Temperature-selectable fret cassette signaling

Abstract

To provide a method for detecting the presence of multiple specific nucleic acid sequences or single nucleotide polymorphisms in a temperature-dependent fashion.SOLUTION: Provided is a method, in which an invasive oligonucleotide and a primary probe oligo (a 5' flap oligonucleotide) hybridize to a target strand nucleic acid to form an invasive cleavage structure cleavable by a FEN-1 endonuclease to release a cleaved 5' flap (primary cleaved flap); in a secondary reaction, the primary cleaved flap from the primary reaction hybridizes to a FRET cassette 1, a hairpin oligonucleotide labeled with a fluorescent dye and a quencher molecule, to form a second invasive cleavage structure that is cleavable by a FEN-1 endonuclease at a site between the fluorophore and a quencher; and cleavage of FRET cassette 1 separates the quencher from the fluorophore, such that fluorescence signal from the fluorophore can be detected.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit under 35 U.S.C. § 119(e) of U.S. Provisional Application No. 63 / 250,894, filed September 30, 2021. The entire disclosure of this prior application is incorporated herein by reference.

[0002] Sequence Listing The text of the computer readable sequence listing submitted herewith, entitled "DIA0105-PCT_SEQUENCE_LISTING", created on April 16, 2022, with a file size of 27,886 bytes, is hereby incorporated by reference in its entirety.

[0003] Technical Field The present disclosure relates generally to the field of biotechnology. More specifically, the present disclosure relates to compositions, methods, kits, and systems for detecting and distinguishing different analyte nucleic acids using an invasive cleavage reaction and a single fluorescent detection channel. [Background technology]

[0004] background Nucleic acid quantification plays an important role in the fields of biology and medicine, for example, it is used in cancer diagnosis and prognosis, as well as in the diagnosis and monitoring of infectious diseases caused by bacterial, fungal and viral pathogens.

[0005] There is great value in detecting multiple nucleic acid analytes in a single reaction mixture. Indeed, so-called "multiplexed" detection significantly increases the value of nucleic acid assays while reducing the associated reagent costs. However, different assay formats accommodate multiplexing capabilities to different extents, which means that practical trade-offs exist. For example, next-generation sequencing techniques allow for the acquisition of vast amounts of information, but the techniques are technically very challenging and generally require highly specialized equipment. Another technique, called "invasive cleavage assays," allows for nucleic acid sequence detection down to the level of single nucleotide differences ("SNPs") and is already used in conjunction with several multiplex assay formats.

[0006] For example, Hall et al., in U.S. Pat. No. 5,994,069, described two multiplexing approaches useful in the context of invasive cleavage detection methods. First, the presence of specific target sequences (or an internal control) can be designed to trigger different cascades coupled to different detectable moieties, such as different dyes in a fluorescent energy transfer format. The contribution of each specific target sequence to the final product can be tallied, allowing for quantitative detection of different nucleic acid sequences in a mixture of nucleic acid sequences. In the second configuration, it is desirable to determine whether any of several analytes are present in a sample, but it is not necessary to know the exact identity of each. For example, in a blood bank, it is desirable to know whether any one of a host of infectious agents is present in a blood sample. Since blood is discarded regardless of which agents are present, the different signals produced by different probes are not needed in such applications, and may in fact be undesirable for confidentiality reasons. Thus, a single detectable label can be used when it is unnecessary or undesirable to distinguish between analytes.

[0007] Elsewhere, Peterson et al., in published US Patent Application No. 2018 / 0163259 A1, describe the detection of different nucleic acid analytes by performing secondary invasive cleavage reactions at different temperatures and for different times, where a first secondary invasive cleavage reaction is completed by the time a second secondary invasive cleavage reaction begins. By performing the reactions at different temperatures and for different times, it is possible to distinguish between nucleic acid analytes in a single multiplex reaction.

[0008] Using a different assay format, Kozlov et al. teach in US Pat. No. 11,034,997 how to perform multiplex real-time PCR for the detection and quantification of target nucleic acids using tagged hydrolysis probes. A single probe cleavage event occurs in each cycle of the PCR reaction, as a polymerase with 5' to 3' exonuclease activity cleaves the tag portion and hydrolyzes the remaining portion of the probe. If the tag has a fluorophore separate from the quencher on the annealing portion of the probe, a fluorescent signal can be generated. The fluorescent label associated with the oligonucleotide probe hybridized to the target nucleic acid can be cleaved from the target-complementary portion of the probe during one cycle of the primer extension (i.e., polymerization) reaction and then decomposed. As a result, the increase in signal intensity is dependent on the performance of additional cycles of the PCR reaction. Similarly, Kozlov et al. teach that a "quenching molecule" (e.g., an oligo) hybridizes to the tag portion of the uncleaved hydrolysis probe at the temperature used to extend the primer in the primer extension reaction of the PCR. Prior to the primer extension cycle in the PCR reaction, fluorescence is quenched by the annealing portion of the probe and a quencher moiety linked to the quenching molecule. Despite the availability of existing nucleic acid multiplex detection platforms, there remains a need for additional approaches that can be easily adapted to automated testing platforms. More specifically, there is a need to maximize the detection capabilities of deployed testing instruments without requiring hardware modifications. [Prior art documents] [Patent documents]

[0009] [Patent Document 1] U.S. Pat. No. 5,994,069 [Patent Document 2] US Patent Application Publication No. 2018 / 0163259 [Patent Document 3] U.S. Pat. No. 11,034,997 Summary of the Invention [Means for solving the problem]

[0010] overview The following numbered embodiments are provided herein:

[0011] Embodiment 1 is a composition comprising a FRET cassette reporter system, the composition comprising: (i) a 5' flap FRET cassette oligonucleotide, the 5' flap portion comprising a first fluorophore moiety, a stem loop portion comprising a first quencher moiety, and a 3' portion comprising a cleaved flap hybridizing sequence, wherein hybridization of a cassette-specific invasive oligonucleotide complementary to the cleaved flap hybridizing sequence of the 5' flap FRET cassette oligonucleotide forms an invasive cleavage structure cleavable by FEN-1 endonuclease at a cleavage site between the first fluorophore moiety and the first quencher moiety, and hybridization of the 5' flap FRET cassette oligonucleotide at the cleavage site forms an invasive cleavage structure cleavable by FEN-1 endonuclease at the cleavage site. and (ii) a masking oligonucleotide comprising a second quencher moiety, wherein at least a portion of the masking oligonucleotide is capable of specifically hybridizing to the 5' flap portion of the FRET cassette oligonucleotide, and wherein hybridization of the masking oligonucleotide to the cassette cleavage flap forms a duplex having a first melting temperature exhibiting a first melting peak, and wherein fluorescent emission from the first fluorophore moiety in the duplex is quenched by the second quencher moiety.

[0012] Embodiment 2 is the composition of embodiment 1, wherein the first quencher moiety and the second quencher moiety are the same as one another.

[0013] Embodiment 3 is the composition of either embodiment 1 or embodiment 2, further comprising a FEN-1 endonuclease.

[0014] Embodiment 4 is the composition of embodiment 3, wherein the FEN-1 endonuclease is a thermostable FEN-1 endonuclease.

[0015] Embodiment 5 is the composition of embodiment 4, wherein the thermostable FEN-1 endonuclease is from an archaeal organism.

[0016] Embodiment 6 is the composition of any one of embodiments 1-5, further comprising a first target-specific invasive oligonucleotide and a first target-specific primary probe oligonucleotide, each of the first target-specific invasive oligonucleotide and the first target-specific primary probe oligonucleotide comprising a sequence configured to hybridize to a target nucleic acid to form an invasive cleavage structure cleavable by FEN-1 endonuclease to produce a primary cleavage flap, the primary cleavage flap being a cassette-specific invasive oligonucleotide configured to hybridize to a cleaved flap hybridizing sequence of the 5' flap FRET cassette oligonucleotide to form an invasive cleavage structure cleavable by FEN-1 endonuclease.

[0017] Embodiment 7 is the composition of embodiment 6, further comprising a target nucleic acid.

[0018] Example 8 is the composition of Example 7, further comprising deoxynucleoside triphosphates (dNTPs), a thermostable DNA polymerase, and a primer having a 3' end extendable by the thermostable DNA polymerase using the target nucleic acid as a template in a template-dependent nucleic acid amplification reaction.

[0019] Embodiment 9 is the composition of any one of embodiments 1-8, further comprising: (iii) a second FRET cassette oligonucleotide, the 5' portion comprising the second fluorophore moiety, a stem-loop portion comprising a third quencher moiety, and a 3' portion comprising a second cleaved flap hybridizing sequence, wherein hybridization of the second cassette-specific invasive oligonucleotide to the second cleaved flap hybridizing sequence of the second FRET cassette oligonucleotide forms an invasive cleavage structure cleavable by FEN-1 endonuclease at a cleavage site between the second fluorophore moiety and the third quencher moiety, and cleavage of the second FRET cassette oligonucleotide at the cleavage site produces a cassette cleavage product comprising the second fluorophore moiety.

[0020] Embodiment 10 is the composition of embodiment 9, wherein the second FRET cassette oligonucleotide is a second 5' flap FRET cassette comprising a 5' flap portion, and the cassette cleavage product is a second cassette cleavage flap comprising a second fluorophore, and the composition further comprises: (iv) a second masking oligonucleotide comprising a fourth quencher moiety, at least a portion of the second masking oligonucleotide is specifically hybridizable to the 5' flap portion of the second FRET cassette oligonucleotide, and hybridization of the second masking oligonucleotide to the second cassette cleavage flap forms a second duplex having a second melting temperature higher than the first melting temperature, and fluorescent emission from the second fluorophore moiety in the second duplex is quenched by the fourth quencher moiety.

[0021] Embodiment 11 is the composition of embodiment 9, wherein the second cassette cleavage product comprises no more than 5, preferably no more than 4, preferably no more than 3, preferably no more than 2 nucleotides.

[0022] Embodiment 12 is the composition of any one of embodiments 9-11, wherein the emission signals from the first fluorophore moiety and the second fluorophore moiety are detectable in the same fluorescence detection channel of a fluorescence monitoring device.

[0023] Embodiment 13 is the composition of any one of embodiments 9-12, wherein the first fluorophore moiety and the second fluorophore moiety are the same as one another.

[0024] Embodiment 14 is the composition of any one of embodiments 9-12, wherein the first fluorophore moiety and the second fluorophore moiety are not the same as one another.

[0025] Embodiment 15 is the composition of any one of embodiments 10 and 12-14, wherein the third quencher moiety and the fourth quencher moiety are the same as one another.

[0026] Embodiment 16 is a method of determining which of two different FRET cassettes in a reaction mixture has been cleaved to generate a fluorescent signal, the method comprising: (a) performing a multiple invasive cleavage reaction in the reaction mixture to cleave one or both of a first FRET cassette and a second FRET cassette to produce two different fluorescent cleavage products if cleavage occurs, the first FRET cassette comprising a first 5' flap portion having a fluorophore attached thereto, the attachment of the fluorophore being arranged such that cleavage of the first FRET cassette by a FEN-1 endonuclease in the multiple invasive cleavage reaction produces a first cassette cleavage flap comprising the fluorophore, and the reaction mixture is stably hybridized to the first cassette cleavage flap to produce a first 5' flap comprising the fluorophore. wherein the first masking oligonucleotide forms a first duplex at a temperature below the Tm of the first cassette but not above the first Tm, and wherein fluorescence emission from a fluorophore of a first cassette cleavage flap of the first duplex is quenched, and each of two different fluorescent cleavage products produced in the multiple invasive cleavage reaction is characterized by a different temperature-dependent fluorescence quenching profile in the reaction mixture; (b) measuring the fluorescent signals produced in the reaction mixture using a single channel of a fluorescence monitoring device under temperature conditions that differentially quench the fluorescence produced by the different fluorescent cleavage products of the multiple invasive cleavage reaction; and (c) determining which of the different FRET cassettes was cleaved in the multiple invasive cleavage reaction from the result of step (b).

[0027] Embodiment 17 is the method of embodiment 16, wherein the second FRET cassette in step (a) produces a fluorescent cleavage product, when cleaved, that does not hybridize to any masking oligo in the reaction mixture resulting in fluorescence quenching.

[0028] Embodiment 18 is the method of embodiment 17, wherein step (c) comprises comparing the fluorescent signals measured at a temperature below the first Tm and above the first Tm.

[0029] Embodiment 19 is the method of embodiment 18, wherein step (c) comprises comparing the fluorescent signals by calculating the difference between the measured fluorescent signals.

[0030] Embodiment 20 is the method of embodiment 17, wherein step (b) comprises measuring any of the fluorescent signals at a temperature below the first Tm, where the fluorescent emission from the fluorophore of the first cassette cleavage flap of the first duplex is quenched, and step (c) comprises determining that the second FRET cassette is cleaved in the reaction mixture if measurable fluorescence is detected in step (b).

[0031] Embodiment 21 is the method of embodiment 17, wherein step (b) comprises measuring any of the fluorescent signals at each of a temperature below the first Tm and a temperature above the first Tm, and step (c) comprises determining that the first FRET cassette is cleaved in the reaction mixture if the fluorescent signal measured at the temperature above the first Tm is greater than the fluorescent signal measured at the temperature below the first Tm.

[0032] Embodiment 22 is the method of any one of embodiments 16 to 21, wherein step (b) comprises measuring any of the fluorescent signals produced in the reaction mixture as a function of temperature to generate a melting / annealing curve.

[0033] Embodiment 23 is the method of embodiment 22, wherein step (c) comprises calculating a derivative of the melting / annealing curve and then determining from the calculated derivative whether the reaction mixture contains a first duplex characterized by a first Tm as an indication that the first FRET cassette has been cleaved in the reaction mixture.

[0034] Embodiment 24 is the method of embodiment 16, wherein the second FRET cassette comprises a second 5' flap sequence having a fluorophore attached thereto, the attachment of the fluorophore being arranged such that cleavage of the second FRET cassette by the FEN-1 endonuclease in a multiple invasive cleavage reaction produces a second cassette cleavage flap comprising the fluorophore, the reaction mixture comprises a second masking oligo that hybridizes to the second cassette cleavage flap to form a second duplex at a temperature below the second Tm but does not hybridize at a temperature above the second Tm, and wherein fluorescence emission from the fluorophore of the second cassette cleavage flap of the second duplex is quenched, and the first Tm and the second Tm differ by at least 5°C.

[0035] Embodiment 25 is the method of embodiment 24, wherein the first Tm is greater than the second Tm, step (b) comprises measuring either the fluorescent signal at a temperature lower than the second Tm and at a temperature higher than the first Tm, and step (c) comprises determining that at least one of the first FRET cassette and the second FRET cassette is cleaved in the reaction mixture if the fluorescent signal measured at the temperature higher than the first Tm is greater than the fluorescent signal measured at a temperature lower than the second Tm.

[0036] Embodiment 26 is the method of any of embodiments 24 or 25, wherein step (b) comprises measuring any of the fluorescent signals produced in the reaction mixture as a function of temperature to generate a melting / annealing curve.

[0037] Embodiment 27 is the method of embodiment 26, wherein step (c) comprises calculating a derivative of the melting / annealing curve and then determining from the calculated derivative whether the reaction mixture contains a first duplex characterized by a first Tm as an indication that the first FRET cassette has been cleaved in the reaction mixture.

[0038] Embodiment 28 is the method of embodiment 26, wherein step (c) comprises calculating a derivative of the melting / annealing curve and then determining from the calculated derivative whether the reaction mixture contains a second duplex characterized by a second Tm as an indication that the second FRET cassette has been cleaved in the reaction mixture.

[0039] Embodiment 29 is the method of any one of embodiments 16-28, wherein the first and second FRET cassettes are labeled with the same fluorophore.

[0040] Embodiment 30 is the method of any one of embodiments 16-28, wherein the first and second FRET cassettes are not labeled with the same fluorophore.

[0041] Embodiment 31 is the method of any one of embodiments 16 to 30, wherein the FEN-1 endonuclease of the multiple invasive cleavage reaction in step (a) comprises a thermostable FEN-1 endonuclease.

[0042] Embodiment 32 is the method of any one of embodiments 16-31, wherein step (c) comprises determining using a computer programmed with software.

[0043]

[0036] Embodiment 33 is a method of analyzing a sample containing a target nucleic acid, comprising: (a) contacting in a reaction mixture any of a first target nucleic acid of the sample with a first primary probe oligonucleotide comprising a sequence complementary thereto and a FEN-1 endonuclease under conditions such that if the first primary probe oligonucleotide hybridizes to the first target nucleic acid, the first primary probe is cleaved by the FEN-1 endonuclease to generate a first primary cleavage flap, wherein the first primary cleavage flap hybridizes to a cleaved flap hybridizing sequence of a first FRET cassette oligonucleotide contained in the reaction mixture and is cleaved by the FEN-1 endonuclease at a cleavage site between a first fluorophore moiety and a first quencher moiety of the first FRET cassette oligonucleotide to form an invasive cleavage structure that releases a first cassette cleavage flap comprising a first fluorophore moiety. (b) hybridizing a first masking oligonucleotide comprising a second quencher moiety to the first cassette cleavage flap to form a duplex at a temperature lower than a first Tm of the first masking oligonucleotide and the first cassette cleavage flap, wherein fluorescent emission from the first fluorophore moiety of the duplex is quenched by the second quencher moiety, and at a second temperature higher than the first Tm, the first masking oligonucleotide and the first cassette cleavage flap do not form a stable duplex; (b) detecting any fluorescence emitted from the first fluorophore moiety at the second temperature; and (c) determining whether the sample contains the first target nucleic acid if fluorescence emitted from the first fluorophore moiety is detected in step (b), or the sample does not contain the first target nucleic acid if fluorescence emitted from the first fluorophore moiety is not detected in step (b).

[0044] Embodiment 34 further comprises contacting any of the second target nucleic acids of the sample in the reaction mixture with a second primary probe oligonucleotide comprising a sequence complementary thereto and a FEN-1 endonuclease under conditions such that if the second primary probe oligonucleotide hybridizes to the second target nucleic acid, the second primary probe is cleaved by the FEN-1 endonuclease to generate a second primary cleavage flap different from the first primary cleavage flap, the second primary cleavage flap hybridizing to a cleaved flap hybridizing sequence of a second FRET cassette oligonucleotide contained in the reaction mixture and cleaved by the FEN-1 endonuclease at a cleavage site between the second fluorophore moiety and a third quencher moiety of the second FRET cassette oligonucleotide to form an invasive cleavage structure releasing a second cassette cleavage flap comprising a second fluorophore moiety, and at a third temperature lower than the second Tm, a second masking oligonucleotide comprising a fourth quencher moiety. 34. The method of embodiment 33, wherein the second masking oligonucleotide hybridizes to the second cassette cleavage flap to form a duplex, and fluorescent emission from the second fluorophore moiety of the duplex is quenched by the fourth quencher moiety, and at a fourth temperature higher than the second Tm, the second masking oligonucleotide and the second cassette cleavage flap do not form a stable duplex, and the first Tm and the second Tm differ from each other by at least 5°C, step (b) further comprises detecting any fluorescence emitted from the second fluorophore moiety at the fourth temperature, and step (c) further comprises determining whether the sample contains the second target nucleic acid if fluorescence emitted from the second fluorophore moiety of the 5' flap cleavage product of the second FRET cassette oligonucleotide is detected in step (b), or the sample does not contain the first target nucleic acid if fluorescence emitted from the second fluorophore moiety of the 5' flap cleavage product of the second FRET cassette oligonucleotide is not detected in step (b).

[0045] Embodiment 35 further comprises contacting any of the second target nucleic acids of the sample in the reaction mixture with a second primary probe oligonucleotide comprising a sequence complementary thereto and a FEN-1 endonuclease under conditions such that if the second primary probe oligonucleotide hybridizes to the second target nucleic acid, the second primary probe is cleaved by the FEN-1 endonuclease to generate a second primary cleavage flap, the second primary cleavage flap hybridizing to a cleaved flap hybridizing sequence of a second FRET cassette oligonucleotide contained in the reaction mixture to form a cleavage flap between the second fluorophore moiety and the third quencher moiety of the second FRET cassette oligonucleotide. 34. The method of embodiment 33, wherein the first target nucleic acid is cleaved by the FEN-1 endonuclease at a site to form an invasive cleavage structure that releases a cleavage product comprising a second fluorophore moiety, and the cleavage product does not hybridize to any masking oligonucleotide in the reaction mixture resulting in fluorescence quenching, step (b) further comprises detecting any fluorescence emitted from the second fluorophore moiety of the cleavage product, and step (c) further comprises determining whether the sample contains the second target nucleic acid if fluorescence emitted from the second fluorophore moiety is detected in step (b), or the sample does not contain the first target nucleic acid if fluorescence emitted from the second fluorophore moiety is not detected in step (b).

[0046] Embodiment 36 is the method of either embodiment 34 or embodiment 35, wherein step (b) comprises detecting any fluorescence emitted from the first and second fluorophore moieties using a single channel of a fluorescence monitoring device.

[0047] Embodiment 37 is the method of embodiment 36, wherein step (b) is performed while a nucleic acid amplification reaction is occurring in the reaction mixture, and the product of the nucleic acid amplification reaction comprises the first target nucleic acid and the second target nucleic acid.

[0048] Embodiment 38 is the method of embodiment 37, wherein the nucleic acid amplification reaction includes a step for thermal cycling and the reaction mixture further comprises a thermostable DNA polymerase.

[0049] Embodiment 39 is the method of any one of embodiments 33 to 36, wherein step (b) is performed when the temperature of the reaction mixture is reduced to allow annealing of the masking oligonucleotide and the complementary cassette cleavage flap.

[0050] Embodiment 40 is the method of 36, wherein the first and second fluorophore moieties are the same as one another.

[0051] Embodiment 41 is the method of embodiment 36, wherein the step of (b) detecting any fluorescence comprises measuring any fluorescence.

[0052] Embodiment 42 is the method of embodiment 41, further comprising any step of detecting or measuring fluorescence at the first temperature.

[0053] Embodiment 43 is the method of any one of embodiments 34 or 35, wherein both the first fluorophore moiety and the second fluorophore moiety are detectable in the same fluorescence detection channel of the energy sensor device.

[0054] Embodiment 44 is the method of embodiment 43, wherein the second fluorophore moiety is the same as the first fluorophore moiety.

[0055] Embodiment 45 is the method of embodiment 43, wherein the second fluorophore moiety is not the same as the first fluorophore moiety.

[0056] Embodiment 46 is the method of any one of embodiments 33-43, wherein fluorescence from the second fluorophore is detected and / or measured at the first temperature.

[0057] Embodiment 47 is the method of any one of embodiments 33-44, wherein the third quencher moiety is the same as the first quencher moiety and / or the second quencher moiety.

[0058] Embodiment 48 is the method of any one of embodiments 33 to 47, wherein the reaction mixture comprises primer oligonucleotides that amplify a target nucleic acid, and at least one primer oligonucleotide acts as an invasive oligonucleotide in the presence of the primary probe oligonucleotide and the target nucleic acid and / or target amplicon to form an invasive cleavage structure that is cleaved by the thermostable FEN-1 endonuclease.

[0059] Embodiment 49 is the method of any one of embodiments 33-48, wherein step (c) comprises determining using a computer programmed with software.

[0060] Embodiment 50 is a system for determining which of a plurality of target nucleic acid analytes are present in a reaction mixture, each of the plurality of target nucleic acid analytes being detectable by a fluorescent signal, the system comprising: a thermocycler; a fluorometer in optical communication with the thermocycler, the fluorometer measuring a fluorescent signal indicative of production of a nucleic acid amplification product by the thermocycler in a single optical channel; and a computer in communication with the fluorometer, the system causing the computer to (a) obtain a melting / annealing curve dataset generated from measurements made by the fluorometer; and (b) measure in the melting / annealing curve dataset a fluorescent signal indicative of production of a nucleic acid amplification product by the reaction mixture. (c) determining the presence of a first target nucleic acid in the reaction mixture by detecting a fluorescent signal from a first fluorescent cleavage product at a temperature at which fluorescence in the reaction mixture is maximally quenched; (d) determining the presence of a second target nucleic acid in the reaction mixture if the derivative plot contains a feature characteristic of the first duplex, wherein the first duplex comprises a first masking oligonucleotide and a second fluorescent cleavage product that is produced in the reaction mixture when the second target nucleic acid is present.

[0061] Embodiment 51 is the system of embodiment 50, wherein the computer is programmed with software instructions to cause the computer to determine that a third target nucleic acid is present in the reaction mixture if (e) the derivative plot comprises a feature characteristic of a second duplex, the second duplex comprising a second masking oligonucleotide and a third fluorescent cleavage product that is produced in the reaction mixture when the third target nucleic acid is present.

[0062] Embodiment 52 is the system of embodiment 50 or embodiment 51, wherein the characteristic feature of the first duplex comprises a maximum, a minimum, or a zero crossing point of the calculated derivative.

[0063] Embodiment 53 is the system of embodiment 52, wherein the calculated derivative comprises a calculated first derivative, the feature characteristic of the first duplex comprises a first maximum of the calculated first derivative of the melt / annealing curve dataset as a first melt peak, and the feature characteristic of the second duplex comprises a second maximum of the calculated first derivative of the melt / annealing curve dataset as a second melt peak.

[0064] Embodiment 54 is a system of any one of embodiments 50 to 53, wherein the thermocycler, the fluorometer, and the computer are all components of a real-time PCR instrument.

[0065] Embodiment 55 is the system of embodiment 50, wherein the computer-generated melting / annealing curve data set comprises data points showing fluorescence as a function of temperature. [Brief description of the drawings]

[0066] [Figure 1]FIG. 1 provides a schematic of an assay that employs two invasive cleavage reactions performed sequentially in the same reaction mixture. In the primary reaction, an "invasive oligonucleotide" (SEQ ID NO: 1) and a "primary probe oligo" (SEQ ID NO: 2) (i.e., a 5' flap oligonucleotide) hybridize to a "target strand" nucleic acid (SEQ ID NO: 3) to form an invasive cleavage structure cleavable by FEN-1 endonuclease to release a cleaved 5' flap ("primary cleavage flap") (SEQ ID NO: 4). In the secondary reaction, the primary cleavage flap from the primary reaction hybridizes to a FRET cassette ("FRET cassette 1") (SEQ ID NO: 5), a hairpin oligonucleotide labeled with a fluorescent dye and a quencher molecule, to form a second invasive cleavage structure cleavable by FEN-1 endonuclease at a site between the fluorophore (shown as "HEX") and the quencher ("Q"). Cleavage of "FRET cassette 1" (SEQ ID NO: 5) produces "truncated FRET cassette 1" (SEQ ID NO: 6), separating the quencher from the fluorophore so that fluorescent signal from the fluorophore can be detected. Each primary cleavage flap can hybridize to a series of new uncleaved FRET cassettes to form further fluorescent cleavage products.

[0067] [Diagram 2] Figure 2 is a series of schematic diagrams showing the structure and use of 5' flap FRET cassettes. Figure 2A shows the structure of an example "5' flap FRET cassette" (SEQ ID NO: 7). Figure 2B shows an example "5' flap FRET cassette 1" (SEQ ID NO: 7) hybridized to the "flap cleaved from primary probe" (SEQ ID NO: 8) (i.e., the primary cleaved flap functions as an invasive oligonucleotide). Figure 2C shows the temperature-dependent interaction between the "cassette cleaved flap" (SEQ ID NO: 9) (i.e., the 5' flap cleaved from the 5' flap FRET cassette) and the complementary "masking oligo" (SEQ ID NO: 10).

[0068] [Diagram 3]FIG. 3 shows diagrammatically how the fluorescent signals produced by cleavage of three different FRET cassettes ("Flapless FRET Cassette" (SEQ ID NO: 11), "5' Flap FRET Cassette 1" (SEQ ID NO: 7), and "5' Flap FRET Cassette 2" (SEQ ID NO: 13)) can be distinguished by temperature-dependent quenching. "Flap cleaved from target 2 primary probe" (SEQ ID NO: 8) hybridizes to "5' Flap FRET Cassette 1" (SEQ ID NO: 7) to facilitate the enzyme-dependent cleavage reaction. "Cassette cleavage flap 1" (SEQ ID NO: 9), or the 5' flap cleaved from "5' Flap FRET Cassette 1" (SEQ ID NO: 7), can hybridize to "Masking Oligo 1" (SEQ ID NO: 10) to form a first hybrid duplex that exhibits quenched fluorescence. "Flap cleaved from target 3 primary probe" (SEQ ID NO: 14) hybridizes to "5' Flap FRET Cassette 2" (SEQ ID NO: 13) to facilitate the enzyme-dependent cleavage reaction. "Cassette Cleavage Flap 2" (SEQ ID NO: 15), or the 5' flap cleaved from "5' Flap FRET Cassette 2" (SEQ ID NO: 13), can hybridize to "Masking Oligo 2" (SEQ ID NO: 16) to form a second duplex that exhibits quenched fluorescence. The two hybrid duplexes are designed to have unique melting / annealing properties, which allows one to be distinguished from the other. The signal produced by cleavage of the "Flapless FRET Cassette" (SEQ ID NO: 11) remains detectable under all temperature conditions.

[0069] [Figure 4]FIG. 4 is a schematic diagram of cleavage products prepared using a flapless FRET cassette and two different 5′ flap FRET cassettes, where all three FRET cassettes are labeled with the same reporter dye (HEX). The different FRET cassettes allow for the detection of different nucleic acid target sequences using only a single fluorescent detection channel of a nucleic acid analyzer. In the illustrated embodiment, temperature 1 is lower than temperature 2 (e.g., temperature 1 can be 40° C. and temperature 2 can be 50° C.). Detection of target 1 is performed using a flapless FRET cassette (e.g., SEQ ID NO: 11 in FIG. 3) that does not have a corresponding masking oligonucleotide, so that the signal from target 1 is detectable at all temperatures. Detection of target 2 is performed using a 5′ flap FRET cassette (e.g., SEQ ID NO: 7 in FIG. 3) that produces “cassette cleavage flap 1” (SEQ ID NO: 9), which is masked by hybridization with “masking oligo 1” (SEQ ID NO: 10) when the reaction mixture is lower than temperature 1. Above temperature 1 but lower than temperature 2, signals reflecting the detection of both target 1 and target 2 are detectable. Detection of target 3 is performed using a 5' flap FRET cassette (e.g., SEQ ID NO: 13 in FIG. 3) that produces "Cassette Cleavage Flap 2" (SEQ ID NO: 15), which is masked by hybridization with "Masking Oligo 2" (SEQ ID NO: 16) when the reaction mixture is below temperature 2. Above temperature 2, signals from all three targets 1, 2, and 3 are detectable. In the illustrated embodiment, the temperatures selected for detection of cleavage of the different FRET cassettes are independent of the sequences of the three different target nucleic acids.

[0070] [Diagram 5] 5 provides a set of graphs showing fluorescence measured in the HEX channel as a function of cycle number. Reactions contain target analyte A (FIG. 5A), target analyte B (FIG. 5B), or target analytes A and B together (FIG. 5C), as described in Example 1.

[0071] [Figure 6]FIG. 6 is a graph showing melting curve analysis using fluorescence detected in the HEX channel for reactions containing target analyte A, target analyte B, and target analytes A and B together, as described in Example 2.

[0072] [Figure 7] 7A-7D provide a set of graphs showing the fluorescence measured in the HEX channel as a function of cycle number and temperature at which the fluorescence measurements were made. FIG. 7A and FIG. 7B show the results obtained for a reaction containing only target analyte B (using a 5' flap FRET cassette and a masking oligonucleotide that is quenched at low temperature) when the fluorescence was measured at 63° C. and 39° C., respectively. FIG. 7C and FIG. 7D show the results obtained for a reaction containing target analytes A and B together, where analyte A was detected using a flapless FRET cassette (without a 5' flap), producing a product that fluoresces at both temperatures, and the fluorescence was measured at 63° C. and 39° C., respectively. The procedure is described in Example 3.

[0073] [Figure 8] FIG. 8 is a graph showing melting curve analysis detected in the HEX channel for reactions containing target analyte A, target analyte B, target analyte C, and a combination of all three target analytes, as described in Example 4.

[0074] [Figure 9] 9 provides a graph showing the unique melting / annealing curve profiles observed for additional analyte combinations, as described in Example 4. The left panel of the figure shows the melting / annealing curve results for reactions that amplified either analyte B or analyte C. The right panel of the figure shows the melting / annealing curve analysis results for reactions that amplified analyte B alone, analyte C alone, or a combination of analytes B and C.

[0075] [Figure 10] FIG. 10 provides a first derivative plot of the measured fluorescence data shown in two panels of FIG. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0076] definition To facilitate the understanding of this disclosure, several terms and phrases are defined below. Further definitions are set forth throughout the detailed description. Throughout the specification and claims, the following terms shall take the meanings expressly associated therewith herein, unless the context clearly dictates otherwise.

[0077] As used herein, the phrase "in one embodiment" may refer to the same embodiment, but does not necessarily refer to the same embodiment. Additionally, as used herein, the phrase "in another embodiment" does not necessarily refer to a different embodiment, but may. Thus, as described below, various embodiments of the present technology can be readily combined without departing from the scope or spirit of the present technology.

[0078] The term "based on" is not exclusive and allows for based on additional factors not listed, unless the context clearly dictates otherwise. Additionally, throughout this specification, the meanings of "a," "an," and "the" include plural references. The meaning of "in" includes "in" and "on."

[0079] As used in the claims of this application, the transitional phrase "consisting essentially of" extends the claim to As discussed in Herz, 537 F.2d 549,551-52,190 USPQ 461,463 (CCPA 1976), limitations are placed on the specific materials or steps of the claimed invention "as well as those that do not substantially affect the basic and novel characteristic(s)." For example, a composition "consisting essentially of" recited elements may contain unrecited contaminants, which are present but at levels such that the contaminants do not alter the function of the recited composition compared to the pure composition (i.e., a composition "consisting of" the recited ingredients). As used herein, the term "sample" refers to a specimen that may contain an analyte of interest (e.g., nucleic acid, such as a microorganism, virus, gene, or a component thereof, including a nucleic acid sequence in or derived from the specimen). A sample may be from any source, such as a biological specimen or an environmental source. A biological specimen includes any tissue or material from a living or dead organism that may contain an analyte or nucleic acid in or derived from the specimen. Examples of biological samples include nasal swab samples, vaginal swab samples, respiratory tissue, exudates (e.g., bronchoalveolar lavage), biopsies, sputum, peripheral blood, plasma, serum, lymph nodes, gastrointestinal tissue, feces, urine, or other fluids, tissues, or materials. Examples of environmental samples include water, ice, soil, slurries, debris, biofilms, suspended particles, and aerosols. A sample may be a treated specimen or material, such as obtained from treating a sample by using filtration, centrifugation, sedimentation, or adhesion to a medium such as a matrix or support. Other treatments of a sample may include treatments that physically or mechanically disrupt tissues, cell aggregates, or cells to release intracellular components, including nucleic acids, into a solution that may contain other components, such as enzymes, buffers, salts, detergents, etc. A sample being tested for the presence of an analyte may be referred to as a "test sample."

[0080] The terms "target nucleic acid" and "target sequence" refer to the nucleic acid to be detected or analyzed. Thus, the "target" nucleic acid is required to be distinguished from other nucleic acids or nucleic acid sequences. For example, when used in relation to an amplification reaction, these terms may refer to the nucleic acid or part of the nucleic acid that is amplified by the reaction, and when used in relation to a polymorphism, they may refer to the locus in the nucleic acid of the suspected polymorphism. When used in relation to an invasive cleavage reaction, these terms typically refer to a nucleic acid molecule that includes a sequence that selectively hybridizes to a first nucleic acid molecule (e.g., a probe oligonucleotide) and a second nucleic acid molecule (an invasive oligonucleotide) to form an overlapping invasive cleavage structure. In general, a target nucleic acid (e.g., present in a sample, isolated from a sample, enriched from a sample, or amplified from or within a sample) is located within a target region and is identifiable by the successful formation of an invasive cleavage structure in combination with a first and second nucleic acid molecule (e.g., a probe oligonucleotide and an invasive oligonucleotide) that can be cleaved by a cleavage agent. Target nucleic acids from organisms are not limited to genomic DNA and RNA. Target nucleic acids from organisms can include any nucleic acid species, including but not limited to genomic DNA and RNA, messenger RNA, structural RNA, ribosomal and tRNA, and small RNA such as snRNA, siRNA and microRNA (miRNA).See, for example, U.S. Patent No. 7,851,150, which is incorporated herein by reference in its entirety.A "segment" is defined as a region of nucleic acid within a target sequence.

[0081] Since mononucleotides react to make oligonucleotides such that the 5' phosphate attached to one mononucleotide pentose ring is attached unidirectionally to the 3' oxygen of its neighbor via a phosphodiester bond, the terminus of an oligonucleotide is called the "5' end" if its 5' phosphate is not linked to the 3' oxygen of a mononucleotide pentose ring, and the terminus of an oligonucleotide is called the "3' end" if its 3' oxygen is not linked to the 5' phosphate of the next mononucleotide pentose ring. As used herein, a nucleic acid sequence may also be said to have a 5' end and a 3' end, even if it is internal to a larger oligonucleotide. A first region along a nucleic acid strand is said to be upstream of another region if, moving in the 5' to 3' direction along the strand of the nucleic acid, the 3' end of the first region precedes the 5' end of the second region.

[0082] As used herein, the term "5' end" refers to a portion of a nucleic acid having a 5' end (i.e., a 5' end where the 5' phosphate is not linked to the 3' oxygen of a mononucleotide pentose ring). The term "3' end" refers to a portion of a nucleic acid having a 3' end (i.e., a 3' end where the 3' oxygen is not linked to the 5' phosphate of a subsequent mononucleotide pentose ring).

[0083] As used herein, the terms "hybridization" or "hybridize" (and grammatical equivalents) are used in reference to the pairing of complementary nucleic acids. Hybridization and the strength of hybridization (i.e., the strength of the association between the nucleic acid strands involved in a duplex) depend on the degree of complementarity between the nucleic acids, the stringency of the conditions involved, the T of the hybrid formed, and the degree of complementarity between the nucleic acids. m and the G:C ratio within the nucleic acid.

[0084] When two different non-overlapping oligonucleotides anneal to different regions of the same linear complementary nucleic acid, with the 3' end of one oligonucleotide adjacent to the 5' end of the other, the former can be called the "upstream" oligonucleotide and the latter can be called the "downstream" oligonucleotide. Similarly, when two overlapping oligonucleotides hybridize to the same linear complementary nucleic acid, with the first oligonucleotide positioned such that its 5' end is upstream of the 5' end of the second oligonucleotide and the 3' end of the first oligonucleotide is upstream of the 3' end of the second oligonucleotide, the first oligonucleotide can be called the "upstream" oligonucleotide and the second oligonucleotide can be called the "downstream" oligonucleotide.

[0085] As used herein, the term "Tm" is used in reference to the "melting temperature" of a nucleic acid strand relative to a complementary nucleic acid strand. The melting temperature of a nucleic acid duplex is the temperature at which a population of double-stranded nucleic acid molecules becomes half-dissociated into single strands. Measuring the melting temperature of a labeled nucleic acid duplex typically involves plotting fluorescence as a function of temperature to generate a melting curve characteristic of the dissociation of the duplex. Graphing the negative first derivative of the melting curve as a function of temperature gives the T m are identifiable as peaks. See, e.g., KM Ririe et al., Analytical Biochemistry 245:154-160 (1997).

[0086] "Calculated T m " refers to the melting temperature that is determined by calculation from the physical sequence of complementary nucleic acids together with factors of the reaction conditions (e.g., salt concentration, concentration of complementary strands in the mixture). m Several formulas for calculating T are well known in the art. As indicated by standard references, mA simple estimate of the value, when the nucleic acid is in an aqueous solution of 1M NaCl (see, e.g., Young and Anderson, (1985) in Nucleic Acid Hybridisation: A Practical Approach (Hames & Higgins, Eds.) pp 47-71, IRL Press, Oxford), is given by the formula: T m = 81.5 + 0.41 (% G + C). m Other calculations for calculating are known in the art and take into account structural and environmental as well as sequence characteristics (see, e.g., Allawi, HT and SantaLucia, J., Jr. Biochemistry 36, 10581-94 (1997)); and SantaLucia, Proc Natl Acad Sci US A., 95(4):1460 (1998)).

[0087] As used herein, the term "cycling hybridization" refers to the process of changing the T of a hybridized nucleic acid strand such that an oligonucleotide is constantly annealed to and dissociated from a target strand without temperature cycling (i.e., the probe-target nucleic acid duplex is alternately melted and annealed without shifting the temperature of the reaction mixture). m It refers to incubation of a reaction mixture containing nucleic acids (e.g., probe oligonucleotides and their complementary target nucleic acids) at or near (e.g., within 4°C, more preferably within 3°C, even more preferably within 2°C, and even more preferably within 1°C) the temperature.

[0088] As used herein, an "invasive cleavage assay" is a procedure for detecting or quantifying a target nucleic acid by enzymatic cleavage of one or more different invasive cleavage structures, where at least one cleavage structure comprises a FRET cassette. In a preferred embodiment, an invasive cleavage assay combines two invasive signal amplification reactions (e.g., a "primary reaction" and a "secondary reaction") in series in a single reaction mixture. Reagents for an invasive cleavage assay may include a structure-specific 5' nuclease (e.g., FEN-1 endonuclease), an "invasive oligonucleotide", a "primary probe" and a "FRET cassette".

[0089] As used herein, the term "INVADER assay" refers to the structure-specific flap endonuclease cleavage assay (Hologic, Inc.), and is described, e.g., in U.S. Pat. Nos. 5,846,717; 5,985,557; 5,994,069; 6,001,567; 6,090,543; 6,872,816; 7,935,800; 9,133, 503; 9,096,893; Lyamichev et al., Nat. Biotech., 17:292 (1999); Hall et al., Proc. Natl. Acad. Sci. USA, 97:8272 (2000); Allawi et al., RNA (2004), 10:1153-1161 (2004), each of which is incorporated by reference in its entirety for all purposes.

[0090] As used herein, the term "invasive cleavage structure" (sometimes simply "cleavage structure") refers to an overlapping nucleic acid duplex structure that is a substrate for cleavage by a flap endonuclease (e.g., FEN-1 endonuclease). The cleavage reaction catalyzed by the enzyme does not require any extension of the nucleic acid strand. In some embodiments, the invasive cleavage structure includes (i) a continuous nucleic acid strand (e.g., a target DNA or a target RNA); (ii) an upstream nucleic acid that hybridizes to a first portion of the target strand to form an upstream duplex (e.g., an invasive oligonucleotide, sometimes referred to as an INVADER oligonucleotide); and (iii) a downstream nucleic acid that hybridizes to form a downstream duplex (e.g., a 5' flap probe, or a primary probe oligonucleotide with a 5' flap that is not complementary to the target strand). The upstream and downstream nucleic acids anneal to adjacent regions of the target nucleic acid, forming an overlap between the 3' portion of the upstream nucleic acid and the duplex formed between the downstream nucleic acid and the target nucleic acid. When one or more bases from the upstream and downstream nucleic acids occupy the same position relative to the target nucleic acid base, overlapping occurs regardless of whether the overlapping base(s) of the upstream nucleic acid are complementary to the target nucleic acid and whether the bases are natural or non-natural bases. In some embodiments, the 3' portion of the upstream nucleic acid that overlaps with the downstream duplex is a non-basic chemical moiety such as an aromatic ring structure (e.g., as disclosed in U.S. Patent No. 6,090,543, the entirety of which is incorporated herein by reference). In some embodiments, one or more of the nucleic acids can be attached to each other by a covalent linkage such as a nucleic acid stem-loop or by a non-nucleic acid chemical linkage (e.g., a multi-carbon chain). When the cleaved 5' flap hybridizes to the FRET cassette, an invasive cleavage structure is also created (e.g., the "target nucleic acid" and the "downstream nucleic acid" are covalently linked in a stem-loop configuration). The "target nucleic acid" sequence of the FRET cassette that hybridizes to the cleaved 5' flap can be referred to as the "cleaved flap hybridizing sequence."

[0091] In some embodiments, the target nucleic acid is amplified (e.g., by PCR) and the amplification product is detected using an invasive cleavage assay while the amplification reaction is occurring. Assays configured to perform a detection assay (e.g., an invasive cleavage assay) in combination with an amplification assay are described in U.S. Patent No. 9,096,893, which is incorporated herein by reference in its entirety for all purposes. In further embodiments, RNA target nucleic acid can be reverse transcribed, amplified and detected in a single reaction using an invasive cleavage assay, as described in International Publication No. WO 2006 / 050499, which is incorporated herein by reference in its entirety for all purposes.

[0092] As used herein, the term "probe oligonucleotide" refers to an oligonucleotide that interacts with a target nucleic acid to form a detectable complex. In some embodiments, the complex between the probe and the target is detected while it exists. In other embodiments, the formation of the complex can be detected when it no longer exists (e.g., by detecting an event, such as a cleavage event, that occurs as a result of the formation of the probe / target complex).

[0093] As used herein, the term "flap probe" refers to a probe oligonucleotide that includes a target-specific portion that specifically hybridizes to a target nucleic acid and a 5' flap portion that does not hybridize to the target nucleic acid. Typically, the 5' flap portion is not complementary to a region of the target nucleic acid adjacent to the duplex formed between the target nucleic acid and the target-specific portion of the flap probe.

[0094] As used herein with respect to the sequential invasive cleavage assay, a "primary probe" is a flap probe that includes a 3' sequence or portion that is complementary to the target nucleic acid to be detected and a 5' flap portion that is not complementary to the target nucleic acid (i.e., the non-complementary 5' flap portion does not hybridize to the target nucleic acid). The 5' flap portion is configured such that upon cleavage of the primary probe involved in the invasive cleavage structure in the "primary" reaction of the sequential invasive cleavage assay, the cleaved 5' flap released from the primary probe can hybridize to a FRET cassette to facilitate the secondary reaction of the sequential invasive cleavage assay.

[0095] As used herein, the term "primary reaction" generally refers to flap endonuclease cleavage of a primary probe, thereby generating a cleaved 5' flap. When a primary probe is cleaved with FEN-1 endonuclease, the sequence of the 5' flap cleaved from the primary probe typically comprises the 5' flap portion of the primary probe plus the first (5'-most) nucleotide of the target-specific portion of the primary probe.

[0096] As used herein, the term "secondary reaction" generally refers to hybridization of a cleaved 5' flap from a primary reaction to a FRET cassette to form a secondary invasive cleavage structure, and cleavage of the secondary invasive cleavage structure by a flap endonuclease to produce a detectable signal.

[0097] As used herein, a reaction is "active" if a reaction product is produced. For example, a secondary reaction is active when a reaction mixture containing the necessary components (e.g., including a FRET cassette, a cleaved 5' flap specific to the FRET cassette, and a FEN enzyme) is incubated at a temperature that allows cycling hybridization of the cleaved 5' flap to the FRET cassette in the reaction mixture, resulting in cleavage of the FRET cassette and separation of the donor (e.g., fluorophore) and acceptor (e.g., quencher) moieties.

[0098] The term "invasive oligonucleotide" (sometimes "INVADER oligonucleotide") refers to an oligonucleotide that hybridizes to a target nucleic acid at a position near the hybridization region between the probe and the target nucleic acid, and the invasive oligonucleotide includes a portion (e.g., a chemical moiety or a nucleotide, whether or not complementary to the target) that overlaps the hybridization region between the probe and the target. In some embodiments, the invasive oligonucleotide contains a sequence at its 3' end that is substantially the same as a sequence located at the 5' end of the probe oligonucleotide.

[0099] As used herein, the term "FRET" refers to fluorescence resonance energy transfer, a process in which chemical moieties (e.g., fluorophores) transfer energy between themselves or from a fluorophore to a non-fluorophore (e.g., a quencher molecule). In some situations, FRET involves an excited donor fluorophore transferring energy to a low-energy acceptor fluorophore via a short-range (e.g., about 10 nm or less) dipole-dipole interaction. In other situations, FRET involves a loss of fluorescence energy from the donor and an increase in fluorescence in the acceptor fluorophore. In yet other forms of FRET, energy can be exchanged from an excited donor fluorophore to a non-fluorescent molecule (e.g., a quencher molecule). FRET is known to those of skill in the art and has been described (see Stryer et al., 1978, Ann. Rev. Biochem., 47:819; Selvin, 1995, Methods Enzymol., 246:300; Orpana, 2004 Biomol Eng 21, 45-50; Olivier, 2005 Mutant Res 573, 103-110, each of which is incorporated herein by reference in its entirety).

[0100] As used herein, the term "FRET cassette" refers to an oligonucleotide that includes a stem-loop or hairpin structure (i.e., a region of nucleobases that are base-paired intramolecularly to form a double helical stem, with a loop of nucleotides connecting the strands of base pairs at one end of the stem) that includes a donor moiety (e.g., a "fluorophore") and a nearby acceptor moiety (e.g., a "quencher"), where attachment of the donor moiety and the acceptor moiety to the same FRET cassette substantially suppresses (e.g., quenches) detectable energy emission from the donor moiety (e.g., fluorescence emission). The FEN-1 enzyme catalyzes the hydrolytic cleavage of the phosphodiester bond 3' adjacent to the junction of single-stranded and double-stranded DNA, generally one nucleotide, to the 5' end of the stem-loop portion of the oligonucleotide, releasing a 5' nucleotide or 5' flap from the stem-loop portion of the FRET cassette. The fluorophore moiety is typically attached to the 5' end or 5' flap of the FRET cassette oligonucleotide, and the quencher moiety is typically attached to the stem-loop portion of the oligonucleotide.

[0101] A "cleaved flap hybridizing sequence" of a FRET cassette refers to a nucleotide base sequence in the 3' portion of a complementary nucleic acid or oligonucleotide, e.g., a 5' flap cleaved from a primary probe (the "primary cleavage flap"), such that when the flap cleavage product hybridizes to the FRET cassette, the 3' end of the complementary oligonucleotide is positioned to form an invasive cleavage structure (i.e., a substrate for a FEN enzyme) such that the cleavage site on the FRET cassette is located between the fluorophore and quencher moieties.

[0102] As used herein, the term "5' flap FRET cassette" refers to a FRET cassette oligonucleotide having a single-stranded 5' flap portion, a stem-loop portion, and a single-stranded 3' portion that includes a cleaved flap-hybridizing sequence, wherein the 5' flap portion and the cleaved flap-hybridizing sequence are not complementary to each other.

[0103] As used herein, the term "flapless FRET cassette" refers to a FRET cassette that does not have a single-stranded 5' portion, e.g., one or more non-complementary 5' nucleotides, and in which the 5' terminal nucleotide of the oligonucleotide is capable of base pairing as the last base pair on the non-looped end of the stem-loop portion of the FRET cassette.

[0104] Since the amplification of the fluorescent signal from the cleavage of the FRET cassette is due to the repeated or cycling hybridization of a complementary oligonucleotide (e.g., the 5' flap cleaved from the primary probe) to the cleaved flap hybridizing sequence in the population of FRET cassettes, the primary probe is typically designed so that the cleaved 5' flap is not extendable by a polymerase using the FRET cassette as a template. For example, the primary probe is typically designed to generate a 5' cleaved flap product having a 3' end that is not complementary to the cleaved flap hybridizing sequence in the FRET cassette when hybridized to the FRET cassette.

[0105] In some embodiments, a mixture of FRET cassettes with two or more different cleaved flap hybridizing sequences may be used (e.g., in a multiple invasive cleavage reaction). Two cleaved flap hybridizing sequences are said to be "different" from each other if the cleaved flap product (e.g., under invasive cleavage assay conditions) is hybridizable to one cleaved flap hybridizing sequence and not measurably hybridizable to the other cleaved flap hybridizing sequence, or vice versa. Cleavage of the FRET cassette by a FEN enzyme (e.g., FEN-1 endonuclease) in a secondary reaction separates the donor and acceptor moieties, such that inhibition is relieved and signal generation is possible. In some embodiments, the donor and acceptor moieties interact by fluorescence resonance energy transfer (e.g., "FRET"). In other embodiments, the donor and acceptor of the FRET cassette interact by a non-FRET mechanism.

[0106] As used herein, an "interactive" label pair refers to a donor moiety and an acceptor moiety that are attached to the same FRET cassette and are in an energy transfer relationship with each other (i.e., by a FRET mechanism or a non-FRET mechanism). A signal (e.g., a fluorescent signal) can be generated when the donor moiety and the acceptor moiety are separated, for example, by cleavage of the FRET cassette in a secondary reaction. Different FRET cassettes that specifically hybridize to different cleaved 5' flaps can each contain the same interactive label pair.

[0107] As used herein, the term "unlabeled" in reference to a probe oligonucleotide refers to a probe oligonucleotide that does not contain a chromophore or fluorophore to facilitate detection. Unlabeled probes may contain modifications to prevent extension by a polymerase, such as a 3' blocking group.

[0108] As used herein, the term "donor" refers to a moiety (e.g., a fluorophore) that absorbs at a first wavelength and emits at a second, longer wavelength. The term "acceptor" refers to a moiety, such as a fluorophore, chromophore, or quencher, that can absorb some or most of the energy emitted from the donor when the donor is in close proximity (typically 1-100 nm) to the donor group. The acceptor may have an absorption spectrum that overlaps with the emission spectrum of the donor. Generally, if the acceptor is a fluorophore, it re-emits at an even longer third wavelength. If the acceptor is a chromophore or quencher, it emits the energy absorbed from the donor without emitting a photon. In some preferred embodiments, a change in the energy levels of the donor and / or acceptor moieties is detected (e.g., by measuring energy transfer between or from the donor and / or acceptor moieties). This can include detecting emission. In some preferred embodiments, the emission spectrum of the acceptor moiety is different from the emission spectrum of the donor moiety such that emissions (eg, of light and / or energy) from the moieties can be distinguished (eg, spectrally resolved) from one another.

[0109] In some embodiments, a donor moiety is used in combination with multiple acceptor moieties. In a preferred embodiment, a donor moiety is used in combination with a non-fluorescent quencher moiety and an acceptor moiety, such that when the donor moiety is close to the quencher (e.g., between 1-100 nm, or more preferably between 1-25 nm, or even more preferably about 10 nm or less), its excitation is transferred to the quencher moiety rather than the acceptor moiety, and when the quencher moiety is removed (e.g., by cleavage of the probe), the excitation of the donor moiety is transferred to the acceptor moiety. In some preferred embodiments, emission from the acceptor moiety is detected (e.g., using wavelength-shifting molecular beacons) (see Tyagi et al., Nature Biotechnology 18:1191 (2000); Mhlanga and Malmberg, 2001 Methods 25, 463-471; Olivier, 2005 Mutant Res 573, 103-110, and U.S. Patent Application No. 20030228703, each of which is incorporated herein by reference in its entirety).

[0110] As used herein, the term "distinct" with respect to signals (e.g., one or more labels) refers to signals that can be distinguished from one another by spectral properties, such as, for example, fluorescence emission wavelength, color, absorbance, mass, size, fluorescence polarization characteristics, charge, etc., or by their ability to interact with another moiety, such as with a chemical reagent, enzyme, antibody, etc.

[0111] As used herein, the term "synthetic" used in reference to polynucleotides or oligonucleotides (e.g., probes) refers to nucleic acids made in cell-free in vitro reactions (e.g., enzymatic or chemical synthesis reactions). Examples of enzymatic formation of synthetic nucleic acids include formation by restriction enzyme digestion, polymerization (templated or non-templated), ligation, etc. Examples of chemical synthesis of nucleic acids include, but are not limited to, phosphodiester and phosphotriester chemistry, phosphoramidite and H-phosphonate chemistry, etc. See, for example, Methods in Molecular Biology, Vol 20: Protocols for Oligonucleotides and Analogues pp. 165-189 (S. Agrawal, Ed., Humana Press, 1993).; Oligonucleotides and Analogues: A Practical Approach, pp. 87-108 (F. Eckstein, Ed., 1991); and Uhlmann and Peyman, supra. See Agrawal and Iyer, Curr. Op. in Biotech. 6:12 (1995); as well as Anti-sense Research and Applications (Crooke and Lebleu; Eds., CRC Press, Boca Raton, 1993), Beaucage and Caruthers, Tetrahedron Lett. 22:1859-1862 (1981), and Agrawal and Zamecnik, U.S. Patent No. 5,149,798 (1992). In some embodiments, preformed synthetic oligonucleotides are introduced into the reaction, while in other embodiments, synthetic oligonucleotides are formed or modified in-situ (e.g., by the action of a polymerase, ligase, cleavage enzyme, etc.).

[0112] As used herein, the term "flap endonuclease" or "FEN" (e.g., "FEN enzyme") refers to a class of nucleolytic enzymes that act as structure-specific endonucleases on DNA structures that have a duplex with a single-stranded 5' overhang or 5' flap on one of the strands that is displaced by another strand of the nucleic acid such that there are overlapping nucleotides at the junction between the single-stranded and double-stranded DNA. FEN enzymes catalyze the hydrolytic cleavage of the phosphodiester bond adjacent 3' to the junction of the single-stranded and double-stranded DNA, releasing the overhang or "flap" (see Trends Biochem. Sci. 23:331-336 (1998) and Annu. Rev. Biochem. 73:589-615 (2004)). FEN enzymes can be individual enzymes or multi-subunit enzymes. In some particular embodiments, FEN enzyme activity can exist as the activity of another enzyme or protein complex, such as a DNA polymerase. In some preferred embodiments, the FEN enzyme does not have DNA polymerization activity (e.g., does not polymerize DNA even in the presence of a template, primer, and dNTPs). In other preferred embodiments, the FEN enzyme has DNA polymerization activity, but does not demonstrate this activity by extending an oligo (e.g., a 5' flap cleaved from a primary probe or derived from another source) in a manner that substantially precludes cycling hybridization to the FRET cassette. For example, the FEN enzyme involved in the secondary invasive cleavage reaction to cleave a fluorophore or fluorescent 5' flap from a FRET cassette does not extend the cleaved 5' flap from the primary probe (i.e., the invasive probe that reversibly hybridizes to the FRET cassette and catalyzes the cleavage reaction). The flap endonuclease may be thermostable. Examples of FEN enzymes useful in the methods disclosed herein are described in U.S. Pat. Nos. 5,614,402; 5,795,763; 6,090,606, and published PCT applications identified in WO 98 / 23774; WO 02 / 070755; WO 01 / 90337; and WO 03 / 073067, each of which is incorporated by reference in its entirety.A specific example of a commercially available FEN enzyme is Cleavase® enzyme (Hologic, Inc.).

[0113] As used herein, "FEN-1" refers to the non-polymerase flap endonuclease from eukaryotic or archaeal organisms that is encoded by FEN-1 (flap structure-specific endonuclease 1) gene.See, for example, U.S. Patent No. 6,562,611 to Kaiser et al., and Kaiser MW et al., (1999) J.Biol.Chem., 274:21387; International Publication No. WO 02 / 070755 and U.S. Patent No. 7,122,364, which are incorporated herein by reference in their entirety for all purposes.The term "FEN-1 activity" refers to any enzymatic activity of FEN-1 enzyme. FEN-1 endonucleases also include modified FEN-1 proteins (e.g., chimeric proteins containing portions of FEN-1 enzymes from different organisms) and enzymes that contain one or more mutations (e.g., substitutions, deletions, insertions, etc.), as described in WO 02 / 070755 and U.S. Patent No. 7,122,364. Archaeal organisms are any generally unicellular organism of the biological kingdom Archaea.

[0114] References to "first" and "second" and "third" etc. (e.g., target nucleic acid, FRET cassette, invasive cleavage assay, etc.) do not necessarily indicate that one precedes the other, but merely provide identifiers to distinguish one from the other.

[0115] A "reaction mixture" is a combination of reagents (eg, oligonucleotides, target nucleic acids, enzymes, etc.) in a single reaction vessel.

[0116] As used herein, a "multiplex" assay is a type of assay that detects or measures multiple analytes (two or more) in a single run assay. This is distinct from procedures that measure one analyte per reaction mixture. A multiplex invasive cleavage assay is performed by combining reagents for detecting or measuring two or more different analytes using independent invasive cleavage assays in a single reaction vessel. In some embodiments, the same species of fluorescent reporter is detected in each of the multiplex assays. In other embodiments, different species of fluorescent reporters are used, but the different reporters are detectable in the same channel of an instrument that detects a range of fluorescent wavelengths.

[0117] As used herein, the term "complementary" refers to a nucleobase sequence that can form a double-stranded hydrogen-bonding region. The nucleobase sequence can be "fully complementary" (i.e., each nucleobase in one sequence can pair with a corresponding nucleobase in a second sequence) or "partially complementary" (i.e., at least one nucleobase in one sequence cannot hydrogen bond with a corresponding nucleobase in a second sequence). The nucleobase sequences can be in the same or different polynucleotides.

[0118] As used herein, the terms "duplex" and "hybrid duplex" refer to a nucleic acid structure that includes a double-stranded hydrogen-bonded region. Such structures can be fully or partially double-stranded and include RNA:RNA, RNA:DNA and DNA:DNA molecules and their analogs. As an example, a "duplex" includes a cleaved 5' flap sequence (e.g., a primary cleavage flap) hybridized to a complementary cleaved flap hybridization sequence of a FRET cassette, and a cassette cleavage flap hybridized to a complementary masking oligonucleotide.

[0119] As used herein, the term "cleaved" refers to a portion of a polynucleotide that has been cleaved from the remainder of the polynucleotide by the action of one or more nucleases. As an example, a 5' flap sequence is "cleaved" when the primary probe has been cleaved by an endonuclease (e.g., a FEN enzyme), thereby separating the 5' flap portion from the target hybridizing portion of the flap probe.

[0120] As used herein with respect to polynucleotides (e.g., oligonucleotides, target nucleic acids, etc.), the term "single-stranded state" refers to the region of a polynucleotide that is available for base pairing. In the case of a single-stranded polynucleotide having a self-complementary region, the term "single-stranded state" refers to the region of the self-complementary polynucleotide that is available for base pairing. As an example, a cassette cleavage flap is in a single-stranded state before hybridizing to a masking oligonucleotide or after melting that separates the cassette cleavage flap from the masking oligonucleotide.

[0121] As used herein, the "temperature conditions" used to carry out a reaction refer to the temperature or temperature range that allows the reaction to occur. Different temperature profiles of different reactions mean that the temperature conditions that allow one reaction to occur may not allow a different reaction to occur. This term also applies to the temperature profile that allows the hybridization of masking oligo to the complementary oligo sequence that contains the fluorophore moiety.

[0122] As used herein, "optimal" (and grammatical variations thereof) reaction conditions refer to the most favorable reaction conditions for promoting or allowing a reaction to occur. For example, the optimal temperature for carrying out a secondary reaction would be the temperature at which the FRET cassette was most efficiently cleaved in the reaction mixture (e.g., corresponding to a peak on a plot of the fluorescent signal as a function of reaction temperature). Similarly, an optimal temperature range is the range of most favorable temperature conditions for promoting or allowing a reaction to occur. In certain exemplary embodiments, the preferred optimal temperature range may include the optimal temperature plus or minus 5°C, more preferably plus or minus 4°C, more preferably plus or minus 3°C, even more preferably plus or minus 2°C, and even more preferably plus or minus 1°C.

[0123] As used herein, "attached" (e.g., two things are "attached") means chemically linked together. For example, a fluorophore moiety is "attached" to a FRET cassette if it is chemically bound to the structure of the FRET cassette.

[0124] As used herein, "equivalent" (e.g., in the context of "equivalent donor-acceptor pairs" or "equivalent donors" or "equivalent fluorophore" moieties) means that the excitation and emission spectra of the detectable chemical species are sufficiently similar or overlapping in wavelength range to permit detection of the fluorescent emission wavelengths within the same channel of an instrument used to monitor the signal.

[0125] As used herein, the term "spectral overlap" refers to two or more optical spectra that have at least one common wavelength.

[0126] As used herein, the emission from a donor moiety (e.g., a fluorophore) is "quenched" when an acceptor moiety (e.g., a quencher) is close enough that the detectable emission of photons from the donor is suppressed or prevented.For example, when both the donor moiety and the acceptor moiety are attached to the same FRET cassette, the emission from the donor moiety is quenched.Similarly, the emission from the donor or fluorophore moiety attached to the 5' flap cleaved from the FRET cassette can be quenched when the cleaved 5' flap (cassette cleavage flap) hybridizes to a complementary oligo that contains a quencher moiety.

[0127] As used herein, "specific" means relating to only one (or only the specifically indicated group), such as having a specific effect on only one (or only the specifically indicated group) or affecting only one (or only the specifically indicated group) in a specific way. For example, a cleavage 5' flap (e.g., a primary cleavage flap) specific to a FRET cassette can hybridize to that FRET cassette, form an invasive cleavage structure, and promote the cleavage reaction, but cannot hybridize to a different FRET cassette (e.g., a FRET cassette with a different cleaved flap hybridization sequence) and promote the cleavage reaction. Similarly, a fluorescent 5' flap (cassette cleavage flap) cleaved from a FRET cassette can hybridize to a masking oligonucleotide specific to that cassette cleavage flap if the sequences of the two oligonucleotides are complementary to each other.

[0128] As used herein, the term "specifically hybridize" means that under given hybridization conditions, a probe or primer detectably hybridizes substantially only to a target sequence in a sample that contains the target sequence (i.e., there is little or no detectable hybridization to non-target sequences). Similarly, a cleavage 5' flap that is "specific" for a FRET cassette can specifically hybridize to that FRET cassette to form an invasive cleavage structure and promote the cleavage reaction, but does not hybridize to a different FRET cassette in a manner that forms an invasive cleavage structure.

[0129] The term "thermostable," when used with respect to an enzyme, such as a FEN enzyme, indicates that the enzyme is functional or active (i.e., capable of performing catalysis) at elevated temperatures (e.g., about 55° C. or higher). In some embodiments, the enzyme is functional or active at elevated temperatures of 65° C. or higher (e.g., 75° C., 85° C., or even 95° C.).

[0130] As used herein, the term "amplified" refers to an increase in the abundance of a molecule, moiety or effect. A target nucleic acid can be amplified by in vitro replication, such as by PCR.

[0131] As used herein, the term "amplification method" used in reference to nucleic acid amplification refers to a process that specifically amplifies the abundance of a nucleic acid of interest. Some amplification methods (e.g., polymerase chain reaction or PCR) involve repeated cycles of heat denaturation, oligonucleotide primer annealing to a template molecule, and nucleic acid polymerase extension of the annealed primer. The conditions and times required for each of these steps are well known in the art. Some amplification methods are performed at a single temperature and are considered "isothermal". The accumulation of amplification products can be exponential or linear. Some amplification methods (e.g., "target amplification" methods) amplify the abundance of a target sequence by copying the target sequence many times (e.g., PCR, NASBA, TMA, strand displacement amplification, ligase chain reaction, LAMP, ICAN, RPA, SPA, HAD, etc.), while some amplification methods amplify the abundance of a nucleic acid species that may or may not contain a target sequence, but whose amplification indicates the presence of a particular target sequence in the reaction. Some signal amplification methods may increase the abundance of a nucleic acid species by converting the starting nucleic acid, for example by cleaving it to form a cleavage product, or by extending it, for example by polymerization or ligation. Target amplification methods may be applied to the signal molecule (e.g., PCR can be used to produce more copies of the product of a ligation, cleavage, or non-target copy reaction), or vice versa.

[0132] As used herein, the terms "polymerase chain reaction" and "PCR" refer to an enzymatic reaction in which a segment of DNA is replicated in vitro from a target nucleic acid. The reaction generally involves extending a primer on each strand of the target nucleic acid with a template-dependent DNA polymerase to produce a complementary copy of a portion of that strand. The chain reaction involves repeated cycles of denaturation of the DNA strands, for example by heating, followed by cooling to allow primer annealing and extension, resulting in the exponential accumulation of copies of the region of the target nucleic acid adjacent to and including the primer binding site. When an RNA target nucleic acid is amplified by PCR, it is generally converted into a DNA copy strand by an enzyme capable of reverse transcription. Exemplary enzymes include MMLV reverse transcriptase, AMV reverse transcriptase, as well as other enzymes well known to those skilled in the art.

[0133] As used herein, the term "oligonucleotide" (e.g., deoxyribonucleotide or ribonucleotide) is defined as a molecule that contains two or more nucleotides, preferably at least 5 nucleotides, more preferably at least about 10-15 nucleotides, more preferably at least about 15-30 nucleotides or longer. Oligonucleotides are typically less than 200 residues in length (e.g., between 15-100 nucleotides), but as used herein, the term is intended to encompass longer polynucleotide chains as well. The exact size depends on many factors, which in turn depend on the ultimate function or use of the oligonucleotide. Oligonucleotides are often referred to by their length. For example, a 24 nucleotide oligonucleotide is referred to as a "24mer." Oligonucleotides can form secondary and tertiary structures by self-hybridizing or by hybridizing to other polynucleotides. Such structures can include, but are not limited to, duplexes, hairpins, cruciforms, bends, and triplexes. Oligonucleotides can be generated in any manner, including chemical synthesis, DNA replication, reverse transcription, PCR, or combinations thereof. In some embodiments, the oligonucleotide that forms the invasive cleavage structure is generated in a reaction (e.g., by extension of a primer in an enzymatic extension reaction). As used herein, the terms "oligonucleotide" and "polynucleotide" can be used interchangeably and can include non-naturally occurring monomers or portions thereof. More specifically, oligonucleotides can include linear or cyclic oligomers of natural and / or modified monomers or linkages, including, for example, deoxyribonucleosides, ribonucleosides, their substitutions and alpha-anomeric forms, peptide nucleic acids (PNAs), locked nucleic acids (LNAs), 2'-O-methyl modifications, phosphorothioates, methylphosphonates, spacers, etc.

[0134] As used herein, a "signal" is a detectable quantity or impulse of energy, such as electromagnetic energy (e.g., light). The emission of light from an appropriately stimulated fluorophore is an example of a fluorescent signal. In some embodiments, a "signal" refers to the aggregate energy detected in a single channel of a detection instrument (e.g., a fluorometer).

[0135] As used herein, a "background" signal is a signal (e.g., a fluorescent signal) generated under conditions in which no target nucleic acid-specific reaction occurs.For example, the signal generated in a secondary reaction that contains a FRET cassette and a FEN enzyme but does not contain a cassette-specific invasive oligonucleotide (e.g., a primary cleavage flap) is considered to be a background signal.In some examples, background signal is measured in a "negative control" reaction or test that omits target nucleic acid.

[0136] As used herein, a "channel" of an energy sensor device, such as a device equipped with a light energy sensor, refers to a given wavelength band that can be detected or quantified to the exclusion of other wavelength bands. For example, one detection channel of a fluorometer can detect light energy emitted by one or more fluorescent labels over a range of wavelengths as a single event. The light emitted as a result of fluorescence can be quantified as relative fluorescence units (RFU) over a given wavelength or wavelength band. Examples of common fluorescence detection channels include those that detect fluorescence emission wavelengths in the range of about 510-530 nm (e.g., common to the "FAM" detection channel), about 560-580 nm (e.g., common to the "HEX" detection channel), about 610-650 nm (e.g., common to the "Texas Red" detection channel), about 675-690 nm (e.g., common to the "Cy5" detection channel), and about 705-730 nm (e.g., common to the "Quasar 705" detection channel). Cy5 and Alexa Fluor® 647 are examples of two different fluorophores that can be detected in the Cy5 channel of a fluorometer.

[0137] As used herein, "threshold" or "threshold cutoff" refers to a quantitative limit used to interpret experimental results, where results above and below the cutoff lead to different conclusions.For example, a measurement signal below a cutoff can indicate the absence of a particular target, while a measurement signal above the same cutoff can indicate the presence of that target.By convention, results that meet the cutoff (i.e., have exactly the cutoff value) are given the same interpretation as results that exceed the cutoff.

[0138] As used herein, "threshold cycle number" refers to an indication of amplification that measures the time or cycle number when a real-time trial curve signal crosses a given value or threshold. Determination of "TTime" and "Ct" are examples of threshold-based indications of amplification. Other methods include performing derivative analysis of the real-time trial curve. For purposes of this disclosure, TArc and OTArc can also be used to determine when a real-time trial curve signal crosses a given value (e.g., corresponding to a maximum or minimum angle of curvature, respectively). A method for TTime determination is disclosed in U.S. Pat. No. 8,615,368, a method for Ct determination is disclosed in European Patent No. 0640828 B1, a derivative-based method is disclosed in U.S. Pat. No. 6,303,305, and a method for TArc and OTArc determination is disclosed in U.S. Pat. No. 7,739,054. Those skilled in the art will recognize variations that can also be used to determine threshold cycle number.

[0139] As used herein, an "internal calibrator" nucleic acid is a nucleic acid that can be amplified in an in vitro nucleic acid amplification reaction and is distinguishable from the analyte nucleic acid (e.g., a target nucleic acid from a test sample) that is co-amplified in the same reaction. By "internal" it is meant that the calibrator nucleic acid is amplified and detected in the same reaction mixture as the analyte target nucleic acid or a fragment thereof. In some embodiments, the internal calibrator nucleic acid is amplified by the same primers used to amplify the analyte nucleic acid to be quantified. In other embodiments, different primers are used for this purpose. Generally speaking, the analyte nucleic acid and the internal calibrator nucleic acid differ by at least one nucleotide position that can be distinguished by an invasive cleavage reaction. This allows the multiplexed invasive cleavage assay to independently detect the amplified internal calibrator and analyte target nucleic acid.

[0140] As used herein, a "calibration standard" is a composition that contains a known or predetermined amount of an internal calibrator nucleic acid.

[0141] As used herein, a "reaction vessel" or "reaction receptacle" is a container for containing a reaction mixture. Examples include individual wells of a multi-well plate, and plastic tubes (including, for example, individual tubes within a formed linear array of a multi-tube unit, etc.). However, it should be understood that any suitable container may be used to contain the reaction mixture.

[0142] As used herein, "allowing" a reaction means that the reaction mixture provides reagents and conditions to test for the presence of a particular nucleic acid (e.g., target DNA, or cleaved 5' flap) that may or may not be present in the reaction mixture. For example, "allowing" a primary reaction of an invasive cleavage assay to occur means that the reaction mixture contains an invasive probe, a primary probe containing a 5' flap sequence, and a FEN enzyme under appropriate buffer and temperature conditions to allow cleavage of the primary probe and release of the cleaved 5' flap if the target DNA is also available in the reaction mixture to participate in the primary reaction. Similarly, "allowing" a secondary reaction of an invasive cleavage assay to occur means that the reaction mixture contains a FRET cassette and a FEN enzyme under appropriate buffer and temperature conditions to allow cleavage of the FRET cassette if the cleaved 5' flap specific to the FRET cassette is also available in the reaction mixture to participate in the secondary reaction. Furthermore, temperature conditions that are "permitting" (or "permit" or "permissive") for a reaction to occur are temperature conditions that are conducive to carrying out or proceeding with a reaction.

[0143] "Kit" means a packaged combination of materials intended to be used in combination with one another. Kits useful in accordance with the disclosed technology may include one or more containers or tubes containing various reagents. The kit may further include instructions or other information in "tangible" form (e.g., printed information, information recorded electronically on a computer-readable medium, or information recorded on a machine-readable medium such as a bar code).

[0144] Detailed Description Disclosed herein is a multiplexed nucleic acid amplification and detection system that can be used to detect the presence of multiple specific nucleic acid sequences in a temperature-dependent manner using only a single fluorescent detection channel of a nucleic acid analyzer. This technology simplifies the multiplex detection of nucleic acid analytes such as single nucleotide polymorphisms (i.e., "SNPs") so that it can be used in diagnostic applications. Conveniently, this technology can be performed using standard PCR equipment equipped for fluorescent detection or monitoring.

[0145] The disclosed procedure uses a "masking oligo" that hybridizes by complementary base pairing to a fluorescently labeled 5' flap cleaved from a probe or FRET cassette (e.g., a cassette cleavage flap) in an invasive cleavage assay. The masking oligonucleotide includes a fluorescent quenching moiety (sometimes referred to herein as a "quencher") attached thereto. When hybridization between the masking oligonucleotide and the labeled cleaved flap occurs, the quenching moiety of the masking oligo is brought into close proximity with the fluorophore moiety of the cleaved 5' flap. The fluorescent signal emitted by the fluorophore of the cleaved 5' flap is then quenched. Different cleavage products (e.g., cleaved flaps of different lengths and / or different sequences) are hybridized to the cognate masking oligo at an appropriate temperature, allowing the identity of the secondary reaction that liberated the cassette cleavage flap to be determined, and therefore, which target sequence was present in the reaction mixture.

[0146] Invasive cleavage reactions and assays Unlike other invasive cleavage assays in which cleavage of a FRET cassette produces a fluorescent signal indicative of the presence of an analyte nucleic acid, the present technique does not require the persistence of a fluorescent signal to determine whether a particular FRET cassette has been cleaved. In fact, the technique described herein actually requires that cleavage of at least one FRET cassette in a multiplex assay produces a fluorescent signal that is quenched or extinguished as a function of the temperature of the reaction mixture. This is accomplished by including in the reaction mixture a masking oligonucleotide that is complementary to the fluorescent cleavage product of at least one FRET cassette. Hybridization to form a duplex containing the fluorescent cleavage product and the complementary masking oligonucleotide occurs at a melting temperature (T m ) is characterized by T m At higher temperatures, the fluorescent cleavage products are in a single-stranded state from which a fluorescent signal can be produced and detected or measured. m At lower temperatures, duplexes form and quench the fluorescence emitted from the fluorophore attached to the 5' flap cleaved from the 5' flap FRET cassette. By including different masking oligos complementary to the different fluorescent 5' flap cleavage products in the reaction mixture, the different duplexes resulting from hybridization of the masking oligonucleotides with the fluorescent cleavage products have different T m When the temperature-dependent difference in fluorescence signal is detected, it is possible to determine which FRET cassette has been cleaved to generate a signal among multiple FRET cassettes labeled with fluorophores detectable in the same channel of a fluorometer. This ability to quench fluorescence, or temperature-dependent difference in fluorescence signal, is essential to the function of the disclosed technology.

[0147] The invasive cleavage assay disclosed herein comprises the formation of an invasive cleavage structure and the enzymatic cleavage of the invasive cleavage structure by a flap endonuclease (e.g., FEN-1) enzyme. In some embodiments, the invasive cleavage structure comprises: (1) a FRET cassette; and (2) an invasive oligonucleotide hybridized to the FRET cassette. In some embodiments, the invasive oligonucleotide hybridized to the FRET cassette is a 5' flap cleaved from a primary probe. In some embodiments, the invasive cleavage assay comprises: (1) a target nucleic acid to be detected; (2) a primary probe having a 5' flap, where the target-complementary sequence of the primary probe hybridizes to the target nucleic acid; and (3) an invasive oligonucleotide hybridized to a target nucleic acid adjacent to and upstream of the hybridized primary probe. In some embodiments, the invasive cleavage assay sequentially combines a first and a second invasive cleavage reaction (see FIG. 1 ), such that the 5′ flap cleaved from the primary probe (e.g., sometimes the “primary cleavage flap”) serves as an invasive oligonucleotide to facilitate enzymatic cleavage of the FRET cassette in the secondary reaction.

[0148] The invasive oligonucleotide upstream of the primary reaction is typically incubated at a temperature substantially higher than the assay temperature (e.g., T m The primary probe oligonucleotides are designed to anneal essentially permanently to the target DNA at the assay temperature, whereas the primary probe oligonucleotides do not anneal. The portion of the primary probe that anneals to the target strand has a T m so that the primary probe oligonucleotides in the reaction mixture will always anneal to and dissociate from the target strand at the reaction temperature without temperature cycling. mis within about 4° C., more preferably within 3° C., even more preferably within 2° C., and even more preferably within 1° C., of the assay temperature. A cleavage structure is formed upon annealing of the primary probe oligonucleotide next to and downstream of the invasive oligonucleotide. This cleavage structure can be cleaved by a flap endonuclease.

[0149] The key feature and working principle of the invasive cleavage reaction according to the present disclosure is that the number of cleavage products can increase and accumulate under isothermal conditions (i.e., without temperature cycling). For example, the primary probe oligonucleotide hybridized to the target nucleic acid can repeatedly anneal and dissociate without temperature cycling. A single site on the target DNA can be reused or recycled to hybridize to a series of new uncleaved probes without temperature cycling, generating thousands of cleaved probes for each target molecule. See, for example, Olivier, Mutat Res, 573:103-110 (2005). Similarly, the fluorescent signal resulting from FEN-1 cleavage of the FRET cassette after cycling hybridization from the primary probe to the cleaved 5' flap also increases and accumulates under isothermal conditions.

[0150] Invasive cleavage assays can also be configured to operate in a sequential manner, where the cleaved flap from the primary invasive cleavage reaction is used to form a second cleavage structure. For example, Hall's SISAR assay uses two sequential signal amplification reactions to increase the total amount of signal generated by the assay (see Hall et al., Proc. Natl. Acad. Sci., USA 97 (2000) 8272-8277). In the SISAR assay shown in Figure 1, cleavage of each primary probe releases a cleaved 5' flap ("primary cleavage flap" in Figure 1). The cleaved flap hybridizes to a hairpin "FRET cassette" oligonucleotide to form a second cleavage structure. Cleavage of the second cleavage structure separates the fluorophore dye from the quenching moiety of the FRET cassette, thereby allowing fluorescence from the fluorophore to be detected.

[0151] Invasive cleavage assays using FRET cassettes (e.g., sequential invasive cleavage assays) typically involve assays in which the cleaved flap-FRET cassette complex is at a T close to the assay temperature (e.g., within 4°C, more preferably within 3°C, even more preferably within 2°C, and even more preferably within 1°C of the assay temperature). m The secondary reaction, which involves cleavage of the FRET cassette, is designed to have a constant annealing time of 100 ms, so that the cleaved flap always anneals and dissociates from the FRET cassette at the reaction temperature, without temperature cycling. Annealing the cleaved flap to the FRET cassette forms a cleavage structure that can be cleaved by a flap endonuclease (e.g., FEN-1 endonuclease). Thus, the secondary reaction, which involves cleavage of the FRET cassette, generates a signal using the same recycling principle as the primary reaction. Each cleaved flap can hybridize to a series of new uncleaved FRET cassettes without temperature cycling, thereby generating thousands of unquenched fluorophores for each cleaved flap.

[0152] A flap endonuclease (e.g., FEN-1) enzyme present in a reaction mixture further comprising a sample nucleic acid, an invasive probe, a primary probe, and a FRET cassette cleaves the 5' flap from the remaining portion of the primary probe when there is a one-base overlap between the invasive probe and the primary probe when both are hybridized to the sample nucleic acid. This cleavage reaction is called the "primary" reaction. The 5' flap released from the primary probe then undergoes cycling hybridization to a FRET cassette having a sequence complementary thereto, whereupon a FEN-mediated cleavage reaction separates the fluorophore from the quencher moiety present on the same FRET cassette, resulting in detectable fluorescence emission. This cleavage reaction, which generates a fluorescent signal by cleavage of the FRET cassette, is called the "secondary" reaction. As described above, the secondary reaction is performed to remove the T of the duplex between the 5' flap cleaved from the primary probe to facilitate cycling hybridization and the FRET cassette. mIf performed at temperatures close to the melting temperature (i.e., the melting temperature), the same 5' flap is free to interact with a similar cognate FRET cassette to further catalyze the cleavage reaction. This linear amplification, which can occur at a fixed temperature, is detectable by an increase in fluorescence as a function of time. Each of the primary and secondary reactions is essentially an isothermal process. In fact, the FEN-1-dependent secondary invasive cleavage reaction generates a detectable fluorescent signal without the requirement of polymerization, even under thermal conditions where thermostable DNA polymerases (e.g., Taq DNA polymerase) exhibit significantly impaired polymerization activity. In fact, polymerase-based extension of the cleaved 5' flap using the FRET cassette as a template would impair cycling hybridization, which is beneficial for fluorescent signal accumulation. Thus, extension of the cleaved 5' flap is contraindicated.

[0153] Useful fluorophores and quenchers In some embodiments, the multiplexed invasive cleavage assay according to the disclosed technology can use only a single detection channel to detect the fluorescent signals generated by the multiplexed invasive cleavage assay, and preferably uses the same chemical species of fluorophore for signal generation from different targets in the multiplexed reaction. In other embodiments, multiple different fluorophores may be combined for signal generation from different targets in the multiplexed reaction.Exemplary fluorophores for use in the FRET cassette system of the disclosed technology include, but are not limited to, fluorescein, rhodamine, REDMOND RED dye, YAKIMA YELLOW dye, hexachloro-fluorescein, TAMRA dye, ROX dye, Cy3, Cy3.5, Cy5, Cy5.5, and Cy7, 4,4-difluoro-5,7-diphenyl-4-bora-3a,4a-diaza-s-indacene-3-propionic acid, 4,4-difluoro-5,p-methoxyphenyl-4-bora-3a,4a-diaza-s-indacene-3-propionic acid, 4,4-difluoro-5,p-methoxyphenyl-4-bora-3a,4a-diaza-s-indacene-3-propionic acid, 4,4-difluoro-5-styryl-4-bora-3a,4a-di Aza-S-indacene-propionic acid, 6-carboxy-X-rhodamine, N,N,N',N'-tetramethyl-6-carboxyrhodamine, Texas Red, Eosin, Fluorescein, 4,4-difluoro-5,7-diphenyl-4-bora-3a,4a-diaza-S-indacene-3-propionic acid, 4,4-difluoro-5,p-ethoxyphenyl-4-bora-3a,4a-diaza-S-indacene-3-propionic acid and 4,4-difluoro -5-Styryl-4-bora-3a,4a-diaza-S-indacene-propionic acid, 6-carboxyfluorescein (6-FAM), 2',4',1,4-tetrachlorofluorescein (TET), 21,4',51,7',1,4-hexachlorofluorescein (HEX), 2',7'-dimethoxy-4',5'-dichloro-6-carboxyrhodamine (JOE), 2'-chloro-5'-fluoro-7',8'-fused phenyl-1,4-dichloro -6-carboxyfluorescein (NED), 2'-chloro-7'-phenyl-1,4-dichloro-6-carboxyfluorescein (VIC), fluorescein isothiocyanate (FITC), 5,6-carboxymethylfluorescein, Texas Red, nitrobenzo-2-oxa-1,3-diazol-4-yl (NBD), coumarin, dansyl chloride, amino-methylcoumarin (AMCA), erythrosine, BODIPY dyes, CASCADE BLUE dyes, OREGON GREEN dyes, pyrene, lissamine, xanthene, acridine, oxazine, phycoerythrin, QUANTUM dyes, thiazole orange-ethidium heterodimer, and the like.

[0154] Exemplary quenchers for use in the FRET cassette system of the present technology include, but are not limited to, cyanine dyes such as Cy3, Cy3.5, Cy5, Cy5.5, and Cy7, rhodamine dyes such as tetramethyl-6-carboxyrhodamine (TAMRA) and tetrapropano-6-carboxyrhodamine (ROX), DABSYL dyes, DABCYL dyes, cyanine dyes, nitrothiazole blue (NTB), anthraquinone, malachite green, nitrothiazole, or nitroimidazole compounds, QSY7 (Molecular Probes, Eugene, OR), ECLIPSE quencher (Epoch Biosciences, Inc., Logan, UT), and the like. Alternative quenchers include Black Hole quencher dyes, particularly BHQ-1, BHQ-2 and BHQ-3 (Biosearch Technologies, Petaluma, Calif.); BLACKBERRY® quenchers (Berry & Associates, East Dexter, Mich.); IOWA BLACK® (Integrated DNA Technologies, Coralville, Iowa). Analysis of factors such as absorbance and emission spectra of various molecules in selecting pairs or groups of moieties for use in FRET configurations is well known to those of skill in the art.

[0155] One of skill in the art will recognize the wavelength ranges that can be detected by different channels of a fluorometer and can readily select fluorophores for different FRET cassettes where emission from the different fluorophores can be detected in the same fluorometer channel.

[0156] Characteristics of temperature-dependent multiplexing technology Unlike previous invasive cleavage assays that benefit from the uninterrupted persistence of fluorescent signals, the disclosed technology benefits from selective suppression of fluorescence following FEN-1-mediated cleavage of the FRET cassette. Peterson et al., in their published application 2018 / 0163259 A1, disclosed a method in which different FRET cassettes labeled with the same fluorophore were cleaved at different temperatures, the different temperatures essentially isolating one reaction from the other. Monitoring the accumulated fluorescent signal as a function of time or cycle number allowed resolution of the activity of the FRET cassettes. In this way, multiple nucleic acid targets could be detected in a multiplexed format using only a single channel of a fluorometer, and even using only a single type of fluorescent label. As disclosed herein, the disclosed technology relies on temperature-dependent fluorescence quenching to effectively remove the contribution of fluorescent signals arising from different FRET cassettes, even when the different FRET cassettes have identical fluorescent labels. Thus, procedures or events that take place after FRET cassette cleavage are beneficial in the disclosed technology.

[0157] The present technique uses at least one FRET cassette structured to include a 5' flap portion that retains a fluorophore after cleavage by the FEN-1 enzyme in a secondary reaction. Preferably, the length of the 5' flap is in the range of 8-30 nucleotides, more preferably 8-25 nucleotides, even more preferably 8-16 nucleotides. Particularly preferred 5' flap lengths are 9 nucleotides, 10 nucleotides, 11 nucleotides, 12 nucleotides, 13 nucleotides, 14 nucleotides, or 15 nucleotides. Figure 1 shows a sequential invasive cleavage reaction in which the FRET cassette is a flapless FRET cassette that does not include a 5' flap portion, and cleavage in the secondary reaction liberates a fluorescent dye attached to a single nucleotide. In contrast, a 5' flap FRET cassette (see Figure 2A) can hybridize by cycling hybridization to a flap cleaved from a primary probe (see Figure 2B) to produce a structure that is cleavable by the FEN-1 enzyme. The resulting cassette cleavage flap emits a fluorescent signal when in a single-stranded state, but the signal can be reversibly suppressed or quenched by hybridization of the cassette cleavage flap to a masking oligo that contains a quenching moiety (see FIG. 2C). The hybrid interaction between the cassette cleavage flap and the masking oligo can be controlled in a temperature-dependent manner. When used in the same reaction mixture, the flapless FRET cassette and the 5' flap FRET cassette can produce a signal that can be resolved by temperature change after the cleavage reaction has occurred.

[0158] In addition to distinguishing the origin of fluorescence arising from a flapless FRET cassette (without a 5' flap portion) and a 5' flap FRET cassette, the disclosed technique allows for the resolution of signals arising from the same fluorescent dye of different 5' flap FRET cassettes. Figure 3 shows diagrammatically how the fluorescent signals produced by three different FRET cassettes (flapless FRET cassette; 5' flap FRET cassette 1; and 5' flap FRET cassette 2), each labeled with a fluorophore (e.g., the same fluorophore) detectable in the same channel of a fluorometer, can be distinguished from each other under different temperature conditions. The top part of the figure shows a flapless FRET cassette that does not contain a 5' flap. This FRET cassette can be cleaved by FEN-1 endonuclease to release a fluorescent dye after cycling hybridization to the complementary cleavage flap from the primary probe, and the fluorescence emitted from the released dye cannot be quenched by interaction with any masking oligonucleotide used in the reaction. Fluorescence emitted from this unquenched dye is detectable at all temperatures. The center part of the diagram in Figure 3 shows a first 5' flap FRET cassette (5' flap FRET cassette 1) that can be cleaved by the FEN-1 enzyme to release a 5' flap ("cassette cleavage flap 1") after cycling hybridization to a complementary cleavage 5' flap from the primary probe. Cassette cleavage flap 1 remains attached to the fluorophore after the cleavage reaction, but the fluorescent signal is quenched when cassette cleavage flap 1 hybridizes to a complementary masking oligo. This is the T of the hybrid duplex containing cassette flap 1 and the complementary masking oligo. m This can occur at lower temperatures. m ("T mWhen the FRET cassette cleavage flap 1 is in a single-stranded state ("unquenched cassette cleavage flap 1" in the diagram), a fluorescent signal can be released by the fluorophore in cassette cleavage flap 1. The bottom part of the diagram in Figure 3 shows a second 5' flap FRET cassette (5' flap FRET cassette 2) that can be cleaved by the FEN-1 enzyme to release a 5' flap ("cassette cleavage flap 2") after cycling hybridization to a complementary cleavage 5' flap from the primary probe. Cassette cleavage flap 2 remains attached to the fluorophore after the cleavage reaction, but the fluorescent signal is quenched when cassette cleavage flap 2 hybridizes to a complementary masking oligo. This is the T of the hybrid duplex containing cassette cleavage flap 2 and the complementary masking oligo. m (This can occur at temperatures lower than T m ("T m When the T rises above the T threshold and cassette cleavage flap 2 becomes single-stranded ("unquenched cassette cleavage flap 2" in the figure), a fluorescent signal can be emitted by the fluorophore of cassette cleavage flap 2. m By designing different 5' flap FRET cassettes to have 5' flap portions with different lengths and / or G:C content, the signals resulting from different cleavage flaps can be distinguished from each other. For example, (1) the fluorophore of only one of the cassette cleavage flaps can be designed to emit a signal at a time, or (2) the signals emitted by the fluorophores of the different cassette cleavage flaps can be monitored as a function of temperature to determine a melting profile. Both of these alternatives are shown in the working examples herein.

[0159] In particular, in procedures using multiple different 5' flap FRET cassettes and masking oligos, the T of a hybrid duplex containing the 5' flap cleavage products ("cassette cleavage flaps") and the cognate masking oligos is generated. mPreferably, the T of different hybrid duplexes analyzed in the same multiplex reaction (e.g., real-time nucleic acid amplification reaction or end-point melting / annealing analysis procedure) should be different. m The T of the hybrid duplex containing the cassette cleavage flap and the cognate masking oligo differs by at least 2° C., more preferably at least 3° C., more preferably at least 5° C., more preferably at least 7° C., more preferably at least 10° C., or even at least 15° C. m Preferred differences between the T and T of the masking oligonucleotide-cassette cleavage flap duplex are in the range of 2° C. to 20° C. difference, more preferably 5° C. to 20° C. difference, more preferably 5° C. to 15° C. difference, or even more preferably 5° C. to 10° C. difference. In some embodiments, the 5′ flap of the masking oligonucleotide and / or the FRET cassette oligonucleotide is in the range of 2° C. to 20° C. difference, more preferably 5° C. to 15° C. difference, or even more preferably 5° C. to 10° C. difference. m For example, in some embodiments, the one or more nucleotide modifications are selected from the group consisting of locked nucleic acid (LNA), peptide nucleic acid (PNA), bridged nucleic acid (BNA), 2'-O alkyl substitution, L-enantiomeric nucleotides, or combinations thereof.

[0160] FIG. 4 shows a schematic of the identification of different target sequences by detecting FEN-1-mediated cleavage products of different FRET cassettes, each FRET cassette having a fluorescent dye that can be detected in the same channel of a fluorometer (e.g., shown as identical HEX fluorophores). The cleavage products and masking oligos shown correspond to those shown in FIG. 3. The fluorophore-containing cleavage products from the flapless FRET cassette (top) indicate the presence of target 1 and remain unquenched at all temperatures. Thus, below temperature 1, where cassette cleavage flap 1 and cassette cleavage flap 2 hybridize to their respective masking oligos, detectable fluorescence indicates the presence of target 1. The fluorophore-containing cleavage products from the 5' flap FRET cassette 1, indicating the presence of target 2 (middle), are unquenched at temperatures above temperature 1. Above temperature 1 and below temperature 2, detectable fluorescence can indicate the presence of target 1 and target 2. Since the fluorescence signals measured in the procedure are additive, the difference between the fluorescence signal measured at a temperature higher than temperature 1 but lower than temperature 2 (middle part of Figure 4) and the fluorescence signal measured at a temperature lower than temperature 1 (top part of Figure 4) indicates the signal due to the presence of target 2. The fluorophore-containing cleavage product from 5' flap FRET cassette 2, which indicates the presence of target 3 (bottom part), is not quenched at temperatures higher than temperature 2. At temperatures higher than temperature 2, detectable fluorescence can indicate the presence of target 1 and target 2 and target 3. Thus, the difference between the fluorescence signal measured at a temperature higher than temperature 2 (bottom part of Figure 4) and the fluorescence signal measured at a temperature higher than temperature 1 but lower than temperature 2 (middle part of Figure 4) indicates the signal due to the presence of target 3. In this figure, temperature 1<temperature 2<temperature 3. Comparison of the results of fluorescence detection at different temperatures can be used to determine which target was involved in promoting the cleavage reaction(s). In some embodiments, this involves comparing the magnitude of the fluorescent signals, where the signals measured at different temperatures are an additive result of the known detectable fluorescence, as described above. In some embodiments, determining which targets were involved in promoting the cleavage reaction(s) involves performing a melting / annealing curve analysis.

[0161] Result Processing and Equipment The procedures disclosed herein can be performed using conventional laboratory equipment (including equipment with integrated or stand-alone computers or processors programmed with appropriate software) to amplify nucleic acids and monitor amplicon production. Included within the meaning of "computer" is an embedded processor controlled by software. The computer can be programmed by either the manufacturer or the end user to perform one or more temperature changes or steps, preferably allowing monitoring of fluorescence in the reaction mixture as cycles of the amplification reaction are occurring. Preferably, the reaction mixture is contained in a reaction vessel (e.g., a tube or a well of a multi-well plate) held within the nucleic acid amplification device. However, it is also possible to complete analysis of the amplification products after the amplification reaction is completed (e.g., to establish melting / annealing curves of the amplification products). This latter analysis can even be completed outside the device that amplifies the nucleic acids.

[0162] The computer component of the device useful for carrying out the disclosed technology can be programmed with software instructions that "make" the computer carry out certain steps. These steps can include either controlling a thermocycler that amplifies the nucleic acid; receiving input from a fluorometer that monitors the fluorescence emission in the reaction mixture in which FRET cassette cleavage occurs in the invasive cleavage reaction; or processing the results to determine which of two or more FRET cassettes have been cleaved to produce a fluorescent signal. In a preferred embodiment, the FRET cassettes are cleaved to produce fluorescent cleavage products, and the fluorescence produced by the different cleavage products can be detected or measured in a single channel of the fluorometer. In some embodiments, the different FRET cassettes are labeled with the same fluorophore. The computer can also be used to carry out mathematical steps (e.g., addition, subtraction, multiplication, and / or division) that lead to a determination of which FRET cassette in the mixture has been cleaved to produce a detectable or measurable fluorescent signal.

[0163] The methods disclosed herein can be performed using automated nucleic acid analysis equipment, such as devices that amplify nucleic acids and monitor the production of nucleic acid amplification products. These analysis equipment include PCR equipment, or real-time PCR equipment that can be programmed to perform a series of temperature cycling steps. Preferably, the PCR equipment is equipped with a fluorometer that monitors the progress of the reaction that occurs in the tubes or wells of a multi-well plate (generally, the reaction "receptacle"). Equipment configured to perform and monitor real-time PCR reactions is particularly preferred for use with the disclosed technology. One example of a preferred equipment for performing, monitoring, and evaluating the results obtained using the disclosed technology is the Panther Fusion System (Hologic, Inc.; San Diego, Calif.), which advantageously automates the steps of the procedure. Another preferred equipment is the ABI 7500 Real-Time PCR System (ThermoFisher Scientific; New York).

[0164] One common approach to assess the state of fluorescent cleavage products in a mixture of a flapless FRET cassette and one or more 5' flap FRET cassettes involves evaluating the cleavage of the two types of FRET cassettes separately. This procedure involves raising the temperature of the reaction mixture to the lowest T for any duplex formed between a masking oligonucleotide and the fluorescent cleavage product of a 5' flap FRET cassette in the mixture. m The second step may include reducing the fluorescence signal to less than 0.5%. As a result, the residual fluorescence signal is derived from the fluorescent cleavage product that cannot be quenched and therefore exhibits a constant value in the first derivative plot of the change in fluorescence as a function of temperature. Thus, the presence of the unquenched fluorescent cleavage product can be indicated by measuring or detecting the residual fluorescence signal (e.g., a specific signal above background fluorescence) when the fluorescence from the other fluorescent cleavage products is quenched as a result of duplex formation with the masking oligonucleotide. Detecting the fluorescent signal under this condition can indicate that the unquenched fluorescent cleavage product is present in the reaction mixture and, as a result, the corresponding FRET cassette has been cleaved. In a second step, a temperature-dependent melting / annealing curve (sometimes a "quenching profile") can be generated for the reaction mixture containing the fluorescent cleavage product, from which a derivative plot can be generated. For example, the first derivative plot of the fluorescence change as a function of temperature contains peaks or maxima corresponding to the different duplexes present in the reaction mixture, and duplex formation quenches the signal from the fluorescent cleavage product contained therein. In this way, it is possible to determine which FRET cassette has been cleaved to produce the fluorescent cleavage product.

[0165] An exemplary system is shown herein by multiplex detection of either two or three analytes using invasive cleavage of different FRET cassettes, where the FRET cassettes have fluorescent labels that can be detected or monitored in a single channel of a fluorometer or fluorescence monitoring device. In some embodiments, multiple FRET cassettes were used in the multiplex procedure. For example, two different FRET cassettes can be combined in a single reaction mixture, where only one of the FRET cassettes has a 5' flap sequence that is complementary to a masking oligonucleotide included in the same reaction mixture. The other FRET cassette can be a flapless FRET cassette that does not contain any 5' flap sequence, or alternatively, a 5' flap FRET cassette in a reaction mixture that does not contain a complementary masking oligonucleotide. In such a case, the fluorescent signal resulting from the cleavage of only one of the two FRET cassettes will undergo temperature-dependent fluorescence quenching. As described above, the fluorescent signal generated by the cleavage of the FRET cassettes remains substantially constant when the temperature of the reaction mixture is changed if the fluorescent cleavage product does not interact with any masking oligonucleotide to quench the fluorescence. Detection of the unquenched fluorescent cleavage product can include detecting a fluorescent signal (e.g., a fluorescent signal above background) at a temperature at which formation of a duplex containing the masking oligonucleotide quenches the fluorescent signals from all other quenched fluorescent cleavage products monitored in the same fluorescent channel in the reaction mixture. Detection of the quenched fluorescent cleavage product in the multiplex reaction mixture can include detecting a T at which fluorescence quenching is maximal. m T for masking oligoduplex formation compared to lower temperatures m This may involve simply establishing that at higher temperatures there is a greater measurable fluorescent signal.

[0166] In a different embodiment, the multiplex reaction mixture comprises two different FRET cassettes, each of which comprises a cleavable 5' flap sequence, and the fluorescent signal emitted by each cleaved 5' flap can be quenched by hybridization to a different complementary masking oligonucleotide. Determining which FRET cassette has been cleaved in the reaction mixture can include derivative analysis, preferably including a first derivative plot of the melting / annealing curve as described above. This approach allows multiple cleavage products to be resolved and identified in a single procedure.

[0167] In some embodiments, the multiple invasive cleavage reaction comprises three or more different FRET cassettes, each labeled with a fluorescent label (e.g., all fluorescent labels may be the same) detectable in a single channel of a fluorometer. Each FRET cassette may have a different cleavable 5' flap sequence that emits a fluorescent signal after cleavage, and may quench the fluorescent signal in a temperature-dependent manner after hybridization of a complementary masking oligonucleotide. The duplexes formed by hybridization of the different fluorescent cassette cleavage flaps have different T mIf the duplex is characterized by, the identity of the duplex can be readily determined by assessing the melting / annealing characteristics (e.g., using derivative analysis). This is shown below using a first derivative plot to detect and identify duplexes containing fluorescent cleavage products hybridized to a masking oligonucleotide. Furthermore, one of the FRET cassettes used in the multiplex reaction can produce a fluorescent cleavage product that does not quench in the reaction mixture. In this situation, it can be said that the summed cleavage products still exhibit different temperature-dependent fluorescence quenching profiles, since some exhibit fluorescence quenching at different temperatures, while one does not. In practical terms, the different cleavage products can be distinguished by monitoring the fluorescence signal as the temperature of the reaction mixture is changed. This may involve monitoring the fluorescence signal when the temperature is changed from a high state to a low state (e.g., to allow annealing of complementary strands to form a duplex) or when it is changed from a low state to a high state (e.g., to facilitate melting of a preformed duplex). For this reason, the temperature-dependent fluorescence quenching profile is sometimes referred to as a "melting / annealing" curve or profile.

[0168] The results from the differential quenching of the fluorescent cleavage products produced in the multiple invasive cleavage reactions can be analyzed by different approaches to determine which alternate FRET cassettes were cleaved to produce a fluorescent signal detectable in a single channel of a fluorometer or fluorescence detection device. Two preferred analysis approaches that can be automated (e.g., by a computer, processor, or controller) include: (1) assessing the difference between the fluorescence readings or measurements at temperatures at which the different fluorescent cleavage products undergo greater or less fluorescence quenching due to masking oligonucleotide hybridization; (2) assessing the difference between the fluorescence readings or measurements at temperatures at which the different fluorescent cleavage products undergo greater or less fluorescence quenching due to masking oligonucleotide hybridization; e.g., the characteristic melting temperatures of the duplexes (i.e., "T m") to evaluate the fluorescence quenching profile (i.e., fluorescence measured as a function of temperature in the presence of a complementary masking oligonucleotide). In certain preferred embodiments, a combination of these different approaches can be used to resolve which of multiple different FRET cassettes have been cleaved in a reaction mixture, where the different cleavage products have fluorophores that are detected in the same channel of a fluorometer. In some embodiments, the different FRET cassettes have identical fluorophores. For example, a reaction mixture containing fluorescent cleavage products that do not interact with the masking oligonucleotide to provide quenching, along with one or more fluorescent cleavage products that hybridize to a cognate masking oligonucleotide, preferably includes a T of a duplex comprising the masking oligonucleotide and the complementary fluorescent cleavage product of the 5' flap FRET cassette. m Fluorescence measured at higher temperatures and its T m The fluorescence is analyzed by evaluating the difference from the fluorescence measured at a lower temperature, as well as a derivative plot (eg, a first derivative plot) of the change in fluorescence as a function of temperature.

[0169] Cleavage of a FRET cassette in a multiplex invasive cleavage assay to generate two fluorescent cleavage products can be assessed by evaluating fluorescence emission at two temperatures, if only one of them undergoes temperature-dependent quenching, or alternatively by using this evaluation approach together with curve analysis to determine the T m The fluorescent cleavage products that cannot be quenched in the reaction mixture remain uniformly fluorescent over the measured temperature range and therefore have a constant slope (i.e., a slope of zero) on a plot of fluorescence as a function of temperature. A plot of the first derivative of the change in fluorescence as a function of temperature can be used to determine the T of any duplex. m In contrast, the first derivative plot, which indicates the presence of fluorescent cleavage products that have undergone temperature-dependent quenching (i.e., due to masking oligonucleotide hybridization), does not show any maximum indicating Tm Furthermore, a simple evaluation of the fluorescence signal measured at the two temperatures can indicate the presence or absence of each of the two fluorescent cleavage products. More specifically, the fluorescence indicates the T m One higher temperature (i.e., the temperature at which there is no fluorescence quenching) and the T of the duplex in the reaction mixture m It can be measured or detected at a second, lower temperature (ie, the temperature at which the duplex is formed; complete fluorescence quenching).

[0170] In some instances, a single unquenched fluorescent cleavage product (e.g., resulting from cleavage of a flapless FRET cassette or a cassette cleavage flap in the absence of a complementary masking oligonucleotide) is present in a reaction mixture with one or more quenchable fluorescent cleavage products. A fluorescent cleavage product that is not subject to quenching by masking oligonucleotide hybridization will have a detectable fluorescent signal in a fluorometer channel greater than the T of a duplex containing other fluorescent cleavage products that have maximal fluorescent quenching in the fluorometer channel. m If it is measured at a lower temperature, it is determined to be present. In other words, measurable fluorescence (i.e., a specific signal above a background signal threshold) at the point where the duplex quenches the fluorescence from other cleavage products in the reaction mixture indicates the presence of a fluorescent cleavage product that is not subject to quenching by masking oligonucleotide hybridization. m The fluorescence signal measured at a higher temperature (e.g., the temperature at which fluorescence quenching is minimal) is T m If the fluorescent signal measured at the lower temperature (ie, the temperature at which fluorescence quenching is complete) exceeds that measured at the lower temperature, then it can be determined that a fluorescent cleavage product is present that is quenched by masking oligonucleotide hybridization.

[0171] In some embodiments, it may be desirable to use a combination of both approaches, particularly when multiple distinct fluorescent cleavage products that are quenched by masking oligonucleotide hybridization are combined with one fluorescent cleavage product that is not quenched. In such cases, melting / annealing curves are generated and evaluated by derivative analysis to determine the T of the fluorescence quenching duplex. m The presence of a quenchable fluorescent cleavage product can be determined by detecting the T of the single quenchable fluorescent cleavage product. Similarly, points on the melting / annealing curves corresponding to complete fluorescence quenching and / or the absence of fluorescence quenching can be used in the above evaluation. More specifically, the presence of a fluorescent cleavage product that does not undergo quenching can be evaluated at a low temperature where quenching by masking oligonucleotide hybridization is complete. Detecting a residual fluorescent signal when quenching is complete (i.e., maximum) in the reaction mixture indicates the presence of a non-quenched fluorescent cleavage product. A single quenchable fluorescent cleavage product indicates the T of duplex formation. m The T of duplex formation compared to the fluorescence reading at a lower temperature (e.g., when the duplex is formed and stable). m A higher fluorescence reading at higher temperatures (eg, in the absence of a duplex) indicates the presence of a quenchable fluorescent cleavage product.

[0172] A reaction mixture containing more than one distinct fluorescent cleavage product, each of which is quenched by masking oligonucleotide hybridization, is prepared by isolating any T of duplexes that may be present in the mixture. m The melting / annealing curve results can be conveniently evaluated by processing the results using derivative analysis to identify the T of the duplex between the masking oligonucleotide and the complementary fluorescent cleavage product. For example, the melting / annealing curves can be processed to calculate the first derivative, and then a first derivative plot of the change in fluorescence as a function of temperature can be established. A peak or maximum on the first derivative plot represents the T of the duplex between the masking oligonucleotide and the complementary fluorescent cleavage product. m Each of the duplexes corresponds to a different T mWhen the FRET cassette is characterized by: , it becomes possible to detect the duplexes independently of each other. Higher order derivatives are also contemplated to identify the duplexes and determine which of the multiple FRET cassettes are cleaved to generate a fluorescent signal. As described elsewhere herein, T for different duplexes can be used to facilitate the distinction between one duplex and the other duplex(es). m It is desirable to have the electrodes spaced apart by a minimum temperature difference.

[0173] Software that allows the computer to process the results and determine which FRET cassettes in the mixture of FRET cassettes that produced cleavage products are encompassed by the above description. EXAMPLES

[0174] Working Example Described herein is a technique for multiplex detection using at least one 5' flap FRET cassette (i.e., a FRET cassette with a 5' flap portion), the 5' flap portion having a fluorescent label. The signal emitted from the fluorescent label after cleavage of the 5' flap FRET cassette by FEN-1 endonuclease in an invasive cleavage assay can be selectively quenched based on temperature. In some embodiments, the invasive cleavage assay includes both a primary and secondary invasive cleavage reaction. In some other embodiments, the invasive cleavage assay includes a secondary invasive cleavage reaction without a primary invasive cleavage reaction. It should be understood that FEN-1 mediated cleavage physically separates the fluorophore and quencher portions of the 5' flap FRET cassette onto different oligonucleotide molecules, thereby relieving the fluorescence quenching characteristic of intact 5' flap FRET cassettes. Quenching of the fluorescence emitted from the cleaved 5' flap is mediated by the temperature-dependent hybridization of the cleaved 5' flap to a complementary masking oligonucleotide with a quencher moiety.With this approach, the quenchable flap is not part of any probe that hybridizes to the target nucleic acid to be detected.Instead, the quenchable flap can be a product of a linear amplification reaction that occurs without polymerization under isothermal conditions.

[0175] According to the present disclosure, 5' flap FRET cassettes reporting different target molecules can be detected using single channel fluorescence detection. The fluorescent labels of different FRET cassettes can be the same fluorescent labels. In some embodiments, the same fluorescent label is used in FRET cassettes with different 5' flaps. Different fluorescent labels can be used instead of the same label, provided that the different labels can be detected in the same fluorescent channel of an optical detector (e.g., a fluorometer). In all cases, the cleavage of the FRET cassette to produce a fluorescent signal was mediated by FEN-1 enzyme (i.e., a non-polymerizing flap endonuclease). The 5' flap cleaved from the primary probe plays a catalytic function in the cleavage of the FRET cassette, which means that the 5' flap hybridizes transiently to the FRET cassette, promotes cleavage to release the fluorescent signal, and then dehybridizes to allow the interaction of the cleaved flap with a new FRET cassette. The 5' flap cleaved from the primary probe hybridized to the nucleic acid target to be detected preferably does not have a fluorophore moiety.

[0176] Example 1 shows how two different nucleic acid sequences (analyte A and sample B) were amplified and detected in the same reaction mixture using either single-channel or dual-channel fluorescence detection. Amplification was by polymerase chain reaction (PCR). The products of the PCR reaction were detected using an invasive cleavage reaction using a fluorescently labeled FRET cassette. Sample A was detected using a FRET cassette with a first label (hexachloro-fluorescein or "HEX") that was detectable in the HEX channel of the fluorometer component of the real-time PCR instrument. Sample B was detected using one of two different FRET cassettes in the same reaction mixture, each FRET cassette having a different label. The first 5' flap FRET cassette used to detect sample B had a second label that was also detectable in the HEX channel of the PCR instrument. The signal emitted by the label attached to the 5' flap cleaved from this FRET cassette, indicating the presence of analyte B, was subjected to quenching after hybridizing the cleaved 5' flap of the FRET cassette to a complementary masking oligo. The second FRET cassette for detecting analyte B contained a label that was detectable in the ROX channel of the PCR instrument, and the fluorescent signal emitted after cleavage was not subject to quenching. In particular, the signal detected in the ROX channel of the real-time amplification and detection instrument was substantially undetectable in the HEX channel of the instrument, and vice versa. In other words, the fluorescent HEX signal was substantially undetectable in the ROX channel, and the fluorescent ROX signal was substantially undetectable in the HEX channel. The results established that each of the two analyte nucleic acids could be amplified and the synthesis of different amplification products could be monitored using fluorophores detected in the same or different optical channels of the PCR instrument.

[0177] Example 1 Single- or dual-channel fluorescence detection of two analyte nucleic acids in one reaction The reaction mixtures were prepared in duplicate, and each reaction mixture contained the lyophilized composition incorporated in an aqueous reconstitution buffer. The lyophilized composition contained dNTPs, a thermostable DNA polymerase (e.g., Taq DNA polymerase; Promega Corporation, Madison, WI), a FEN-1 flap endonuclease enzyme (e.g., Cleavase 2.0; Hologic Inc., Marlborough, MA), an oligonucleotide, and trehalose. The oligonucleotides in the lyophilized composition contained a pair of primers for each of the two analyte nucleic acids to be detected, a primary probe with a 5' flap that is non-complementary to the analyte sequence to be amplified and / or detected, and a FRET cassette that can be cleaved by the FEN-1 enzyme after hybridization of the 5' flap cleaved from the primary probe. In this example, the oligonucleotide that facilitated the cleavage of the 5' flap from the primary probe also served as a primer in the nucleic acid amplification reaction. In an isothermal cycling hybridization reaction, two different primary cleavage flaps (one from each of the primary probes complementary to analyte A and analyte B) were reversibly hybridized to three different FRET cassettes. FRET cassette 1 (used to detect analyte A) was a flapless FRET cassette labeled with a HEX fluorophore and a BlackBerry quencher moiety (Berry & Associates; Dexter, MI). After cleavage of FRET cassette 1, the emission signal from the HEX fluorophore was detectable in the HEX channel of the instrument used to amplify the nucleic acid and monitor the progress of the amplification reaction. FRET cassette 1 did not have a 5' flap sequence. FRET cassette 2 also does not contain a 5′ flap but is a flapless FRET cassette labeled with a CAL Fluor Red 610 fluorophore (Biosearch Technologies, Inc.; Novato, CA) and a BHQ®-2 quencher moiety (Biosearch Technologies, Inc.) and was used to detect analyte B.After the cleavage reaction, the emission signal from the CAL Fluor Red 610 fluorophore was detectable in the ROX channel but not the HEX channel of the instrument used to amplify the nucleic acid and monitor the progress of the amplification reaction. FRET cassette 3, which was also used to detect analyte B, contained a BHQ® quenching moiety (Biosearch) attached to the hairpin portion. The 5' flap FRET cassette was labeled on the 5' flap with a CAL Fluor® Orange 560 fluorophore quenched with Biosearch Technologies, Inc. After the cleavage reaction, the emission signal from the CAL Fluor® Orange 560 fluorophore was detectable in the HEX channel of the instrument used to amplify the nucleic acid and monitor the progress of the amplification reaction. A masking oligonucleotide complementary to the 5' flap of FRET Cassette 3 and containing a BHQ® quenching moiety (Biosearch Technologies, Inc.) was included in the reaction mixture at a 3-fold molar excess over FRET Cassette 3.

[0178] The target analytes, FRET cassette configurations, and detection channels used in this procedure are summarized in Table 1. [Table 1]

[0179] Amplification reactions using the detection system shown in Table 1 included either the analyte A target alone, the analyte B target alone, or a combination of analyte A and analyte B targets. A "no target" negative control reaction included all reagents but no added template. Each reaction included all three FRET cassettes and masking oligonucleotides. Thermal cycling and fluorescence monitoring were performed using an ABI 7500 Real-Time PCR System instrument (ThermoFisher Scientific; Grand Island, NY). Reaction conditions included 10 cycles of 95°C for 120 s, 69°C for 5 s, 67°C for 5 s, 65°C for 6 s, and 72C for 5 s. This was followed by 40 cycles of 95°C for 10 s, 69°C for 5 s, 67°C for 5 s, and 65°C for 25 s. Fluorescence emission data was collected for the ROX and HEX channels of the real-time PCR instrument at a temperature where the masking oligo remained unhybridized to the 5' flap released from FRET cassette 3. Fluorescence signal was measured in the HEX and ROX channels as a function of cycle number for reactions containing both analyte B and analyte A. The signal in the ROX channel (data not shown) yielded a sigmoidal curve reflecting a cycle-dependent increase in signal indicating that analyte B was amplified in the PCR reaction. The HEX signal reflected the combined signal from the amplification of analyte B and analyte A but did not distinguish one from the other (data not shown). HEX and ROX fluorescent signals were independently detectable in multiplex reactions amplifying analyte B and analyte A.

[0180] Figures 5A-5C display the signal measured in the HEX channel as a function of cycle number for reactions containing the analytes individually or in combination, as indicated. The graph in Figure 5A confirmed that amplified analyte A could be detected in the absence of added analyte B template, exhibiting a characteristic sigmoidal curve for signal accumulation for this analyte. Figure 5B displays the signal measured in the HEX channel showing amplification of analyte B template in the absence of analyte A template. The monotonic signal accumulation curve in this case lacked the sigmoidal characteristics of the curve shown in Figure 5A. Figure 5C shows the signal detected in an amplification reaction containing both analyte A and analyte B templates in a single reaction. Here, the signal measured in the HEX channel increased to produce an extended sigmoidal curve, representing the combined signal produced by the analyte A and analyte B FRET cassettes measured in a single channel of the fluorometer. In this case, signals produced by non-identical fluorophores were detected in a single (i.e., same) channel of the fluorometer. As discussed elsewhere herein, melting / annealing curve analysis and analysis of the curve shape (eg, first derivative analysis) can be used to infer the identity of the analytes giving rise to each different result.

[0181] Example 2 shows a procedure for resolving the identity of different analyte nucleic acids in a multiplex amplification reaction mixture using a masking oligonucleotide to quench the signal from the 5' flap FRET cassette used to detect analyte B. Only the fluorescence emitted from the label of the cleaved 5' flap complementary to the masking oligo was subject to quenching.

[0182] Example 2 End-point melting / annealing curve analysis distinguishes amplified targets The post-PCR reaction mixture from Example 1 was used for melting / annealing curve analysis on the same real-time PCR instrument used for nucleic acid amplification. This involved monitoring the magnitude of the fluorescent signal in the HEX channel of the real-time PCR instrument as the temperature was varied from 90° C. to 21.4° C.

[0183] The results of the post-amplification melting / annealing curve analysis are shown in Figure 6. These data confirmed that the cleavage products of the FRET cassette specific for the detection of analyte A, with the HEX channel signal produced from the flapless FRET cassette that cannot be quenched by the masking oligonucleotide, remained uniformly fluorescent as a function of temperature (i.e., across the temperature range shown). In contrast, the HEX channel signal produced after cleavage of the 5' flap FRET cassette 3 specific for the detection of analyte B in the presence of a masking oligonucleotide containing a quenching moiety, exhibited temperature-dependent fluorescence quenching. More specifically, the fluorescence in the reaction mixture spiked with only the analyte B template was effectively quenched as the temperature approached 20°C, resulting in a decrease in the fluorescence signal toward 500,000 RLU. Experimentally, it was observed that virtually all of the fluorescence was quenched at temperatures below 40°C. In reactions containing both analyte A and analyte B templates, the signal in the HEX channel was essentially a combination of the individual temperature profiles. More specifically, the melting / annealing curve profile followed that of the analyte B template reaction, but with an overall increase in signal due to the addition of unaltered fluorescence due to the presence of analyte A in the template reaction. As the temperature approached 20°C, the signal indicative of the presence of analyte B was quenched, so that the fluorescence in the mixed reaction approached the signal level observed in the analyte A only reaction. As shown, the fluorescence was maximized at approximately 63°C in the reaction mixture containing the analyte B template and decreased above 63°C. Without wishing to be limited by a particular theory of operation, masking oligo hybridization is believed to be a step in which the T of the 5' flap masking oligonucleotide duplex is increased by 100%. m This decrease in fluorescence above 63 °C is likely due to a temperature-dependent buffering effect on the fluorophore, as it should be reduced or completely lost at substantially higher temperatures (e.g., 10 °C-20 °C higher).

[0184] The results obtained using the invasive cleavage system for multiplex detection of nucleic acid analytes disclosed herein can be processed in different ways to determine the presence or absence of the analytes in the test sample. For example, the fluorescence signals measured at two different temperatures (e.g., 63°C and 30°C) in a single channel (e.g., the HEX channel of the fluorometer in this figure) can be compared to a threshold value to establish the presence or absence of each of the two different analytes. The threshold value may be established in advance (i.e., before performing the assay) or at the time the assay is performed (e.g., using one or more calibration standards with one or more analytes to be detected). This analytical method can be used to determine the presence or absence of analytes in a test sample. 6 RFU and 1 × 10 6 An exemplary threshold in RFU can be shown using the results in Figure 6. Under these parameters, 2.2 x 10 6 A fluorescence reading above RFU indicated detection of analyte B, while a reading below this threshold indicated the absence of analyte B. 6 RFU and 2.2 × 10 6 A fluorescence reading between RFU indicated the presence of analyte A and the absence of analyte B. Similarly, or alternatively, at 30° C., 1×10 6 A fluorescence reading above 1 × 10 RFU indicated the presence of analyte A. Such a reading would not inform about the presence of analyte B, since the fluorescence from the cleaved 5' flap, which indicates the presence of that analyte, was virtually completely quenched at that temperature. 6 A fluorescence reading below the RFU indicates the absence of analyte A.

[0185] Derivative-based data analysis techniques can be used in place of or in combination with the threshold-based analysis described above. For example, a data plot relating to the presence of analyte A (see FIG. 6) may have a constant slope (e.g., 0 slope) and a slope of approximately 5×10 5The fluorescence magnitude is at least two times greater than the background fluorescence in RFU. This fluorescence magnitude (e.g., measured at the temperature at which fluorescence quenching in the reaction mixture is at a maximum) can be used to indicate the presence of analyte A. A data plot relating to the presence of analyte B exhibited a first derivative maximum in the range of about 50°C to 58°C and a zero point crossing (i.e., an x-axis crossing indicating zero slope) at about 63°C. Any data set exhibiting these characteristics can be interpreted as indicating the presence of analyte B. In some embodiments, the fluorescence magnitude at a particular temperature can be used for threshold-based analysis, the temperature-dependent rate of change of fluorescence can be used for derivative-based data analysis, and the two analyses can be combined to make a determination regarding the presence or absence of each of multiple analytes that may be present in the assay reaction.

[0186] Taken together, the above results and discussion demonstrated that unique profiles characterized the melting / annealing curves for each of the three different starting target conditions (i.e., analyte A only, analyte B only, or a combination of analytes A and B). These data indicate that end-point melting / annealing curve analysis using a single detection channel readily resolved the presence of one or more targets in the reaction mixture.

[0187] The preceding examples have demonstrated the detection of two different amplified nucleic acid target sequences using invasive cleavage reactions using real-time and end-point format nucleic acid analysis. Fluorescent signals for the different amplified targets were detected in a single optical channel (i.e., the HEX channel) of the fluorometer component of the instrument that monitored the nucleic acid amplification as the reaction was occurring (e.g., as a function of time or cycle number). This procedure demonstrated that a hybrid duplex containing a cleaved flap containing a masking oligonucleotide and a fluorescent label was cleaved at the T of the duplex. m It is stable at lower temperatures and has a duplex T mThe fact that fluorophores are unstable at higher temperatures was taken advantage of. The results established that maximum quenching was observed at approximately 39°C, and minimum quenching was observed at approximately 63°C. Taken together, the results shown in Figures 5 and 6 demonstrate how multiple different nucleic acid analytes can be detected and resolved from one another in a multiplex reaction mixture using only a single optical channel of a fluorometer. This was accomplished using different fluorophore species detected in the same optical channel of a fluorometer, although a single species of fluorophore could be used instead (e.g., as demonstrated below). Additionally, one of skill in the art will understand how different fluorescent labels can be substituted for the exemplary labels listed above, and how instrument channels other than the HEX channel can be used for detection of those labels.

[0188] In Example 3, a procedure is described for detecting the presence or absence of multiple analytes using real-time monitoring and only a single optical channel of the PCR instrument's fluorometer. Here, the cycling procedure included a temperature step where fluorescence quenching by masking oligonucleotides allowed for the determination of the presence or absence of each of the two analytes. More specifically, fluorescence readings were determined at 63°C and 39°C during the cycles of the PCR procedure. As shown below, this technique differentiated in a real-time format the dual signals measured in a single optical channel. This procedure can be advantageously used for quantification of each of the detected analytes according to standard procedures for processing real-time amplification trial curves by determining the cycle number at which a threshold level of amplification is achieved (e.g., Ct value). The determined Ct value can then be compared to a calibration plot or formula relating a threshold and the amount or concentration of the analyte nucleic acid. An alternative real-time procedure can establish whether the analyte nucleic acid is present in a multiplex reaction mixture above or below a specified (e.g., predetermined) amount or concentration level. This can include establishing whether a particular level of reaction progression (e.g., measured by Ct value) is achieved by a specified number of cycles. In particular, where the previous examples used two different fluorophores detected in the same light channel of a fluorometer linked to a PCR instrument, here the same fluorophore is used to detect two different analytes.

[0189] Example 3 Real-time monitoring of multiplex amplification using a single fluorophore species The reaction mixture for the real-time amplification protocol was prepared as follows: Serial dilutions of plasmid DNA containing the target sequence of specimen A were prepared from a standard stock solution. Wild-type bacterial genomic DNA carrying the target sequence of specimen B served as the source of template nucleic acid for the amplification of that specimen. The lyophilized pellet described in Example 1 was reconstituted with an aqueous buffer and then spiked with each of the 5' flap FRET cassettes and corresponding masking oligonucleotides specific for the detection of specimen B. Again, the cleavage of the FRET cassette in the reaction mixture was catalyzed by the primary cleavage flap from the primary probe.

[0190] Four reaction mixtures were prepared in duplicate using multiwell PCR plates (one plate each to demonstrate high and low temperature monitoring). The negative control mixture received no nucleic acid template for either analyte A or analyte B. A second set of mixtures received only bacterial genomic DNA containing the analyte B template, but not the analyte A plasmid. A third set of mixtures received only the analyte A plasmid and no bacterial genomic DNA containing the analyte B template. A fourth set of mixtures received both bacterial genomic DNA containing the analyte B template and the analyte A plasmid. All runs contained a three-fold excess of masking oligo relative to the corresponding 5' flap FRET cassette used to detect analyte B. PCR reactions with monitoring of the fluorescent signal generated by invasive cleavage of the FRET cassette were performed on an ABI 7500 real-time PCR instrument using either a first set of cycling conditions with fluorescence monitoring at 63 °C ("high" temperature monitoring) or a second set of cycling conditions with fluorescence monitoring at 39 °C ("low" temperature monitoring). The cycling conditions used for high temperature (63°C) fluorescence monitoring were as follows: (1) 95°C for 120 seconds; (2) 95°C for 15 seconds; 69°C for 5 seconds; 67°C for 5 seconds; 65°C for 6 seconds; and 72°C for 25 seconds x 10 cycles (initial Taq optimization stage), (3) 95°C for 10 seconds; 69°C for 5 seconds; 67°C for 5 seconds; 65°C for 5 seconds; and 63°C for 25 seconds x 40 cycles. The cycling conditions used for low temperature (39°C) fluorescence monitoring were as follows: (1) 95°C for 120 seconds; (2) 95°C for 15 seconds; 69°C for 5 seconds; 67°C for 5 seconds; 65°C for 6 seconds; and 72°C for 25 seconds x 10 cycles (initial Taq optimization stage), (3) 95°C for 10 seconds; 69°C for 5 seconds; 67°C for 5 seconds; 65°C for 5 seconds; and 39°C for 25 seconds x 40 cycles. A total of 50 cycles of fluorescence monitoring at high and low temperatures were performed.

[0191] The results of the procedure are shown in the real-time PCR amplification plots in Figures 7A-7D. The most interesting results were the runs performed using sample B template alone or sample B template in combination with sample A template. Reactions using sample A template alone were treated as controls and are not shown in the figures.

[0192] Figures 7A and 7B show analyte B-specific fluorescence as a function of cycle number, where the fluorescence was determined in the HEX channel of a PCR instrument at 63°C (no fluorescence quenching) or 39°C (fluorescence quenched by the masking oligonucleotide). An increase in fluorescence above background was evident at 63°C starting at about cycle 12 and continuing through cycle 40, as shown in Figure 7A. Fluorescence measurements taken at 39°C remained at background levels throughout the procedure, as shown in Figure 7B. These data confirmed that quenching of the fluorescence signal of analyte B was essentially complete at lower temperatures due to hybridization of the cleaved flap to the complementary masking oligonucleotide. The fluorescence observed at lower temperatures (39°C) in the plot of Figure 7B was due to background fluorescence and not due to a signal indicative of the presence of analyte B.

[0193] Figures 7C and 7D show real-time trial curve results obtained by monitoring the fluorescent signal produced in a reaction that amplified and detected a combination of analyte B and analyte A template nucleic acids. The curve shown in Figure 7C, where the fluorescent readout was measured during a temperature step at 63°C, reflects the combined contribution from the fluorescence produced from cleavage of both FRET cassettes. Only cleavage of the FRET cassette, indicating the presence of analyte B, resulted in a fluorescent flap sequence that could be hybridized by a masking oligonucleotide, which has the effect of quenching the fluorescent signal at 39°C. As shown in Figure 7C, the fluorescent signal measured at the 63°C temperature step (i.e., without fluorescence quenching) began to rise above background levels at about cycle 11 and entered a log-linear phase by about cycle 19. The rate of fluorescence increase began to taper off by about cycle 29, while the magnitude of the signal continued to increase. The curve shown in Figure 7D reflects the fluorescent readout measured during a temperature step at 39°C. The signal from the cleaved flap, indicating the presence of analyte B, was efficiently quenched at this temperature, so the fluorescence measured in Figure 7D was solely from the cleavage reaction, indicating the presence of analyte A.

[0194] Taken together, the results presented in Figures 7A-7D show that a single reaction mixture can be used to detect multiple target nucleic acids using real-time monitoring of only a single fluorescence detection channel (e.g., monitoring emission from a single fluorophore species) and that each target can be distinguished by monitoring the fluorescence emission at different temperatures. When the disclosed technique was carried out, one temperature corresponded to the condition of fluorescence quenching (i.e., substantially complete or maximal quenching of the fluorescence resulting from the cleaved flap). Detection of analyte B demonstrated this condition. Different temperatures in the procedure did not quench the fluorescence resulting from the cleaved flap.

[0195] Although the reactions used to produce the results shown in Figures 7A-7D were run sequentially on the same instrument (i.e., fluorescence measurements were taken during either the 63 °C step or the 39 °C step), it is preferable to monitor a single reaction mixture for fluorescence at both temperature steps to simplify detection of multiple analytes in the system. Again, this is possible because detection of analyte B signal was effectively removed from double positive samples (i.e., samples with analyte B and analyte A) by fluorescence quenching, thereby leaving only a signal indicating the presence of analyte A.

[0196] As described above, results from real-time monitoring of a multiplex reaction mixture can be used to quantify the analyte nucleic acid or determine whether the analyte nucleic acid is present in an amount greater than or less than a threshold amount or concentration (which may be, for example, zero concentration). In some embodiments, quantifying the analyte nucleic acid can include comparing the determined Ct value to a calibration plot or formula relating Ct value and analyte concentration. In other embodiments, determining whether the analyte nucleic acid is present in an amount greater than or less than a threshold amount or concentration can include determining whether a time-dependent fluorescence value reaches a certain level of reaction progress by a specified time or number of cycles. Using the data from Figures 7A-7D as an example, a qualitative determination regarding the presence or absence of an analyte can include determining whether a fluorescence reading of at least 500,000 RFU (relative fluorescence units) is achieved by 30 PCR cycles. The fluorescent signal in Figures 7B and 7D due to detection of analyte B has been effectively eliminated by fluorescence quenching. The increase in signal observed in the plot of Figure 7A compared to the plot of Figure 7B indicates a contribution of fluorescence due to detection of analyte B, thus confirming the presence of that target in the reaction mixture. Thus, comparing the results obtained from Figures 7A and 7B indicates that the reaction mixture contained only analyte B and no analyte A. The increase in signal observed in the plot of Figure 7C compared to the plot of Figure 7D indicates a contribution of fluorescence due to the detection of analyte B in that reaction mixture. Again, the remaining fluorescence plotted in Figure 7D is attributable to the signal resulting from the detection of analyte A. Thus, comparing the results obtained from Figures 7C and 7D indicates that the reaction mixture contained both analyte A and analyte B. A similar process can be used to assess the presence or absence of three different analytes.

[0197] Example 4 describes a procedure in which an invasive cleavage reaction was used to detect three different analyte nucleic acid sequences in a single reaction mixture, in which the three FRET cassettes were labeled with the same fluorophore (i.e., HEX). Of course, other fluorophores that can be detected in the same or different single optical channels of a fluorometer in optical communication with the instrument that amplifies the nucleic acids can be used in place of the HEX fluorophore.

[0198] Example 4 Multiplexed detection of three analyte nucleic acids using a single type of fluorophore Invasive cleavage detection of three analyte nucleic acids amplified by PCR was performed using three different sets of oligonucleotides in the same reaction mixture. Each set of detection oligonucleotides was used to detect a different one of the three analytes (Analyte A, Sample B, and Sample C). Each assay reaction used: (1) a unique primary probe with a target-specific binding sequence and a unique 5' flap that was not complementary to the target amplification product to be detected; (2) a unique oligonucleotide (referred to as the "invasive primer") that served as an invasive oligonucleotide to cleave the 5' flap from the primary probe when the primary probe hybridized to its cognate target nucleic acid and further served as a primer in the amplification reaction; and (3) a unique FRET cassette. Each of the three different FRET cassettes was labeled with a HEX fluorophore and a quencher moiety. The FRET cassette used to indicate the presence of Sample A did not contain a 5' flap hybridized by any masking oligo in the reaction mixture. More specifically, the invasive primers, primary probe and flapless FRET cassette of Example 1 were used to detect analyte A. The FRET cassettes for detecting analytes B and C contained 5' flap sequences. The FRET cassettes are shown in Figure 3 along with the associated cleavage flaps from the primary probe and masking oligos used in the procedure above. The FRET cassette systems for detecting analytes A, C and B appear at the top, middle and bottom of the figure, respectively.

[0199] The multiplex reaction mixture contained two different masking oligonucleotides, one complementary to the 5' flap of the FRET cassette used to detect analyte B and one complementary to the 5' flap of the FRET cassette used to detect analyte C. Each masking oligonucleotide contained a quenching moiety at its 5' end. The FRET 5' flap masking oligonucleotide duplex for analyte B had a GC content of approximately 60% and the FRET 5' flap masking oligonucleotide duplex for analyte C had a GC content of approximately 40%. The two duplexes contained different T m The cleavage product of the 5' flap FRET cassette and the complementary masking oligonucleotide used to detect analyte B formed a more stable duplex for "high temperature" detection, whereas the cleavage product of the 5' flap FRET cassette and the complementary masking oligonucleotide used to detect analyte C formed a less stable duplex for "low temperature" detection.

[0200] Individual reaction mixtures contained all assay oligonucleotides (including all three FRET cassettes) along with all reagents necessary to perform PCR amplification of the three analyte nucleic acids, cleavage of the primary probes specific for the different analyte nucleic acids, and cleavage of the corresponding FRET cassettes. Reactions contained either analyte A alone, analyte B alone, analyte C alone, or a combination of analytes A, B, and C. Thermal cycling conditions were as follows: (1) 95°C for 120 s × 1 cycle; (2) 95°C for 15 s, 69°C for 5 s, 67°C for 5 s, 65°C for 6 s, and 72°C for 25 s × 10 cycles (initial Taq optimization stage); and (3) 95°C for 10 s, 69°C for 5 s, 67°C for 5 s, and 65°C for 25 s × 40 cycles. Melting / annealing curve analysis after amplification over a range of 90°C to 20.7°C was performed on an ABI 7500 real-time PCR instrument.

[0201] Figure 8 shows the post-amplification melting / annealing curve results for reactions performed using each of the three analyte polynucleotides individually or in combination with one another. The results confirmed that each of the four reactions yielded a unique melting / annealing curve profile, thereby demonstrating successful single-channel multiplexing of three analytes using a single type of fluorophore.

[0202] The results shown in the two panels of FIG. 9 show the unique melting / annealing curve profiles observed for the additional analyte combinations. The left panel of FIG. 9 shows the melting / annealing curve results of the reactions in which analyte B and analyte C were amplified individually. The results show how the cleaved 5' flaps from each of the FRET cassettes (where the cleaved 5' flaps hybridized to different masking oligos) can be distinguished from each other using only a single fluorescence monitoring channel of the real-time PCR instrument. The right panel of FIG. 9 shows the melting / annealing curve analysis results of the reactions in which either analyte B alone, analyte C alone, or a combination of analyte B and analyte C. Again, the results show how the cleaved 5' flaps from each of the FRET cassettes (where the cleaved 5' flaps hybridized to different masking oligos) can be distinguished from each other using only a single fluorescence monitoring channel of the real-time PCR instrument.

[0203] FIG. 10 presents a first derivative plot of the measured fluorescence data shown in the two panels of FIG. 9. The left panel of FIG. 10 shows a first derivative plot of the melting / annealing curve shown in the left panel of FIG. 9. The maximum of the first derivative plot of fluorescence as a function of temperature corresponds to the melting temperature (T m ) is shown. In this case, the T mwere separated from each other by about 10°C (i.e., about 46°C for the detection of analyte C and about 56°C for the detection of analyte B). This distinction between the two melting / annealing curves made it possible to resolve which of the two analytes were present in the reaction mixture being analyzed. A single maximum observed or detected at about 46°C indicates the presence of analyte C, and a single maximum at about 56°C indicates the detection of analyte B). The right panel of Figure 10 shows the first derivative of the melting plot that appears in the right panel of Figure 9. Each of the different curves had unique features that allowed distinguishing reaction mixtures containing analyte B, analyte C, or a combination of analyte B and analyte C. The fluorescence signal resulting from the presence of analyte A is not quenchable as a function of temperature (i.e., there is no masking oligo complementary to the FRET cassette cleavage product), so there is no effect on the first derivative (i.e., a constant first derivative is 0).

[0204] In the above example, a test sample was analyzed for the presence of two different analytes using derivative analysis of the melting / annealing curves. Here, a first derivative calculation was used for illustrative purposes. However, second or even higher order derivative analysis is contemplated for this purpose. This procedure involved detecting or monitoring a fluorescent signal in an amplification reaction mixture using only a single channel of the fluorometer component of a real-time nucleic acid amplification device. The plot can present the first derivative of the raw data, but alternatively, can present the first derivative of processed data (e.g., smoothed, normalized to a constant maximum reading, etc.). Detection of a peak or maximum in the first derivative plot at 46° C. indicated the presence of analyte C or its amplification product. Detection of a peak or maximum in the first derivative plot at 56° C. indicated the presence of analyte B or its amplification product. Detection of peaks at both 46° C. and 56° C. (e.g., high level signal at both of these temperatures) indicated the presence of both analyte C and analyte B, or the amplification products of these analytes. In some embodiments, the analysis can include identifying signals greater than a threshold (e.g., a pre-established threshold, or a threshold based on a fraction or percentage of the normalized maximum). Although the examples show the use of first derivative analysis, second derivative analysis can be used instead. Here, the maximum on the first derivative plot corresponds to the zero crossing point on the second derivative plot. Detecting the presence of analyte A did not rely on derivative analysis because the FRET cassette used to detect this analyte did not contain a 5' flap sequence complementary to any masking oligo. Cleavage of this FRET cassette resulted in a fluorescent signal that remained stable over the temperature range used for melting / annealing curve analysis. The presence of analyte A in the test sample was reflected by the magnitude of fluorescence at the temperature at which the signal resulting from cleavage of the other FRET cassettes (e.g., to detect analyte B and / or analyte C) was quenched (e.g., about 30°C in the plot of Figure 10).

[0205] All literature and similar materials cited in this application, including but not limited to patents, patent applications, papers, books, articles, and Internet web pages, are expressly incorporated by reference in their entirety for any purpose.Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the various embodiments described herein belong.If the definition of a term in the incorporated reference appears to be different from the definition provided in the present teachings, the definition provided in the present teachings shall prevail.

[0206] Various modifications and variations of the described compositions, methods, and uses of the technology will be apparent to those skilled in the art without departing from the scope and spirit of the described technology. Although the technology has been described in connection with certain exemplary embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention that are obvious to those skilled in the art of biochemistry, molecular biology, or related fields are intended to be within the scope of the following claims. In certain embodiments, for example, the following are provided: (Item 1) A composition comprising a FRET cassette reporter system, said composition comprising: (i) a 5' flap FRET cassette oligonucleotide, a 5' flap portion comprising a first fluorophore moiety; a stem loop portion comprising a first quencher moiety; a 3' portion comprising a cleaved flap hybridizing sequence, hybridization of a cassette-specific invasive oligonucleotide complementary to the cleaved flap hybridizing sequence of the 5' flap FRET cassette oligonucleotide forms an invasive cleavage structure cleavable by a FEN-1 endonuclease at a cleavage site between the first fluorophore moiety and the first quencher moiety; a 5' flap FRET cassette oligonucleotide, wherein cleavage of the 5' flap FRET cassette oligonucleotide at the cleavage site produces a cassette cleavage flap comprising the 5' flap portion and the first fluorophore moiety; and (ii) a masking oligonucleotide comprising a second quencher moiety, at least a portion of the masking oligonucleotide is capable of specifically hybridizing to the 5' flap portion of the FRET cassette oligonucleotide; hybridization of the masking oligonucleotide to the cassette cleavage flap forms a duplex having a first melting temperature exhibiting a first melting peak; a masking oligonucleotide, wherein the fluorescent emission from the first fluorophore moiety in the duplex is quenched by the second quencher moiety; A composition comprising: (Item 2) 2. The composition of claim 1, wherein the first quencher moiety and the second quencher moiety are the same as each other. (Item 3) 3. The composition of either item 1 or item 2, further comprising a FEN-1 endonuclease. (Item 4) 4. The composition of claim 3, wherein the FEN-1 endonuclease is a thermostable FEN-1 endonuclease. (Item 5) 5. The composition of claim 4, wherein the thermostable FEN-1 endonuclease is derived from an archaeal organism. (Item 6) further comprising a first target-specific invasive oligonucleotide and a first target-specific primary probe oligonucleotide; each of the first target-specific invasive oligonucleotide and the first target-specific primary probe oligonucleotide comprises a sequence configured to hybridize to a target nucleic acid to form an invasive cleavage structure cleavable by a FEN-1 endonuclease to generate a primary cleavage flap; 6. The composition of any one of items 1 to 5, wherein the primary cleavage flap is a cassette-specific invasive oligonucleotide configured to hybridize to the cleaved flap hybridizing sequence of the 5' flap FRET cassette oligonucleotide to form an invasive cleavage structure cleavable by the FEN-1 endonuclease. (Item 7) 7. The composition according to item 6, further comprising the target nucleic acid. (Item 8) 8. The composition of claim 7, further comprising deoxynucleoside triphosphates (dNTPs), a thermostable DNA polymerase, and a primer having a 3' end extendable by the thermostable DNA polymerase in a template-dependent nucleic acid amplification reaction using the target nucleic acid as a template. (Item 9) (iii) Below: a 5' portion comprising a second fluorophore moiety; a stem loop portion including a third quencher portion; a 3' portion containing a second cleaved flap hybridizing sequence; and a second FRET cassette oligonucleotide comprising: hybridization of a second cassette-specific invasive oligonucleotide to the second cleaved flap hybridizing sequence of the second FRET cassette oligonucleotide forms an invasive cleavage structure cleavable by a FEN-1 endonuclease at a cleavage site between the second fluorophore moiety and the third quencher moiety; cleavage of the second FRET cassette oligonucleotide at the cleavage site produces a cassette cleavage product that includes the second fluorophore moiety. The composition according to any one of items 1 to 8. (Item 10) the second FRET cassette oligonucleotide is a second 5' flap FRET cassette comprising a 5' flap portion, the cassette cleavage product is a second cassette cleavage flap comprising the second fluorophore, and the composition comprises: (iv) a second masking oligonucleotide comprising a fourth quencher moiety, at least a portion of the second masking oligonucleotide is capable of specifically hybridizing to the 5' flap portion of the second FRET cassette oligonucleotide; hybridization of the second masking oligonucleotide to the second cassette cleavage flap forms a second duplex having a second melting temperature higher than the first melting temperature; 10. The composition of claim 9, further comprising a second masking oligonucleotide, wherein the fluorescent emission from the second fluorophore moiety in the second duplex is quenched by the fourth quencher moiety. (Item 11) 10. The composition of claim 9, wherein the second cassette cleavage product comprises 5 or less, preferably 4 or less, preferably 3 or less, preferably 2 or less nucleotides. (Item 12) 12. The composition according to any one of items 9 to 11, wherein the emission signals from the first fluorophore moiety and the second fluorophore moiety are detectable in the same fluorescence detection channel of a fluorescence monitoring device. (Item 13) 13. The composition according to any one of items 9 to 12, wherein the first fluorophore moiety and the second fluorophore moiety are the same as each other. (Item 14) 13. The composition according to any one of items 9 to 12, wherein the first fluorophore moiety and the second fluorophore moiety are not the same as each other. (Item 15) 15. The composition according to any one of items 10 and 12-14, wherein the third quencher moiety and the fourth quencher moiety are the same as each other. (Item 16) 1. A method for determining which of two different FRET cassettes in a reaction mixture has been cleaved to generate a fluorescent signal, the method comprising: (a) performing a multiple invasive cleavage reaction in said reaction mixture to cleave one or both of the first FRET cassette and the second FRET cassette to produce two distinct fluorescent cleavage products, if cleavage occurs; the first FRET cassette comprises a first 5' flap portion having a fluorophore attached thereto, the attachment of the fluorophore being arranged such that cleavage of the first FRET cassette by a FEN-1 endonuclease in the multiple invasive cleavage reaction produces a first cassette cleavage flap comprising the fluorophore; the reaction mixture comprises a first masking oligonucleotide that stably hybridizes to the first cassette cleavage flap to form a first duplex at a temperature below a first Tm but not above the first Tm, and fluorescent emission from the fluorophore of the first cassette cleavage flap of the first duplex is quenched; each of the two different fluorescent cleavage products produced in the multiple invasive cleavage reactions is characterized by a different temperature-dependent fluorescence quenching profile in the reaction mixture; (b) measuring fluorescent signals produced in the reaction mixture using a single channel of a fluorescence monitoring device under temperature conditions that differentially quench fluorescence produced by the different fluorescent cleavage products of the multiple invasive cleavage reactions; (c) determining from the result of step (b) which of the different FRET cassettes was cleaved in the multiple invasive cleavage reaction; A method comprising: (Item 17) 17. The method of claim 16, wherein the second FRET cassette in step (a), when cleaved, produces a fluorescent cleavage product that does not hybridize to any masking oligo in the reaction mixture resulting in fluorescence quenching. (Item 18) 18. The method of claim 17, wherein step (c) comprises comparing the fluorescent signals measured at a temperature below the first Tm and at a temperature above the first Tm. (Item 19) 20. The method according to claim 18, wherein step (c) comprises comparing the fluorescent signals by calculating the difference between the measured fluorescent signals. (Item 20) step (b) comprises measuring any of the fluorescent signals at a temperature below the first Tm, whereby fluorescent emission from the fluorophore of the first cassette cleavage flap of the first duplex is quenched; step (c) comprises determining that the second FRET cassette has been cleaved in the reaction mixture if measurable fluorescence is detected in step (b); Item 18. The method according to item 17. (Item 21) step (b) comprises measuring any of said fluorescent signals at a temperature below said first Tm and at a temperature above said first Tm, 18. The method of claim 17, wherein step (c) comprises determining that the first FRET cassette is cleaved in the reaction mixture if the fluorescent signal measured at a temperature higher than the first Tm is greater than the fluorescent signal measured at a temperature lower than the first Tm. (Item 22) 22. The method according to any one of items 16 to 21, wherein step (b) comprises measuring any of the fluorescent signals produced in the reaction mixture as a function of temperature to generate a melting / annealing curve. (Item 23) 23. The method of claim 22, wherein step (c) comprises calculating a derivative of the melting / annealing curve and then determining from the calculated derivative whether the reaction mixture contains the first duplex characterized by the first Tm as an indication that the first FRET cassette has been cleaved in the reaction mixture. (Item 24) the second FRET cassette comprises a second 5' flap sequence having a fluorophore attached thereto, the attachment of the fluorophore being arranged such that cleavage of the second FRET cassette by the FEN-1 endonuclease in the multiple invasive cleavage reaction produces a second cassette cleavage flap comprising the fluorophore; the reaction mixture comprises a second masking oligo that hybridizes to the second cassette cleavage flap to form a second duplex at a temperature below a second Tm but does not hybridize at a temperature above the second Tm, and fluorescent emission from the fluorophore of the second cassette cleavage flap of the second duplex is quenched; 17. The method of claim 16, wherein the first Tm and the second Tm differ by at least 5°C. (Item 25) the first Tm is greater than the second Tm; step (b) comprises measuring any of said fluorescent signals at a temperature below said second Tm and above said first Tm; 25. The method of claim 24, wherein step (c) comprises determining that at least one of the first FRET cassette and the second FRET cassette is cleaved in the reaction mixture if the fluorescent signal measured at a temperature higher than the first Tm is greater than the fluorescent signal measured at a temperature lower than the second Tm. (Item 26) 26. The method of any of items 24 or 25, wherein step (b) comprises measuring any of the fluorescent signals produced in the reaction mixture as a function of temperature to generate a melting / annealing curve. (Item 27) 27. The method of claim 26, wherein step (c) comprises calculating a derivative of the melting / annealing curve and then determining from the calculated derivative whether the reaction mixture contains the first duplex characterized by the first Tm as an indication that the first FRET cassette has been cleaved in the reaction mixture. (Item 28) 27. The method of claim 26, wherein step (c) comprises calculating a derivative of the melting / annealing curve and then determining from the calculated derivative whether the reaction mixture contains the second duplex characterized by the second Tm as an indication that the second FRET cassette has been cleaved in the reaction mixture. (Item 29) 29. The method of any one of items 16 to 28, wherein the first and second FRET cassettes are labeled with the same fluorophore. (Item 30) 29. The method of any one of items 16 to 28, wherein the first and second FRET cassettes are not labeled with the same fluorophore. (Item 31) 31. The method of any one of items 16 to 30, wherein the FEN-1 endonuclease of the multiple invasive cleavage reaction in step (a) comprises a thermostable FEN-1 endonuclease. (Item 32) 32. The method according to any one of items 16 to 31, wherein step (c) comprises determining using a computer programmed with software. (Item 33) 1. A method for analyzing a sample containing a target nucleic acid, comprising: (a) contacting in a reaction mixture any of the first target nucleic acids of the sample with a first primary probe oligonucleotide comprising a sequence complementary thereto and a FEN-1 endonuclease under conditions such that, if the first primary probe oligonucleotide hybridizes to the first target nucleic acid, the first primary probe is cleaved by the FEN-1 endonuclease to generate a first primary cleavage flap; the first primary cleavage flap hybridizes to a cleaved flap hybridizing sequence of a first FRET cassette oligonucleotide included in the reaction mixture to form an invasive cleavage structure that is cleaved by the FEN-1 endonuclease at a cleavage site between a first fluorophore moiety and a first quencher moiety of the first FRET cassette oligonucleotide to release a first cassette cleavage flap that includes the first fluorophore moiety; a first masking oligonucleotide comprising a second quencher moiety hybridizes to the first cassette cleavage flap to form a duplex at a temperature below a first Tm of the first masking oligonucleotide and the first cassette cleavage flap; fluorescent emission from the first fluorophore moiety of the duplex is quenched by the second quencher moiety; at a second temperature higher than the first Tm, the first masking oligonucleotide and the first cassette cleavage flap do not form a stable duplex; (b) detecting any fluorescence emitted from the first fluorophore moiety at the second temperature; and (c) Below: if fluorescence emitted from the first fluorophore moiety is detected in step (b), then the sample contains the first target nucleic acid; or If no fluorescence emitted from the first fluorophore moiety is detected in step (b), the sample does not contain the first target nucleic acid. determining whether the A method comprising: (Item 34) step (a) further comprises contacting in the reaction mixture any of the second target nucleic acids of the sample with a second primary probe oligonucleotide comprising a sequence complementary thereto and the FEN-1 endonuclease under conditions such that, if the second primary probe oligonucleotide hybridizes to the second target nucleic acid, the second primary probe is cleaved by the FEN-1 endonuclease to generate a second primary cleavage flap that is different from the first primary cleavage flap; said second primary cleavage flap hybridizes to a cleaved flap hybridizing sequence of a second FRET cassette oligonucleotide included in said reaction mixture to form an invasive cleavage structure that is cleaved by said FEN-1 endonuclease at a cleavage site between a second fluorophore moiety and a third quencher moiety of said second FRET cassette oligonucleotide to release a second cassette cleavage flap that includes said second fluorophore moiety; at a third temperature lower than the second Tm, a second masking oligonucleotide comprising a fourth quencher moiety hybridizes to the second cassette cleavage flap to form a duplex; fluorescent emission from the second fluorophore moiety of the duplex is quenched by the fourth quencher moiety; at a fourth temperature, higher than the second Tm, the second masking oligonucleotide and the second cassette cleavage flap do not form a stable duplex; the first Tm and the second Tm differ from each other by at least 5°C; step (b) further comprises detecting any fluorescence emitted from said second fluorophore moiety at said fourth temperature; Step (c) if fluorescence emitted from the second fluorophore moiety of the 5' flap cleavage product of the second FRET cassette oligonucleotide is detected in step (b), the sample contains the second target nucleic acid; or If no fluorescence emitted from the second fluorophore moiety of the 5' flap cleavage product of the second FRET cassette oligonucleotide is detected in step (b), the sample does not contain the first target nucleic acid. Item 34. The method of item 33, further comprising determining whether the patient is a (Item 35) step (a) further comprises contacting in the reaction mixture any of the second target nucleic acids of the sample with a second primary probe oligonucleotide comprising a sequence complementary thereto and the FEN-1 endonuclease under conditions such that, if the second primary probe oligonucleotide hybridizes to the second target nucleic acid, the second primary probe is cleaved by the FEN-1 endonuclease to generate a second primary cleavage flap; the second primary cleavage flap hybridizes to a cleaved flap hybridizing sequence of a second FRET cassette oligonucleotide included in the reaction mixture to form an invasive cleavage structure that is cleaved by the FEN-1 endonuclease at a cleavage site between the second fluorophore moiety and a third quencher moiety of the second FRET cassette oligonucleotide to release a cleavage product that includes the second fluorophore moiety; the cleavage products do not hybridize to any masking oligonucleotides in the reaction mixture resulting in fluorescence quenching; step (b) further comprises detecting any fluorescence emitted from the second fluorophore moiety of the cleavage product; Step (c) if fluorescence emitted from the second fluorophore moiety is detected in step (b), the sample contains the second target nucleic acid; or if no fluorescence emitted from the second fluorophore moiety is detected in step (b), the sample does not contain the first target nucleic acid; Item 34. The method of item 33, further comprising determining whether the patient is a (Item 36) 36. The method of any one of claims 34 and 35, wherein step (b) comprises detecting any fluorescence emitted from the first and second fluorophore moieties using a single channel of a fluorescence monitoring device. (Item 37) 37. The method of claim 36, wherein step (b) is performed while a nucleic acid amplification reaction is occurring in the reaction mixture, and a product of the nucleic acid amplification reaction comprises the first target nucleic acid and the second target nucleic acid. (Item 38) 38. The method of claim 37, wherein the nucleic acid amplification reaction comprises a step for thermal cycling and the reaction mixture further comprises a thermostable DNA polymerase. (Item 39) 37. The method according to any one of items 33 to 36, wherein step (b) is carried out when the temperature of the reaction mixture is reduced to allow annealing of the masking oligonucleotide and the complementary cassette cleavage flap. (Item 40) 37. The method of claim 36, wherein the first and second fluorophore moieties are the same as each other. (Item 41) 37. The method of claim 36, wherein the step of (b) detecting any fluorescence comprises measuring any fluorescence. (Item 42) 42. The method of claim 41, further comprising either detecting or measuring fluorescence at the first temperature. (Item 43) 36. The method of any one of items 34 or 35, wherein both the first fluorophore moiety and the second fluorophore moiety are detectable in the same fluorescence detection channel of an energy sensor device. (Item 44) Item 44. The method of item 43, wherein the second fluorophore moiety is the same as the first fluorophore moiety. (Item 45) Item 44. The method of item 43, wherein the second fluorophore moiety is not the same as the first fluorophore moiety. (Item 46) 44. The method of any one of items 33 to 43, wherein fluorescence from the second fluorophore is detected and / or measured at the first temperature. (Item 47) 45. The method according to any one of items 33 to 44, wherein the third quencher moiety is the same as the first quencher moiety and / or the second quencher moiety. (Item 48) 48. The method of any one of items 33 to 47, wherein the reaction mixture comprises primer oligonucleotides that amplify a target nucleic acid, and at least one primer oligonucleotide acts as an invasive oligonucleotide in the presence of a primary probe oligonucleotide and a target nucleic acid and / or a target amplicon to form an invasive cleavage structure that is cleaved by the thermostable FEN-1 endonuclease. (Item 49) 49. The method of any one of items 33 to 48, wherein step (c) comprises determining using a computer programmed with software. (Item 50) 1. A system for determining which of a plurality of target nucleic acid analytes are present in a reaction mixture, wherein each target nucleic acid analyte of the plurality of target nucleic acid analytes is detectable by a fluorescent signal, the system comprising: A thermocycler and a fluorometer in optical communication with the thermocycler, a fluorometer that measures a fluorescent signal indicative of production of a nucleic acid amplification product by the thermocycler in a single optical channel; a computer in communication with the fluorometer; The computer includes: (a) obtaining a melting / annealing curve data set generated from measurements performed by said fluorometer; (b) determining the presence of a first target nucleic acid in the reaction mixture by detecting a fluorescent signal from a first fluorescent cleavage product in the melting / annealing curve data set at a temperature at which fluorescence in the reaction mixture is maximally quenched; (c) generating a derivative plot from the melting / annealing curve data set; and (d) determining that a second target nucleic acid is present in the reaction mixture if the derivative plot contains a feature characteristic of a first duplex; said computer being programmed with software instructions; wherein the first duplex comprises a first masking oligonucleotide and a second fluorescent cleavage product produced in the reaction mixture when the second target nucleic acid is present. system. (Item 51) The computer includes: (e) determining that a third target nucleic acid is present in the reaction mixture if the derivative plot contains a feature characteristic of a second duplex; said computer being programmed with software instructions; wherein the second duplex comprises a second masking oligonucleotide and a third fluorescent cleavage product produced in the reaction mixture when the third target nucleic acid is present. Item 51. The system according to item 50. (Item 52) 52. The system of either claim 50 or claim 51, wherein the characteristic feature of the first duplex comprises a maximum, minimum, or zero crossing point of a calculated derivative. (Item 53) the calculated derivatives include calculated first derivatives; the unique feature of the first duplex comprises a first maximum of a calculated first derivative of the melting / annealing curve data set as a first melting peak; the characteristic feature of the second duplex comprises a second maximum of the calculated first derivative of the melting / annealing curve data set as a second melting peak; Item 53. The system according to item 52. (Item 54) 54. The system of any one of items 50 to 53, wherein the thermocycler, the fluorometer, and the computer are all components of a real-time PCR instrument. (Item 55) 51. The system of claim 50, wherein the melting / annealing curve data set generated by the computer comprises data points showing fluorescence as a function of temperature.

Claims

1. 1. A method for determining which of two different FRET cassettes in a reaction mixture has been cleaved to generate a fluorescent signal, the method comprising: (a) performing a multiple invasive cleavage reaction in said reaction mixture to cleave one or both of the first FRET cassette and the second FRET cassette, if cleavage occurs, to produce two different fluorescent cleavage products; the first FRET cassette comprises a first 5' flap portion having a fluorophore attached thereto, the attachment of the fluorophore being arranged such that cleavage of the first FRET cassette by a FEN-I endonuclease in the multiple invasive cleavage reaction produces a first cassette cleavage flap comprising the fluorophore; the reaction mixture comprises a first masking oligonucleotide that stably hybridizes to the first cassette cleavage flap to form a first duplex at a temperature below a first Tm but not above the first Tm, and fluorescent emission from the fluorophore of the first cassette cleavage flap of the first duplex is quenched; each of the two different fluorescent cleavage products produced in the multiple invasive cleavage reactions is characterized by a different temperature-dependent fluorescence quenching profile in the reaction mixture; (b) measuring fluorescent signals produced in the reaction mixture using a single channel of a fluorescence monitoring device under temperature conditions that differentially quench fluorescence produced by the different fluorescent cleavage products of the multiple invasive cleavage reactions; (c) determining from the results of step (b) which of the different FRET cassettes were cleaved in the multiple invasive cleavage reaction; A method comprising:

2. 2. The method of claim 1, wherein the second FRET cassette in step (a), when cleaved, produces a fluorescent cleavage product that does not hybridize to any masking oligo in the reaction mixture resulting in fluorescence quenching.

3. 3. The method of claim 2, wherein step (c) comprises comparing the fluorescent signals measured at a temperature below the first Tm and above the first Tm.

4. 4. The method of claim 3, wherein step (c) comprises comparing the fluorescent signals by calculating the difference between the measured fluorescent signals.

5. step (b) comprises measuring any of said fluorescent signals at a temperature below said first Tm, wherein fluorescent emission from said fluorophore of said first cassette cleavage flap of said first duplex is quenched; step (c) comprises determining that the second FRET cassette has been cleaved in the reaction mixture if measurable fluorescence is detected in step (b); The method of claim 2.

6. step (b) comprises measuring any of said fluorescent signals at a temperature below said first Tm and at a temperature above said first Tm, 3. The method of claim 2, wherein step (c) comprises determining that the first FRET cassette is cleaved in the reaction mixture if the fluorescent signal measured at a temperature higher than the first Tm is greater than the fluorescent signal measured at a temperature lower than the first Tm.

7. 7. The method of any one of claims 1 to 6, wherein step (b) comprises measuring any of the fluorescent signals produced in the reaction mixture as a function of temperature to generate a melting / annealing curve.

8. 8. The method of claim 7, wherein step (c) comprises calculating a derivative of the melting / annealing curve and then determining from the calculated derivative whether the reaction mixture contains the first duplex characterized by the first Tm as an indication that the first FRET cassette has been cleaved in the reaction mixture.

9. the second FRET cassette comprises a second 5' flap sequence having a fluorophore attached thereto, the attachment of the fluorophore being arranged such that cleavage of the second FRET cassette by the FEN-I endonuclease in the multiple invasive cleavage reaction produces a second cassette cleavage flap comprising the fluorophore; the reaction mixture comprises a second masking oligo that hybridizes to the second cassette cleavage flap to form a second duplex at a temperature below a second Tm but does not hybridize at a temperature above the second Tm, and fluorescent emission from the fluorophore of the second cassette cleavage flap of the second duplex is quenched; 2. The method of claim 1, wherein the first Tm and the second Tm differ by at least 5°C.

10. the first Tm is greater than the second Tm; step (b) comprises measuring any of said fluorescent signals at a temperature below said second Tm and above said first Tm; 10. The method of claim 9, wherein step (c) comprises determining that at least one of the first FRET cassette and the second FRET cassette has been cleaved in the reaction mixture if the fluorescent signal measured at a temperature higher than the first Tm is greater than the fluorescent signal measured at a temperature lower than the second Tm.

11. 11. The method of claim 9 or 10, wherein step (b) comprises measuring any of the fluorescent signals produced in the reaction mixture as a function of temperature to generate a melting / annealing curve.

12. 12. The method of claim 11 , wherein step (c) comprises calculating a derivative of the melting / annealing curve and then determining from the calculated derivative whether the reaction mixture contains the first duplex characterized by the first Tm as an indication that the first FRET cassette has been cleaved in the reaction mixture.

13. 12. The method of claim 11 , wherein step (c) comprises calculating a derivative of the melting / annealing curve and then determining from the calculated derivative whether the reaction mixture contains the second duplex characterized by the second Tm as an indication that the second FRET cassette has been cleaved in the reaction mixture.

14. The method of any one of claims 1 to 13, wherein the first and second FRET cassettes are labeled with the same fluorophore.

15. The method of any one of claims 1 to 13, wherein the first and second FRET cassettes are not labeled with the same fluorophore.

16. The method of any one of claims 1 to 15, wherein the FEN-I endonuclease of the multiply invasive cleavage reaction in step (a) comprises a thermostable FEN-I endonuclease.

17. The method of any one of claims 1 to 16, wherein step (c) comprises determining using a computer programmed with software.

18. 1. A method for analyzing a sample containing a target nucleic acid, comprising: (a) contacting in a reaction mixture any of the first target nucleic acids of said sample with a first primary probe oligonucleotide comprising a sequence complementary thereto and a FEN-I endonuclease under conditions such that, if said first primary probe oligonucleotide hybridizes to said first target nucleic acid, said first primary probe is cleaved by said FEN-I endonuclease to generate a first primary cleavage flap; said first primary cleavage flap hybridizes to a cleaved flap hybridizing sequence of a first FRET cassette oligonucleotide included in said reaction mixture to form an invasive cleavage structure that is cleaved by said FEN-1 endonuclease at a cleavage site between a first fluorophore moiety and a first quencher moiety of said first FRET cassette oligonucleotide to release a first cassette cleavage flap that includes said first fluorophore moiety; a first masking oligonucleotide comprising a second quencher moiety hybridizes to the first cassette cleavage flap to form a duplex at a temperature below a first Tm of the first masking oligonucleotide and the first cassette cleavage flap; the fluorescence emission from the first fluorophore moiety of the duplex is quenched by the second quencher moiety; at a second temperature higher than the first Tm, the first masking oligonucleotide and the first cassette cleavage flap do not form a stable duplex; (b) detecting any fluorescence emitted from the first fluorophore moiety at the second temperature; and (c) Below: if fluorescence emitted from the first fluorophore moiety is detected in step (b), then the sample contains the first target nucleic acid; or if no fluorescence emitted from the first fluorophore moiety is detected in step (b), the sample does not contain the first target nucleic acid; determining whether the A method comprising:

19. step (a) further comprises contacting in the reaction mixture any of the second target nucleic acids of the sample with a second primary probe oligonucleotide comprising a sequence complementary thereto and with the FEN-I endonuclease under conditions such that, if the second primary probe oligonucleotide hybridizes to the second target nucleic acid, the second primary probe is cleaved by the FEN-I endonuclease to generate a second primary cleavage flap that is different from the first primary cleavage flap; said second primary cleavage flap hybridizes to a cleaved flap hybridizing sequence of a second FRET cassette oligonucleotide contained in said reaction mixture and forms an invasive cleavage structure that is cleaved by said FEN-1 endonuclease at a cleavage site between a second fluorophore moiety and a third quencher moiety of said second FRET cassette oligonucleotide to release a second cassette cleavage flap that includes said second fluorophore moiety; at a third temperature lower than the second Tm, a second masking oligonucleotide comprising a fourth quencher moiety hybridizes to the second cassette cleavage flap to form a duplex; fluorescent emission from the second fluorophore moiety of the duplex is quenched by the fourth quencher moiety; at a fourth temperature, higher than the second Tm, the second masking oligonucleotide and the second cassette cleavage flap do not form a stable duplex; the first Tm and the second Tm differ from each other by at least 5°C; step (b) further comprises detecting any fluorescence emitted from said second fluorophore moiety at said fourth temperature; Step (c) is if fluorescence emitted from the second fluorophore moiety of the 5' flap cleavage product of the second FRET cassette oligonucleotide is detected in step (b), the sample contains the second target nucleic acid; or If no fluorescence emitted from the second fluorophore moiety of the 5' flap cleavage product of the second FRET cassette oligonucleotide is detected in step (b), the sample does not contain the first target nucleic acid.

20. The method of claim 18, further comprising determining whether

20. step (a) further comprises contacting in said reaction mixture any of said sample second target nucleic acids with a second primary probe oligonucleotide comprising a sequence complementary thereto and said FEN-I endonuclease under conditions such that if said second primary probe oligonucleotide hybridizes to said second target nucleic acid, said second primary probe is cleaved by said FEN-I endonuclease to generate a second primary cleavage flap; said second primary cleavage flap hybridizes to a cleaved flap hybridizing sequence of a second FRET cassette oligonucleotide included in said reaction mixture to form an invasive cleavage structure that is cleaved by said FEN-1 endonuclease at a cleavage site between a second fluorophore moiety and a third quencher moiety of said second FRET cassette oligonucleotide to release a cleavage product comprising said second fluorophore moiety; the cleavage products do not hybridize to any masking oligonucleotides in the reaction mixture resulting in fluorescence quenching; step (b) further comprises detecting any fluorescence emitted from the second fluorophore moiety of the cleavage product; Step (c) is if fluorescence emitted from the second fluorophore moiety is detected in step (b), then the sample contains the second target nucleic acid; or if no fluorescence emitted from the second fluorophore moiety is detected in step (b), the sample does not contain the first target nucleic acid; 20. The method of claim 18, further comprising determining whether

21. 21. The method of claim 19 or claim 20, wherein step (b) comprises detecting any fluorescence emitted from the first and second fluorophore moieties using a single channel of a fluorescence monitoring device.

22. 22. The method of claim 21 , wherein step (b) is performed while a nucleic acid amplification reaction is occurring in the reaction mixture, and a product of the nucleic acid amplification reaction comprises the first target nucleic acid and the second target nucleic acid.

23. 23. The method of claim 22, wherein the nucleic acid amplification reaction includes a step for thermal cycling and the reaction mixture further includes a thermostable DNA polymerase.

24. 22. The method of any one of claims 18 to 21, wherein step (b) is performed when the temperature of the reaction mixture is reduced to allow annealing of the masking oligonucleotide and the complementary cassette cleavage flap.

25. 22. The method of claim 21 , wherein the first and second fluorophore moieties are the same as one another.

26. 22. The method of claim 21, wherein the step of (b) detecting any fluorescence comprises measuring any fluorescence.

27. 27. The method of claim 26, further comprising either detecting or measuring fluorescence at the first temperature.

28. 21. The method of claim 19, wherein both the first fluorophore moiety and the second fluorophore moiety are detectable in the same fluorescence detection channel of an energy sensor device.

29. 30. The method of claim 28, wherein the second fluorophore moiety is the same as the first fluorophore moiety.

30. 30. The method of claim 28, wherein the second fluorophore moiety is not the same as the first fluorophore moiety.

31. 29. The method of any one of claims 18 to 28, wherein fluorescence from the second fluorophore is detected and / or measured at the first temperature.

32. 30. The method of any one of claims 18 to 29, wherein the third quencher moiety is the same as the first quencher moiety and / or the second quencher moiety.

33. 33. The method of any one of claims 18 to 32, wherein the reaction mixture comprises primer oligonucleotides that amplify a target nucleic acid, and at least one primer oligonucleotide acts as an invasive oligonucleotide in the presence of a primary probe oligonucleotide and a target nucleic acid and / or a target amplicon to form an invasive cleavage structure that is cleaved by the thermostable FEN-I endonuclease.

34. 34. The method of any one of claims 18 to 33, wherein step (c) comprises determining using a computer programmed with software.

35. 1. A system for determining which of a plurality of target nucleic acid analytes are present in a reaction mixture, wherein each target nucleic acid analyte of the plurality of target nucleic acid analytes is detectable by a fluorescent signal, the system comprising: A thermocycler and a fluorometer in optical communication with the thermocycler, a fluorometer that measures a fluorescent signal indicative of production of a nucleic acid amplification product by the thermocycler in a single optical channel; a computer in communication with the fluorometer; The computer includes: (a) obtaining a melting / annealing curve data set generated from measurements performed by said fluorometer; (b) determining the presence of a first target nucleic acid in the reaction mixture by detecting a fluorescent signal from a first fluorescent cleavage product in the melting / annealing curve data set at a temperature at which fluorescence in the reaction mixture is maximally quenched; (c) generating a derivative plot from the melting / annealing curve data set; and (d) determining that a second target nucleic acid is present in the reaction mixture if the derivative plot contains a feature characteristic of a first duplex; said computer being programmed with software instructions; wherein the first duplex comprises a first masking oligonucleotide and a second fluorescent cleavage product produced in the reaction mixture when the second target nucleic acid is present. system.

36. The computer includes: (e) determining that a third target nucleic acid is present in the reaction mixture if the derivative plot contains a feature characteristic of a second duplex; said computer being programmed with software instructions; wherein the second duplex comprises a second masking oligonucleotide and a third fluorescent cleavage product produced in the reaction mixture when the third target nucleic acid is present.

36. The system of claim 35.

37. 37. The system of claim 35 or claim 36, wherein the characteristic feature of the first duplex comprises a maximum, minimum, or zero crossing point of a calculated derivative.

38. the calculated derivatives include calculated first derivatives; the unique features of the first duplex include a first maximum of a calculated first derivative of the melting / annealing curve data set as a first melting peak; the characteristic feature of the second duplex comprises a second maximum of the calculated first derivative of the melting / annealing curve data set as a second melting peak; 38. The system of claim 37.

39. The system of any one of claims 35 to 38, wherein the thermocycler, the fluorometer, and the computer are all components of a real-time PCR instrument.

40. 36. The system of claim 35, wherein the computer-generated melting / annealing curve data set comprises data points showing fluorescence as a function of temperature.

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