REAL-TIME MULTIPLEXED HYDROLYSIS PROBE ASSAY USING SPECTRALLY IDENTIFIABLE MICROSPHERES
20210310056 · 2021-10-07
Assignee
Inventors
Cpc classification
C12Q1/6818
CHEMISTRY; METALLURGY
C12Q1/6818
CHEMISTRY; METALLURGY
C12Q1/6834
CHEMISTRY; METALLURGY
International classification
Abstract
Methods and compositions for the detection and quantification of nucleic acids are provided. In one embodiment, a sample is contacted with a primer and a quencher-probe complementary to a target nucleic acid. The quencher-probe is complementary to an anti-probe that comprises a reporter and is attached to a solid support. Thus, hybridized probe is cleaved with a nucleic acid polymerase having exonuclease activity to release the quencher from the probe. The presence of the target nucleic acid is then detected and/or optionally quantified by detecting an increase in signal from the fluorescent reporter on the solid support.
Claims
1. A method for detecting a target nucleic acid in a sample comprising: (a) contacting the sample with a first target-specific primer complementary to a first region on a first strand of the target nucleic acid, a second target-specific primer complementary to a region on a second strand of the target nucleic and oriented such that the first and second target-specific primers can amplify the target nucleic acid by polymerase chain reaction (PCR), and a target-specific probe complementary to a second region on the first strand of the target nucleic acid downstream of the first region, under conditions suitable for hybridization of the target nucleic acid with the first target-specific primer, the target-specific probe and the second target-specific primer, wherein the target-specific probe comprises a quencher; (b) performing multiple PCR cycles with a nucleic acid polymerase having exonuclease activity; (c) cleaving target-specific probe that is hybridized to the target nucleic acid by extension of the target-specific primer with said nucleic acid polymerase to release the quencher from the target-specific probe; (d) hybridizing any uncleaved target-specific probe to a reporter probe that is complementary to at least a portion of the target-specific probe, said reporter probe comprising a fluorophore and being attached to a solid support, wherein the hybridization temperature of the target-specific probe for the target nucleic acid is higher than the hybridization temperature of the target-specific probe for the reporter probe; and (e) detecting a signal from the fluorophore two or more times, and detecting the target nucleic acid by detecting a change in signal detected at the two or more times.
2. The method of claim 1, wherein signal is detected two or more times during the multiple PCR cycles.
3. The method of claim 1, wherein signal is detected before and after performing the multiple PCR cycles.
4. The method of claim 1, wherein the reporter probe and the target-specific probe comprise both natural bases and isobases.
5. The method of claim 1, wherein the solid support is an encoded bead.
6. The method of claim 1, further comprising detecting a reference signal from a fluorophore on a non-hybridizing probe at the two or more times, wherein the fluorophore on the non-hybridizing probe is the same fluorophore as the reporter probe fluorophore and is attached to a solid support, and using the reference signal to normalize the change in signal from the reporter probe detected at the two or more times.
7. The method of claim 6, wherein the non-hybridizing probe is attached to a spatially discrete location on the same solid support to which the reporter probe is attached.
8. The method of claim 6, wherein the non-hybridizing probe is attached to a different solid support than that to which the reporter probe is attached.
9. The method of claim 8, wherein the different solid supports are different encoded beads.
10. The method of claim 1, wherein the target nucleic acid is a first target nucleic acid, the quencher is a first quencher, the reporter probe is a first reporter probe, the fluorophore is a first fluorophore, the solid support is a first solid support, and the method further comprises: (a) including in the contacting step, a third target-specific primer complementary to a first region on a first strand of a second target nucleic acid, a fourth target-specific primer complementary to a region on a second strand of the second target nucleic acid and oriented such that the third and fourth target-specific primers can amplify the second target nucleic acid by PCR, and a second target-specific probe complementary to a second region on the first strand of the second target nucleic acid downstream of the first region, under conditions suitable for hybridization of the second target nucleic acid with the third target-specific primer, the second target-specific probe, and the fourth target-specific primer, wherein the second target-specific probe comprises a second quencher; (b) during the cleaving step cleaving second target-specific probe that is hybridized to the second target nucleic acid, with said nucleic acid polymerase to release the second quencher from the second target-specific probe; (c) during the hybridizing step, hybridizing any uncleaved second target-specific probe to a second reporter probe that is complementary to at least a portion of the second target-specific probe, said second reporter probe comprising a second fluorophore and being attached to a second solid support; and (d) detecting a signal from the second fluorophore two or more times and detecting the second target nucleic acid by detecting a change in signal from the second fluorophore at the two or more times.
11. The method of claim 10, wherein the first solid support and the second solid support are spatially discrete locations on one solid support.
12. The method of claim 10, wherein the first solid support is physically separate from the second solid support.
13. The method of claim 10, wherein the first fluorophore and the second fluorophore are the same.
14. A method for quantifying an amount of a target nucleic acid in a sample, comprising: (a) amplifying by PCR the target nucleic acid in the presence of a nucleic acid polymerase having exonuclease activity, a target-specific primer pair comprising a first primer complementary to a first region on a first strand of the target nucleic acid and a second primer complementary to a region on a second strand of the target nucleic acid, and a target-specific probe complementary to a second region on the first strand of the target nucleic acid downstream of the first region, wherein the target-specific probe comprises a quencher, and further wherein the nucleic acid polymerase cleaves target-specific probe hybridized to the target nucleic acid and releases the quencher from the target-specific probe when extending the first primer along the first strand of the target nucleic acid; (b) hybridizing uncleaved target-specific probe to a reporter probe that is complementary to at least a portion of the target-specific probe, said reporter probe comprising a fluorophore reporter and being attached to a solid support; (c) detecting a first signal from the fluorophore reporter on the solid support at a first time during the PCR and a second signal from the reporter on the solid support at a second time during the PCR; and (d) correlating a change in signal detected at the first time and the second time with the amount of the target nucleic acid in the sample.
15. The method of claim 14, wherein quantifying the amount of the target nucleic acid in the sample comprises using a standard curve.
16. The method of claim 14, wherein quantifying the amount of the target nucleic acid in the sample comprises determining a relative amount of the target nucleic acid.
17. The method of claim 14, further comprising detecting at least a third signal from the reporter on the solid support at a third time.
18. The method of claim 14, comprising detecting a signal from the reporter on the solid support prior to extending the target-specific primer with the nucleic acid polymerase having exonuclease activity to cleave the hybridized target-specific probe and release the quencher from the target-specific probe.
19. A method for detecting the presence or absence of a target nucleic acid in a sample comprising: (a) contacting the sample with a first target-specific primer complementary to a first region on a first strand of the target nucleic acid, a second target-specific primer complementary to a region on a second strand of the target nucleic and oriented such that the first and second target-specific primers can amplify the target nucleic acid by polymerase chain reaction (PCR), and a target-specific probe complementary to a second region on the first strand of the target nucleic acid downstream of the first region, under conditions suitable for hybridization of the target nucleic acid if present, with the first target-specific primer, the target-specific probe and the second target-specific primer, wherein the target-specific probe comprises a quencher; (b) performing multiple PCR cycles with a nucleic acid polymerase having exonuclease activity; (c) cleaving target-specific probe that is hybridized to the target nucleic acid by extension of the target-specific primer with said nucleic acid polymerase to release the quencher from the target-specific probe; (d) hybridizing any uncleaved target-specific probe to a reporter probe that is complementary to at least a portion of the target-specific probe, said reporter probe comprising a fluorophore and being attached to a solid support, wherein the hybridization temperature of the target-specific probe for the target nucleic acid is higher than the hybridization temperature of the target-specific probe for the reporter probe; and (e) detecting the presence or absence of the target nucleic acid by detecting a signal from the reporter probe after performing the multiple PCR cycles and comparing the detected signal to a reference signal from the reporter on a non-hybridizing probe attached to a solid support, wherein a change in the detected signal indicates the presence of the target nucleic acid.
20. The method of claim 19, wherein the ratio of the detected signal from the reporter probe and the reference signal is compared to a predetermined ratio of the signal from the reporter probe and the reference signal and wherein determining that the ratio has changed indicates the presence of the target nucleic acid.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
[0058] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.
[0059]
[0060]
[0061]
[0062]
DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS
[0063] Certain aspects of the present disclosure employ hydrolysis probes for the detection of nucleic acids. Hydrolysis probes take advantage of the 5′ exonuclease activity of some polymerases. During the extension or elongation phase of a PCR reaction, a polymerase, such as Taq polymerase, uses an upstream primer as a binding site and then extends. The hydrolysis probe is then cleaved during polymerase extension at its 5′ end by the 5′-exonuclease activity of the polymerase (see, e.g.,
[0064] Since the fluorophore is located only on the microsphere and the hydrolysis probe comprises a quencher, there will not be an excess of fluorophore in the solution, which eliminates the need for washing steps prior to imaging. Also, since the probes are not extendable primers, they will not be susceptible to mispriming events or primer dimer formation, making them more specific than an extendable primer.
I. Definitions
[0065] The terms “upstream” and “downstream” are used herein in relation to the synthesis of the nascent strand that is primed by a target-specific primer. Thus, for example, a target-specific probe hybridized to a region of the target nucleic acid that is “downstream” of the region of the target nucleic acid to which the primer is hybridized is located 3′ of the primer and will be in the path of a polymerase extending the primer in a 5′ to 3′ direction.
[0066] A primer is a nucleic acid that is capable of priming the synthesis of a nascent nucleic acid in a template-dependent process. A target-specific primer refers to a primer that has been designed to prime the synthesis of a particular target nucleic acid. A primer pair refers to two primers, commonly known as a forward primer and a reverse primer or as an upstream primer and a downstream primer, which are designed to amplify a target sequence between the binding sites of the two primers on a template nucleic acid molecule. In certain embodiments, the primer has a target-specific sequence that is between 10-40, 15-30, or 18-26 nucleotides in length.
[0067] A probe is a nucleic acid that is capable of hybridizing to a complementary nucleic acid. A target-specific probe refers to a probe that has been designed to hybridize to a particular target nucleic acid. Probes present in the reaction may comprise a blocked 3′ hydroxyl group to prevent extension of the probes by the polymerase. The 3′ hydroxyl group may be blocked with, for example, a phosphate group, a 3′ inverted dT, or a reporter. High stringency hybridization conditions may be selected that will only allow hybridization between sequences that are completely complementary.
[0068] Various aspects of the present invention use sets of complementary tag and anti-tag sequences. Which sequence in a complementary pair is called the “tag” and which is called the “anti-tag” is arbitrary. The tags and anti-tags are preferably non-cross hybridizing, i.e., each tag and anti-tag should hybridize only to its complementary partner, and not to other tags or anti-tags in the same reaction. Preferably, the tags and anti-tags also will not hybridize to other nucleic acids in the sample during a reaction. The tag and anti-tag sequences are also preferably designed to be isothermic, i.e., of similar optimal hybridization temperature, whereby all of the tag and anti-tag sequences in a multiplex reaction will have approximately the same Tm. The proper selection of non-cross hybridizing tag and anti-tag sequences is useful in assays, particularly assays in a highly parallel hybridization environment, that require stringent non-cross hybridizing behavior. In certain embodiments, the tag and anti-tag sequences are between 6 to 60, 8 to 50, 10 to 40, 10 to 20, 12 to 24, or 20 to 30 nucleotides in length. In some embodiments, the tag and anti-tag sequences are 12, 14, 16, or 24 nucleotides in length. A number of tag and tag complement (i.e., anti-tag) sequences are known in the art and may be used in the present invention. For example, U.S. Patent 7,226,737, incorporated herein by reference, describes a set of 210 non-cross hybridizing tags and anti-tags. In addition, U.S. Pat. No. 7,645,868, incorporated herein by reference, discloses a family of 1168 tag sequences with a demonstrated ability to correctly hybridize to their complementary sequences with minimal cross hybridization. A “universal” tag or anti-tag refers to a tag or anti-tag that has the same sequence across all reactions in a multiplex reaction.
[0069] As used herein, “hybridization,” “hybridizes” or “capable of hybridizing” is understood to mean the forming of a double or triple stranded molecule or a molecule with partial double or triple stranded nature. The term “anneal” as used herein is synonymous with “hybridize.” As used herein “stringent conditions” or “high stringency” are those conditions that allow hybridization between or within one or more nucleic acid strands containing complementary sequences, but preclude hybridization of non-complementary sequences. Such conditions are well known to those of ordinary skill in the art, and are preferred for applications requiring high selectivity. Stringent conditions may comprise low salt and/or high temperature conditions. It is understood that the temperature and ionic strength of a desired stringency are determined in part by the length of the particular nucleic acids, the length and nucleobase content of the target sequences, the charge composition of the nucleic acids, and to the presence or concentration of formamide, tetramethylammonium chloride or other solvents in a hybridization mixture.
II. PCR
[0070] The polymerase chain reaction (PCR) is a technique widely used in molecular biology to amplify a piece of DNA by in vitro enzymatic replication. Typically, PCR applications employ a heat-stable DNA polymerase, such as Taq polymerase. This DNA polymerase enzymatically assembles a new DNA strand from nucleotides (dNTPs) using single-stranded DNA as template and DNA primers to initiate DNA synthesis. A basic PCR reaction requires several components and reagents including: a DNA template that contains the target sequence to be amplified; one or more primers, which are complementary to the DNA regions at the 5′ and 3′ ends of the target sequence; a DNA polymerase (e.g., Taq polymerase) that preferably has a temperature optimum at around 70° C.; deoxynucleotide triphosphates (dNTPs); a buffer solution providing a suitable chemical environment for optimum activity and stability of the DNA polymerase; divalent cations, typically magnesium ions (Mg2.sup.+); and monovalent cation potassium ions.
[0071] The majority of PCR methods use thermal cycling to subject the PCR sample to a defined series of temperature steps. Each cycle typically has 2 or 3 discrete temperature steps. The cycling is often preceded by a single temperature step (“initiation”) at a high temperature (>90° C.), and followed by one or two temperature steps at the end for final product extension (“final extension”) or brief storage (“final hold”). The temperatures used and the length of time they are applied in each cycle depend on a variety of parameters. These include the enzyme used for DNA synthesis, the concentration of divalent ions and dNTPs in the reaction, and the melting temperature (Tm) of the primers. Commonly used temperatures for the various steps in PCR methods are: initialization step −94-96° C.; denaturation step −94-98° C.; annealing step −50-65° C.; extension/elongation step −70-74° C.; final elongation −70-74° C.; final hold −4-10° C.
[0072] Real-time polymerase chain reaction, also called quantitative real time polymerase chain reaction (qPCR) or kinetic polymerase chain reaction, is used to amplify and simultaneously quantify a targeted DNA molecule. It enables both detection and quantification (as absolute number of copies or relative amount when normalized to DNA input or additional normalizing genes) of a specific sequence in a DNA sample. Real-time PCR may be combined with reverse transcription polymerase chain reaction to quantify low abundance RNAs. Relative concentrations of DNA present during the exponential phase of real-time PCR are determined by plotting fluorescence against cycle number on a logarithmic scale. Amounts of DNA may then be determined by comparing the results to a standard curve produced by real-time PCR of serial dilutions of a known amount of DNA. Various PCR and real-time PCR methods are disclosed in U.S. Pat. Nos. 5,656,493; 5,994,056; 6,174,670; 5,716,784; 6,030,787; 6,174,670, and 7,955,802, which are incorporated herein by reference.
[0073] Digital PCR (dPCR) involves partitioning the sample such that individual nucleic acid molecules contained in the sample are localized in many separate regions, such as in individual wells in microwell plates, in the dispersed phase of an emulsion, or arrays of nucleic acid binding surfaces. Each partition will contain 0 or 1 molecule, providing a negative or positive reaction, respectively. Unlike conventional PCR, dPCR is not dependent on the number of amplification cycles to determine the initial amount of the target nucleic acid in the sample. Accordingly, dPCR eliminates the reliance on exponential data to quantify target nucleic acids and provides absolute quantification.
[0074] Multiplex-PCR and multiplex real-time PCR use of multiple, unique primer sets within a single PCR reaction to produce amplicons of different DNA sequences. By targeting multiple genes at once, additional information may be gained from a single test run that otherwise would require several times the reagents and more time to perform. Annealing temperatures for each of the primer sets should be optimized to work within a single reaction.
III. Complementary Tags
[0075] Some embodiments of the present invention employ complementary tag sequences (i.e., tags and anti-tags) in the primers and/or probes. The proper selection of non-hybridizing tag and anti-tag sequences is useful in assays, particularly assays in a highly parallel hybridization environment, that require stringent non-cross hybridizing behavior.
[0076] Certain thermodynamic properties of forming nucleic acid hybrids are considered in the design of tag and anti-tag sequences. The temperature at which oligonucleotides form duplexes with their complementary sequences known as the T.sub.m (the temperature at which 50% of the nucleic acid duplex is dissociated) varies according to a number of sequence dependent properties including the hydrogen bonding energies of the canonical pairs A-T and G-C (reflected in GC or base composition), stacking free energy and, to a lesser extent, nearest neighbor interactions. These energies vary widely among oligonucleotides that are typically used in hybridization assays. For example, hybridization of two probe sequences composed of 24 nucleotides, one with a 40% GC content and the other with a 60% GC content, with its complementary target under standard conditions theoretically may have a 10° C. difference in melting temperature (Mueller et al., 1993). Problems in hybridization occur when the hybrids are allowed to form under hybridization conditions that include a single hybridization temperature that is not optimal for correct hybridization of all oligonucleotide sequences of a set. Mismatch hybridization of non-complementary probes can occur, forming duplexes with measurable mismatch stability (Peyret et al., 1999). Mismatching of duplexes in a particular set of oligonucleotides can occur under hybridization conditions where the mismatch results in a decrease in duplex stability that results in a higher T.sub.m than the least stable correct duplex of that particular set. For example, if hybridization is carried out under conditions that favor the AT-rich perfect match duplex sequence, the possibility exists for hybridizing a GC-rich duplex sequence that contains a mismatched base having a melting temperature that is still above the correctly formed AT-rich duplex. Therefore, design of families of oligonucleotide sequences that can be used in multiplexed hybridization reactions must include consideration for the thermodynamic properties of oligonucleotides and duplex formation that will reduce or eliminate cross hybridization behavior within the designed oligonucleotide set.
[0077] There are a number of different approaches for selecting tag and anti-tag sequences for use in multiplexed hybridization assays. The selection of sequences that can be used as zip codes or tags in an addressable array has been described in the patent literature in an approach taken by Brenner and co-workers (U.S. Pat. No. 5,654,413, incorporated herein by reference). Chetverin et al. (WO 93/17126, U.S. Pat. Nos. 6,103,463 and 6,322,971, incorporated herein by reference) discloses sectioned, binary oligonucleotide arrays to sort and survey nucleic acids. These arrays have a constant nucleotide sequence attached to an adjacent variable nucleotide sequence, both bound to a solid support by a covalent linking moiety. Parameters used in the design of tags based on subunits are discussed in Barany et al. (WO 9,731,256, incorporated herein by reference). A multiplex sequencing method has been described in U.S. Pat. 4,942,124, incorporated herein by reference. This method uses at least two vectors that differ from each other at a tag sequence.
[0078] U.S. Pat. 7,226,737, incorporated herein by reference, describes a set of 210 non-cross hybridizing tags and anti-tags. U.S. Published Application No. 2005/0191625, incorporated herein by reference, discloses a family of 1168 tag sequences with a demonstrated ability to correctly hybridize to their complementary sequences with minimal cross hybridization. U.S. Publication No. 2009/0148849, incorporated herein by reference, describes the use of tags, anti-tags, and capture complexes in the amplification of nucleic acid sequences.
[0079] A population of oligonucleotide tag or anti-tag sequences may be conjugated to a population of primers or other polynucleotide sequences in several different ways including, but not limited to, direct chemical synthesis, chemical coupling, ligation, amplification, and the like. Sequence tags that have been synthesized with target specific primer sequences can be used for enzymatic extension of the primer on the target for example in PCR amplification. A population of oligonucleotide tag or anti-tag sequences may be conjugated to a solid support by, for example, surface chemistries on the surface of the support.
IV. Solid Supports
[0080] In certain embodiments, the probes and/or primers may be attached to a solid support. Such solid supports may be, for example, microspheres (i.e., beads) or other particles such as microparticles, gold or other metal nanoparticles, quantum dots, or nanodots. In certain aspects, the particles may be magnetic, paramagnetic, or super paramagnetic. Examples of microspheres, beads, and particles are illustrated in U.S. Pat. No. 5,736,330 to Fulton, U.S. Pat. No. 5,981,180 to Chandler et al., U.S. Pat. No. 6,057,107 to Fulton, U.S. Pat. No. 6,268,222 to Chandler et al., U.S. Pat. No. 6,449,562 to Chandler et al., U.S. Pat. No. 6,514,295 to Chandler et al., U.S. Pat. No. 6,524,793 to Chandler et al., and U.S. Pat. No. 6,528,165 to Chandler, which are incorporated by reference herein.
[0081] The particles may be encoded with a label. In certain embodiments, the present invention is used in conjunction with Luminex® xMAP® and MagPlex™ technologies. The Luminex xMAP technology allows the detection of nucleic acid products immobilized on fluorescently encoded microspheres. By dyeing microspheres with 10 different intensities of each of two spectrally distinct fluorochromes, 100 fluorescently distinct populations of microspheres are produced. These individual populations (sets) can represent individual detection sequences and the magnitude of hybridization on each set can be detected individually. The magnitude of the hybridization reaction is measured using a third reporter, which is typically a third spectrally distinct fluorophore. In embodiments in which a labeled hydrolysis probe is attached to the microsphere, hybridization and hydrolysis of the probe results in a decrease in signal from the third reporter. As both the microspheres and the reporter molecules are labeled, digital signal processing allows the translation of signals into real-time, quantitative data for each reaction. The Luminex technology is described, for example, in U.S. Pat. Nos. 5,736,330, 5,981,180, and 6,057,107, all of which are specifically incorporated by reference. Luminex® MagPlex™ microspheres are superparamagnetic microspheres that are fluorescently encoded using the xMAP® technology discussed above. The microspheres contain surface carboxyl groups for covalent attachment of ligands (or biomolecules).
[0082] Alternatively, the solid support may be a planar array such as a gene chip or microarray (see, e.g., Pease et al., 1994; Fodor et al., 1991). The identity of nucleic acids on a planar array is typically determined by it spatial location on the array. Microsphere based assays may also be analyzed on bead array platforms. In general, bead array platforms image beads and analytes distributed on a substantially planar array. In this way, imaging of bead arrays is similar to the gene chips discussed above. However, in contrast to gene chips where the analyte is typically identified by its spatial position on the array, bead arrays typically identify the analyte by the encoded microsphere to which it is bound.
[0083] The ability to directly synthesize on or attach polynucleotide probes to solid substrates is well known in the art. See U.S. Pat. Nos. 5,837,832 and 5,837,860, both of which are incorporated by reference. A variety of methods have been utilized to either permanently or removably attach the probes to the substrate. Exemplary methods include: the immobilization of biotinylated nucleic acid molecules to avidin/streptavidin coated supports (Holmstrom, 1993), the direct covalent attachment of short, 5′-phosphorylated primers to chemically modified polystyrene plates (Rasmussen et al., 1991), or the precoating of the polystyrene or glass solid phases with poly-L-Lys or poly L-Lys, Phe, followed by the covalent attachment of either amino- or sulfhydryl-modified oligonucleotides using bi-functional crosslinking reagents (Running et al., 1990; Newton et al., 1993). Numerous materials may be used as solid supports, including reinforced nitrocellulose membrane, activated quartz, activated glass, polyvinylidene difluoride (PVDF) membrane, polystyrene substrates, polyacrylamide-based substrate, other polymers such as poly(vinyl chloride), poly(methyl methacrylate), poly(dimethyl siloxane), photopolymers (which contain photoreactive species such as nitrenes, carbenes and ketyl radicals capable of forming covalent links with target molecules.
V. Detection
[0084] Various aspects of the present invention relate to the direct or indirect detection of one or more target nucleic acids by detecting an increase or decrease in a signal. The detection techniques employed will depend on the type of reporter and platform (e.g., spectrally encoded beads, microarray, etc.). Flow cytometry, for example, is particularly useful in the analysis of microsphere based assays. Flow cytometry involves the separation of cells or other particles, such as microspheres, in a liquid sample. Generally, the purpose of flow cytometry is to analyze the separated particles for one or more characteristics. The basic steps of flow cytometry involve the direction of a fluid sample through an apparatus such that a liquid stream passes through a sensing region. The particles should pass one at a time by the sensor and are categorized based on size, refraction, light scattering, opacity, roughness, shape, fluorescence, etc.
[0085] In the context of the Luminex xMAP® system, flow cytometry can be used for simultaneous sequence identification and hybridization quantification. Internal dyes in the microspheres are detected by flow cytometry and used to identify the specific nucleic acid sequence to which a microsphere is coupled. The label on the target nucleic acid molecule or probe is also detected by flow cytometry and used to determine hybridization to the microsphere.
[0086] Methods of flow cytometry are well known in the art and are described, for example, in U.S. patents, all of which are specifically incorporated by reference. U.S. Pat. Nos. 5,981,180, 4,284,412; 4,989,977; 4,498,766; 5,478,722; 4,857,451; 4,774,189; 4,767,206; 4,714,682; 5,160,974; and 4,661,913. The measurements described herein may include image processing for analyzing one or more images of particles to determine one or more characteristics of the particles such as numerical values representing the magnitude of fluorescence emission of the particles at multiple detection wavelengths. Subsequent processing of the one or more characteristics of the particles such as using one or more of the numerical values to determine a token ID representing the multiplex subset to which the particles belong and/or a reporter value representing a presence and/or a quantity of analyte bound to the surface of the particles can be performed according to the methods described in U.S. Pat. No. 5,736,330 to Fulton, U.S. Pat. No. 5,981,180 to Chandler et al., U.S. Pat. No. 6,449,562 to Chandler et al., U.S. Pat No. 6,524,793 to Chandler et al., U.S. Pat. No. 6,592,822 to Chandler, and U.S. Pat. No. 6,939,720 to Chandler et al., which are incorporated by reference herein.
[0087] In one example, techniques described in U.S. Pat. No. 5,981,180 to Chandler et al. may be used with the fluorescent measurements described herein in a multiplexing scheme in which the particles are classified into subsets for analysis of multiple analytes in a single sample. Additional examples of systems that may be configured as described herein (e.g., by inclusion of an embodiment of an illumination subsystem described herein) are illustrated in U.S. Pat. No. 5,981,180 to Chandler et al., U.S. Pat. No. 6,046,807 to Chandler, U.S. Pat. No. 6,139,800 to Chandler, U.S. Pat. No. 6,366,354 to Chandler, U.S. Pat. No. 6,411,904 to Chandler, U.S. Pat. No. 6,449,562 to Chandler et al., and 6,524,793 to Chandler et al., which are incorporated by reference herein.
[0088] Microspheres may also be analyzed on array platforms that image beads and analytes distributed on a substantially planar array. In this way, imaging of bead arrays is similar to imaging of gene chips. However, in contrast to gene chips where the analyte is identified by its spatial position (i.e., x, y coordinate) on the array, bead arrays typically identify the analyte by the encoded microsphere to which it is bound. Examples of commercially available bead array systems include Luminex's MAGPIX®, and Illumina's BeadXpress™ Reader and BeadStation 500™. Once beads are in a planar layer, they can be identified by their “coding” (either in the form of embedded dyes, or other methods that create unique signals for each bead type). Following or preceding the resolution of the “code” of the bead, the signal can be measured and these two measurements coupled to determine the hybridization of a particular nucleic acid to the bead.
VI. Kits
[0089] The present invention also provides kits containing components for use with the amplification and detection methods disclosed herein. Any of the components disclosed here in may be combined in a kit. In certain embodiments the kits comprise a plurality of primers for priming amplification of a plurality of nucleic acid targets, and a plurality of probes complementary to the plurality of nucleic acid targets. In some embodiments, the probes are immobilized on a solid support(s). In one embodiment, a plurality of probes are attached to a plurality of encoded magnetic beads such that the identity of each probe is known from the encoded magnetic bead on which it is immobilized. In certain embodiments, the kit also comprises a labeling agent. In certain embodiments the kits comprise probes that are not attached to a solid support. In some embodiments the kit comprises an imaging chamber, which may be a disposable imaging chamber, for use in an imaging system.
[0090] The kits will generally include at least one vial, test tube, flask, bottle, syringe or other container, into which a component may be placed, and preferably, suitably aliquoted. Where there is more than one component in the kit, the kit also will generally contain a second, third or other additional containers into which the additional components may be separately placed. However, various combinations of components may be comprised in a container. The kits of the present invention also will typically include packaging for containing the various containers in close confinement for commercial sale. Such packaging may include cardboard or injection or blow molded plastic packaging into which the desired containers are retained.
[0091] A kit may also include instructions for employing the kit components. Instructions may include variations that can be implemented.
VII. Examples
[0092] The following examples are included to demonstrate preferred embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventors to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.
Example 1
[0093] As shown in
[0094] In this assay chemistry, the fluorophore is located only on the microspheres so that there is no fluorophore to image through in solution. Second, the probes are hydrolysis probes and are not extendable.
[0095] In order for the assay to detect a depleting probe in solution, the amount of probe to start with must be under the saturation of hybridization to the microspheres. Determining this saturation limit will be performed with a quencher-fluorophore combination in PCR solution.
[0096] Next, whether the saturation limit is effective for hybridization to the amplicon will be tested. However, the hydrolysis of the probes in cumulative over the course of the reaction, which may counteract any concentration dynamic range limitations.
[0097] Luminex MagPlex® Microspheres are coupled to amine-modified oligonucleotide probes according to the manufacturer's instructions.
[0098] Microsphere region 25 is coupled to a probe specific for Staphylococcus epidermidis: 5′-/5AmMC12/GTA ATA ATG GCG GTG GTC/3Cy3Sp/-3′ (SEQ ID NO: 1)
[0099] Microsphere region 54 is coupled to a probe that was designed to not hybridize to Staphylococcus epidermidis: 5′-/5AmMC12/GAT TGT AAG ATT TGA TAA AGT GTA/3Cy3Sp/-3′ (SEQ ID NO: 2)
[0100] A solution phase probe includes a probe that is partly complementary to the probe on microsphere region 25 and fully complementary to the Staphylococcus epidermidis target amplicon: 5′/BHQ2/GAC CAC CGC CAT TAT TAC GAA CAG CTG-3′ (SEQ ID NO: 3)
[0101] An additional solution phase probe includes a probe that is complementary to the probe on region 54: 5′/BHQ2/TAC ACT TTA TCA AAT CTT ACA ATC-3′ (SEQ ID NO: 4)
[0102] Next a PCR Master mix is made for each reaction including:
TABLE-US-00001 2x TaqMan ® Master Mix (Applied Biosystems) 12.5 μL Water 5.7 μL 50 mM MgCl.sub.2 2.0 μL 20x Primer Mix 1.3 μL 2500 beads/μL per region 1.0 μL
The 20× Primer Mix contains the following ratios per μL:
TABLE-US-00002 TE pH 8.0 0.64 μL 100 μM Forward Primer 0.18 μL 100 μM Reverse Primer 0.18 μL
[0103] The Forward Primer has the following oligonucleotide sequence: 5′-TCA GCA GTT GAA GGG ACA GAT-3′ (SEQ ID NO: 5)
[0104] The Reverse Primer has the following oligonucleotide sequence: 5′-CCA GAA CAA TGA ATG GTT AAG G-3′ (SEQ ID NO: 6)
[0105] The template can be purchased from ATCC # 12228D-5 (S. epidermidis purified DNA). 2.5 μL of template in water are added to each “template” PCR reaction (2 ng per reaction), and 2.5 μL water alone are added to the “no template” PCR reactions.
[0106] The following thermal cycling protocol is used on an ABI Step One Plus ThermalCycler: [0107] 50° C. for 2 min. [0108] 95° C. for 10 min. [0109] Followed by 35 cycles of a two step PCR [0110] 95° C. for 15 sec. [0111] 60° C. for 1 min.
[0112] After PCR, the reaction mix is taken directly to a Luminex instrument, allowed to hybridize for 10 minutes at room temperature and analyzed for Median Fluorescent Intensity (MFI) values using 100 microspheres per MFI data point. The delta MFI between the control microsphere (region 54) and the target specific microsphere (region 25) is used to determine positivity or negativity of the reaction based on predetermined cutoff thresholds.
Example 2—Dual-Phase Chemistry Studies
[0113] Studies were performed to assess dual-phase PCR chemistry. A PCR reaction including beads coupled with Cy3 labeled fluorescent probes was used to assess whether complimentary probes labeled with a BHQ2 quencher would be consumed in the reaction by hybridization or hydrolysis to the target amplicon generated. In the case where template is present, the signal on the particle should increase because the complementary quenching probe is consumed.
[0114] The following beads with their respective probe sets were used in the PCR reaction:
TABLE-US-00003 Bead 33: (SEQ ID NO: 7) 5′-/5Cy3/GAC CAC CGC CAT TAT TAC G/3AmMC6T/-3′ Bead 45: (SEQ ID NO: 2) 5′-/5Cy3/GAT TGT AAG ATT TGA TAA AGT GTA /3AmMO/-3′
Bead 33 is partially complimentary to a probe that is specific for the S. epidermidis amplicon:
TABLE-US-00004 (SEQ ID NO: 8) 5′- CAG CTG TTC GTA ATA ATG GCG GTG GTC /3BHQ_2/ -3′
There was no complimentary quenching oligonucleotide to hybridize to the probe on bead 45.
[0115] Results were obtained by reading the results of the PCR reaction on a MAGPIX® instrument after 15 minutes of hybridization at 40° C. Two conditions were tested: with beads in PCR and leaving the beads out of the PCR reaction, but adding them directly after the cycling was complete. As shown below in Table 1, the signal on the non-quenched, non-specific, bead 45 was diminished when beads were present during PCR as compared to when the beads were added after PCR, indicating that there was some general degradation or quenching as a result of the PCR process. Table 1 also shows that the no-template reactions for bead 33 in the beads after PCR scenario was lower than in the beads in PCR scenario, indicating that something was partially inhibiting the hybridization of the quencher to bead 33 in the beads in PCR scenario. The quencher was intact, however, because it hybridized as expected in the beads after PCR scenario. Despite these issues, the assay was able to detect the presence of 40 k copies of S. epidermidis by comparing the template to no template signals. This level of sensitivity was achievable both when PCR was performed in the presence of beads or when the beads were added after amplification.
TABLE-US-00005 TABLE 1 PCR Results. Bead 33- Bead 45 - Quenched and no quencher and specific (MFI) non-specific (MFI) Beads in PCR template 3650 4017 no template 3173 3972.5 template 3491 3966 no template 3059 3966 template 3507 3948 no template 3078 3935.5 Beads put in after PCR template 2486 5690 no template 1454.5 5721 template 2551 5655 no template 1450 5682 template 2593 5616 no template 1421 5630
[0116] The forward and reverse primers hybridized to the amplicon and had melting temperatures (T.sub.ms) as shown in
[0117] The PCR mix formula for each reaction was:
TABLE-US-00006 Stock Final Vol (μL) PCR buffer 10x 1x 4.0 dNTP 10 mM 300 μM 1.2 MgCl2 40 mM 2 mM 2.0 Hot start taq 0.25 (NEB M0495L) primer mix 20x 400/50 nM 2.0 beads 2500/μL 1.0 probe 20x 600 fmol/rxn 2.0 Water 17.55 template 10 Total volume: 40
[0118] The following thermal cycling protocol was used on a thermalcycling instrument for 40 cycles in slow mode: [0119] 95° C. for 3 min. [0120] 95° C. for 15 sec. [0121] 60° C. for 45 sec.
[0122] All of the methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.
REFERENCES
[0123] The following references, to the extent that they provide exemplary procedural or other details supplementary to those set forth herein, are specifically incorporated herein by reference. [0124] U.S. Pat. Nos. 4,942,124; 4,284,412; 4,989,977; 4,498,766; 5,478,722; 4,857,451; 4,774,189; 4,767,206; 4,714,682; 5,160,974; 4,661,913; 5,654,413; 5,656,493; 5,716,784; 5,736,330; 5,837,832; 5,837,860; 5,981,180; 5,994,056; 5,736,330; 5,981,180; 6,057,107; 6,030,787; 6,046,807; 6,057,107; 6,103,463; 6,139,800; 6,174,670; 6,268,222; 6,322,971; 6,366,354; 6,411,904; 6,449,562; 6,514,295; 6,524,793; 6,528,165; 6,592,822; 6,939,720; 6,977,161; 7,226,737; 7,645,868; and 7,955,802 [0125] U.S. Published Application Nos. 2005/0191625; and 2009/0148849 [0126] Fodor et al., “Light-directed, spatially addressable parallel chemical synthesis,” Science, 251(4995):767-73, 1991. [0127] Holmstrom et al., “A highly sensitive and fast nonradioactive method for detection of polymerase chain reaction products,” Anal. Biochem., 209(2):278-83, 1993. [0128] Running et al., “A procedure for productive coupling of synthetic oligonucleotides to polystyrene microtiter wells for hybridization capture,” Biotechniques, 8(3):276, 279, 1990. [0129] Pease et al., “Light-generated oligonucleotide arrays for rapid DNA sequence analysis,” Proc. Natl. Acad. Sci. USA, 91(11):5022-6, 1994. [0130] Peyret et al., “Nearest-neighbor thermodynamics and NMR of DNA sequences with internal A.A, C.C, G.G, and T.T mismatches,” Biochemstry, 38(12):3468-77, 1999. [0131] International (PCT) Publication Nos. WO 93/17126; and WO 97/31256