WO2013074163A1 - Détection quantitative, fortement multiplexée d'acides nucléiques - Google Patents

Détection quantitative, fortement multiplexée d'acides nucléiques Download PDF

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Publication number
WO2013074163A1
WO2013074163A1 PCT/US2012/051236 US2012051236W WO2013074163A1 WO 2013074163 A1 WO2013074163 A1 WO 2013074163A1 US 2012051236 W US2012051236 W US 2012051236W WO 2013074163 A1 WO2013074163 A1 WO 2013074163A1
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Prior art keywords
probe
array
capture
nucleic acid
probes
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PCT/US2012/051236
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English (en)
Inventor
Kris SCABOO
Patrick Martin
Brad TAFT
Jason LA
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NVS Technologies, Inc.
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Priority claimed from US13/399,872 external-priority patent/US20120214686A1/en
Priority to CN201280067273.7A priority Critical patent/CN104093857A/zh
Priority to KR1020147016411A priority patent/KR20140094007A/ko
Priority to EP12849697.3A priority patent/EP2780479A4/fr
Priority to SG11201402341QA priority patent/SG11201402341QA/en
Priority to BR112014011937A priority patent/BR112014011937A2/pt
Application filed by NVS Technologies, Inc. filed Critical NVS Technologies, Inc.
Priority to JP2014542300A priority patent/JP2014533502A/ja
Priority to CA2855953A priority patent/CA2855953A1/fr
Priority to MX2014005912A priority patent/MX338076B/es
Priority to AU2012337389A priority patent/AU2012337389A1/en
Publication of WO2013074163A1 publication Critical patent/WO2013074163A1/fr
Priority to IL232586A priority patent/IL232586A0/en
Priority to HK15100214.3A priority patent/HK1199746A1/xx

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6834—Enzymatic or biochemical coupling of nucleic acids to a solid phase
    • C12Q1/6837—Enzymatic or biochemical coupling of nucleic acids to a solid phase using probe arrays or probe chips
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays

Definitions

  • the invention is in the field of real-time DNA amplification, detection and quantification, as well as associated consumables, devices, and systems, including arrays.
  • Real time PCR is routinely used for detection of nucleic acids of interest in a biological sample.
  • M Tevfik Dorak Editor
  • single well multiplexing used to detect more than 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.
  • 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.
  • amplification reactions are performed on a sample, and amplicons are separately detected on a nucleic acid array.
  • amplicons are separately detected on a nucleic acid array.
  • 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.
  • 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 ;
  • 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.
  • methods of detecting a target nucleic acid include 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 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.
  • 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 500 ⁇ 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.
  • 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.
  • the precise configuration of the detection chamber can vary.
  • the configuration is selected to reduce signal background in the chamber proximal to the array.
  • 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.
  • levels of 95% or less total background are achieved by optimizing the configuration of the chamber proximal to the array. This configuration optimization is achieved, e.g., by keeping the depth of the chamber above the array to a minimum.
  • the chamber is less than about 1 mm in depth or other dimension proximal to the array, more typically about 500 ⁇ or less in at least one dimension proximal to the array, preferably less than about 250 ⁇ or less, e.g., between about 10 ⁇ and about 200 ⁇ and in some embodiments the chamber is about 150 ⁇ in a dimension proximal to the array.
  • the chamber is about 142 ⁇ in depth above the array. In another example herein, the chamber is about 100 ⁇ 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.
  • 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.
  • background noise components e.g., detector responses unrelated to the specific detection of array spot signal and background signal from the reaction fluid.
  • 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.
  • the background noise 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.
  • 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.
  • 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)).
  • TIRF total internal reflection fluorescence microscopy
  • 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.
  • the reactants employed in the analytical methods of the invention are configured to reduce background signal relative to actual probe/array binding signal.
  • 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.
  • 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.
  • the donor and acceptor 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.
  • the optical system 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.
  • 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.).
  • the capture probe is labeled with a fluorophore such that the signal producing label is tethered to the surface of the array.
  • the labeled probe and labeled probe fragment carries a quencher group complementary to the fluorophore, i.e., able to quench the fluorescence of the 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.
  • the capture probe will bear the fluorophore at a 3' or other complementary position.
  • 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.
  • the capture probes on the array produce signal in the unhybridized state.
  • the quencher bearing probe fragment is released to hybridize to the complementary capture probe on the array, quenching the signal from its associated
  • 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.
  • the array typically includes a non-rate limiting number of capture nucleic acids that hybridize to the labeled probe fragment.
  • Typical array densities are between about 350 fmol/cm or greater, e.g., about 2,000 fmol/cm 2 or greater, 2,500 fmol/cm 2 or greater, 3,000 fmol/cm 2 or greater, 4,000 fmol/cm 2 or greater, 4,500 fmol/cm 2 or greater, or 5,000 fmol/cm 2 or greater.
  • the number of sites that bind probe on the array is at least IX the number of sites that would be saturated by the concentration of probe fragments produced during amplification, and is optionally 5X, 10X, 5 OX 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 T m 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.
  • 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.
  • 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.
  • 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.
  • the hybridization temperature is less than the temperature of the amplification reaction, so the T m of the probe fragment for the capture nucleic acid can be less than an intra-molecular T m of the probe (e.g., where the probe comprises a quencher to reduce background), and/or lower than the T m 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 have a higher T m 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 T m for binding to the first flap than the first flap has for binding to the array.
  • the extension reaction occurs at a first temperature, i.e., below the T m of the intact probe for the target nucleic acid, but above both the intra-molecular T m of the intact probe, and the T m of the probe fragment for the capture probe on the array).
  • a first temperature i.e., below the T m of the intact probe for the target nucleic acid, but above both the intra-molecular T m of the intact probe, and the T m of the probe fragment for the capture probe on the array.
  • 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.
  • 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.
  • the labeled probe fragment and its complementary capture nucleic acid is designed or selected to have a T m that is higher than the extension reaction temperature, e.g., 10 or more degrees higher.
  • the T m of the labeled probe fragment and capture nucleic acid will typically be, e.g., 71°C.
  • 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 the array and detect the resulting signal.
  • a two-step temperature profile may be used rather than a three step profile.
  • the orientation of the orthogonal labeled probe fragment relative to the probe portion that binds to the target sequence may be varied.
  • 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.
  • 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.
  • Sample can be loaded into the chamber by any of a variety of mechanisms, depending on the precise configuration of the consumable.
  • the sample is loaded through at least one port or fluidic channel in operable communication with the chamber.
  • 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.
  • fluidic or microfluidic channels, capillaries, or the like can be used for sample delivery.
  • 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.
  • 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.
  • 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.
  • 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.
  • the labeled probe typically comprises a fluorescent or luminescent label, although other labels such as quantum dots can also be used.
  • 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.
  • quenchers are not required in the practice of the invention.
  • 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).
  • intercalating dyes typically provide a detectable signal event upon incorporation, or intercalation, into double stranded nucleic acids.
  • 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, el27, which is incorporated herein by reference for all purposes.
  • the probe configurations and assay methods may also 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.
  • Local background can be detected for one or more regions of the array, with signal intensity measurements being normalized by correcting for said background.
  • 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.
  • 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.
  • 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.
  • 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).
  • 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 signals can be detected based upon positioning of the signals on the array after hybridization of the probe fragments to the array.
  • 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.
  • capture arrays having larger numbers of different capture probes may also be employed in some circumstances, e.g., where amplification reactions are pooled for interrogation by the array, or the like.
  • An advantage of the present invention is that one capture array configuration may be used for multiple different target nucleic acid sequence panels.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the method includes incubating a plurality of labeled probes, each specific for a different 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the device can be less than about 500 ⁇ in depth in at least one dimension proximal to the array, e.g., between about ⁇ and about 200 ⁇ 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.
  • 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 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 ⁇ , ⁇ -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.
  • a chemically reactive group an electrophilic group
  • an NHS ester a tetra- or pentafluorophenyl ester,
  • 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.
  • thermo regulatory module has a feedback enabled control system operably coupled to a computer which controls or is part of the module.
  • 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.
  • 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.
  • 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 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.
  • 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.
  • 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 500 ⁇ 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.
  • the consumable is configured to permit thermocycling of fluid within the chamber.
  • 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/cm or greater, 2,500 fmol/cm 2 or greater, 3,000 fmol/cm 2 or greater, 4,000 fmol/cm 2 or greater, 4,500 fmol/cm 2 or greater, or 5,000 fmol/cm or greater.
  • 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.
  • 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.
  • 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.
  • the upper and lower surfaces can be ultrasonically fused together, with the gasket or feature delimiting regions that are fused.
  • 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.
  • the upper and lower surfaces can be laser welded together by directing laser light through the transparent region and onto the shaded region.
  • the capture nucleic acid array is typically coupled to a thermally stable coating on the window.
  • 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 ⁇ , ⁇ -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
  • the chamber can be between about ⁇ and about 200 ⁇ in depth, e.g., about 140 ⁇ 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 10 mm and about 20 mm in average diameter.
  • kits e.g., comprising the consumable of the invention.
  • 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).
  • control reagents e.g., control templates, probes, or primers, e.g., which bind to control sites on an array of the consumable.
  • kits providing the consumable for use in a system or device of the invention, e.g., to practice the methods of the invention.
  • steps of the methods optionally have corresponding structural features in the systems, devices, consumables or kits, and vice-versa.
  • Figure 1 A and IB are schematic illustrations of PCR probes of the invention.
  • Figure 2 is a schematic of a PCR chamber of the invention.
  • Figure 3 is a graph showing array based real time PCR curves for copy number titration for a three step amplification reaction.
  • Figure 4 is a graph showing solution based real time PCR curves generated from aliquots of solutions.
  • Figure 5 is a graph showing an array based real time PCR curve generated with an unquenched probe.
  • Figure 6 is a graph showing real time PCR curves for a multiplexed
  • Figure 7 is a graph showing array-based real time PCR curves for a 10-plex reaction with no target added.
  • Figure 8 is a graph showing array-based real time PCR curves with a 10-plex panel and 3 targets present at 10 4 copies each.
  • Figure 9 is a graph showing real-time kinetics of hybridization of a 5 ' flap mimic.
  • Figure 10A and 10B show schematics of aspects of the invention.
  • Figure 11 is a schematic system illustration.
  • Figure 12 is a graph showing array based real time PCR curves for copy number titration for a two step amplification reaction.
  • Figure 13 is a plot showing the relation of reaction chamber thickness to the signal to background ratio.
  • 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.
  • Methods of performing target nucleic acid amplification, detection and real-time quantification are a feature of the invention.
  • amplification of the target nucleic acid releases a target-specific labeled probe fragment that hybridizes to an array; the array is 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.
  • 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.
  • 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.
  • 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.
  • Figure 10A provides a partial overview of the method.
  • 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).
  • 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.
  • 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.
  • 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.
  • 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).
  • PCR polymerase chain reaction
  • 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)
  • 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.
  • appropriate reaction conditions including use of standard amplification buffers, enzymes, temperatures, and cycle times.
  • 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.
  • 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.
  • 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.
  • Optional region C comprising label quencher D, is
  • label groups 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.
  • fluorescent labeling groups may include individual fluorophores or interactive dye pairs or groups, e.g., FRET pairs, as well as donor/quencher pairs.
  • Nucleic acid sequence based amplification (NASBA), or the ligase chain reaction (LCR) can be used.
  • 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.
  • NASBA 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.
  • the probe can be cleaved using an endonuclease, rather than relying on nuclease activity of the amplification enzyme.
  • a detection chamber that has at least one high efficiency nucleic acid detection array on at least one inner surface of the chamber.
  • the high efficiency array typically has a non-rate limiting number of capture nucleic acids that permits 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.
  • 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 500 ⁇ or shallower above or below the array, although detection in chambers as deep as 1mm or larger can work. Further details regarding the reaction chamber and array is described below with reference to the consumable useful in the methods.
  • 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.
  • 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.
  • 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.
  • the target specific probe is again provided with an orthogonal flap portion 104b, as noted in Figure 1 OA, above.
  • the flap 104b bears a quencher group 116.
  • 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.
  • the flap portion 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.
  • 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.
  • 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.
  • 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.
  • the capture probes may optionally be coupled to a mobile substrate, such as beads, resins, particles or the like (generally referred to
  • 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.
  • 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.
  • 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.
  • 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.
  • a variety of different bead types may be used in conjunction with this aspect of the invention.
  • polystyrene, cellulosic, acrylic, vinyl, silica, paramagnetic or other inorganic particles, or any of a variety of other bead types may be employed.
  • the beads with typically be differentially labeled with a unique label signature.
  • label types 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.
  • 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.
  • 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.
  • 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.
  • signal intensity from the array can be used to both detect and quantify a nucleic acid of interest, in real time.
  • 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. Typical desirable chamber depths are in the range of about ⁇ to about 500 ⁇ .
  • the chamber is often in the range of about ⁇ to about 250 ⁇ in depth above the array, e.g., about ⁇ to about 150 ⁇ 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.
  • Figure 2 provides a blow-up schematic of an example consumable.
  • 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.
  • 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.
  • the top or bottom surface will include a window through which detection optics can view the array.
  • Middle layer 3 can take any of a variety of forms, depending on the consumable assembly method to be used.
  • 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
  • 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.
  • 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.
  • 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.
  • UV light or radiation e.g., electron beam, or "EB” radiation
  • 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)
  • 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.
  • 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:
  • 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.).
  • 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 ⁇ , ⁇ -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.
  • an electrophilic group an NHS ester, a tetra- or pentafluorophenyl ester, a mono- or dinitropheny
  • 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; Miiller 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
  • 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.
  • control reagents e.g., a control template, control probe, control primers, etc.
  • buffers or the like.
  • 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.
  • 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.
  • FIG. 11 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.
  • 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.
  • 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 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.
  • 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
  • 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.
  • 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 metal plate configured to mate with a portion of an outer surface of the chamber, a fluid bath, etc.
  • 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
  • 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.
  • 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)
  • 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.
  • 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.
  • light e.g., a fluorescent or luminescent signal
  • 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.
  • 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.
  • 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.
  • 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.
  • a fused silica or glass capillary or other transparent microfluidic channel is used as the detection channel.
  • 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.
  • Figure 14 provides a schematic illustration of an overall detection system 1400.
  • the system includes first and second excitation light sources, such as lasers 1402 and 1404, that each provide excitation light at different wavelengths.
  • first and second excitation light sources such as lasers 1402 and 1404, that each provide excitation light at different wavelengths.
  • 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.
  • 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 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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).
  • a polymerase e.g., by a thermostable polymerase in a polymerase chain reaction
  • a ligase e.g., as in a ligase chain reaction.
  • 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).
  • 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.
  • the chamber is less than about 1mm deep above the array; desirably the chamber is less than about 500 ⁇ in depth. Typically, the chamber is less than about 400 ⁇ , less than about 300 ⁇ , less than about 200 ⁇ , or less than about 150 ⁇ in depth above the array. In one example provided herein, the chamber is about 142 ⁇ in depth.
  • 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.
  • 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).
  • a linking sequence or structure between the capture site and the surface can be used.
  • nucleic acid sequences or polyethylene glycol linkers 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.
  • 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.
  • 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.
  • the probe will include a "flap" region. This flap region does not base-pair with 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.
  • 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.
  • 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.
  • 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.
  • 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 [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.
  • the PCR chamber used in most of the experiments is shown in Figure 2.
  • 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. (Coral ville, 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 ⁇ in diameter with a 200 ⁇ center to center pitch between spots.
  • the PCR chamber was assembled using a pressure sensitive adhesive (PSA) and a polycarbonate top piece with inlet and outlet ports as shown in Figure 2.
  • PSA pressure sensitive adhesive
  • the chamber had a final depth/thickness (or height) of 142 ⁇ and a diameter of 15 mm.
  • the chamber holds a volume of approximately 45 uL of PCR reagents.
  • 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.
  • an excitation light source e.g., a mercury arc lamp or an LED
  • 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
  • a photo-detector which is a CCD or CMOS camera.
  • 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.
  • 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.
  • 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 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.
  • the amplification reagent mix contained standard PCR reagents including two
  • 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.
  • 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 T m 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.
  • PCR was performed with the following reagent formulation: 200 nM primers, IX FAST START Tm 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.
  • the PCR Probe sequence that was used in this example was:
  • TaqMan sequence is shown in bold.
  • the double underlined sequences denote the homologous 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.
  • 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 T m of 42 °C using the PCR buffer conditions.
  • DNA plasmids comprising a target sequence were added to each PCR reaction at a concentration of 10 6 , 10 4 , and 10 2 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.
  • 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 IB.
  • 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).
  • the orthogonal flap (B) comprises a sequence that was designed to have a T m with its complement on the capture array of 70° C. Typically the sequence length is 25 to 27 bases.
  • the overall probe is designed so that the most stable secondary structure has a T m 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:
  • 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 T m 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
  • a capture probe was spotted that was homologous to the flap portion of the PCR Probe. PCR was performed, as above, except with the following cycling conditions:
  • Figure 12 shows the copy number titrations for the array based PCR for two targets where the first was present at the outset at 10 4 copies of target DNA plasmid while the second was present at 10 6 copies.
  • EXAMPLE 3 ARRAY BASED PCR CURVE USING AN UNQUENCHED PCR PROBE.
  • the PCR probe sequence used is as follows:
  • This sequence was labeled with a 5' Cy3 fluorophore, but did not include a 3' quencher.
  • 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 [0140] The PCR probe and capture probe sequences are shown below:
  • CTCCCTTTTAAGCTTCTNNNNNNNN (SEQ ID NO:4) (T m of 50.3°)
  • A/H3 NNNNNNNNNNNNNNCCTGTT GCCAATTT CAGAGTGTT
  • A/H3 NNN NNN NNN NNN NNN N (SEQ ID NO: 10) (T m of 42°)
  • phiMS2 NNN NNN NNN NNN NNN N (SEQ ID NO: 12) (T m of 43°)
  • 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.
  • CTTCTNNNNNNNN (SEQ ID NO: 13) (T m of 50.3°)
  • A/HI NNNNNNNNNNNNNNACCTTGGCGCT ATTAGATTTC
  • A/H3 NNNNNNNNNNNCCTGTTGCCA ATTTCAGAG TGTTTTGCT
  • GCCTNNNNNNNNNNN (SEP ID NO: 19) (T m of 50.4°)
  • PIV2 NNNNNNNNNNNNNCCATTT ACCTAAGTGA TGGAATCAAT
  • CTGCACNNNNNNNNN (SEP ID NO: 21) (T m of 49.9°)
  • RSV NNNNNNNNNN NNNTTCGAAGGCTC CACATACACAG CTGCTGNNNNNNNNN (SEQ ID NP: 22) (T m of 49.9°)
  • RSV-v2 NNNNNNNNNNNNNTCGAAGGC TCCACATACA CAGCTGCTGNNNNNNNN (SEQ ID NP: 23) (T m of 51°)
  • PPC1 NNNNNNNNNNNTTCGGCAT TTCCTGGATTGAGT
  • 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.
  • 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).
  • 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).
  • 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.
  • 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.
  • Thermocycling conditions included a hot start step at 95C for 85s, followed by
  • Figure 15 shows a 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.
  • 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.
  • 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.
  • 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.
  • the system can work very well in a single chamber where surface hybridization has to be coupled with optimal solution phase PCR.
  • 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.
  • 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
  • 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.
  • 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.

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Abstract

Cette invention concerne des procédés de détection et de quantification d'acides nucléiques cibles dans des échantillons, lesdits procédés faisant appel à des réactions multiplexées mises en œuvre dans une seule et même chambre. Des chambres contenant des consommables optimisées pour réduire le bruit de fond du signal à proximité de puces à haut rendement sont décrites, ainsi que des procédés pour les utiliser. Les dispositifs et les systèmes conçus pour utiliser les consommables pour la mise en œuvre desdits procédés constituent une caractéristique de l'invention.
PCT/US2012/051236 2011-11-17 2012-08-16 Détection quantitative, fortement multiplexée d'acides nucléiques WO2013074163A1 (fr)

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EP12849697.3A EP2780479A4 (fr) 2011-11-17 2012-08-16 Détection quantitative, fortement multiplexée d'acides nucléiques
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BR112014011937A BR112014011937A2 (pt) 2011-11-17 2012-08-16 detecção altamente multiplexada quantitativa de ácidos nucléicos
CN201280067273.7A CN104093857A (zh) 2011-11-17 2012-08-16 核酸的定量、高度多重检测
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IL232586A IL232586A0 (en) 2011-11-17 2014-05-13 Highly multiplexed quantitative detection of nucleic acids
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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP3022319A4 (fr) * 2013-07-15 2017-03-22 Seegene, Inc. Détection d'une séquence cible d'acides nucléiques par clivage par pto et hybridation d'oligonucléotides immobilisés dépendant de l'extension

Families Citing this family (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP3283501B1 (fr) 2015-04-16 2023-01-04 William Marsh Rice University Ajustement st chiométrique de sondes d'hybridation d'acide nucléique par des espèces oligonucléotidiques auxiliaires
CN110016500A (zh) * 2018-01-10 2019-07-16 深圳闪量科技有限公司 表面探针定量pcr方法
WO2021102039A1 (fr) * 2019-11-21 2021-05-27 10X Genomics, Inc, Analyse spatiale d'analytes
CN111808989A (zh) * 2020-06-18 2020-10-23 重庆浦洛通基因医学研究院有限公司 一种冠状病毒/流感病毒/鼻病毒核酸联合检测试剂盒及使用方法
EP4442838A2 (fr) * 2020-10-15 2024-10-09 Genentech, Inc. Analyse de criblage de candidats de liaison à une cible multiplexée

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7189508B2 (en) * 2001-07-17 2007-03-13 Stratagene California Methods for detection of a target nucleic acid by capture using multi-subunit probes
US20080241838A1 (en) * 2006-12-29 2008-10-02 Applera Corporation, Applied Biosystems Group Methods and systems for detecting nucleic acids
US20100047784A1 (en) * 2006-06-30 2010-02-25 Rosetta Genomics Ltd. Method for detecting nucleic acids
WO2010058342A1 (fr) * 2008-11-21 2010-05-27 Koninklijke Philips Electronics N.V. Détermination par pcr multiplexée en temps réel sur des surfaces solides utilisant des colorants spécifiques d’acide nucléique bicaténaire
US20110039720A1 (en) * 2007-12-19 2011-02-17 Biocartis S.A. Device and method for parallel quantitative analysis of multiple nucleic acids

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5422253A (en) * 1992-12-07 1995-06-06 Wisconsin Alumni Research Foundation Method of site specific nucleic acid cleavage
US6350580B1 (en) * 2000-10-11 2002-02-26 Stratagene Methods for detection of a target nucleic acid using a probe comprising secondary structure
WO2005010199A2 (fr) * 2003-07-16 2005-02-03 Geneohm Sciences, Inc. Reaction de clivage invasive associee a une lecture electrochimique
WO2005068660A1 (fr) * 2003-12-19 2005-07-28 Beckman Coulter, Inc. Essai envahisseur multiplexe en phase solide

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7189508B2 (en) * 2001-07-17 2007-03-13 Stratagene California Methods for detection of a target nucleic acid by capture using multi-subunit probes
US20100047784A1 (en) * 2006-06-30 2010-02-25 Rosetta Genomics Ltd. Method for detecting nucleic acids
US20080241838A1 (en) * 2006-12-29 2008-10-02 Applera Corporation, Applied Biosystems Group Methods and systems for detecting nucleic acids
US20110039720A1 (en) * 2007-12-19 2011-02-17 Biocartis S.A. Device and method for parallel quantitative analysis of multiple nucleic acids
WO2010058342A1 (fr) * 2008-11-21 2010-05-27 Koninklijke Philips Electronics N.V. Détermination par pcr multiplexée en temps réel sur des surfaces solides utilisant des colorants spécifiques d’acide nucléique bicaténaire

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See also references of EP2780479A4 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP3022319A4 (fr) * 2013-07-15 2017-03-22 Seegene, Inc. Détection d'une séquence cible d'acides nucléiques par clivage par pto et hybridation d'oligonucléotides immobilisés dépendant de l'extension

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IL232586A0 (en) 2014-06-30
SG11201402341QA (en) 2014-06-27
BR112014011937A2 (pt) 2017-05-30
JP2014533502A (ja) 2014-12-15
EP2780479A4 (fr) 2015-04-08
MX338076B (es) 2016-04-01
CA2855953A1 (fr) 2013-05-23
EP2780479A1 (fr) 2014-09-24
HK1199746A1 (en) 2015-07-17
MX2014005912A (es) 2014-09-04
KR20140094007A (ko) 2014-07-29
CN104093857A (zh) 2014-10-08
AU2012337389A1 (en) 2014-06-19

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