U.S. patent application number 16/043002 was filed with the patent office on 2018-11-15 for method of performing digital pcr.
The applicant listed for this patent is LIFE TECHNOLOGIES CORPORATION. Invention is credited to Caifu Chen, David Keys, Casey McFarland.
Application Number | 20180327813 16/043002 |
Document ID | / |
Family ID | 49384101 |
Filed Date | 2018-11-15 |
United States Patent
Application |
20180327813 |
Kind Code |
A1 |
Chen; Caifu ; et
al. |
November 15, 2018 |
Method of Performing Digital PCR
Abstract
A method of detection of a target nucleic acid is provided. The
method includes fractionating a sample into a plurality of sample
volumes wherein more than 50% of the fractions contain no more than
1 target nucleic acid molecule per sample volumes, and subjecting
the plurality of sample volumes to conditions for amplification.
The method further includes detecting a change in ion concentration
in a sample volume wherein a target nucleic acid is present,
counting the number of fractions with an amplified target nucleic
acid, and determining the quantity of target nucleic acid in the
sample.
Inventors: |
Chen; Caifu; (Palo Alto,
CA) ; McFarland; Casey; (San Francisco, CA) ;
Keys; David; (Alameda, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
LIFE TECHNOLOGIES CORPORATION |
Carlsbad |
CA |
US |
|
|
Family ID: |
49384101 |
Appl. No.: |
16/043002 |
Filed: |
July 23, 2018 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14395456 |
Oct 17, 2014 |
10030262 |
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PCT/US13/37352 |
Apr 19, 2013 |
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16043002 |
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PCT/US13/32598 |
Mar 15, 2013 |
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14395456 |
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61635584 |
Apr 19, 2012 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12Q 1/6851 20130101;
C12Q 1/6858 20130101; C12Q 1/6858 20130101; C12Q 1/6858 20130101;
C12Q 1/6851 20130101; C12Q 2565/301 20130101; C12Q 2563/159
20130101; C12Q 2563/159 20130101; C12Q 2565/607 20130101; C12Q
1/686 20130101; C12Q 1/6851 20130101; C12Q 2565/607 20130101; C12Q
2527/119 20130101; C12Q 2563/159 20130101; C12Q 2565/301 20130101;
C12Q 2563/159 20130101; C12Q 2527/119 20130101; C12Q 2565/607
20130101; C12Q 2565/607 20130101 |
International
Class: |
C12Q 1/686 20060101
C12Q001/686; C12Q 1/6858 20060101 C12Q001/6858; C12Q 1/6851
20060101 C12Q001/6851 |
Claims
1. A device for performing PCR comprising: a sample holder
configured to fractionate a sample into a plurality of sample
volumes wherein more than 50% of the fractions contain no more than
1 target nucleic molecules per sample volume and further configured
to hold a sample; a sensing layer in chemical communication with
the sample holding device; and at least one sensor in electrical
communication with the sensing layer.
2. The device of claim 1, wherein the sample holding device is a
matrix.
3. The device of claim 2, wherein the matrix is an extracellular
matrix.
4. The device of claim 1, wherein the sample holding device is a
gel.
5. The device of claim 4, wherein the gel is a hydrogel.
6. The device of claim 1, wherein the sample holding device is
located around at least a portion of the periphery of the
sensor.
7. A device for performing PCR comprising: a sample holder
configured hold a sample and to fractionate the sample into a
plurality of sample volumes, wherein the sample contains an initial
quantity of a target nucleic acid and wherein more than 50% of the
fractions contain no more than 1 target nucleic molecule of the
target nucleic acid per sample volume; a thermal cycler configured
to amplify or enrich the sample to produce an amplified quantity of
the target nucleic acid molecule so that an ion concentration in
one or more of the sample volumes is changed; and an ion detector
comprising a plurality of sensors in communication with the sample
volumes, the ion detector configured to detect a change in ion
concentration in the one or more of sample volumes; wherein the
system is configured to determine the initial quantity of the
target nucleic acid from the detected change(s) in the one or more
sample volumes.
8. The device of claim 7, wherein the sample holding device is a
matrix.
9. The device of claim 8, wherein the matrix is an extracellular
matrix.
10. The device of claim 7, wherein the sample holding device is a
gel.
11. The device of claim 10, wherein the gel is a hydrogel.
12. The device of claim 7, wherein the sample holding device is
located around at least a portion of the periphery of the
sensor.
13. A device for performing PCR comprising: a sample holder
configured hold a sample and to fractionate the sample into a
plurality of sample volumes, wherein more than 50% of the fractions
contain no more than 1 target nucleic molecules per sample volume;
a thermal cycler configured to amplify or enrich the sample to
produce an amplified quantity of the target nucleic acid molecule
so that an ion concentration in one or more of the sample volumes
is changed; a sensing layer in chemical communication with the
sample holding device; and at least one sensor in electrical
communication with the sensing layer; wherein the system is
configured to determine the initial quantity of the target nucleic
acid from ion concentration change(s) sensed in the at least one
sensor.
14. The device of claim 13, wherein the sample holding device is a
matrix.
15. The device of claim 14, wherein the matrix is an extracellular
matrix.
16. The device of claim 13, wherein the sample holding device is a
gel.
17. The device of claim 16, wherein the gel is a hydrogel.
18. The device of claim 13, wherein the sample holding device is
located around at least a portion of the periphery of the sensor.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a Divisional application of U.S. patent
application Ser. No. 14/395,456 filed Oct. 17, 2014, which is a 371
of International Application No. PCT/US2013/37352 filed Apr. 19,
2013, which is a Continuation of International Application No.
PCT/US2013/32598 filed Mar. 15, 2013, which claims benefit of U.S.
Provisional Application No. 61/635,584 filed Apr. 19, 2012. All
publications, patents, and patent applications mentioned in this
specification are herein incorporated by reference to the same
extent as if each individual publication, patent, or patent
application was specifically and individually indicated to be
incorporated by reference.
BACKGROUND
[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, or RNA). One difference between dPCR and
traditional PCR lays in the method of measuring nucleic acids
amounts. Both PCR and dPCR carry out one reaction per single
sample, dPCR also carries out a single reaction within a sample,
however the sample is separated into a large number of partitions
and the reaction is carried out in each partition individually.
This separation allows for sensitive measurement of nucleic acid
amounts. 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 within the sample are localized and concentrated
within many separate regions. The sample is fractionated by the
simple process of dilution so that each fraction contains
approximately one copy of DNA template or less. By isolating
individual DNA templates this process effectively enriches DNA
molecules that were present at very low levels in the original
sample. The partitioning of the sample facilitates counting of
molecules using Poisson statistics. As a result, each partition
will contain "0" or "1" molecule(s), or a negative or positive
reaction, respectively. While the starting copy number of a
molecule is proportional to the number of amplification cycles in
conventional PCR, dPCR is not dependent on the number of
amplification cycles to determine the initial sample amount.
[0004] Current methods of dPCR analysis utilize fluorescent probes
and light based detection methods to identify the products of
amplification. Such approaches require sufficient amplification of
the target molecules to generate enough signal to be detectable but
can lead to additional error or bias. It is therefore desirable to
provide an improved method for detection of nucleic acids of
interest within a sample using alternate methods of analysis having
increased accuracy and precision and which has a sensitivity that
can be used in connection with dPCR-based approaches.
SUMMARY
[0005] Provided herein is a method of detection of a target nucleic
acid comprising: fractionating a sample into a plurality of sample
volumes wherein more than 50% of the fractions contain no more than
1 target nucleic acid molecule per sample volumes; subjecting the
plurality of sample volumes to conditions for amplification;
detecting a change in ion concentration in a sample volume wherein
a target nucleic acid is present; counting the number of fractions
with an amplified target nucleic acid; and determining the quantity
of target nucleic acid in the sample. In some embodiments, the
method further comprises combining a sample with primers and probes
for amplification. The change in ion concentration may be an
increase in ion concentration or may be a decrease in ion
concentration. In some embodiments, the method may further include
combining a sample with bead. In some embodiments, the method may
include loading the sample on a substrate wherein the substrate
includes at least one well. The substrate may be a glass, metal,
metal oxide, silicon, ceramic, polymer coating or any combination
thereof. The well may or may not be sealed with a sealing layer
which may be solid or liquid such as, a cover slip, glass, plastic,
composite material, optically transparent material, an immiscible
fluid, or any other suitable sealing structure. Additionally, the
surface of the well may be a treated surface. The treated surface
may include a surface treatment to facilitate binding of a target
molecule of interest including coating the surface with a
hydrophilic coating, antibodies, streptavidin, avidin, thin-film
coatings, nanofibers, oligonucleotides, any combination thereof or
any other suitable surface treatment. Alternatively, the sample may
be loaded onto a matrix, such as an extracellular matrix, a polymer
matrix, or a gel, such as a polyacrylamide gel, agarose gel, or a
hydrogel. The method may further include positioning each of the
plurality of samples in a plurality of isolated positions, wherein
each of the plurality of isolated positions is in chemical
communication with a single sensor or wherein each of the plurality
of isolated positions is in chemical communication with their own
individual sensor. In some embodiments of the method, the change in
ion concentration is an increase in ion concentration or a decrease
in ion concentration. In some embodiments, the ion may be a
positive ion such as a hydrogen ion or may be a negative ion such
as a pyrophosphate molecule. The change in ion concentration may be
indicated by a change in pH or may be converted to an electrical
signal. In some embodiments, the method may include quantifying the
amount of a target nucleic acid in a starting sample.
[0006] Further provided herein is a method for performing absolute
quantification of a nucleic acid comprising: diluting a sample
containing an initial amount of a target nucleic acid into a
plurality of sample volumes wherein the percentage of reaction
areas containing one or more target nucleic acid molecules is
greater than 50% and less than 100%; subjecting the plurality of
sample volumes to at least one amplification cycle; detecting a
change in ion concentration in at least one of the plurality of
sample volumes as a result of the at least one amplification cycle;
and quantitating an initial amount of target nucleic acid. The
change in ion concentration may be an increase in ion
concentration, a decrease in ion concentration, a change in pH, may
involve the detection of a positive ion such as a hydrogen ion, a
negative ion such as a pyrophosphate molecule, or both positive and
negative ion.
BRIEF DESCRIPTION OF THE DRAWINGS
[0007] The novel features of the invention are set forth with
particularity in the appended claims. A better understanding of the
features and advantages of the present invention will be obtained
by reference to the following detailed description that sets forth
illustrative embodiments, in which the principles of the invention
are utilized, and the accompanying drawings of which:
[0008] FIG. 1 shows an example of the extension phase of PCR;
[0009] FIG. 2 shows one embodiment of a device for use with the
method described herein;
[0010] FIG. 3 shows one embodiment of the device for use with the
method described herein;
[0011] FIG. 4A & 4B show one embodiment of the device for use
with the method described herein;
[0012] FIG. 5A & 5B show one embodiment of the device for use
with the method described herein; and
[0013] FIGS. 6A-6B show one embodiment of the device for use with
the method described herein.
DETAILED DESCRIPTION
[0014] Polymerase chain reaction (PCR) relies on thermal cycling,
which consists of cycles of repeated heating and cooling of the
reaction for DNA melting and enzymatic replication of DNA. The vast
majority of PCR methods use thermal cycling, i.e., alternately
heating and cooling the PCR sample to a defined series of
temperature steps. These thermal cycling steps are necessary first
to physically separate the two strands in a DNA double helix at a
high temperature in a process called DNA melting. At a lower
temperature, each strand is then used as the template in DNA
synthesis by the DNA polymerase to selectively amplify the target
DNA during the annealing phase and extension phases. Polymerases
include heat-stable DNA polymerase such as, for example, Taq
polymerase. The selectivity of PCR results from the use of primers
that are complementary to the DNA region targeted for amplification
under specific thermal cycling conditions. Primers (short DNA
fragments) containing sequences complementary to the target region
along with a DNA polymerase, are key components to enable selective
and repeated amplification. FIG. 1 illustrates the extension of a
complementary strand of DNA. During DNA replication as the
deoxyribose-phosphate backbone is lengthened, a pyrophosphate
molecule is liberated to drive the reaction forward. Additionally,
the reaction also releases a single hydrogen ion, H.sup.+, from the
hydroxide group on the complimentary strand. In the methods
provided herein, detecting a change in chemical concentration may
indicate that amplification is occurring, and therefore, that the
template DNA of interest is present in the reaction vessel.
[0015] Provided herein is a method of detection of a target nucleic
acid comprising: fractionating a sample into a plurality of sample
volumes wherein more than 50% of the fractions contain no more than
1 target nucleic acid molecule per sample volumes; subjecting the
plurality of sample volumes to conditions for amplification;
detecting a change in ion concentration in a sample volume wherein
a target nucleic acid is present; counting the number of fractions
with an amplified target nucleic acid; and determining the quantity
of target nucleic acid in the sample. In some embodiments, the
method further comprises combining a sample with primers and probes
for amplification. The change in ion concentration may be an
increase in ion concentration or may be a decrease in ion
concentration. In some embodiments, the method may further include
combining a sample with bead. In some embodiments, the method may
include loading the sample on a substrate wherein the substrate
includes at least one well. The substrate may be a glass, metal,
metal oxide, silicon, ceramic, polymer coating or any combination
thereof. The well may or may not be sealed with a sealing layer
which may be solid or liquid such as, a cover slip, glass, plastic,
composite material, optically transparent material, an immiscible
fluid, or any other suitable sealing structure. Additionally, the
surface of the well may be a treated surface. The treated surface
may include a surface treatment to facilitate binding of a target
molecule of interest including coating the surface with a
hydrophilic coating, antibodies, streptavidin, avidin, thin-film
coatings, nanofibers, oligonucleotides, any combination thereof or
any other suitable surface treatment. Alternatively, the sample may
be loaded onto a matrix, such as an extracellular matrix, a polymer
matrix, or a gel, such as a polyacrylamide gel, agarose gel, or a
hydrogel. The method may further include positioning each of the
plurality of samples in a plurality of isolated positions, wherein
each of the plurality of isolated positions is in chemical
communication with a single sensor or wherein each of the plurality
of isolated positions is in chemical communication with their own
individual sensor. In some embodiments of the method, the change in
ion concentration is an increase in ion concentration or a decrease
in ion concentration. In some embodiments, the ion may be a
positive ion such as a hydrogen ion or may be a negative ion such
as a pyrophosphate molecule. The change in ion concentration may be
indicated by a change in pH or may be converted to an electrical
signal. In some embodiments, the method may include quantifying the
amount of a target nucleic acid in a starting sample.
[0016] Further provided herein is a method for performing absolute
quantification of a nucleic acid comprising: diluting a sample
containing an initial amount of a target nucleic acid into a
plurality of sample volumes wherein the percentage of reaction
areas containing one or more target nucleic acid molecules is
greater than 50% and less than 100%; subjecting the plurality of
sample volumes to at least one amplification cycle; detecting a
change in ion concentration in at least one of the plurality of
sample volumes as a result of the at least one amplification cycle;
and quantitating an initial amount of target nucleic acid. The
change in ion concentration may be an increase in ion
concentration, a decrease in ion concentration, a change in pH, may
involve the detection of a positive ion such as a hydrogen ion, a
negative ion such as a pyrophosphate molecule, or both positive and
negative ion.
[0017] FIG. 2A shows one embodiment of a device in which a single
molecule of a target nucleic acid may be detected. As shown in FIG.
2A, a representative device 200 may include an etched substrate 202
including at least one well 204, which well 204 may be configured
to contain and confine a volume of sample. An ion sensitive layer
206 may be in chemical communication with the well 204 and is in
electrical communication with a sensor 208. In some embodiments,
the well 204, or wells in an embodiment where there is more than
one well, is in chemical communication with a single sensor, or
each well 204 may be in chemical communication with its own
individual sensor, as shown in FIG. 2A. In some embodiments the
sensor may be a chemical field-effect transistor (chemFET) sensor,
an ion-sensitive field effect transistor (ISFET), or any other
suitable biosensor. In some embodiments, the sensor detects changes
to ion concentration, pH, heat, enzyme activity, or any other form
of energy emitted in response to a reaction occurring in the well.
In some embodiments, the sensor can be configured to detect the
release of a single hydrogen ion during a reaction. For example, as
the template strand is being replicated, with each nucleotide
binding occurrence the system may detect a change in ion
concentration and report such change as a spike. In some
embodiments, a bead 207 may be used to bind the DNA strands, as
shown in FIG. 2A. Other embodiments of a suitable device are
described in further detail in published U.S. Patent Publication
No. 2010/0301398, which publication is incorporated by reference in
its entirety.
[0018] During use, as amplification occurs and as incorporation of
a nucleotide occurs, hydrogen ions are released, effectively
dropping the pH in the well. The ion sensing layer then detects the
pH change as a rise in charge. If enough charge builds up, the
sensor reads out this change in voltage built up across the sensing
plate. In some embodiments, the sample may undergo PCR using any
suitable method for performing PCR. Such methods may include, but
are not limited to, the use of a thermal cycler or isothermal
amplification, such as loop-mediated isothermal amplification
(LAMP), nicking enzyme amplification reaction (NEAR),
helicase-dependant amplification, recombinase polymerase
amplification (RPA), or any other suitable method of performing a
reaction including a detectable reaction byproduct. In some
embodiments, thermal convection, such as microscale thermal
convection or infrared-mediated temperature control may be used. In
some embodiments, a heating element 220 may be fabricated into a
substrate 202 as show in FIG. 2B. The heating element may be
located in the substrate or under the sensing layer, or in any
other suitable location on the device. In some embodiments, the
heating element may be located in a cover or lid placed over the
opening of the well which may serve to provide heat to the well and
may or may not at least partially seal the well. In some
embodiments, the heating element is an infrared heat source, such
as for example, a tungsten lamp, radiation (the red part of the
spectrum). In such an embodiment, cooling is achieved using heat
dissipation or by compressed air.
[0019] In some embodiments, active heating/sensing elements may be
integrated into a device or chip to perform independent PCR
reactions (reactions which do not require the use of equipment
outside of the device/chip). In some embodiments, conductor and
semiconductor materials may be used to generate heat and/or other
forms of electromagnetic radiation. In some embodiments,
temperature sensing and heating may be accomplished through
deposition of platinum, doped polysilicon, or any other suitable
material. In some embodiments, an energy source may be coupled to a
device. In some embodiments, the device may be coupled to a Peltier
or other thermal source such as a thermal block, heat pad, or any
other suitable heating source.
[0020] Fabrication of one or an array of wells in silicon with
integrated actuators (heaters) for PCR monitoring has been
described for example in U.S. Publication No. 2010/0301398 and
Iordanov et al., Sensorised Nanoliter Reactor Chamber for DNA
Multiplication, IEEE (2004) 229-232, both of which are incorporated
by reference in thier entirety. Wells or chambers thus fabricated
might each be provided with an integrated ISFET for monitoring of
nucleic acid amplification. As noted by Iordanov et al. in their
above-noted paper, untreated silicon and standard silicon-related
materials are inhibitors of Taq polymerase. Therefore, when silicon
or a silicon-related material, e.g. silicon germanium or strained
silicon (all such materials will hereinafter be referred to as a
silicon substrate) is employed for fabrication of a microchip
chamber or channel for nucleic acid amplification it will usually
be covered with material to prevent reduction of polymerase
efficiency by the silicon, such as, for example, SU8,
polymethyl-methacrylate (PMMA), Perspex.TM. or glass.
[0021] Surface passivation of microfabricated silicon-glass chips
for PCR is also described by Shoffner et al. in Nucleic Acid Res.
(1996) 24, 375-379. In their studies, silicon chips were fabricated
using standard photolithographic procedures and etched to a depth
of 115 .mu.m. Pyrex.TM. glass covers were placed on top of each
silicon chip and the silicon and glass were anodically bonded.
Several types of surface passivations were investigated with a view
to improving PCR amplification efficiency with thermo-cycling in
the provided chamber. An oxidised silicon surface (SiO2) was found
to give consistent amplifications comparable with reactions
performed in a conventional PCR tube. Such a surface may also be
favoured in fabricating a microfluidic device for carrying out
nucleic acid amplification with ISFET pH sensing according to the
invention. For further discussion of surface passivation in the
fabrication of PCR microfluidic devices reference may be made to
Zhang et al., PCR microfluidic devices for DNA amplification,
Biotechnology Advances (2006) 24, 243-284. As described in that
review article, as an alternative to static surface passivation by
substrate coating, it may be possible to include a passivation
agent in the sample (dynamic passivation).
[0022] As an alternative to low reaction volume chambers as
described above for carrying out PCR monitoring in a stationary
sample, the sample for PCR monitoring may be caused to flow through
a channel or chamber of a microfluidic device and as it flows is
subjected consecutively to different temperatures whereby
thermo-cycling for PCR 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
PCR 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 PCR 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 and may be combined with ISFET
monitoring of amplification. 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 with integrated ISFET 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.
Alternatively, the base of the channel(s) may be formed by a
printed circuit board (PCB) housing ISFET and 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 enzmes for amplification and all four dNTPs in
excess.
[0023] 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.
[0024] 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. It will be appreciated that nucleic acid
amplification resulting from such on chip sample-shunting PCR
(described in the above-noted review article of Auroux et al.) may
be monitored by providing an ISFET in a wall of the microfluidic
chamber, or in any suitable location for measuring pH.
[0025] For further details of microfluidic devices for PCR, which
may be modified for ISFET sensing in accordance with the invention,
reference may again be made to Zhang et al. (2006) Biotech. Adv.
24, 243-284. As discussed in that review article, while such
devices may preferably take the form of silicon chips, other
materials for the chip substrate may be employed such as glass,
various polymers and ceramics. As an alternative to contact heating
for thermo-cycling, various non-contact heating methods may be
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.
[0026] In some embodiments, the device provided herein may be used
to perform DPCR. FIG. 3 shows one embodiment of a device for use
with DPCR. The device 300 may consist of a substrate 302 having a
well 304, in some embodiments, at least 1 well, at least 100 wells,
at least 1000 wells, at least 10,000 wells, at least 30,000 wells.
In some embodiments, the substrate 302 may have less than 50,000
wells, less than 40,000 wells, less than 30,000 wells, less than
20,000 wells, less than 5,000 wells, less than 1000 wells, less
than 500 wells, less than 100 wells, less than 10 wells. As shown
in FIG. 3, the at least one well 304 may be located in chemical
communication with a sensing layer 306 which may be in electrical
communication with at least one sensor 308. In some embodiments,
each well is in chemical and electrical communication with its own
respective sensor 308. The sample 312, which may include, for
example, the sample, PCR primers and reagents, buffers, or any
other suitable reagent for amplification may be loaded on the chip.
In some embodiments, a sealing layer 310, such as for example an
immiscible fluid layer, a plastic layer, a glass layer, or any
other suitable sealing layer, may then be positioned over the wells
to isolate the reaction mixture in each of the individual wells. In
some embodiments, at least a portion of the surface of the well may
be coated with a hydrophilic coating or material. In some
embodiments, at least a portion of the surface of the well may be
coated with a hydrophobic coating or material. Additionally, the
reaction mixture may then be subjected to conditions necessary for
performing PCR. As previously described, as PCR occurs, hydrogen
ions will be released each time the amplicon is extended/replicated
thereby changing the pH of the reaction mixture in the well. This
change in the pH of the reaction mixture may then be detected by
the sensor in chemical communication with the well.
[0027] In some embodiments, the sample may be loaded onto the chip
prior to amplification as previously described. In some
embodiments, the sample may be amplified outside of the chip, or
enriched, and then the sample with amplified template may be loaded
onto the chip. Once on the chip, the sample may undergo any
suitable reaction to release a detectable by-product. In some
embodiments, small reaction volumes are formed by shearing a sample
into smaller reaction volumes using an immiscible fluid, which are
then loaded onto the chip.
[0028] FIG. 4A shows an alternate embodiment of the device provided
herein. As shown in FIG. 4A, in some embodiments of the device and
methods provided herein, instead of dividing a sample among the
wells in a substrate, a sample 412, such as DNA, may be contained
and confined in a matrix 414 where individual molecules of DNA are
isolated from each other. The matrix may be any suitable matrix
including, but not limited to, an extracellular matrix, a polymer
matrix, or gel, such as a polyacrylamide gel. The matrix should be
of sufficient material to reduce the signal interference between
DNA fragments, colonies, clusters, polonies. In some embodiments
the matrix may be a continuous layer spread over the surface of the
sensing layer 406, as shown in FIG. 4A. In some embodiments, the
sample may be sandwiched between two glass slides and placed on the
sensing plate. In some embodiments, a support 416 may be in
structural communication with the matrix. In an alternate
embodiment of the device 400, the matrix 412 may be used in
conjunction with a substrate 402 containing at least one well 404,
as shown in FIG. 4B. In such an embodiment, the wells 404 may be
used to further reduce the signal noise between sample clusters and
reducing the number of sensors detecting the signal from an
individual cluster. The sensors below the individual colonies,
clusters, polonies, fragments may then detect the change in pH for
areas or wells with amplification each time an amplicon is extended
and/or replicated as hydrogen molecules are released. The detection
may depend on the proximity of the sample to a sensor or
alternatively may be time based detection to identify the number of
amplified polonies across the entire surface area. The
quantification using the time based approach may then be done by
calculating the number of colonies per area or volume.
[0029] In some embodiments, the device may be used to perform
emulsion dPCR. FIGS. 5A & 5B depict one embodiment of using the
device with an emulsion. In such an embodiment, an emulsion may be
created in which the emulsion droplets contain a sample of
interest. In some embodiments, an emulsion may be created from a
solution containing sample, primers, probes, reagents, buffers, or
any other suitable component necessary for PCR. In some
embodiments, a first emulsion may be created from a sample. A
second emulsion containing reagents, probes, primers buffers, or
any other suitable component for PCR, such as 5' nucleases or
TaqMan, may be created. In such an embodiment, the droplets from
the sample emulsion and the droplets from the reagents emulsion may
be coalesced at some point prior to amplification using any
suitable form of energy including heat, light, current, chemical
properties, charge, or any other suitable form for coalescing
droplets. In some embodiments, the emulsion droplets may be formed
using shaking, stirring, agitating, sonicating, or any other
suitable method for forming an emulsion. In some embodiments, the
unamplified emulsion droplets containing a sample 512 may be loaded
onto a substrate 502 containing at least one well 504. FIG. 5A is a
top view of a substrate showing samples 512 loaded into wells 504,
wherein the sample 512 has been diluted down to contain a single
copy of target nucleic acid. In such an embodiment, some of the
wells may contain at least one template of a target nucleic acid
and some of the well may contain zero templates of a target nucleic
acid. In some embodiments, the surface of the wells may be treated
with a surface coating, such as for example, a hydrophilic surface
coating, a DNA binding agent or any other suitable treatment for
retaining DNA molecules. Once the sample has been loaded on the
substrate an immiscible fluid layer or a sealing layer 510 (as
shown in FIG. 5B) may be placed over the wells to isolate the
individual reaction volumes and may also act as an overlay. FIG. 5B
shows the device 500 as seen from the side. In some embodiments, as
shown in FIG. 5B, the sealing layer 510 may also fill any empty
wells 504 which are not filled with sample. Once the samples have
been loaded on the substrate 502, the sample may then be subjected
to conditions necessary for PCR amplification. Wells containing
amplified sample, or positive sample wells 516, may then be
differentiated from wells with no target DNA, or negative sample
wells 518. Additionally, non sample wells 520 may be distinguished
from the positive sample wells 516 and the negative sample wells
518, since the non sample wells 520 will have different electrical
properties due to the sealing layer
[0030] In some embodiments, the device may be used with emulsion
dPCR using beads, as shown in FIGS. 6A & 6B. In such an
embodiment, an emulsion may be created which the emulsion droplets
contain at least one template of a sample of interest and necessary
PCR mixtures and/or reagents, probes, primers and a bead 607,
wherein the bead 607 may include primer sites for binding DNA or
any other target nucleic acid of interest. In some embodiments, the
emulsion droplets may be formed using shaking, stirring, agitating,
sonicating, or any other suitable method for forming an emulsion.
Any beads 607 with DNA present will have DNA extended from the
beads, or template positive beads 622. In some embodiments,
enrichment of template positive beads may be done. The amplified
emulsion droplets containing the sample 612 and the beads 607 may
then be loaded onto a substrate 602 containing at least one well
604, as shown in FIG. 6A. In some embodiments, the surface of the
wells may be treated with a surface coating, such as for example, a
hydrophilic surface coating, a DNA binding agent or any other
suitable treatment for retaining DNA molecules. In some
embodiments, once the sample has been loaded on the substrate an
immiscible fluid layer or a sealing layer may be placed over the
wells to isolate the individual reaction volumes and may also act
as an overlay. FIG. 6B shows the device 600 as seen from the side.
Once the samples and/or beads have been loaded into the wells 604,
the sample may then be subjected to conditions necessary for PCR
amplification. In some embodiments, new primers and/or probes may
be used to interrogate for an assay of interest. Wells containing
beads, as opposed to empty wells 628, with the sequence
corresponding to the assay of interest, or positive wells 624, will
give a signal, otherwise the wells will be negative wells 628.
[0031] In some embodiments, an emulsion can be created with
necessary reagents, primers, probes, and beads with primer sites.
PCR may then be conducted on the emulsion so that beads with DNA
present will have DNA extended from the beads to form template
positive beads. The emulsion may then be broken. Positive beads may
then be identified and enrichment of the positive beads may or may
not be done. The beads may then be loaded onto chips which may or
may not include wells. PCR may then be conducted on the chip and
may be loaded with new primers to interrogate for an assay of
interest. Wells with beads with the sequence corresponding to the
assay of interest may then give a signal. Wells without beads
corresponding to an assay of interest will then be negative.
[0032] In some embodiments of the method provided herein detection
of hydrogen ions may occur without true sequencing. A
non-sequencing hydrogen ion detection method may involve generating
an amplicon with two target specific PCR primers, binding the
amplicon to beads, hybridizing a target specific detection primer,
loading the beads into the wells, doing bead detection on the chip
to identify the wells with beads, supplying a reaction mixture with
polymerase and all 4 dNTPs, and detecting hydrogen ions. In such a
method the system may provide a count of how many beads have
amplicons and match the detection primer, but would not necessarily
provide any sequence information. The hydrogen ion spike would then
indicate that polymerization of a DNA strand has occurred.
[0033] In some embodiments of the method detection of hydrogen ions
may occur with partial sequencing of a target molecule, for example
by identifying a sequence of the bead bound amplicon but which
would not require a traditional 1:1 read out of the bases. In such
an embodiment, a sequencing run may be performed using dNTP pools
each of which was lacking of one of the four bases. This would give
sequence patterns that could be used to positively identify a
particular target molecule and which would also give more hydrogen
ions per sequencing cycle and predictable variation in peak
height.
[0034] Further provided herein is a method of performing ion
detection without beads or wells. In such an embodiment, single
amplicons would be bound to individual regions of hydrogel film
located around the periphery of the hydrogen ion detector. A
reaction may then be run to saturate the ring and then sequencing
could be performed which would then release hydrogen ions to be
detected by the hydrogen ion detector.
[0035] Provided herein is a method for performing digital castPCR,
PAP, or TPAP extension assay. Competitive allele-specific
TaqMan.RTM. PCR (castPCR) is a method of detecting and quantitating
rare mutations in a sample that contains large amounts of normal,
wild type genomic DNA (gDNA). castPCR.TM. technology combines
allele-specific TaqMan.RTM. qPCR with allele-specific MGB blockers
in order to suppress non-specific amplification from wild type
alleles, resulting in better specificity than traditional
allele-specific PCR, and is discussed further in copending
application U.S. Ser. No. 13/350,764, Methods, Compositions, and
Kits for Detecting Rare Cells, which application is incorporated by
reference in its entirety. Phosphorylated activated polymerization
(PAP) is a process involving pyrophosphorolysis-mediated
primer-deblockings when hybridized with target or template nucleic
acids followed by extension of the activated primers. The primers
used in PAP typically include terminator nucleotides, such as
dideoxynucleotides (ddNMPs) at 3'. TPAP refers to the
polymerization of non-extensible prier in the presence of
triphosphate. Usually, the non-extensible nucleotide at the 3' end
of the primer is first removed to generate an extensible primer
before the polymerization could occur. Digital castPCR, PAP, or
TPAP extension assays may be also be used for rare mutation
detection however detection sensitivities of thousands of hydrogens
per well may be required. In such an embodiment, universal
attachment of sheared gDNA (.about.100 kb each) including a rare
mutant allele may be bound to a bead. During allele specific
extension using castPCR or TPAP, as extension of the rare mutant
allele occurs, hydrogen ions may be released. The released hydrogen
ions may then be detected by an ion sensitive detection system.
[0036] In some embodiments, a nucleic acid may be amplified using
activation by polyphosphorolysis (APP). APP may be carried out
using the steps of: (a) annealing to a nucleic acid a first
oligonucleotide which has a non-extendable 3' end ("P*") that is
removable by polyphosphorolysis (i.e., activatable); (b) removing
that 3' non-extendable terminus using a polyphosphorolyzing agent
and a biocatalyst (i.e., a DNA polymerase) having
polyphosphorolysis activity to produce an unblocked
oligonucleotide; and, (c) extending the unblocked oligonucleotide
to produce a desired nucleic acid strand. The APP method may also
be used to amplify a desired nucleic acid strand by, for example,
adding the following additional steps: (d) separating the desired
nucleic acid strand of step (c) from the template strand, and (e)
repeating steps (a)-(d) until a desired level of amplification of
the desired nucleic acid strand is achieved. Steps (a) to (c) of
APP can be conducted sequentially as two or more temperature stages
on a thermocycler, or they can be conducted as one temperature
stage on a thermocycler.
[0037] Further provided herein is a method for performing multiplex
digital mutation detection assay using an ion sensitive detection
system. In such an embodiment, allele specific primers may be
attached to a bead. Each bead may be capable of analyzing 100-500
mutations. Multiplex emulsion TPAP PCR may then be performed in the
presence of 0-1 molecules per bead. The beads and bound allele are
then subjected to conditions for amplification. The presence or
absence of mutations may then be counted and the frequency of these
100-500 rare mutations may then be determined. Other examples of
TPAP or APP may be found in copending application U.S. Ser. No.
13/324,676, Polymerization of Nucleic Acids Using Activation by
Polyphosphorolysis (APP) Reactions, which is incorporated by
reference in its entirety.
[0038] Alternatively, a mixture of allele specific primers may be
attached to beads. In some embodiments, at least 2 allele specific
primers may be attached, at least 3 allele specific primers may be
attached, at least 50, at least 100, at least 500, at least 1000.
The beads may then be mixed with the sample and PCR reagents
[0039] Further provided herein is a method for performing multiplex
digital PCR using pre-amplification partitioning and dual-stage
emulsion PC in a single step or single amplification reaction.
Performing partitioning and amplification in a single step
eliminates random noise or error and biases which may interfere
with accurate quantification. In some embodiments, the partitioning
of a sample, or the partitioning of a template molecule or nucleic
acid occurs before any processing of the sample occurs. Processing
may include for example, tailing, targeting, amplification,
bead-loading, or any other suitable processing.
[0040] In some embodiments of the method of single amplification
provided herein, the method may comprise partitioning a sample with
a multiplex of targeting oligos which each contain a sequence
specific 3' end and a universal 5' end, and wherein the targeting
oligos are present in concentrations sufficient to ensure that
several rounds of amplification of any target in the reaction
volume may occur to produce a tailed amplicon of the target.
Additionally, universal oligos may be added to the reaction volumes
wherein the universal oligos are identical to the tails present on
all targeting oligos, and wherein the universal oligos are present
in sufficient concentration to continue amplification of any tailed
amplicon beyond the first round. Furthermore, beads, such as
sequencing bead for example, may or may not be present in the
reaction volumes. In some embodiments, the reaction volumes may be
monodisperse droplets, polydispersed droplets, and/or emulsions. In
some embodiments, one step RT-PCR in emulsion may be possible and
may allow for digital analysis of RNA.
EXAMPLES
Multiplex Digital PCR Using Pre-Amplification Partitioning and
Dual-Stage Emulsion PCR
[0041] Materials: A multiplex pool of target specific oligos each
tailed with the same universal sequence is combined in low
concentrations with sample, and PCR MasterMix. For example: allele
specific forward primers with desired number of targets, allele
specific reverse primer with desired number of targets, universal
forward primers or tail, universal reverse primer or tail, may be
combined with the sample and PCR MasterMix. Additionally, beads
preloaded with universal forward primers/tails may be added, as
well as a higher concentration of matching universal forward
primers or tails and matching universal reverse primers or tail.
The amount of universal reverse primers or tails maybe in a
slightly higher concentration than the universal forward primer or
tail to drive bead loading.
[0042] Methods: Once the multiplex pool, sample, and PCR MasterMix
have been combined, the reaction mixture may then be partitioned by
emulsification into thousands or millions of droplets. Each droplet
will therefore contain sufficient targeting primers to initiate
amplification of every target. Amplification may then be started by
whichever allele specific primers match the template in a
particular droplet. All other allele specific primers are
unproductive. After the first stage of amplification, the allele
specific primers for the target may then be exhausted.
Amplification may then be continued by the universal primers which
are the same in every reaction or droplet. The universal forward
primer on the bead may also be extended generating sequencing
templates.
[0043] Analysis: Quantification of the droplet is digital (positive
for amplification or negative for amplification).
[0044] While preferred embodiments of the present invention have
been shown and described herein, it will be obvious to those
skilled in the art that such embodiments are provided by way of
example only. Numerous variations, changes, and substitutions will
now occur to those skilled in the art without departing from the
invention. It should be understood that various alternatives to the
embodiments of the invention described herein may be employed in
practicing the invention. It is intended that the following claims
define the scope of the invention and that methods and structures
within the scope of these claims and their equivalents be covered
thereby
* * * * *