U.S. patent application number 17/385853 was filed with the patent office on 2022-04-21 for systems and methods for performing digital pcr.
This patent application is currently assigned to LIFE TECHNOLOGIES CORPORATION. The applicant listed for this patent is LIFE TECHNOLOGIES CORPORATION. Invention is credited to David N. Keys, Nivedita Sumi Majumdar, Theodore E. Straub.
Application Number | 20220119859 17/385853 |
Document ID | / |
Family ID | 1000006062238 |
Filed Date | 2022-04-21 |
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United States Patent
Application |
20220119859 |
Kind Code |
A1 |
Keys; David N. ; et
al. |
April 21, 2022 |
SYSTEMS AND METHODS FOR PERFORMING DIGITAL PCR
Abstract
Systems and methods are described for quantifying a target
nucleic acid. A sample comprising a target nucleic acid is
segregated into a first plurality of the reaction volumes
containing at least one target nucleic acid molecule and a second
plurality of the reaction volumes containing no target nucleic acid
molecules. The reaction volumes are subjected to an amplification
assay, wherein the amplification assay is configured to amplify the
target nucleic acid. An indicator of amplification is detected or
measured in at least some of the plurality of reaction volumes. The
target nucleic acid is quantified based on the detection or
measurement. After discontinuing the amplification assay, the
plurality of reaction volumes may be heated and changes in the
indicators of amplification of two or more of the at least some of
the reaction volumes may be detected or measured.
Inventors: |
Keys; David N.; (Alameda,
CA) ; Majumdar; Nivedita Sumi; (San Bruno, CA)
; Straub; Theodore E.; (Lexington, MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
LIFE TECHNOLOGIES CORPORATION |
Carlsbad |
CA |
US |
|
|
Assignee: |
LIFE TECHNOLOGIES
CORPORATION
Carlsbad
CA
|
Family ID: |
1000006062238 |
Appl. No.: |
17/385853 |
Filed: |
July 26, 2021 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15280160 |
Sep 29, 2016 |
11085074 |
|
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17385853 |
|
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62234158 |
Sep 29, 2015 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
B01L 2300/024 20130101;
C12Q 1/686 20130101; C12Q 1/6851 20130101; B01L 2300/0829 20130101;
B01L 7/52 20130101 |
International
Class: |
C12Q 1/686 20060101
C12Q001/686; C12Q 1/6851 20060101 C12Q001/6851 |
Claims
1.-39. (canceled)
40. A system for detecting or quantifying a nucleic acid in a
sample, the system comprising: a reaction device; an electronic
processor; a memory comprising instructions for performing steps
comprising: segregating a sample comprising a target nucleic acid
into a plurality of sample reaction volumes, wherein the plurality
of sample reaction volumes include a first plurality of the sample
reaction volumes each containing at least one molecule of the
target nucleic acid and a second plurality of the sample reaction
volumes each containing no molecules of the target nucleic acid;
subjecting the plurality of sample reaction volumes to an
amplification assay, wherein the amplification assay is configured
to amplify the target nucleic acid; detecting or measuring
indicators of amplification for at least some of the sample
reaction volumes of the plurality of sample reaction volumes;
discontinuing the amplification assay; and after discontinuing the
amplification assay, heating the plurality of sample reaction
volumes and detecting or measuring changes in the indicators of
amplification for two or more of the at least some of the sample
reaction volumes; and an input/output device comprising an input
device and a display; wherein the electronic processor performs the
instructions and displays an amount of the target nucleic acid on
the display.
41. The system of claim 40, wherein the memory is a random access
memory.
42. A system for detecting or quantifying a nucleic acid in a
sample, the system comprising: an electronic processor; a memory
comprising instructions for performing steps comprising:
segregating a sample comprising a target nucleic acid into a
plurality of sample reaction volumes, wherein the plurality of
sample reaction volumes include a first plurality of the sample
reaction volumes each containing at least one molecule of the
target nucleic acid and a second plurality of the sample reaction
volumes each containing no molecules of the target nucleic acid;
subjecting the plurality of sample reaction volumes to an
amplification assay, wherein the amplification assay is configured
to amplify the target nucleic acid; detecting or measuring
indicators of amplification for at least some of the sample
reaction volumes of the plurality of sample reaction volumes;
discontinuing the amplification assay; and after discontinuing the
amplification assay, heating the plurality of sample reaction
volumes and detecting or measuring changes in the indicators of
amplification for two or more of the at least some of the sample
reaction volumes; and an input/output device comprising an input
device and a display; wherein the electronic processor performs the
instructions and displays information regarding a detection of the
target nucleic acid on the display.
43. The system of claim 42, wherein the memory is a random access
memory.
44. A system for detecting or quantifying a nucleic acid in a
sample, the system comprising: a reaction device configured to
contain or provide a plurality of sample reaction volumes from a
sample solution containing a nucleic acid; an electronic processor;
a temperature controller; a memory comprising instructions for (1)
detecting or measuring an indicator of amplification for at least
some of the sample reaction volumes, (2) heating the plurality of
sample reaction volumes, and (3) detecting or measuring a change in
the indicator of amplification for one or more of the at least some
of the sample reaction volumes; an output device comprising a
display; wherein the electronic processor performs the instructions
and displays at least one of (1) a detection and/or an amount of
the indicator of amplification and/or (2) the change in the
indicator of amplification of one or more of the at least some of
the sample reaction volumes.
45. The system of claim 44, wherein the memory is a random access
memory.
46. The method of claim 1, wherein detecting or measuring
indicators of amplification comprises detecting or measuring one
indicator of amplification for each of the at least some of the
sample reaction volumes.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of priority of U.S.
Provisional Patent Application No. 62/234,158, filed on Sep. 29,
2015, which is incorporated herein in its entirety by
reference.
INTRODUCTION
[0002] Digital PCR (dPCR) is a refinement of conventional
polymerase chain reaction (PCR) methods which can be used to
directly quantify and clonally amplify nucleic acids (including
DNA, cDNA, methylated DNA, RNA, or the like). One difference
between dPCR and traditional PCR lays in the method of measuring
nucleic acids amounts. In dPCR, a sample is separated into a large
number of individual sample volumes or portions and respective PCR
reactions are carried out in each sample portion individually. This
separation allows for sensitive measurement of very small amounts
of a nucleic acid. dPCR has been demonstrated as useful for
studying variations in gene sequences, such as copy number
variation or point mutations.
[0003] In dPCR, a sample is partitioned so that individual nucleic
acid molecules to be assessed within the sample are localized and
concentrated within many separate regions. While the starting
number of copies of a molecule is proportional to the number of
amplification cycles in conventional PCR, dPCR does not dependent
determining a number of amplification cycles to determine the
initial sample amount. Instead, the initial sample is partitioned
into a large number of relatively small sample portions containing
one copy, or approximately one copy, or no copy of the nucleic acid
template or target. As a result, each partitioned sample portion
may be characterized as a "0" or "1" for containing at least one of
a type of target nucleic acid molecule, resulting in a negative
("0") or positive ("1") PCR reaction, respectively. The
partitioning of the sample in this way may use Poisson statistics
to provide an estimate of molecules in the initial sample. However,
the accuracy of this estimate varies, depend on the number of "0"
and "1" produced.
[0004] There exists a need to improve upon the information and data
obtained during dPCR, and the analysis based upon such information,
so as to enhance the accuracy of the results obtained from dPCR.
For instance, techniques for differentiating between partitioned
samples that initially include a single sample cell and those
containing more than one sample cell may provide more accurate dPCR
results.
SUMMARY
[0005] Embodiments of the present invention are generally directed
to systems and methods for quantifying one or mo nucleic acids. In
certain embodiments, a sample or reaction solution is segregated,
distributed, or divided into a plurality of sample reaction volumes
or reaction sites associated with a sample reaction device, fluidic
device, sample holder, or other such device. The plurality of
sample reaction volumes may include a first, plurality of the
sample reaction volumes or reaction sites each containing one
molecule, or approximately one molecule, of a target nucleic acid
and a second plurality of the sample reaction volumes each
containing no molecules of the target nucleic acid. The plurality
of sample reaction volumes or reaction sites are subjected to an
amplification assay using, for example, at least a primer and a
probe or indicator dye, wherein the amplification assay is
configured to amplify the target nucleic acid. An indicator of
amplification presented by the target nucleic acid present in any
of the plurality of sample reaction volumes may be detected or
measured during the amplification assay. After the amplification
assay is discontinued, the plurality of sample reaction volumes may
be further processed, for example, by heating the sample reaction
volumes and detecting or measuring changes in the indicator from
the reaction volumes. In some embodiments, the indicator may also
be detected or measured during one or more cycles of the
amplification assay. A sample reaction volume may include a
segregated sample (e.g., nucleic acid sample) and one or more
reagents for supporting an amplification reaction. The one or more
reagents may be incorporated into the sample either before or after
loading the sample into the reaction volumes or reaction sites.
[0006] In certain embodiments, after the amplification assay is
completed, a first detection or measurement may be taken of an
indicator of amplification at a first temperature for at least some
of the plurality of the sample reaction volumes, wherein the
indicator provides an indication of the existence and/or amount of
the amplified product. Optionally, one or more additional
detections or measurements of the indicator may be taken after the
amplification assay completed at one or more additional
temperatures that are different than the first temperature, for
example, at one or mare temperatures that are higher than the first
temperature. Optionally, one or more detections or measurements of
the indicator may also be taken during one or more cycles of the
amplification assay, for example, at a same predetermined assay
temperature in two or more cycles of the amplification, wherein the
assay temperature may be higher than the first temperature.
[0007] Additional objects, features, and/or advantages will be set
forth in part in the description which follows, and in part will be
obvious from the description, or may be learned by practice of the
present disclosure and/or claims. At least sonic of these objects
and advantages may be realized and attained by the elements and
combinations particularly pointed out in the appended claims.
[0008] It is to be understood that both the foregoing general
description and the following detailed description are exemplary
and explanatory only and are not restrictive of the claims; rather
the claims should be entitled to their full breadth of scope,
including equivalents.
BRIEF DESCRIPTION OF THE DRAWINGS
[0009] The present disclosure can be understood from the following
detailed description, either alone or together with the
accompanying drawings. The drawings are included to provide a
further understanding of the present disclosure, and are
incorporated in and constitute a part of this specification. The
drawings illustrate one or more exemplary embodiments of the
present teachings and together with the description serve to
explain certain principles and operation.
[0010] FIG. 1A illustrates a system according to an embodiment of
the present invention.
[0011] FIG. 1B illustrates exemplary techniques useful for
detecting amplification of a target nucleic acid according to
embodiments of the current invention.
[0012] FIGS. 2A-2B illustrate exemplary methods of performing
amplification that include a melt stage.
[0013] FIGS. 3A-3D illustrate exemplary methods of performing
amplification with detection techniques.
[0014] FIG. 4 illustrates a graph of illustrative, prophetic
exemplary amplification detection data taken at interval
temperatures.
[0015] FIG. 5 illustrates a graph of illustrative, prophetic
exemplary real time amplification detection measurements.
[0016] FIGS. 6A and 6B illustrate a graph of illustrative,
prophetic exemplary real time amplification detection measurements
with angle of launch depictions.
[0017] FIGS. 7A and 7B illustrate graphs of illustrative, prophetic
exemplary melt stage detection measurements.
[0018] FIG. 8 illustrates a scatter plot of illustrative, prophetic
exemplary melt stage detection measurements.
[0019] FIG. 9 illustrates a chip including a plurality of reaction
sites in accordance with various embodiment described herein.
[0020] FIG. 10 illustrates a block diagram of a computer system in
accordance with various embodiments described herein.
[0021] FIG. 11 illustrates a block diagram of exemplary instruments
in accordance with various embodiments described herein.
DETAILED DESCRIPTION
[0022] This description and the accompanying drawings that
illustrate exemplary embodiments should not be taken as limiting.
Various mechanical, compositional, structural, electrical, and
operational changes may be made without departing from the scope of
this description and claims, including equivalents. In some
instances, well-known structures and techniques have not been shown
or described in detail so as not to obscure the disclosure. Like
numbers in two or more figures represent the same or similar
elements. Furthermore, elements and their associated features that
are described in detail with reference to one embodiment may,
whenever practical, be included in other embodiments in which they
are not specifically shown or described. For example, if an element
is described in detail with reference to one embodiment and is not
described with reference to a second embodiment, the element may
nevertheless be claimed as included in the second embodiment.
[0023] For the purposes of this specification and appended claims,
unless otherwise indicated, all numbers expressing quantities,
percentages, or proportions, and other numerical values used in the
specification and claims, are to be understood as being modified in
all instances by the term "about," to the extent they are not
already so modified. Accordingly, unless indicated to the contrary,
the numerical parameters set forth in the following specification
and attached claims are approximations that may vary depending upon
the desired properties sought to be obtained. At the very least,
and not as an attempt to limit the application of the doctrine of
equivalents to the scope of the claims each numerical parameter
should at least be construed in light of the number of reported
significant digits and by applying ordinary rounding
techniques.
[0024] It is noted that, as used in this specification and the
appended claims, the singular forms "a," "an," and "the," and any
singular use of any word, include plural referents unless expressly
and unequivocally limited to one referent. As used herein, the term
"include" and its grammatical variants are intended to be
non-limiting, such that recitation of items in a list is not to the
exclusion of other like items that can be substituted or added to
the listed items.
[0025] As used herein, the term "biological sample" "sample" means
a material, substance, or solution comprising one or more
biological molecules, chemicals, components, and/or compounds
(e.g., a nucleic acid, DNA molecule, or RNA molecule) of interest
to a user, manufacturer, or distributor of the various embodiments
of the present invention described or implied herein. A sample may
include, but is not limited to, one or more of a DNA sequence
(including cell-free DNA), an RNA sequence, a gene, an
oligonucleotide, an amino acid sequence, a protein, a biomarker, or
a cell (e.g., circulating tumor cell), or any other suitable target
biomolecule. As used herein, the term "sample solution" means a
liquid or fluid comprising at least one sample.
[0026] As used herein, the term "reagent" means a material or
substance (e.g., in solid and/or liquid form) containing chemicals
or compounds to be used in combination with a sample in order to
facilitate a biological assay, test, process, or experiment (e.g.,
a PCR assay, test, process, or experiment). A reagent may comprise
a combination of at least one nucleotide, at least one
oligonucleotides, at least one primer, at least one polymerase, at
least one salt, at least one buffering agent, at least one dye
(e.g., control dye and/or binding dye), at least one marker, at
least one probe, at least one enhancing agent, at least one enzyme,
at least one detergent, and/or at least one blocking agent. The
reagent may comprise at least one master mix (MMx) containing, for
example, a combination of at least one polymerase, at least one
nucleotide, at least one salt, at least one buffering agent, at
least one dye (e.g., control dye and/or binding dye), and/or at
least one enhancing agent. In some cases, the MMx may include one
or more DNA binding dyes (e.g., a SYBR dye) or other chemicals.
[0027] As used herein, the term "reaction solution", "reaction
mix", and "reaction build" means a solution or mixture containing
both a biological sample and one or more reagents. The reaction
solution, reaction mix, or reaction build may be used in
conjunction with one or more of PCR (e.g., qPCR, dPCR, multiplex
dPCR), fetal diagnostics, viral detection, quantification
standards, genotyping, sequencing, sequencing validation, mutation
detection, detection of genetically modified organisms, rare allele
detection, and/or copy number variation, or the like.
[0028] As used herein, an "indicator" means a physical, electrical,
magnetic, chemical, and/or optical property or effect produced by a
sample that may be used in determining the existence and/or in
determining, measuring, or estimating the amount of a target
nucleic acid. An indicator may comprise one or more of luminescence
(e.g., fluorescence, chemiluminescence, bioluminescence), color,
transmissivity, opacity, reflectivity, or polarization, pH, charge,
surface potential, current, or voltage changes.
[0029] As used herein, the term "amplification product" means any
product produced by an amplification assay or process, for example,
an increased number of target nucleic acid molecules or other
nucleic acid molecules produced during a PCR assay or process
(e.g., during a qPCR or dPCR assay or process). As used herein, an
"indicator of amplification" means a physical, electrical,
magnetic, chemical, and/or optical property or effect produced by a
sample that may be used in determining the existence, and/or in
determining, measuring, or estimating an amount of amplification of
a target nucleic acid in a biological assay, test, process, or
experiment (e.g., a PCR assay, test, process, or experiment). An
indicator of amplification may comprise one or more of luminescence
(e.g., fluorescence, chemiluminescence, bioluminescence), color,
transmissivity, opacity, reflectivity, or polarization, pH, charge,
surface potential, current, or voltage changes.
[0030] Polymerase chain reaction (PCR) may comprise a thermal
cycling process, in which cycles of heating and cooling are used to
provide repeated cycles of nucleic acid melting and enzymatic
replication of nucleic acids. A number of PCR methods use thermal
cycling involving alternately heating and cooling the PCR sample to
a defined series of temperature steps. These thermal cycling steps
may be used first to physically separate nucleic acids, such as
separating the two strands in a nucleic acid double helix, at a
high temperature in a process called melting. At a lower
temperature, each strand is then used as the template in synthesis
by the polymerase to selectively amplify a target nucleic acid
during an annealing phase and extension phases. Example polymerases
include heat-stable polymerase such as, for example, Taq
polymerase. The selectivity of PCR results from the use of pruners
that are complementary to nucleic acid regions targeted for
amplification under specific thermal cycling conditions. Primers
(short nucleic acid fragments) containing sequences complementary
to the target region along with a polymerase, are used to enable
selective and repeated amplification.
[0031] Referring to FIG. 1A, in certain embodiments of the present
invention, a system or instrument 10 for detecting or quantifying a
nucleic acid in a sample or sample solution comprises base and/or
housing 20. The base and/or housing 20 comprises, or is configured
to receive, contain, or hold, a reaction device 25. The reaction
device 25 comprises, or is configured to provide, a plurality of
sample reaction volumes 30 receiving, containing, holding, and/or
segregating all or a portion of the sample or sample solution.
Optionally, the system 10 may additionally comprise a temperature
controller 35 such as a thermal cycler, or the like (e.g., for
performing a qPCR assay).
[0032] With additional reference to FIG. 10, the system 10 may also
comprise computer system 1000. The computer system 1000 may be
configured or employed to carry out processing functionality,
according to various embodiments, upon which embodiments of
temperature controller 35 may be utilize, when present. Computing
system 1000 can include one or more processors, such as a processor
or electronic processor 1004. Processor 1004 can be implemented
using a general or special purpose processing engine such as, for
example, a microprocessor, controller or other control logic.
[0033] In certain embodiments, a digital amplification technique is
performed. For example, the digital amplification technique may
comprise a digital PCR (dPCR) assay, process, experiment, or test,
wherein a sample or reaction solution is segregated, distributed,
or divided, into a plurality of sample reaction volumes or reaction
sites associated with a reaction device, fluidic device, sample
holder, or other such device. The plurality of sample reaction
volumes may include a first plurality of the sample reaction
volumes each containing one molecule or approximately one molecule
of a target nucleic acid and a second plurality of the sample
reaction volumes each containing no molecules of the target nucleic
acid. The plurality of sample reaction volumes or reaction sites
are subjected to an amplification assay using, for example, at
least a primer and probe or indicator dye, wherein the
amplification assay is configured to amplify the target nucleic
acid. During the dPCR assay, an indicator of the target present in
any of the plurality of sample reaction volumes may be detected or
measured. Similar to other types of PCR, dPCR may progress by
exposing the partitioned sample reaction volumes, which contain
reagents for amplification, to an amplification assay designed to
amplify the target nucleic acid. For example, thermal cycling may
be performed such that the template nucleic acid is amplified
within the reaction volumes that include an initial one, or
approximately one, copy of the template nucleic acid molecule.
[0034] In order to quantify the nucleic acid amplification, an
indicator of amplification exhibited by the reaction volumes may be
detected. In some exemplary embodiments in accordance with the
present disclosure, one or more fluorescent dyes or probes may be
used such that the dyes or probes bond to nucleic acids and exhibit
fluorescence to indicate presence of a nucleic acid.
[0035] For example, amplified target nucleic acids can be detected
using a detectable nucleic acid binding agent which can be, for
example, an intercalating agent or a non-intercalating agent. As
used herein, an intercalating agent is an agent or moiety capable
of non-covalent insertion between stacked base pairs of a
double-stranded nucleic acid molecule. A non-intercalating agent is
one that does not insert into the double-stranded nucleic acid
molecule. The nucleic acid binding agent can produce a detectable
signal directly or indirectly. The signal can be detectable
directly using, for example, fluorescence or absorbance, or
indirectly using, for example, any moiety or ligand that is
detectably affected by its proximity to double-stranded nucleic
acid is suitable, for example a substituted label moiety or binding
ligand attached to the nucleic acid binding agent. It is typical
for the nucleic acid binding agent to produce a detectable signal
when bound to a double-stranded nucleic acid that is
distinguishable from the signal produced when that same agent is in
solution or bound to a single-stranded nucleic acid. For example,
intercalating agents such as ethidium bromide fluoresce more
intensely when intercalated into double-stranded DNA than when
bound to single-stranded DNA, RNA, or in solution (see, e.g., U.S.
Pat. Nos. 5,994,056; 6,171,785; and 6,814,934). Similarly,
actinomycin D fluoresces red when bound to single-stranded nucleic
acids, and green when bound to double-stranded nucleic acids. And
in another example, the photoreactive psoralen
4-aminomethyle-4-5',8-trimethylpsoralen (AMT) has been reported to
exhibit decreased absorption at long wavelengths and fluorescence
upon intercalation into double-stranded DNA (Johnston et al.
Photochem. Photobiol. 33:785-791 (1981). For example, U.S. Pat. No.
4,257,774 describes the direct binding of fluorescent intercalators
to DNA (e.g., ethidium salts, daunomycin, mepacrine and acridine
orange, 4',6-diamidino-.alpha.-phenylindole). Non-intercalating
agents (e.g., minor groove binders such as Hoechst 33258,
distamycin, netropsin) may also be suitable for use. For example,
Hoechst 33258 (Searle, et al. Nucleic Acids Res. 18:3753-3762
(1990)) exhibits altered fluorescence with an creasing amount of
target. Exemplary detectable DNA binding agents may include, for
example, acridine derivatives (e.g., acridine homodimer, acridine
orange, acridine yellow, 9-amino-6-chloro-2-methoxyacridine (ACMA),
proflavin,), actinomycins (e.g., actinomycin D (Jain, et al. J.
Mol. Biol. 68:1-10 (1972), 7-amino-actinomycin D (7-AAD)),
anthramycin, auramine, azure B, BOBO.TM.-1, BOBO.TM.-3,
BO-PRO.TM.-1, BO-PRO.TM.-3, chromomycin (e.g., A3) crystal violet,
cyanine dyes, DAPI (Kapu ci ski, et al. Nucleic Acids Res.
6:3519-3534 (1979)), 4',6-diamidino-2-phenylindole (DAPI),
daunomycin, distamycin (e.g., distamycin D), dyes described in U.S.
Pat. No. 7,387,887, ellipticine, ethidium salts (e.g., ethidium
bromide, ethidium homdimer-1, ethidium homdimer-2, dihydroethidium
(also known as hydroethidine), ethidium monoazide), fluorcoumanin,
fluorescent intercalators as described in U.S. Pat. No. 4,257,774,
GelStar.RTM. (Cambrex Bio Science Rockland Inc., Rockland, Me.),
hexidium iodide, Hoechst 33258 (Searle, et al., (supra)), Hoechst
33342, Hoechst 34580, homidium, hydroxystilbamidine, JO-JO-1,
JO-PRO.TM.-1, LDS 751, LOLO-1, LO-PRO.TM.-1, malachite green,
mepacrine (e.g., orange), mithramycin, netropsin, the Nissl
substance, 4',6-diamidino-.alpha.-phenylindole, proflavine,
POPO.TM.-1, POPO.TM.-3, PO-PRO.TM.-1, propidium iodide, ruthenium
polypyridyls, Sevron dyes (e.g., Brilliant Red 2B, Brilliant Red
4G, Brilliant Red B, Orange, Yellow L), SYBR 101, SYBR 102, SYBER
103, SYBR.RTM. Gold, SYBR.RTM. Green I (U.S. Pat. No. 5,436,134 and
5,658,751), SYBR.RTM. Green II, SYTOX.RTM. Blue, SYTOX.RTM. Green,
SYTOX.RTM. Orange, SYTO.RTM. 1, SYTO.RTM. 11, SYTO.RTM. 13,
SYTO.RTM. 14, SYTO.RTM. 15, SYTO.RTM. 16, SYTO.RTM. 17, SYTO.RTM.
18, SYTO.RTM. 20, SYTO.RTM. 21, SYTO.RTM. 22, SYTO.RTM. 23,
SYTO.RTM. 24, SYTO.RTM. 25, SYTO.RTM. 40, SYTO.RTM. 43, SYTO.RTM.
44, SYTO.RTM. 45, SYTO.RTM. 59, SYTO.RTM. 60, SYTO.RTM. 61,
SYTO.RTM. 62, SYTO.RTM. 63, SYTO.RTM. 64, SYTO.RTM. 80, SYTO.RTM.
81, SYTO.RTM. 82, SYTO.RTM. 83, SYTO.RTM. 84, SYTO.RTM. 85,
thiazole orange (Aldrich Chemical Co., Milwaukee, Wis.), TO-PRO-1,
TO-PRO-3, TO-PRO-5, TOTO-1, TOTO-2, TOTO.TM.-3, YO-PRO.RTM.-1,
YO-PRO.RTM.-3, YOYO-1, and YOYO.RTM.-3 (Molecular Probes, Inc.,
Eugene, Oreg.), among others.
[0036] SYBR.RTM. Green I (see, e.g., U.S. Pat. Nos. 5,436,134;
5,658,751; and/or 6,569,927), for example, has been used to monitor
an amplification (e.g., PCR) reaction by amplifying the target
sequence in the presence of the dye, exciting the biological sample
with light at a wavelength absorbed by the dye and detecting the
emission therefrom. It is to be understood that the use of the
SYBR.RTM. Green dye is presented as an example and that many such
dyes may be used in the methods described herein. Other nucleic
acid binding agents can also be suitable as would be understood by
one of skill in the art.
[0037] In certain embodiments, detection or measurement of the
indicator of amplification in a digital amplification assay may be
performed at the endpoint of the amplification reaction. The
digital amplification may be detected or measured at an ambient
temperature after one or more thermal cycles have been completed.
It may be beneficial to detect indicators of amplification at other
times (such as during amplification and/or during a melt stage) in
order to better determine the amplicons produced.
[0038] FIG. 1B illustrates a graph illustrating various
amplification detection schemes contemplated by the present
disclosure, for example during an amplification assay or
post-amplification (i.e., after completion of an amplification
assay). For example, an indicator of amplification exhibited by
reaction sites or reaction volumes hosting dPCR amplification may
be subject to detection in each of these reaction sites or reaction
volumes. As illustrated in FIG. 1B, detection point 102 corresponds
to the endpoint detection at the ambient temperature described
above.
[0039] In one exemplary embodiment, monitoring for an indicator of
amplification occurring in the reaction sites may occur via a
real-time detection scheme in which detection data is taken during
the dPCR amplification assay. For example, the detection data may
be obtained for each reaction site at a predetermined point during
a thermal cycling process or procedure (e.g., at a predetermined
temperature for one or more cycles of the thermal cycling process
or procedure). Detection points 104 in FIG. 1B graphically
represents the collection of such real time detection during
amplification. As depicted, detection for an indicator of
amplification under this real time technique relies on detection
data taken during numerous PCR thermal cycles at the same
temperature, e.g., at 60.degree. C. In at least one exemplary
embodiment, the real time detection occurs for each cycle of the
total number of PCR cycles. In other exemplary embodiments, the
real time detection occurs during a subset of cycles (e.g.,
predetermined subset, user selectable subset, or dynamic subset
based on detected results), in accordance with a predetermined
pattern of cycles based on the reagents used for amplification, or
at any other suitable cycles.
[0040] In one exemplary embodiment, the present disclosure
contemplates performing a melt stage on the reaction sites after
the amplification assay and an end point reading has been
performed. During such a melt stage, the sample reaction volumes in
the reaction sites are heated at a constant rate over a
predetermined time and changes to an indicator of amplification are
detected. For example, the plurality of sample reaction volumes may
be heated at a constant rate over a period of time, such as 10
minutes, 15 minutes, 30 minutes, 1 hour, or any other suitable
period of time. During the heating, changes in an indicator of
amplification for the sample reaction volumes may be detected, and
changes in the indicators may be identified. For example, the bonds
of a nucleic acid molecule may melt causing disassociation during
heating. This disassociation may trigger a change (decrease) in the
indicator of amplification exhibited.
[0041] Various exemplary post-amplification measurements are
depicted as 106, 018, and 110 in FIG. 1B using various melt stage
detection techniques. Detection point 106 comprises single point
detection at a predetermined temperature, such as 60.degree.,
wherein the detection occurs during a melt stage
post-amplification. For example, as a temperature for the sample in
each of the reaction volumes is raised to 60.degree., the indicator
of amplification exhibited by each reaction volume may be detected
and changes determined. Detection points 108 correspond to interval
detection at a plurality of predetermined target temperatures, for
example starting at 60.degree. and at certain higher temperatures
as the heating increases. Detection points 110 correspond to a
rapid detection technique over the predetermined time for the melt
stage.
[0042] In exemplary embodiments, results for the detection point
102 may be combined with one or more of results for detection
points 104, detection point 106, detection points 108, and/or
detection points 110. The combined results may be analyzed to
improve or enhance the accuracy of the dPCR detection or assay
results. For example, the combined results may be used to correct
for or eliminate contributions to an indicator signal that are
produced by substances or molecules besides the target of interest
produced by a primer dimer). It will appreciate that not all of the
various techniques 104, 106, 108, 110 need be performed for
improving or enhancing the accuracy of a dPCR assay results, but
rather one or more can be utilized and in various combinations to
assist in improving or enhancing the accuracy of dPCR amplification
detection or assay results.
[0043] FIGS. 2A, 2B, 3A, 3B, and 3C illustrate various exemplary
methods that can be used to quantify dPCR amplification of a target
nucleic acid based on data collected from one or more of the
detection techniques illustrated in FIG. 1B.
[0044] FIGS. 2A-2B illustrate exemplary methods for performing dPCR
in accordance with embodiments of the present disclosure. The
methods described herein may be performed with a variety of
different reaction devices including, but not limited to a sample
chip, an electronic chip, a circuit board, a TLDA card, droplets in
a free solution, droplets on a planar surface, droplets over a
temperature gradient, droplets in a capillary tube or flow system,
a microfluidic device with individual chambers, a 384-well or
higher density microliter plate, an array of reaction wells, an
array of through-holes in a substrate, or any other suitable
reaction device. The method may comprise any of the detection
techniques for dPCR with which those having ordinary skill in the
art are familiar (e.g., optical detection or electrical detection).
Various exemplary devices that may be utilized to implement the
dPCR detection methods described herein are explained in further
detail below.
[0045] At 202A of FIG. 2A, a sample is segregated, distributed, or
divided into a plurality of sample reaction volumes. For example,
the plurality of sample reaction volumes may be segregated such
that a first plurality of the sample reaction volumes contain at
least one molecule of a target nucleic acid and a second plurality
of the sample reaction volumes contain no molecules of the target
nucleic acid. The sample may be fractionated by a dilution process
so that each sample reaction volume contains one copy,
approximately one copy, or no copy of the target nucleic acid. In
an embodiment, the segregated sample reaction volumes may include
one or more reagents for amplifying the target nucleic acid
molecules. The reagent(s) may be incorporated, mixed, or added into
the sample prior to segregation or after segregation.
[0046] In one exemplary embodiment, the plurality of sample
reaction volumes may be segregated on a sample holder 900 of FIG.
9, or the like, described in further detail below, although various
other reaction devices may be used to segregate the sample reaction
volumes and implement the amplification detection techniques
described herein and/or known in the art. Accordingly, the sample
reaction volumes may be segregated among the plurality of reaction
sites (e.g., through-holes or wells) of sample holder 900.
[0047] At 204A, the plurality of sample reaction volumes are
subjected to an amplification assay. For example, the plurality of
sample reaction volumes may be simultaneously subjected to an
amplification assay, wherein the amplification assay is designed to
amplify the target nucleic acid to produce amplified product (i.e.,
one or more amplicons). The assay may utilize at least a primer,
probe and/or dye, and an enzyme, such as a Taqman.TM. assay or any
other suitable assay, as those having ordinary skill in the art are
familiar with. Accordingly, the sample reaction volumes contain the
sample portion and the reagents for amplification and
detection.
[0048] In some embodiments, an assay may include two probes, such
as a FAM.TM. dye-labeled probe and a VIC.RTM. dye-labeled probe,
and amplification detection measurements based on each dye may be
utilized in order to determine quantities for amplified target
nucleic acid(s). For instance, multiple indicators of amplification
may be exhibited from a sample reaction volume based on each of the
dye-labeled probes. An assay may also include a variety of primers,
such as ELITe.RTM. primers. In an embodiment, one ELITe.RTM. primer
may overlap a target sequence (i.e., an allele specific primer)
while one ELITe.RTM. primer may not (i.e., a locus specific
primer). Some implementations may leverage a standard primer rather
than an ELITe.RTM. primer for the locus specific primer. In some
embodiments, a multiplexing assay may be used where multiple allele
specific primers may generate amplicons with a single locus
specific primer.
[0049] In some embodiments, an assay may include primers with
target specific 3' domains and non-target specific 5' tails to
generate amplicons with adjusted target melt temperatures. In
another example, an assay may include primers with target specific
3' domains and universal 5' tails to generate amplicons with
adjusted target melt temperatures. In this example, the assay
formulations may utilize universal primers such that initial
amplification is caused by target specific domains (e.g., target
specific 3' domain) while further amplification can be caused by
the universal primers. These amplicons may be later differentiated
by target melt temperatures. In some embodiments, an assay may
include primers designed to identify amplification reactions
involving normal (wild-type) nucleic acids and non-normal (mutant)
nucleic acids. An assay may also include primers designed to
identify certain types of mutations (i.e., single nucleotide
polymorphisms (SNPs) and inDels at locus within amplicons). For
instance, the identification may be based on target melt
temperatures for the produced amplicons, In some embodiments, use
of known spike-in concentrations may also be leveraged for
identification. Various embodiments may utilize ELITe.RTM. primers,
non-ELITe.RTM. (standard) primers, or any suitable combination.
[0050] In an exemplary embodiment, the plurality of sample reaction
volumes subjected to the amplification assay may be subjected to a
plurality of PCR steps, such as thermal cycling, as described
herein. For example, a temperature of the sample reaction volumes
may be increased to physically separate strands of the target
nucleic acid (i.e. strands of a nucleic acid molecule). The
temperature may then be decreased and each strand may be used as a
template for synthesis by an enzyme (i.e., polymerase) to
selectively amplify the target nucleic acid, for instance during
annealing and extension phases of the PCR process. In an
embodiment, a plurality of PCR cycles may be performed that result
in amplification of the target nucleic acid molecule.
[0051] At 206A, an indicator of amplification presented by the
plurality of sample reaction volumes may be detected or measured.
For example, an indicator of amplification may be presented by each
of the plurality of sample reaction volumes that host amplification
of a nucleic acid molecule (e.g., amplification of the target
nucleic acid molecule).
[0052] In an embodiment, one or more dyes may be used that
fluoresce when bound to double-stranded nucleic acids, and this
fluorescence may be detected as an indicator of amplification. For
example, the nucleic acid binding agent (dye) may produce a
detectable signal when bound to double-stranded nucleic acids that
is distinguishable from the signal produced when that same agent is
in solution or bound to a single-stranded nucleic acid. The
fluorescence may be detected using a fluorescence detector, for
example mounted over a chip that houses the segregated sample
reaction volumes, or may be detected in any other suitable
manner.
[0053] In an exemplary embodiment, an indicator of amplification
may be detected for each of the plurality of reaction volumes at a
first temperature. For example, an initial detection for an
indicator of amplification may occur while the volumes are at a
first temperature, which according to various exemplary embodiments
may be ambient temperature. FIG. 4 illustrates a graph of
exemplary, prophetic detection results. An indicator of
amplification (e.g., fluorescence) for the sample reaction volumes
may be represented by the "Property" attribute on the y-axis and
the temperature may be represented by the x-axis. Accordingly, the
indicator of amplification presented by the reaction volumes
detected at an ambient temperature are illustrated in the graph of
FIG. 4.
[0054] At 208A, a melt stage is performed, for example, after
discontinuing the amplification assay during 204A. During the melt
stage, the plurality of sample reaction volumes may be heated a
constant rate over a predetermined time and changes to an indicator
for the sample reaction volumes are identified based on the
heating. For example, the plurality of sample reaction volumes may
be heated at a constant rate over a period of time, such as 10
minutes, 15 minutes, 30 minutes, 1 hour, or any other suitable
period of time. During the heating, the results for the indicator
of amplifications (e.g., fluorescence) for the sample reaction
volumes may be detected, and changes in the indicator may be
identified. For example, the bonds of a nucleic acid molecule may
melt causing disassociation during heating of the sample reaction
volumes. This disassociation triggers a change (e.g., a decrease)
in the indicator of amplification exhibited by a sample reaction
volume. For example, one or more dyes that fluoresce to produce the
indicator of amplification while bound to double-stranded nucleic
acid molecules may cease to produce such an indicator (or produce
less of the indicator) when the molecules undergo
disassociation.
[0055] In an embodiment, the indicator of amplification (e.g.,
fluorescence) for the sample reaction volumes may be detected at a
series of intervals during the melt. As discussed above, detection
points 108 of FIG. 1B illustrate this type of interval detection
scheme, and FIG. 4 further illustrates a graph 400 of illustrative,
prophetic exemplary detection results, where the indicator may be
detected at various target temperatures. In an embodiment, the
target temperatures may be based on temperature intervals (e.g.,
5.degree. C. 10.degree. C., and the like), or may be a
predetermined set of target temperatures. In FIG. 4, the target
temperatures include 55.degree. C., 65.degree. C., 75.degree. C.,
85.degree. C., and 95.degree. C. At each target temperature,
detection of the reactions in each of the plurality of reaction
volumes can occur such that changes in the indicator of
amplification may he identified. For example, the results for
detection at ambient temperature (e.g., initial detection) may he
compared to the results detected at 55.degree. C. The comparison
shows that a set of sample reaction volumes from the plurality of
reaction volumes exhibits a decrease in the indicator of
amplification between ambient temperature and 55.degree. C., e.g.,
as melting begins. Similarly, comparisons between results detected
for target temperatures 55.degree. C. and 65.degree. C., 65.degree.
C. and 75.degree. C., 75.degree. C. and 85.degree. C. and
85.degree. C. and 95.degree. C. show decreases in the indicators of
amplification for the sample reaction volumes.
[0056] In an embodiment, the results for the indicator of
amplification (e.g., fluorescence) for the sample reaction volumes
may be detected rapidly during the melt. For example, detection may
occur at closely spaced intervals such that a continuous data
function may be generated. As discussed above, detection points 110
of FIG. 1B illustrate this type of rapid detection scheme. In
analyzing the results detected rapidly, trends for the indicators
exhibited by the plurality of reaction volumes may be determined.
For example, the analysis may show a trend that a set of sample
reaction volumes from the plurality of reaction volumes exhibits a
decrease in the indicator of amplification as the melt progresses
(e.g., temperature of the reaction volumes increases). The trends
may be similar to results determined from comparisons during
interval detection during a melt. In an example, trends between
temperatures similar to the target temperatures of an interval
detection method, such as ambient and 55.degree. C., 55.degree. C.
and 65.degree. C., 65.degree. C. and 75.degree. C., 75.degree. C.
and 85.degree. C., and 85.degree. C. and 95.degree. C., may be
determined based on the analysis.
[0057] In an exemplary embodiment, the rapid detection also may
enable identification of trends at specific temperatures (e.g.,
temperatures other than the target temperatures of the interval
detection). For example, during the melt, a temperature (or
temperature window) may trigger a noticeable change (e.g., change
beyond a threshold) for the indicators exhibited by the reaction
volumes. The rapid detection may enable identification of these
temperatures, or temperature window, via analysis of the changes in
the indicators. Accordingly, the rapid detection methodology may
provide enhanced sensitivity to the analysis.
[0058] At 210A, the amplification of the target nucleic acid may be
quantified based on the initial detected indicators and the
detected changes in the indicators. For example, the indicators
exhibited by the reaction volumes at ambient temperature may
suggest a level of amplification that occurred in each reaction
volume over the PCR process. However not all of the detected
amplified product is a result of amplification of the target
nucleic acid. In other words, the indicators presented by the
reaction volumes at ambient temperature may indicate amplification
of something other than the target nucleic acid, or may be
triggered by some other source, yet erroneously detected as the
amplified product of the target nucleic acid. Such erroneously
detected amplified product can include, for instance,
primer-dimers, misincorporations, dust/debris, sample nucleic acid,
or various other sources. As used herein, the term "erroneous
amplification product" means an amplification product produced by
nucleic acid molecules that are not a target nucleic acid.
Performance of the melt and a subsequent analysis of the detected
results may enable identification of indicators that are not the
results of the desired amplification of the target nucleic acid.
After considering the indicators that are related to some other
source, the amplification for the target nucleic acid may be
quantified with greater accuracy.
[0059] In an embodiment, a single point detection algorithm may be
used with the endpoint detection (e.g., at ambient temperature), as
illustrated by detection point 106 of FIG. 1B described above. For
example, an identified change may be determined based on the
detection results at ambient temperature (e.g., detection point
102) and the predetermined temperature for single point detection
(e.g., detection point 106). One of identified changes 402 in FIG.
4 may include the identified change based on the single point
detection.
[0060] For example, the detected change at this temperature may
include a further separation of the reaction volumes that exhibit
indicators of amplification triggered by nucleic acid amplification
from the reaction volumes that do not. Reaction volumes that
exhibit some indicator of amplification may show a decrease such
that the background noise of the results is reduced. For example,
when a dye, such a SYBR dye (or other intercalating dye), is used
to produce the indicator of amplification, the dye may bond with
various reaction products (e.g., nucleic acids) due to the
non-specific design of the dye. Accordingly, background noise
(e.g., fluorescence) may be caused by dye binding to anilities
which are not products of the reaction, or binding to products
which are not the intended product of the reaction. In an
embodiment, reaction volumes that exhibit the indicator of
amplification may be discernible from those that do not based on
the further separation, and the amplification of the target nucleic
acid may be more accurately quantified based on the discerned
reaction volumes.
[0061] In an embodiment, an interval detection algorithm may be
used with the endpoint detection (e.g., at ambient temperature), as
illustrated by detection points 108 of FIG. 1B. For example,
identified changes may be determined based on the detection results
at ambient temperature (e.g., detection point 102) and the
predetermined temperatures for interval detection (e.g., detection
points 108).
[0062] In one embodiment, the identified changes in the indicators
for the plurality of reaction volumes may be analyzed to determine
indicators triggered by a source other than amplification of the
target nucleic acid. For example, based on the particular target
nucleic acid, an expected melt temperature (or expected melt
temperature range) may be determined such that the target nucleic
acid would be expected to melt at the temperature. Therefore, an
indicator exhibited by a reaction volume based on amplification of
the target nucleic acid would be expected to decrease at the
expected melt temperature or temperature range (e.g., fluorescence
exhibited by a reaction volume would be expected to decrease at the
expected melt temperature). The expected melt temperature may be
specific to a target nucleic acid, the amplification assay used
during amplification, particular primers, and any other suitable
factor for amplification. In an example, the expected melt
temperature for a target nucleic acid (being amplified using a
specific amplification assay) may be determined using empirical
analysis (e.g., amplified target nucleic acid molecules may be
heated until disassociation, and the temperature when
disassociation occurs may be the melt temperature).
[0063] In an embodiment, during performance of the melt, indicator
detection may occur at various temperature intervals. FIG. 4
illustrates a graph of illustrative, prophetic exemplary detection
results based on interval temperatures. In an example, the expected
melt temperature for the target nucleic acid here gray comprise
70.degree. C., or an expected melt temperature range may comprise
65.degree. C. to 75.degree. C.,
[0064] Identified change 404, detected at temperature 75.degree.
C., may indicate changes to indicators triggered by amplification
of the target nucleic acid. Because the expected melt temperature
for the target nucleic acid comprises 70.degree. C., or a range
between 65.degree. C. to 75.degree. C., the decreases of indicators
detected at 75.degree. C. correspond to indicators triggered by
amplification of the target nucleic acid. For instance, indicator
406 illustrates one or more indicators exhibited by reaction
volumes where the indicators decrease (e.g., a decrease in
fluorescence) during the detection at 75.degree. C. Accordingly,
quantifying the decrease in indicators exhibited by the plurality
of reaction volumes between 65.degree. C. and 75.degree. C. (e.g.,
identified change 404) may enable the quantification of amplicon
(amount of amplified target nucleic acid) that resulted from the
dPCR amplification.
[0065] Identified change 408, detected at temperature 85.degree.
C., may indicate changes to indicators triggered by a source other
than amplification of the target nucleic acid. Because the expected
melt temperature for the target nucleic acid comprises 70.degree.
C., or a range between 65.degree. C. to 75.degree. C., the
decreases of indicators detected at 85.degree. C. correspond to
indicators triggered by other sources. Indicator 410, detected at
temperature 95.degree. C., also corresponds to indicators triggered
by a source other than amplification of the target nucleic acid
sequence. At such a high temperature, nucleic acids would be
expected to melt, and therefore indicator 410 may correspond to a
source like dust, or some other source that may cause an indicator
to persist above certain temperature thresholds.
[0066] In an embodiment, during performance of the melt, indicator
detection also may occur rapidly in lieu of or in addition to set
temperature point interval detection. For example, the rapid
detection scheme may be used with the endpoint detection (e.g., at
ambient temperature), as illustrated by detection points 110 of
FIG. 1B. In this way, identified changes may be determined based on
the detection results at ambient temperature (e.g., detection paint
102) and rapidly over the melt temperature range (e.g., detection
points 110).
[0067] Similar to the interval detection analysis, the identified
changes to indicators for the plurality of reaction volumes at
various temperatures may be compared to an expected melt
temperature for the target nucleic acid. For example, indicators
that show changes (e.g., decreases) at tempera other than the
expected melt temperature (or expected melt temperature range) for
the target nucleic acid may correspond to sources other than target
nucleic acid amplification (e.g., the indicators may be triggered
by something other than target nucleic acid amplification). On the
outer hand, indicators that show changes (e.g., decreases) at the
expected melt temperature (or expected melt temperature range) for
the target nucleic acid may correspond to target nucleic acid
amplification (e.g., the indicators may be triggered by target
nucleic acid amplification).
[0068] In an embodiment, based on the detected changes in the
indicators during the melt stage, the amount of amplified product
of the target nucleic acid resulting from the amplification process
may be quantified. For example, the amount of amplified product may
be directly proportional to the change detected for indicators
(e.g., detected decrease in fluorescence) at the expected melt
temperature (or expected melt temperature range). In an embodiment,
the quantification may be based on identification of indicators
triggered by a source other than target nucleic acid amplification.
For example, it may be determined that indicators that changed
(e.g., decreased) at temperatures other than the expected melt
temperature (or expected melt temperature range) correspond to
sources other than target nucleic acid amplification. Accordingly,
these indicators may be discounted when quantifying the
amplification of the target nucleic acid.
[0069] In an embodiment, detected changes for indicators at an
expected melt temperature (or an expected melt temperature range)
may confirm expected amplification, and amplification of the target
nucleic acid may be quantified based on these confirmed indicators.
For example, a Poisson model may be used along with a total number
of sample reaction volumes and total number of non-amplifying
sample reaction volumes, distinguished using indicator changes at
an expected melt temperature, to calculate a mean number of
reactions per sample reaction volume. The result can be divided by
the mean volume of each sample reaction volume to arrive at the
copies per unit volume for the reaction product melting at the
expected melt temperature or temperature range.
[0070] FIG. 2B illustrates an exemplary method for performing
digital amplification (dPCR) using multiplexing in accordance with
at least one exemplary embodiment of the present disclosure. The
method described herein may be performed with the sample chip, a
circuit board comprising through holes, or with any other suitable
device and detection scheme for dPCR with which those having
ordinary skill in the art are familiar.
[0071] At 202B of FIG. 2B, a sample is segregated, distributed, or
divided into a plurality of sample reaction volumes. In one
exemplary embodiment, the sample may comprise two or more different
target nucleic acids. For example, the dPCR process of the method
may comprise a multiplexed amplification such that more than one
target nucleic acid is amplified. The plurality of sample reaction
volumes may be segregated such that a first plurality of the sample
reaction volumes contain at least one molecule of one of the target
nucleic acids and a second plurality of the sample reaction volumes
contain no molecules of the target nucleic acids. The sample may be
fractionated by a dilution process so that each sample reaction
volume contains one copy, approximately one copy, or no copy of the
nucleic acid template or target. In an embodiment, the segregated
sample reaction volumes may include a plurality of reagents for
amplifying one or more target nucleic acid molecules. The reagents
may be incorporated into the sample prior to segregation or after
segregation.
[0072] In one exemplary embodiment, the plurality of sample
reaction volumes may be segregated on a sample holder similar to
sample holder 900 of FIG. 9, described in further detail below.
Accordingly, the sample reaction volumes may be segregated among
the plurality of reaction sites (e.g., a plurality of wells or
through-holes) of chip 900. In an embodiment, a circuit board may
comprise a plurality of through-holes, and the plurality of sample
reaction volumes may be segregated among the plurality of
through-holes.
[0073] Referring again to FIG. 2B, at 204B, the plurality of sample
reaction volumes are subjected to an amplification assay. For
example, the plurality of sample reaction volumes may be
simultaneously subjected to a multiplexing amplification assay,
wherein the multiplexing amplification assay is designed to amplify
multiple target nucleic acids to produce amplified product (i.e.,
one or more amplicons). The multiplexing assay may comprise at
least a probe, a primer, and an enzyme, such as a Taqman.TM. assay
or any other suitable assay. In one exemplary embodiment, the
multiplexing assay includes reagents (e.g., primers and/or probes)
specific to each of the multiple types of target nucleic acids such
that each target nucleic acid sequence, if present in a reaction
site, may be amplified by exposure to the assay. Reagents (e.g.,
primers and/or probes) of the multiplexing assay may be designed
such that each target nucleic acid is amplified, and the resultant
amplicons may later be differentiated based on target melt
temperatures. In some instances, expected target melt temperatures
for amplicons may be based on the designed primers pairs used to
amplify the target nucleic acids. The multiplexing assay may be
designed for two, three, four, or more target nucleic acids, and
the resultant amplicons may be differentiated based on the results
of a melt, as described herein.
[0074] In some embodiments, a multiplexing assay may include two
probes, such as a FAM.TM. dye-labeled probe and a VIC.RTM.
dye-labeled probe, and amplification detection results based on
each dye may be utilized in order to determine quantities for
amplified target nucleic acid(s). For instance, multiple indicators
of amplification may be exhibited from a sample reaction volume
based on each of the dye-labeled probes. A multiplexing assay may
also include a variety of primers, such as ELITe.RTM. primers. In
an embodiment, one ELITe.RTM. primer may overlap a target sequence
(i.e., an allele specific primer) while one ELITe.RTM. primer may
not (i.e., a locus specific primer). Some implementations may
leverage a standard primer rather than an ELITe.RTM. primer for the
locus specific primer. In some embodiments, a multiplexing assay
may be used where multiple allele specific primers may generate
amplicons with a single locus specific primer.
[0075] In some embodiments, a multiplexing assay may include
primers with target specific 3' domains and non-target specific 5'
tails to generate amplicons with adjusted target melt temperatures.
In another example, a multiplexing assay may include primers with
target specific 3' domains and universal 5' tails to generate
amplicons with adjusted target melt temperatures. In this example,
the assay formulations may utilize universal primers such that
initial amplification is caused by target specific domains (e.g.,
target specific 3' domain) while further amplification can be
caused by the universal primers. These amplicons may be later
differentiated by target melt temperatures. In some embodiments, a
multiplexing assay may include primers designed to identify
amplification reactions involving normal (wild-type) nucleic acids
and non-normal (mutant) nucleic acids. An assay may also include
primers designed to identify certain types of mutations (i.e., SNPs
and inDels at locus within amplicons). For instance, the
identification may be based on target melt temperatures for the
produced amplicons. In some embodiments, use of known spike-in
concentrations may also be leveraged for identification. Various
embodiments may utilize ELITe.RTM. primers, non-ELITe.RTM.
(standard) primers, or any suitable combination. In an embodiment,
the plurality of sample reaction volumes subjected to the
multiplexing amplification assay may be further subjected to a
plurality of amplification steps, such as thermal cycling, as
described herein. For example, a temperature of the sample reaction
volumes may be increased to physically separate strands of the
target nucleic acid (i.e. strands of a nucleic acid molecule). The
temperature may then be decreased and each strand may be used as a
template for synthesis by an enzyme (i.e., polymerase) to
selectively amplify the target nucleic acids, for instance during
annealing and extension phases of the amplification process. In an
embodiment, a plurality of amplification cycles may be performed
that result in amplification of the target nucleic acid
molecules.
[0076] At 206B, an indicator of amplification presented by the
plurality of sample reaction volumes may be detected or measured.
For example, an indicator of amplification may be presented by each
of the plurality of sample reaction volumes that host amplification
of a nucleic acid molecule (e.g., amplification of one of the
target nucleic acid molecules).
[0077] In an embodiment, one or more dyes may be used that
fluoresce when bound to double-stranded nucleic acids, and this
fluorescence may be detected as an indicator of amplification. For
example, the nucleic acid binding agent (dye) may produce a
detectable signal when bound to double-stranded nucleic acids that
is distinguishable from the signal produced when that same agent is
in solution or bound to a single-stranded nucleic acid. The
fluorescence may be detected using a fluorescence detector, for
example mounted over a chip that houses the segregated sample
reaction volumes, or may be detected in any other suitable manner.
In an exemplary embodiment, indicator(s) of amplification presented
by the plurality of reaction volumes may be detected at a first
temperature. For example, an initial detection may comprise the
indicators for amplification of a plurality of sample reaction
volumes while the volumes are at a first temperature, which
according to various exemplary embodiments may be ambient
temperature. Detection point 102 of FIG. 1B may illustrate
detection of the indicator presented by the plurality of reaction
site at an ambient temperature.
[0078] At 208B, a melt stage is performed, for example, after
discontinuing the amplification assay during 204A. During the melt
stage, the plurality of sample reaction volumes are heated at a
constant rate over a predetermined time and changes in the
indicators for the plurality of sample reaction volumes are
identified based on the heating. For example, the plurality of
sample reaction volumes may be heated at a constant rate over a
period of time, such as 10 minutes, 15 minutes, 30 minutes, 1 hour,
or any other suitable period of tine. During the heating, the
results for the indicators of amplifications (e.g., fluorescence)
for the plurality of sample reaction volumes may be detected, and
changes in the indicators may be identified.
[0079] In an embodiment, the indicators of amplifications (e.g.,
fluorescence) for the plurality of sample reaction volumes may be
detected at a series of intervals during the melt. Detection points
108 of FIG. 1B illustrate this type of interval detection
algorithm. In an embodiment, the indicators may be detected at
various target temperatures based on temperature intervals (e.g.,
5.degree. C., 10.degree. C. and the like), or a predetermined set
of target temperatures. At each target temperature, detection of
the reactions in each of the plurality of reaction volumes can
occur such that changes in the indicators of amplification may be
identified. For example, the results may be similar to the
detection results illustrated in FIG. 4 for singleplex digital
amplification.
[0080] In an embodiment, the results for the indicators of
amplifications (e.g., fluorescence) for the plurality of sample
reaction volumes may be detected rapidly during the melt such that
a continuous function may be generated. Detection points 110 of
FIG. 1B illustrate this type of rapid detection algorithm. In
analyzing the results detected rapidly, trends for the indicators
exhibited by the plurality of reaction volumes may be determined.
For example, the analysis may show a trend that a set of sample
reaction volumes from the plurality of reaction volumes exhibits a
decrease in the indicator of amplification as the melt progresses
(e.g., temperature of the reaction volumes increases). The trends
may be similar to results determined from comparisons during
interval detection during a melt.
[0081] At 210A, the amplification of the target nucleic acid may be
quantified based on the initial detected indicators and the
detected changes in the indicators. For example, the indicators
exhibited by the reaction volumes at ambient temperature may
suggest a level of amplification that occurred in each reaction
volume over the amplification process, however not all of the
detected amplified product is a result of amplification of one of
the target nucleic acids. In other words, the indicators presented
by the reaction volumes at ambient temperature may indicate
amplification of something other than one of the target nucleic
acids, or may be triggered by some other source, yet erroneously
detected as the amplified product. Such erroneously detected
amplified product can include, for instance, primer-dimers,
misincorporations, dust/debris, or various other sources.
[0082] Moreover, in one exemplary embodiment, indicators of
amplification exhibited by the reaction site triggered by
amplification of one of the target nucleic acids are not
discernible based on the particular target nucleic acid amplified.
In other words, in a multiplexing digital amplification process,
indicators of amplification triggered by an expected amplicon are
not specific to one of the target nucleic acids, and therefore it
cannot be determined which target nucleic acid triggered the
indicator of amplification. Performance of the melt and a
subsequent analysis of the detected results may enable
identification of indicators that are not the results of
amplification of the target nucleic acid and identification of
indicators specific to each of the multiple target nucleic acids.
After considering the indicators that are related to some other
source, the amplification for each target nucleic acid may be
quantified with greater accuracy.
[0083] In an embodiment, an interval detection algorithm may be
used with the endpoint detection (e.g., at ambient temperature), as
illustrated by detection points 108 of FIG. 1B. For example,
identified changes may be determined based on the detection results
at ambient temperature (e.g., detection point 102) and the
predetermined temperatures for interval detection (e.g., detection
points 108).
[0084] In one embodiment, the identified changes in the indicators
for the plurality of reaction volumes may be analyzed to determine
indicators triggered by a source other than amplification of the
target nucleic acid. For example, based on the particular target
nucleic acids, expected melt temperatures (or expected melt
temperature ranges) may be determined such that the amplified
target nucleic acids would be expected to melt at the temperatures.
Therefore, an indicator exhibited by a reaction volume based on
amplification of the target nucleic acid would be expected to
decrease at one of the expected melt temperatures or temperature
ranges (e.g., fluorescence exhibited by a reaction volume would be
expected to decrease at one of expected melt temperatures).
[0085] In an example, the expected melt temperatures for a first
type of target nucleic acid may be 60.degree. C., or an expected
melt temperature range be from 55.degree. C. to 65.degree. C., and
an expected melt temperature for a second type of target nucleic
acid may be 70.degree. C., or an expected melt temperature range
may be from 65.degree. C. to 75.degree. C. Therefore, an identified
change to an indicator of amplification at 60.degree. C., or
between 55.degree. C. to 65.degree. C., may indicate changes to
indicators triggered by amplification of the first type of target
nucleic acid. Similarly, an identified change to an indicator of
amplification at 70.degree. C., or between 65.degree. C. to
75.degree. C., may indicate changes to indicators triggered by
amplification of the second type of target nucleic acid. Identified
changes to indicators at other temperatures, or temperature ranges,
may indicate changes to indicators triggered by a source other than
amplification of a target nucleic acid.
[0086] In an embodiment, during performance of the melt, indicator
detection also may occur rapidly in lieu of or in addition to set
temperature point interval detection. For example, rapid detection
algorithm may be used with the endpoint detection (e.g., at ambient
temperature), as illustrated by detection points 110 of FIG. 1B.
For example, identified changes may be determined based on the
detection results at ambient temperature (e.g., detection point
102) and rapidly over the melt temperatures (e.g., detection points
110).
[0087] Similar to the interval detection analysis, the identified
changes to indicators for the plurality of reaction volumes at
various temperatures may be compared to expected melt temperatures
for the multiple target nucleic acids. For example, indicators that
show changes (e.g., decreases) at temperatures other than the
expected melt temperatures (or expected melt temperature ranges)
for the target nucleic acids may correspond to sources other than
target nucleic acid amplification (e.g., the indicators may be
triggered by something other than target nucleic acid
amplification). On the other hand, indicators that show changes
(e.g., decreases) at the expected melt temperatures (or expected
melt temperature ranges) for the target nucleic acids may
correspond to target nucleic acid amplification (e.g., the
indicators may be triggered by target nucleic acid
amplification).
[0088] In an embodiment, based on the detected changes in the
indicators during the melt, the amplified product of the target
nucleic acid resulting from the amplification process may be
quantified. For example, the amplified product of the first type of
target nucleic acid may be directly proportional to the change
detected for indicators (e.g., detected decrease in fluorescence)
at the expected melt temperature (or expected melt temperature
range) for the first type of target nucleic acid and the amplified
product of the second type of target nucleic acid may be directly
proportional to the change detected for indicators (e.g., detected
decrease in fluorescence) at the expected melt temperature (or
expected melt temperature range) for the second type of target
nucleic acid. In an embodiment, the quantification may be based on
identification of indicators triggered by a source other than
target nucleic acid amplification. For example, it may be
determined that indicators that changed (e.g., decreased) at
temperatures other than the expected melt temperatures (or expected
melt temperature ranges) correspond to sources other than target
nucleic acid amplification. Accordingly, these indicators may be
discounted when quantifying the amplification of the target nucleic
acids.
[0089] In an embodiment, clustering of measured melt temperatures
may be used to identify target nucleic acid amplification and
quantify amplified target nucleic acids. For example, decreases to
a plurality of indicators of amplification associated with a
plurality of sample reaction volumes may be measured at a
particular temperature (or within a temperature range) to determine
melt temperatures for these indicators, as described herein. These
measured melt temperatures may be clustered, for example, based on
calculated Euclidean distances and/or calculated silhouette values
such that clusters of indicators with similar melt temperatures may
be determined. Reference is made to HRM Experiments, Using
MeltDoctor.TM. HRM Reagents and High Resolution Melt Software
v.3.0, Life Technologies, 2010, which reviews the use of silhouette
scores for clustering when performing a melt using available
software tools. Those of ordinary skill in the art will recognize
various additional techniques exist for clustering data that may be
implemented to obtain clusters for the purposes of the present
embodiments. Accordingly, one or more clusters may be identified
that comprise indicators of amplification with similar melt
temperatures.
[0090] In some embodiments, an indicator of amplification may be
confirmed as indicating target nucleic acid amplification when the
indicator is determined to be within an identified cluster of melt
temperatures. For example, a first identified cluster may be
associated with a first target nucleic acid based on a comparison
between the measured melt temperature (or melt temperature range)
for the identified cluster and the expected melt temperature for
the first target nucleic acid. Similarly, a second identified
cluster may be associated with a second target nucleic acid based
on a comparison between the measured melt temperature (or melt
temperature range) for the identified cluster and the expected melt
temperature for the second target nucleic acid. In such an
embodiment, indicators may be confirmed for each target nucleic
acid based on the identified melt temperature cluster for the
indicators. In some embodiments, it may be determined that
indicators that are not within one of the identified clusters do
not comprise amplified target nucleic acid. Accordingly, these
indicators may be discounted when quantifying the amplification of
the target nucleic acids.
[0091] In an embodiment, amplification of each target nucleic acid
may be quantified based on indicators of amplification confirmed by
clustering for each target nucleic acid, as described herein, or
indicators of amplification confirmed based on an expected melt
temperature (or an expected melt temperature range) for each target
nucleic acid. For example, a Poisson model may be used along with a
total number of sample reaction volumes and total number of
non-amplifying sample reaction volumes, distinguished using
indicators changes at an expected melt temperature or indicators
that are not part of a cluster, to calculate a mean number of
reactions per sample reaction volume. The result can he divided by
the mean volume of each sample reaction volume to arrive at the
copies per unit volume for the reaction product melting at the
expected melt temperature or temperature range.
[0092] FIGS. 3A-3D illustrate another exemplary method for
performing dPCR. The method described herein may be performed with
the sample chip, as described further below, a circuit hoard
comprising through holes, or with any other suitable device, such
as the exemplary devices described herein. For example, FIGS. 3A-3D
may describe methods for performing dPCR and quantifying a target
nucleic acid using an end point detection scheme together with one
or more of real-time detection, single point detection, interval
detection, and rapid detection, as illustrated in FIG. 1B.
[0093] At 302A of FIG. 3A, a sample may be segregated, distributed,
or divided into a plurality of sample reaction volumes. For
example, the plurality of sample reaction volumes may be segregated
such that a first plurality of the sample reaction volumes contain
at least one molecule of a target nucleic acid and a second
plurality of the sample reaction volumes contain no molecules of
the target nucleic acid. The sample may be fractionated by a
dilution process so that each sample reaction volume contains one
copy, approximately one copy, or no copy of the nucleic acid
template a target, copy of the target nucleic acid or less. In an
embodiment, the sample reaction volumes may range from about 1 aL
to 50 uL. In other embodiments, the reaction volumes may be
approximately 1 nL, 1 pL, 33 nL, or any other suitable volume.
[0094] In one exemplary embodiment, the plurality of sample
reaction volumes may be segregated on a sample holder similar to
sample holder 900 of FIG. 9, described in further detail below,
although various other devices may be used to segregate the sample
reaction volumes and implement the amplification detection
techniques described herein. Accordingly, the sample reaction
volumes may be segregated among the plurality of rear sites (e.g.,
wells or through-holes) of sample holder 900.
[0095] At 304A, the plurality of sample reaction volumes are
subjected to an amplification assay. For example, the plurality of
sample reaction volumes may be simultaneously subjected to an
amplification assay, wherein the amplification assay is designed to
amplify a target nucleic acid to produce amplified product (i.e.,
amplicons). The assay may comprise at least a probe, a primer, and
an enzyme, such as a Taqman.TM. assay or any other suitable
assay.
[0096] In one embodiment, the method of FIG. 3A may progress to
306B of FIG. 3B, where real time detection may be used to quantify
target nucleic acid amplification. For example, detection points
104 of FIG. 1B may illustrate real time detection during digital
amplification, and this real time detection may be used to quantify
the amplification of the target nucleic acid.
[0097] In one embodiment, the method of FIG. 3A may progress to
3160 of FIG. 3C, where a melt stage may be used to quantify target
nucleic acid amplification. For example, one or more of detection
point 106, detection points 108, and detection points 110 of FIG.
1B may illustrate detection schemes for detecting an indicator of
amplification, and this detection may be used to quantify the
amplification of the target nucleic acid.
[0098] In one embodiment, the method of FIG. 3A may progress to
328D of FIG. 3D, where an angle of launch may be used to quantify
target nucleic acid amplification. For example, one or more of
detection point 106, detection points 108, and detection points 110
of FIG. 1B may illustrate detection schemes for detecting an
indicator of amplification, and this detection may be used to
quantify the amplification of the target nucleic acid.
[0099] FIG. 3B illustrates exemplary elements for a real time
detection scheme of an indicator of amplification during digital
amplification (e.g., dPCR assay). At 306B, a plurality of
measurements of an indicator of amplification may be taken for each
of the plurality of sample reaction volumes at a predetermined
amplification assay temperature while subjecting the reaction
volumes to the amplification assay. For example, the indicator of
amplification exhibited by a reaction volume may indicate the
presence of amplified product (e.g., amplification of the target
nucleic acid molecule).
[0100] In an embodiment, one or more dyes may be used that
fluoresce when bound to double-stranded nucleic acids, and this
fluorescence may be detected as an indicator of amplification. For
example, the nucleic acid binding agent (dye) may produce a
detectable signal when bound to double-stranded nucleic acids that
is distinguishable from the signal produced when that same agent is
in solution or bound to a single-stranded nucleic acid. The
fluorescence may be detected using a fluorescence detector, for
example mounted over a chip that houses the segregated sample
reaction volumes, or may be detected in any other suitable manner.
In an embodiment, the plurality of sample reaction volumes
subjected to the amplification assay may be further subjected to a
plurality or PCR steps, such as thermal cycling, as described
herein. In one exemplary embodiment, the plurality of measurements
may be taken during each PCR cycle at the predetermined
amplification assay temperature. FIG. 5 illustrates a graph of
illustrative, prophetic exemplary measurement results for the
indicator of amplification. The measured indicator of amplification
for the plurality of sample reaction volumes is represented by the
"Property" attribute on the y-axis and the PCR thermal cycles are
represented on the x-axis.
[0101] Referring again to FIG. 3B, at 308B, quantification cycle
(Cq) or cycle threshold (Ct) values may be determined for the
plurality of sample reaction volumes based on the obtained
measurements. A Cq value may be the cycle in which an indicator of
amplification can be measured and Ct value may be the value at
which the measured indicator of amplification reaches a threshold
value (e.g., predetermined threshold value). For example, with
reference to FIG. 5, reaction volumes represented by the lines 502
have approximately the same Ct values. Line 504 represents reaction
volumes with a later Ct value, and lines 506 represent additional
reaction volumes with the latest Ct values. In an embodiment, the
Cq values meas ed indicator values at a cycle) may be deterermined
at each cycle at a predetermined point during the cycle, at a
plurality of predetermined cycles, or based on any suitable
period.
[0102] In embodiments that leverage a multiplexing assay, reagents
may be implemented that exhibit multiple indicators of
amplification. For example, a multiplexing assay may include two
probes, such as a FAM.TM. dye-labeled probe and a VIC.RTM.
dye-labeled probe, and each probe may designed to exhibit an
indicator of amplification based on one of a plurality of target
nucleic acids. In some embodiments, an indicator of amplification
based on the FAM dye-labeled probe may fluoresce at a different
wavelength than an indicator of amplification based on the VIC
dye-labeled probe. Accordingly, measured indicators (e.g.,
fluorescence) may be associated with a particular target nucleic
acid based on the wavelength emitted. In these embodiments,
measured Cq or Ct values may be attributed to a particular target
nucleic acid based on the wavelength for the measured
indicator.
[0103] At 310B, the determined Cq or Ct values may be compared to
an expected Cq or Ct value. For example, based on the particular
amplification assay implemented and the target nucleic acid, an
expected Cq value or expected Ct value may be determined. The Cq or
Ct values determined for the indicators exhibited by the reaction
volumes based on measurements taken may be compared to the expected
CQ or Ct value.
[0104] In embodiments that leverage multiple indicators of
amplification, determined Cq or Ct values may be associated with
particular target nucleic acids, and the comparison may include
comparing associated Cq or Ct values with expected Cq or Ct values
for the associated target nucleic acid. For example, a first target
nucleic acid may be associated with an indicator of amplification
that fluoresces at a first wave length and a second target nucleic
acid may be associated with an indicator of amplification that
fluoresces at a second wave length. Cq or Ct values for each target
nucleic acid may be determined based on fluorescence at each wave
length, and the determined Cq or Ct values may be compared to
expected Cq or Ct values for the target nucleic acid associated
with the determined values. At step 312B, erroneous amplification
may be detected based on the comparisons. For example, the expected
Ct value may match (or substantially match) the Ct value for the
indicators exhibited by the reaction volumes represented by lines
502. Accordingly, the measured indicator of amplification for the
reaction volumes represented by lines 502 may indicate
amplification of target nucleic acids. On the other hand, the Ct
values for reaction volumes represented by lines 504 and 506 do not
match the expected Ct value. Accordingly, the measured indicator of
amplification for the reaction volumes represented by lines 504 and
506 indicates amplification of something other than the target
nucleic acid or detection of amplification that is triggered by
some other source (e.g., primer dimer, dust, misincorporation, or
any other suitable source) and not by actual detection of amplified
target nucleic acids. Comparison of determined Cq values and
expected Cq values may be similarly implemented to detect erroneous
amplification.
[0105] In embodiments that leverage multiple indicators of
amplification, Ct or Cq values associated with a particular target
nucleic acid (e.g., based on the wavelength for indicators measured
to determine the Ct or Cq values) may be used to detect erroneous
amplification. For example, a first set of Ct or Cq values may be
associated with a first target nucleic acid and a second set of Ct
or Cq values may be associated with a second target nucleic. A
comparison between the first set of Ct or Cq values and the
expected Ct or Cq values for the first target nucleic acid may be
used to detect erroneous amplification measured as a result of
indicators of amplification associated with the first target
nucleic acid (e.g., based on a wavelength). Similarly, a comparison
between the second set of Ct or Cq values and the expected Ct or Cq
values for the second target nucleic acid may be used to detect
erroneous amplification measured as a result of indicators of
amplification associated with the second target nucleic acid. Thus,
depending on the wavelength for an exhibited indicator, a
comparison may be made to one of the expected Ct or Cq values to
identify erroneous amplification (or an indicator exhibited based
on something other than amplified target nucleic acid).
[0106] As 314B in FIG. 3B, the amplified product the target nucleic
acid resulting from the amplification process may be quantified.
For example, the amount of amplification may be directly
proportional to the measured indicator of amplification for the
reaction volumes that comprised a Cq or Ct value substantially
similar to the expected Cq or Ct value. In an embodiment, the
quantification also may he based on identification of the measured
indicator of amplification triggered by a source other than target
nucleic acid amplification. For example, reaction volumes that
comprise a Cq or Ct value that do not substantially match the
expected Cq or Ct value may include a measured indicator of
amplification that corresponds to sources other than target nucleic
acid amplification. Accordingly, the measured indicator of
amplification for these reaction volumes may be discounted when
quantifying the amplification for the target nucleic acid.
[0107] In embodiments that leverage a multiplexing assay, amplified
product of each of a plurality of target nucleic acids may be
quantified. For example, the amount of amplified product for a
first target nucleic acid may be directly proportional to the
measured indicators of amplification associated with the first
target nucleic acid that comprised a Cq or Ct value substantially
similar to the expected Cq or Ct value for the first target nucleic
acid. An amount of amplified product for a second target nucleic
acid may be similarly quantified. Indicators exhibited by reaction
volumes that do not substantially match the expected Cq or Ct
values for either of the target nucleic acids may include measured
indicators of amplification that correspond to sources other than
target nucleic acid amplification.
[0108] In an embodiment, measured indicators of amplification for
the reaction volumes that included a Cq or Ct value substantially
similar to the expected Cq or Ct value (for at least one target
nucleic acid) may confirm expected amplification, and amplification
of the target nucleic acid may be quantified based on these
confirmed indicators. For example, a Poisson model may be used
along with a total number of sample reaction volumes and total
number of non-amplifying sample reaction volumes, distinguished
using Cq car Ct value substantially similar to the expected Cq or
Ct value, to calculate a mean number of reactions per sample
reaction volume. The result can be divided by the mean volume of
each sample reaction volume to arrive at the copies per unit volume
for the reaction product that includes a Cq or Ct value
substantially similar to the expected Cq or Ct value.
[0109] FIG. 3C illustrates an exemplary method that utilizes a melt
stage and detection of an indicator of post digital amplification.
At 316C, after subjecting the reaction volumes to the amplification
assay, a set of first measurements of an indicator of amplification
may be taken for each of the plurality of sample reaction volumes
at a first temperature. In an embodiment, the measurement of the
indicator of amplification may be taken for each of the plurality
of sample reaction volumes at a first temperature
post-amplification (e.g., ambient temperature). For example, the
set of first measurements may comprise end point detection 102 of
FIG. 1B, taken at ambient temperature after performance of an
amplification assay.
[0110] In an embodiment, the indicator of amplification measured
may be based on an intercalating dye (e.g., SYBR.RTM. dye) that
binds to double stranded nucleic acids, as described herein. In
some embodiments, the indicators measured for real time detection
of amplified nucleic acids (e.g., based on Cq or Ct values or based
on angle of launch analysis) may be different from the indicators
measured for the melt stage analysis. For example, the real time
detection techniques may leverage a non-intercalating dye or probe
based indicators that are not useful to a melt analysis. In other
embodiments, the indicator measured may be consistent throughout
the various detection techniques.
[0111] At 318C, at least one set of additional measurements may be
taken of the indicator of amplification at a post-amplification
temperature that is higher than the first temperature. For example,
an initial measurement of the indicator of amplification may
include measuring the properties of sample reaction volumes while
the volumes are at a first temperature (e.g., ambient temperature)
after amplification (e.g., PCR amplification). FIG. 4 illustrates a
graph of illustrative, prophetic exemplary detection results. The
indicator of amplification (e.g., fluorescence) for the sample
reaction volumes may be represented by the "Property" attribute on
the y-axis and the temperature may be represented by the x-axis.
Accordingly, the measured indicator of amplification for the
plurality of reaction volumes measured at an ambient temperature is
illustrated in the graph of FIG. 4.
[0112] In an embodiment, the sample reaction volumes may be heated
at a constant rate over a period of time, such as 10 minutes, 15
minutes, 30 minutes, 1 hour, or any other suitable period of time.
During the heating, the indicator of amplification exhibited by the
plurality of sample reaction volumes may be measured at least once,
and, in some embodiment, a plurality of times.
[0113] In an embodiment, indicator of amplification (e.g.,
fluorescence) exhibited by the plurality of sample reaction volumes
may be measured at a series of intervals during the heating. For
example, FIG. 4 illustrates a graph of illustrative, prophetic
exemplary detection results, where the indicator of amplification
may be measured at various target temperatures. In an embodiment,
the target temperatures may be based on temperature intervals
(e.g., 5.degree. C., 10.degree. C., and the like), or may include a
predetermined set of target temperatures. At each target
temperature, the indicator of amplification may be measured such
that changes in the indicator may be identified.
[0114] In an embodiment, the indicator of amplification exhibited
by the plurality of sample reaction volumes may be measured rapidly
at closely spaced intervals during the heating. In analyzing the
rapidly measured property, trends for the indicator of
amplification exhibited by the plurality of reaction volumes may be
determined. For example, the analysis may show a trend that a set
of sample reaction volumes from the plurality of reaction volumes
exhibits a decrease in the indicator of amplification as the
temperature increases. The trends may be similar to results
determined from comparisons during interval detection during the
temperature increase.
[0115] In an embodiment, the rapid detection may enable
identification of trends at specific temperatures (e.g,
temperatures other than the target temperatures of the interval
detection). For example, a temperature (or temperature window) may
trigger a noticeable change (e.g., change beyond a threshold) in
the indicator of amplification exhibited by some of the reaction
volumes. The rapid detection may enable identification of these
temperatures, or temperature windows, via analysis of the changes
in the indicator of amplification. Accordingly, the rapid detection
methodology may provide enhanced sensitivity to the analysis.
[0116] At 320C, identified changes in the indicator of
amplification may be associated with the post-amplification
temperature for the change. For example, referring again to FIG. 4,
identified change 404 may be associated with the temperature
75.degree. C. and identified change 408 may be associated with the
temperature 85.degree. C. Identified changes 402 may comprise
multiple changes, and each change may be associated with one of
temperature 55.degree. C. and 65.degree. C., as illustrated.
[0117] In an embodiment, during heating, measurement of the
indicator of amplification may occur rapidly. Similar to the
interval detection analysis, the identified changes to measured
indicator of amplification for the plurality of reaction volumes at
various temperatures may be associated with each temperature for
the change.
[0118] At 322C, the associated temperatures may be compared to an
expected melt temperature. For example, based on the particular
amplification assay implemented and the target nucleic acid, an
expected melt temperature (or expected melt temperature range) may
be predetermined such that the target nucleic acid would be
expected to melt at the temperature. The associated temperatures
for changes in the indicator of amplification based on the
measurements taken may be compared to the expected melt
temperature.
[0119] At 324C, erroneous amplification may be identified based on
the comparisons. In an embodiment, the identified changes in the
indicator of amplification for the plurality of reaction volumes
may be analyzed to determine changes triggered by a source other
than amplification of the target nucleic acid. A change in the
indicator of amplification exhibited by a reaction volume based on
amplification of the target nucleic acid would be expected to
decrease at the expected melt temperature or temperature range
(e.g., fluorescence exhibited by a reaction volume would be
expected to decrease at the expected melt temperature). For
example, the expected melt temperature for the target nucleic acid
here may be 70.degree. C., or an expected melt temperature range
may be from 65.degree. C. to 75.degree. C.
[0120] In an embodiment, a single point detection algorithm may be
used with the endpoint detection (e.g., at ambient temperature), as
illustrated by detection point 106 of FIG. 1B. For example, an
identified change may be determined based on the detection results
at ambient temperature (e.g., detection point 102) and the
predetermined temperature for single point detection (e.g.,
detection point 106). One of identified changes 402 may comprise
the identified change based on the single point detection.
[0121] For example, the detected change at these temperatures may
include a further separation of the reaction volumes that exhibit
indicators of amplification triggered by nucleic acid amplification
from the reaction volumes that do not. Reaction volumes that
exhibit some indicator of amplification may show a decrease such
that the background noise of the results is reduced. For example,
when a dye, such a SYBR dye (or other intercalating dye), is used
to produce the indicator of amplification, the dye may bond with
various reaction products (e.g., nucleic acids) due to the
non-specific design of the dye. Accordingly, background noise
(e.g., fluorescence) may be caused by reaction products bonding to
a non-specific dye. In an embodiment, reaction volumes that exhibit
the indicator of amplification may be discernible from reaction
volumes that do not based on the further separation, and the
amplification of the target nucleic acid may be more accurately
quantified based on the discerned reaction volumes.
[0122] In an embodiment, an interval detection algorithm may be
used with the endpoint detection (e.g., at ambient temperature), as
illustrated by detection points 108 of FIG. 1B. For example,
identified changes may be determined based on the detection results
at ambient temperature (e.g., detection point 102) and the
predetermined temperatures for interval detection (e.g., detection
points 108). FIG. 4 may illustrate changes 402 (as discussed
above), 404, and 406 based on interval detection.
[0123] Identified change 404, associated with temperature
75.degree. C., may indicate changes to indicators triggered by
amplification of the target nucleic acid. Because the expected melt
temperature for the target nucleic acid comprises 70.degree. C., or
a range between 65.degree. C. to 75.degree. C., the decrease of the
exhibited indicator detected at 75.degree. C. correspond to
indicator triggered by amplification of the target nucleic acid.
Identified change 408, associated with temperature 85.degree. C.,
may indicate changes to indicators triggered by a source other than
amplification of the target nucleic acid. Because the expected melt
temperature for the target nucleic acid comprises 70.degree. C., or
a range between 65.degree. C. to 75.degree. C., the changes
detected at 85.degree. C. correspond to indicators triggered by
other sources. Indicators of amplification 410, measured at
temperature 95.degree. C., also corresponds to indicators triggered
by a source other than amplification of the target nucleic acid. At
such a high temperature, nucleic acids would be expected to melt,
and therefore indicators 410 may correspond to a source like dust,
or some other source that may cause the indicator to persist above
certain temperature thresholds.
[0124] In an embodiment, during heating, indicator measurement may
occur rapidly in lieu of or in addition to set temperature point
interval measurement. For example, rapid measurement algorithm may
be used with the endpoint measurement (e.g., at ambient
temperature), as illustrated by detection points 110 of FIG. 1B.
Identified changes may be determined based on the detection results
at ambient temperature (e.g., detection point 102) and rapidly over
the melt temperatures (e.g., detection points 110).
[0125] For example, changes in the indicators (e.g., decreases) at
temperatures other than the expected melt temperature (or expected
melt temperature range) for the target nucleic acid may correspond
to sources other than target nucleic acid amplification (e.g.,
indicators triggered by something other than target nucleic acid
amplification). On the other hand, changes to indicators (e.g.,
decreases) at the expected melt temperature (or expected melt
temperature range) for the target nucleic acid may correspond to
target nucleic acid amplification (e.g., indicators triggered by
target nucleic acid amplification).
[0126] As 326C, the amount of amplified product of the target
nucleic acid resulting from the amplification process may be
quantified. For example, indicators exhibited by the reaction
volumes at ambient temperature may suggest a level of amplification
that occurred in each reaction volume, however not all of the
amplification may comprise the target nucleic acid. In other words,
the indicators of amplification presented by the reaction volumes
at ambient temperature may indicate amplification of something
other than the target nucleic acid, or may be triggered by some
other source. These may comprise, for instance, primer dimer,
misincorporation, dust, or any other suitable source. Performance
of the heating and a subsequent analysis of the measured property
may enable identification of indicators that are not the results of
amplification of the target nucleic acid. After considering the
indicators that are related to some other source, the amplification
for the target nucleic acid be quantified with greater
accuracy.
[0127] In an embodiment, the identified changes in the indicators
for the plurality of reaction volumes may be analyzed to determine
indicators triggered by a source other than amplification of the
target nucleic acid. For example, based on the particular target
nucleic acid, an expected melt temperature (or expected melt
temperature range) may be determined such that the target nucleic
acid would be expected to melt at the temperature. Therefore, an
indicator exhibited by a reaction volume based on amplification of
the target nucleic acid would be expected to decrease at the
expected melt temperature temperature range (e.g., fluorescence
exhibited by a reaction volume would be expected to decrease at the
expected melt temperature).
[0128] In an embodiment the amplification may he directly
proportional to the change detected to indicators (e.g., detected
decrease in fluorescence) at the expected melt temperature (or
expected melt temperature range). In an embodiment, the
quantification may also be based on identification of indicators
triggered by a source other than target nucleic acid amplification.
For example, it may be determined that indicators of amplification
that changed (e.g., decreased) at temperatures other than the
expected melt temperature (or expected melt temperature range)
correspond to sources other than target nucleic acid amplification.
Accordingly, these indicators may be discounted when quantifying
the amplification for the target nucleic acid.
[0129] In an embodiment, clustering of measured melt temperatures
may be used to identify target nucleic acid amplification and
quantify amplified target nucleic acids. For example, decreases to
a plurality of indicators of amplification associated with a
plurality of sample reaction volumes may be measured at a
particular temperature (or within a temperature range) to determine
melt temperatures for these indicators, as described herein. These
measured melt temperatures may be clustered, for example, based on
calculated Euclidean distances and/or calculated silhouette values
such that clusters of indicators with similar melt temperatures may
be determined. Reference is made to HRM Experiments, Using
MeltDoctor.TM. HRM Reagents and High Resolution Melt Software
v.3.0, Life Technologies, 2010, which reviews the use of silhouette
scores for clustering when performing a melt using available
software tools. Those of ordinary skill in the art will recognize
various additional techniques exist for clustering data that may be
implemented to obtain clusters for the purposes of the present
embodiments. Accordingly, one or more clusters may be identified
that comprise indicators of amplification with similar melt
temperatures.
[0130] In some embodiments, an indicator of amplification may be
confirmed as indicating target nucleic acid amplification when the
indicator is determined to be within an identified cluster of melt
temperatures. For example, a first identified cluster may be
associated with a first target nucleic acid based on a comparison
between the measured melt temperature (or melt temperature range)
for the identified cluster and the expected melt temperature for
the first target nucleic acid. Similarly, a second identified
cluster may be associated with a second target nucleic acid based
on a comparison between the measured melt temperature (or melt
temperature range) for the identified cluster and the expected melt
temperature for the second target nucleic acid. In such an
embodiment, indicators may be confirmed for each target nucleic
acid based on the identified melt temperature cluster for the
indicators. In some embodiments, it may be determined that
indicators that are not within one of the identified clusters do
not comprise amplified target nucleic acid. Accordingly, these
indicators may be discounted when quantifying the amplification of
the target nucleic acids.
[0131] In an embodiment, amplification of each target nucleic acid
may be quantified based on indicators of amplification confirmed by
clustering for each target nucleic acid, as described herein, or
indicators of amplification confirmed based on an expected melt
temperature (or an expected melt temperature range) for each target
nucleic acid. For example, a Poisson model may be used along with a
total number of sample reaction volumes and total number of
non-amplifying sample reaction volumes, distinguished using
indicators changes at an expected melt temperature or indicators
that are not part of a cluster, to calculate a mean number of
reactions per sample reaction volume. The result can be divided by
the mean volume of each sample reaction volume to arrive at the
copies per unit volume for the reaction product melting at the
expected melt temperature or temperature range.
[0132] FIG. 3D illustrates exemplary elements for an angle of
launch detection scheme to identify amplification during digital
amplification. At 328D, a plurality of measurements of an indicator
of amplification may be taken for each of the plurality of sample
reaction volumes at a predetermined amplification assay temperature
while subjecting the sample reaction volumes to the amplification
assay. For example, the indicator of amplification exhibited by a
sample reaction volume may indicate the presence of amplified
product (e.g., amplification of the target nucleic acid
molecule).
[0133] In an embodiment, the plurality of measurements of an
indicator of amplification taken for each of the plurality of
sample reaction volumes may be performed similarly to 306B of FIG.
3B. For example, one or more dyes and/or probes may be used that
fluoresce when bound to double-stranded nucleic acids, and this
fluorescence may be detected as an indicator of amplification. The
fluorescence may be detected using a fluorescence detector angled
to detect fluorescence from the segregated reaction volumes. In an
embodiment, the plurality of reaction volumes subjected to the
amplification assay may be further subjected to a plurality of PCR
steps, such as thermal cycling, as described herein. In one
exemplary embodiment, the plurality of measurements may be taken
during each thermal cycle at a predetermined amplification assay
temperature.
[0134] FIGS. 6A and 6B illustrate graphs of illustrative, prophetic
exemplary measurement results for the indicator of amplification.
Graphs 600A and 600E illustrate angle of launch analysis based on
the plurality of measurements at different cycle numbers during
amplification. In an embodiment, the measurements may be taken at
each cycle at a predetermined point during the cycle, at a
plurality of predetermined cycles, or based on any suitable
period.
[0135] In some embodiments, a sample reaction volume may exhibit
multiple indicators of amplification. For example, multiples dyes
and/or probes may be implemented such that a sample reaction volume
exhibits one or more indicators based on the amplification
reactions occurring in the sample reaction volume. In some
embodiments, a first indicator may indicate amplification of a
first target nucleic acid while a second indicator may indicate
amplification of a second target nucleic acid. For instance, Graphs
600A and 600B may illustrate amplification results for reactions
that leverage two probes (e.g., FAM.TM. and VIC.RTM. probes), or
any other suitable probes and/or dyes. Here, measured indicators of
amplification based on a first of the probes are shown on the
x-axis and measured indicators of amplification for a second of the
probes are shown on the y-axis. In an embodiment, the first probe
may be designed to amplify a first target nucleic acid while the
second probe may be designed to amplify a second target nucleic
acid.
[0136] In an embodiment where the plurality of measurements were
previously taken, for instance an embodiment where the method of
FIG. 3B is performed prior to the method of FIG. 3D, step 328D may
be omitted. For example, data for the plurality of measurements may
be available based on the measurements taken during the method of
FIG. 3B, and thus the data may be analyzed, as described herein,
without need for obtaining additional measurements.
[0137] Referring again to FIG. 3D, at 330D, an angle of launch for
measured indicators of each of the plurality of sample reaction
volumes may be determined based on the measurements taken at
various cycles. For example, graph 600A of FIG. 6A illustrates
measurement values for indicators of amplification taken at a first
cycle during the amplification process and graph 600B of FIG. 6B
illustrates measurement values for indicators of amplification
taken at a second cycle during the amplification process, where the
second cycle is some time after the first cycle. In an embodiment,
the first cycle may be at cycle 27 of the amplification process and
the second cycle may be at cycle 40 of the amplification
process.
[0138] In an embodiment, for each cycle at which measurements were
taken (e.g., at 328D or any previous elements in FIG. 3D at which
real time measurements were taken) values may be stored for the
indicator measurement values of each of the plurality of sample
reaction volumes at that cycle. Here, the measurements taken at
cycle 27 are illustrated in graph 600A of FIG. 6A and the
measurements taken a cycle 40 are illustrated in graph 600B of FIG.
6B.
[0139] The measurements at each measured cycle may be analyzed to
determine the angle of launch (or trajectory) for each reaction
volume. For instance, a measured data point associated with a
reaction volume at a given measured cycle may be compared to a
reference point (e.g., origin point (0,0)) such that an angle may
be determined. In some embodiments, the particular angles for given
data points may be determined based on the tan.sup.-1 function and
the x and y values for the data point. Over the duration of
amplification (e.g., over the totality of cycles or up to a
predetermined cycle), the angles determined at each measured cycle
for each reaction volume (e.g., each data point) may be averaged
(or smoothed) such that an angle of launch may he determined for
indicators measured from the individual reaction volumes. In some
embodiments, a slope for the angles of launch determined for the
given reaction volumes may be calculated using a regression
analysis or by some other suitable manner.
[0140] In an embodiment, graph 600A of FIG. 6A illustrates
exemplary measured values 602A for the plurality of sample reaction
volumes at cycle 27. In this example, the measured values for
indicators of amplification have not separated such that the values
may be distinguished or such that the angles of launch determined
for sample reaction volumes may be distinguished. In an embodiment,
graph 600B of FIG. 6B illustrates exemplary measured values for the
plurality of sample reaction volumes at cycle 40. In this example,
the indicators have separated such that the values may be
distinguished or such that the angles of launch determined for
sample reaction volumes may be distinguished. Accordingly, the
sample reaction volumes associated with the illustrated data points
in graphs 600A and 600B may comprise determined angles of
launch.
[0141] Referring again to FIG. 3D, at 332D, the determined angles
of launch may be analyzed to determine differences and similarities
between the plurality of sample reaction volumes. In an embodiment,
data points 604B in FIG. 6B may illustrate measurements taken for a
plurality of sample reaction volumes that share a similar indicator
measurement at cycle 40 and that share a similar determined angle
of launch (e.g., based on the measurements taken at each measured
cycle up to cycle 40). Similarly, data points 606B may illustrate
measurements taken for a plurality of sample reaction volumes that
share a similar indicator measurement at cycle 40 and that share a
similar determined angle of launch. These data points may be
clustered, as described herein or using known clustering
algorithms, based on one or both of indicator measurements at cycle
40 and/or calculated angles of launch. In an embodiment, the angles
of launch may be compared based on the calculated slopes, where
angles may be declared similar when a calculated slope for a first
angle of launch is within a threshold value of a calculated slope
for a second angle of launch.
[0142] In an embodiment, the determined angles of launch and/or
calculated slopes may confirm whether an indicator exhibited by a
sample reaction volume corresponds to amplification of a first
target nucleic acid, a second target nucleic acid, or both. For
instance, in graphs 600A and 600B of FIGS. 6A and 6B measured
indicators of amplification based on a first probe are shown on the
x-axis and measured indicators of amplification based on a second
probe are shown on the y-axis. In an embodiment, the first probe
may be designed to amplify a first target nucleic acid while the
second probe may be designed to amplify a second target nucleic
acid. Thus, an angle of launch within a first range (e.g.,
approximately 60.degree. to 90.degree.) may confirm that a data
point is based on amplification of the first target nucleic acid.
Similarly, an angle of launch within a second range (e.g.,
approximately 0.degree. to 30.degree.) may confirm that a data
point is based on amplification of the second target nucleic acid.
An angle of launch within a third range (e.g., approximately
30.degree. to 60.degree.) may confirm that a data point is based on
amplification of both the first target nucleic acid and the second
target nucleic acid.
[0143] In some embodiments, the angle of launch analysis may flag
the sample reaction volumes associated with these data points as
containing validated target nucleic acid amplification for one or
more target nucleic acids. For instance, sample reaction volumes
associated with clustered data points 604B may be flagged as
containing validated amplified first target nucleic acid based on
the angle of launch for the cluster. Similarly, sample reaction
volumes associated with clustered data points 604B may be flagged
as containing validated amplified first target nucleic acid and
amplified second target nucleic acid based on the angle of launch
for the cluster.
[0144] Data points 608B may illustrate measurements taken for two
sample reaction volumes that share a similar indicator measurement
at cycle 40 and that share a similar determined angle of launch,
however, these sample reaction volumes may not share these values
with other sample reaction volumes. For instance, data points 604B
and 606B each form a cluster, while data points 608B do not. In
some examples, these values may indicate false indicators of
amplification. For instance, the angle of launch analysis may flag
the sample reaction volumes associated with these data points as
containing off-target amplicons (amplicons that are something other
than amplified target nucleic acid). Detection techniques as
described with reference to FIGS. 3A-3C may be implemented with the
angle of launch analysis to further confirm whether these sample
reaction volumes contain off-target amplicons.
[0145] In an embodiment, data point 610B may illustrate a
measurement taken for a sample reaction volume at cycle 40 that is
not similar to other sample reaction volumes. For instance, the
measurement value at cycle 40 for the sample reaction volume
associated with data point 610B may be different from the
measurement values at cycle 40 for the dusters of sample reaction
es associated with data points 604B and 606B. However, the
determined angle of launch for the reaction volume associated with
data point 610B may be similar to the determined angles of launch
for the cluster of sample reaction volumes associated with data
points 604B. This determination may be based on compared slopes for
the determined angles of launch, as described herein. In this
embodiment, the similar angles of launch may indicate that the
sample reaction volume associated with data point 610B contains
amplified target nucleic acid consistent with the cluster of sample
reaction volumes associated with data points 604B.
[0146] The difference between the measurements take at cycle 40 for
these data points may be due to detection technique failures rather
than amplification errors (e.g., off-target amplification). For
instance, reagents used to produce the indicator of amplification
may be present in low quantities, or other issues may he
experienced by the amplification detection processes. In an
embodiment, the angle of launch analysis may flag the sample
reaction volume associated with data point 610B as containing
validated target nucleic acid amplification consistent with the
duster of sample reaction volumes associated with data points 604B.
Detection techniques as described with reference to FIGS. 3A-3C may
be implemented with the angle of launch analysis to further confirm
whether this sample reaction volume contains amplified target
nucleic acid.
[0147] In an embodiment, data point 612B may also illustrate a
measurement taken for a sample reaction volume at cycle 40 that is
not similar to other sample reaction volumes. For instance, the
measurement value at cycle 40 for the sample reaction volume
associated with data point 612B may be different from the
measurement values at cycle 40 for the clusters of sample reaction
volumes associated with data points 604B and 606B. However, the
determined angle of launch for the sample reaction volume
associated with data point 612B may be similar to the determined
angles of launch for the cluster of sample reaction volumes
associated with data points 606B. In this embodiment, the shared
angle of launch may indicate that the sample reaction volume
associated with data point 612B contains amplified target nucleic
acid consistent with the cluster of sample reaction volumes
associated with data points 606B.
[0148] The difference between the measurements take at cycle 40 may
also be due to detection technique failures rather than
amplification errors (e.g., off-target amplification). The angle of
launch analysis may flag the sample reaction volume associated with
data point 612B as containing validated target nucleic acid
amplification consistent with the cluster of sample reaction
volumes associated with data points 606B. Detection techniques as
described with reference to FIGS. 3A-3C may be implemented with the
angle of launch analysis to further confirm whether this sample
reaction volume contains amplified target nucleic acid.
[0149] Referring again to FIG. 3D, at 334D, erroneous amplification
may be identified based on the determined angles of launch and
additional amplification data. For example, data points 610B and
612B may each comprise conflicting factors as to whether the sample
reaction volumes associated with the data points contain amplified
target nucleic acid. For these data points, the clustering for
measurements taken at cycle 40 may indicate erroneous amplification
while the analyzed angle of launch may indicate on-target
amplification (amplification of target nucleic acids). Here,
additional data for the sample reaction volumes, such as Cq or Ct
values as determined by the method of FIG. 3B or melt profile
values as &tel r fined by the method of FIG. 3C, may be
leveraged to determine whether the sample reaction volumes contain
amplified target nucleic acid.
[0150] Graph 700A of FIG. 7A illustrates melt curves for a
plurality of sample reaction volumes based on data derived from a
melt technique, for instance a melt methodology as described in the
method of FIG. 2A, 2B, or 3C. The line style depicts the class of
the curves (e.g., amplified (solid), un-amplified (dotted),
determined as questionable (dashed)), based on the identification
technique described herein. Graph 700B of FIG. 7B illustrates a
first derivate of the melt curves illustrated in graph 700A of FIG.
7A. Here, the line style also depicts the class of the curves
(e.g., amplified (solid), un-amplified (dotted), determined as
questionable (dashed)), based on the identification technique
described herein, or other suitable identification techniques.
[0151] FIG. 8 illustrates a scatter plot of data points that depict
values for measured indicators of amplification prior to the
performance of a melt for a plurality of sample reaction volumes.
In this visualization, the data points have been classified based
on data from a melt detection technique. For instance, melt
temperatures associated with the indicator of amplification
exhibited by sample reaction volumes may be used to classify the
associated data points. Here, the data points are classified as
non-amplified (e.g., no or minimal detected indicator of
amplification prior to melt and after amplification), no clear melt
profile (e.g., the melt based technique did not identify a clear
melt temperature), and shade based melt temperature (e.g., melt
temperature for the measured indicator exhibited by a sample
reaction volume associated with a data point corresponds to shade
and accompanying temperature key).
[0152] In an embodiment, the identification from the melt curve
analysis (e.g., as amplified, un-amplified, or questionable) may be
used in combination with the angle of launch analysis to confirm
whether a sample reaction volume contains amplified target nucleic
acid or erroneous (or off-target) amplification (or emits an
erroneous indicator of amplification). When combining an angle of
launch detection technique with a melt detection technique, the
confirmation may be based on the comparison between expected melt
temperature and measured melt temperate for sample reaction
volumes. For example, the identification based on the melt curve
profiles for the sample reaction volumes associated with data
points 610B and 612B of FIG. 6B may be used in order to confirm the
amplification status of the sample reaction volumes.
[0153] In an embodiment, the identification from the real time
detection technique, as described with reference to FIG. 3B, may be
used in combination with the angle of launch analysis to determine
whether a sample reaction volume contains amplified target nucleic
acid or erroneous (or off-target) amplification (or emits an
erroneous indicator of amplification). Here, the confirmation may
be based on the comparison between expected Cq or Ct values and
measured Cq or Ct values for a sample reaction volume. For example,
the identification based on the expected Cq or Ct values and
measured Cq or Ct values for the sample reaction volumes associated
with data points 610B and 612B of FIG. 6B may be used in order to
confirm the amplification status of the sample reaction
volumes.
[0154] In an embodiment, the melt curve profile, the real time
detection technique based on the expected Cq or Ct values, and
detection technique based on a determined angle of launch may be
used in combination in order to determine the amplification status
of the sample reaction volumes. For example, combinations of two or
more of these described techniques may be leveraged to arrive at an
amplification status. In another embodiment, a sample reaction
volume may be determined as containing amplified target nucleic
acid when each of the detection techniques identifies the sample
reaction volume as containing amplified target nucleic acid (e.g.,
all three techniques confirm that the sample reaction volume
contains amplified target nucleic acid).
[0155] In embodiments where a multiplexing assay is implemented,
confirmation may be specific to a particular target nucleic acid.
For example, the melt curve profile, the real time detection
technique based on the expected Cq or Ct values, and the detection
technique based on a determined angle of launch may each correlate
indicators of amplification to one of a plurality or target nucleic
acids. Accordingly, a combination may be implemented to determine
the amplification status of the sample reaction volumes and which
particular amplified target nucleic acids are present (if any) in
the sample reaction volumes.
[0156] In an exemplary embodiment, quantification of the target
nucleic acid amplification may be based on both a real time
detection method (as illustrated in FIG. 3B) and a melt detection
technique (as illustrated in FIG. 3C). For example, quantification
of a target nucleic acid may be based end point detection (as
illustrated by detection point 102 of FIG. 1) and one or more of
real time detection (as illustrated by detection points 104 of FIG.
1), single point detection (as illustrated by detection point 106
of FIG. 1), interval detection (as illustrated by detection points
108 of FIG. 1) and rapid detection (as illustrated by detection
points 110 of FIG. 1). In exemplary embodiments, identification of
erroneous amplification and accurate target nucleic acid
amplification using real time detection techniques and/or malt
stage techniques (as described with respect to FIGS. 3B and 3C) may
each be used to quantify the amplification of a target nucleic
acid. For example, based on the type of target nucleic acid being
amplified, the amplification assay, the time/throughput desired for
the analysis, and any other suitable consideration, a combination
of end point analysis with one or more of the real time detection
and melt stage may be selected.
[0157] In an embodiment, real time detection of an indicator of
amplification during an amplification assay (as illustrated in FIG.
3) may also be used to determine a number of molecules present in a
reaction volume prior to digital amplification. For example, based
on the real time measurement of indicators of amplification for the
plurality reaction volumes, it may be determined whether each
reaction volume comprised zero, one, or more than one target
nucleic acid molecule prior to dPCR amplification.
[0158] In some embodiments, combinations of dyes or probes may be
used such that multiple indicators of amplification are exhibited
by a sample reaction volume. For instance, a sample reaction volume
may comprise detection reagents such that a first indicator of
amplification may indicate amplification of a first target nucleic
acid based on a first dye or probe, a second indicator of
amplification (different from the first indicator of amplification)
may indicate amplification of a second target nucleic acid based on
a second dye or probe, and a third indicator of amplification
(different from the first or second indicators of amplification)
may indicate amplification of a third target nucleic acid based on
a third dye or probe. In this example, particular indicators of
amplification may be measured and analyzed based on the real time
detection techniques, angle of launch detection techniques, and
melt detection techniques described herein, such that
quantification of amplified product for the individual first,
second, and third target nucleic acids may be achieved.
[0159] In some embodiments, a first probe designed to indicate
amplification of a first target nucleic acid and a second probe
designed to indicate amplification of a second target nucleic acid
may be implemented. The first and second indicators of
amplification may be used as part of a real time detection
technique (i.e., based on expected and measured Cq or Ct values)
and/or angle of launch detection technique. In addition, a dye may
be used along with the first and second probe such that a melt
stage may be performed in order to implement a melt detection
technique. Quantification of amplified product for the individual
first and second target nucleic acids may be achieved using a
combination of techniques.
[0160] In various embodiments, a sample holder may have a plurality
of sample sites or volumes, configured for receiving a plurality of
sample reaction volumes. Some examples of a sample holder may
include, but are not limited to, a multi-well plate, such as a
standard microtiter 96-well plate, a 384-well plate, a microcard, a
through-hole array, or a substantially planar holder, such as a
glass or plastic slide. The reaction sites in various embodiments
of a sample holder may include depressions, indentations, ridges,
through-holes, and combinations thereof, patterned in regular or
irregular arrays formed on the surface of the sample holder.
[0161] Referring to FIG. 9, in certain embodiments, a sample
holder, an article, chip, device, substrate, slide, or plate 900
comprises a substrate 902 containing a plurality of reaction sites
or reaction chambers 904 located in or on substrate 902. The
plurality of reaction sites 901 may comprise a plurality of
through-holes, wells, surface indentations, treated surface areas,
or the like. In certain embodiments, sample holder 900 may comprise
an article. Additionally or alternatively, sample holder 900 may
comprise a microfluidic device which, for example, may further
include a plurality of channels or paths for transferring reagents
and/or test solutions to reaction sites 904. In other embodiments,
reaction sites 904 comprise a plurality of droplets or beads and
sample holder 900 may comprise one or more chambers and/or channels
containing some or all of the droplets or beads 904. In such
embodiments, the droplets or beads may form an emulsion, where some
or all of the droplets or beads contain one or more target of at
least one polynucleotide or nucleotide sequence. Where reaction
sites 904 include beads, the beads may optionally include an
attached optical signature or label. Droplets or beads may be
inspected, monitored, or measured either one at time or in groups
containing one or more droplets or beads, for example using an
imaging system according to embodiments.
[0162] Reaction sites 904 may include reaction volumes located
within through-holes, wells or indentations formed in substrate
902, spots of solution distributed on the surface 910, or other
types of reaction chambers or formats, such as samples or solutions
located within test sites or volumes of a microfluidic system, or
within or on small beads or spheres.
[0163] Reaction sites 904 may be configured to provide sufficient
surface tension by capillary action to draw in respective amounts
of liquid or sample containing a biological components of interest.
Sample holder 900 may have a general form or construction as
disclosed in any of U.S. Pat. Nos. 6,306,578; 7,332,271; 7,604,983;
7,682,565; 6,387,331; or 6,893,877, which are herein incorporated
by reference in their entirety as if fully set forth herein.
Substrate 902 may be a flat plate or comprise any form suitable for
a particular application, assay, or experiment. Substrate 602 may
comprise any of the various materials known in the fabrication arts
including, but not limited to, a metal, glass, ceramic, silicon, or
the like. Additionally or alternatively, substrate 902 may comprise
a polymer material such as an acrylic, styrene, polyethylene,
polycarbonate, and polypropylene material. Substrate 902 and
reaction sites 904 may be formed by one or more of machining,
injection molding, hot embossing, laser drilling, photolithography,
or the like.
[0164] According to various embodiments of the present teachings,
each reaction site 904 may have a volume of about 1.3 nanoliters.
Alternatively, the volume of each reaction site may be less than
1.3 nanoliters. This may be achieved, for example, by decreasing
the diameter of reaction site 904 and/or the thickness of the
sample holder. For example, each reaction chamber 904 may have a
volume that is less than or equal to 1 nanoliter, less than or
equal to 100 picoliters, less than or equal to 30 picoliters, or
less than or equal to 10 picoliters. In other embodiments, the
volume some or all of the reaction sites 904 is in a range of 1 to
20 nanoliters.
[0165] In some embodiments, the reaction sites 904 are
through-holes. In these examples, each through-hole has a volume of
about 1.3 nanoliters. Alternatively, the volume each through-hole
may be less than 1.3 nanoliters. This may be achieved, for example,
by decreasing the diameter of through-hole and/or the thickness of
the sample holder. For example, each through-hole may have a volume
that is less than or equal to 1 nanoliter, less than or equal to
100 picoliters, less than or equal to 30 picoliters, or less than
or equal to 10 picoliters. In other embodiments, the volume some or
all of the through-holes is in a range of 1 to 20 nanoliters.
[0166] In various embodiments, a density of reaction sites 904 may
be at least 100 reaction sites per square millimeter. In other
embodiments, there may be higher densities of reaction chambers.
For example, a density of reaction sites 904 within chip 100 may be
greater than or equal to 150 reaction sites per square millimeter,
greater than or equal to 200 reaction sites per square millimeter,
greater than or equal to 500 reaction sites per square millimeter,
greater than or equal to 1,000 reaction sites per square
millimeter, greater than or equal to 10,000 reaction sites per
square millimeter.
[0167] In some embodiments, the reaction sites 904 comprise a
plurality of through-holes. Accordingly, a density of through-holes
within a sample holder may be greater than or equal to 150
through-holes per square millimeter, greater than or equal to 200
through-holes per square millimeter, greater than or equal to 500
through-holes per square millimeter, greater than or equal to 1,000
through-holes per square millimeter, greater than or equal to
10,000 through-holes per square millimeter.
[0168] In some embodiments, reaction volumes may be segregated
using through-holes, wells, or droplets. An exemplary volume range
for reaction volumes is 1 aL to 50 uL. In other embodiments, the
reaction volumes may be approximately 1 nL, 1 pL, 33 nL, or any
other suitable volume.
[0169] In certain embodiments, surface 910 may comprise a
hydrophobic material, for example, as described in US Patent
Application Publication Numbers 2006/0057209 or 2006/0105453, which
are herein incorporated by reference in their entirety as if fully
set forth herein. In such embodiments, reaction sites 904 may
comprise a hydrophilic material that attracts water or other liquid
solutions. An array of such hydrophilic regions may comprise
hydrophilic islands on a hydrophobic surface and may be formed on
or within substrate 902 using any of various micro-fabrication
techniques including, but are not limited to, depositions, plasmas,
masking methods, transfer printing, screen printing, spotting, or
the like.
[0170] Sample holder 900 may also include an identifier 918. In
this example, identifier 918 may be an alpha-numeric sequence.
However, it should be recognized that an identifier may be another
type of symbol or characters according to various embodiments
described herein. Identifier 918 may be, for example, a barcode, a
QR code, a symbol, a numeric sequence, an RFID identifier, or an
alpha sequence. Furthermore, although identifier 918 is shown in
the bottom right corner of sample holder 900, identifier 918 may be
located in any position on the sample holder as long as the
position is known and stored in memory of the system according to
various embodiments described herein.
[0171] FIG. 10 is a block diagram that illustrates the computer
system 1000. Instruments (e.g., the system or instrument 10 shown
in FIG. 1A and discussed above herein) to perform experiments may
be connected to the exemplary computing system 1000. According to
various embodiments, the instruments describer with reference to
FIGS. 9 and 11 may utilized with computing system 1000. Computing
system 1000 can include one or more processors, such as a processor
1004. Processor 1004 can be implemented using a general or special
purpose processing engine such as, for example, a microprocessor,
controller or other control logic. In this example, processor 1004
is connected to a bus 1002 or other communication medium.
[0172] Computing system 1000 may include bus 1002 or other
communication mechanism for communicating information, and
processor 1004 coupled with bus 1002 for processing
information.
[0173] Computing system 1000 also includes a memory 1006, which can
be a random access memory (RAM) or other dynamic memory, coupled to
bus 1002 for storing instructions to be executed by processor 1004.
Memory 1006 also may be used for storing temporary variables or
other intermediate information during execution of instructions to
be executed by processor 1004. Computing system 1000 may further
include a read only memory (ROM) 1008 or other static storage
device coupled to bus 1002 for storing static information and
instructions for processor 1004.
[0174] Computing system 1000 may also include a storage device
1010, such as a magnetic disk, optical disk, or solid state drive
(SSD) is provided and coupled to bus 1002 for storing information
and instructions. Storage device 1010 may include a media drive and
a removable storage interface. A media drive may include a drive or
other mechanism to support fixed or removable storage media, such
as a hard disk drive, a floppy disk drive, a magnetic tape drive,
an optical disk drive, a CD or DVD drive (R or RW), flash drive, or
other removable or fixed media drive. As these examples illustrate,
the storage media may include a computer-readable storage medium
having stored therein particular computer software, instructions,
or data.
[0175] In alternative embodiments, storage device 1010 may include
other similar instrumentalities for allowing computer programs or
other instructions or data to be loaded into computing system 1000.
Such instrumentalities may include, for example, a removable
storage unit and an interface, such as a program cartridge and
cartridge interface, a removable memory (for example, a flash
memory or other removable memory module) and memory slot, and other
removable storage units and interfaces that allow software and data
to be transferred from the storage device 1010 to computing system
1000.
[0176] Computing system 1000 can also include a communications
interface 1018. Communications interface 1018 can be used to allow
software and data to be transferred between computing system 1000
and external devices. Examples of communications interface 1018 can
include, a modem, a network interface (such as an Ethernet or other
NIC card), a communications port (such as for example, a USB port,
a RS-232C serial port), a PCMCIA slot and card, Bluetooth, etc.
Software and data transferred via communications interface 1018 are
in the form of signals which can be electronic, electromagnetic,
optical or other signals capable of being received by
communications interface 1018. These signals may be transmitted and
received by communications interface 1018 via a channel such as a
wireless medium, wire or cable, fiber optics, or other
communications medium. Some examples of a channel include a phone
line, a cellular phone link, an RF link, a network interface, a
local or wide area network, and other communications channels.
[0177] Computing system 1000 may be coupled via bus 1002 to a
display 1012, such as a cathode ray tube (CRT) or liquid crystal
display (LCD), for displaying information to a computer user. An
input device 1014, including alphanumeric and other keys, is
coupled to bus 1002 for communicating information and command
selections to processor 1004, for example. An input device may also
be a display, such as an LCD display, configured with touchscreen
input capabilities.
[0178] It will be appreciated that, for clarity purposes, the above
description has described embodiments with reference to different
functional units and processors. However, it will be apparent that
any suitable distribution of functionality between different
functional units, processors or domains may be used without
detracting from the disclosure. For example, functionality
illustrated to be performed by separate processors or controllers
may be performed by the same processor or controller. Hence,
references to specific functional units are only to be seen as
references to suitable means for providing the described
functionality, rather than indicative of a strict logical or
physical structure or organization. As mentioned above, an
instrument that may be utilized according to various embodiments,
but is not limited to, is a polymerase chain reaction (PCR)
instrument. FIG. 11 is a block diagram that illustrates an
amplification instrument 1100, upon which embodiments of the
present teachings may be implemented. Amplification instrument 1100
may include a heated cover 1110 that is placed over a plurality of
samples 1112 contained in chip or a consumable, for example. In
various embodiments, a consumable may be a glass or plastic slide
with a plurality of sample regions, which sample regions have a
cover between the sample regions and heated cover 1110. Some
examples of a consumable may include, but are not limited to, a
multi-well plate, such as a standard microtiter 96-well, a 384-well
plate, or a microcard, or a substantially planar support, such as a
glass or plastic slide. The reaction sites in various embodiments
of a consumable may include depressions, indentations, ridges, and
combinations thereof, patterned in regular or irregular arrays
formed on the surface of the consumable. Various embodiments of
amplification instruments include a sample block 1114, elements for
heating and cooling 1116, a heat exchanger 1118, control system
1120, and user interface 1122. Various embodiments of a thermal
block assembly according to the present teachings comprise
components 1114-1118 of amplification instrument 1100 of FIG.
11.
[0179] Real-time amplification instrument 1100 has an optical
system 1124. In FIG. 11, an optical system 1124 may have an
illumination source (not shown) that emits electromagnetic energy,
an optical sensor, detector, or imager (not shown), for receiving
electromagnetic energy from samples 1112 in a consumable, and
optics 1140 used to guide the electromagnetic energy from each DNA
sample to the imager. For embodiments of amplification instrument
1100 in FIG. 11 and real-time amplification instrument 1100 in FIG.
11, control system 1120, may be used to control the functions of
the detection system, heated cover, and thermal block assembly.
Control system 1120 may be accessible to an end user through user
interface 1122 of amplification instrument 1100 in FIG. 8 and
real-time amplification instrument 1100 in FIG. 11. Also a computer
system 1000, as depicted in FIG. 10, may serve as to provide the
control the function of amplification instrument 1100 in FIG. 11,
as well as the user interface function. Additionally, computer
system 1000 of FIG. 10 may provide data processing, display and
report preparation functions. Such instrument control functions may
be dedicated locally to the amplification instrument, or computer
system 1000 of FIG. 10 may provide remote control of part or all of
the control, analysis, and reporting functions, as will be
discussed in more detail subsequently.
[0180] As an alternative to low reaction volume chambers as
described above for carrying out nucleic acid amplification
monitoring in a stationary sample, the sample may be caused to flow
through a channel or chamber of a microfluidic device and as it
flows it may be subjected consecutively to different temperatures
whereby thermo-cycling is achieved. Thus, for example, the sample
may be caused to flow through a channel or chamber which passes
consecutively through different temperature zones suitable for the
amplification stages of denaturing, primer annealing and primer
extension, e.g. a channel in a microfluidic device, such as, for
example, a silicon chip device, which passes consecutively through
zones of different temperature provided in the base suitable for
successive repeats along the channel of the stages of denaturing,
primer annealing and primer extension. Such microfluidic structures
for performing continuous flow nucleic acid amplification on a chip
are described, for example, in Auroux et al., Minaturised Nucleic
Acid Analysis Lab Chip (2004) 4, 534-546. Structures of this type
may be fabricated through the use of standard microfabrication
techniques using for example photolithography to define the fluidic
network and then an etching or deposition step to create the
required channel or channels, for example in a PMMA, acrylic,
Perspex.TM. or glass substrate. A cover plate in glass or PMMA or
other material may or may not be overlaid to cover the channels.
The base of the channel or channels may be formed by substrate
bonding to a silicon chip and temperature sensors as well as
heating or heat pump (Peltier) elements, such that the reaction
mixture is in direct contact with these sensors and actuators, and
may or may not include circuitry for temperature control.
[0181] Alternatively, the base of the channel(s) may be formed by a
printed circuit board (PCB) housing temperature sensors such that
these are in direct contact with the reaction mixture. The PCB may
also house heating or heat pump elements, sensor interface and
temperature control circuitry. Reagents present within the
microfluidic channel or chamber may be those of the buffered
amplification reaction mixture, which may include the primers
chosen for ability to hybridize to the target at sites suitable for
amplification of the chosen sequence, the required enzyme or
enzymes for amplification and all four dNTPs in excess.
[0182] Temperature control may be achieved by a
proportional-integral-derivative (PID) controller, which is one of
the most common closed-loop feedback control systems. Errors
between the measured temperature and the target temperature may be
then used to calculate the level of heating required. Calculation
of this output level may be performed based on the current error
directly (proportional), the history of the error (integral), and
the predicted future error based on its rate of change
(derivative). Similarly, a PI controller may stabilize temperature
based on present and historical values of the error as described in
Iordanov et al. (2004) ibid. Alternatively, techniques such as
pulse-width modulation or duty-cycling may be implemented.
[0183] It may alternatively be chosen to have a reciprocating
system whereby the amplification mixture is moved backwards and
forwards in a microchamber between the required temperature zones
for thermo-cycling. As an alternative to contact heating for
thermo-cycling, various noncontact heating methods may he employed
as also discussed in the same review article, including by way of
example hot-air mediated heating, utilization of IR light,
laser-mediated heating, induction heating and microwave
irradiation.
[0184] In an embodiment, a flow based melt may similarly be
performed. For example, a sample comprising amplified product may
be disposed to flow along an axis of a temperature gradient and
monitored by a detector, such as a fluorescence detector. As
described herein, changes to indicators of amplification (e.g.,
fluorescence), may be detected as the sample flows over the
temperature gradient. Based on the location of the sample along the
gradient and the known temperature conditions along the gradient,
detected changes in the indicators of amplification may be
associated with a particular melt temperature (or temperature
window). The sample may be flowed using devices, such as a chip or
circuit board, and channels, as described herein with reference to
flow based PCR amplification. The temperature gradient may be
achieved using one or more heater, as described herein with
reference to the flow based PCR amplification.
[0185] In various exemplary embodiments in accordance with the
present disclosure, digital nucleic acid amplification (e.g., dPCR)
may be performed using a microfabricated chip that includes an
array of reaction sites or chambers into which the sample is
segregated into separate reaction volumes (sample portions) upon
being introduced to the device. In such a device, the sample
portions remain in their individual reaction sites or chambers
while subjected to the amplification assay, including for example
the various stages of thermal cycling.
[0186] In other embodiments, reaction volumes may be segregated
using droplets. For example, a plurality of droplets may be
generated using a device, for instance, by drawing a sample and oil
through a nozzle. The droplets may be approximately an embodiment.
The droplets may then be transferred for thermal cycling such that
PCR amplification may be achieved. For example, the droplets may be
transferred to a PCR plate or a chip with reaction sites or
chambers, and a thermal cycler may be used to cycle the droplets
through phases of amplification. The droplets may then be exposed
to a reader in order to determine amplification results. For
instance, the PCR plate or chip may be loaded onto a reader that
draws the droplets from each reaction site or chamber and exposes
them to a reader (such as a detector that measures
fluorescence).
[0187] In another embodiment, after generation of the droplets, a
flow based technique may be used to perform thermal cycling. For
example, the droplets may be caused to flow through a channel or
chamber which passes consecutively through different temperature
zones suitable for the amplification stages of denaturing, primer
annealing and primer extension, e.g. a channel in a microfluidic
device, such as, for example, a silicon chip device, which passes
consecutively through zones of different temperature provided in
the base suitable for successive repeats along the channel of the
stages of denaturing, primer annealing and primer extension.
Similarly, the droplets may then be exposed to a reader in order to
determine amplification results.
[0188] In order to perform the described melt analysis, the
droplets may be loaded on to a device, such as a plate or a chip,
and a heater may be used to systematically heat the droplets, as
described herein with reference to melt performance on a segregated
sample within reaction sites or chambers. In another embodiment,
the droplets may be caused to flow down a temperature gradient
while also being exposed to a detector, such as a fluorescence
detector, as described herein with reference to the flow based melt
performance embodiment. In such a configuration, known temperatures
may be associated with locations along the gradient, and the
location at which indicators of amplification change (e.g.,
decrease) along the gradient may be used to determine a melt
temperature.
[0189] Those skilled in the art will appreciate that the features
described above can be combined in various ways to form multiple
variations of exemplary embodiments in accordance with the present
disclosure, and that various modifications may be made to the
configuration and methodology of the exemplary embodiments
disclosed herein without departing from the scope of the present
disclosure and claims. Those skilled in the art also will
appreciate that various features disclosed with respect to one
exemplary embodiment herein may be used in combination with other
exemplary embodiments with appropriate modifications, even if such
combinations are not explicitly disclosed herein.
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