Note: Descriptions are shown in the official language in which they were submitted.
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QUANTITATIVE, HIGHLY MULTIPLEXED DETECTION OF NUCLEIC ACIDS
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority to U.S. Patent Application No.
13/399,872, filed
February 17, 2012, which claims priority to Provisional U.S. Patent
Application No.
61/463,580, filed February 18, 2011, and Provisional U.S. Patent Application
No. 61/561,198,
filed November 17, 2011, the full disclosures of each of which are hereby
incorporated herein
by reference in their entirety for all purposes.
STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY
SPONSORED RESEARCH AND DEVELOPMENT
[0002] This invention was made with support of a U.S. Dept. of Homeland
Security
grant, Contract Number HSHQDC-10-C-00053. The government may have certain
rights in
the invention.
FIELD OF THE INVENTION
[0003] The invention is in the field of real-time DNA amplification,
detection and
quantification, as well as associated consumables, devices, and systems,
including arrays.
BACKGROUND OF THE INVENTION
[0004] Real time PCR is routinely used for detection of nucleic acids of
interest in a
biological sample. For a review of real time PCR see, e.g., M Tevfik Dorak
(Editor) (2006)
Real-time PCR (Advanced Methods) Taylor & Francis, 1st edition ISBN-10:
041537734X
ISBN-13: 978-0415377348, and Logan et al. (eds.) (2009) Real-Time PCR: Current
Technology and Applications, Caister Academic Press, 1st edition ISBN-10:
1904455395,
ISBN-13: 978-1904455394. For additional details, see also, e.g., Gelfand et
al. "Homogeneous
Assay System Using The Nuclease Activity of A Nucleic Acid Polymerase" USP
5,210,015;
Leone et al. (1995) "Molecular beacon probes combined with amplification by
NASBA enable
homogenous real-time detection of RNA." Nucleic Acids Res. 26:2150-2155; and
Tyagi and
Kramer (1996) "Molecular beacons: probes that fluoresce upon hybridization"
Nature
Biotechnology 14:303-308. Traditionally, single well multiplexing, used to
detect more than
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one target nucleic acid per sample in a single reaction container (e.g., well
of a multiwell plate),
is achieved using self-quenched PCR probes such as TAQMANTm or Molecular
Beacon probes
that are specific for each amplicon. Upon binding to the amplicon in solution,
or upon
degradation of the probes during PCR, the probes unquench, producing a
detectable signal.
The probes are labeled with fluorophores of different wavelengths, permitting
a multiplexing
capability of up to about 5 targets in a single "one pot" reaction. More than
about 5 probes per
reaction is difficult to achieve, due to practical spectral range and label
emission limitations.
This severely limits multiplexing of a single reaction, which, in turn,
significantly limits how
many targets can be screened per sample and drives up reagent cost and
instrument complexity
in detecting multiple targets of interest.
[0005] Nucleic acid arrays represent another approach to multiplexing the
detection of
amplification products. Most typically, amplification reactions are performed
on a sample, and
amplicons are separately detected on a nucleic acid array. For example, Sorge
"Methods for
Detection of a Target Nucleic Acid Using A Probe Comprising Secondary
Structure" US
6,350,580 propose the capture of a probe that is released upon amplification
by purifying the
probe out of the amplification mixture and then detecting it. This multiple-
step approach to
making and detecting amplicons makes real time analysis of the amplification
mixture
impractical.
[0006] Various approaches that amplify the reactants in the presence of
the capture
nucleic acids have also been proposed. For example, Kleiber et al. "Integrated
Method and
System for Amplifying And Detecting Nucleic Acids," US 6,270,965, propose
detection of an
amplicon via evanescence induced fluorescence. Similarly, Alexandre et al
"Identification and
Quantification of a Plurality of Biological (Micro) Organisms or Their
Components," US
7,829,313, proposes detection of amplicons on arrays. In another example,
target
polynucleotides are detected by detecting a probe fragment that is produced as
a result of
amplification, e.g., by binding to an electrode, followed by electrochemical
detection. See,
e.g., Aivazachvilli et al. "Detection of Nucleic Acid Amplification" US
2007/0099211;
Aivazachvilli et al. "Systems and Methods for Detecting Nucleic Acids US
2008/0193940, and
Scaboo et al. "Methods And Systems for Detecting Nucleic Acids" US
2008/0241838.
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[0007] These methods all suffer from practical limitations that limit
their use for
multiplex target nucleic acid detection. For example, Kleiber (US 6270,965)
relies on
evanescence induced fluorescence to detect fluorescence of amplicons at the
array surface, and
requires complex and expensive optics and arrays. Alexandre (7,829,313)
propose detection of
amplicons on an array; as in Kleiber this increases array costs significantly,
because each array
has to be custom designed to detect each amplicon. In practice, it can be
difficult to achieve
similar hybridization kinetics for disparate amplicons on an array,
particularly where the
amplicons are relatively large, as in Alexandre. Furthermore, this art
provides little guidance
regarding how to detect signal on an array where there is an accompanying
solution phase that
also comprises high levels of signal background, or of arrays that remain
stable through in situ
thermal cycling.
[0008] The present invention overcomes these and other problems in the
art. A more
complete understanding of the invention will be obtained upon complete review
of the
following.
SUMMARY OF THE INVENTION
[0009] The invention provides methods and associated devices, systems and
consumables that permit highly multiplexed detection of nucleic acids of
interest, e.g., for the
detection of viruses, bacteria, plasmodium, fungi, or other pathogens in a
biological sample.
The consumable comprises a signal-optimized chamber having a high-efficiency
thermo-stable
nucleic acid detection array on an interior surface of the chamber. The array
is configured to
detect up to about 100 or more different universal labeled probes. The methods
generate the
labeled universal probes (as "probe fragments") during amplification of a
portion of a nucleic
acid of interest, with the amplification reaction being performed in the
chamber. The universal
probes are hybridized to the array after a few amplification cycles, and
subsequent to selected
amplification cycles thereafter, allowing for both detection and
quantification of one or more
nucleic acids of interest in the sample, in real time.
[0010] Accordingly, in a first aspect, methods of detecting a target
nucleic acid are
provided. This includes providing a detection chamber that has at least one
high efficiency
nucleic acid detection array on at least one surface of the chamber. The high
efficiency array
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typically has a non-rate limiting number of capture nucleic acids that permits
an increased
capture rate of detectable probe fragments produced by a reaction in the
chamber, and the
capture nucleic acids are configured to capture relatively small probe nucleic
acids, which also
increases array efficiency. Detection of binding of the probes to the array is
preferably carried
out under conditions that are selected or configured to reduce background
signal levels
proximal to the array, e.g.,resulting from unbound free probe. For example, in
certain
embodiments, the chamber itself is configured to reduce signal background
proximal to the
array, e.g., by shaping the chamber to reduce background (e.g., by making the
chamber
relatively thin proximal to the array, e.g., the chamber is typically about
500pm or shallower
above the array). Thinner chambers also have less thermal mass, and can be
temperature
cycled more rapidly and more efficiently than thicker chambers. Other ways in
which the
system and methods are configured to reduce the level of background signal are
described in
greater detail, below.
[0011] A sample that has one or more copies of the target nucleic acid to
be detected is
loaded into the detection chamber. An amplification primer and a labeled probe
are hybridized
to the one or more target nucleic acid copies. At least a portion of one or
more of the target
nucleic acid copies is amplified in an amplification primer dependent
amplification reaction.
The amplification reaction results in cleavage of the labeled probe, e.g., due
to nuclease activity
of an amplification enzyme. This results in release of a labeled probe
fragment, which is to be
detected by the array. The labeled probe fragment is hybridized to the high-
efficiency array
(typically after a few amplification cycles are run to amplify the amount of
probe fragment
released in the chamber). A label signal produced by binding the labeled probe
fragment to the
array is then detected, thereby detecting the target nucleic acid.
[0012] The precise configuration of the detection chamber can vary. The
configuration
is selected to reduce signal background in the chamber proximal to the array.
Generally, at
least 1%, and often, about 5% or more of the signal in the chamber is
concentrated at the array
(e.g., about 6%, 8%, or even 10% or more) in the region of the array.
Background of 99% or
less total signal can be normalized by the system, though lower levels are
often desirable. In
typical embodiments described herein, levels of 95% or less total background
are achieved by
optimizing the configuration of the chamber proximal to the array. This
configuration
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optimization is achieved, e.g., by keeping the depth of the chamber above the
array to a
minimum. In typical embodiments, the chamber is less than about 1 mm in depth
or other
dimension proximal to the array, more typically about 500 pm or less in at
least one dimension
proximal to the array, preferably less than about 250 pm or less, e.g.,
between about 10 pm and
about 200 pm and in some embodiments the chamber is about 150pm in a dimension
proximal
to the array. In one example herein, the chamber is about 142 pm in depth
above the array. In
another example herein, the chamber is about 100 pm in depth. The relevant
chamber
dimension depends on the signal detection path of the detection system, for
example, where the
signal is generated by passing light onto the array, where some of the light
escapes through the
array and into the fluid above the array, the relevant dimension is the depth
of the chamber
above the array. In addition to reducing the level of background signal
detected, reducing
chamber thickness also has the benefit of reducing the contribution of
background noise
components, e.g., detector responses unrelated to the specific detection of
array spot signal and
background signal from the reaction fluid. In particular, one major noise
contributor is the shot
noise from the detectors used, which generally increases with the square root
of the total
amount of signal detected, which, in turn, scales with the thickness of the
reaction chamber.
Accordingly, by providing reduced thickness of the reaction chamber, one
reduces the
background noise, and consequently increases the signal to background noise
ratio (SNR) of
the overall system. Other potential noise contributors include detection of
excess light, e.g.,
unfiltered excitation light, unintended ambient light, scattered fluorescence,
autofluorescence of
system components, or the like. A number of these noise contributors may be
mitigated
through the conventional approaches, such as through the use of appropriate
optical filters, e.g.,
to eliminate or reduce excess excitation light, sealed optical systems that
reduce or prevent
ambient light at the detector, and through the configuration of array spot
size and spacing to
reduce or eliminate signal cross talk at the detector. In particularly
preferred aspects, the SNR
for the assay methods and systems of the invention will typically be 2.5 or
greater, preferably
greater than 3, greater than 4, greater than 5, greater than 10, and in some
cases, greater than 20
or more.
[0013] Alternative or additional approaches for configuring the system and
methods of
the invention to reduce background signal can also be employed in conjunction
with the
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devices and methods of the invention. For example, the devices and systems of
the invention
may be configured to provide excitation illumination to the capture array
using a total internal
reflection fluorescence microscopy ("TIRF") configuration, where excitation
light is directed
into the substrate underlying the capture array such that it is entirely
internally reflected (see,
M. Tokunaga et al., Biochem. and Biophys. Res. Comm. 235, 47 (1997) and P.
Ambrose,
Cytometry, 36, 244 (1999)). Notwithstanding this, an evanescent wave is
generated at the
substrate-fluid interface of the array that decays exponentially away from the
surface, resulting
in effective illumination adjacent to the surface, e.g., to a depth of 100 nm,
without exciting
fluorophores in the remainder of the solution.
[0014] In still another alternative or additional approach, the reactants
employed in the
analytical methods of the invention are configured to reduce background signal
relative to
actual probe/array binding signal. For example, background signal may be
reduced through the
use of cooperative fluorophores on both the capture array probes and the
labeled probe
fragment, e.g., in a FRET construct. In particular, a donor fluorophore,
having a first excitation
spectrum and a first emission spectrum may be coupled to one of the capture
probe or the
labeled probe fragment. An acceptor fluorophore that has an excitation
spectrum that overlaps
the emission spectrum of the donor, and that is different from the donor's
excitation spectrum,
is coupled to the other probe. When the capture probe and labeled probe
fragment hybridize,
the donor and acceptor are brought into sufficient proximity for energy
transfer, yielding a
distinctive fluorescent signal corresponding to the emission spectrum of the
acceptor
fluorophore. By configuring the optical system to excite only within the
donor's excitation
spectrum, and filter the emission spectrum of the donor, one can selectively
detect signal
arising from the energy transfer signal from the acceptor, upon hybridization.
A wide variety
of FRET label pairs have been described previously (See, e.g., U.S. Patent
Nos. 6,008,373, to
Waggoner, and 7,449,298, to Lee et al.).
[0015] In an alternative configuration, interactive labeling groups are
employed to
further reduce the potential for background signals. In particular, in an
aspect of the invention,
the capture probe is labeled with a fluorophore such that the signal producing
label is tethered
to the surface of the array. In this context, the labeled probe and labeled
probe fragment carries
a quencher group complementary to the fluorophore, i.e., able to quench the
fluorescence of the
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capture probe label. The quencher is provided on a position of the labeled
probe fragment such
that when hybridized to the capture probe, it will be sufficiently proximal to
the fluorophore on
the capture probe to quench the fluorescent signal. For example, where the
labeled probe
fragment is labeled with a quencher at its 5' end, the capture probe will bear
the fluorophore at
a 3' or other complementary position. In the context of the assays of the
invention,
amplification of the target sequence results in production of labeled probe
fragments that
quench the fluorescent signals from the array when it hybridizes with the
labeled capture probe,
resulting in a negative signal event as indicative of the presence of target
signal. In particular,
the capture probes on the array produce signal in the unhybridized state. Upon
amplification of
the target sequence, the quencher bearing probe fragment is released to
hybridize to the
complementary capture probe on the array, quenching the signal from its
associated
fluorophore, and resulting in a dark location on the array, as compared to
unhybiridized array
locations. By providing a quencher group that does not produce a fluorescent
signal under
detection conditions for the assay, any background fluorescence from the
uncleaved probe or
unbound labeled probe fragment is eliminated.
[0016] As will be appreciated, a number of methods may be employed in the
context of
the invention for reducing the contribution of signal from unbound, intact
labeled probe in the
reaction solution, or background signal, relative to the signal detected from
bound labeled
probe fragment, including, for example, configuring the reaction chamber to
concentrate signal
within the focal plane of the detector, employing interactive labeling
techniques that either
present different emission spectra when bound to the array versus when unbound
in solution, or
self quenching probes that have reduced fluorescence when present in the same
intact probe,
versus when separated in a cleaved probe fragment.
[0017] As noted, the array typically includes a non-rate limiting number
of capture
nucleic acids that hybridize to the labeled probe fragment. This means that
the amplification
reaction produces a number of probe fragments during amplification that
results in a probe
fragment concentration in the reaction mixture that is not saturating for the
number of sites on
the array (e.g., accessible complimentary capture nucleic acids) available to
bind the probe
fragments. Restated, the number of binding sites on the array is maintained in
excess, and
preferably well in excess, of what would be saturated at the concentration of
probe fragments
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produced in an amplification reaction. Because the number of sites on the
array is not rate
limiting, the ratio of probe fragments on the array to background probe
fragments in solution is
optimized. Typical array densities are between about 350 fmol/cm2 or greater,
e.g., about 2,000
fmol/cm2 or greater, 2,500 fmol/cm2 or greater, 3,000 fmol/cm2 or greater,
4,000 fmol/cm2 or
greater, 4,500 fmol/cm2 or greater, or 5,000 fmol/cm2 or greater. In some
embodiments, the
number of sites that bind probe on the array is at least 1X the number of
sites that would be
saturated by the concentration of probe fragments produced during
amplification, and is
optionally 5X, 10X, 50X or more. The ratio will vary with the number of
amplification cycles
and the amount of probe produced. The efficiency of the array is also a
function of the length
of the probe fragment to be captured. Shorter fragments typically display more
efficient
hybridization, although the probes do have to be long enough to bind at a
given Tm during
hybridization. Typical probe fragments to be captured by the array are about
50 nucleotides in
length or less; the arrays comprise sites that have corresponding
complimentary capture nucleic
acid sequences (the capture nucleic acids can optionally also include
additional sequences, e.g.,
to space the complimentary site above the surface, e.g., to reduce surface
effects). More
typically, the probes and capture sequences are about 40 nucleotides or less
in length, e.g.,
about 30, about 20, or about 15 nucleotides or shorter in length.
[0018] In some cases, the capture array probes, and complementary labeled
probe
fragments used in a given analysis are selected such that they provide a
narrow range of Tm
over all members of the array. In particular, to ensure optimal and consistent
hybridization to
the capture array, the capture probes in a given array will each have a Tm
within about 10 C of
every other member of the array, and preferably, within about 7 C, 5 C, or 3
C of every
other probe in the array. Such a narrow Tm range allows for consistent
hybridization and
resulting signal generation across all members of the array.
[0019] In typical embodiments, the hybridization temperature is less than
the
temperature of the amplification reaction, so the Tm of the probe fragment for
the capture
nucleic acid can be less than an intra-molecular Tm of the probe (e.g., where
the probe
comprises a quencher to reduce background), and/or lower than the Tm of the
probe for the
target nucleic acid. That is, in typical thermocycling embodiments,
amplification reactions are
performed at higher temperatures than hybridization steps; accordingly, the
probe will typically
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have a higher Tm for the target nucleic acid than the probe fragment has for
the array. The
labeled probe typically comprises a first orthogonal flap that is not
complimentary to the target
nucleic acid; this flap is cleaved from the labeled probe to produce the
labeled probe fragment.
The labeled probe optionally comprises a second orthogonal flap, e.g., coupled
to a quencher
moiety, which is at least partially complimentary to the first flap (e.g., to
provide proximity-
based quenching of a label on the first flap). Background is reduced where the
second flap has
a higher Tm for binding to the first flap than the first flap has for binding
to the array. In such a
configuration, the extension reaction occurs at a first temperature, i.e.,
below the Tm of the
intact probe for the target nucleic acid, but above both the intra-molecular
Tm of the intact
probe, and the Tm of the probe fragment for the capture probe on the array).
Following
extension, as the reaction temperature is lowered, it crosses below the intra-
molecular Tm of the
probe, allowing for the formation of the secondary structure of the probe, and
resultant
quenching of the fluorophore. Further cooling to below the Tm of the probe
fragment to the
capture probe allows hybridization of the probe fragment to the array and
detection of its
associated fluorophore. Because the intact probe has previously formed into
its secondary
structure, it is both less likely to bind to the capture probe, and is
quenched, thus reducing both
unintended capture of intact probe, and background signal from the
fluorophores present on the
intact probe in solution (or that may have bound to the capture probe array.
Although in certain
aspects, quenchers are employed on the intact probes of the invention, in
certain embodiments
it has been surprisingly determined that, quenchers are not required on the
probe, because the
optimized chamber design and high efficiency array achieve discrimination of
array signal from
background, even where background is increased by omitting a quencher from the
probe.
[0020] In other embodiments, the labeled probe fragment and its
complementary
capture nucleic acid is designed or selected to have a Tm that is higher than
the extension
reaction temperature, e.g., 10 or more degrees higher. Thus, where the
extension reaction is
carried out, for example, at between 55 and 60 C, the Tm of the labeled
probe fragment and
capture nucleic acid will typically be, e.g., 71 C. In such cases,
hybridization of the labeled
probe fragment to the capture nucleic acid on the array occurs at the same
temperature as the
extension reaction, obviating the need to further reduce the temperature in
order to hybridize to
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the array and detect the resulting signal. As a result, a two-step temperature
profile may be
used rather than a three step profile.
[0021] In the context of the intact labeled probe, the orientation of the
orthogonal
labeled probe fragment relative to the probe portion that binds to the target
sequence, may be
varied. In particular, a release labeled probe fragment may hybridize to a
capture probe on the
array in an orientation where the end cleaved from the target specific portion
of the probe is
either proximal or distal to the point at which the capture probe is coupled
to the array surface.
In some cases, for example, by ensuring that any intact probe would only bind
to the capture
probe in an orientation that projected the target specific portion of the
probe toward the surface
of the array, one could then take advantage of potential surface interference
with that binding,
to further reduce the potential for undesired capture of intact probe by the
array. Such methods
are particularly useful in the case of solid surfaces on the arrays, e.g.,
silica substrates and the
like.
[0022] Sample can be loaded into the chamber by any of a variety of
mechanisms,
depending on the precise configuration of the consumable. In one convenient
application, the
sample is loaded through at least one port or fluidic channel in operable
communication with
the chamber. For example, ports can be fabricated in a top surface of the
consumable, with the
ports leading into the chamber. This provides for simplified loading, e.g.,
via a pipette or other
fluid delivery device. Alternatively, fluidic or microfluidic channels,
capillaries, or the like,
can be used for sample delivery.
[0023] The methods can be used for detection of a nucleic acid of interest
in a sample
and/or quantification of the nucleic acid, e.g., in real time. Thus, in one
aspect, the target
nucleic acid is optionally amplified in a plurality of amplification cycles
prior to detecting
signal, with the target nucleic acid portion additionally being amplified
after signal detection,
i.e., in the presence of additional copies of the labeled probe. Resulting
released labeled probe
fragments are subsequently hybridized to the array and detected, with detected
signal intensity
being correlated to presence and/or quantity of the target nucleic acid
present in the sample.
Typically, the sample is amplified for more than 1 cycle before initial
detection, to increase the
level of signal by increasing the number of probe fragments released by the
amplification. For
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example, the target nucleic acid can optionally be amplified for at least,
e.g., 2, 3, 4, 5 or more
amplification cycles prior to detecting signal from the array.
[0024] The labeled probe typically comprises a fluorescent or luminescent
label,
although other labels such as quantum dots can also be used. In one preferred
embodiment, the
label is a fluorescent dye. The signal produced by the probe fragment is
typically an optical
signal. The labeled probe optionally comprises a label and a label quencher;
cleavage of the
labeled probe results in separation of the label and the quencher, thereby
unquenching the label.
However, as noted above, quenchers are not required in the practice of the
invention.
[0025] Signal is typically detected by detecting one or more optical
signal wavelengths
corresponding to optical labels on the probes or probe fragments. Because
binding position of
probe fragments on the array can be used to discriminate between different
probes, it is not
necessary to use different labels on the different probes to distinguish the
probes in a
multiplexed amplification reaction (an amplification reaction designed to
amplify multiple
target nucleic acids, if more than one of the targets is present in the
sample). However,
multiple probes labels can be used to enhance multiplexing capabilities. Where
multiple probes
are used, detecting the signal can include detecting a plurality of optical
signal wavelengths
from a plurality of signals generated by a plurality of different labels
(e.g., different fluorescent
dye moieties on different probes).
[0026] Although generally described in terms of label groups that are
attached to the
probe fragment that binds to the array, e.g., the labeled probe fragment, it
will be appreciated
that other detection schemes may be employed that do not require the use of
pre-labeled probes.
For example, in some embodiments, intercalating dyes may be used.
Intercalating dyes
typically provide a detectable signal event upon incorporation, or
intercalation, into double
stranded nucleic acids. In the context of the invention, the hybridization of
the cleaved probe
fragment to the complementary probe on the array creates a double stranded
duplex at the array
surface which could incorporate an intercalating dye, and provide a unique
signal indiciative of
that hybridization. Intercalating dyes are well known in the art and include
those described in,
e.g., Gudnason et al., Nucleic Acids Research, (2007) Vol. 35, No. 19, e127,
which is
incorporated herein by reference for all purposes. Similarly, although optical
signal detection
methods are particularly preferred, the probe configurations and assay methods
may also
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generally be practiced using non-optical labeling and/or detection methods,
e.g., using
electrochemical detection methods, e.g., ChemFETS, ISFETS, etc., optionally in
conjunction
which electrochemical labeling groups, e.g., possessing large charged groups
to amplify
detection of hybridization of the probe fragment to an array probe at or near
a detector surface.
[0027] Local
background can be detected for one or more regions of the array, with
signal intensity measurements being normalized by correcting for said
background. Typically,
the normalized signal intensity is less than about 10% of total signal, e.g.,
between about 1 and
about 10% of the total signal. In one example class of embodiments, the
normalized signal
intensity is between about 4 and about 7% of total signal. Typically, where
approximately 1%
or more of the signal is localized to the array, e.g., where about 1%, 2%, 3%,
4%, 5%, 6%, 7%,
8%, 9%, 10% or more of the signal is localized to the array for a region of
the chamber, it is
possible to discriminate the array signal from background. It is possible to
discriminate even
lower levels of signal to background, but this is not generally preferred. The
methods can also
include normalizing signal intensity by correcting for variability in array
capture nucleic acid
spotting (e.g., by correcting for spot size, spot density, or both), or by
correcting for uneven
field of view of different regions of the array.
[0028] The
ability to simultaneously detect multiple target nucleic acids in a sample
represents a preferred aspect of the invention. The sample may have one or a
plurality of target
nucleic acids, with the array comprising a plurality of capture nucleic acid
types that are
capable of detecting more than one target per sample. The capture nucleic acid
types are
spatially separated on the array, eliminating the need for the use of multiple
labels (although, as
noted, multiple labels can be used). In multiplex approaches, a plurality of
amplification
probes, each specific for a different nucleic acid target, is incubated with
the sample, which can
include one or more target nucleic acids. For example, there can be between
about 5 and about
100 or more capture nucleic acid types. Each potential target to be detected
will utilize a
different probe as well, e.g., there are optionally between about 5 and about
100 or more
labeled probe types in the amplification reaction, each specific for a
potential target of interest.
The array includes corresponding capture nucleic acids, e.g., between about 5
and about 100 or
more capture nucleic acid types. This permits a corresponding number of
signals to be detected
and processed by the array. For example, between about 5 and about 100 or more
different
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signals can be detected based upon positioning of the signals on the array
after hybridization of
the probe fragments to the array. As will be appreciated, the number of
capture probe types on
an array will generally be dictated by the number of distinct amplification
reactions that can be
multiplexed within a single reaction volume. However, capture arrays having
larger numbers
of different capture probes, e.g., greater than 100, greater than 1000, 10,000
or more capture
probe types, may also be employed in some circumstances, e.g., where
amplification reactions
are pooled for interrogation by the array, or the like.
[0029] An advantage of the present invention is that one capture array
configuration
may be used for multiple different target nucleic acid sequence panels. In
particular, a probe
set for a first panel will include probes that have first target specific
portions specific for the
targets in the panel, and second capture portions complementary to individual
probes on the
capture array. A probe set for a second different panel (whether partially
overlapping, or
completely different) will include target specific portions for that panel,
while the capture
portions will be the same as for the first panel's probe set. Restated, for
any panel of targets,
the probe set will include a semi-fixed portion of the probes used for that
panel, which will
always be complementary to a member of the capture array. The probes will also
include
variable portions that are selected for the specific panel of target nucleic
acids. For example, in
an analytical process, a first set of probes is employed where each probe in
the first set has a
first fixed portion that corresponds to a different capture probe on the
capture probe array.
Each probe also includes a target specific portion that is complementary to a
given target
sequence in the first panel. For a second panel, a second set of probes is
employed where each
probe in the set includes the same first fixed portion, but has a second
target specific portion
that is specific for the targets in that panel.
[0030] With reference to Figure 1A, portion A of the labeled probe
corresponds to the
variable portion, while portion B would correspond to the fixed portion that
would be
complementary to the probes on the array. The use of a universal or common
capture array and
capture probe set allows for more efficient and lower cost manufacturing of
the consumables
used in the invention.
[0031] Thus, in one embodiment in which the sample comprises multiple
target nucleic
acids, the method includes incubating a plurality of labeled probes, each
specific for a different
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target nucleic acid, with the target nucleic acids. Amplifying at least a
portion of the target
nucleic acids in the amplification primer dependent amplification reaction
results in cleavage of
a plurality of labeled probe types and resulting release of a plurality of
labeled probe fragment
types. The plurality of probe fragment types are hybridized to the array. Each
of the different
probe fragment types hybridizes to a spatially discrete capture nucleic acid
type. Detecting the
label signal includes detecting a plurality of label signals from a plurality
of spatially discrete
regions corresponding to the spatially discrete capture nucleic acids on the
array. Optionally,
and in several preferred embodiments, the labeled probe types comprise the
same label moiety,
but additional multiplexing and/or use of differentially controls or
registration probes can
include using a plurality of different label moieties. Typically, the labeled
probe types can
include one or more different label moieties, with the number of different
moieties being less
than the number of labeled probe types.
[0032] Devices and systems for performing the methods are a feature of the
invention.
The devices or systems can include a detection chamber that comprises at least
one high
efficiency nucleic acid detection array on at least one surface of the
chamber. As noted with
reference to the methods, the chamber is configured to reduce signal
background for signals
detected from the array. The device or system typically includes a thermo-
regulatory module
operably coupled to the detection chamber, which regulates temperature within
the chamber
during operation of the device. An optical train detects signal(s) produced at
the array during
operation of the device.
[0033] All of the dimensional features of the chamber to reduce background
noted with
reference to the methods optionally apply to the device. For example, the
device can be less
than about 500pm in depth in at least one dimension proximal to the array,
e.g., between about
lOpm and about 200pm in depth in at least one dimension proximal to the array.
The chamber
surface on which the array is formed can be composed of any suitable material,
e.g., a ceramic,
glass, quartz, or a polymer. In several embodiments, e.g., those utilizing epi-
fluorescence, the
surface will be at least partially transparent.
[0034] As noted with reference to the methods, the capture nucleic acids
on the array
are typically present at a non-rate limiting density during operation of the
device. The array
optionally includes a plurality of capture nucleic acid types, e.g., localized
to spatially distinct
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regions of the array. For example, 5 or more different capture nucleic acid
types can be present
on the array, e.g., up to about 100 or more different types. The capture
nucleic acids are
optionally coupled to a thermostable coating on the surface of the chamber,
facilitating
thermocycling of the array. Example coating can optionally include: a
chemically reactive
group, an electrophilic group, an NHS ester, a tetra- or pentafluorophenyl
ester, a mono- or
dinitrophenyl ester, a thioester, an isocyanate, an isothiocyanate, an acyl
azide, an epoxide, an
aziridine, an aldehyde, an a,13-unsaturated ketone or amide comprising a vinyl
ketone or a
maleimide, an acyl halide, a sulfonyl halide, an imidate, a cyclic acid
anhydride, a group active
in a cycloaddition reaction, an alkene, a diene, an alkyne, an azide, or a
combination thereof.
[0035] The thermo-regulatory module optionally includes features that
facilitate
thermocycling, such as a thermoelectric module, a Peltier device, a cooling
fan, a heat sink, a
metal plate configured to mate with a portion of an outer surface of the
chamber, etc.
Typically, the thermo regulatory module has a feedback enabled control system
operably
coupled to a computer which controls or is part of the module.
[0036] The optical train can include or be operably coupled to an
epifluorescent
detection system. Typical optical train components include any of: an
excitation light source,
an arc lamp, a mercury arc lamp, an LED, a lens, an optical filter, a prism, a
camera, a
photodetector, a CMOS camera, and/or a CCD array. The device can also include
or be
coupled to an array reader module, which correlates a position of the signal
in the array to a
nucleic acid to be detected.
[0037] The device or system can include or be operably coupled to system
instructions,
e.g., embodied in a computer or computer readable medium. The instructions can
control any
aspect of the device or system, e.g., to correlate one or more measurements of
signal intensity
and a number of amplification cycles performed by the thermo-regulatory module
to determine
a concentration of a target nucleic acid detected by the device.
[0038] A system can include the device, e.g., operably coupled to a
computer. The
computer can include, e.g., instructions that control thermocycling by the
thermo-regulatory
module, and/or that specify when images are taken or viewed by the optical
train, and/or can
convert image information into signal intensity curves as a function of time,
determine
concentration of a target nucleic acid analyzed by the device, and/or the
like. The computer
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can include instructions for normalizing signal intensity to account for
background, e.g., for
detecting local background for one or more regions of the array, and for
normalizing array
signal intensity measurements by correcting for said background. Similarly,
the computer can
include instructions for normalizing signal intensity by correcting for
variability in array
capture nucleic acid spotting, uneven field of view of different regions of
the array, or the like.
[0039] The invention includes, in one aspect, a nucleic acid detection
consumable, e.g.,
for use with the devices and systems of the invention, e.g., to practice the
methods of the
invention. The consumable can include, e.g., a thin chamber less than about
500pm in depth,
where the chamber includes an optically transparent window that has a high
efficiency capture
nucleic acid array disposed on an inner surface of the window. The consumable
can also
include at least one reagent delivery port, e.g., fluidly coupled to the
chamber. Typically, the
consumable is configured to permit thermocycling of fluid within the chamber.
[0040] All of the features noted above with reference to the array and
chamber in the
context of the devices, systems and methods of the invention apply to the
consumable as well
(and vice-versa). For example, the nucleic acid array can include a plurality
of different
capture nucleic acid types, which types are located in spatially distinct
regions of the array.
The density of the capture nucleic acids can be about, e.g., 2,000 fmol/cm2 or
greater, 2,500
fmol/cm2 or greater, 3,000 fmol/cm2 or greater, 4,000 fmol/cm2 or greater,
4,500 fmol/cm2 or
greater, or 5,000 fmol/cm2 or greater.
[0041] Similarly, the chamber can include a first upper surface comprising
the reagent
delivery port, and a bottom transparent surface comprising the window, e.g.,
where the top and
bottom surface are joined by sidewalls formed of a pressure-sensitive adhesive
material. Other
structures for joining the top and bottom surface to form the chamber can also
be used. For
example, the top and bottom surface can be joined together by a gasket or
shaped feature on the
upper or lower surface, or both. The gasket or feature is optionally fused or
adhered to a
corresponding region of the upper or lower surface, or both. In some
embodiments, the gasket
or feature directs flow of a UV curable adhesive, which adhesive is flowed
between the upper
and lower surfaces and exposed to UV light, thereby joining the upper and
lower surfaces. In
other embodiments, the upper and lower surfaces can be ultrasonically fused
together, with the
gasket or feature delimiting regions that are fused. In another example, the
feature is a
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transparent region on either the upper or lower surface and a corresponding
shaded region on a
cognate upper or lower surface. In this embodiment, the upper and lower
surfaces can be laser
welded together by directing laser light through the transparent region and
onto the shaded
region.
[0042] The capture nucleic acid array is typically coupled to a thermally
stable coating
on the window. For example, the coating can include a chemically reactive
group, an
electrophilic group, an NHS ester, a tetra- or pentafluorophenyl ester, a mono-
or dinitrophenyl
ester, a thioester, an isocyanate, an isothiocyanate, an acyl azide, an
epoxide, an aziridine, an
aldehyde, an a,13-unsaturated ketone or amide comprising a vinyl ketone or a
maleimide, an
acyl halide, a sulfonyl halide, an imidate, a cyclic acid anhydride, a group
active in a
cycloaddition reaction, an alkene, a diene, an alkyne, an azide, or a
combination thereof. The
window itself can include, e.g., glass, quartz, a ceramic, a polymer or other
transparent
material.
[0043] All of the features noted above with respect to the methods,
systems and devices
apply with respect to the configuration of the chamber in the consumable. For
example, the
chamber can be between about lOpm and about 200pm in depth, e.g., about 140 pm
in depth.
The chamber can be significantly wider in other dimensions, e.g., between
about 1 mm and
about 50 mm in average diameter. In one specific embodiment, the chamber is
between about
mm and about 20 mm in average diameter.
[0044] The invention includes kits, e.g., comprising the consumable of the
invention.
The kits can also include packaging materials, instructions for practicing the
methods, control
reagents (e.g., control templates, probes, or primers, e.g., which bind to
control sites on an array
of the consumable).
[0045] The methods, systems, devices, consumables and kits can be used in
combination, e.g., with the kit providing the consumable for use in a system
or device of the
invention, e.g., to practice the methods of the invention. Unless stated
otherwise, steps of the
methods optionally have corresponding structural features in the systems,
devices, consumables
or kits, and vice-versa.
BRIEF DESCRIPTION OF THE FIGURES
[0046] Figure 1A and 1B are schematic illustrations of PCR probes of the
invention.
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[0047] Figure 2 is a schematic of a PCR chamber of the invention.
[0048] Figure 3 is a graph showing array based real time PCR curves for
copy number
titration for a three step amplification reaction.
[0049] Figure 4 is a graph showing solution based real time PCR curves
generated from
aliquots of solutions.
[0050] Figure 5 is a graph showing an array based real time PCR curve
generated with
an unquenched probe.
[0051] Figure 6 is a graph showing real time PCR curves for a multiplexed
amplification.
[0052] Figure 7 is a graph showing array-based real time PCR curves for a
10-plex
reaction with no target added.
[0053] Figure 8 is a graph showing array-based real time PCR curves with a
10-plex
panel and 3 targets present at 104 copies each.
[0054] Figure 9 is a graph showing real-time kinetics of hybridization of
a 5' flap
mimic.
[0055] Figure 10A and 10B show schematics of aspects of the invention.
[0056] Figure 11 is a schematic system illustration.
[0057] Figure 12 is a graph showing array based real time PCR curves for
copy number
titration for a two step amplification reaction.
[0058] Figure 13 is a plot showing the relation of reaction chamber
thickness to the
signal to background ratio.
[0059] Figure 14 is a schematic illustration of an overall detection
system for mobile
substrate embodiments of the invention.
Figure 15 is a plot of real time PCR using quencher bearing probes, with
decreasing fluorescent signal over the reaction's progress.
DETAILED DESCRIPTION
[0060] Methods of performing target nucleic acid amplification, detection
and real-time
quantification are a feature of the invention. In the methods, amplification
of the target nucleic
acid releases a target-specific labeled probe fragment that hybridizes to an
array; the array is
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distributed in the chamber where the amplification takes place. Signal is
detected from the
array, providing both detection and real-time quantification of the target
nucleic acid.
[0061] The invention also provides reaction chambers, typically formatted
as
consumables that comprise nucleic acid detection arrays within the chamber, as
well as devices
and systems that interact with the consumables.
METHODS
[0062] The invention provides methods of detecting and quantifying one or
more target
nucleic acids in a sample, in real time. The methods are highly amenable to
multiplexing,
enabling specific detection and quantification of a larger number of different
target nucleic
acids using one chamber reaction and detection than can be achieved using
available solution-
based real-time nucleic acid detection methods. This is because the invention
utilizes array-
based detection of analytes (with the array being in contact with the
analytes), rather than
solution-phase spectral detection. A nucleic acid detection array has
significantly greater
ability to resolve analytes via array position discrimination as compared to
discrimination of,
e.g., different dye labels in solution. By way of comparison, it is possible
to construct arrays
that simultaneously detect thousands of different analytes, while it is
typically not possible to
detect more than about 5 differently labeled fluorophores in solution.
[0063] Figure 10A provides a partial overview of the method. As shown, a
primer 102
is hybridized to a template or target sequence 100 along with a labeled probe
104. The probe
104 comprises a portion 104a that is complementary to the target or template
sequence 100, and
an orthogonal sequence 104b that is not complimentary to the template (a
"flap"), coupled to a
label moiety 106. The orthogonal sequence 104b or probe fragment is cleaved
during an
amplification reaction (e.g., a PCR amplification cycle). In one convenient
approach, the
natural nuclease activity of a polymerase is used to cleave the flap¨in this
approach, primer
extension by the polymerase results in cleavage of the flap 104b by nuclease
action of the
polymerase as it encounters the junction between the flap 104b and the
template 100. This
releases the flap as a labeled probe fragment 104b, which is then hybridized
to the array 110
bearing capture probes 112 that are complementary to the released probe
fragment 104b, e.g.,
by adjusting the temperature to conditions that permit specific hybridization.
Detection of the
label on the array provides for detection and quantification of the template,
in real time.
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[0064] In general, a sample that is to be tested for the presence (or
absence) of one or
more target nucleic acid(s) is subjected to an amplification reaction. The
reaction can easily be
multiplexed to amplify, detect and quantify between about 10 and about 100 or
more different
nucleic acids, in a single reaction chamber. For example, about 10, about 20,
about 30, about
40, about 50, about 60, about 70, about 80, about 90, or about 100 or more
nucleic acids can be
detected in a single amplification/ detection chamber. A working example
herein
demonstrating simultaneous amplification, detection and quantification of 10
different target
nucleic acids in a reaction/ detection chamber is shown below. This example,
and the
capabilities of the method herein, exceeds the capabilities of typical
spectrally-limited,
solution-based multiplex detection.
[0065] In the methods, each target nucleic acid to be detected is
specifically amplified
using at least one, and generally two amplification primers (the use of two
primers adds
specificity to the reaction, and speeds the rate of product formation, as
compared to a single
primer). The primers are typically specifically hybridized to the target
nucleic acid(s) in the
sample, and extended using a polymerase, e.g., in a standard polymerase chain
reaction (PCR).
The design and construction of amplification primers that can be used to
amplify a target
nucleic acid of interest follows known methods. For details regarding PCR
primer design, see
e.g., Anton Yuryev (Editor) (2007) PCR Primer Design (Methods in Molecular
Biology)
[Hardcover] Humana Press; 1st edition ISBN-10: 158829725X, ISBN-13: 978-
1588297259, as
well as the references noted below.
[0066] PCR amplification using the primers on the target template nucleic
acids can be
performed using appropriate reaction conditions, including use of standard
amplification
buffers, enzymes, temperatures, and cycle times. For a review of PCR
techniques, including
hybridization conditions, buffers, reagents, reaction cycle times, and the
like, see, e.g., Yuryev
(above), van Pelt-Verkuil et al. (2010) Principles and Technical Aspects of
PCR Amplification
Springer; 1st Edition ISBN-10: 9048175798, ISBN-13: 978-9048175796; Bustin
(Ed) (2009)
The PCR Revolution: Basic Technologies and Applications Cambridge University
Press; 1st
edition ISBN-10: 0521882311, ISBN-13: 978-0521882316; Viljoen et al. (2005)
Molecular
Diagnostic PCR Handbook Springer, ISBN 1402034032; Kaufman et al. (2003)
Handbook of
Molecular and Cellular Methods in Biology and Medicine Second Edition Ceske
(ed) CRC
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Press (Kaufman); The Nucleic Acid Protocols Handbook Ralph Rapley (ed) (2000)
Cold
Spring Harbor, Humana Press Inc (Rapley); Chen et al. (ed) PCR Cloning
Protocols, Second
Edition (Methods in Molecular Biology, volume 192) Humana Press; PCR Protocols
A Guide
to Methods and Applications (Innis et al. eds) Academic Press Inc. San Diego,
CA (1990)
(Innis). Amplification conditions, primer design, and other details applicable
to real-time based
PCR methods are described, e.g., in Logan et al. (eds.) (2009) Real-Time PCR:
Current
Technology and Applications, Caister Academic Press, 1st edition ISBN-10:
1904455395,
ISBN-13: 978-1904455394, and M Tevfik Dorak (Editor) (2006) Real-time PCR
(Advanced
Methods) Taylor & Francis, 1st edition ISBN-10: 041537734X ISBN-13: 978-
0415377348.
[0067] A labeled probe specific for each target nucleic acid in the sample
is hybridized
along with the amplification primer(s) to the target nucleic acid(s). The
amplification reaction
cleaves the template-hybridized labeled probe to release a labeled probe
fragment. This labeled
fragment then hybridizes to the array in the reaction chamber, as shown in
Figure 10A.
[0068] Figure 1A schematically shows a probe useful in the methods of the
invention.
The probe comprises region A that is complimentary to a target nucleic acid.
The probe also
comprises "flap" B, which is not complimentary to the target nucleic acid.
Label E is attached
to flap B. Label E is shown in the terminal position, but the label can, in
fact, be formatted at
any point along Flap B. For example, any of a variety of nucleotides can be
labeled, and used
in standard or slightly modified nucleic acid synthesis protocols to provide a
label at any
desired position on the probe.
[0069] In Figure 1A, Optional region C, comprising label quencher D, is
complimentary to a portion of flap B. Under appropriate solution conditions,
region C base-
pairs with flap B, bringing label E and quencher D into proximity, thereby
quenching label E.
This reduces signal background of the solution phase in the reaction/
detection chamber, but
probe quenching is not required for practice of the invention. One surprising
aspect of the
invention is that it is possible to specifically detect probe fragments bound
to the array, even
where there is unquenched probe in solution proximal to the array. A working
example of this
embodiment is described herein. In general, the use of high efficiency arrays
in reaction/
detection chambers that are configured to reduce solution-phase background
permits
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discrimination of signal at the array from signal background in solution in
the methods,
consumables, devices and systems of the invention.
[0070] Depending upon the assay configuration, a wide variety of different
label groups
may be employed for labeling the labeled probe. As noted, such labels
typically include
fluorescent labeling groups, which may include individual fluorophores or
interactive dye pairs
or groups, e.g., FRET pairs, as well as donor/quencher pairs. A range of
different fluorescent
labeling groups suitable for labeling nucleic acid probes are described in,
e.g., the Molecular
Probes Handbook, 11th Edition (Life Technologies, Inc.).
[0071] While much of the discussion herein is directed to PCR based
amplification,
other amplification reactions can be substituted. For example, multienzyme
systems involving
cleavage reactions coupled to amplification reactions, such as those including
the cleavage of
scissile bonds (see, e.g., US 5,011,769; US 5,660,988; US 5,403,711; US
6,251,600) and
forked nucleic acid structures (US 7,361,467; US 5,422,253; US 7,122,364; US
6,692,917) can
be used. Helicase dependent amplification coupled to TaqMan like cleavage
(Tong, Y et > al
2008 BioTechniques 45:543-557) can also be used. Nucleic acid sequence based
amplification
(NASBA), or the ligase chain reaction (LCR) can be used. In NASBA-based
approaches, the
probe can be hybridized to a template along with amplification primer(s), as
in PCR. The
probe can be cleaved by the nuclease action of reverse transcriptase, or an
added endonuclease,
releasing the probe fragment in a manner similar to the release by a
polymerase in PCR. One
potential advantage of NASBA is that no thermocycling is required. This
simplifies overall
device and system requirements. For a description of NASBA, see, e.g., Compton
(1991),
"Nucleic acid sequence-based amplification," Nature 350 (6313): 91-2. For the
use of NASBA
to detect, e.g., pathogenic nucleic acids, see, e.g., Keightley et al. (2005)
"Real-time NASBA
detection of SARS-associated coronavirus and comparison with real-time reverse
transcription-
PCR," Journal of Medical Virology 77 (4): 602-8. When an LCR-style reaction is
used, the
probe can be cleaved using an endonuclease, rather than relying on nuclease
activity of the
amplification enzyme.
[0072] In the methods herein, a detection chamber that has at least one
high efficiency
nucleic acid detection array on at least one inner surface of the chamber is
provided. The high
efficiency array typically has a non-rate limiting number of capture nucleic
acids that permits
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an efficient capture of probe fragments produced by the amplification reaction
in the chamber.
The capture nucleic acids are configured to capture relatively small probe
nucleic acids, which
also increases array efficiency. The chamber is configured to reduce signal
background
proximal to the array, e.g., by shaping the chamber to reduce background. For
example,
background is reduced by making the chamber thin (shallow) proximal to (e.g.,
above or
below) the array; e.g., the chamber is typically about 500pm or shallower
above or below the
array, although detection in chambers as deep as lmm or larger can work.
Further details
regarding the reaction chamber and array is described below with reference to
the consumable
useful in the methods.
[0073] Signals captured by the array are detected and signal intensity is
measured.
Signal intensity is correlated to the presence and/or quantity of the target
nucleic acid present in
the sample. Typically, the sample is amplified for more than 1 cycle before
initial detection, to
increase the level of signal by increasing the number of probe fragments
released by the
amplification. For example, the target nucleic acid can optionally be
amplified for at least, e.g.,
2, 3, 4, 5, 6, 7, 8, 9, 10 or more amplification cycles prior to detecting
signal from the array.
Figure 10B provides an alternative configuration of the assays of the
invention.
As shown, the target specific probe is again provided with an orthogonal flap
portion 104b, as
noted in Figure 10A, above. However, rather than being labeled with a
fluorescent labeling
moiety 106 as in Figure 10A, that produces a fluorescent signal, the flap 104b
bears a quencher
group 116. In contrast, the capture probe 112 on the array 110 is labeled with
a corresponding
fluorescent group 114, i.e., that is quenched by the quencher group 116 on the
flap 104b. When
the flap portion 104b is cleaved and released from the full length probe 104
upon amplification
of the target sequence 100, the flap is able to hybridize to the capture probe
112 and quenches
the signal from its associated fluorophore 114. As a result, presence of a
target sequence of
interest, results in amplification of that sequence and cleavage of the
quencher-flap 104b from
the probe 104, which in turn, is able to hybridize to the capture probe 112 on
the array 110,
resulting in a reduced or absent fluorescent signal for a given capture probe
location on the
array. By locating the quencher and fluorophore on complementary portions of
the flap or
probe fragment and capture probe, respectively, one can ensure that these
groups are within
sufficient proximity for energy transfer and quenching.
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Fluorophore-quencher group pairs that may be coupled to the flap and capture
probes are well known in the art, for example, fluorophore-quencher pairs such
as black hole
quencher 2/Cy 3 and Iowa Black RQ/Cy3.
[0074] As will be appreciated, the foregoing quencher probe assay
configuration
eliminates a fluid borne fluorescent component and consequently, any
background fluorescent
signal that might emanate from the fluid. Instead, the signaling event is the
loss of fluorescent
signal due to quenching of surface bound fluorophores.
[0075] In one typical embodiment, fluorescent or other optical images are
captured
from the array at selected times, temperatures, and amplification cycle
intervals, during the
amplification reactions. These images are analyzed to determine whether the
target nucleic
acid(s) are present in the sample, and to provide quantification of starting
target nucleic acid
concentrations in the sample. The images are analyzed using a combination of
mean gray
intensity measurements, background correction and baseline adjustments. The
background can
be measured locally for each spot in the array. The background is computed by
measuring the
image intensity of a concentric annulus of the solution surrounding the array
region (e.g., array
spot) of interest. The signal from each region is then corrected to account
for local background
in the region. The corrected signal from each region can be further normalized
to account for
variability in spotting, as well as uneven illumination in the field of view.
The average of the
corrected intensity measurements obtained from the first few cycles, typically
between cycle 5
and 15, are used to adjust the baseline and normalize measurements from each
region.
[0076] Further details regarding methods of quantifying nucleic acids
based upon signal
intensity measurements following amplification can be found, e.g., in the
references noted in
above this section and in Jang B. Rampal (Editor) (2010) Microarrays: Volume
2, Applications
and Data Analysis (Methods in Molecular Biology) Humana Press; 2nd Edition
ISBN-10:
1617378526, ISBN-13: 978-1617378522; Stephen A. Bustin (Editor) (2004) A-Z of
Quantitative PCR (IUL Biotechnology, No. 5) (IUL Biotechnology Series)
International
University Line; 1st edition ISBN-10: 0963681788, ISBN-13: 978-0963681782; and
in
Kamberova and Shah (2002) DNA Array Image Analysis: Nuts & Bolts (Nuts & Bolts
series)
DNA Press; 2nd edition ISBN-10: 0966402758, ISBN-13: 978-0966402759.
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[0077] In an alternate configuration, the capture probes may optionally be
coupled to a
mobile substrate, such as beads, resins, particles or the like (generally
referred to
interchangeably herein as "beads"), rather than a static substrate. For
example, as noted
elsewhere herein, a planar substrate may be used to provide arrayed capture
probes that will
hybridize with the cleaved probe fragments produced during the amplification
of the target
nucleic acid sequence or sequences within the sample material. The presence of
a given target
nucleic acid sequence is detected by detecting which capture probe position on
the array the
probe fragments hybridize. Because each probe fragment is specific to a
particular target
sequence, if that probe fragment is present, it is indicative that the target
was present and
amplified. In a mobile phase substrate, each different type of capture probe
in a given analysis
is coupled to a different mobile substrate that also bears a unique label. The
mobile substrates
are then passed through a detection channel in order to identify both the
bead, and by
implication, the capture probe, and whether the labeled probe fragment is
present. If the
labeled probe is detected on a given bead that corresponds to a particular
capture probe, it is
indicative that the target sequence associated with that probe fragment (and
complementary
capture probe) was present in the sample and amplified. This aspect of the
invention may be
employed in endpoint detection, e.g., after completion of the overall
amplification reaction, but
may also be employed in quantitative analysis, e.g., siphoning a fraction of
beads from the
amplification mixture after one or more amplification cycles, and measuring
the labeled probe
fragment signal intensity from the beads.
[0078] The concentration of captured labeled probe fragments on a given
bead will
provide a sufficiently high signal to background ratio in the detection
channel such that
separation of the beads from the reaction mixture is not necessary. In
addition, as with the
array based substrates, the inclusion of a secondary structure in the intact
probe and/or optional
quenching group permits greater ability to distinguish between probe fragment
and intact probe
background signal, whether in solution or from unintended binding to the
mobile substrate. In
some cases, the nature of the intact probe's secondary structure would also be
expected to give
rise to steric hindrance in binding with the capture probes on the mobile
substrate, resulting in
some cases in a reduced likelihood that the intact probe would bind to the
beads.
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[0079] A variety of different bead types may be used in conjunction with
this aspect of
the invention. For example, polystyrene, cellulosic, acrylic, vinyl, silica,
paramagnetic or other
inorganic particles, or any of a variety of other bead types may be employed.
As noted, the
beads with typically be differentially labeled with a unique label signature.
Again, a variety of
different label types may be used, including organic fluorescent labels,
inorganic fluorescent
labels (e.g., quantum dots), luminescent labels, electrochemical labels, or
the like. Such labels
are widely commercially available and configured to be readily coupled to
appropriately
activated beads. In the case of fluorescent labeling groups, a large number of
label signatures
may be provided by providing different combinations of 2, 3, 4 or more
spectrally distinct
fluorescent labeling groups and different levels of each label, so as to
provide a broad range of
unique label signatures without having to use a broad spectrum of excitation
radiation, e.g.,
multiple lasers.
[0080] The method is typically performed using the devices, systems,
consumables and
kits herein. All features of the devices, systems and consumables can be
provided to practice
the methods herein, and the methods herein can be practiced in combination
with the devices,
systems, consumables and kits.
CONSUMABLES
[0081] The one-pot reaction chambers of the invention are configured to
reduce signal
background. High-efficiency arrays are formed on at least one inner surface of
the chambers.
The arrays typically are in contact with amplification reactants and products
during both
amplification and array hybridization steps of the methods. This allows a user
to run one or
more amplification reaction cycles, detect the results by monitoring signal
from the array in
real time, and to then run one or more additional amplification cycles, again
followed by
detection. Thus, signal intensity from the array can be used to both detect
and quantify a
nucleic acid of interest, in real time.
[0082] The consumables of the invention include a chamber and a high
efficiency array
on an inner surface of the chamber. The chamber is typically thin (shallow),
e.g., less than
about 1 mm in depth. In general, the thinner the chamber, the less solution
above the array,
which reduces signal background from labeled probes or probe fragments in the
solution.
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Typical desirable chamber depths are in the range of about lpm to about 500pm.
For ease of
fabrication of the consumable, the chamber is often in the range of about lOpm
to about 250pm
in depth above the array, e.g., about 100pm to about 150 pm in depth. The
chamber can
include a surface that has a reagent delivery port, e.g., for delivery of a
sample by manual or
automated pipettor.
[0083] Figure 2 provides a blow-up schematic of an example consumable. In
this
example, bottom surface layer 1 and upper surface layer 2, are joined by
middle layer 3.
Cutout 4 forms a chamber upon assembly of layers 1, 2, and 3. Port(s) 5
form(s) a convenient
way to deliver buffer and reagents to the chamber upon assembly. A high
efficiency array can
be formed on the top or bottom layer in the region that forms the top or
bottom surface of the
cutout. In one convenient embodiment, where epifluorescent detection is used
for detection of
label bound to the array, the array is fabricated on the lower surface, with
the consumable being
configured to be viewed by detection optics located in the devices and systems
of the invention
below the lower surface. Generally, either the top or bottom surface (or both)
will include a
window through which detection optics can view the array.
[0084] Middle layer 3 can take any of a variety of forms, depending on the
consumable
assembly method to be used. In one convenient embodiment, top and bottom
surfaces 1 and 2
are joined by layer 3 formed of a pressure-sensitive adhesive material.
Pressure sensitive
adhesive layers (e.g., tape) are well known and widely available. See, e.g.,
Benedek and
Feldstein (Editors) (2008) Handbook of Pressure-Sensitive Adhesives and
Products: Volume 1:
Fundamentals of Pressure Sensitivity, Volume 2: Technology of Pressure-
Sensitive Adhesives
and Products, Volume 3: Applications of Pressure-Sensitive Products, CRC
Press; 1st edition
ISBN-10: 1420059343, ISBN-13: 978-1420059342.
[0085] Other fabrication methods for joining the top and bottom surface to
form the
chamber can also be used. For example, the top and bottom surfaces can be
joined together by
a gasket or shaped feature on the upper or lower surface, or both. The gasket
or feature is
optionally fused or adhered to a corresponding region of the upper or lower
surface, or both.
Silicon and polymer chip fabrication methods can be applied to form features
in the top or
bottom surface. For an introduction to feature fabrication methods, including
micro-feature
fabrication, see, e.g., Franssila (2010) Introduction to Microfabrication
Wiley; 2nd edition
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ISBN-10: 0470749830, ISBN-13: 978-0470749838; Shen and Lin (2009) "Analysis of
mold
insert fabrication for the processing of microfluidic chip" Polymer
Engineering and Science
Publisher: Society of Plastics Engineers, Inc. Volume: 49 Issue: 1 Page:
104(11); Abgrall
(2009) Nanofluidics ISBN-10: 159693350X, ISBN-13: 978-1596933507; Kaajakari
(2009)
Practical MEMS: Design of microsystems, accelerometers, gyroscopes, RF MEMS,
optical
MEMS, and microfluidic systems Small Gear Publishing ISBN-10: 0982299109, ISBN-
13:
978-0982299104; Saliterman (2006) Fundamentals of BioMEMS and Medical
Microdevices
SPIE Publications ISBN-10: 0819459771, ISBN-13: 978-0819459770; Madou (2002)
Fundamentals of Microfabrication: The Science of Miniaturization, Second
Edition CRC Press;
ISBN-10: 0849308267, ISBN-13: 978-0849308260. These fabrication methods can be
used to
form essentially any feature that is desired on the top or bottom surface,
eliminating the need
for an intermediate layer. For example, a depression can be formed in the top
or bottom
surface (or both) and the two layers joined, thereby forming the chamber.
[0086] In some embodiments, the gasket or feature directs flow of a UV or
radiation
curable adhesive. This adhesive is flowed between the upper and lower surfaces
and exposed
to UV light or radiation (e.g., electron beam, or "EB" radiation), thereby
joining the upper and
lower surfaces. For a description of available adhesives, including UV and
radiation curable
adhesives, see, e.g., Ebnesajjad (2010) Handbook of Adhesives and Surface
Preparation:
Technology, Applications and Manufacturing William Andrew; 1st edition ISBN-
10:
1437744613, ISBN-13: 978-1437744613; Drobny (2010) Radiation Technology for
Polymers,
Second Edition CRC Press; 2 edition ISBN-10: 1420094041, ISBN-13: 978-
1420094046.
[0087] In other embodiments, the upper and lower surfaces can be
ultrasonically fused
together, with the gasket or surface feature delimiting regions that are fused
and the chamber or
other structural features to be produced in the consumable. Ultrasonic welding
and related
techniques useful for fusing materials are taught, e.g., in Astashev and
Babitsky (2010)
Ultrasonic Processes and Machines: Dynamics, Control and Applications
(Foundations of
Engineering Mechanics) Springer; 1st Edition. edition ISBN-10: 3642091245,
ISBN-13: 978-
3642091247; and Leaversuch (2002) "How to use those fancy ultrasonic welding
controls,"
Plastics Technology 48(10): 70-76.
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[0088] In another example, the feature is a transparent region on either
the upper or
lower surface and a corresponding shaded region on a cognate upper or lower
surface. In this
embodiment, the upper and lower surfaces can be laser welded together by
directing laser light
through the transparent region and onto the shaded region. Laser welding
methods are taught,
e.g., in Steen et al. (2010) Laser Material Processing Springer; 4th ed.
edition ISBN-10:
1849960615, ISBN-13: 978-1849960618; Kannatey-Asibu (2009) Principles of Laser
Materials
Processino (Wiley Series on Processing of Engineering Materials) Wiley ISBN-
10:
0470177985, ISBN-13: 978-0470177983; and Duley (1998) Laser Welding Wiley-
Interscience
ISBN-10: 0471246794, ISBN-13: 978-0471246794.
[0089] The capture nucleic acid array is typically coupled to a thermally
stable coating
on the window. The window itself can include, e.g., glass, quartz, a ceramic,
a polymer or
other transparent material. A variety of coatings suitable for coating the
window are available.
In general, the coating is selected based upon compatibility with the array
substrate (e.g.,
whether the chamber surface that the array is attached to is glass or a
polymer), ability to be
derivatized or treated to include reactive groups suitable for attaching array
members, and
compatibility with process conditions (e.g., thermostability, photostability,
etc.). For example,
the coating can include a chemically reactive group, an electrophilic group,
an NHS ester, a
tetra- or pentafluorophenyl ester, a mono- or dinitrophenyl ester, a
thioester, an isocyanate, an
isothiocyanate, an acyl azide, an epoxide, an aziridine, an aldehyde, an ct,13-
unsaturated ketone
or amide comprising a vinyl ketone or a maleimide, an acyl halide, a sulfonyl
halide, an
imidate, a cyclic acid anhydride, a group active in a cycloaddition reaction,
an alkene, a diene,
an alkyne, an azide, or a combination thereof. For a description of surface
coatings and their
use in attaching biomolecules to surfaces see, e.g., Plackett (Editor) (2011)
Biopolymers: New
Materials for Sustainable Films and Coatings Wiley ISBN-10: 0470683414, ISBN-
13: 978-
0470683415; Niemeyer (Editor) (2010) Bioconjugation Protocols: Strategies and
Methods
(Methods in Molecular Biology) Humana Press; 1st Edition. edition ISBN-10:
1617373540,
ISBN-13: 978-1617373541; Lahann (Editor) (2009) Click Chemistry for
Biotechnology and
Materials Science Wiley ISBN-10: 0470699701, ISBN-13: 978-0470699706;
Hermanson
(2008) Bioconjugate Techniques, Second Edition Academic Press; 2nd edition
ISBN-10:
0123705010, ISBN-13: 978-0123705013. Wuts and Greene (2006) Greene's
Protective Groups
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in Organic Synthesis Wiley-Interscience; 4th edition ISBN-10: 0471697540, #
ISBN-13: 978-
0471697541; Wittmann (Editor) (2006) Immobilisation of DNA on Chips II (Topics
in Current
Chemistry) Springer; 1st edition ISBN-10: 3540284362, ISBN-13: 978-3540284369;
Licari
(2003) Coating Materials for Electronic Applications: Polymers, Processing,
Reliability,
Testing (Materials and Processes for Electronic Applications) William Andrew
ISBN-10:
0815514921, ISBN-13: 978-0815514923; Conk (2002) Fabrication Techniques for
Micro-
Optical Device Arrays Storming Media ISBN-10: 1423509641, ISBN-13: 978-
1423509646,
and Oil and Colour Chemists Association (1993) Surface Coatings - Raw
materials and their
usage, Third Edition Springer; 3rd edition, ISBN-10: 0412552108, ISBN-13: 978-
0412552106.
[0090] Methods of making nucleic acid arrays are available and can be
adapted to the
invention by forming the arrays on an inner chamber surface. Techniques for
forming nucleic
acid microarrays that can be used to form arrays on an inner chamber surface
are described,
e.g., in Rampal (Editor) Microarrays: Volume I: Synthesis Methods (Methods in
Molecular
Biology) Humana Press; 2nd Edition ISBN-10: 1617376639, ISBN-13: 978-
1617376634;
Miller and Nicolau (Editors) (2010) Microarray Technology and Its Applications
(Biological
and Medical Physics, Biomedical Engineering) Springer; 1st Edition. ISBN-10:
3642061826,
ISBN-13: 978-3642061820; Xing and Cheng (Eds.) (2010) Biochips: Technology and
Applications (Biological and Medical Physics, Biomedical Engineering)
Springer; 1st Edition.
ISBN-10: 3642055850, ISBN-13: 978-3642055850; Dill et al. (eds) (2010)
Microarrays:
Preparation, Microfluidics, Detection Methods, and Biological Applications
(Integrated
Analytical Systems) Springer ISBN-10: 1441924906, ISBN-13: 978-1441924902;
Whittmann
(2010) Immobilisation of DNA on Chips II (Topics in Current Chemistry)
Springer; 1st Edition
ISBN-10: 3642066666, ISBN-13: 978-3642066665; Rampal (2010) DNA Arrays:
Methods and
Protocols (Methods in Molecular Biology) Humana Press; 1st Edition ISBN-10:
1617372048,
ISBN-13: 978-1617372049; Schena (Author, Editor) (2007) DNA Microarrays
(Methods
Express) Scion Publishing; 1st edition, ISBN-10: 1904842151, ISBN-13: 978-
1904842156;
Appasani (Editor) (2007) Bioarrays: From Basics to Diagnostics Humana Press;
1st edition
ISBN-10: 1588294765, ISBN-13: 978-1588294760; and Ulrike Nuber (Editor) (2007)
DNA
Microarrays (Advanced Methods) Taylor & Francis ISBN-10: 0415358663, ISBN-13:
978-
0415358668. Techniques for attaching DNA to a surface to form an array can
include any of a
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variety of spotting methods, use of chemically reactive surfaces or coatings,
light-directed
synthesis, DNA printing techniques, and many other methods available in the
art.
[0091] Methods of quantifying array densities are provided in the
references noted
above and in Gong et al. (2006) "Multi-technique Comparisons of Immobilized
and Hybridized
Oligonucleotide Surface Density on Commercial Amine-Reactive Microarray
Slides" Anal.
Chem. 78:2342-2351.
[0092] The consumable can be packaged in a container or packaging
materials to form
a kit. The kit can also include components useful in using the consumable,
e.g., control
reagents (e.g., a control template, control probe, control primers, etc.),
buffers, or the like.
DEVICES AND SYSTEMS
[0093] Devices and systems that use the consumable and/or practice the
methods of the
invention are a feature of the invention as well. The device or system can
include the features
of the consumable, e.g., a reaction chamber and array (whether formatted as a
consumable, or
as dedicated portion of the device). Most typically, the device will typically
have a receiver,
e.g., a stage that mounts the consumable noted above, along with detection
optics for
monitoring the array, modules for thermocycling the chamber, and a computer
with system
instructions that control thermocycling, detection, and post-signal
processing.
[0094] An example schematic system is illustrated in Figure 11. As shown,
consumable 10 is mounted on stage 20. Environmental control module (ECM) 30
(e.g.,
comprising a Peltier device, cooling fans, etc.) provides environmental
control (e.g.,
thermocycling of temperature). Illumination light is provided by source 40
(e.g., a lamp, arc
lamp, LED, laser, or the like). Optical train 50 directs light from
illumination source 40 to
consumable 10. Signals from consumable 10 are detected by the optical train
and signal
information is transmitted to computer 60. Computer 60 optionally also
controls ECM 30
Signal information can be processed by computer 60, and outputted to user
viewable display
70, or to a printer, or both. ECM 30 can be mounted above or below consumable
10 and
additional viewing optics 80 (located above or below stage 20) can be
included.
[0095] The stage/ receiver is configured to mount the consumable for
thermocycling
and analysis. The stage can include registration and alignment features such
as alignment arms,
detents, holes, pegs, etc., that mate with corresponding features of the
consumable. The stage
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can include a cassette that receives and orients the consumable, placing it in
operable linkage
with other device elements, although this is not necessary in many
embodiments, e.g., where
the consumable mounts directly to the stage. Device elements are configured to
operate with
the consumable and can include a fluidic delivery system for delivering
buffers and reagents to
the consumable, a thermocycling or other temperature control or environmental
control
module, detection optics, etc. In embodiments where the chamber is build into
the device,
rather being incorporated into the consumable, the device elements are
typically configured to
operate on or proximal to the chamber.
[0096] Fluid delivery to the consumable can be done by the device or
system, or can be
performed prior to loading the consumable into the device or system. Fluid
handling elements
can be integrated into the device or system, or can be formatted into a
separate processing
station discrete from the device or system. Fluid handling elements can
include pipettors
(manual or automated) that deliver reagents or buffers to ports in the
consumable, or can
include capillaries, microfabricated device channels, or the like. Manual and
automated
pipettors and pipettor systems that can be used to load the consumable are
available from a
variety of sources, including Thermo Scientific (USA), Eppendorf (Germany),
Labtronics
(Canada) and many others. Generally speaking, a variety of fluidic handling
systems are
available and can be incorporated into the devices and systems of the
invention. See, e.g.,
Kirby (2010) Micro- and Nanoscale Fluid Mechanics: Transport in Microfluidic
Devices
ISBN-10: 0521119030, ISBN-13: 978-0521119030; Bruus (2007) Theoretical
Microfluidics
(Oxford Master Series in Physics) Oxford University Press, USA ISBN-10:
0199235090,
ISBN-13: 978-0199235094; Nguyen (2006) Fundamentals And Applications of
Microfluidics,
Second Edition (Integrated Microsystems) ISBN-10: 1580539726, ISBN-13: 978-
1580539722;
Wells (2003) High Throughput Bioanalytical Sample Preparation: Methods and
Automation
Strategies (Progress in Pharmaceutical and Biomedical Analysis) Elsevier
Science; 1st edition
ISBN-10: 044451029X, ISBN-13: 978-0444510297. The consumable optionally
comprises
ports that are configured to mate with the delivery system, e.g., ports of an
appropriate
dimension for loading by a pipette or capillary delivery device.
[0097] The ECM or thermo-regulatory module can include features that
facilitate
thermocycling, such as a thermoelectric module, a Peltier device, a cooling
fan, a heat sink, a
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metal plate configured to mate with a portion of an outer surface of the
chamber, a fluid bath,
etc. Many such thermo-regulatory components are available for incorporation
into the devices
and systems of the invention. See, for example, Kennedy and Oswald (Editors)
(2011) PCR
Troubleshooting and Optimization: The Essential Guide, Caister Academic Press
ISBN-10:
1904455727; ISBN-13: 978-1904455721; Bustin (2009) The PCR Revolution: Basic
Technologies and Applications Cambridge University Press; 1st edition ISBN-10:
0521882311,
ISBN-13: 978-0521882316; Wittwer et al. (eds.) (2004) Rapid Cycle Real-Time
PCR-Methods
and Applications Springer; 1 edition, ISBN-10: 3540206299, ISBN-13: 978-
3540206293;
Goldsmid (2009) Introduction to Thermoelectricity (Springer Series in
Materials Science)
Springer; 1st edition, ISBN-10: 3642007155, ISBN-13: 978-3642007156; Rowe
(ed.) (2005)
Thermoelectrics Handbook: Macro to Nano CRC Press; 1 edition, ISBN-10:
0849322642,
ISBN-13: 978-0849322648. The thermo regulatory module can, e.g., be formatted
into a
cassette that receives the consumable, or can be mounted on the stage in
operable proximity to
the consumable.
[0098] Typically, the ECM or thermo regulatory module has a feedback
enabled control
system operably coupled to a computer which controls or is part of the module.
Computer
directed feedback enabled control is an available approach to instrument
control. See, e.g.,
Tooley (2005) PC Based Instrumentation and Control, Third Edition, ISBN-10:
0750647167,
ISBN-13: 978-0750647168; Dix et al. (2003) Human-Computer Interaction (3rd
Edition)
Prentice Hall, 3rd edition ISBN-10: 0130461091, ISBN-13: 978-0130461094. In
general,
system control is performed by a computer, which can use, e.g., a script file
as an input to
generate target temperatures and cycle time periods as well as to specify when
images are to be
viewed/ taken by the detection optics. Photo images are typically taken at
different times during
a reaction and are analyzed by the computer to generate intensity curves as a
function of time
and thereby derive the concentration of the target.
[0099] The optical train can include any typical optical train components,
or can be
operably coupled to such components. The optical train directs illumination to
the consumable,
e.g., focused on an array of the consumable, or an array region. The optical
train can also
detect light (e.g., a fluorescent or luminescent signal) emitted from the
array. For a description
of available optical components, See, e.g., Kasap et al. (2009) Cambridge
Illustrated Handbook
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of Optoelectronics and Photonics Cambridge University Press; 1st edition ISBN-
10:
0521815967, ISBN-13: 978-0521815963; Bass et al. (2009) Handbook of Optics,
Third Edition
Volume I: Geometrical and Physical Optics, Polarized Light, Components and
Instruments(set)
McGraw-Hill Professional; 3rd edition, ISBN-10: 0071498893, ISBN-13: 978-
0071498890;
Bass et al. (2009) Handbook of Optics, Third Edition Volume II: Design,
Fabrication and
Testing, Sources and Detectors, Radiometry and Photometry McGraw-Hill
Professional; 3rd
edition ISBN-10: 0071498907, ISBN-13: 978-0071498906; Bass et al. (2009)
Handbook of
Optics, Third Edition Volume III: Vision and Vision Optics McGraw-Hill
Professional, ISBN-
10: 0071498915, ISBN-13: 978-0071498913; Bass et al. (2009) Handbook of
Optics, Third
Edition Volume IV: Optical Properties of Materials, Nonlinear Optics, Quantum
Optics
McGraw-Hill Professional, 3rd edition, ISBN-10: 0071498923, ISBN-13: 978-
0071498920;
Bass et al. (2009) Handbook of Optics, Third Edition Volume V: Atmospheric
Optics,
Modulators, Fiber Optics, X-Ray and Neutron Optics McGraw-Hill Professional;
3rd edition,
ISBN-10: 0071633138, ISBN-13: 978-0071633130; and Gupta and Ballato (2006) The
Handbook of Photonics, Second Edition, CRC Press, 2nd edition ISBN-10:
0849330955,
ISBN-13: 978-0849330957. Typical optical train components include any of: an
excitation
light source, an arc lamp, a mercury arc lamp, an LED, a lens, an optical
filter, a prism, a
camera, a photodetector, a CMOS camera, and/or a CCD array. In one desirable
embodiment,
an epifluorescent detection system is used. The device can also include or be
coupled to an
array reader module, which correlates a position of the signal in the array to
a nucleic acid to be
detected.
[0100] In the context of the mobile substrate embodiments of the
invention, in certain
aspects, the reaction vessel may be coupled directly to a detection channel,
e.g., within an
integrated microfluidic channel system, or through an appropriate fluidic
interface between the
amplification mixture and the detection channel. Alternatively, a fluidic
interface, such as are
present in conventional flow cytometers, may be provided on the detection
channel in order to
sample the amplification reaction mixture. The detection channel is typically
configured to
have a dimension that permits substantially only single beads to traverse the
channel at a given
time. The detection channel will typically include a detection window allowing
excitation of
the beads and collection of the fluorescent signals emanating from the beads.
In many cases, a
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fused silica or glass capillary or other transparent microfluidic channel is
used as the detection
channel.
[0101] The optical detection systems of the invention will typically
include one or more
excitation light sources capable of delivering excitation light at one or more
excitation
wavelengths. Also included will be an optical train that is configured to
collect the light
emanating from the detection channel, and filter excitation light from the
fluorescent signals.
The optical train also typically includes additional separation elements for
transmitting the
fluorescent signals, and for separating the fluorescent signal component(s)
emanating from the
bead and the signal component(s) emanating from the captured probe fragment.
[0102] Figure 14 provides a schematic illustration of an overall detection
system 1400.
As shown, the system includes first and second excitation light sources, such
as lasers 1402 and
1404, that each provide excitation light at different wavelengths.
Alternatively, a single broad
spectrum light source or multiple narrow spectrum light sources may be used to
deliver
excitation light at the appropriate wavelength range or ranges to excite the
detectable labels in
the sample, e.g., those associated with the beads, and those associated with
the labeled probe
fragments.
[0103] Excitation beams, shown as the solid arrows, from each laser are
directed to the
detection channel 1408, e.g., through the use of directional optics, such as
dichroic 1406. Light
that emanates from the beads 1410 in the detection channel 1408, is collected
by collection
optics, e.g., objective lens 1412. The collected light is then passed through
filter 1414 that is
configured to pass the emitted fluorescence, shown as the dashed arrows, while
rejecting the
collected excitation radiation. The collected fluorescence includes
fluorescence emitted from
the label on the captured probe fragments at a first emission spectrum, as
well as fluorescent
signals from the bead label signature, at one or more different emission
spectra, depending
upon the number of labels used in the beads. The collected fluorescence is
then passed through
dichroic 1416 that reflects the fluorescence from the captured probe fragments
to a first
detector 1420. The remaining fluorescent signature from the beads is then
subjected to further
separation by passing the signal through a second dichroic 1418, that reflects
a first bead signal
component to a second detector 1422, and passes a second bead signal component
to the third
detector 1424. The detectors are typically coupled to an appropriate processor
or computer for
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storing signal data associated with detected beads, and analyzing the signal
data to determine
the identity of the bead, and thus the capture probe and associated target
nucleic acid sequence.
Additionally, the processor or computer may include programming to quantify
signal data and
originating target sequence copy number, where time course experiments are
performed, e.g.,
beads are sampled after one or more amplification cycles in an overall
amplification reaction.
[0104] The device or system can include or be operably coupled to system
instructions,
e.g., embodied in a computer or computer readable medium. The instructions can
control any
aspect of the device or system, e.g., to correlate one or more measurements of
signal intensity
and a number of amplification cycles performed by the thermo-regulatory module
to determine
a concentration of a target nucleic acid detected by the device.
[0105] A system can include a computer operably coupled to the other
device
components, e.g., through appropriate wiring, or through wireless connections.
The computer
can include, e.g., instructions that control thermocycling by the thermo-
regulatory module, e.g.,
using feedback control as noted above, and/or that specify when images are
taken or viewed by
the optical train. The computer can receive or convert image information into
digital
information and/or signal intensity curves as a function of time, determine
concentration of a
target nucleic acid analyzed by the device, and/or the like. The computer can
include
instructions for normalizing signal intensity to account for background, e.g.,
for detecting local
background for one or more regions of the array, and for normalizing array
signal intensity
measurements by correcting for said background. Similarly, the computer can
include
instructions for normalizing signal intensity by correcting for variability in
array capture
nucleic acid spotting, uneven field of view of different regions of the array,
or the like.
ADDITIONAL DEFINITIONS
[0106] Before describing the present invention in detail, it is to be
understood that this
invention is not limited to particular devices or biological systems, which
can, of course, vary.
It is also to be understood that the terminology used herein is for the
purpose of describing
particular embodiments only, and is not intended to be limiting. As used in
this specification
and the appended claims, the singular forms "a", an and the include plural
referents unless
the content clearly dictates otherwise. Thus, for example, reference to "a
surface" e.g., of the
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consumable chamber discussed herein, optionally includes a combination of two
or more
surfaces, and the like.
[0107] Unless defined otherwise, all technical and scientific terms used
herein have the
same meaning as commonly understood by one of ordinary skill in the art to
which the
invention pertains. Although any methods and materials similar or equivalent
to those
described herein can be used in the practice for testing of the present
invention, the preferred
materials and methods are described herein. In describing and claiming the
present invention,
the following terminology is used in accordance with the definitions set out
below.
[0108] An "amplification primer" is a moiety (e.g., a molecule) that can
be extended in
a template-dependent amplification reaction. Most typically, the primer will
include or will be
a nucleic acid that binds to the template under amplification conditions.
Typically, the primer
will comprise a terminus that can be extended by a polymerase (e.g., by a
thermostable
polymerase in a polymerase chain reaction), or by a ligase (e.g., as in a
ligase chain reaction).
[0109] A "detection chamber" is a partly or fully enclosed structure in
which a sample
is analyzed or a target nucleic acids is detected. The chamber can be entirely
closed, or can
include ports or channels fluidly coupled to the chamber, e.g., for the
delivery of reagents or
reactants. The shape of the chamber can vary, depending, e.g., on the
application and available
system equipment. A chamber is "configured to reduce signal background
proximal to the
array" when it is dimensionally shaped to reduce signal background, e.g., by
including a narrow
dimension (e.g., chamber depth) near the array (thereby reducing the amount of
solution-
generated signal proximal to the array), or when the chamber is otherwise
configured to reduce
background, e.g., by the use of coatings (e.g., optical coatings) or
structures (e.g., baffles or
other shaped structures proximal to the array). Typically, the chamber is
configured to have a
dimension (e.g., depth) proximal to the array, such that signal in solution is
low enough to
permit signal differences at the array to be detected. For example, in one
embodiment, the
chamber is less than about lmm deep above the array; desirably the chamber is
less than about
500pm in depth. Typically, the chamber is less than about 400pm, less than
about 300pm, less
than about 200pm, or less than about 150pm in depth above the array. In one
example
provided herein, the chamber is about 142pm in depth.
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[0110] A "high-efficiency nucleic acid array" is an array of capture
nucleic acids that
efficiently hybridize to a probe or probe fragment under hybridization
conditions. In typical
embodiments, the array is formatted on an inner surface of a reaction/
detection chamber. The
array can be formed by any conventional array technology, from spotting to
chemical or
photochemical synthesis on the surface. High efficiency is achieved by
controlling the length
of the region of the capture probe that recognizes the probe or fragment
(shorter probes
hybridize more efficiently than long probes, down to a minimum hybridization
length for the
hybridization conditions), and by controlling the number of capture nucleic
acids in each array
region. Capture sites can be made more efficient/ available for hybridization
by including a
linking sequence or structure between the capture site and the surface (thus
formatting the
capture sites at a selected distance from the surface, which can reduce
surface effects on
hybridization). For example, nucleic acid sequences or polyethylene glycol
linkers (or both)
can be used. The number of capture nucleic acids in each array region are
distributed such that
the number of sites available for hybridization for a given probe or fragment
produced as a
result of a typical amplification reaction is not rate limiting. As noted
previously, this means
that the number of sites available for binding labeled probe fragments
produced during the
amplification reaction is in excess, and preferably substantially in excess of
the number of sites
that would be saturated by the concentration of probe fragments in the
reaction mixture after
the amplification reaction.
[0111] A "labeled probe" is a molecule or compound that specifically
hybridizes to a
target nucleic acid under amplification conditions, and that comprises a
moiety that is
detectable, or that can be made detectable. Most typically, the labeled probe
is a nucleic acid
that comprises an optical label such as a fluorophore, dye, lumophore, quantum
dot, or the like.
The label can be directly detectable, or can be in a quenched state, e.g.,
where the probe
comprises a quencher moiety. In many embodiments herein, the labeled probe is
cleaved
during target nucleic acid amplification to release a probe fragment
comprising a detectable
label. For example, the labeled probe can include a fluorophore and a
quencher, e.g., where an
amplification reaction results in cleavage of the probe to release the labeled
probe fragment.
Most typically, the probe will include a "flap" region. This flap region does
not base-pair with
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the target during hybridization, and is cleaved from the rest of the probe by
a nuclease (e.g.,
nuclease activity of a polymerase), thereby forming the probe fragment.
EXAMPLES
[0112] The following examples are offered to illustrate, but not to limit
the claimed
invention. It is understood that the examples and embodiments described herein
are for
illustrative purposes only and that various modifications or changes in light
thereof will be
suggested to persons skilled in the art and are to be included within the
spirit and purview of
this application and scope of the appended claims.
EXAMPLE DETECTION SYSTEM
[0113] The detection system of this example allows for single chamber,
multiplexed,
real time PCR detection of a target nucleic acid. The system extends the
multiplexing
capability of real time PCR by moving from traditional spectral discrimination
to array-based
spatial discrimination to generate real time information specific to each
target being amplified.
[0114] Traditionally, single well multiplexing is achieved by using PCR
probes such as
TAQMANTm probes that are specific to each amplicon and that are labeled with
fluorophores
of different wavelengths. This approach limits single-reaction multiplexing
capability to a
maximum of about 5 targets, due to limits on dye emission spectra and the
spectral window.
[0115] The approach described in this example uses a labeled PCR probe
that acts as a
surrogate for the amplicon to transfer information about the progression of
amplification to a
surface bound array during the process. Information about the kinetics of
amplification is
preserved, allowing for both detection and quantitative information to be
obtained, based on a
cycle number thresholding method.
[0116] During the extension step in the PCR cycle, the 5'-3' nuclease
activity of Taq
Polymerase cleaves the PCR probe to release a flap nucleic acid that can then
preferentially
hybridize to a capture probe on the array surface. Each flap and corresponding
capture probe is
unique to a potential target within the test panel.
Reaction Chamber Depth
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[0117] An experiment was conducted to evaluate the relationship of chamber
thickness
to the Signal to Background Noise Ratio for a given array. Substrates were
machined with
chambers of varying depths and coated with functionalized polymer. Actual
depths of the
chambers were measured. The substrates were then spotted with the capture
probes and
assembled into enclosed reaction chamber using UV cured epoxy. A solution
containing 45nM
of a synthetic mimic of the labeled probe fragment complementary to each
capture probe on the
array and 255nM of the corresponding intact probe (to mimic 15% cleavage) was
pipetted into
each enclosed reaction chamber and the signal vs. background signal was
measured after a 3
min hybridization at 30 C. Results for one of the assays is shown in Figure
13. As can be seen,
reduction in the thickness of the reaction chamber from 600 microns to below
200 microns
showed a dramatic increase in the signal to background noise ratio, with
optimal ratios below
300 microns and preferably below 200 microns thickness.
PCR Chamber and Array
[0118] The PCR chamber used in most of the experiments is shown in Figure
2. As
shown, the chamber consists of a bottom surface containing an array of capture
oligos
complimentary to the flap sequence of the PCR probes. The capture probes were
synthesized
by Integrated DNA Technologies Inc. (Coralville, Iowa) and have a 5' terminal
amine group
for covalent attachment to the substrate forming the bottom of the PCR
chamber, along with a
polyethylene glycol linker between the attachment chemistry and the oligo
sequence. The
length of the sequence is the same as the corresponding PCR probe flap. The
bottom of the
PCR chamber was formed from a commercially available slide. This slide came
with a
polymeric coating containing active NHS esters for subsequent attachment of
the capture
probes. Slides included both glass and plastic substrates coated with the
polymeric coating.
Both types of slides result in similar experimental data. The capture probes
are spotted using a
SPOTBOTTm (Arrayit Technologies (Sunnyvale, CA)) according to standard array
protocols.
The capture probe spots were typically 100pm in diameter with a 200pm center
to center pitch
between spots.
[0119] After spotting and washing of the capture probes, the PCR chamber
was
assembled using a pressure sensitive adhesive (PSA) and a polycarbonate top
piece with inlet
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and outlet ports as shown in Figure 2. The chamber had a final depth/thickness
(or height) of
142 pM and a diameter of 15 mm. The chamber holds a volume of approximately 45
uL of
PCR reagents.
Thermocycler and Optics Breadboard.
[0120] The thermocycling and optical detection system included an
epifluorescent,
single-channel detection system that includes (1) an excitation light source
(e.g., a mercury arc
lamp or an LED), (2) interference optical filters that are used for the
excitation light and for the
emission light so that specific combination of fluorophores are detected, such
as Cy3, Cy5 or
others, and (3) a photo-detector, which is a CCD or CMOS camera.
[0121] The system also included thermocycling components such as a pair of
thermoelectric modules, metal plates, heat sinks and powerful cooling fans
that were used to
rapidly thermocycle an enclosed consumable (e.g., the array and chamber
described above) to
desired temperatures and for desired times. The thermoelectric modules were
controlled to
specific temperatures for specific time periods by the use of a feedback
enabled control system
that used thermistors proximate to the consumable as the feedback to the
control system.
[0122] System control was performed by a computer, which used a script
file as input
to generate the target temperatures and time periods as well as to specify
when an image was to
be taken by the photo-detector. The resulting images taken at different times
during the thermal
reaction were analyzed by the computer to generate intensity curves as a
function of time and
thereby derive the concentration of the target.
[0123] Single-channel fluorescent images were captured from the consumable
at
various times and temperatures during the progress of the thermal reactions.
These fluorescent
images were then analyzed to yield quantification of starting target nucleic
acid concentrations.
The fluorescent images were analyzed using a combination of mean gray
intensity
measurements, background correction and baseline adjustments. The background
was
measured locally for each spot in the array. The background was computed by
measuring the
fluorescent intensity of a concentric annulus of the solution surrounding the
spot of interest.
The signal from each spot was then corrected to account for the local
background. The
corrected signal from each spot was further normalized to account for
variability in spotting as
well as uneven illumination in the field of view. The average of the corrected
intensity
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measurements obtained from the first few cycles, typically between cycle 5 and
15, were then
used to adjust the baseline and normalize measurements from each spot.
EXAMPLE 1: THREE STEP AMPLIFICATION REACTION
[0124] The amplification reagent mix contained standard PCR reagents
including two
PCR amplification primers specific to each target being amplified, as well as
a PCR probe
specific to each target being amplified. The structure of typical probe is
schematically shown
in Figure 1A. As shown, Figure 1A probe region (A) represents a nucleic acid
region of the
probe that is complimentary to a target amplicon, designed using the same
rules as is typical for
a traditional real time PCR probe (e.g., as in a TAQMANTm probe). Probe region
(B)
represents an orthogonal nucleic acid "flap" sequence that is complimentary to
a corresponding
capture probe (discussed below), but not the target nucleic acid. For purposes
of illustration,
this sequence is designed in one example to have a Tm of between 40 and 46
C, although
other probe designs can be substituted. In one example, the sequence length is
about 13 or 14
bases. Probe region (C) represents a nucleic acid with a sequence that is
complimentary to a
portion of the sequence of nucleic acid region (B). This sequence is designed
to facilitate the
formation of a secondary structure of the full-length probe, e.g., with a Tm
of between 47 and
51 C. Quencher (D) represents an optional quencher molecule. Label (E)
represents a
fluorophore or other optically detectable label. The fluorophore Cy3 was used
for the data
presented below.
[0125] For the data in this example, PCR was performed with the following
reagent
formulation: 200 nM primers, 1X FAST STARTTm PCR buffer (available from
Roche), 2-6
mM MgC12, 0.5 mg/mL BSA, 0.2 units/uL FAST START Taq polymerase (Roche), and
150
nM of PCR Probe.
[0126] 100 uL PCR reactions were prepared using the formulation described
above.
The PCR Probe sequence that was used in this example was:
NNNNNNNNNNNNNCCTGTTGCCAATTTCAGAGTGTTTTGCTTAACNNNNNNNN
NGAT (SEQ ID NO:1).
[0127] The 5' and 3' flaps are denoted in underline/double underline and
the traditional
TaqMan sequence is shown in bold. The double underlined sequences denote the
homologous
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regions designed to form secondary structure. The predicted melting
temperature of the
secondary structure was 51 C, as determined by mFold (idtdna.com) using the
PCR buffer
conditions. The PCR probe was labeled with a 5' Cy3 fluorophore and a Black
Hole Quencher
2 moiety on the 3' end.
[0128] The capture probe sequence attached to the bottom substrate of the
PCR
chamber was: NNN NNN NNN NNN N (SEQ ID NO:2) with a Tm of 42 C using the PCR
buffer conditions.
[0129] DNA plasmids comprising a target sequence were added to each PCR
reaction at
a concentration of 106, 104, and 102 copies/uL. The solution was then degassed
by heating to
95 C. After degassing, polymerase was added and the reaction was loaded into
the PCR
chamber using a pipette. Left over solution was loaded onto an Applied
Biosystems 7500 for
parallel analysis.
[0130] The cycling conditions for the array based PCR were as follows:
Temp Time Purpose
95 C 120 sec Fast Start Enzyme activation
95C 15 sec denaturation
60C 60 sec polymerase extension
30C 120 sec Flap hybridization and optical reading
(denaturation and extension were performed for 5 cycles, and then
denaturation/ extension/ flap
hybridization and optical reading are repeated for 8 cycles).
[0131] The Cycling conditions for the ABI 7500 were as follows:
Temp Time Purpose
95 C 120 sec Fast Start Enzyme activation
95 C 15 sec denaturation
60 C 60sec polymerase extension and optical reading
Denaturation/ extension and reading were performed for 40 cycles.
[0132] The results for the copy number titrations are shown in Figure 3
for the array
based PCR and Figure 4 for the solution phase PCR. As can be seen from the
figures, the
results are comparable, giving similar behavior for the titrations.
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EXAMPLE 2: TWO STEP AMPLIFICATION REACTION
[0133] As with Example 1, above, the amplification reagent mix contained
standard
PCR reagents including two PCR primers (200nM) complementary to each target
being
amplified as well as a PCR probe (300nM) having a sequence complementary to
each target
being amplified. The structure of typical probe is shown in Figure 1B. As
shown, the labeled
probe again includes a nucleic acid fragment (A) that is complimentary to a
target amplicon
designed using the same rules as is typical for a traditional real time PCR
probe (i.e. TaqMan).
Also included is orthogonal nucleic acid "flap" sequence (B) that is
complimentary to a
corresponding capture probe on the capture array. The probe also includes a
fluorescent label
(C) coupled to the flap portion B and a quencher moiety (D) coupled to the
target specific
portion (A).
[0134] For the two step amplification, the orthogonal flap (B) comprises a
sequence
that was designed to have a Tm with its complement on the capture array of 70
C. Typically
the sequence length is 25 to 27 bases. As with Example 1, above, the overall
probe is designed
so that the most stable secondary structure has a Tm no higher than 10 C
below the extension
and measurement temperature in the buffer conditions used for PCR. The oligo
was designed
using the unafold software available at www.idtdna.com. The following PCR
probe sequence
was used in this example:
NNN NNN NNN NNN NNN NNN N/Cy3/NN NNN NNN ATG GCC GTT AGC TTC AGT
CAA TTC AAC AG/BHQ_2/ (SEQ ID NO:40)
[0135] Where the double underlined sequence constitutes the orthogonal
flap and the
non-underlined sequence is homologous to the amplicon. The most stable
secondary structure
of the probe has a melting temperature of 45 C. The Tm of the orthogonal flap
is 71 C. The
PCR probe was labeled with an internal Cy3 fluorophore C (available from GE
Healthcare
Biosciences, Piscataway, NJ) and a Black Hole Quencher 2 moiety D (available
from
Biosearch, Inc., Novato, CA) on the 3' end.
[0136] A capture probe was spotted that was homologous to the flap portion
of the PCR
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Probe. PCR was performed, as above, except with the following cycling
conditions:
Temp Time Purpose
95 C 60 sec Fast Start Enzyme activation
95C 15 sec denaturation
55C 60 sec polymerase extension, flap hybridization and
optical
reading
[0137] Forty cycles were carried out and with the fluorescent signal being
measured at
the end of each extension step. Figure 12 shows the copy number titrations for
the array based
PCR for two targets where the first was present at the outset at 104 copies of
target DNA
plasmid while the second was present at 106 copies.
EXAMPLE 3: ARRAY BASED PCR CURVE USING AN UNQUENCHED PCR PROBE.
[0138] The same protocol was used as in Example 1 with the following
exceptions.
The PCR probe sequence used is as follows:
NNNNNNNNNNNNNTCGCTGAACAAGCAACCGTTACCCNNNNNNNN (SEQ ID NO: 3)
This sequence was labeled with a 5' Cy3 fluorophore, but did not include a 3'
quencher.
106 copies of target were added and the PCR was run. The real time data is
shown in Figure 5.
EXAMPLE 4: MULTIPLEXED ARRAY BASED AMPLIFICATION
[0139] This experiment establishes the ability to interrogate for and
amplify multiple
targets within the same PCR chamber. PCR conditions are the same as shown in
Example 1
except for the following exceptions: first, 5 sets of primers and 5 separate
PCR probes are
added to the PCR reaction specific to each target to be interrogated. Second,
5 unique capture
probes are deposited onto the bottom substrate of the PCR chamber
corresponding to the 5' flap
sequence of each of the 5 PCR probes. Third, after the 10th PCR cycle the
temperature was
dropped to the surface hybridization temperature of 30 C every 2 cycles
instead of every 5
cycles as in Example 1. This allows for a higher frequency of optical
interrogation during the
PCR amplification.
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[0140] The PCR probe and capture probe sequences are shown below:
PCR Probes:
Flu A: NNNNNNNNNNNNNCCCCAT GGAATGTTAT
CTCCCTTTTAAGCTTCTNNNNNNNN (SEQ ID NO:4) (Tm of 50.3 )
A/H1: NNNNNNNNNNNNNACCTTGGC GCTATTAGAT TTCCATTTGC CNNNNNNNN
(SEQ ID NO:5) (Tm of 51.2 )
A/H3: NNNNNNNNNNNNNNCCTGTT GCCAATTT CAGAGTGTT
TTGCTTAACNNNNNNNNNNNN (SEQ ID NO:6) (Tm of 51 )
HuB: NNNNNNNNNNNNNNTCAAAGC CAATTCGAG CAGCTGAAAC TNNNNNNNN
(SEQ ID NO: 7) (Tm of 51 )
phiMS2: NNNNNNNNNNNNNTCGCTGAA CAAGCAACC
GTTACCCNNNNNNNNNNNN (SEQ ID NO:8) (Tm of 52 )
Capture Probes
HuA: NNN NNN NNN NNN N (SEQ ID NO:8) (Tm of 46 )
A/H1: NNN NNN NNN NNN N (SEQ ID NO: 9) (Tm of 45 )
A/H3: NNN NNN NNN NNN N (SEQ ID NO: 10) (Tm of 42 )
HuB: NNN NNN NNN NNN N (SEQ ID NO: 11) (Tm of 46 )
phiMS2: NNN NNN NNN NNN N (SEQ ID NO: 12) (Tm of 43 )
[0141] 5 targets plasmids encompassing the sequences specific to the
primers and PCR
probes above were added to a 100uL PCR reaction and the solution was prepped
and loaded as
described in above. The resulting real time array based PCR data is shown in
Figure 6.
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EXAMPLE 5: HIGH LEVEL MULTIPLEXING
[0142] This example demonstrates single chamber multiplexing for detection
of
multiple targets that can be any of the ten potential targets included in the
panel of this
example. This level of multiplexing ¨ a panel of more than five potential
targets ¨ cannot be
achieved in traditional solution phase PCR.
[0143] The experimental materials and procedures were the same as that
above except
for the following: first, 10 Primer sets and PCR probes were incorporated into
PCR reaction in
the same concentrations as above. The sequences of the PCR probes and capture
probes are
given below:
PCR Probes:
HuA: NNNNNNNNNNN NNCCCCATGG AATGTTATCT CCCTTTTAAG
CTTCTNNNNNNNN (SEQ ID NO: 13) (Tm of 50.3 )
A/H1: NNNNNNNNNNNNNNACCTTGGCGCT ATTAGATTTC
CATTTGCCNNNNNNNN (SEQ ID NO: 14) (Tm of 51.2 )
A/H3: NNNNNNNNNNNNNCCTGTTGCCA ATTTCAGAG TGTTTTGCT
TAACNNNNNNNNNNN (SEQ ID NO: 15) (Tm of 51 )
HuB-v2: NNNNNNNNNNNNNTCAAAGCC AATTCGAGCA GCTGAAAC TNNNNNNNN
(SEQ ID NO: 16) (Tm of 51 )
phiMS2: NNNNNNNNN NNNNTCGCTG AACAAGCAA CCGTTACCC NNNNNNNN
(SEQ ID NO: 17) (Tm of 52 )
MPV: NNNNNNNNNNNNNNATGG CCGTTAGCTT CAGTCAATTC AACAGNNNNNNN
(SEQ ID NO: 18) (Tm of 48.4 )
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PIV1: NNNNNNNNNNNNNTTGGAATT GTCTCGACA ACAATCTTTG
GCCTNNNNNNNNN (SEQ ID NO: 19) (T. of 50.4 )
PIV2: NNNNNNNNNNNNNCCATTT ACCTAAGTGA TGGAATCAAT
CGCAAAAGNNNNNNNN (SEQ ID NO: 20) (T. of 48.8 )
PIV3: NNNNNNNNNNNNNNNACATAA GCTTTGATC AACCCTATG
CTGCACNNNNNNNNN (SEQ ID NO: 21) (T. of 49.9 )
RSV: NNNNNNNNNN NNNTTCGAAGGCTC CACATACACAG CTGCTGNNNNNNNNN
(SEQ ID NO: 22) (T. of 49.9 )
R5V-v2: NNNNNNNNNNNNNTCGAAGGC TCCACATACA CAGCTGCTGNNNNNNNN
(SEQ ID NO: 23) (T. of 51 )
OPC1: NNNNNNNNNNNNNTTCGGCAT TTCCTGGATTGAGT
CGGTACTANNNNNNNN (SEQ ID NO: 24) (T. of 48.7 )
Capture Probes
Capture Probe T.
FluA NNN NNN NNN NNN N SEQ ID NO:26 (T. of 46 )
A/H1 NNN NNN NNN NNN N SEQ ID NO:27 (T. of 45 )
A/H3 NNN NNN NNN NNN N SEQ ID NO:28 (T. of 42 )
HuB-v2 NNN NNN NNN NNN N SEQ ID NO:29 (T. of 46 )
phiMS2 NNN NNN NNN NNN N SEQ ID NO:30 (T. of 43 )
MPV NNN NNN NNN NNN N SEQ ID NO:31
PIV1 NNN NNN NNN NNN N SEQ ID NO:32
PIV2 NNN NNN NNN NNN N SEQ ID NO:33
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PIV3 NNN NNN NNN NNN N SEQ ID NO:34
RSV NNN NNN NNN NNN N SEQ ID NO:35
RSV-v2 NNN NNN NNN NNN N SEQ ID NO:36
OPC1 NNN NNN NNN NNN N SEQ ID NO:37
[0144] Figure 7 shows the resulting real time array-based PCR curves when
no target
was added to the PCR reactions (No Template Control). As can be seen from the
figure, no
signal was obtained from a solution containing all PCR components except
target. Figure 8
shows the same experiment with 3 plasmid targets (MPV, OPC-1, PIV2) added at
10,000
copies/uL.
EXAMPLE 6: DEMONSTRATION OF FAST HYBRIDIZATION KINETICS
[0145] A PCR chamber was constructed as described above. The following
amine,
pegylated capture probe sequence was deposited on the bottom substrate: NNN
NNN NNN
NNN N (SEQ ID NO:38).
[0146] A solution containing the PCR buffer described above was prepared
containing
the following oligo sequence (100 nM) which mimics the 5' flap portion of the
PCR probe,
labeled with a Cy3 fluorophore on the 5' end and complimentary to the capture
probe: NNN
NNN NNN NNN N (SEQ ID NO:39).
[0147] The solution was loaded into the PCR chamber and the chamber was
heated to
60 C (15 degrees above the Tm of the duplex) and then cooled back down to 30
C. This
mimics the conditions during the hybridization step of the PCR protocol. An
optical reading
was taken every 20 sec for 2 min. The resulting data is shown in Figure 9.
[0148] One interesting aspect of the data shows that there is already
significant
hybridization occurring at the instant that the internal temperature reaches
30 C.
EXAMPLE 7: DARK ASSAY CONFIGURATION
[0149] The three step amplification assay described in Example 1, above,
was repeated
using a flap portion of the probe that included an Iowa Black RQ quencher
group available
from Integrated DNA Technologies, coupled to its 5' end, and without any other
labeling or
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quenching groups attached to the probe. The capture probes deposited upon the
array surface
carried a Cy3 fluorophore coupled to the 3' terminus.
[0150] All primer and probe sequences were ordered from IDT and received
lyophilized. They were then resuspended in water to stock concentrations (100
uM to 200 uM)
and used to prepare primer/probe stock solutions for PCR reactions. H03 NVS
PCR buffer,
was combined with primers and probes to make a PCR master mix.
[0151] Functionalized surfaces (COP substrates coated with functionalized
polymer)
were spotted using traditional microarray spotting techniques via an Array-It
spotbot 2.
Spotted slides were incubated at 75% humidity for 8-15 hours and were then
rinsed with DI
water and dried with argon. Labeled capture probes were spotted in
concentrations ranging
from 100 nM to 50 uM in 50mM pH 8.5 spotting buffer, producing a range of
signal
intensities.
[0152] As with the examples above, the chip based reaction chambers were
built on top
of functionalized, arrayed surfaces using double sided pressure sensitive
adhesive gaskets,
polycarbonate lids and optically clear seals. Total volume of the reaction
chamber is
approximately 30 uL with a height of 150 um.
[0153] Target sequences of a known concentration are added to PCR master
mix and
the resulting solution is loaded in the reaction vessel. The vessel was sealed
and loaded on a
the above-described thermocycling instrument. The target sequences were
obtained from
plasmid stocks or the resulting amplicons from previous reactions involving
those amplicon
stocks. All target molecules were quantitated using UV-Vis spectrometry on a
NanoDrop
instrument.
[0154] Thermocycling conditions included a hot start step at 95C for 85s,
followed by
40 cycles of melt (95C) and extension (55C) of Ss and 20s respectively. At
cycles 10, 15, 18,
and every 2 cycles thereafter, an additional step is added to the cycle for
data collection. The
temperature is brought to 30C following extension, and held for 30s. Images of
the surface are
collected at the end of these hybridization steps. Average pixel intensity in
the spots for each
assay is calculated and plotted vs cycle number to generate real-time PCR
curves. The
solution and surface probe designs are as depicted in assay format 3. For
duplex PCR reactions:
600 nM primers and 300 nM probes for the H3 and OPC1 assays are added. Figure
15 shows a
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plot of the progress of the real time PCR reaction based upon the increasing
hybridization of
flap portions and their associated quenchers to the Cy3 labeled capture
probes. The fluorescent
signal is plotted as an absolute value of the negative signal, showing the
decrease in signal as
the reaction progresses.
[0155] The approaches outlined herein have multiple advantages not found
in previous
approaches. The systems of the invention allow for single chamber, highly
multiplexed,
quantitative PCR by efficiently transferring information from solution phase
PCR to a surface
confined array in real time during the amplification process. This allows for
a much higher
level of multiplexing while preserving the efficiency and leveraging the
immense body of
knowledge accumulated for solution phase real time PCR. In order to achieve
this, multiple
novel aspects of the system had to be developed.
[0156] For example, one feature of the invention is the use of an amplicon
surrogate to
bridge the solution phase and the solid phase. Previous teachings aimed
towards the goal of
array based real time PCR have generally relied upon hybridization of the
amplicon itself to the
solid phase array. This presents multiple issues that complicate the system,
hinder the
efficiency, and require more expensive components to elucidate the required
information. In a
multiplexed PCR environment, it is very hard to design amplicons of similar
lengths and
hybridization efficiencies. The use of a PCR probe with a cleavable 5' flap
homogenizes the
species that transfers the information from each amplicon to the surface by
employing a very
short sequence that is ideal for hybridization kinetics. This approach also
makes the capture
probe sequences on the array independent of the sequence of the amplicon to be
detected. This
allows for selection of the most advantageous capture sequences and the
possibility of a
universal array that can be used for many different target panels, simplifying
the design and
manufacturing approach.
[0157] As disclosed here, the PCR probe also leverages the design rules
that have
already been developed for probe based solution phase real time PCR. The use
of a very short
sequence for hybridization (e.g., 13-14 bases) makes the hybridization very
efficient, allowing
for high signal on the array in the low salt environment of standard PCR
buffers. Thus the
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system can work very well in a single chamber where surface hybridization has
to be coupled
with optimal solution phase PCR.
[0158] Another feature of the invention is the discrimination of surface
hybridized
signal from solution phase background fluorescence. This aspect of the
invention is important
in extracting relevant information from the array. Previous teachings employ
complicated or
expensive optical approaches to overcome this problem, such the use of total
internal
reflectance or confocal microscopy to isolate the surface signal from the
solution background.
By contrast, this invention provides the use of simple standard optical
equipment that require
no optical "tricks" to achieve discrimination. The ability to discriminate the
signal arises from
multiple sources. The surface chemistry that is used in the array provides a
very high capture
probe density and thus hybridized target density. It has been shown that the
surface can
approach 100% capture efficiency of target nucleic acid. This high capture
density and
efficiency serves to concentrate the surface signal, aiding in
surface/solution phase
discrimination. The use of a short target nucleic acid serves to dramatically
enhance this effect.
[0159] Another aspect of the invention that aids in the signal
discrimination is the use
of a very thin PCR chamber. The background signal from the solution is
linearly related to the
height of solution above the array. The use of a thin chamber takes advantage
of this effect.
[0160] Another aspect of the invention is the use of fast hybridization
kinetics to allow
for real time transfer of the solution phase information to the surface array.
The system
described in these examples demonstrates extremely fast solid phase
hybridization. This
phenomenon facilitates the technology and can be attributed to multiple
aspects of the
invention, including the short 5' flap target, the optimal solid phase surface
chemistry, the thin
consumable and the temperature gradient produced during the thermocycling
temperature
program.
[0161] While the foregoing invention has been described in some detail for
purposes of
clarity and understanding, it will be clear to one skilled in the art from a
reading of this
disclosure that various changes in form and detail can be made without
departing from the true
scope of the invention. For example, all the techniques and apparatus
described above can be
used in various combinations. All publications, patents, patent applications,
and/or other
documents cited in this application are incorporated by reference in their
entirety for all
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purposes to the same extent as if each individual publication, patent, patent
application, and/or
other document were individually indicated to be incorporated by reference for
all purposes.
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