far red correspond to a certain gene specific primer
pair. See, e.g., Table 1.
[0072] Evaluation of antimicrobial drug efficacy. To evaluate the
antimicrobial drug's efficacy, the number of gene specific mRNA
from the sample, with and without antibiotic treatment, was
detected, and the numbers between these two samples were
compared.
TABLE-US-00001 TABLE 1 Primers for amplifying bacteria RNA
ECHS_A2938_QUEF F-5'-GCCTCTGTTCGTCTCGACATC-3'
R-5'-ACCACGTTTTCGCCCTCTTT-3' Taqman: FAM-CACCGCGCCGATACG-MGB
ECHS_A2835_RECA F-5'-GACGCGTTTTAATAACTGGGATGAG
R-5-GCAGAAGCGTAACAGGTCATTAAAG Taqman: FAM-CTGGAGCGCGACTTAA-MGB
ECHS_A0486_RIBH F-5'-GCACTGCCCACTTTGAATATGTC
R-5'-GCTGTCCTGGGCAACATG Taqman: FAM-CCAGGCCGTTGCTTG-MGB
Example 4
[0073] For the detection of rarer populations of cells, such as one
in a million cells, this example uses two rounds of drop-based
digital PCR to perform detection. After the first round of
drop-based digital PCR, pooled mutated amplicons are
re-encapsulated using a Poisson distribution to ensure <30% of
droplets contain templates, are subjected to digital PCR and the
bright droplets are counted by fluorescence detection (FIG. 2).
[0074] Using two rounds of drop-based digital PCR, a standard curve
is generated from PBMC spiked with known numbers of cells from the
30019 cell line, engineered to stably express mutated PLCG2-M1141R,
and reliable detection of 1 in 10.sup.4, 10.sup.5 and 10.sup.6
cells with the PLCG2 mutation was observed, compared to 10.sup.6
cells without the mutation, or the negative water control (FIG.
3A).
[0075] In this fashion, 1 in 500,000 pretreatment cells of Patient
1 were detected with mutated PLCG2-M1141R, of similar order of
magnitude as mathematical calculations (FIG. 3B). Altogether, these
results confirm that pretreatment samples carry the capacity to
contain resistant subclones prior to the initiation of targeted
inhibition of BTK, albeit at rare frequencies.
[0076] The data in FIG. 3A were generated as follows:
[0077] 1. Cells carrying wild-type PLCG2-M1141R and mutant
PLCG2-M1141R were mixed at 10,000:1, 100,000:1 and 1,000,000:1
ratio, respectively.
[0078] 2. The cell mixture was encapsulated with a RT-PCR cocktail
into drops and RT-PCR performed. The primers used for RT-PCR are
mutation specific primers. The number of the cells encapsulated
into drops is 3,000,000 in total per sample, which means there are
300, 30, and 3 cells in each sample.
[0079] 3. The emulsions were broken, e.g., with a surfactant. The
amplicons were collected and re-emulsified the amplicons with a PCR
cocktail into drops. The primers used for PCR are also mutation
specific primers. After amplification, droplets were counted.
[0080] 4. From the counting result, the number of amplicons from
the cells originally added were calculated.
[0081] 5. A standard curve was prepared and used to show that there
is a linear correlation between the number of amplicons generated
(Y axis) and the number of cells that were added (X axis). This
could be used to determine how many amplicons were generated from a
single mutant cell.
[0082] For FIG. 3B, with a real sample and CLL pretreatment, steps
1-4 were used. This could be used to determine how many amplicons
were generated in total. Finally, this total number divided by the
number of amplicons one mutant cell can generate was used to
determine how many mutant cells were in the sample. For example, if
there are 6 mutant cells in 3,000,000 cells, the ratio of mutant to
wild-type is 1:500,000, shown in FIG. 3B as a number (0.0002%) and
FIG. 3A as a data point on the standard curve. In contrast, the
PBMC control did not show mutant amplicons.
[0083] While several embodiments of the present invention have been
described and illustrated herein, those of ordinary skill in the
art will readily envision a variety of other means and/or
structures for performing the functions and/or obtaining the
results and/or one or more of the advantages described herein, and
each of such variations and/or modifications is deemed to be within
the scope of the present invention. More generally, those skilled
in the art will readily appreciate that all parameters, dimensions,
materials, and configurations described herein are meant to be
exemplary and that the actual parameters, dimensions, materials,
and/or configurations will depend upon the specific application or
applications for which the teachings of the present invention
is/are used. Those skilled in the art will recognize, or be able to
ascertain using no more than routine experimentation, many
equivalents to the specific embodiments of the invention described
herein. It is, therefore, to be understood that the foregoing
embodiments are presented by way of example only and that, within
the scope of the appended claims and equivalents thereto, the
invention may be practiced otherwise than as specifically described
and claimed. The present invention is directed to each individual
feature, system, article, material, kit, and/or method described
herein. In addition, any combination of two or more such features,
systems, articles, materials, kits, and/or methods, if such
features, systems, articles, materials, kits, and/or methods are
not mutually inconsistent, is included within the scope of the
present invention.
[0084] All definitions, as defined and used herein, should be
understood to control over dictionary definitions, definitions in
documents incorporated by reference, and/or ordinary meanings of
the defined terms.
[0085] The indefinite articles "a" and "an," as used herein in the
specification and in the claims, unless clearly indicated to the
contrary, should be understood to mean "at least one."
[0086] The phrase "and/or," as used herein in the specification and
in the claims, should be understood to mean "either or both" of the
elements so conjoined, i.e., elements that are conjunctively
present in some cases and disjunctively present in other cases.
Multiple elements listed with "and/or" should be construed in the
same fashion, i.e., "one or more" of the elements so conjoined.
Other elements may optionally be present other than the elements
specifically identified by the "and/or" clause, whether related or
unrelated to those elements specifically identified. Thus, as a
non-limiting example, a reference to "A and/or B", when used in
conjunction with open-ended language such as "comprising" can
refer, in one embodiment, to A only (optionally including elements
other than B); in another embodiment, to B only (optionally
including elements other than A); in yet another embodiment, to
both A and B (optionally including other elements); etc.
[0087] As used herein in the specification and in the claims, "or"
should be understood to have the same meaning as "and/or" as
defined above. For example, when separating items in a list, "or"
or "and/or" shall be interpreted as being inclusive, i.e., the
inclusion of at least one, but also including more than one, of a
number or list of elements, and, optionally, additional unlisted
items. Only terms clearly indicated to the contrary, such as "only
one of" or "exactly one of," or, when used in the claims,
"consisting of," will refer to the inclusion of exactly one element
of a number or list of elements. In general, the term "or" as used
herein shall only be interpreted as indicating exclusive
alternatives (i.e. "one or the other but not both") when preceded
by terms of exclusivity, such as "either," "one of," "only one of,"
or "exactly one of." "Consisting essentially of," when used in the
claims, shall have its ordinary meaning as used in the field of
patent law.
[0088] As used herein in the specification and in the claims, the
phrase "at least one," in reference to a list of one or more
elements, should be understood to mean at least one element
selected from any one or more of the elements in the list of
elements, but not necessarily including at least one of each and
every element specifically listed within the list of elements and
not excluding any combinations of elements in the list of elements.
This definition also allows that elements may optionally be present
other than the elements specifically identified within the list of
elements to which the phrase "at least one" refers, whether related
or unrelated to those elements specifically identified. Thus, as a
non-limiting example, "at least one of A and B" (or, equivalently,
"at least one of A or B," or, equivalently "at least one of A
and/or B") can refer, in one embodiment, to at least one,
optionally including more than one, A, with no B present (and
optionally including elements other than B); in another embodiment,
to at least one, optionally including more than one, B, with no A
present (and optionally including elements other than A); in yet
another embodiment, to at least one, optionally including more than
one, A, and at least one, optionally including more than one, B
(and optionally including other elements); etc.
[0089] When the word "about" is used herein in reference to a
number, it should be understood that still another embodiment of
the invention includes that number not modified by the presence of
the word "about."
[0090] It should also be understood that, unless clearly indicated
to the contrary, in any methods claimed herein that include more
than one step or act, the order of the steps or acts of the method
is not necessarily limited to the order in which the steps or acts
of the method are recited.
[0091] In the claims, as well as in the specification above, all
transitional phrases such as "comprising," "including," "carrying,"
"having," "containing," "involving," "holding," "composed of," and
the like are to be understood to be open-ended, i.e., to mean
including but not limited to. Only the transitional phrases
"consisting of" and "consisting essentially of" shall be closed or
semi-closed transitional phrases, respectively, as set forth in the
United States Patent Office Manual of Patent Examining Procedures,
Section 2111.03.
Sequence CWU 1
1
12128DNAArtificial SequenceSynthetic Polynucleotide 1gggtaagtgg
catgagcaag aaagaacc 28224DNAArtificial SequenceSynthetic
Polynucleotide 2tttctgcgct ttgtggttta tgaa 24324DNAArtificial
SequenceSynthetic Polynucleotide 3acacaggaga aggtgacatt tgaa
24421DNAArtificial SequenceSynthetic Polynucleotide 4gcctctgttc
gtctcgacat c 21520DNAArtificial SequenceSynthetic Polynucleotide
5accacgtttt cgccctcttt 20615DNAArtificial SequenceSynthetic
Polynucleotide 6caccgcgccg atacg 15725DNAArtificial
SequenceSynthetic Polynucleotide 7gacgcgtttt aataactggg atgag
25825DNAArtificial SequenceSynthetic Polynucleotide 8gcagaagcgt
aacaggtcat taaag 25916DNAArtificial SequenceSynthetic
Polynucleotide 9ctggagcgcg acttaa 161023DNAArtificial
SequenceSynthetic Polynucleotide 10gcactgccca ctttgaatat gtc
231118DNAArtificial SequenceSynthetic Polynucleotide 11gctgtcctgg
gcaacatg 181215DNAArtificial SequenceSynthetic Polynucleotide
12ccaggccgtt gcttg 15
* * * * *