Temperature-selectable FRET cartridge signal conduction

Abstract

The present invention relates to a multiplexed nucleic acid amplification and detection system associated with temperature selectable FRET cartridge signaling that can be used to detect the presence of a plurality of specific nucleic acid sequences or single nucleotide polymorphisms (i.e., "SNPs") in a temperature dependent manner using only a single fluorescence detection channel of a nucleic acid analyzer. The techniques may be performed using a standard PCR instrument equipped for fluorescence detection or monitoring.

Description

This application is a divisional application of the invention application with the application date of September 29, 2022, Chinese application number 202280066372.7, and invention name “Temperature-selective FRET box signal transduction”. CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit under 35 U.S.C. §119(e) of U.S. Provisional Application No. 63 / 250,894, filed on September 30, 2021. The entire disclosure of this prior application is hereby incorporated by reference. Sequence Listing

[0002] The text of the computer-readable sequence listing filed herewith, entitled "DIA0105-PCT_SEQUENCE_LISTING," was created on April 16, 2022, has a file size of 27,886 bytes, and is hereby incorporated by reference in its entirety. Technical Field

[0003] 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 fluorescence detection channel. Background Art

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

[0005] In a single reaction mixture, detecting multiple nucleic acid analytes has great value. In fact, so-called "multiple" detection has greatly improved the value of nucleic acid assays while reducing associated reagent costs. However, different assay formats are adapted to multiplexing capabilities to varying degrees, which means that there is actual trade-off. For example, next-generation sequencing technology allows obtaining a large amount of information, but the technology is technically very challenging and requires highly specialized equipment usually. Another type of technology that is referred to as "invasive cutting assay" allows nucleic acid sequence detection to reach the level of single nucleotide differences (" SNPs "), and has been used in conjunction with some multiple assay formats.

[0006] For example, Hall et al. describe two multiplexing methods that can be used in conjunction with invasive cleavage detection methods in U.S. Patent No. 5,994,069. First, the presence of a specific target sequence (or internal control) can be designed to trigger different cascades coupled to different detectable parts (such as different dyes) in the form of fluorescent energy transfer. The contribution of each specific target sequence to the final product can be counted, thereby allowing quantitative detection of different nucleic acid sequences contained in a mixture of nucleic acid sequences. In the second configuration, it is desirable to determine whether any of several analytes is present in a sample, but it is not necessary to know the exact identity of each analyte. For example, in a blood bank, it is desirable to know whether any of a variety of infectious agents is present in a blood sample. Because the blood is discarded regardless of which infectious agent is present, different signals do not need to be generated by different probes in this application, and this may actually be undesirable for confidentiality reasons. Therefore, when it is not necessary or desirable to distinguish between analytes, a single detectable label can be used.

[0007] Elsewhere, Peterson et al., in published U.S. patent application 2018 / 0163259A1, describe detecting different nucleic acid analytes by performing secondary invasive cleavage reactions at different temperatures and times. Here, the first secondary invasive cleavage reaction is completed before the second secondary invasive cleavage reaction begins. By performing the reactions at different temperatures and times, nucleic acid analytes can be distinguished in a single multiplex reaction.

[0008] Using different assay formats, Kozlov et al., in U.S. Patent No. 11,034,997, indicate a method for multiplexing real-time PCR using labeled hydrolysis probes to detect and quantify target nucleic acids. Because a polymerase with 5' to 3' exonuclease activity cuts the tag portion and hydrolyzes the remaining portion of the probe, a single probe cleavage event occurs in each cycle of the PCR reaction. When the tag comprises a fluorophore separated 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 cut from the target complementary portion of the probe that is subsequently degraded during one cycle of the primer extension (i.e., polymerization) reaction. Therefore, the increasing signal intensity depends on performing additional PCR reaction cycles. Similarly, Kozlov et al. teach that a "quencher molecule" (e.g., an oligonucleotide) hybridizes to the tag portion of the uncut hydrolysis probe at a temperature used to extend the primer in the primer extension reaction of PCR. Before the primer extension cycle in the PCR reaction, the fluorescence is partially quenched by the quencher attached to the annealing portion of the probe and the quencher molecule.

[0009] Despite the availability of existing nucleic acid multiplex detection platforms, there remains a need for additional methods that can be easily adapted to automated testing platforms. More particularly, there is a need to maximize the detection capabilities of the employed testing instruments without requiring hardware changes. Summary of the Invention

[0010] 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 comprising: a 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 hybridization sequence, wherein hybridization of a cassette-specific invasive oligonucleotide complementary to the cleaved flap hybridization 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, wherein the cleaved flap hybridization sequence is complementary to the cleaved flap hybridization sequence of the 5' flap FRET cassette oligonucleotide. said cleavage site to produce a box-cleaved flap comprising the 5' flap portion and the first fluorophore portion; and (ii) a masked oligonucleotide comprising a second quencher portion, wherein at least a portion of the masked oligonucleotide can specifically hybridize to the 5' flap portion of the FRET box oligonucleotide, wherein hybridization of the masked oligonucleotide to the box-cleaved flap forms a duplex having a first melting temperature exhibiting a first melting peak, and wherein fluorescence emission from the first fluorophore portion in the duplex is quenched by the second quencher portion.

[0012] Embodiment 2 is a composition according to embodiment 1, wherein the first quencher moiety and the second quencher moiety are identical to each other.

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

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

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

[0016] Embodiment 6 is a composition according to any one of embodiments 1 to 5, further comprising a first target-specific invasive oligonucleotide and a first target-specific primary probe oligonucleotide, wherein each of the first target-specific invasive oligonucleotide and the first target-specific primary probe oligonucleotide comprises a sequence configured to hybridize with the target nucleic acid to form an invasive cleavage structure sequence that can be cleaved by a FEN-1 nuclease to produce a primary cleavage flap, and wherein the primary cleavage flap is a box-specific invasive oligonucleotide that is configured to hybridize with the cleavage flap hybridization sequence of the 5' flap FRET box oligonucleotide to form an invasive cleavage structure that can be cleaved by the FEN-1 nuclease.

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

[0018] Embodiment 8 is a composition according to embodiment 7, further comprising deoxynucleoside triphosphates (dNTPs), a thermostable DNA polymerase, and a primer having a 3' end that can be extended by the thermostable DNA polymerase in a template-dependent nucleic acid amplification reaction using the target nucleic acid as a template.

[0019] Embodiment 9 is a composition according to any one of embodiments 1 to 8, further comprising: (iii) a second FRET cassette oligonucleotide comprising a 5' portion comprising a second fluorophore moiety, a stem-loop portion comprising a third quencher moiety, and a 3' portion comprising a second cleaved flap hybridization sequence, wherein hybridization of a second cassette-specific invasive oligonucleotide to the second cleaved flap hybridization 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, and wherein 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 a composition according to embodiment 9, wherein the second FRET box oligonucleotide is a second 5' flap FRET box comprising a 5' flap portion, wherein the box cleavage product is a second box-cleaved flap comprising the second fluorophore, and wherein the composition further comprises: (iv) a second masked oligonucleotide comprising a fourth quencher portion, wherein at least a portion of the second masked oligonucleotide can specifically hybridize to the 5' flap portion of the second FRET box oligonucleotide, wherein hybridization of the second masked oligonucleotide to the second box-cleaved flap forms a second duplex having a second melting temperature higher than the first melting temperature, and wherein fluorescence emission from the second fluorophore portion in the second duplex is quenched by the fourth quencher portion.

[0021] Embodiment 11 is a composition according to 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 a composition according to any one of embodiments 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.

[0023] Embodiment 13 is a composition according to any one of embodiments 9 to 12, wherein the first fluorophore moiety and the second fluorophore moiety are identical to each other.

[0024] Embodiment 14 is a composition according to any one of embodiments 9 to 12, wherein the first fluorophore moiety and the second fluorophore moiety are different from each other.

[0025] Embodiment 15 is a composition according to any one of embodiments 10 and 12 to 14, wherein the third quencher moiety and the fourth quencher moiety are the same as each other.

[0026] Embodiment 16 is a method for determining which of two different FRET cassettes in a reaction mixture is cleaved to produce a fluorescent signal, the method comprising the steps of: (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, such that if cleavage occurs, two different fluorescent cleavage products are produced, wherein 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 cleaved flap comprising the fluorophore, the reaction mixture comprising a first masked oligonucleotide, the first masked oligonucleotide being cleaved at a temperature below a first Tm; (b) stably hybridizes with the first box-cleaved flap to form a first duplex at a temperature of 100°C, but not at a temperature above the first Tm, wherein fluorescence emission from the fluorophore of the first box-cleaved flap of the first duplex is quenched, and each of the two different fluorescent cleavage products produced in the multiple invasive cleavage reaction is characterized by a different temperature-dependent fluorescence quenching spectrum in the reaction mixture; (b) measuring the fluorescent signal produced in the reaction mixture using a single channel of a fluorescence monitoring device under temperature conditions that differentially quench fluorescence produced by different fluorescent cleavage products of the multiple invasive cleavage reactions; and (c) determining which of the different FRET boxes was cleaved in the multiple invasive cleavage reaction based on the result of step (b).

[0027] Embodiment 17 is a method according to embodiment 16, wherein the second FRET box in step (a) produces a fluorescent cleavage product if cleaved, and the fluorescent cleavage product does not hybridize to any masking oligonucleotide in the reaction mixture to cause fluorescence quenching.

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

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

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

[0031] Embodiment 21 is a method according to 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 wherein step (c) comprises determining that the first FRET box is cleaved in the reaction mixture if the fluorescent signal measured at a temperature above the first Tm is greater than the fluorescent signal measured at a temperature below the first Tm.

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

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

[0034] Embodiment 24 is a method according to embodiment 16, wherein the second FRET box 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 box by the FEN-1 nuclease in the multiple invasive cleavage reaction produces a second box-cleaved flap comprising the fluorophore, wherein the reaction mixture comprises a second masked oligonucleotide that hybridizes to the second box-cleaved flap to form a second duplex at a temperature below a second Tm, but not above the second Tm, wherein fluorescence emission from the fluorophore of the second box-cleaved flap of the second duplex is quenched, and wherein the first Tm differs from the second Tm by at least 5°C.

[0035] Embodiment 25 is a method according to embodiment 24, wherein the first Tm is greater than the second Tm, wherein step (b) comprises measuring any of the fluorescent signals at a temperature below the second Tm and at a temperature above the first Tm, and wherein step (c) comprises determining that at least one of the first FRET box and the second FRET box is cleaved in the reaction mixture if the fluorescent signal measured at a temperature above the first Tm is greater than the fluorescent signal measured at a temperature below the second Tm.

[0036] Embodiment 26 is a method according to embodiment 24 or 25, wherein step (b) comprises measuring any said fluorescent signal generated in said reaction mixture as a function of temperature to generate a melting / annealing curve.

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

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

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

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

[0041] Embodiment 31 is a method according to 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 to 31, wherein step (c) comprises determining using a computer programmed with software.

[0043] Embodiment 33 is a method for analyzing a sample containing target nucleic acids, the method comprising the following steps: (a) contacting any first target nucleic acid of the sample with a first primary probe oligonucleotide containing a sequence complementary thereto and a FEN-1 endonuclease in a reaction mixture 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 produce a first primary cleaved flap, wherein the first primary cleaved flap hybridizes to a cleaved flap hybridization sequence of a first FRET cassette oligonucleotide contained in the reaction mixture to form an invasive cleavage structure, the invasive cleavage structure being cleaved by the FEN-1 endonuclease at a cleavage site between a first fluorophore portion and a first quencher portion of the first FRET cassette oligonucleotide to release a first fluorophore portion comprising the first quencher portion; portion of a first box-cleaved flap, wherein a first masked oligonucleotide comprising a second quencher portion hybridizes with the first box-cleaved flap to form a duplex at a temperature below a first Tm of the first masked oligonucleotide and the first box-cleaved flap, wherein fluorescence emission from the first fluorophore portion in the duplex is quenched by the second quencher portion, and wherein at a second temperature above the first Tm, the first masked oligonucleotide and the first box-cleaved flap do not form a stable duplex; (b) detecting any fluorescence emitted from the first fluorophore portion at the second temperature; and (c) determining that the sample contains the first target nucleic acid if fluorescence emitted from the first fluorophore portion is detected in step (b), or determining that the sample does not contain the first target nucleic acid if fluorescence emitted from the first fluorophore portion is not detected in step (b).

[0044] Embodiment 34 is a method according to embodiment 33, wherein step (a) further comprises contacting any second target nucleic acid of the sample with a second primary probe oligonucleotide comprising a sequence complementary thereto and the FEN-1 endonuclease in the reaction mixture 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 produce a second primary cleaved flap that is different from the first primary cleaved flap, wherein the second primary cleaved flap hybridizes to a cleaved flap hybridization sequence of a second FRET cassette oligonucleotide contained in the reaction mixture to form an invasive cleavage structure, which is cleaved by the FEN-1 endonuclease at a cleavage site between the second fluorophore moiety and the third quencher moiety of the second FRET cassette oligonucleotide to release the second cassette cleaved flap comprising the second fluorophore moiety, wherein at a third temperature below a second Tm, A second masked oligonucleotide comprising a fourth quencher portion hybridizes to the second box-cleaved flap to form a duplex, wherein fluorescence emission from the second fluorophore portion of the duplex is quenched by the fourth quencher portion, wherein at a fourth temperature greater than the second Tm, the second masked oligonucleotide and the second box-cleaved flap do not form a stable duplex, and wherein the first Tm and the second Tm differ from each other by at least 5°C; wherein step (b) further comprises detecting any fluorescence emitted from the second fluorophore portion at the fourth temperature; and wherein step (c) further comprises determining that the sample contains the second target nucleic acid if fluorescence emitted from the second fluorophore portion of the 5' flap cleavage product of the second FRET box oligonucleotide is detected in step (b), or determining that the sample does not contain the first target nucleic acid if fluorescence emitted from the second fluorophore portion of the 5' flap cleavage product of the second FRET box oligonucleotide is not detected in step (b).

[0045] Embodiment 35 is a method according to embodiment 33, wherein step (a) further comprises contacting any second target nucleic acid of the sample with a second primary probe oligonucleotide comprising a sequence complementary thereto and the FEN-1 endonuclease in the reaction mixture 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 produce a second primary cleaved flap, wherein the second primary cleaved flap hybridizes to a cleaved flap hybridization sequence of a second FRET cassette oligonucleotide contained in the reaction mixture to form an invasive cleavage structure, the invasive cleavage structure being formed at the second FRET cassette oligonucleotide. The nucleotide is cleaved by the FEN-1 endonuclease at a cleavage site between the second fluorophore moiety and the third quencher moiety to release a cleavage product comprising the second fluorophore moiety, wherein the cleavage product does not hybridize to any masking oligonucleotide in the reaction mixture to result in fluorescence quenching; wherein step (b) further comprises detecting any fluorescence emitted from the second fluorophore moiety of the cleavage product; and wherein step (c) further comprises determining that the sample comprises the second target nucleic acid if fluorescence emitted from the second fluorophore moiety is detected in step (b), or determining that the sample does not comprise the first target nucleic acid if fluorescence emitted from the second fluorophore moiety is not detected in step (b).

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

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

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

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

[0050] Embodiment 40 is a method according to embodiment 36, wherein the first fluorophore moiety and the second fluorophore moiety are identical to each other.

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

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

[0053] Embodiment 43 is a method according to 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 a method according to embodiment 43, wherein the second fluorophore moiety is the same as the first fluorophore moiety.

[0055] Embodiment 45 is a method according to embodiment 43, wherein the second fluorophore moiety is different from the first fluorophore moiety.

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

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

[0058] Embodiment 48 is a method according to any one of embodiments 33 to 47, wherein the reaction mixture comprises primer oligonucleotides that amplify the target nucleic acid, wherein 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 to 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 is present in a reaction mixture, wherein each of the plurality of target nucleic acid analytes is detectable by a fluorescent signal, the system comprising: a thermal cycler; a fluorometer in optical communication with the thermal cycler, wherein the fluorometer measures a fluorescent signal indicative of production of a nucleic acid amplification product achieved by the thermal cycler using a single optical channel; and a computer in communication with the fluorometer, wherein the computer is programmed with software instructions such that the computer: (a) obtains a melting / annealing curve dataset based on measurements made by the fluorometer, (b) determines 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 dataset at a temperature at which fluorescence in the reaction mixture is maximally quenched, (c) generates a derivative plot based on the melting / annealing curve dataset, and (d) determines the presence of a second target nucleic acid in the reaction mixture if the derivative plot comprises features characteristic of a first duplex, wherein the first duplex comprises a first masked oligonucleotide and a second fluorescent cleavage product produced in the reaction mixture when the second target nucleic acid is present.

[0061] Embodiment 51 is a system according to embodiment 50, wherein the computer is further programmed with software instructions so that the computer: (e) determines that a third target nucleic acid is present in the reaction mixture if the derivative graph comprises features that are characteristic of a second duplex, wherein the second duplex comprises a second masked oligonucleotide and a third fluorescent cleavage product produced in the reaction mixture when the third target nucleic acid is present.

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

[0063] Embodiment 53 is a system according to embodiment 52, wherein the calculated derivative includes a calculated first-order derivative, wherein the first duplex-specific feature includes a first maximum of the calculated first-order derivative of the melting / annealing curve data set as a first melting peak, and wherein the second duplex-specific feature includes a second maximum of the calculated first-order derivative of the melting / annealing curve data set as a second melting peak.

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

[0065] Embodiment 55 is a system according to embodiment 50, wherein the melting / annealing curve data set obtained by the computer comprises data points indicative of fluorescence as a function of temperature. BRIEF DESCRIPTION OF THE DRAWINGS

[0066] Figure 1 A schematic diagram of an assay employing two invasive cleavage reactions performed sequentially in the same reaction mixture is provided. In the primary reaction, an "invasive oligonucleotide" (SEQ ID NO: 1) and a "primary probe oligonucleotide" (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 that can be cleaved by a FEN-1 endonuclease, thereby releasing a cleaved 5' flap ("primary cleaved 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 that can be cleaved by the 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 "Cleaved FRET Cassette 1" (SEQ ID NO: 6) and separates the quencher from the fluorophore, allowing detection of a fluorescent signal from the fluorophore. Each primary cleaved flap can hybridize with a series of new uncleaved FRET cassettes to form additional fluorescent cleavage products.

[0067] Figure 2 A series of schematics illustrating the structure and use of the 5' flap FRET cassette are presented. Figure 2 Panel A of FIG. 5 presents the structure of an example “5′ flap FRET cassette” (SEQ ID NO: 7). Figure 2 Panel B of presents an example "5' flap FRET cassette 1" (SEQ ID NO: 7) hybridized to the "cleaved flap from primary probe" (SEQ ID NO: 8) (ie, the primary cleaved flap used as the invasive oligonucleotide). Figure 2 Panel C shows the temperature-dependent interaction between the "box-cleaved flap" (SEQ ID NO: 9) (ie, the 5' flap cleaved from the 5' flap FRET box) and the complementary "masking oligonucleotide" (SEQ ID NO: 10).

[0068] Figure 3Schematic illustration of how the fluorescence signals generated 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. "Cleaved flap from target 2 primary probe" (SEQ ID NO: 8) hybridizes with "5' Flap FRET Cassette 1" (SEQ ID NO: 7) to promote the enzyme-dependent cleavage reaction. "Cassette cleaved flap 1" (SEQ ID NO: 9) or the 5' flap cleaved from "5' Flap FRET Cassette 1" (SEQ ID NO: 7) can hybridize with "Masking Oligonucleotide 1" (SEQ ID NO: 10) to form a first hybridization duplex exhibiting quenched fluorescence. "Cleaved flap from target 3 primary probe" (SEQ ID NO: 14) hybridizes with "5' Flap FRET Cassette 2" (SEQ ID NO: 13) to promote the enzyme-dependent cleavage reaction. "Cassette cleaved flap 2" (SEQ ID NO: 15) or the 5' flap cleaved from "5' flap FRET cassette 2" (SEQ ID NO: 13) can hybridize with "masking oligonucleotide 2" (SEQ ID NO: 16) to form a second duplex that exhibits quenched fluorescence. These two hybrid duplexes are designed to have unique melting / annealing properties, which allows one hybrid duplex to be distinguished from the other. The signal generated by cleaving the "flapless FRET cassette" (SEQ ID NO: 11) is detectable under all temperature conditions.

[0069] Figure 4 is a schematic diagram of cleavage products prepared using a flapless FRET cassette and two different 5' flap FRET cassettes, wherein all three FRET cassettes are labeled with the same reporter dye (HEX). Different FRET cassettes allow different nucleic acid target sequences to be detected using only a single fluorescence detection channel of a nucleic acid analyzer. In the embodiment shown, Temperature 1 is lower than Temperature 2 (e.g., Temperature 1 can be 40°C and Temperature 2 can be 50°C). Using a flapless FRET cassette without a corresponding masking oligonucleotide (e.g., from Figure 3 Detection of target 1 was performed using a 5' flap FRET cassette (e.g., from Figure 3When the reaction mixture is below temperature 1, the "cassette cleaved flap 1" (SEQ ID NO: 7) is masked by hybridization with the "masking oligonucleotide 1" (SEQ ID NO: 10). At temperatures above temperature 1 but below temperature 2, signals reflecting the detection of both targets 1 and 2 can be detected. A 5' flap FRET cassette (e.g., from Figure 3 In the embodiment shown, the detection of target 3 is performed using "Cassette Cleavage Flap 2" (SEQ ID NO: 13), and when the reaction mixture is below temperature 2, the "Cassette Cleavage Flap 2" (SEQ ID NO: 15) is masked by hybridization with "Masking Oligonucleotide 1" (SEQ ID NO: 16). Above temperature 2, signals from all three of targets 1, 2, and 3 are detectable. In the illustrated embodiment, the temperature selected for detecting cleavage of the different FRET cassettes is independent of the sequences of the three different target nucleic acids.

[0070] Figure 5 A set of graphs showing fluorescence as a function of cycle number measured in the HEX channel is provided. The reaction contains target analyte A ( Figure 5 Panel A), target analyte B ( Figure 5 B) or contains both target analytes A and B ( Figure 5 Small Figure C), as described in Example 1.

[0071] Figure 6 Graphs are provided showing melting curve analysis performed as described in Example 2 for reactions containing target analyte A, target analyte B, and both target analytes A and B using fluorescence detected in the HEX channel.

[0072] Figure 7 Panels AD provide a set of graphs showing fluorescence measured in the HEX channel as a function of cycle number and the temperature at which the fluorescence measurements were taken. Figure 7 Small Figure A and Figure 7 Panel B shows the results obtained for a reaction containing only target analyte B (using a 5' flap FRET cassette and a masked oligonucleotide quenched at low temperature) when fluorescence was measured at 63°C and 39°C, respectively. Figure 7 Small Figure C and Figure 7 Panel D shows the results obtained for a reaction containing both target analytes A and B, where analyte A was detected using a flapless FRET cassette (containing no 5' flap) and the reaction produced a product that fluoresced at both temperatures, where fluorescence was measured at 63°C and 39°C, respectively. The procedure is described in Example 3.

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

[0074] Figure 9 Graphs are provided showing the unique melting / annealing profiles observed for additional analyte combinations as described in Example 4. The left panel of the graph shows the melting / annealing curve results for reactions amplifying Analyte B or Analyte C. The right panel of the graph shows the melting / annealing curve analysis results for reactions amplifying Analyte B alone, Analyte C alone, or a combination of Analyte B and Analyte C.

[0075] Figure 10 Provided Figure 9 The two panels show first derivative plots of the measured fluorescence data. DETAILED DESCRIPTION definition

[0076] For ease of understanding of this disclosure, a number of terms and phrases are defined below. Additional definitions are set forth throughout the detailed description. Throughout the specification and claims, unless the context clearly provides otherwise, the following terms have the meanings clearly associated herein.

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

[0078] Unless the context clearly dictates otherwise, the term "based on" is not exclusive and allows for being based on additional factors not described. In addition, throughout the specification, the meanings of "a," "an," and "the" include plural referents. The meaning of "in" includes "in" and "on."

[0079] The transition phrase “consisting essentially of” as used in the claims of this application limits the scope of the claim to specified materials or steps “and those materials or steps that do not materially affect one or more basic and novel characteristics of the claimed invention,” as discussed in In re Herz, 537 F.2d 549, 551-52, 190 USPQ 461, 463 (CCPA 1976). For example, a composition that “consists essentially of the elements” may contain unspecified contaminants at levels such that, although present, the contaminants do not alter the function of the composition compared to a pure composition (i.e., a composition “consisting of the components”). As used herein, the term “sample” refers to a sample that may contain an analyte of interest (e.g., a microorganism, a virus, a nucleic acid such as a gene, or a component thereof that includes a nucleic acid sequence in the analyte or a nucleic acid sequence derived from the analyte). The sample can be from any source, such as a biological sample or an environmental source. A biological sample includes any tissue or material derived from a living or dead organism that may contain an analyte or a nucleic acid in the analyte or a nucleic acid derived from the analyte. Examples of biological samples include: nasal swab samples, vaginal swab samples, respiratory tissue, exudates (e.g., bronchoalveolar lavage fluid), biopsies, sputum, peripheral blood, plasma, serum, lymph nodes, gastrointestinal tissue, feces, urine, or other liquids, tissues or materials. Examples of environmental samples include water, ice, soil, mud, debris, biofilms, airborne particulate matter and aerosols. The sample can be a processed specimen or material, such as obtained by treating the sample using filtration, centrifugation, sedimentation or adhesion to a medium (e.g., a matrix or support). Other processing of the sample can include physical or mechanical destruction of tissue, cell aggregates or cells to release intracellular components including nucleic acids into a solution that may contain other components (e.g., enzymes, buffers, salts, detergents, etc.). The sample being tested for the presence of an analyte can sometimes be referred to as a "test sample."

[0080] The terms "target nucleic acid" and "target sequence" refer to nucleic acids to be detected or analyzed. Therefore, attempts are made to distinguish "target" nucleic acids from other nucleic acids or nucleic acid sequences. For example, when used to refer to an amplification reaction, these terms can refer to a portion of a nucleic acid or nucleic acid that will be amplified by the reaction, and when used to refer to a polymorphism, they can refer to a locus in a nucleic acid suspected of being polymorphic. When used to refer to an invasive cleavage reaction, these terms generally refer to nucleic acid molecules containing a sequence that selectively hybridizes with a first nucleic acid molecule (e.g., a probe oligonucleotide) and a second nucleic acid molecule (invasive oligonucleotide) to form an overlapping invasive cleavage structure. Typically, a target nucleic acid (e.g., present in a sample, separated from a sample, enriched or amplified) is located within a target region and can be identified via 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 cut by a cutting agent. Target nucleic acids from organisms are not limited to genomic DNA and RNA. Target nucleic acids from an organism can include any nucleic acid species, including but not limited to genomic DNA and RNA, messenger RNA, structural RNA, ribosomal and tRNA, and small RNAs 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 nucleic acid region within a target sequence.

[0081] Because mononucleotides are reacted in a certain manner to prepare oligonucleotides so that the 5' phosphate linked to one mononucleotide pentose ring is attached to the 3' oxygen of its neighbor in one direction via a phosphodiester linkage, one end of an oligonucleotide is referred to as the "5' end" if its 5' phosphate is not linked to the 3' oxygen of a mononucleotide pentose ring, and as the "3' end" if its 3' oxygen is not linked to the 5' phosphate of a subsequent mononucleotide pentose ring. As used herein, a nucleic acid sequence, even within a larger oligonucleotide, may be said to have a 5' and a 3' end. A first region along a nucleic acid chain is said to be upstream of another region if the 3' end of the first region precedes the 5' end of the second region when moving along the nucleic acid chain in the 5' to 3' direction.

[0082] As used herein, the term "5' terminal portion" refers to a portion of a nucleic acid having a 5' terminus (i.e., a 5' terminus whose 5' phosphate is not linked to the 3' oxygen of a mononucleotide pentose ring). The term "3' terminal portion" refers to a portion of a nucleic acid having a 3' terminus (i.e., a 3' terminus whose 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 to refer 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 participating in the duplex) are affected by factors such as the degree of complementarity between the nucleic acids, the stringency of the conditions involved, the T of the hybrid formed, and the strength of the hybridization. m and the G:C ratio within nucleic acids.

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

[0085] As used herein, the term " m " is used to refer to the "melting temperature" of a nucleic acid strand with respect 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 is halfway dissociated into single strands. Measuring the melting temperature of a labeled nucleic acid duplex typically involves plotting fluorescence versus temperature to generate a melting curve characteristic of duplex dissociation. When the negative first derivative of the melting curve is plotted as a function of temperature, T m Can be identified as peaks. See, eg, KM Ririe et al., Analytical Biochemistry 245: 154-160 (1997).

[0086] "Calculated T m " refers to the melting temperature determined by calculation based on the physical sequence of the complementary nucleic acid and factors such as the reaction conditions (e.g., salt concentration, concentration of complementary chains in the mixture). m Several equations are well known in the art. As shown in standard references, when nucleic acid is in 1M NaCl in water, T m A simple estimate of the value can be calculated by the following equation: m = 81.5 + 0.41 (G + C%) (see, for example, Young and Anderson, (1985) Nucleic Acid Hybridisation: A Practical Approach (Hames and Higgins eds.) pp. 47-71, IRL Press, Oxford). Used to calculate T mOther calculations of are known in the art and take into account structure and environment as well as sequence features (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 "cyclic hybridization" refers to a situation in which the T of a hybridized nucleic acid strand (e.g., a probe oligonucleotide and its complementary target nucleic acid) is m Below or close to T m Next (for example, at T m within 4°C, more preferably within T m within 3°C, still more preferably within T m 2°C and still more preferably within T m The reaction mixture containing the nucleic acids is incubated at a temperature within 1° C. of the reaction mixture so that the oligonucleotides continuously anneal and dissociate from the target strand without temperature cycling (i.e., without changing the temperature of the reaction mixture to alternatively melt and anneal the probe-target nucleic acid duplex).

[0088] As used herein, an "invasive cleavage assay" is a method for detecting or quantifying a target nucleic acid by enzymatic cleavage of one or more distinct invasive cleavage structures, wherein at least one of the cleavage structures 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 within a single reaction mixture. Reagents for an invasive cleavage assay can 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 a structure-specific flap endonuclease cleavage assay (Hologic, Inc.) and is described in, e.g., U.S. Patent 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 herein by reference in its entirety for all purposes.

[0090] As used herein, the term "invasive cleavage structure" (sometimes referred to simply as "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 extension of any nucleic acid chain. In some embodiments, the invasive cleavage structure includes: (i) a continuous nucleic acid chain (e.g., a target DNA or RNA); (ii) an upstream nucleic acid (e.g., an invasive oligonucleotide, sometimes referred to as an INVADER oligonucleotide) that hybridizes with the first portion of the target chain to form an upstream duplex; and (iii) a downstream nucleic acid (e.g., a 5' flap probe, or a primary probe oligonucleotide having a 5' flap that is not complementary to the target chain) that hybridizes to form a downstream duplex. The upstream and downstream nucleic acids anneal to a continuous region of the target nucleic acid, and wherein an overlap is formed between the 3' portion of the upstream nucleic acid and the duplex formed between the downstream nucleic acid and the target nucleic acid. Whether or not one or more overlapping bases of the upstream nucleic acid are complementary to the target nucleic acid, and whether or not these bases are natural bases or non-natural bases, when one or more bases from the upstream nucleic acid and the downstream nucleic acid occupy the same position relative to the target nucleic acid base, the overlap occurs. In some embodiments, the 3' portion of the upstream nucleic acid that overlaps with the downstream duplex is a non-base chemical portion, such as an aromatic ring structure (e.g., as disclosed in U.S. Patent No. 6,090,543, which is incorporated herein by reference in its entirety). In some embodiments, one or more nucleic acids can be attached to each other by covalent linkage (e.g., nucleic acid stem-loop) or by non-nucleic acid chemical linkage (e.g., polycarbon chain). When the cut 5' flap hybridizes with the FRET box (e.g., wherein the "target nucleic acid" and the "downstream nucleic acid" are covalently linked in a stem-loop configuration), an invasive cleavage structure can also be produced. The "target nucleic acid" sequence of the FRET box that hybridizes with the cut 5' flap can be referred to as a "cut flap hybridization sequence."

[0091] In some embodiments, the target nucleic acid is amplified (e.g., by PCR), and the amplified product is detected using an invasive cleavage assay when the amplification reaction occurs. Configurations for performing 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, an invasive cleavage assay can be used in a single reaction to reverse transcribe, amplify, and detect an RNA target nucleic acid, as described in 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 when it is present. In other embodiments, the formation of the complex can be detected when the complex is no longer present (e.g., by detecting an event that occurs as a result of the formation of the probe / target complex, such as a cleavage event).

[0093] As used herein, the term "flap probe" refers to a probe oligonucleotide that comprises 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 in reference to a serial invasive cleavage assay, a "primary probe" is a flap probe comprising a 3' sequence or portion that is complementary to a target nucleic acid to be detected and a 5' flap portion that is non-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, after the primary probe that participates in the invasive cleavage structure in the "primary" reaction of the sequential invasive cleavage assay is cleaved, the cleaved 5' flap released from the primary probe can hybridize to the FRET cassette to facilitate the secondary reaction of the sequential invasive cleavage assay.

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

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

[0097] As used herein, a reaction is "active" when 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 for the FRET cassette, and a FEN enzyme) is incubated at a temperature that allows cyclic hybridization of the cleaved 5' flap in the reaction mixture to the FRET cassette, thereby 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 referred to as "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, wherein the invasive oligonucleotide comprises a portion (e.g., a chemical moiety or 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 comprises a sequence at its 3' end that is substantially identical to a sequence located at the 5' end of the probe oligonucleotide.

[0099] As used herein, the term "FRET" refers to fluorescence resonance energy transfer, which is a process in which a chemical moiety (e.g., a fluorophore) transfers energy between them or from a fluorophore to a non-fluorophore (e.g., a quencher molecule). In some cases, FRET involves an excited donor fluorophore transferring energy to a lower-energy acceptor fluorophore via a short-range (e.g., about 10 nm or less) dipole-dipole interaction. In other cases, FRET involves a loss of donor fluorescence energy and an increase in fluorescence in the acceptor fluorophore. In still 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 skilled 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 herein incorporated by reference in its entirety).

[0100] As used herein, the term "FRET box" refers to an oligonucleotide comprising a stem-loop or hairpin structure (i.e., a region where nucleic acid bases pair intramolecularly to form a double helical stem having a nucleotide loop connecting the base-paired strands at one end of the stem), the oligonucleotide comprising a donor portion (e.g., a "fluorophore") and a nearby acceptor portion (e.g., a "quencher"), wherein the attachment of the donor and acceptor portions to the same FRET box significantly inhibits (e.g., quenches) detectable energy emission (e.g., fluorescence emission) from the donor portion. The FEN-1 enzyme catalyzes the hydrolytic cleavage of the 3' phosphodiester bond adjacent to the junction of single-stranded and double-stranded DNA, typically with one nucleotide entering the 5' end of the oligonucleotide stem-loop portion, thereby releasing the 5' nucleotide or 5' flap from the stem-loop portion of the FRET box. The fluorophore portion is typically attached to the FRET box oligonucleotide at the 5' end or in the 5' flap, while the quencher portion is typically attached to the stem-loop portion of the oligonucleotide.

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

[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 comprising a cleaved flap hybridization sequence, and wherein the 5' flap portion and the cleaved flap hybridization 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 can base pair to the last base pair on the non-loop end of the stem-loop portion of the FRET cassette.

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

[0105] In some embodiments, (e.g., in a multiplex invasive cleavage reaction) a mixture of FRET boxes with two or more different cleaved flap hybridization sequences can be used. Two cleaved flap hybridization sequences are said to be "different" from one another when the cleaved flap product can hybridize to one cleaved flap hybridization sequence and cannot measurably hybridize to another cleaved flap hybridization sequence (e.g., under invasive cleavage assay conditions), and vice versa. Cleavage of the FRET box by a FEN enzyme (e.g., FEN-1 endonuclease) in a secondary reaction separates the donor and acceptor moieties, which results in relief of inhibition and allows signal generation. In some embodiments, the donor and acceptor moieties interact via fluorescence resonance energy transfer (e.g., FRET). In other embodiments, the donor and acceptor of the FRET box interact via a non-FRET mechanism.

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

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

[0108] As used herein, the term "donor" refers to a portion (e.g., a fluorophore) that absorbs at a first wavelength and emits at a second, longer wavelength. The term "acceptor" refers to a portion, such as a fluorophore, chromophore, or quencher, that is capable of absorbing some or most of the energy emitted by the donor when it is close to the donor group (usually between 1-100 nm). The acceptor can have an absorption spectrum that overlaps with the emission spectrum of the donor. Typically, if the acceptor is a fluorophore, it re-emitted at a third, still longer wavelength. If the acceptor is a chromophore or quencher, it releases the energy absorbed from the donor without emitting photons. In some preferred embodiments, changes in the energy levels of the donor and / or acceptor moieties are detected (e.g., via measuring energy transfer between or from the donor and / or acceptor moieties). This may involve detecting light emission. In some preferred embodiments, the emission spectrum of the acceptor moiety is different from the emission spectrum of the donor moiety, so that the emissions from these moieties (e.g., the emission of light and / or energy) can be distinguished from each other (e.g., spectrally resolved).

[0109] In some embodiments, the donor moiety is used in combination with multiple acceptor moieties. In a preferred embodiment, the 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 instead of the acceptor moiety, and when the quencher moiety is removed (e.g., by cleavage of the probe), the donor moiety excitation is transferred to the acceptor moiety. In some preferred embodiments, emission from the acceptor moiety is detected (e.g., using a wavelength-shifted molecular beacon) (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 20030228703, each of which is incorporated herein by reference in its entirety).

[0110] As used herein, the term "different" in reference to signals (e.g., signals of one or more labels) means that the signals can be distinguished from each other by, for example, spectral properties (such as fluorescence emission wavelength, color, absorbance, mass, size, fluorescence polarization properties, charge, etc.) or by the 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 a polynucleotide or oligonucleotide (e.g., a probe) refers to a nucleic acid produced in an in vitro cell-free reaction (e.g., an enzymatic or chemical synthesis reaction). Examples of enzymatic formation of synthetic nucleic acids include formation by restriction enzyme digestion, polymerization (templated or non-templated), ligation, and the like. Examples of chemical synthesis of nucleic acids include, but are not limited to, phosphodiester and phosphotriester chemistry, phosphoramidite and H-phosphonate chemistry, and the like. See, e.g., Methods in Molecular Biology, Vol. 20: Protocols for Oligonucleotides and Analogs 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, Agrawal and Iyer, Curr. Op. Biotech. 6: 12 (1995); and Anti-sense Research and Applications (Crooke and Lebleu; eds., CRC Press, Boca Raton, 1993), Beaucage and Caruthers, Tetrahedron Lett. 22: 1859-1862 (1981), and U.S. Pat. No. 5,149,798 to Agrawal and Zamecnik (1992). In some embodiments, preformed synthetic oligonucleotides are introduced into the reaction, while in other embodiments, synthetic oligonucleotides are formed or modified during the reaction (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 nuclear enzymes that act as structure-specific endonucleases on DNA structures having duplexes containing a single-stranded 5' overhang or 5' flap on one strand that is displaced by another nucleic acid strand, resulting in overlapping nucleotides at the junction between the single-stranded and double-stranded DNA. FEN enzymes catalyze the hydrolytic cleavage of the 3' phosphodiester bond near the junction of single-stranded and double-stranded DNA, thereby 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 separate enzymes or multiple subunit enzymes. In some specific embodiments, FEN enzyme activity can exist as an activity of another enzyme or protein complex (such as 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, primers, and dNTPs). In other preferred embodiments, the FEN enzyme has DNA polymerization activity, but does not exhibit such activity by extending an oligonucleotide (e.g., a 5' flap cleaved from a primary probe or derived from another source) in a manner that substantially excludes cyclic hybridization with the FRET box. For example, a FEN enzyme that participates in a secondary invasive cleavage reaction to cleave a fluorophore or fluorescent 5' flap from the FRET box will not extend a cleaved 5' flap from a primary probe (i.e., an invasive probe that reversibly hybridizes to the FRET box to catalyze the cleavage reaction). The flap endonuclease may be thermostable. Examples of FEN enzymes that can be used in the methods disclosed herein are described in U.S. Patent Nos. 5,614,402; 5,795,763; 6,090,606; and in published PCT applications identified by WO 98 / 23774; WO 02 / 070755; WO 01 / 90337; and WO 03 / 073067, each of which is incorporated by reference in its entirety. Specific examples of commercially available FEN enzymes include enzyme (Hologic, Inc.).

[0113] As used herein, "FEN-1" refers to a non-polymerase flap-like endonuclease from a eukaryotic or archaeal organism, as encoded by the FEN-1 (flap-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; WO 02 / 070755, and U.S. Patent No. 7,122,364, which are incorporated herein by reference in their entireties for all purposes. The term "FEN-1 activity" refers to any enzymatic activity of a FEN-1 enzyme. As described in WO 02 / 070755 and U.S. Patent No. 7,122,364, FEN-1 endonucleases also include modified FEN-1 proteins (e.g., chimeric proteins comprising portions of FEN-1 enzymes from different organisms) and enzymes comprising one or more mutations (e.g., substitutions, deletions, insertions, etc.). An archaeon is any of the usually single-celled organisms of the kingdom Archaea.

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

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

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

[0117] As used herein, the term "complementary" refers to nucleobase sequences that are capable of forming double-stranded hydrogen bonding regions. Nucleobase sequences can be "fully complementary" (i.e., each nucleobase in one sequence is capable of pairing with the corresponding nucleobase in a second sequence), or they can be "partially complementary" (i.e., at least one nucleobase in one sequence is not capable of hydrogen bonding with the corresponding nucleobase in the 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 nucleic acid structures that contain double-stranded hydrogen bonding regions. Such structures can be fully or partially double-stranded and include RNA:RNA, RNA:DNA, and DNA:DNA molecules and their analogs. For example, a "duplex" includes a cleaved 5' flap sequence (e.g., a primary cleaved flap) that hybridizes to a complementary cleaved flap hybridization sequence of a FRET cassette; and a cassette cleaved flap that hybridizes to a complementary masking oligonucleotide.

[0119] As used herein, the term "cleaved form" 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. For example, a 5' flap sequence is in a "cleaved form" 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 reference to polynucleotides (e.g., oligonucleotides, target nucleic acids, etc.), the term "single-stranded state" refers to a region of the 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 a region of the self-complementary polynucleotide that is available for base pairing. For example, the cassette-cleaved flap is in a single-stranded state prior to hybridization with a masking oligonucleotide, or after melting to separate the cassette-cleaved flap from the masking oligonucleotide.

[0121] As used herein, "temperature conditions" for carrying out a reaction refer to the temperature or temperature range that allows a reaction to occur. Different temperature profiles for different reactions mean that the temperature conditions that allow one reaction to occur may not allow a different reaction to occur. The term is also applicable to temperature profiles that allow hybridization of a masking oligonucleotide with a complementary oligonucleotide sequence comprising a fluorophore moiety.

[0122] As used herein, "optimal" (and grammatical variants thereof) reaction conditions refer to the most favorable reaction conditions that promote or allow a reaction to occur. For example, the optimal temperature for performing a secondary reaction is the temperature at which the FRET box is most effectively cut in the reaction mixture (e.g., corresponding to a peak on a graph of the fluorescence signal as a function of the reaction temperature). Similarly, an optimal temperature range is a range of the most favorable temperature conditions for promoting or allowing a reaction to occur. In certain exemplary embodiments, a 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, still more preferably plus or minus 2°C, and yet more preferably plus or minus 1°C.

[0123] As used herein, "attached" (eg, two things are "attached") refers to chemically bonded together. For example, a fluorophore moiety is "attached" to a FRET cassette when the fluorophore moiety is chemically bonded 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 fluorophores") means that the excitation and emission spectra of the detectable chemicals are sufficiently similar or overlapping over a wavelength range to allow detection of the fluorescence emission wavelength in the same channel of an instrument used to monitor the signal.

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

[0126] As used herein, emission from a donor moiety (e.g., a fluorophore) is "quenched" when detectable emission of photons from the donor is suppressed or prevented because the acceptor moiety (e.g., a quencher) is in close enough proximity. For example, when both the donor moiety and the acceptor moiety are attached to the same FRET cassette, emission from the donor moiety is quenched. Similarly, 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 (the flap cleaved by the cassette) hybridizes to a complementary oligonucleotide comprising a quencher moiety.

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

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

[0129] The term "thermostable" when used in reference to an enzyme such as a FEN enzyme means that the enzyme is functional or active (i.e., can catalyze) at elevated temperatures (e.g., at 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, for example, by in vitro replication, such as by PCR.

[0131] As used herein, the term "amplification method" used in reference to nucleic acid amplification means the process of the abundance of specific amplification related nucleic acids. Some amplification methods (for example, polymerase chain reaction or PCR) include iterative cycles of thermal denaturation, annealing of oligonucleotide primers to template molecules and nucleic acid polymerase extension of annealed primers. The conditions and time required for each of these steps are well known in the art. Some amplification methods are carried out at a single temperature and are considered to be "isothermal". The accumulation of amplified products can be exponential or linear. Some amplification methods (for example, "target amplification" methods) amplify the abundance of target sequences by repeatedly replicating the target sequence (for example, PCR, NASBA, TMA, chain displacement amplification, ligase chain reaction, LAMP, ICAN, RPA, SPA, HAD, etc.), and some amplification methods amplify the abundance of nucleic acid substances that may or may not include the target sequence, but its amplification indicates the presence of specific target sequences in the reaction. Some signal amplification methods can increase the abundance of nucleic acid substances by converting the starting nucleic acid (for example, by cutting the starting nucleic acid to form a cleavage product, or by, for example, polymerization or connection to extend it). Target amplification methods can be applied to signal molecules (eg, PCR can be used to generate more copies of a 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 that replicates DNA segments from a target nucleic acid in vitro. The reaction typically involves extending primers on each strand of the target nucleic acid with a template-dependent DNA polymerase to produce complementary copies of a portion of the strand. The chain reaction comprises iterative cycles of DNA strand denaturation, for example by heating followed by cooling to allow primer annealing and extension, resulting in an exponential accumulation of copies of regions of the target nucleic acid that flank and include the primer binding sites. When an RNA target nucleic acid is amplified by PCR, it is typically converted into a DNA copy strand by an enzyme capable of reverse transcription. Exemplary enzymes include MMLV reverse transcriptase, AMV reverse transcriptase, and other enzymes familiar to those of ordinary skill in the art.

[0133] As used herein, the term "oligonucleotide" (sometimes referred to as "oligonucleotide") is defined as a molecule comprising two or more nucleotides (e.g., deoxyribonucleotides or ribonucleotides) (preferably at least 5 nucleotides, more preferably at least about 10-15 nucleotides, and more preferably at least about 15 to 30 nucleotides, or longer). The length of an oligonucleotide is typically less than 200 residues (e.g., between 15 and 100 nucleotides), however, as used herein, the term is also intended to encompass longer polynucleotide chains. The exact size will depend on many factors, which in turn depend on the ultimate function or purpose of the oligonucleotide. Oligonucleotides are typically referred to by their length. For example, a 24-nucleotide oligonucleotide is referred to as a "24-mer." Oligonucleotides can form secondary and tertiary structures by self-hybridization or hybridization with 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 a combination thereof. In some embodiments, the oligonucleotide that forms the invasive cleavage structure is generated in the reaction (e.g., by extending the primer in an enzymatic extension reaction). As used herein, the terms "oligonucleotide" and "polynucleotide" are used interchangeably and may include non-naturally occurring monomers or portions thereof. More particularly, the oligonucleotide may include, for example, natural and / or modified monomers or linked linear or cyclic oligomers, including deoxyribonucleic acid, ribonucleic acid, substituted and α-anomeric forms thereof, peptide nucleic acids (PNA), locked nucleic acids (LNA), 2'-O-methyl modifications, phosphorothioates, methylphosphonates, spacers, and the like.

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

[0135] As used herein, a "background" signal is a signal (e.g., a fluorescent signal) produced under conditions that do not allow a target nucleic acid-specific reaction to occur. For example, the signal produced in a secondary reaction that includes a FRET cassette and a FEN enzyme but does not include a cassette-specific invasive oligonucleotide (e.g., a flap that is cleaved by the primary reaction) is considered a background signal. In some cases, background signal is measured in a "negative control" reaction or assay in which the target nucleic acid is omitted.

[0136] As used herein, a "channel" of an energy sensor device (such as a device equipped with an optical energy sensor) refers to a predefined wavelength band that can be detected or quantified, and does not include other wavelength bands. For example, a detection channel of a fluorometer may be able to 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 (RFUs) at a given wavelength or over a wavelength band. Examples of common fluorescence detection channels include channels that detect fluorescence emission wavelengths in the following ranges: about 510-530 nm (e.g., commonly used in "FAM" detection channels), about 560-580 nm (e.g., commonly used in "HEX" detection channels), about 610-650 nm (e.g., commonly used in "Texas Red" detection channels), about 675-690 nm (e.g., commonly found in "Cy5" detection channels), and about 705-730 nm (e.g., commonly found in "Quasar705" detection channels). Cy5 and Alexa 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 value" refers to a quantitative limit for interpreting experimental results, where results above and below the cutoff value lead to different conclusions. For example, a measured signal below the cutoff value may indicate the absence of a particular target, but a measured signal exceeding the same cutoff value may indicate the presence of that target. By convention, a result that meets the cutoff value (i.e., exactly has the cutoff value) has the same interpretation as a result that exceeds the cutoff value.

[0138] As used herein, "threshold cycle number" refers to an amplification marker that measures the time or cycle number when the real-time run curve signal intersects an arbitrary value or threshold value. "TTime" and "Ct" determinations are examples of threshold-based amplification markers. Other methods involve performing derivative analysis of the real-time run curve. For the purposes of this disclosure, TArc and OTArc can also be used to determine when the real-time run curve signal intersects an arbitrary value (e.g., corresponding to the maximum or minimum angle of curvature, respectively). Methods for TTime determination are disclosed in US8,615,368; methods for Ct determination are disclosed in EP 0640828 B1; derivative-based methods are disclosed in US6,303,305; and methods for TArc and OTArc determinations are disclosed in US7,739,054. One of ordinary skill in the art will appreciate that changes can also be used to determine the 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 can be distinguished from an analyte nucleic acid (e.g., a target nucleic acid from a test sample) co-amplified in the same reaction. "Internal" means 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 one or more of the same primers used to amplify the analyte nucleic acid to be quantified. In other embodiments, different primers are used for this purpose. Generally, the analyte nucleic acid and the internal calibrator nucleic acid differ by at least one nucleotide position that can be distinguished by the invasive cleavage reaction. This allows multiplexed invasive cleavage assays to independently detect amplified internal calibrator and analyte target nucleic acids.

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

[0141] As used herein, a "reaction vessel" or "reaction receiving vessel" is a container for holding a reaction mixture. Examples include individual wells of a multiwell plate and plastic tubes (e.g., including individual tubes within a linear array of formed multi-tube units, etc.). However, it should be understood that any suitable container can be used to hold the reaction mixture.

[0142] As used herein, "permitting" a reaction to occur means that the reaction mixture provides reagents and conditions to test for the presence of a specific nucleic acid (e.g., target DNA or cleaved 5' flap), which may or may not be present in the reaction mixture. For example, "permitting" 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, and if the target DNA is also present in the reaction mixture to participate in the primary reaction, the primary probe is allowed to be cleaved and the cleaved 5' flap is released. Similarly, "permitting" a secondary reaction of an invasive cleavage assay to occur means that the reaction mixture contains a FRET box and a FEN enzyme under appropriate buffer and temperature conditions, and if the cleaved 5' flap specific for the FRET box is also present in the reaction mixture to participate in the secondary reaction, the FRET box is allowed to be cleaved. Still further, the temperature conditions that "permitting" (or "permitting" or "are permissive for") a reaction to occur are temperature conditions that are conducive to or allow the reaction to proceed.

[0143] "Kit" means a packaged combination of materials intended for use in conjunction with one another. A kit useful according to the disclosed technology may comprise one or more vessels or tubes containing the various reagents. The kit may further comprise instructions or other information in a "tangible" form (e.g., printed information, electronically recorded on a computer-readable medium, or otherwise recorded on a machine-readable medium such as a barcode).

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

[0145] The disclosed procedure uses a "masked oligonucleotide" that hybridizes to a fluorescently labeled 5' flap (e.g., a flap cut from a cassette) that is cut from a probe or FRET cassette through complementary base pairing in an invasive cleavage assay. The masked oligonucleotide contains a fluorescence quenching portion (sometimes referred to herein as a "quencher") attached to it. When hybridization occurs between the masked oligonucleotide and the labeled cut flap, the quenching portion of the masked oligonucleotide is close to the fluorophore portion of the cut 5' flap. The fluorescent signal emitted by the fluorophore of the cut 5' flap is then quenched. When different cleavage products (e.g., cut flaps of different lengths and / or different sequences) hybridize to the homologous masked oligonucleotide at an appropriate temperature, the identity of the secondary reaction that releases the flap cut from the cassette can be determined, and thereby which target sequences are present in the reaction mixture. Invasive cleavage reactions and assays

[0146] Unlike other invasive cleavage assays that generate a fluorescent signal to indicate the presence of analyte nucleic acid by cutting a FRET box, the present technology does not require the persistence of the fluorescent signal to determine whether a specific FRET box is cut. In fact, the technology described herein actually requires that the cutting of at least one FRET box in a multiplex assay generates a fluorescent signal that is quenched or extinguished as the temperature of the reaction mixture changes. This is achieved by including a masked oligonucleotide complementary to the fluorescent cleavage product of the at least one FRET box in the reaction mixture. The hybridization interaction for forming a duplex comprising the fluorescent cleavage product and the complementary masked oligonucleotide is characterized by a melting temperature (T m ). Above T m At a temperature below T, the fluorescent cleavage product is in a single-stranded state, where a fluorescent signal can be generated and detected or measured. mAt a temperature of 1 , a duplex forms and quenches the fluorescence emitted from the fluorophore attached to the 5' flap cleaved from the 5' flap FRET cassette. By including different masking oligonucleotides complementary to different fluorescent 5' flap cleavage products in the reaction mixture (wherein the different duplexes generated by hybridization of the masking oligonucleotides to the fluorescent cleavage products exhibit different T values), the fluorescence emitted from the fluorophore attached to the 5' flap cleaved from the 5' flap FRET cassette is quenched. m ), it is possible to determine which of multiple FRET cassettes labeled with fluorophores detectable in the same channel of a fluorimeter is cleaved to generate a signal. This fluorescence quenching ability, or the difference in the temperature dependence of the fluorescence signal, is essential for the function of the disclosed technology.

[0147] The invasive cleavage assays disclosed herein involve the formation of an invasive cleavage structure, and enzymatic cleavage of the invasive cleavage structure by a flap endonuclease (e.g., FEN-1). In some embodiments, the invasive cleavage structure comprises: (1) a FRET box; and (2) an invasive oligonucleotide that hybridizes to the FRET box. In some embodiments, the invasive oligonucleotide that hybridizes to the FRET box 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, wherein a target-complementary sequence of the primary probe hybridizes to the target nucleic acid; and (3) an invasive oligonucleotide that hybridizes to a target nucleic acid proximal to and upstream of the hybridized primary probe. In some embodiments, the invasive cleavage assay combines a first and a second invasive cleavage reaction in tandem (see Figure 1 ), such that the cleaved 5' flap from the primary probe (eg, sometimes referred to as the "primary cleaved flap") serves as an invasive oligonucleotide to promote enzymatic cleavage of the FRET cassette in the secondary reaction.

[0148] The upstream invasive oligonucleotide of the primary reaction is typically designed to anneal to the target DNA essentially permanently at the assay temperature (e.g., to have a T significantly above the assay temperature). m ), while this is not the case for the primary probe oligonucleotide. The portion of the primary probe that anneals to the target strand is designed to have a T close to the assay temperature with respect to the target strand. m , such that the primary probe oligonucleotides in the reaction mixture will continuously anneal and dissociate from the target strand at the reaction temperature without temperature cycling. In some embodiments, T m Within about 4°C of the assay temperature, more preferably within 3°C of the assay temperature, still more preferably within 2°C of the assay temperature, and still more preferably within 1°C of the assay temperature, a cleavage structure is formed upon annealing of the primary probe oligonucleotide adjacent to and downstream of the invasive oligonucleotide. This cleavage structure can be cleaved by a flap endonuclease.

[0149] A key feature and operating principle of the invasive cleavage reaction according to the present disclosure is that the cleavage product can increase quantity and accumulate under isothermal conditions (that is, without temperature cycling). For example, the primary probe oligonucleotide hybridized with the target nucleic acid can repeat annealing and dissociation in the absence of temperature cycling. The single site on the target DNA can be reused or recycled to hybridize with a series of new uncut probes in the absence of temperature cycling, thereby producing thousands of cut probes for each target molecule. See, for example, Olivier, Mutat Res, 573:103-110 (2005). Similarly, after cyclic hybridization with the 5' flap cut from the primary probe, the fluorescent signal generated by the FEN-1 cutting FRET box 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 secondary cleavage structure. For example, Hall's SISAR assay uses two sequential signal amplification reactions to multiply the total amount of signal generated by the assay (see Hall et al., Proc. Natl. Acad. Sci., USA 97 (2000) 8272-8277). Figure 1 In the SISAR assay presented, cleavage of each primary probe releases a cleaved 5' flap ( Figure 1 The cleaved flap hybridizes to the hairpin "FRET cassette" oligonucleotide to form a secondary cleavage structure. Cleavage of the secondary cleavage structure separates the fluorophore dye from the quenching portion of the FRET cassette, making fluorescence from the fluorophore detectable.

[0151] Invasive cleavage assays using a FRET cassette (e.g., serial invasive cleavage assays) are typically designed so that the cleaved flap-FRET cassette complex has a T close to the assay temperature. m (e.g., within 4°C of the assay temperature, more preferably within 3°C of the assay temperature, still more preferably within 2°C of the assay temperature, and yet more preferably within 1°C of the assay temperature) such that the cleaved flap will continuously anneal and dissociate from the FRET box at the reaction temperature and without temperature cycling. Upon annealing the cleaved flap to the FRET box, a cleavage structure is formed, and this structure can be cleaved by a flap endonuclease (e.g., FEN-1 endonuclease). Thus, the secondary reaction involving cleavage of the FRET box generates a signal using the same recycling principle as the primary reaction. Each cleaved flap can hybridize to a series of new uncleaved FRET boxes without temperature cycling, thereby generating thousands of unquenched fluorophores for each cleaved flap.

[0152] When there is a single base overlap between the invasive probe and the primary probe and when both the invasive probe and the primary probe are hybridized to the analyte nucleic acid, a flap endonuclease (e.g., FEN-1) present in a reaction mixture further comprising the analyte nucleic acid, the invasive probe, the primary probe, and the FRET box will cut the 5' flap from the remainder of the primary probe. This cleavage reaction is referred to as a "primary" reaction. The 5' flap released from the primary probe is then subjected to cyclic hybridization with a FRET box having a sequence complementary thereto, whereby a FEN-mediated cleavage reaction separates the fluorophore from the quencher portion present on the same FRET box, thereby generating detectable fluorescence emission. This cleavage reaction that generates a fluorescent signal by cutting the FRET box is referred to as a "secondary" reaction. As described above, if the secondary reaction occurs near the T of the duplex between the 5' flap cut from the primary probe and the FRET box, the secondary reaction will be detected. m (i.e., the melting temperature) to promote cyclic hybridization, the same 5' flap is free to interact with similar cognate FRET boxes to further catalyze the cleavage reaction. This linear amplification, which can occur at a fixed temperature, can be detected by an increase in fluorescence that varies with time. Each of the primary and secondary reactions is essentially an isothermal process. In fact, even under thermal conditions where thermostable DNA polymerases (e.g., Taq DNA polymerase) exhibit severely impaired polymerization activity, the FEN-1-dependent secondary invasive cleavage reaction can produce a detectable fluorescent signal without the need for polymerization. In fact, polymerase-based extension of the cleaved 5' flap using the FRET box as a template will impair cyclic hybridization that is conducive to the accumulation of fluorescent signal. Therefore, extension of the cleaved 5' flap is prohibited. Useful fluorophores and quenchers

[0153] In some embodiments, a multiplexed invasive cleavage assay according to the presently disclosed technology may use only a single detection channel to detect the fluorescent signal generated by the multiplexed invasive cleavage assay, and preferably will use fluorophores of the same chemical class to generate signals from different targets in the multiplexed reaction. In other embodiments, multiple different fluorophores may be combined to generate signals from different targets in the multiplexed reaction. Exemplary fluorophores used in the FRET box system of the presently disclosed technology include, but are not limited to, fluorescein, rhodamine, REDMOND RED dye, YAKIMA YELLOW dye, hexachlorofluorescein, TAMRA dye, ROX dye, Cy3, Cy3.5, Cy5, Cy5.5, and Cy7, 4,4-difluoro-5,7-diphenyl-4-boron-3a,4a-diaza-indacene-3-propionic acid, 4,4-difluoro-5,4-methoxyphenyl-4-boron-3a,4a-diaza-indacene-3-propionic acid, 4,4-difluoro-5-phenyl-4-boron-3a,4a-diaza-indacene-3-propionic acid, -boron-3a,4-adiaza-sym-indacenyl-propionic acid, 6-carboxy-X-rhodamine, N,N,N',N'-tetramethyl-6-carboxyrhodamine, Texas Red, eosin, fluorescein, 4,4-difluoro-5,7-diphenyl-4-boron-3a,4a-diaza-sym-indacenyl-3-propionic acid, 4,4-difluoro-5,4-ethoxyphenyl-4-boron-3a,4a-diaza-sym-indacenyl-3-propionic acid, and 4,4 -difluoro-5-phenylvinyl-4-boron-3a,4a-diaza-symmetrical 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, nitrobenzyl-2-oxa-1,3-diazo-4-yl (NBD), coumarin, dansyl chloride, aminomethylcoumarin (AMCA), erythrosine, BODIPY dye, CASCADE BLUE dye, OREGON GREEN dye, pyrene, lissamine, xanthene, acridine, oxazine, phycoerythrin, QUANTUM DYE, thiazole orange-ethidium heterodimer, etc.

[0154] Exemplary quenchers used in the FRET cell systems of the technology include, but are not limited to, cyanine dyes (e.g., Cy3, Cy3.5, Cy5, Cy5.5, and Cy7), rhodamine dyes (e.g., tetramethyl-6-carboxyrhodamine (TAMRA) and tetrapropyl-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 quenchers (Epoch Biosciences, Inc., Logan, UT), etc. Alternative quenchers include Black Hole Quencher dyes, particularly BHQ-1, BHQ-2, and BHQ-3 (Biosearch Technologies, Petaluma, CA); Quencher (Berry & Associates, East Dexter, MI); and IOWA (Integrated DNA Technologies, Coralville, IA) Analysis of the absorption and emission spectra of various molecules, among other factors, when selecting pairs or groups of moieties for use in FRET configurations is well within the skill of the art.

[0155] One of ordinary skill in the art will be aware of the wavelength ranges that can be detected by different channels of a fluorometer and will therefore be able to readily select fluorophores for use in different FRET cassettes, where emission from different fluorophores can be detected in the same fluorometer channel. Characteristics of temperature-dependent multiplexing technology

[0156] Unlike existing invasive cleavage assays that benefit from the uninterrupted persistence of the fluorescent signal, the technology disclosed in the present invention benefits from the selective inhibition of fluorescence after FEN-1-mediated cleavage of the FRET box. Peterson et al. disclosed in published U.S. patent application 2018 / 0163259A1 a method in which different FRET boxes labeled with the same fluorophore are cleaved at different temperatures, where the different temperatures essentially separate one reaction from another. Monitoring the cumulative fluorescence signal over time or cycle number allows the activity of the FRET box to be resolved. In this way, multiple nucleic acid targets can be detected in a multiplexed format using only a single channel of a fluorometer, or even using only a single type of fluorescent label. As disclosed herein, the technology disclosed in the present invention relies on temperature-dependent fluorescence quenching to effectively remove the contribution of the fluorescent signal generated by different FRET boxes, even if the different FRET boxes have the same fluorescent label. Therefore, in the technology disclosed in the present invention, the process or event that occurs after the FRET box is cleaved provides useful information.

[0157] The present technology utilizes at least one FRET cassette configured to include a 5' flap portion that retains the fluorophore after cleavage by the FEN-1 enzyme in the secondary reaction. Preferably, the 5' flap has a length ranging from 8 to 30 nucleotides (more preferably, from 8 to 25 nucleotides, and even more preferably, from 8 to 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 A series of invasive cleavage reactions are shown in which the FRET cassette is a flapless FRET cassette that does not contain a 5' flap portion and in which cleavage in the secondary reaction releases a fluorescent dye attached to a single nucleotide. In contrast, the 5' flap FRET cassette (see Figure 2 Panel A) can be hybridized with the cleaved flap from the primary probe (see Figure 2 B) to generate a structure that can be cleaved by the FEN-1 enzyme. When in a single-stranded state, the resulting cassette-cleaved flap emits a fluorescent signal, but this signal can be reversibly inhibited or quenched by hybridizing the cassette-cleaved flap to a masking oligonucleotide containing a quenching moiety (see Figure 2 (Panel C). The hybridization interaction between the flap of the cassette cleavage and the masking oligonucleotide 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 generate a signal that can be resolved by temperature changes after the cleavage reaction occurs.

[0158] In addition to distinguishing the origin of fluorescence generated by a flapless FRET cassette (without a 5' flap portion) and a 5' flap FRET cassette, the disclosed technology also allows resolution of signals generated by the same fluorescent dye of different 5' flap FRET cassettes. Figure 3 Schematic illustration of how the fluorescence signals generated 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 fluorimeter, can be distinguished from one another under different temperature conditions. The upper portion of the figure shows a flapless FRET cassette that does not contain a 5' flap. After cyclic hybridization with a complementary cleaved flap from a primary probe, this FRET cassette can be cleaved by the FEN-1 endonuclease to release the fluorescent dye, wherein 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 any temperature. Figure 3The middle portion of the figure shows the first 5' flap FRET cassette (5' flap FRET cassette 1), which can be cleaved by the FEN-1 enzyme to release the 5' flap ("cassette cleaved flap 1") after cyclic hybridization with the complementary cleaved 5' flap from the primary probe. After the cleavage reaction, cassette cleaved flap 1 remains attached to the fluorophore, but if cassette cleaved flap 1 hybridizes to a complementary masking oligonucleotide, the fluorescence signal is quenched. For a hybridized duplex comprising cassette flap 1 and a complementary masking oligonucleotide, this may occur at temperatures below T. m If the temperature rises above T m (“T m 1”), the fluorophore of the box-cleaved flap 1 can emit a fluorescent signal, thereby placing the box-cleaved flap 1 in a single-stranded state (“unquenched box-cleaved flap 1” in the figure). Figure 3 The lower portion of the figure in FIG shows a second 5' flap FRET cassette (5' flap FRET cassette 2), which can be cleaved by the FEN-1 enzyme to release the 5' flap ("cassette-cleaved flap 2") after cyclic hybridization with the complementary cleaved 5' flap from the primary probe. After the cleavage reaction, the cassette-cleaved flap 2 remains attached to the fluorophore, but if the cassette-cleaved flap 2 hybridizes to the complementary masking oligonucleotide, the fluorescence signal is quenched. For a hybridized duplex comprising the cassette-cleaved flap 2 and the complementary masking oligonucleotide, this may occur at temperatures below T. m If the temperature rises above T m (“T m 2”), the fluorophore of the cassette-cleaved flap 2 can emit a fluorescent signal, thereby placing the cassette-cleaved flap 2 in a single-stranded state (“unquenched cassette-cleaved flap 2” in the figure). Different 5’ flap FRET cassettes are designed to contain different T m The 5' flap portion of the cassette can be distinguished from each other by different lengths and / or G:C contents. For example, the signals emitted by the fluorophores of the different cassette-cleaved flaps can be monitored: (1) so that only the fluorophore of one cassette-cleaved flap emits a signal at a time; or (2) by determining the melting profile based on its temperature dependence. Both of these alternatives are demonstrated in the working examples herein.

[0159] Notably, in procedures using a variety of different 5' flap FRET cassettes and masking oligonucleotides, the T values for the hybrid duplex comprising the 5' flap cleavage product ("cassette cleaved flap") and the homology masking oligonucleotide were calculated. m Preferably, the T values of different hybridization duplexes analyzed in the same multiplex reaction (e.g., whether a real-time nucleic acid amplification reaction or an end-point melting / annealing analysis procedure) are different. mThe difference is 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. T for the hybrid duplex comprising the cassette-cleaved flap and the homology masking oligonucleotide m The preferred difference between the two is in the range of 2°C to 20°C, more preferably 5°C to 20°C, more preferably 5°C to 15°C, or even more preferably 5°C to 10°C. In some embodiments, the 5' flap of the masking oligonucleotide and / or the FRET cassette oligonucleotide may comprise one or more nucleotide modifications to adjust or alter the T of the masking oligonucleotide-cassette cleaved flap duplex. m For example, in some embodiments, the one or more nucleotide modifications are selected from locked nucleic acid (LNA), peptide nucleic acid (PNA), bridged nucleic acid (BNA), 2'-O alkyl substitutions, L-enantiomer nucleotides, or a combination thereof.

[0160] Figure 4 Schematic illustration of the identification of different target sequences by detecting FEN-1-mediated cleavage products of different FRET cassettes, where each FRET cassette contains a fluorescent dye (e.g., displayed as the same HEX fluorophore) that can be detected in the same channel of a fluorimeter. The indicated cleavage products and masking oligonucleotides correspond to Figure 3 Those shown in . The fluorophore-containing cleavage product from the flapless FRET cassette (indicating the presence of target 1) (top) remains unquenched at all temperatures. Thus, at temperatures below temperature 1, where cassette-cleaved flap 1 and cassette-cleaved flap 2 are hybridized to their respective masking oligonucleotides, detectable fluorescence indicates the presence of target 1. The fluorophore-containing cleavage product from the 5' flap FRET cassette 1 (indicating the presence of target 2) (middle) remains unquenched at temperatures above temperature 1. At temperatures above temperature 1 and below temperature 2, detectable fluorescence can indicate the presence of both target 1 and target 2. Because the fluorescence signals measured in this procedure are additive, at temperatures above temperature 1 but below temperature 2 ( Figure 4 The fluorescence signal measured at a temperature lower than 1 ( Figure 4 The difference between the fluorescence signals measured at temperatures above 2 (upper) indicates the signal due to the presence of target 2. The fluorophore-containing cleavage product from the 5' flap FRET cassette 2 (indicating the presence of target 3) (lower) remains unquenched at temperatures above temperature 2. At temperatures above temperature 2, detectable fluorescence can indicate the presence of target 1, target 2, and target 3. Therefore, at temperatures above 2 ( Figure 4 The fluorescence signal measured at a temperature higher than temperature 1 but lower than temperature 2 ( Figure 4The difference between the fluorescence signals measured at different temperatures (middle of the figure) indicates the signal generated due to the presence of target 3. In this figure, temperature 1 < temperature 2 < temperature 3. The fluorescence detection results at different temperatures can be compared to determine which target is responsible for promoting one or more cleavage reactions. In some embodiments, this involves comparing the size of the fluorescence signals, where the signals measured at different temperatures are the sum of known detectable fluorescence, as described immediately above. In some embodiments, determining which target is responsible for promoting one or more cleavage reactions involves performing a melting / annealing curve analysis. Results processing and equipment

[0161] Amplified nucleic acid can be used and the conventional laboratory equipment that monitors amplicon generation can be used to carry out program disclosed herein, and the equipment includes the equipment with an integrated or independent computer or processor programmed with appropriate software. The implication of "computer" includes an embedded processor controlled by software. The computer can be programmed by a manufacturer or end user to perform one or more temperature changes or steps, preferably allowing the fluorescence in the monitoring reaction mixture to be monitored when the circulation of amplified reaction occurs. Preferably, the reaction mixture is included in the reaction vessel (for example, the hole of a pipe or a multi-well plate) maintained in the nucleic acid amplification device. However, it is also possible to complete the analysis of the amplified product (for example, to set up the melting / annealing curve of the amplified product) after the amplified reaction is complete. The latter analysis can even be completed outside the equipment of amplified nucleic acid.

[0162] The computer components of the apparatus for performing the disclosed technology can be programmed with software instructions that "cause" the computer to perform certain steps. These steps can involve any of the following steps: controlling a thermal cycler that amplifies nucleic acids; receiving input from a fluorometer that monitors fluorescence emission in a reaction mixture where FRET box cleavage occurs in an invasive cleavage reaction; or processing the results to determine which of two or more FRET boxes is cut to produce a fluorescent signal. In a preferred embodiment, the FRET box is cut to produce fluorescent cleavage products, wherein the fluorescence produced by the different cleavage products can be detected or measured in a single channel of the fluorometer. In some embodiments, different FRET boxes are labeled with the same fluorophore. A computer can also be used to perform mathematical steps (e.g., addition, subtraction, multiplication, and / or division) to determine which FRET box in the mixture is cut to produce a detectable or measurable fluorescent signal.

[0163] The method disclosed herein can be carried out using an automated nucleic acid analyzer (e.g., a device that amplifies nucleic acid and monitors the generation of nucleic acid amplification products). These analyzers include PCR instruments or real-time PCR instruments that can be programmed to perform a series of temperature cycling steps. Preferably, the PCR instrument is equipped with a fluorometer that monitors the progress of the reaction that occurs in the hole of a tube or multi-well plate (typically a reaction "receiving vessel"). An instrument configured to carry out and monitor real-time PCR reactions is particularly preferably used in conjunction with the disclosed technology. An example of a preferred device for performing, monitoring, and evaluating the results that can be obtained by the disclosed technology is the Panther Fusion System (Hologic, Inc.; San Diego, California), which advantageously automates the steps of the program. Another preferred device is the ABI 7500 real-time PCR system (ThermoFisher Scientific; New York).

[0164] One general method for evaluating the status of fluorescent cleavage products in a mixture of a flapless FRET cassette and one or more 5' flap FRET cassettes involves evaluating cleavage of the two types of FRET cassettes separately. This procedure may involve lowering the temperature of the reaction mixture to below the lowest T for any duplex formed between the masking oligonucleotide and the fluorescent cleavage products of the 5' flap FRET cassette in the mixture. m . The result is that the residual fluorescence signal comes from the fluorescent cleavage product that can not be quenched, and therefore exhibits a constant value on the first derivative diagram of fluorescence varying with temperature. Therefore, when the fluorescence from other fluorescent cleavage products is quenched due to forming a duplex with the masking oligonucleotide, measuring or detecting the residual fluorescence signal (for example, a specific signal exceeding background fluorescence) can indicate that non-quenchable fluorescent cleavage products exist. Detecting the fluorescent signal under such conditions can indicate that there is non-quenchable fluorescent cleavage products in the reaction mixture, so the corresponding FRET box is cut. In a second step, a temperature-dependent melting / annealing curve (sometimes "quenching spectrum") for the reaction mixture containing the fluorescent cleavage product can be obtained, and a derivative diagram is thus obtained. For example, the first derivative diagram of the fluorescence variation that varies with temperature will include peaks or maximum values corresponding to the different duplexes present in the reaction mixture, and wherein duplex formation quenches the signal from the fluorescent cleavage product contained therein. In this way, it is possible to determine which one or more FRET boxes are cut to produce fluorescent cleavage products.

[0165] This paper demonstrates an exemplary system by performing multiple detection on two or three analytes using invasive cutting of different FRET boxes, wherein the FRET box comprises a fluorescent label that can be detected or monitored in a single channel of a fluorimeter or fluorescence monitoring device. In some embodiments, multiple FRET boxes are used in a multiplex program. For example, two different FRET boxes can be combined in a single reaction mixture, wherein only one FRET box comprises a 5' flap sequence complementary to the masked oligonucleotide contained in the same reaction mixture. The other FRET box can be a flapless FRET box that does not comprise any 5' flap sequence, or alternatively can be a 5' flap FRET box in a reaction mixture that does not comprise complementary masked oligonucleotides. In such cases, the fluorescent signal generated by cutting only one of the two FRET boxes will undergo temperature-dependent fluorescence quenching. As described above, the fluorescent signal generated by cutting the FRET box remains substantially constant as the temperature of the reaction mixture changes, wherein the fluorescent cleavage product does not interact with any masked oligonucleotide to quench fluorescence. Detection of such non-quenchable fluorescent cleavage products can involve detecting a fluorescent signal (e.g., a fluorescent signal above background) at a temperature at which formation of the duplex comprising the masking oligonucleotide quenches the fluorescent signal from all other quenchable fluorescent cleavage products monitored in the same fluorescent channel in the reaction mixture. Detection of quenchable fluorescent cleavage products in a multiplex reaction mixture can involve simply determining that the fluorescent signal is greater than or equal to the background signal below T. m Compared with the temperature at which fluorescence quenching is maximal, the temperature above the T for the formation of masked oligonucleotide duplexes is m The fluorescence signal that can be measured at a higher temperature is greater.

[0166] In various embodiments, the multiplex reaction mixture comprises two different FRET cassettes, wherein each FRET cassette comprises a cleavable 5' flap sequence, and wherein the fluorescent signal emitted by each cleaved 5' flap can be quenched by hybridization with a different complementary masking oligonucleotide. Determining which FRET cassette is cleaved in the reaction mixture can involve derivative analysis, preferably a first derivative plot of the melting / annealing curve, as also described above. In this way, multiple cleavage products can be resolved and identified in a single procedure.

[0167] In some embodiments, the multiplex invasive cleavage reaction comprises three or more different FRET cassettes, each labeled with a fluorescent label detectable in a single channel of a fluorimeter (e.g., all fluorescent labels can be the same). The FRET cassettes can each contain a different cleavable 5' flap sequence that emits a fluorescent signal upon cleavage, wherein the fluorescent signal can be quenched in a temperature-dependent manner upon hybridization of a complementary masking oligonucleotide. When the duplex formed by hybridization of the flaps cleaved by the different fluorescent cassettes is characterized by different T mWhen, can easily determine the identity of duplex by assessing melting / annealing characteristics (for example, using derivative analysis).Below use first-order derivative figure to detect and identify the duplex comprising the fluorescent cleavage product hybridized with masked oligonucleotide to illustrate this point.Still further, one of the FRET box used in the multiplex reaction can produce the fluorescent cleavage product that is not quenched in the reaction mixture.In this case, the cleavage product of aggregation can still be said to show different temperature-dependent fluorescence quenching spectra, because some products show fluorescence quenching at different temperatures, and a kind of product does not show fluorescence quenching.In fact, along with the temperature change of reaction mixture, different cleavage products can be distinguished by monitoring fluorescent signal.This may relate to when temperature changes from high to low (for example, to allow the annealing of complementary chain to form duplex) or alternatively changes from low to high (for example, to promote the melting of preformed duplex) time monitoring fluorescent signal.Therefore, temperature-dependent fluorescence quenching spectrum is sometimes referred to as " melting / annealing " curve or spectrum.

[0168] The results of differential quenching of fluorescent cleavage products generated in multiple invasive cleavage reactions can be analyzed by different methods to determine which alternative FRET cassettes are cleaved to produce a fluorescent signal detectable in a single channel of a fluorimeter or fluorescence detection device. Two preferred analysis methods that can be automated (e.g., by a computer, processor, or controller) involve: (1) evaluating the difference between fluorescence readings or measurements at temperatures where different fluorescent cleavage products experience greater or lesser fluorescence quenching due to hybridization of the masking oligonucleotide; and (2) evaluating the fluorescence quenching profile (i.e., fluorescence measured as a function of temperature in the presence of the complementary masking oligonucleotide), for example, by using mathematical derivative analysis of the characteristic melting temperature (i.e., "T") of the duplex formed in the reaction mixture. m In certain preferred embodiments, a combination of these different methods can be used to distinguish which of a plurality of different FRET cassettes is cleaved in a reaction mixture, wherein the different cleavage products comprise fluorophores that are detected in the same channel of a fluorometer. In some embodiments, the different FRET cassettes comprise the same fluorophore. For example, a reaction mixture containing a fluorescent cleavage product that does not interact with a masking oligonucleotide to achieve quenching and one or more fluorescent cleavage products that hybridize to a cognate masking oligonucleotide is preferably analyzed by evaluating the fluorescence intensity of a duplex containing a complementary fluorescent cleavage product of the masking oligonucleotide and the 5' flap FRET cassette at a temperature above and below the T of the duplex. m The difference between the fluorescence measured at the temperature and the derivative of the change in fluorescence as a function of temperature (e.g., a first derivative plot) is shown.

[0169] Cleavage of a FRET cassette used in a multiplex invasive cleavage assay to generate two fluorescent cleavage products, only one of which is subject to temperature-dependent quenching, can be analyzed by assessing fluorescence emission at two temperatures, or alternatively using this assessment method in conjunction with curve analysis by T m Identify duplexes. Fluorescent cleavage products that cannot be quenched in the reaction mixture remain consistently fluorescent over the temperature range of the measurement and therefore have a constant slope (i.e., zero slope) on a graph of fluorescence as a function of temperature. A graph of the first derivative of the change in fluorescence as a function of temperature will not exhibit a T value indicative of any duplex. m Conversely, a first derivative plot indicating the presence of a fluorescent cleavage product that undergoes temperature-dependent quenching (ie, due to hybridization of the masking oligonucleotide) would exhibit a T indicative of a duplex containing a fluorescently cleaved 5′ flap. m Still further, a simple evaluation of the fluorescence signal measured at two temperatures can indicate the presence or absence of each of the two fluorescent cleavage products. More particularly, the presence or absence of each of the two fluorescent cleavage products can be determined at a temperature above the T of the duplex in the reaction mixture. m at a temperature below the T of the duplex in the reaction mixture (i.e., a temperature at which no fluorescence quenching occurs). m Fluorescence is measured or detected at a second temperature (ie, a temperature at which duplexes are formed; complete fluorescence quenching).

[0170] In some cases, a single non-quenchable fluorescent cleavage product (e.g., resulting from cleavage of a flapless FRET cassette, or from cleavage of a flap of a cassette in the absence of a complementary masking oligonucleotide) is present in a reaction mixture with one or more quenchable fluorescent cleavage products. m The presence of a fluorescent cleavage product that is not quenched by hybridization of the masking oligonucleotide is determined when a detectable fluorescent signal is measured in the fluorometer channel at a temperature above T (at which fluorescence quenching in the fluorometer channel is maximal). In other words, measurable fluorescence at the point where the duplex quenches fluorescence from other cleavage products in the reaction mixture (i.e., a specific signal above the background signal threshold) indicates the presence of a fluorescent cleavage product that is not quenched by hybridization of the masking oligonucleotide. If the fluorescence signal is not quenched at a temperature above T m The fluorescence signal measured at a temperature below T (e.g., the temperature at which fluorescence quenching is minimal) exceeds that measured at a temperature below T m The presence of fluorescent cleavage products quenched by hybridization of the masking oligonucleotide can be determined by measuring the fluorescence signal at a temperature below 0.05°C (i.e., a temperature at which fluorescence quenching is complete).

[0171] In some embodiments, it may be desirable to combine these two approaches, particularly when multiple different fluorescent cleavage products that undergo quenching by hybridization of the masking oligonucleotide are combined with one fluorescent cleavage product that does not undergo quenching. In such cases, melting / annealing curves can be generated and evaluated by derivative analysis to determine the T of the duplex that quenches fluorescence. m To determine the presence of quenchable fluorescent cleavage products. Similarly, the point corresponding to complete fluorescence quenching and / or the absence of fluorescence quenching on the melting / annealing curve can be used for the above-mentioned assessment. More particularly, the presence of fluorescent cleavage products that do not undergo quenching can be assessed at a reduced temperature when the quenching caused by masked oligonucleotide hybridization is complete. When quenching is complete (that is, maximum) in the reaction mixture, residual fluorescence signal is detected to indicate the presence of non-quenchable fluorescent cleavage products. For a single quenchable fluorescent cleavage product, the presence of a fluorescent cleavage product that is not quenchable is determined by quenching at a temperature lower than the T of the duplex formation. m Compared to the fluorescence readings at a temperature greater than the T for duplex formation (e.g., at which duplexes form and are stable), the fluorescence readings at a temperature greater than the T for duplex formation are m A higher fluorescence reading at a temperature that is less than 100 nm (eg, at which no duplex is present) indicates the presence of a quenchable fluorescent cleavage product.

[0172] Reaction mixtures containing more than one distinct fluorescent cleavage product, each of which undergoes quenching by hybridization with a masking oligonucleotide, can be conveniently evaluated by processing the melting / annealing curve results using derivative analysis to identify the T of duplexes that may be present in the mixture. m 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 with temperature can be constructed. The peak or maximum on the first derivative plot corresponds to the T of the duplex between the masking oligonucleotide and the complementary fluorescent cleavage product. m When each duplex is characterized by a different T m When , duplexes can be detected independently of each other. Higher order derivatives are also considered for identifying duplexes and determining which of the multiple FRET boxes is cleaved to produce the fluorescent signal. As described elsewhere herein, it is desirable to have T for different duplexes separated by minimal temperature differences. m , to facilitate the distinction of one duplex from one or more other duplexes.

[0173] Included in the above description is software that enables a computer to process the results and determine which FRET cassette in the mixture of FRET cassettes produced the cleavage product. Example

[0174] Presented herein are techniques for performing multiplexed detection using at least one 5' flap FRET cassette (i.e., a FRET cassette having a 5' flap portion) wherein the 5' flap portion comprises a fluorescent label. Upon cleavage of the 5' flap FRET cassette by FEN-1 endonuclease in an invasive cleavage assay, the signal emitted from the fluorescent label can be selectively quenched based on temperature. In some embodiments, the invasive cleavage assay comprises both a primary and a secondary invasive cleavage reaction. In some other embodiments, the invasive cleavage assay comprises a secondary invasive cleavage reaction and does not comprise a primary invasive cleavage reaction. It will be appreciated 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 the intact 5' flap FRET cassette. The quenching of fluorescence emitted from the cleaved 5' flap is mediated by temperature-dependent hybridization of the cleaved 5' flap with a complementary masking oligonucleotide comprising a quencher portion. In this way, the quenchable flap is not part of any probe that hybridizes to the target nucleic acid to be detected. In contrast, the quenchable flap can be the product of a linear amplification reaction that occurs under isothermal conditions in the absence of polymerization.

[0175] According to the present disclosure, single-channel fluorescence detection can be used to detect 5' flap FRET boxes that report different target molecules. The fluorescent labels used for different FRET boxes can be the same fluorescent label. In some embodiments, the same fluorescent label is used on FRET boxes with different 5' flaps. Different fluorescent labels can be used to replace the same label, provided that the different labels can be detected in the same fluorescent channel of an optical detector (e.g., a fluorimeter). In all cases, cutting the FRET box to produce a fluorescent signal is mediated by the FEN-1 enzyme (i.e., a non-polymeric flap endonuclease). The 5' flap from the cut of the primary probe plays a catalytic role in the cutting of the FRET box, which means that the 5' flap hybridizes instantaneously with the FRET box, promotes cutting to release the fluorescent signal, and then dehybridizes to allow the cut flap to interact with a new FRET box. The 5' flap cut from the primary probe that hybridizes to the nucleic acid target to be detected preferably does not contain a fluorophore portion.

[0176] Example 1 demonstrates how to amplify and detect two different nucleic acid sequences (analyte A and analyte B) in the same reaction mixture using single-channel or dual-channel fluorescence detection. Amplification is performed by polymerase chain reaction (PCR). The products of the PCR reaction are detected using an invasive cleavage reaction using a fluorescently labeled FRET box. Analyte A is detected using a FRET box having a first label (hexachlorofluorescein or "HEX") that is detectable in the HEX channel of the fluorometer assembly of the real-time PCR instrument. Analyte B is detected using either of two different FRET boxes in the same reaction mixture, wherein each FRET box contains a different label. The first 5' flap FRET box for detecting analyte B contains a second label that is also detectable in the HEX channel of the PCR instrument. The signal emitted by the label connected to the 5' flap cut from this FRET box indicates the presence of analyte B and undergoes quenching after hybridization of the cut 5' flap of the FRET box with a complementary masked oligonucleotide. The second FRET box for detecting analyte B contains a label detectable in the ROX channel of the PCR instrument, wherein the fluorescent signal emitted after cleavage does not undergo quenching. Notably, the signal detected in the ROX channel of the real-time amplification and detection instrument was essentially undetectable in the instrument's HEX channel, and vice versa. In other words, the fluorescent HEX signal was essentially undetectable in the ROX channel, and vice versa. The results demonstrate that each of the two analyte nucleic acids can be amplified, and that the synthesis of different amplification products can be monitored using fluorophores detected in the same or different optical channels of the PCR instrument. Example 1 Single- or dual-channel fluorescence detection of two analyte nucleic acids in a single reaction

[0177] The reaction mixture is prepared as replicates, wherein each reaction mixture comprises a lyophilized composition that has been absorbed into an aqueous reconstitution buffer. The lyophilized composition comprises dNTPs, a thermostable DNA polymerase (e.g., Taq DNA polymerase from Promega Corporation in Madison, Wisconsin), a FEN-1 flap endonuclease (e.g., Cleavase 2.0 enzyme from Hologic Inc. in Marlborough, Massachusetts), oligonucleotides, and trehalose. For each of the two analyte nucleic acids to be detected, the oligonucleotides in the lyophilized composition include: a primer pair, a primary probe with a 5' flap that is non-complementary to the analyte sequence to be amplified and / or to be detected, and a FRET box that can be cut by the FEN-1 enzyme after hybridization of the 5' flap cut from the primary probe. In this embodiment, the oligonucleotide that promotes cutting the 5' flap from the primary probe is also used as a primer in the nucleic acid amplification reaction. In the isothermal cyclic hybridization reaction, two different primary cut flaps (one from each primary probe complementary to analyte A and analyte B) reversibly hybridize with three different FRET boxes. FRET Cassette 1 (used to detect analyte A) is a flapless FRET Cassette labeled with a HEX fluorophore and a BlackBerry Quencher moiety (Berry & Associates; Dexter, MI). Following cleavage of FRET Cassette 1, emission from the HEX fluorophore is detected in the HEX channel of an instrument used to amplify nucleic acids and monitor the progress of the amplification reaction. FRET Cassette 1 does not contain a 5' flap sequence. FRET Cassette 2 is also a flapless FRET Cassette that does not contain a 5' flap but is labeled with a CAL Fluor Red 610 fluorophore (Biosearch Technologies, Inc.; Novato, CA) and The quencher moiety (Biosearch Technologies, Inc.) is labeled and used to detect analyte B. After the cleavage reaction, the emission signal from the CAL Fluor Red 610 fluorophore can be detected in the ROX channel of the instrument used to amplify the nucleic acid and monitor the progress of the amplification reaction, rather than in the HEX channel. The FRET cassette 3, also used to detect analyte B, is labeled with CAL on the 5' flap. The 5' flap FRET cassette of the Orange 560 fluorophore is attached to the hairpin portion with After the cleavage reaction, the CAL quencher can be detected in the HEX channel of the instrument used to amplify the nucleic acid and monitor the progress of the amplification reaction. The emission signal of the Orange560 fluorophore is complementary to the 5' flap of FRET cassette 3 and contains The masked oligonucleotide of the quenching moiety (Biosearch Technologies, Inc.) was included in the reaction mixture at a three-fold molar excess over the FRET cassette 3.

[0178] The target analytes, FRET cassette configurations, and detection channels used in the procedure are summarized in Table 1 . Table 1 Multiplex detection system HEX channel ROX Channel Masked oligonucleotides FRET Cassette 1 (Analyte A) X FRET Cassette 2 (Analyte B) X FRET Cassette 3 (Analyte B) X X

[0179] The amplification reaction using the detection system presented in Table 1 includes a single analyte A target, a single analyte B target, or a combination of analyte A and analyte B targets. The "no target" negative control reaction includes all reagents but does not include an added template. Each reaction includes all three FRET boxes and a masked oligonucleotide. Thermal cycling and fluorescence monitoring are performed using an ABI 7500 real-time PCR system instrument (ThermoFisher Scientific; Grand Island, New York). Reaction conditions include the following 10 cycles: 95°C for 120 seconds, 69°C for 5 seconds, 67°C for 5 seconds, 65°C for 6 seconds, and 72°C for 5 seconds. Subsequently, the following 40 cycles are performed: 95°C for 10 seconds, 69°C for 5 seconds, 67°C for 5 seconds, and 65°C for 25 seconds. At a temperature where the masked oligonucleotide remains unhybridized to release the 5' flap from the FRET box 3, fluorescence emission data for the ROX channel and HEX channel of the real-time PCR instrument are collected. The fluorescence signal that changes with the cycle number of the reaction containing both analyte B and analyte A is measured in the HEX and ROX channels. The signal in the ROX channel (data not shown) results in a sigmoid curve, which reflects the cycle-dependent increase of the signal, indicating that analyte B is amplified in the PCR reaction. The HEX signal reflects the combined signal from the amplification of analyte B and analyte A, but does not distinguish between the two (data not shown). The HEX and ROX fluorescence signals can be independently detected in the multiplex reaction of amplifying analyte B and analyte A.

[0180] Figure 5 Panels AC show the signal measured in the HEX channel as a function of cycle number for reactions containing individual analytes or combinations, as shown. Figure 5 The graph in panel A of demonstrates that amplified Analyte A can be detected in the absence of added Analyte B template and shows a characteristic sigmoidal curve of signal accumulation for this analyte. Figure 5 Panel B shows the signal measured in the HEX channel, which indicates the amplification of Analyte B template in the absence of Analyte A template. In this case, the monotonic signal accumulation curve lacks Figure 5 The S-shaped characteristic of the curve is shown in panel A. Figure 5The small figure C shows the signal detected in the amplified reaction, and the amplified reaction includes both analyte A template and analyte B template in a single reaction.Here, the signal measured in the HEX channel increases in a manner to produce an extended S-shaped curve, representing the combined signal produced by analyte A and analyte B FRET boxes measured in a single channel of a fluorometer.In this case, the signal produced by non-identical fluorophores is detected in a single (i.e., identical) channel of the fluorometer.As discussed elsewhere herein, the identity of the analyte producing each different result can be inferred using the analysis of melting / annealing curve analysis and curve shape (e.g., first derivative analysis).

[0181] Example 2 demonstrates a procedure for resolving the identities 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 that is complementary to the masking oligonucleotide undergoes quenching. Example 2 Endpoint melting / annealing curve analysis differentiates amplified targets

[0182] 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 involves monitoring the magnitude of the fluorescent signal in the HEX channel of the real-time PCR instrument as the temperature is varied from 90°C to 21.4°C.

[0183] The results of the post-amplification melting / annealing curve analysis are presented in Figure 6These data confirm that the cleavage product of the FRET box specifically for detecting analyte A consistently maintains fluorescence that varies with temperature (i.e., within the temperature range shown), and the cleavage product has a HEX channel signal generated by a flapless FRET box that is not quenchable with a masked oligonucleotide. In contrast, in the presence of a masked oligonucleotide comprising a quenching portion, the HEX channel signal generated after cleavage of the 5' flap FRET box 3 specifically for detecting analyte B exhibits temperature-dependent fluorescence quenching. More specifically, when the temperature approaches 20°C, the fluorescence in the reaction mixture into which only the analyte B template is incorporated is effectively quenched, thereby reducing the fluorescence signal to 500,000 RLU. It has been empirically observed that essentially all fluorescence is quenched at temperatures slightly below 40°C. For reactions comprising analyte A and analyte B templates, the signal in the HEX channel is essentially a combination of separate temperature spectra. More specifically, the spectrum of this melting / annealing curve follows the melting / annealing curve of the analyte B templated reaction, but the signal is increased overall by adding the unchanged fluorescence caused by the presence of analyte A in the templated reaction. As the temperature approaches 20°C, the signal indicating the presence of analyte B is quenched, so that the fluorescence in the mixed reaction approaches the signal level observed in the reaction containing only analyte A. As shown in the figure, in the reaction mixture containing analyte B template, the fluorescence is maximum at approximately 63°C and decreases above 63°C. While not wishing to be bound by any particular theory of operation, this decrease in fluorescence above 63°C may be due to a temperature-dependent buffering effect on the fluorophore, as the masking oligonucleotide hybridization may occur at a temperature greater than the T of the 5' flap masking oligonucleotide duplex. m The ions are reduced or completely lost at much higher temperatures (e.g., 10°C-20°C higher).

[0184] As disclosed herein, the results obtained using an invasive cutting system for multiplexed detection of nucleic acid analytes can be processed in different ways to determine the presence or absence of an analyte in a test sample. For example, a fluorescence signal 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 determine the presence or absence of each of two different analytes. The threshold value can be pre-set (i.e., before the assay is performed) or set at the time of the assay (e.g., using one or more calibration standards with one or more analytes to be detected). A threshold value of 2.2 x 10 6 RFU and 1x 10 6 Exemplary thresholds at RFU and Figure 6 Under these parameters, the results at 63 ° C were greater than 2.2 x 10 6Fluorescence readings of RFU indicate detection of analyte B, while readings below this threshold indicate the absence of analyte B. 6 RFU and 2.2x 10 6 Fluorescence readings between RFU indicate the presence of analyte A and the absence of analyte B. Similarly or alternatively, greater than 1x10 6 Fluorescence readings of RFU indicate the presence of analyte A. Such readings do not provide information about the presence of analyte B because at this temperature, the fluorescence from the cleaved 5' flap indicating the presence of this analyte is essentially completely quenched. At 30°C, less than 1 x 10 6 Fluorescence readings of RFU indicate the absence of Analyte A.

[0185] Derivative-based data analysis methods can be used instead of or in combination with the threshold-based analysis described above. For example, a data plot related to the presence of analyte A (see Figure 6 As an example) has a constant slope (e.g., zero slope) and is approximately 5 x 10 5 The fluorescence size of at least twice the background fluorescence of RFU. This fluorescence size (for example, measured at a temperature when fluorescence quenching is maximum in a reaction mixture) can be used to indicate the presence of analyte A. The data graph related to the presence of analyte B shows a first-order derivative maximum value in the range of about 50 DEG C -58 DEG C, wherein at 63 DEG C there is a zero crossing (that is, passing through the x-axis to indicate zero slope). Any data set showing these features can be interpreted as indicating the presence of analyte B. In some embodiments, the fluorescence size at a specific temperature can be used for analysis based on threshold value, and the temperature-dependent rate of change of fluorescence can be used for data analysis based on derivative, and these two analyses can be combined to determine the presence or absence of each of the multiple analytes that may be present in the assay reaction.

[0186] In summary, the above results and discussion indicate that unique spectra characterize 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 Analyte A and Analyte B. These data demonstrate that endpoint melting / annealing curve analysis using a single detection channel readily discerns which target or targets are present in the reaction mixture.

[0187] The previous examples demonstrate the use of real-time and endpoint formats for nucleic acid analysis using invasive cleavage reactions to detect two different amplified nucleic acid target sequences. Fluorescent signals for the different amplified targets are detected in a single optical channel (i.e., a HEX channel) of a fluorometer component of an instrument monitoring nucleic acid amplification as the reaction occurs (e.g., as a function of time or cycle number). The procedure exploits the fact that a hybrid duplex comprising a masking oligonucleotide and a cleaved flap containing a fluorescent label is activated at a temperature below the T of the duplex. m is stable at temperatures above the T m The results showed that the maximum quenching was observed at about 39 °C and the minimum quenching was observed at about 63 °C. In summary, Figure 5 and Figure 6 The results shown show how to detect and resolve multiple different nucleic acid analytes in a multiplex reaction mixture using only a single optical channel of a fluorometer. Although this was achieved using different fluorophore species detected in the same optical channel of the fluorometer, a single type of fluorophore could be used instead (e.g., as shown below). Furthermore, one of ordinary skill in the art would understand how to substitute different fluorescent labels for the exemplary markers described above and would understand how to use instrument channels other than the HEX channel to detect those markers.

[0188] Embodiment 3 describes the program for detecting the presence or absence of multiple analytes using the real-time monitoring of a PCR instrument and only a single optical channel of a fluorometer. Here, the cycling program includes a temperature step, which allows the determination of each presence or absence of two analytes by the fluorescence quenching of a masking oligonucleotide under this temperature step. More specifically, in the cycling process of the PCR program, fluorescence readings are measured at 63°C and 39°C. As shown below, the technology distinguishes the dual signals measured in a single optical channel in real time. According to the standard program for processing the real-time amplification run curve, by determining the cycle number (e.g., Ct value) that reaches the amplification threshold level, this program can be advantageously used for quantifying every kind of analyte detected. The Ct value determined can then be compared with a calibration graph or equation, and the calibration graph or equation associate the threshold value with the amount or concentration of the analyte nucleic acid. Alternative real-time program can determine whether the analyte nucleic acid is present in the multiple reaction mixture with a higher or lower specified (e.g., predetermined) amount or concentration level. This may involve determining whether a specific reaction progress level (e.g., measured by Ct value) is realized by the specified cycle number. It is noteworthy that where the previous examples used two different fluorophores detected in the same optical channel of a fluorometer connected to a PCR instrument, here the same fluorophore species is used to detect two different analytes. Example 3 Real-time monitoring of multiplex amplification using a single fluorophore

[0189] The reaction mixture for the real-time amplification protocol was prepared as follows. Serial dilutions of plasmid DNA containing the analyte A target sequence were prepared from a standard stock solution. Wild-type bacterial genomic DNA containing the analyte B target sequence was used as the source of template nucleic acid for amplification of this analyte. The lyophilized pellet described in Example 1 was reconstituted with an aqueous buffer and then spiked with a 5' flap FRET cassette and corresponding masking oligonucleotide specifically for detecting analyte B. Furthermore, cleavage of the FRET cassette in the reaction mixture was catalyzed by the primary cleavage of the flap from the primary probe.

[0190] Four kinds of reaction mixtures (one plate per portion to display high and low temperature monitoring) were prepared in duplicate using multi-well PCR plates. The negative control mixture did not receive the nucleic acid template of analyte A or analyte B. The second group of mixtures only received the bacterial genomic DNA comprising the analyte B template, and did not receive the analyte A plasmid. The third group of mixtures only received the analyte A plasmid, and did not receive the bacterial genomic DNA containing the analyte B template. The fourth group of mixtures received both the bacterial genomic DNA containing the analyte B template and the analyte A plasmid. All experiments included a three-fold excess of masked oligonucleotides compared to the corresponding 5' flap FRET box for detecting analyte B. Using the first group of cycling conditions for fluorescence monitoring at 63°C ("high" temperature monitoring) or the second group of cycling conditions for fluorescence monitoring at 39°C ("low" temperature monitoring), the PCR reaction with monitoring of the fluorescent signal generated by the invasive cutting of the FRET box was carried out on an ABI 7500 real-time PCR instrument. The cycling conditions 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 optimal stage), and (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 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 optimal stage); and (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. Fluorescence monitoring at high and low temperatures was performed for a total of 50 cycles.

[0191] The results of the program are presented in Figure 7Panels A to D show real-time PCR amplification plots. The most interesting results are from experiments performed using Analyte B template alone or in combination with Analyte A template. Reactions using Analyte A template alone were considered controls and are not presented in the figures.

[0192] Figure 7 Small Figure A and Figure 7 Panel B shows the analyte B-specific fluorescence as a function of cycle number, where fluorescence was measured in the HEX channel of the PCR instrument at 63°C (no fluorescence quenching) or at 39°C (fluorescence quenching by the masking oligonucleotide). At 63°C, starting from approximately cycle 12 and continuing until cycle 40, there was a clear increase in fluorescence above background, as shown in FIG. Figure 7 Throughout the procedure, fluorescence measurements at 39°C remained essentially at background levels, as shown in panel A. Figure 7 These data demonstrate that quenching of the analyte B fluorescence signal is essentially complete at lower temperatures due to hybridization of the cleaved flap to the complementary masking oligonucleotide. Figure 7 In the graph of panel B, the fluorescence observed at the lower temperature (39°C) is due to background fluorescence rather than a signal indicating the presence of analyte B.

[0193] Figure 7 Small Figure C and Figure 7 Panel D shows the real-time run curve results obtained by monitoring the fluorescent signal generated in the reaction of amplifying and detecting the combination of Analyte B and Analyte A template nucleic acids. Figure 7 The curve shown in panel C of reflects the combined contribution of fluorescence generated by cleavage of both FRET cassettes, where the fluorescence reading is measured during a temperature step of 63°C. Only cleavage of the FRET cassette, which indicates the presence of analyte B, generates a fluorescent flap sequence that can hybridize to the masking oligonucleotide, thereby quenching the fluorescence signal at 39°C. Figure 7 As shown in panel C of FIG, the fluorescence signal measured at a temperature step of 63° C. (i.e., without fluorescence quenching) begins to rise above the background level at approximately cycle 11 and enters a log-linear phase at approximately cycle 19. The rate of fluorescence increase begins to gradually decrease at approximately cycle 29, but the magnitude of the signal continues to increase. Figure 7 The curve shown in panel D of reflects the fluorescence readings measured during the temperature step of 39°C. Because the signal from the cleaved flap indicating the presence of analyte B is effectively quenched at this temperature, Figure 7 The fluorescence measured in panel D comes only from the cleavage reaction indicating the presence of analyte A.

[0194] In short, Figure 7The results presented in panels AD of 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 fluorescence emission at different temperatures. As practiced in the disclosed technology, one temperature corresponds to a condition of fluorescence quenching (i.e., substantially complete or maximum quenching of fluorescence due to the cleaved flap). Detection of analyte B demonstrates this condition. The different temperatures in the procedure do not quench the fluorescence generated by the cleaved flap.

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

[0196] As described above, the results from real-time monitoring of the multiplex reaction mixture can be used to quantify the analyte nucleic acid, or to determine whether the analyte nucleic acid is present in an amount greater than or less than a threshold amount or concentration (e.g., even at zero concentration). In some embodiments, quantification of the analyte nucleic acid can involve comparing the determined Ct value to a calibration graph or equation that relates Ct values to analyte concentrations. 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 involve determining whether the time-dependent fluorescence value reaches a reaction progression level at a specific time or cycle number. Figure 7 Taking the data in panels AD as an example, a qualitative determination regarding the presence or absence of an analyte can involve determining whether a fluorescence reading of at least 500,000 RFU (relative fluorescence units) is obtained through 30 cycles of PCR. Figure 7 Thumbnail B and Figure 7 In panel D, the fluorescence signal generated by the detection of analyte B has been effectively removed by fluorescence quenching. Figure 7 Compared with the small picture B, Figure 7 The increased signal observed in the graph of panel A indicates the contribution of fluorescence caused by the detection of analyte B and thus confirms the presence of this target in the reaction mixture. Figure 7 Small Figure A and Figure 7 The result of panel B will indicate that the reaction mixture contains only analyte B and no analyte A. Figure 7 Compared with the small picture D, Figure 7The increased signal observed in the graph of panel C indicates the fluorescence contribution caused by the detection of analyte B in the reaction mixture. Figure 7 The remaining fluorescence plotted in panel D is the signal due to the detection of analyte A. Therefore, comparing Figure 7 Small Figure C and Figure 7 The results of Panel D would indicate that the reaction mixture contains 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 for detecting three different analyte nucleic acid sequences in a single reaction mixture using an invasive cleavage reaction, wherein the three FRET boxes are 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 for amplifying nucleic acids can be used as a substitute for the HEX fluorophore. Example 4 Multiplexed detection of three analyte nucleic acids using a single type of fluorophore

[0198] Three different sets of oligonucleotides were used in the same reaction mixture to perform invasive cleavage detection on three analyte nucleic acids amplified by PCR. Each set of detection oligonucleotides was used to detect a different one of the three analytes (Analyte A, Analyte B, and Analyte C). Each assay reaction employed: (1) a unique primary probe having 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 an "invasive primer") that acts as an invasive oligonucleotide to cleave the 5' flap from the primary probe when the primary probe hybridizes with its cognate target nucleic acid and further acts 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 Analyte A did not contain a 5' flap that hybridized to any masking oligonucleotide in the reaction mixture. More specifically, Analyte A was detected using the invasive primer, primary probe, and flapless FRET cassette of Example 1. The FRET cassettes used to detect Analytes B and C contained a 5' flap sequence. The FRET cassette used in the procedure is shown along with the relevant cleaved flap and masking oligonucleotides from the primary probe. Figure 3 The FRET cassette systems used to detect analytes A, C, and B are shown in the upper, middle, and lower parts of the figure, respectively.

[0199] The multiplex reaction mixture contains two different masked oligonucleotides, one complementary to the 5' flap of the FRET cassette for detecting analyte B, and the other complementary to the 5' flap of the FRET cassette for detecting analyte C. Each masked oligonucleotide contains a quenching moiety at its 5' end. The GC content of the FRET 5' flap-masking oligonucleotide duplex for analyte B is about 60%, and the GC content of the FRET 5' flap-masking oligonucleotide duplex for analyte C is about 40%. Both duplexes are characterized by T m The cleavage product of the 5'-flap FRET cassette used to detect analyte B and the complementary masking oligonucleotide form a more stable duplex for "high temperature" detection, while the cleavage product of the 5'-flap FRET cassette used to detect analyte C and the complementary masking oligonucleotide form a less stable duplex for "low temperature" detection.

[0200] A single reaction mixture contained all assay oligonucleotides (including all three FRET boxes) and all reagents required for 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 boxes. Reactions contained analyte A only, analyte B only, analyte C only, or a combination of analytes A, B, and C. Thermal cycling conditions were as follows: (1) 95°C for 120 seconds x 1 cycle; (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 optimal phase); and (3) 95°C for 10 seconds, 69°C for 5 seconds, 67°C for 5 seconds, and 65°C for 25 seconds x 40 cycles. Post-amplification melting / annealing curve analysis was performed on an ABI 7500 real-time PCR instrument over the range of 90°C to 20.7°C.

[0201] Figure 8 Post-amplification melting / annealing curve results are shown for reactions performed using each of the three analyte polynucleotides alone and in combination with each other. The results demonstrate that each of the four reactions produced a unique melting / annealing curve profile, demonstrating the successful single-channel multiplexing of the three analytes using a single type of fluorophore.

[0202] Figure 9 The results shown in the two panels of FIG5 demonstrate unique melting / annealing profiles observed for additional analyte combinations. Figure 9 The left panel shows the melting / annealing curve results for reactions that individually amplify Analyte B and Analyte C. The results demonstrate how the cleaved 5' flaps from each FRET cassette, hybridized to different masking oligonucleotides, can be distinguished from each other using only a single fluorescence monitoring channel of a real-time PCR instrument. Figure 9The right panels show the results of melting / annealing curve analysis of reactions amplifying analyte B alone, analyte C alone, or a combination of analytes B and C. Furthermore, these results demonstrate how the cleaved 5' flaps from each FRET cassette, hybridized to different masking oligonucleotides, can be distinguished from each other using only a single fluorescence monitoring channel of a real-time PCR instrument.

[0203] Figure 10 Presented Figure 9 The two panels show first derivative plots of the measured fluorescence data. Figure 10 The left panel shows Figure 9 The first derivative of the melting / annealing curve is presented in the left panel. The maximum of the first derivative of the fluorescence as a function of temperature indicates the melting temperature (T m ). In this case, two T m The difference is about 10°C (ie, the T m is about 46°C, and the T for detecting analyte B m The difference between the two melting / annealing curves allows for the resolution of which of the two analytes is present in the reaction mixture being analyzed. A single maximum observed or detected at about 46°C would indicate the presence of analyte C, while a single maximum at about 56°C would indicate the detection of analyte B. Figure 10 The right thumbnail shows Figure 9 The first derivative of the melting plot is shown in the right panel of FIG. Each different curve has unique characteristics that allow differentiation between reaction mixtures containing analyte B, analyte C, or a combination of analytes B and C. Since the fluorescence signal caused by the presence of analyte A is not quenchable with temperature (i.e., there is no masking oligonucleotide complementary to the FRET cassette cleavage product), there is no effect on the first derivative (i.e., the first derivative of the constant is zero).

[0204] In the above situation, the derivative analysis of the melting / annealing curve is used to analyze whether the test sample has two different analytes. For the purpose of illustration, the first-order derivative is used here to calculate. However, it is considered that second-order or even higher-order derivative analysis is used for this purpose. The program relates to a single channel detection or monitoring of the fluorescence signal in the amplification reaction mixture using only the fluorometer component of the real-time nucleic acid amplification device. The figure can present the first-order derivative of the raw data, but alternatively the first-order derivative of the processed data (e.g., smooth, normalized relative to a constant maximum reading, etc.) can be presented. At 46°C, a peak or maximum value is detected in the first-order derivative graph to indicate the presence of analyte C or its amplification product. At 56°C, a peak or maximum value is detected in the first-order derivative graph to indicate the presence of analyte B or its amplification product. At both 46°C and 56°C, a peak (e.g., a high-level signal at these two temperatures) is detected to indicate the presence of both analyte C and analyte B or the amplification product of these analytes. In some embodiments, analysis can involve identifying a signal greater than a threshold value (e.g., a predetermined threshold value or a threshold value based on a fraction or percentage of a normalized maximum value). Although the examples demonstrate the use of first-order derivative analysis, second-order derivative analysis may also be used. Here, the maximum value on the first-order derivative plot will correspond to the zero crossing point on the second-order derivative plot. Detecting the presence of analyte A is independent of derivative analysis because the FRET box used to detect this analyte does not contain a 5' flap sequence that is complementary to any masking oligonucleotide. Cutting of this FRET box results in a fluorescent signal that remains stable over the temperature range used for melting / annealing curve analysis. The presence of analyte A in the test sample is reflected by the magnitude of the fluorescence at a temperature at which the signal generated by the cutting of other FRET boxes (e.g., for detecting analyte B and / or analyte C) will be quenched (e.g., at Figure 10 at about 30°C).

[0205] All documents and similar materials cited in this application (including but not limited to patents, patent applications, articles, books, papers 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 of ordinary skill in the art to which the various embodiments described herein belong. When the definition of a term in an incorporated reference appears to be different from the definition provided in this teaching, the definition provided in this teaching shall prevail.

[0206] Various modifications and variations of the described compositions, methods, and uses of the techniques will be apparent to those skilled in the art without departing from the scope and spirit of the technology as described. Although the technology has been described in conjunction with specific 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 biochemistry, molecular biology, or related fields are intended to fall within the scope of the following claims.

[0207] The present disclosure relates to the following embodiments. 1. A composition comprising a FRET cassette reporter system, the composition comprising: (i) a 5' flap FRET cassette oligonucleotide comprising: a 5' flap portion comprising a first fluorophore portion, a stem-loop portion comprising a first quencher portion, and The 3' portion containing the cleaved flap hybridization sequence, wherein hybridization of a cassette-specific invasive oligonucleotide complementary to said cleaved flap hybridization sequence of said 5' flap FRET cassette oligonucleotide forms an invasive cleavage structure cleavable by a FEN-1 endonuclease at the cleavage site between said first fluorophore moiety and said first quencher moiety, wherein cleavage of the 5' flap FRET cassette oligonucleotide at the cleavage site produces a cassette-cleaved flap comprising the 5' flap portion and the first fluorophore moiety; and (ii) a masked oligonucleotide comprising a second quencher moiety, wherein at least a portion of said masking oligonucleotide is specifically hybridizable to said 5' flap portion of said FRET box oligonucleotide, wherein hybridization of the masking oligonucleotide to the cassette-cleaved flap forms a duplex having a first melting temperature exhibiting a first melting peak, and wherein fluorescence emission from the first fluorophore moiety in the duplex is quenched by the second quencher moiety. 2. The composition of claim 1 , wherein the first quencher moiety and the second quencher moiety are identical to each other. 3. The composition according to item 1 or 2, further comprising FEN-1 endonuclease. 4. The composition according to item 3, wherein the FEN-1 endonuclease is a thermostable FEN-1 endonuclease. 5. The composition of item 4, wherein the thermostable FEN-1 endonuclease is from an archaeal organism. 6. The composition according to any one of items 1 to 5, further comprising a first target-specific invasive oligonucleotide and a first target-specific primary probe oligonucleotide, wherein each of the first target-specific invasive oligonucleotide and the first target-specific primary probe oligonucleotide comprises a sequence configured to hybridize to the target nucleic acid to form an invasive cleavage structure that is cleavable by the FEN-1 endonuclease to produce a primary cleavage flap, and wherein the primary cleavage flap is a cassette-specific invasive oligonucleotide configured to hybridize to the cleavage flap hybridization sequence of the 5' flap FRET cassette oligonucleotide to form an invasive cleavage structure cleavable by the FEN-1 endonuclease. 7. The composition according to item 6, further comprising the target nucleic acid. 8. The composition according to item 7, further comprising deoxynucleoside triphosphates (dNTPs), a thermostable DNA polymerase, and a primer having a 3' end that can be extended by the thermostable DNA polymerase in a template-dependent nucleic acid amplification reaction using the target nucleic acid as a template. 9. The composition according to any one of items 1 to 8, further comprising: (iii) a second FRET cassette oligonucleotide comprising a 5' portion comprising a second fluorophore moiety, a stem-loop portion comprising a third quencher portion, and The 3' portion of the flap hybridization sequence containing the second cut, wherein hybridization of a second cassette-specific invasive oligonucleotide to the second cleaved flap hybridization sequence of the second FRET cassette oligonucleotide forms an invasive cleavage structure cleavable by a FEN-1 endonuclease at the cleavage site between the second fluorophore moiety and the third quencher moiety, and wherein cleavage of the second FRET cassette oligonucleotide at the cleavage site produces a cassette cleavage product comprising the second fluorophore moiety. 10. The composition of claim 9, wherein the second FRET cassette oligonucleotide is a second 5' flap FRET cassette comprising a 5' flap portion, wherein the cassette cleavage product is a second cassette cleaved flap comprising the second fluorophore, and wherein the composition further comprises: (iv) a second masked oligonucleotide comprising a fourth quencher moiety, wherein at least a portion of the second masking oligonucleotide can specifically hybridize to the 5' flap portion of the second FRET box oligonucleotide, wherein hybridization of the second masked oligonucleotide to the second cassette-cleaved flap forms a second duplex having a second melting temperature higher than the first melting temperature, and wherein fluorescence emission from the second fluorophore moiety in the second duplex is quenched by the fourth quencher moiety. 11. The composition according to item 9, wherein the second box cleavage product comprises no more than 5, preferably no more than 4, preferably no more than 3, preferably no more than 2 nucleotides. 12. The composition of 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. 13. The composition of any one of items 9 to 12, wherein the first fluorophore moiety and the second fluorophore moiety are identical to each other. 14. The composition of any one of items 9 to 12, wherein the first fluorophore moiety and the second fluorophore moiety are different from each other. 15. The composition of any one of items 10 and 12 to 14, wherein the third quencher moiety and the fourth quencher moiety are identical to each other. 16. A method for determining which of two different FRET cassettes in a reaction mixture is cleaved to produce a fluorescent signal, the method comprising the steps of: (a) performing multiple invasive cleavage reactions in the reaction mixture to cleave one or both of the first FRET cassette and the second FRET cassette, thereby producing 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 the FEN-1 endonuclease in the multiplex invasive cleavage reaction produces a first cassette-cleaved flap comprising the fluorophore, The reaction mixture comprises a first masked oligonucleotide that stably hybridizes to the first cassette-cleaved flap to form a first duplex at a temperature below a first Tm, but not above the first Tm, wherein fluorescence emission from a fluorophore of the first cassette-cleaved flap of the first duplex is quenched, and 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 a fluorescent signal generated in the reaction mixture using a single channel of a fluorescence monitoring device under temperature conditions that differentially quench fluorescence generated by different fluorescent cleavage products of the multiple invasive cleavage reactions; and (c) determining which of the different FRET cassettes is cleaved in the multiplex invasive cleavage reaction based on the results of step (b). 17. A method according to item 16, wherein the second FRET box in step (a) generates a fluorescent cleavage product if cleaved, and the fluorescent cleavage product does not hybridize with any masking oligonucleotide in the reaction mixture to cause fluorescence quenching. 18. A method according to item 17, wherein step (c) comprises comparing fluorescence signals measured at a temperature below the first Tm and at a temperature above the first Tm. 19. A method according to claim 18, wherein step (c) comprises comparing the fluorescent signals by calculating the difference between the measured fluorescent signals. 20. The method according to claim 17, wherein step (b) comprises measuring any said fluorescent signal at a temperature below said first Tm, wherein fluorescence emission from the fluorophore of said first cassette-cleaved flap of said first duplex is quenched, and wherein step (c) comprises determining that the second FRET cassette is cleaved in the reaction mixture if measurable fluorescence is detected in step (b). 21. The method according to claim 17, wherein step (b) comprises measuring any said fluorescent signal at each of a temperature below said first Tm and a temperature above said first Tm, and Wherein step (c) comprises determining that the first FRET cassette is cleaved in the reaction mixture if the fluorescent signal measured at a temperature above the first Tm is greater than the fluorescent signal measured at a temperature below the first Tm. 22. A method according to any one of items 16 to 21, wherein step (b) comprises measuring any said fluorescent signal generated in the reaction mixture as a function of temperature to generate a melting / annealing curve. 23. A method according to claim 22, wherein step (c) includes calculating the derivative of the melting / annealing curve and then determining whether the reaction mixture contains the first duplex characterized by the first Tm as an indication that the first FRET box is cut in the reaction mixture based on the calculated derivative. 24. The method according to claim 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 the multiplex invasive cleavage reaction produces a second cassette-cleaved flap comprising the fluorophore, wherein the reaction mixture comprises a second masked oligonucleotide that hybridizes to the second cassette-cleaved flap to form a second duplex at a temperature below a second Tm, but not above the second Tm, wherein fluorescence emission from the fluorophore of the second cassette-cleaved flap of the second duplex is quenched, and wherein the first Tm differs from the second Tm by at least 5°C. 25. The method according to claim 24, wherein the first Tm is greater than the second Tm, wherein step (b) comprises measuring any said fluorescent signal at a temperature below said second Tm and above said first Tm, and 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 above the first Tm is greater than the fluorescent signal measured at a temperature below the second Tm. 26. A method according to item 24 or 25, wherein step (b) comprises measuring any said fluorescent signal generated in the reaction mixture as a function of temperature to generate a melting / annealing curve. 27. A method according to claim 26, wherein step (c) includes calculating the derivative of the melting / annealing curve and then determining whether the reaction mixture contains the first duplex characterized by the first Tm as an indication that the first FRET box is cut in the reaction mixture based on the calculated derivative. 28. A method according to claim 26, wherein step (c) includes calculating the derivative of the melting / annealing curve and then determining whether the reaction mixture contains the second duplex characterized by the second Tm as an indication that the second FRET box is cut in the reaction mixture based on the calculated derivative. 29. The method of any one of items 16 to 28, wherein the first FRET box and the second FRET box are labeled with the same fluorophore. 30. The method of any one of items 16 to 28, wherein the first FRET box and the second FRET box are not labeled with the same fluorophore. 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. 32. A method according to any one of items 16 to 31, wherein step (c) comprises determining using a computer programmed with software. 33. A method for analyzing a sample containing a target nucleic acid, the method comprising the steps of: (a) contacting any first target nucleic acid of the sample with a first primary probe oligonucleotide comprising a sequence complementary thereto and a FEN-1 endonuclease in a reaction mixture 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 produce a first primary cleaved flap, wherein the first primary cleaved flap hybridizes to a cleaved flap hybridization sequence of a first FRET cassette oligonucleotide contained in the reaction mixture to form an invasive cleavage structure, which is cleaved by the FEN-1 endonuclease at a cleavage site between the first fluorophore moiety and the first quencher moiety of the first FRET cassette oligonucleotide to release the first cassette cleaved flap comprising the first fluorophore moiety, wherein a first masked oligonucleotide comprising a second quencher moiety hybridizes to the first cassette-cleaved flap to form a duplex at a temperature below a first Tm of the first masked oligonucleotide and the first cassette-cleaved flap, wherein fluorescence emission from the first fluorophore moiety in the duplex is quenched by the second quencher moiety, and wherein at a second temperature greater than the first Tm, the first masked oligonucleotide and the first cassette-cleaved flap do not form a stable duplex; (b) detecting any fluorescence emitted from the first fluorophore moiety at the second temperature; and (c) If In step (b), the fluorescence emitted from the first fluorophore moiety is detected, then the sample is determined to contain the first target nucleic acid, or If no fluorescence emitted from the first fluorophore moiety is detected in step (b), then the sample is determined not to contain the first target nucleic acid. 34. The method according to claim 33, wherein step (a) further comprises contacting any second target nucleic acid of the sample with a second primary probe oligonucleotide comprising a sequence complementary thereto and the FEN-1 endonuclease in the reaction mixture 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 produce a second primary cleaved flap that is different from the first primary cleaved flap, wherein the second primary cleaved flap hybridizes to a cleaved flap hybridization sequence of a second FRET cassette oligonucleotide contained in the reaction mixture to form an invasive cleavage structure, which is cleaved by the FEN-1 endonuclease at a cleavage site between the second fluorophore moiety and the third quencher moiety of the second FRET cassette oligonucleotide to release a second cassette cleaved flap comprising the second fluorophore moiety, wherein at a third temperature below the second Tm, a second masked oligonucleotide comprising a fourth quencher moiety hybridizes to the second cassette-cleaved flap to form a duplex, wherein fluorescence emission from the second fluorophore moiety in the second duplex is quenched by the fourth quencher moiety, wherein at a fourth temperature above the second Tm, the second masked oligonucleotide and the second cassette-cleaved flap do not form a stable duplex, and wherein the first Tm and the second Tm differ from each other by at least 5°C; wherein step (b) further comprises detecting any fluorescence emitted from the second fluorophore moiety at the fourth temperature; and wherein step (c) further comprises If fluorescence emitted from the second fluorophore portion of the 5' flap cleavage product of the second FRET cassette oligonucleotide is detected in step (b), then the sample is determined to contain the second target nucleic acid, or If fluorescence emitted from the second fluorophore portion of the 5' flap cleavage product of the second FRET box oligonucleotide is not detected in step (b), then the sample is determined to not contain the first target nucleic acid. 35. The method according to claim 33, wherein step (a) further comprises contacting any second target nucleic acid of the sample with a second primary probe oligonucleotide comprising a sequence complementary thereto and the FEN-1 endonuclease in the reaction mixture 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 produce a second primary cleaved flap, wherein the second primary cleavage flap hybridizes to a cleavage flap hybridization sequence of a second FRET cassette oligonucleotide contained in the reaction mixture to form an invasive cleavage structure, which is cleaved by the FEN-1 endonuclease at a cleavage site between the second fluorophore moiety and the third quencher moiety of the second FRET cassette oligonucleotide to release a cleavage product comprising the second fluorophore moiety, wherein the cleavage product does not hybridize to any masking oligonucleotide in the reaction mixture to result in fluorescence quenching; wherein step (b) further comprises detecting any fluorescence emitted from said second fluorophore portion of said cleavage product; and wherein step (c) further comprises If fluorescence emitted from the second fluorophore moiety is detected in step (b), then the sample is determined to contain the second target nucleic acid, or If no fluorescence emitted from the second fluorophore moiety is detected in step (b), then the sample is determined not to contain the first target nucleic acid. 36. A method according to item 34 or item 35, wherein step (b) comprises detecting any fluorescence emitted from the first fluorophore portion and the second fluorophore portion using a single channel of a fluorescence monitoring device. 37. A method according to item 36, wherein step (b) is performed while a nucleic acid amplification reaction occurs in the reaction mixture, and wherein the product of the nucleic acid amplification reaction comprises the first target nucleic acid and the second target nucleic acid. 38. A method according to claim 37, wherein the nucleic acid amplification reaction includes a thermal cycling step, and wherein the reaction mixture further comprises a thermostable DNA polymerase. 39. A method according to any one of items 33 to 36, wherein step (b) is performed when the temperature of the reaction mixture is lowered to allow annealing of the masking oligonucleotide and the flap cleaved by the complementary cassette. 40. A method according to claim 36, wherein the first fluorophore portion and the second fluorophore portion are identical to each other. 41. A method according to item 36, wherein step (b) of detecting any fluorescence comprises measuring any fluorescence. 42. The method of claim 41 , further comprising the step of detecting or measuring fluorescence at the first temperature. 43. A method according to 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. 44. A method according to claim 43, wherein the second fluorophore portion is the same as the first fluorophore portion. 45. A method according to claim 43, wherein the second fluorophore portion is different from the first fluorophore portion. 46. A method according to any one of items 33 to 43, wherein fluorescence from the second fluorophore is detected and / or measured at the first temperature. 47. A method according to any one of items 33 to 44, wherein the third quencher portion is the same as the first quencher portion and / or the second quencher portion. 48. A method according to any one of items 33 to 47, wherein the reaction mixture comprises primer oligonucleotides for amplifying the target nucleic acid, wherein 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 nuclease. 49. A method according to any one of items 33 to 48, wherein step (c) comprises determining using a computer programmed with software. 50. A system for determining which of a plurality of target nucleic acid analytes is present in a reaction mixture, wherein each of the plurality of target nucleic acid analytes is detectable by a fluorescent signal, the system comprising: Thermal cycler; a fluorometer in optical communication with the thermal cycler, wherein the fluorometer uses a single optical channel to measure a fluorescent signal indicative of the production of nucleic acid amplification products achieved by the thermal cycler; and a computer in communication with the fluorometer, wherein the computer is programmed with software instructions such that the computer: (a) obtaining a melting / annealing curve data set based on 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) obtaining a derivative plot based on the melting / annealing curve dataset, and (d) determining that a second target nucleic acid is present in the reaction mixture if the derivative pattern comprises features characteristic of the first duplex, The first duplex comprises a first masked oligonucleotide and a second fluorescent cleavage product produced in the reaction mixture when the second target nucleic acid is present. 51. The system of claim 50, wherein the computer is further programmed with software instructions such that the computer: (e) determines that a third target nucleic acid is present in the reaction mixture if the derivative plot comprises features characteristic of a second duplex, The second duplex comprises a second masked oligonucleotide and a third fluorescent cleavage product produced in the reaction mixture when the third target nucleic acid is present. 52. A system according to claim 50 or claim 51, wherein the characteristic feature unique to the first double-strand comprises a maximum, minimum or zero crossing of a calculated derivative. 53. The system according to claim 52, The calculated derivatives include the calculated first-order derivatives, wherein the first duplex-specific feature comprises a first maximum of a calculated first derivative of the melting / annealing curve data set as a first melting peak, and The second duplex-specific feature comprises a second maximum of the calculated first-order derivative of the melting / annealing curve dataset as a second melting peak. 54. A system according to any one of items 50 to 53, wherein the thermal cycler, the fluorometer and the computer are all components of a real-time PCR instrument. 55. A system according to claim 50, wherein the melting / annealing curve data set obtained by the computer comprises data points indicating fluorescence that changes with temperature.

Claims

1. A system for determining which of a plurality of target nucleic acid analytes are present in a reaction mixture, wherein each of the plurality of target nucleic acid analytes is detectable by a fluorescent signal, the system comprising: Thermal cycler; a fluorometer in optical communication with the thermal cycler, wherein the fluorometer uses a single optical channel to measure a fluorescent signal indicative of the production of nucleic acid amplification products achieved by the thermal cycler; and a computer in communication with the fluorometer, wherein the computer is programmed with software instructions such that the computer: (a) obtaining a melting / annealing curve data set based on 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) obtaining a derivative plot based on the melting / annealing curve dataset, and (d) determining that a second target nucleic acid is present in the reaction mixture if the derivative pattern comprises features characteristic of the first duplex, The first duplex comprises a first masked oligonucleotide and a second fluorescent cleavage product produced in the reaction mixture when the second target nucleic acid is present.

2. The system of claim 1 , wherein the computer is further programmed with software instructions such that the computer: (e) determines that a third target nucleic acid is present in the reaction mixture if the derivative plot comprises features characteristic of a second duplex, The second duplex comprises a second masked oligonucleotide and a third fluorescent cleavage product produced in the reaction mixture when the third target nucleic acid is present.

3. The system of claim 1 or claim 2, wherein the characteristic feature unique to the first duplex comprises a maximum, a minimum, or a zero crossing of a calculated derivative.

4. The system according to claim 3, The calculated derivatives include the calculated first-order derivatives, wherein the first duplex-specific feature comprises a first maximum of a calculated first derivative of the melting / annealing curve data set as a first melting peak, and The second duplex-specific feature comprises a second maximum of the calculated first-order derivative of the melting / annealing curve dataset as a second melting peak.

5. The system of any one of claims 1-4, wherein the thermal cycler, the fluorometer, and the computer are components of a real-time PCR instrument.

6. The system of claim 1, wherein the melting / annealing curve data set obtained by the computer comprises data points indicative of fluorescence as a function of temperature.

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