Compositions, kits and methods for detecting nucleic acids using in-channel multiplexing

Abstract

Compositions, kits, and methods are disclosed that enable in-channel multiplexing by enabling determination of individual detectable signals, each associated with a different assay target, within the same detection channel. The plurality of detectable signals may be parsed individually and independently to enable detection and / or quantification of each respective target. Enabling a plurality of targets to be measured in the same detection channel increases the diversity of multiple measurements without relying on the problems of additional dyes and accompanying increase in spectral overlap.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to U.S. Provisional Patent Application No. 63 / 433,546, filed on March 21, 2023; and claims priority to U.S. Provisional Patent Application No. 63 / 408,665, filed on September 21, 2022; and claims priority to U.S. Provisional Patent Application No. 63 / 356,863, filed on June 29, 2022; and claims priority to U.S. Provisional Patent Application No. 63 / 356,874, filed on June 29, 2022, the entire contents of each of which are incorporated herein by reference. Technical Field

[0003] The present disclosure relates to compositions, kits and methods that enable multiplexing by enabling determination of signals having similar or identical spectral characteristics but each associated with a different assay target. Aspects of the present disclosure also relate to compositions, kits and methods that enable multiplexing by enabling determination of signals associated with different assay targets using the same detection channel (e.g., within the same fluorescence channel).

[0004] Introduction

[0005] Nucleic acid detection assay is usually carried out by adding a sample suspected of including one or more target nucleic acids to the reaction mixture. The reaction mixture may include one or more detectable labels, each of which is designed to be relevant to different target nucleic acids, and produces a signal corresponding to the amount of the target nucleic acid in the reaction mixture. In "single weight" assay, the reaction mixture includes a single detectable label designed to be relevant to a single target. On the contrary, in "multiple" assay, the reaction mixture includes a plurality of different detectable labels, each of which is usually designed to have specificity to different target nucleic acids. Therefore, multiple assays can detect a variety of different targets in a single reaction mixture. In some applications, detectable label is a fluorescent dye integrated with nucleic acid probes, primers or some other nucleic acid molecules, and these other nucleic acid molecules are designed to specifically hybridize with the corresponding target nucleic acid designed to be relevant thereto.

[0006] In various multiple nucleic acid detection assays, each detectable label is assigned to different target nucleic acids. Then, the presence and / or amount of each target nucleic acid can be determined by measuring the signal emitted from the detectable label in a separate "detection channel", and the separate detection channel corresponds to the specific characteristics of the corresponding signal emitted. For example, in the context of fluorescent emission dyes as detectable labels, separate detection channels can correspond to the emission wavelength spectrum associated with each dye. However, a large amount of overlaps can be present in the emission spectra of different dyes. The overlap increased in the emission spectrum increases the difficulty of parsing the emission (e.g., fluorescence) signal detected separately, and therefore increases the difficulty of detecting and / or quantifying the corresponding target. Excessive overlap may require, for example, a complex deconvolution algorithm to fully resolve the separate fluorescent signal.

[0007] Although multiple dyes may be selected with the goal of minimizing spectral overlap, the finite nature of emission spectra imposes practical limits on the number of individual dyes that can be combined in the same multiplex assay, at least without resorting to increasingly complex reaction protocols and back-end deconvolution requirements. Therefore, currently, there are significant limitations on the number of different targets that can be detected and / or measured in multiplex assays. Therefore, there is a constant need for compositions, kits, and methods that can increase the "diversity" of detection assays. In addition, it may be desirable to otherwise use dyes that have some degree of overlap in emission spectra and / or to use the same dye for different target nucleic acids.

[0008] Challenges arise when multiplexing is implemented for determining the relative amounts of different target nucleic acids in a sample. Specifically, using detectable labels with overlapping emission spectra can make it difficult to determine the respective contribution of each label individually, and therefore the contribution of the respective different target nucleic acids associated therewith.

[0009] There is a need to provide more robust techniques for performing multiplexed nucleic acid detection assays, such as, for example, nucleic acid detection utilizing various polymerase chain reaction (PCR) assays. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] Various objects, features, characteristics and advantages of the present invention within the scope of the present disclosure will become apparent and more readily understood from the following description of various embodiments taken in conjunction with the accompanying drawings and the appended claims, all of which constitute a part of this specification. In the accompanying drawings, like reference numerals may be used to indicate corresponding or similar parts in the various figures, and the various elements depicted are not necessarily drawn to scale, wherein:

[0011] Figure 1A The emission spectra of various fluorescent dyes and their associated detection channels that can be used in nucleic acid detection assays are shown;

[0012] Figure 1BThe emission spectra of various fluorescent dyes and their associated detection channels that can be used in nucleic acid detection assays are shown, where both of the dyes "Dye 1" and "Dye 2" have emissions detectable in the same channel;

[0013] Figure 2A is a schematic overview of a method for detecting multiple target nucleic acids within the same detection channel according to various embodiments of the present disclosure;

[0014] Figure 2B is to show that when the reaction mixture is cycled between a first set of reaction conditions and a second set of reaction conditions, Figure 2A A plot of the signal response over time for the method outlined in ;

[0015] Figure 3A shows the activity of cleavable and non-cleavable probes during the annealing step, the extension step, and the denaturation step of a thermal cycle according to various embodiments;

[0016] Figure 3B is a graph showing fluorescence signal response over time during thermal cycling of an amplification process utilizing amplification techniques according to various embodiments. Figure 3A of cleavable and non-cleavable probes;

[0017] Figure 4A A process is shown for using a tailed primer specific for a nucleic acid target to form a template to which an extendable fluorescent ("EF") probe can hybridize;

[0018] Figure 4B Exemplary tailed forward primers, reverse primers, and EF probes that may be included in a reaction mixture or composition are shown for implementing Figure 4A process;

[0019] Figure 4C A three-stage thermal cycling method is shown that can be used during an amplification process involving an EF probe and an optional cleavable probe;

[0020] Figure 5A and Figure 5B An overview of methods for detecting multiple target nucleic acids within the same detection channel in polymerase chain reaction (PCR) applications is provided;

[0021] Fig. 6A The fluorescence signals of the TaqMan probe and the extendable fluorescent (EF) probe at the extension step and the denaturation step are shown;

[0022] Figure 6B , Figure 6C and Fig.6DThe results of a duplex assay test are shown, where the TaqMan probe and the EF probe were designed to be in the same dye channel ( Figure 6B ) or different dye channels ( Figure 6C and Fig.6D ) to generate a fluorescent signal;

[0023] Fig. 6E Compare how to use Figure 6B The baseline-adjusted (dRn) EF-related fluorescence signal derived from the assay results is similar to that in Figure 6C The EF-related fluorescence signal is directly measured in the assay;

[0024] Fig. 6F The baseline-adjusted TaqMan relative fluorescence signals (dRn) were compared;

[0025] Figure 7 shows the results of a 9-plex assay test including 5 different detection channels / dyes, wherein four of the channels have corresponding TaqMan probes and EF probes (each channel having a different dye common to the TaqMan probes and EF probes in that channel), and one channel has only a TaqMan probe, according to an embodiment of the present disclosure; and

[0026] Figure 8 is a graph comparing the partitioned endpoint signals at 65°C and 95°C after the dPCR process, showing that the partitioned signals fall into identifiable clusters that allow estimation of the concentrations of the different targets. DETAILED DESCRIPTION

[0027] A selected list of defined terms

[0028] In the context of nucleic acid probes and target nucleic acids, the term "specifically interacts" (and similar terms) indicates that the probe is designed to interact with the target to a greater extent than with non-target nucleic acids also present in the reaction mixture. For example, a specific interaction can include hybridization of the probe in whole or in part with the corresponding target. The hybridization between the probe and the target does not need to be 100%. For example, a functionally effective interaction can be achieved with a probe that has at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 97%, at least about 98%, at least about 99%, or at most 100% homology to its corresponding target.

[0029] As used herein, "detection channel" is a specified subset of the total range of possible values ​​of a detectable signal. For example, in the case where the detectable signal is a fluorescent signal, a detection channel (i.e., a fluorescent channel or a dye channel) can represent a wavelength band of a specified size. The detection channel can, for example, have a band size of about 10nm-60nm, depending on instrument features, such as sensitivity and / or desired signal granularity. The detection channel can also include a discontinuous wavelength or wavelength range. According to the arrangement of filters for measuring the detectable signal, the detection channel can be defined additionally or alternatively. Each different detection channel generally includes a specific filter arrangement, to block non-channel emission. Therefore, as a functional definition, each detectable signal in a given filter arrangement can be considered to be in the same detection channel. In some cases, different fluorescent labels (e.g., different chemical structures) are still detected with the same detection channel. For example, fluorescent dyes Cy5 and Alexa647 provide similar emission wavelengths and can be detected in the same channel.

[0030] As used herein, "substantially identical" signals are signals that cannot be clearly distinguished from each other under the detection conditions used. Optionally, the emission spectra of two substantially identical signals overlap to the extent that each signal cannot be detected individually, such as the composite emission spectrum does not show the presence of two different peaks. Optionally, "substantially identical fluorescence" emission can be in a similar wavelength band. For example, a first fluorescent signal and a second fluorescent signal with substantially identical fluorescence may have an emission peak value that differs by no more than about 10nm, or no more than about 8nm, or no more than about 6nm, or no more than about 4nm, or no more than about 2nm, or no more than about 1nm, or substantially cannot be distinguished from each other by a detection instrument that is used to measure fluorescent emission. In addition, or alternatively, in the application of measuring fluorescent signals using the same detection device, such as the same filter arrangement, it can be considered that the fluorescent signal has "substantially identical fluorescence". In an embodiment, substantially identical signals have substantially identical excitation / absorption spectra so that they cannot be subjected to excitation individually. Optionally, both labels are subjected to excitation during detection. Two labels can be excited and / or detected simultaneously.

[0031] As used herein about signal, "substance" indicates significantly higher than background. For example, "substance signal" and / or detectable signal with "substance fluorescence" is a signal significantly higher than background (i.e., baseline) level, including a fluorescent signal significantly higher than fluorescence background / baseline level. This can be defined by a threshold value separating background fluorescence from substantive fluorescence. The threshold value can vary according to specific test protocols and application requirements. In some embodiments (for example, without passive reference), the threshold value is set to, for example, about 1,000 to about 30,000, or about 2,000 to about 20,000, or about 3,000 to about 15,000, or about 4,000 to about 6,000 ΔRn, or in the range of endpoints defined by any two of the aforementioned values. In some embodiments (for example, using passive reference), the threshold value is set to, for example, about 0.01 to 0.5 ΔRn. In some embodiments, the threshold value is higher than the baseline level in some percentages, such as about 5% to about 10% higher than the baseline level.

[0032] The "background" or "baseline" level of the signal during the amplification process (i.e., the background / baseline level of fluorescence) can be determined according to methods known to those skilled in the art. As a non-limiting example, the baseline level can be determined as the median signal of the amplification cycle before exponential amplification occurs. For example, exponential amplification can be determined when the signal change from one amplification cycle to the next amplification cycle exceeds a certain percentage indicating an exponential change.

[0033] As a corollary, a signal and / or fluorescence level that is not "substantial" according to the foregoing may be described herein as "negligible." Similarly, a probe is "substantially bound" to a target when the probe is significantly above background (e.g., above binding to a non-target) relative to probe binding. Optionally, at least 1%, 5%, 10%, 20%, 50%, or 80% of the probe or target is bound.

[0034] As used herein, a "cleavable" probe is one that is intended to be cleaved as a result of the specific interaction of the probe with its corresponding target, thereby resulting in the release of the corresponding label and an increase in the corresponding detectable signal.

[0035] As used herein, a "non-cleavable" probe is a probe with a label that is intended to be associated with the probe throughout the assay. In a non-cleavable probe, the corresponding detectable signal changes according to the configuration of the probe, rather than changing according to the release of the label on the probe. Extendable fluorescent probes such as those described in various embodiments, such as universal or hairpin extendable fluorescent probes, are examples of non-cleavable probes.

[0036] The terms "detectable signal" and "label signal" are used synonymously in this article. For example, a "first label signal" is a signal emitted by a first label of a first probe type, and a "second label signal" is a signal emitted by a second label of a second probe type. "Total signal" is the total measured signal in a specific detection channel at a given time point or measurement point. A plurality of different "detectable signals" / "label signals" may contribute to the same "total signal". For example, the total signal may include a signal generated by a first label of a first probe type and a signal generated by a second label of a second probe type. In some embodiments, the signal is a fluorescent signal, and terms such as "first fluorescent signal", "second fluorescent signal" and "total fluorescent signal" can be used as specific examples of corresponding broader terms.

[0037] The term "spectral similarity" refers to the emission signals of detectable labels with the same spectral profile or substantially overlapping spectral profiles. Therefore, different probe types carrying the same detectable label or different probe types carrying different detectable labels have substantial spectral overlap in the emission signal, and both probes can be regarded as probes with spectral similarity. In some specific implementations, detectable labels with spectral similarity can be detected in the same optical detection channel, but other techniques can also be used to detect the emission signals of such detectable labels. Reference to substantially overlapping spectra should be understood to mean spectral similarity.

[0038] The term "end point" as used in connection with a cycle is a designated cycle assuming that the PCR process is complete and / or a designated cycle at which a signal threshold value above a specified amount of background signal occurs. In various embodiments, the end point cycle according to the present disclosure may be in the range of 20 to 45 cycles, such as 30 to 40 cycles. However, the number of cycles of the end point cycle may vary. For example, the number of cycles of the end point cycle may be related to the position at which the emission (e.g., fluorescence) signal indicating the amplification product reaches an approximate plateau. "End point signal" refers to the emission signal measured during the end point cycle. The end point signal may be measured at any designated or selected cycle.

[0039] The chemical structures and formulae described herein are constructed according to the standard rules of chemical valency known in the chemical art.

[0040] When substituents are designated by their conventional chemical formula as written left to right, they equally encompass chemically identical substituents that would result from writing the structure right to left, for example, -CH2O- is equivalent to -OCH2-.

[0041] Unless otherwise indicated, the term "alkyl" by itself or as part of another substituent refers to a straight chain (i.e., unbranched) or branched carbon chain (or carbon) or combinations thereof, which may be fully saturated, monounsaturated, or polyunsaturated, and may include monovalent groups, divalent groups, and polyvalent groups. An alkyl group may include a specified number of carbons (e.g., C1-C 10 Meaning one to ten carbons). In embodiments, alkyl is fully saturated. In embodiments, alkyl is monounsaturated. In embodiments, alkyl is polyunsaturated. Alkyl is a non-cyclized chain. Examples of saturated hydrocarbon groups include, but are not limited to, groups such as methyl, ethyl, n-propyl, isopropyl, n-butyl, tert-butyl, isobutyl, sec-butyl, methyl, homologues and isomers thereof, such as n-pentyl, n-hexyl, n-heptyl, n-octyl, etc. Unsaturated alkyl is an alkyl group with one or more double or triple bonds. Examples of unsaturated alkyl groups include, but are not limited to, vinyl, 2-propenyl, crotyl, 2-isopentenyl, 2-(butadienyl), 2,4-pentadienyl, 3-(1,4-pentadienyl), ethynyl, 1-propynyl and 3-propynyl, 3-butynyl and higher homologues and isomers. Hydrocarbyloxy is an alkyl group connected to the rest of the molecule by an oxygen linker (-O-). The alkyl moiety can be an alkenyl moiety. The alkyl moiety may be an alkynyl moiety. Alkenyl groups include one or more double bonds. Alkynyl groups include one or more triple bonds.

[0042] Unless otherwise stated, the term "alkylene" by itself or as part of another substituent refers to a divalent group derived from an alkyl group, such as, but not limited to -CH2CH2CH2CH2-. Typically, an alkyl (or alkylene) group will have 1 to 24 carbon atoms, with those having 10 or fewer carbon atoms preferred herein. "Lower alkyl" or "lower alkylene" is a shorter chain alkyl or alkylene group, typically with eight or fewer carbon atoms. Unless otherwise stated, the term "alkenylene" by itself or as part of another substituent refers to a divalent group derived from an alkene. Unless otherwise stated, the term "alkynylene" by itself or as part of another substituent refers to a divalent group derived from an alkyne. In an embodiment, an alkylene group is fully saturated. In an embodiment, an alkylene group is monounsaturated. In an embodiment, an alkylene group is polyunsaturated. An alkenylene group includes one or more double bonds. An alkynylene group includes one or more triple bonds.

[0043] Unless otherwise indicated, the term "heteroalkyl" by itself or in combination with another term means a stable straight chain or branch or a combination thereof, including at least one carbon atom and at least one heteroatom (e.g., O, N, P, Si, and S), and wherein the nitrogen atom and sulfur atom may be optionally oxidized, and the nitrogen heteroatom may be optionally quaternized. The heteroatom (e.g., N, S, Si, or P) may be placed at any internal position of the heteroalkyl or at the position where the alkyl is connected to the rest of the molecule. The heteroalkyl is a non-cyclized chain. Examples include, but are not limited to, -CH2-CH2-O-CH3, -CH2-CH2-NH-CH3, -CH2-CH2-N(CH3)-CH3, -CH2-S-CH2-CH3, -S-CH2-CH2, -S(O)-CH3, -CH2-CH2-S(O)2-CH3, -CH=CH-O-CH3, -Si(CH3)3, -CH2-CH=N-OCH3, -CH=CH-N(CH3)-CH3, -O-CH3, -O-CH2-CH3, and -CN. Up to two or three heteroatoms may be consecutive, for example, -CH2-NH-OCH3 and -CH2-O-Si(CH3)3. The heteroalkyl moiety may include one heteroatom (e.g., O, N, S, Si, or P). The heteroalkyl moiety may include two optionally different heteroatoms (e.g., O, N, S, Si, or P). The heteroalkyl moiety may include three optionally different heteroatoms (e.g., O, N, S, Si, or P). The heteroalkyl moiety may include four optionally different heteroatoms (e.g., O, N, S, Si, or P). The heteroalkyl moiety may include five optionally different heteroatoms (e.g., O, N, S, Si, or P). The heteroalkyl moiety may include up to 8 optionally different heteroatoms (e.g., O, N, S, Si, or P). Unless otherwise stated, the term "heteroalkenyl" by itself or in combination with another term means a heteroalkyl group including at least one double bond. In addition to one or more double bonds, the heteroalkenyl group may optionally include more than one double bond and / or one or more triple bonds. Unless otherwise stated, the term "heteroalkynyl" by itself or in combination with another term means a heteroalkyl group including at least one triple bond. In addition to one or more triple bonds, the heteroalkynyl group may optionally include more than one triple bond and / or one or more double bonds. In an embodiment, the heteroalkyl group is fully saturated. In an embodiment, the heteroalkyl group is monounsaturated. In an embodiment, the heteroalkyl group is polyunsaturated.

[0044] Similarly, unless otherwise indicated, the term "heteroalkylene" by itself or as part of another substituent means a divalent radical derived from a heteroalkyl group, such as, but not limited to, -CH2-CH2-S-CH2-CH2- and -CH2-S-CH2-CH2-NH-CH2-. For heteroalkylene, heteroatoms may also occupy either or both of the chain termini (e.g., alkyleneoxy, alkylenedioxy, alkylenamino, alkylenediamino, etc.). Still further, for alkylene and heteroalkylene linking groups, the direction in which the formula of the linking group is written does not imply an orientation of the linking group. For example, the formula -C(O)2R'- represents both -C(O)2R'- and -R'C(O)2-. As described above, heteroalkyl groups as used herein include those groups that are attached to the remainder of the molecule through a heteroatom, such as -C(O)R', -C(O)NR', ​​-NR'R", -OR', -SR' and / or -S02R'. When "heteroalkyl" is recorded, followed by a specific heteroalkyl group, such as -NR'R", etc., it should be understood that the terms heteroalkyl and -NR'R" are not redundant or mutually exclusive. Rather, the specific heteroalkyl group is recorded to increase clarity. Therefore, the term "heteroalkyl" should not be interpreted herein to exclude specific heteroalkyl groups, such as -NR'R", etc. Unless otherwise stated, the term "heteroalkenylene" by itself or as part of another substituent means a divalent group derived from a heteroalkenylene. Unless otherwise stated, the term "heteroalkynyl" by itself or as part of another substituent means a divalent group derived from a heteroalkynyl. In an embodiment, a heteroalkylene is fully saturated. In an embodiment, a heteroalkylene is monounsaturated. In an embodiment, a heteroalkylene is polyunsaturated. A heteroalkylene includes one or more double bonds. Heteroalkynyl groups include one or more triple bonds.

[0045] Unless otherwise stated, the terms "cycloalkyl" and "heterocycloalkyl" themselves or in combination with other terms mean the cyclic forms of "alkyl" and "heteroalkyl", respectively. Cycloalkyl and heterocycloalkyl are not aromatic. In addition, for heterocycloalkyl, heteroatoms may occupy the position where the heterocycle is connected to the rest of the molecule. Examples of cycloalkyl include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, 1-cyclohexenyl, 3-cyclohexenyl, cycloheptyl, etc. Examples of heterocycloalkyl include, but are not limited to, 1-(1,2,5,6-tetrahydropyridyl), 1-piperidyl, 2-piperidyl, 3-piperidyl, 4-morpholinyl, 3-morpholinyl, tetrahydrofuran-2-yl, tetrahydrofuran-3-yl, tetrahydrothiophene-2-yl, tetrahydrothiophene-3-yl, 1-piperazinyl, 2-piperazinyl, etc. "Cycloalkylene" and "heterocycloalkylene" by themselves or as part of another substituent refer to divalent radicals derived from cycloalkyl and heterocycloalkyl, respectively. In embodiments, cycloalkyl is fully saturated. In embodiments, cycloalkyl is monounsaturated. In embodiments, cycloalkyl is polyunsaturated. In embodiments, heterocycloalkyl is fully saturated. In embodiments, heterocycloalkyl is monounsaturated. In embodiments, heterocycloalkyl is polyunsaturated.

[0046] In an embodiment, the term "cycloalkyl" means a monocyclic ring system, a bicyclic ring system, or a polycyclic cycloalkyl ring system. In an embodiment, a monocyclic ring system is a cyclic hydrocarbon group containing 3 to 8 carbon atoms, wherein such groups can be saturated or unsaturated, but not aromatic. In an embodiment, the cycloalkyl group is fully saturated. A bicyclic ring system or a polycyclic cycloalkyl ring system refers to a plurality of rings fused together, wherein at least one of the fused rings is a cycloalkyl ring, and wherein the plurality of rings are attached to the parent molecular moiety through any carbon atom contained within the cycloalkyl rings of the plurality of rings.

[0047] In an embodiment, cycloalkyl is a cycloalkenyl. The term "cycloalkenyl" is used according to its ordinary ordinary meaning. In an embodiment, cycloalkenyl is a monocyclic ring system, a bicyclic ring system, or a polycyclic cycloalkenyl ring system. A bicyclic ring system or a polycyclic cycloalkenyl ring system refers to a plurality of rings fused together, wherein at least one of the fused rings is a cycloalkenyl ring, and wherein the plurality of rings is attached to the parent molecular moiety through any carbon atom contained within the cycloalkenyl ring of the plurality of rings.

[0048] In embodiments, the term "heterocycloalkyl" means a monocyclic ring system, a bicyclic ring system, or a polycyclic heterocycloalkyl ring system. In embodiments, the heterocycloalkyl is fully saturated. A bicyclic ring system or a polycyclic heterocycloalkyl ring system refers to multiple rings fused together, wherein at least one of the fused rings is a heterocycloalkyl ring, and wherein the multiple rings are attached to the parent molecular moiety through any atom contained within the heterocycloalkyl rings of the multiple rings.

[0049] Unless otherwise stated, the term "halo" or "halogen" alone or as part of another substituent means a fluorine, chlorine, bromine or iodine atom. In addition, terms such as "haloalkyl" are intended to include monohaloalkyl and polyhaloalkyl. For example, the term "halo (C1-C4) alkyl" includes but is not limited to fluoromethyl, difluoromethyl, trifluoromethyl, 2,2,2-trifluoroethyl, 4-chlorobutyl, 3-bromopropyl, etc.

[0050] Unless otherwise indicated, the term "acyl" means -C(O)R, where R is substituted or unsubstituted alkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl.

[0051] Unless otherwise specified, the term "aryl" means a polyunsaturated aromatic hydrocarbon substituent, which can be a monocyclic or fused together (i.e., fused ring aryl) or multiple rings (preferably 1 to 3 rings) covalently linked. A fused ring aryl refers to multiple rings fused together, wherein at least one of the fused rings is an aryl ring, and wherein multiple rings are connected to the parent molecular moiety by any carbon atom contained in the aryl rings of the multiple rings. The term "heteroaryl" refers to an aryl (or ring) containing at least one heteroatom, such as N, O or S, wherein the nitrogen atom and the sulfur atom are optionally oxidized, and the nitrogen atom is optionally quaternized. Therefore, the term "heteroaryl" includes fused ring heteroaryls (i.e., multiple rings fused together, wherein at least one of the fused rings is a heteroaromatic ring, and wherein multiple rings are connected to the parent molecular moiety by any atom contained in the heteroaromatic rings of the multiple rings). 5,6- fused ring heteroaryl refers to two rings fused together, wherein one ring has 5 members and the other ring has 6 members, and wherein at least one ring is a heteroaryl ring. Likewise, a 6,6-fused ring heteroarylene refers to two rings fused together, one ring having 6 members and the other ring having 6 members, and wherein at least one ring is a heteroaryl ring. And a 6,5-fused ring heteroarylene refers to two rings fused together, one ring having 6 members and the other ring having 5 members, and wherein at least one ring is a heteroaryl ring. A heteroaryl group can be attached to the rest of the molecule through a carbon or heteroatom. Non-limiting examples of aryl and heteroaryl groups include phenyl, naphthyl, pyrrolyl, pyrazolyl, pyridazinyl, triazinyl, pyrimidinyl, imidazolyl, pyrazinyl, purinyl, oxazolyl, isoxazolyl, thiazolyl, furanyl, thienyl, pyridyl, pyrimidinyl, benzothiazolyl, benzoxazolylbenzimidazolyl, benzofuranyl, isobenzofuranyl, indolyl, isoindolyl, benzothienyl, isoquinolyl, quinoxalinyl, quinolyl, 1-naphthyl, 2-naphthyl, 4-biphenyl, 1-pyrrolyl, 2-pyrrolyl, 3-pyrrolyl, 3-pyrazolyl, 2-imidazolyl, 4-imidazolyl, Substituents for each of the above noted aryl and heteroaryl ring systems are selected from the group of acceptable substituents described below. "Arylene" and "heteroarylene" by themselves or as part of another substituent refer to divalent radicals derived from aryl and heteroaryl, respectively. A heteroaryl substituent may be -O-bonded to a ring heteroatom nitrogen.

[0052] Spirocycle is two or more rings, wherein adjacent rings are connected by a single atom.Each ring in a spirocycle may be the same or different.Each ring in a spirocycle may be substituted or unsubstituted, and may have a substituent different from each other ring in a group of spirocycles.The possible substituents of each ring in a spirocycle are possible substituents of the same ring when it is not a part of a spirocycle (e.g., a substituent of a cycloalkyl ring or a heterocycloalkyl ring).Spirocycle may be a substituted or unsubstituted cycloalkyl, a substituted or unsubstituted cycloalkylene, a substituted or unsubstituted heterocycloalkyl or a substituted or unsubstituted heterocycloalkylene, and each ring in a spirocyclic group may be any one of those listed previously, including all rings with one type (e.g., all rings are substituted heterocycloalkylene, wherein each ring may be the same or different substituted heterocycloalkylene).When referring to a spirocycle system, heterocyclic spirocycle means a spirocycle, wherein at least one ring is a heterocycle, and wherein each ring may be a different ring.When referring to a spirocycle system, the substituted spirocycle means that at least one ring is substituted, and each substituent may be optionally different.

[0053] symbol Indicates the point of attachment of a chemical moiety to the rest of a molecule or chemical formula.

[0054] The term "oxo" as used herein means oxygen double-bonded to a carbon atom.

[0055] The term "alkylarylene" is an arylene moiety covalently bonded to an alkylene moiety (also referred to herein as an alkylene linker). In an embodiment, the alkylarylene group has the formula:

[0056]

[0057] The alkylarylene moiety can be substituted on the alkylene moiety or the arylene linker, for example, at carbon 2, 3, 4 or 6, with halogen, oxo, -N, -CF, -CCl, -CBr, -CI, -CN, -CHO, -OH, -NH, -COOH, -CONH, -NO, -SH, -SOCH, -SOH, -OSOH, -SONH, -NHNH, -ONH, -NHC(O)NHNH, substituted or unsubstituted C1-C5 alkyl, or substituted or unsubstituted 2 to 5 membered heteroalkyl. In an embodiment, the alkylarylene is unsubstituted.

[0058] Each of the above terms (e.g., "alkyl," "heteroalkyl," "cycloalkyl," "heterocycloalkyl," "aryl," and "heteroaryl") includes both substituted and unsubstituted forms of the indicated radical. Preferred substituents for each type of radical are provided below.

[0059] Substituents for the alkyl and heteroalkyl groups (including those groups often referred to as alkylene, alkenyl, heteroalkylene, heteroalkenyl, alkynyl, cycloalkyl, heterocycloalkyl, cycloalkenyl, and heterocycloalkenyl) may be one or more groups selected from, but not limited to, the following groups: -OR', =O, =NR', =N-OR', -NR'R", -SR', halogen, -SiR'R"R"', -OC(O)R', -C(O)R', -C02R', -CONR'R", -OC(O)NR'R", -NR"C(O)R', -NR'C(O)NR"R"', -NR "C(O)2R', -NRC(NR'R"R"')=NR"", -NRC(NR'R")=NR"', -S(O)R', -S(O)2R', -S(O)2NR'R", -NRSO2R', -NR'NR"R"', -ONR'R", -NR'C(O)NR"NR"'R"", -CN, -NO2, -NR'SO2R", -NR'C(O)R", -NR'C(O)OR", -NR'OR'', in a number ranging from zero to (2m'+1), where m' is the total number of carbon atoms in such a group. R', R", R'" and R"" each preferably independently refers to hydrogen, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl (e.g., aryl substituted with 1-3 halogens), substituted or unsubstituted heteroaryl, substituted or unsubstituted alkyl, alkoxy or thioalkoxy, or arylalkyl. When the compounds described herein contain more than one R group, for example, each R group is independently selected, as are each R', R", R'" and R"" group when more than one of these groups is present. When R' and R" are attached to the same nitrogen atom, they can be combined with the nitrogen atom to form a 4-membered ring, a 5-membered ring, a 6-membered ring, or a 7-membered ring. For example, -NR'R" includes, but is not limited to, 1-pyrrolidinyl and 4-morpholinyl. From the above discussion of substituents, one skilled in the art will understand that the term "alkyl" is intended to include groups including carbon atoms bonded to groups other than hydrogen groups, such as haloalkyl groups (e.g., -CF3 and -CH2CF3) and acyl groups (e.g., -C(O)CH3, -C(O)CF3, -C(O)CH2OCH3, etc.).

[0060] Similar to the substituents described for alkyl, the substituents for aryl and heteroaryl are different and are selected from, for example: -OR', -NR'R", -SR', halogen, -SiR'R"R"', -OC(O)R', -C(O)R', -CO2R', -CONR'R", -OC(O)NR'R", -NR"C(O)R', -NR'C(O)NR"R"', -NR"C(O)2R', -NR-C(N R'R"R"')=NR"", -NR-C(NR'R")=NR"', -S(O)R', -S(O)2R', -S(O)2NR'R", -NRSO2R', -NR'NR"R "', -ONR'R", -NR'C(O)NR"NR"'R"", -CN, -NO2, -R', -N3, -CH(Ph)2, fluoro(C1-C4)alkoxy, and In some embodiments, the present invention relates to fluoro(C1-C4)alkyl, -NR'SO2R", -NR'C(O)R", -NR'C(O)OR", -NR'OR", and the number ranges from zero to the total number of open valences on the aromatic ring system. And wherein R', R", R'' and R''' are preferably independently selected from hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl and substituted or unsubstituted heteroaryl. When the compounds described herein contain more than one R group, for example, each R group is independently selected, as are each R', R", R'' and R''' groups when more than one of these groups is present.

[0061] The substituents of the ring (e.g., cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkylene, heterocycloalkylene, arylidene or heteroarylidene) can be depicted as substituents on the ring, rather than substituents on specific atoms of the ring (commonly referred to as floating substituents). In this case, the substituent can be connected to any ring atom in the ring atom (following the chemical valence rules), and in the case of condensed rings or spiro rings, the substituents depicted as being associated with one member of the condensed ring or spiro ring (floating substituents on monocycle) can be substituents on any condensed ring or spiro ring (floating substituents on polycycles). When the substituent is connected to the ring, but not a specific atom (floating substituent), and the subscript of the substituent is an integer greater than 1, multiple substituents can be on the same atom, the same ring, different atoms, different condensed rings, different spiro rings, and each substituent can be optionally different. When the point of attachment of the ring to the rest of the molecule is not limited to a single atom (floating substituent), the point of attachment can be any atom of the ring, and in the case of condensed rings or spiro rings, it is any atom of any condensed ring or spiro ring, while following the chemical valence rules. When a ring, fused ring, or spiro ring contains one or more ring heteroatoms, and the ring, fused ring, or spiro ring is shown to have one or more floating substituents (including but not limited to the point of attachment to the rest of the molecule), the floating substituent may be bonded to the heteroatom. In a structure or formula with a floating substituent, when a ring heteroatom is shown to be bonded to one or more hydrogens (e.g., a ring nitrogen having two bonds to ring atoms and a third bonded to hydrogen), when the heteroatom is bonded to the floating substituent, the substituent will be understood to replace the hydrogen while following the rules of chemical valence.

[0062] Two or more substituents can be optionally connected to form aryl, heteroaryl, cycloalkyl or heterocycloalkyl. This so-called cyclic substituent is typically but not necessarily connected to the cyclic base structure. In one embodiment, the cyclic substituent is connected to the adjacent members of the base structure. For example, two cyclic substituents connected to the adjacent members of the cyclic base structure produce condensed ring structure. In another embodiment, the cyclic substituent is connected to the single member of the base structure. For example, two cyclic substituents connected to the single member of the cyclic base structure produce spiro ring structure. In another embodiment, the cyclic substituent is connected to the non-adjacent members of the base structure.

[0063] Two substituents on adjacent atoms of an aryl or heteroaryl ring may optionally form a group of the formula -TC(O)-(CRR') q -U-, wherein T and U are independently -NR-, -O-, -CRR'- or a single bond, and q is an integer from 0 to 3. Alternatively, two substituents on adjacent atoms of the aryl or heteroaryl ring may be optionally replaced by a substituent of the formula -A-(CH2) r-B-, wherein A and B are independently -CRR'-, -O-, -NR-, -S-, -S(O)-, -S(O)2-, -S(O)2NR'- or a single bond, and r is an integer from 1 to 4. One of the single bonds of the new ring so formed may optionally be replaced by a double bond. Alternatively, two substituents on adjacent atoms of an aryl or heteroaryl ring may optionally be replaced by a substituent of the formula -(CRR') s -X'-(C"R"R"') d -substituted with a substituent, wherein s and d are independently integers from 0 to 3, and X' is -O-, -NR'-, -S-, -S(O)-, -S(O)2- or -S(O)2NR'-. The substituents R, R', R" and R'" are preferably independently selected from hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl and substituted or unsubstituted heteroaryl.

[0064] As used herein, the term "heteroatom" or "ring heteroatom" is intended to include oxygen (O), nitrogen (N), sulfur (S), phosphorus (P), selenium (Se), and silicon (Si). In an embodiment, the term "heteroatom" or "ring heteroatom" is intended to include oxygen (O), nitrogen (N), sulfur (S), phosphorus (P), and silicon (Si).

[0065] As used herein, "substituent" means a group selected from the following moieties:

[0066] (A) Oxo, halogen, -CCl3, -CBr3, -CF3, -CI3, -CHCl2, -CHBr2, -CHF2, -CHI2, -CH2Cl, -CH2Br, -CH2F, -CH2I, -OCCl3, -OCF3, -OCBr3, -OCI3, -OCHCl2, -OCHBr2, -OCHI2, -OCHF2, -OCH2Cl, -OCH2Br, -OCH2I, -OCH2F, -CN, -OH, -NH2, -COOH, -CONH2, -NO2, -SH, -SO3H, -OSO3H, -SO2NH2, -NHNH2, -ONH2, -N HC(O)NHNH2, -NHC(O)NH2, -NHC(NH)NH2, -NHSO2H, -NHC(O)H, -NHC(O)OH, -NHOH, -N3, -SF5, unsubstituted alkyl (e.g., C1-C8 alkyl, C1-C6 alkyl, or C1-C4 alkyl), unsubstituted heteroalkyl (e.g., 2- to 8-membered heteroalkyl, 2- to 6-membered heteroalkyl, or 2- to 4-membered heteroalkyl), unsubstituted cycloalkyl (e.g., C3-C8 cycloalkyl, C3-C6 cycloalkyl, or C5-C6 cycloalkyl), unsubstituted heterocycloalkyl (e.g., 3- to 8-membered heterocycloalkyl, 3- to 6-membered heterocycloalkyl, or 5- to 6-membered heterocycloalkyl), unsubstituted aryl (e.g., C6-C 10 Aryl, C 10 aryl or phenyl), or unsubstituted heteroaryl (e.g., 5- to 10-membered heteroaryl, 5- to 9-membered heteroaryl, or 5- to 6-membered heteroaryl), and

[0067] (B) alkyl (e.g., C1-C8 alkyl, C1-C6 alkyl, or C1-C4 alkyl), heteroalkyl (e.g., 2- to 8-membered heteroalkyl, 2- to 6-membered heteroalkyl, or 2- to 4-membered heteroalkyl), cycloalkyl (e.g., C3-C8 cycloalkyl, C3-C6 cycloalkyl, or C5-C6 cycloalkyl), heterocycloalkyl (e.g., 3- to 8-membered heterocycloalkyl, 3- to 6-membered heterocycloalkyl, or 5- to 6-membered heterocycloalkyl), aryl (e.g., C6-C 10 Aryl, C 10 aryl or phenyl), heteroaryl (e.g., 5- to 10-membered heteroaryl, 5- to 9-membered heteroaryl, or 5- to 6-membered heteroaryl) substituted with at least one substituent selected from:

[0068] (i) Oxo, halogen, -CCl3, -CBr3, -CF3, -CI3, -CHCl2, -CHBr2, -CHF2, -CHI2, -CH2Cl, -CH2Br, -CH2F, -CH2I, -OCCl3, -OCF3, -OCBr3, -OCI3, -OCHCl2, -OCHBr2, -OCHI2, -OCHF2, -OCH2Cl, -OCH2Br, -OCH2I, -OCH2F, -CN, -OH, -NH2, -COOH, -CONH2, -NO2, -SH, -SO3H, -OSO3H, -SO2NH2, -NHNH2, -ONH2, -N HC(O)NHNH2, -NHC(O)NH2, -NHC(NH)NH2, -NHSO2H, -NHC(O)H, -NHC(O)OH, -NHOH, -N3, -SF5, unsubstituted alkyl (e.g., C1-C8 alkyl, C1-C6 alkyl, or C1-C4 alkyl), unsubstituted heteroalkyl (e.g., 2- to 8-membered heteroalkyl, 2- to 6-membered heteroalkyl, or 2- to 4-membered heteroalkyl), unsubstituted cycloalkyl (e.g., C3-C8 cycloalkyl, C3-C6 cycloalkyl, or C5-C6 cycloalkyl), unsubstituted heterocycloalkyl (e.g., 3- to 8-membered heterocycloalkyl, 3- to 6-membered heterocycloalkyl, or 5- to 6-membered heterocycloalkyl), unsubstituted aryl (e.g., C6-C 10 Aryl, C 10 aryl or phenyl), or unsubstituted heteroaryl (e.g., 5- to 10-membered heteroaryl, 5- to 9-membered heteroaryl, or 5- to 6-membered heteroaryl), and

[0069] (ii) alkyl (e.g., C1-C8 alkyl, C1-C6 alkyl, or C1-C4 alkyl), heteroalkyl (e.g., 2- to 8-membered heteroalkyl, 2- to 6-membered heteroalkyl, or 2- to 4-membered heteroalkyl), cycloalkyl (e.g., C3-C8 cycloalkyl, C3-C6 cycloalkyl, or C5-C6 cycloalkyl), heterocycloalkyl (e.g., 3- to 8-membered heterocycloalkyl, 3- to 6-membered heterocycloalkyl, or 5- to 6-membered heterocycloalkyl), aryl (e.g., C6-C 10 Aryl, C 10 aryl or phenyl), heteroaryl (e.g., 5- to 10-membered heteroaryl, 5- to 9-membered heteroaryl, or 5- to 6-membered heteroaryl) substituted with at least one substituent selected from:

[0070] (a) Oxo, halogen, -CCl3, -CBr3, -CF3, -CI3, -CHCl2, -CHBr2, -CHF2, -CHI2, -CH2Cl, -CH2Br, -CH2F, -CH2I, -OCCl3, -OCF3, -OCBr3, -OCI3, -OCHCl2, -OCHBr2, -OCHI2, -OCHF2, -OCH2Cl, -OCH2Br, -OCH2I, -OCH2F, -CN, -OH, -NH2, -COOH, -CONH2, -NO2, -SH, -SO3H, -OSO3H, -SO2NH2, -NHNH2, -ONH2, -N HC(O)NHNH2, -NHC(O)NH2, -NHC(NH)NH2, -NHSO2H, -NHC(O)H, -NHC(O)OH, -NHOH, -N3, -SF5, unsubstituted alkyl (e.g., C1-C8 alkyl, C1-C6 alkyl, or C1-C4 alkyl), unsubstituted heteroalkyl (e.g., 2- to 8-membered heteroalkyl, 2- to 6-membered heteroalkyl, or 2- to 4-membered heteroalkyl), unsubstituted cycloalkyl (e.g., C3-C8 cycloalkyl, C3-C6 cycloalkyl, or C5-C6 cycloalkyl), unsubstituted heterocycloalkyl (e.g., 3- to 8-membered heterocycloalkyl, 3- to 6-membered heterocycloalkyl, or 5- to 6-membered heterocycloalkyl), unsubstituted aryl (e.g., C6-C 10 Aryl, C 10 aryl or phenyl), or unsubstituted heteroaryl (e.g., 5- to 10-membered heteroaryl, 5- to 9-membered heteroaryl, or 5- to 6-membered heteroaryl), and

[0071] (b) alkyl (e.g., C1-C8 alkyl, C1-C6 alkyl, or C1-C4 alkyl), heteroalkyl (e.g., 2- to 8-membered heteroalkyl, 2- to 6-membered heteroalkyl, or 2- to 4-membered heteroalkyl), cycloalkyl (e.g., C3-C8 cycloalkyl, C3-C6 cycloalkyl, or C5-C6 cycloalkyl), heterocycloalkyl (e.g., 3- to 8-membered heterocycloalkyl, 3- to 6-membered heterocycloalkyl, or 5- to 6-membered heterocycloalkyl), aryl (e.g., C6-C 10 Aryl, C 10-CHCl, -CHBr, -CHF, -CHI, -CHCl, -CHBr, -CHF, -CHI, -OCCl, -OCF, -OCBr, -OCI, -OCHCl, -OCHBr, -OCHI, -OCHF, -OCHCl, -OCHBr, -OCHI, -OCHF, -OCHCl, -OCHBr, -OCHI, -OCHF, -CN, -OH, -NH, -COOH, -CONH, -NO, -SH, -SO3H, -OS O3H, -SO2NH2, -NHNH2, -ONH2, -NHC(O)NHNH2, -NHC(O)NH2, -NHC(NH)NH2, -NHSO2H, -NHC(O)H, -NHC(O)OH, -NHOH, -N3, -SF5, unsubstituted alkyl (e.g., C1-C8 alkyl, C1-C6 alkyl, or C1-C4 alkyl), unsubstituted heteroalkyl (e.g., 2- to 8-membered heteroalkyl, 2- to 6-membered heteroalkyl, or 2- to 4-membered heteroalkyl), unsubstituted cycloalkyl (e.g., C3-C8 cycloalkyl, C3-C6 cycloalkyl, or C5-C6 cycloalkyl), unsubstituted heterocycloalkyl (e.g., 3- to 8-membered heterocycloalkyl, 3- to 6-membered heterocycloalkyl, or 5- to 6-membered heterocycloalkyl), unsubstituted aryl (e.g., C6-C 10 Aryl, C 10 aryl or phenyl), or an unsubstituted heteroaryl (e.g., a 5- to 10-membered heteroaryl, a 5- to 9-membered heteroaryl, or a 5- to 6-membered heteroaryl).

[0072] As used herein, "a substituent group of limited size" or "a substituent group of limited size" means a group selected from all substituents described above for "a substituent group", wherein each substituted or unsubstituted alkyl group is a substituted or unsubstituted C1-C 20 alkyl, each substituted or unsubstituted heteroalkyl is a substituted or unsubstituted 2 to 20-membered heteroalkyl, each substituted or unsubstituted cycloalkyl is a substituted or unsubstituted C3-C8 cycloalkyl, each substituted or unsubstituted heterocycloalkyl is a substituted or unsubstituted 3 to 8-membered heterocycloalkyl, each substituted or unsubstituted aryl is a substituted or unsubstituted C6-C 10 The aryl group is an aryl group, and each substituted or unsubstituted heteroaryl group is a substituted or unsubstituted 5- to 10-membered heteroaryl group.

[0073] As used herein, "lower substituent" or "lower substituent group" means a group selected from all substituents above for "substituent", wherein each substituted or unsubstituted alkyl is a substituted or unsubstituted C1-C8 alkyl, each substituted or unsubstituted heteroalkyl is a substituted or unsubstituted 2 to 8-membered heteroalkyl, each substituted or unsubstituted cycloalkyl is a substituted or unsubstituted C3-C7 cycloalkyl, each substituted or unsubstituted heterocycloalkyl is a substituted or unsubstituted 3 to 7-membered heterocycloalkyl, each substituted or unsubstituted aryl is a substituted or unsubstituted phenyl, and each substituted or unsubstituted heteroaryl is a substituted or unsubstituted 5 to 6-membered heteroaryl.

[0074] In some embodiments, each substituent described in the compounds herein is substituted by at least one substituent. More specifically, in some embodiments, each substituted alkyl, substituted heteroalkyl, substituted cycloalkyl, substituted heterocycloalkyl, substituted aryl, substituted heteroaryl, substituted alkylene, substituted sub-assorted alkyl, substituted cycloalkylene, substituted sub-heterocycloalkylene, substituted arylene and / or substituted sub-heteroaryl described in the compounds herein is substituted by at least one substituent. In other embodiments, at least one or all of these groups are substituted by at least one size-restricted substituent. In other embodiments, at least one or all of these groups are substituted by at least one low-level substituent.

[0075] In other embodiments of the compounds herein, each substituted or unsubstituted alkyl group may be a substituted or unsubstituted C1-C 20 alkyl, each substituted or unsubstituted heteroalkyl is a substituted or unsubstituted 2 to 20-membered heteroalkyl, each substituted or unsubstituted cycloalkyl is a substituted or unsubstituted C3-C8 cycloalkyl, each substituted or unsubstituted heterocycloalkyl is a substituted or unsubstituted 3 to 8-membered heterocycloalkyl, each substituted or unsubstituted aryl is a substituted or unsubstituted C6-C 10 aryl, and / or each substituted or unsubstituted heteroaryl is a substituted or unsubstituted 5- to 10-membered heteroaryl. In some embodiments of the compounds herein, each substituted or unsubstituted alkylene is a substituted or unsubstituted C1-C 20 alkylene, each substituted or unsubstituted heteroalkylene is a substituted or unsubstituted 2- to 20-membered heteroalkylene, each substituted or unsubstituted cycloalkylene is a substituted or unsubstituted C3-C8 cycloalkylene, each substituted or unsubstituted heterocycloalkylene is a substituted or unsubstituted 3- to 8-membered heterocycloalkylene, each substituted or unsubstituted arylene is a substituted or unsubstituted C6-C 10 The arylene group is an arylene group, and / or each substituted or unsubstituted heteroarylene group is a substituted or unsubstituted 5- to 10-membered heteroarylene group.

[0076] In some embodiments, each substituted or unsubstituted alkyl is a substituted or unsubstituted C1-C8 alkyl, each substituted or unsubstituted heteroalkyl is a substituted or unsubstituted 2-8 membered heteroalkyl, each substituted or unsubstituted cycloalkyl is a substituted or unsubstituted C3-C7 cycloalkyl, each substituted or unsubstituted heterocycloalkyl is a substituted or unsubstituted 3-7 membered heterocycloalkyl, each substituted or unsubstituted aryl is a substituted or unsubstituted C6-C 10 In some embodiments, each substituted or unsubstituted alkylene group is a substituted or unsubstituted C1-C8 alkylene group, each substituted or unsubstituted heteroalkylene group is a substituted or unsubstituted 2-8-membered heteroalkylene group, each substituted or unsubstituted cycloalkylene group is a substituted or unsubstituted C3-C7 cycloalkylene group, each substituted or unsubstituted heteroalkylene group is a substituted or unsubstituted 3-7-membered heteroalkylene group, and each substituted or unsubstituted arylene group is a substituted or unsubstituted C6-C8 alkylene group. 10 arylene, and / or each substituted or unsubstituted heteroarylene is a substituted or unsubstituted 5- to 9-membered heteroarylene. In some embodiments, the compound is a chemical species listed in the embodiments section, figures or tables below.

[0077] In embodiments, a substituted or unsubstituted moiety (e.g., substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted alkylene, substituted or unsubstituted heteroalkylene, substituted or unsubstituted cycloalkylene, substituted or unsubstituted heterocycloalkylene, substituted or unsubstituted arylene, and / or substituted or unsubstituted heteroarylene) is unsubstituted (e.g., unsubstituted alkyl, unsubstituted heteroalkyl, unsubstituted cycloalkyl, unsubstituted heterocycloalkyl, unsubstituted aryl, unsubstituted heteroaryl, unsubstituted alkylene, unsubstituted heteroalkylene, unsubstituted cycloalkylene, unsubstituted heterocycloalkylene, unsubstituted arylene, and / or unsubstituted heteroarylene, respectively). In embodiments, the substituted or unsubstituted moiety (e.g., substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted alkylene, substituted or unsubstituted heteroalkylene, substituted or unsubstituted cycloalkylene, substituted or unsubstituted heterocycloalkylene, substituted or unsubstituted arylene and / or substituted or unsubstituted heteroarylene) is substituted (e.g., substituted alkyl, substituted heteroalkyl, substituted cycloalkyl, substituted heterocycloalkyl, substituted aryl, substituted heteroaryl, substituted alkylene, substituted heteroalkylene, substituted cycloalkylene, substituted heterocycloalkylene, substituted arylene and / or substituted heteroarylene, respectively).

[0078] In an embodiment, a substituted moiety (e.g., substituted alkyl, substituted heteroalkyl, substituted cycloalkyl, substituted heterocycloalkyl, substituted aryl, substituted heteroaryl, substituted alkylene, substituted heteroalkylene, substituted cycloalkylene, substituted heterocycloalkylene, substituted arylene, and / or substituted heteroarylene) is substituted with at least one substituent, wherein if the substituted moiety is substituted with multiple substituents, each substituent is optionally different. In an embodiment, if the substituted moiety is substituted with multiple substituents, each substituent is different.

[0079] In embodiments, a substituted moiety (e.g., substituted alkyl, substituted heteroalkyl, substituted cycloalkyl, substituted heterocycloalkyl, substituted aryl, substituted heteroaryl, substituted alkylene, substituted heteroalkylene, substituted cycloalkylene, substituted heterocycloalkylene, substituted arylene, and / or substituted heteroarylene) is substituted with at least one size-restricted substituent, wherein if the substituted moiety is substituted with multiple size-restricted substituents, each size-restricted substituent is optionally different. In embodiments, if the substituted moiety is substituted with multiple size-restricted substituents, each size-restricted substituent is different.

[0080] In an embodiment, a substituted moiety (e.g., substituted alkyl, substituted heteroalkyl, substituted cycloalkyl, substituted heterocycloalkyl, substituted aryl, substituted heteroaryl, substituted alkylene, substituted heteroalkylene, substituted cycloalkylene, substituted heterocycloalkylene, substituted arylene, and / or substituted heteroarylene) is substituted with at least one lower substituent, wherein if the substituted moiety is substituted with multiple lower substituents, each lower substituent is optionally different. In an embodiment, if the substituted moiety is substituted with multiple lower substituents, each lower substituent is different.

[0081] In an embodiment, a substituted moiety (e.g., substituted alkyl, substituted heteroalkyl, substituted cycloalkyl, substituted heterocycloalkyl, substituted aryl, substituted heteroaryl, substituted alkylene, substituted heteroalkylene, substituted cycloalkylene, substituted heterocycloalkylene, substituted arylene, and / or substituted heteroarylene) is substituted with at least one substituent, a size-restricted substituent, or a lower substituent; wherein if the substituted moiety is substituted with a plurality of groups selected from substituents, size-restricted substituents, and lower substituents; each substituent, size-restricted substituent, and / or lower substituent may be optionally different. In an embodiment, if the substituted moiety is substituted with a plurality of groups selected from substituents, size-restricted substituents, and lower substituents; each substituent, size-restricted substituent, and / or lower substituent is different.

[0082] Certain compounds of the present disclosure have asymmetric carbon atoms (optical or chiral centers) or double bonds; enantiomers, racemates, diastereomers, tautomers, geometric isomers, stereoisomeric forms of amino acids can be defined as (R)- or (S)- or (D)- or (L)- and individual isomers are included within the scope of the present disclosure, based on absolute stereochemistry. The compounds of the present disclosure do not include those known in the art that are too unstable to synthesize and / or separate. The present disclosure is intended to include compounds in racemic and optically pure forms. Optically active (R)- and (S)- or (D)- and (L)-isomers can be prepared using chiral synthons or chiral reagents, or resolved using conventional techniques. When the compounds described herein contain olefinic bonds or other centers of geometric asymmetry, and unless otherwise specified, it is intended that the compounds include E and Z geometric isomers.

[0083] As used herein, the term "isomers" refers to compounds that have the same number and kind of atoms, and therefore the same molecular weight, but differ in the structural arrangement or configuration of the atoms.

[0084] As used herein, the term "tautomer" refers to one structural isomer of two or more structural isomers that exist in equilibrium and are easily converted from one isomeric form to another.

[0085] It will be apparent to those skilled in the art that certain compounds of the present disclosure may exist in tautomeric forms and all such tautomeric forms of compounds are within the scope of the present disclosure.

[0086] Unless otherwise stated, structures depicted herein are also intended to include all stereochemical forms of the structure; i.e., the R and S configurations for each asymmetric center. Therefore, single stereochemical isomers as well as enantiomeric and diastereomeric mixtures of the present compounds are within the scope of the present disclosure.

[0087] Unless otherwise stated, structures depicted herein are also meant to include compounds that differ only in the presence of one or more isotopically enriched atoms. For example, in addition to the replacement of hydrogen by deuterium or tritium, or by 13 C- or 14 Compounds of the present structures having C-enriched carbon substituted carbons are within the scope of the present disclosure.

[0088] The compounds of the present disclosure may also contain unnatural proportions of atomic isotopes at one or more atoms that constitute such compounds. For example, the compounds may be radiolabeled with radioactive isotopes, such as tritium ( 3 H), iodine-125 ( 125 I) or carbon-14 ( 14 C) All isotopic variations of the compounds of the present disclosure, whether radioactive or not, are encompassed within the scope of the present disclosure.

[0089] It should be noted that throughout this application, candidates are written as Markush groups, e.g., each amino acid position contains more than one possible amino acid. It is specifically contemplated that each member of the Markush group should be considered individually, thereby comprising another embodiment, and the Markush group should not be construed as a single unit.

[0090] As used herein, the terms "bioconjugate" and "bioconjugate linker" refer to the binding between atoms or molecules of a bioconjugate reactive group or a bioconjugate reactive moiety. The binding can be direct or indirect. For example, the conjugate between a first bioconjugate reactive group (e.g., -NH2, -COOH, -N-hydroxysuccinimide or -maleimide) and a second bioconjugate reactive group (e.g., a sulfhydryl, a sulfur-containing amino acid, an amine, an amino acid containing an amine side chain, or a carboxylate) provided herein can be direct, such as through a covalent bond or linker (e.g., a first linker of a second linker), or indirect, such as through a non-covalent bond (e.g., electrostatic interactions (e.g., ionic bonds, hydrogen bonds, halogen bonds), van der Waals interactions (e.g., dipole-dipole, dipole-induced dipole, London dispersion), ring stacking (π effect), hydrophobic interactions, etc.). In embodiments, the bioconjugate or bioconjugate linker is formed using bioconjugate chemistry (i.e., the combination of two bioconjugate reactive groups), including but not limited to nucleophilic substitution (e.g., reaction of amines and alcohols with acyl halides, active esters), electrophilic substitution (e.g., enamine reactions), and carbon-carbon and carbon-heteroatom multiple bond additions (e.g., Michael reactions, Diels-Alder additions). In embodiments, a first bioconjugate reactive group (e.g., a maleimide moiety) is covalently linked to a second bioconjugate reactive group (e.g., a sulfhydryl group). In embodiments, a first bioconjugate reactive group (e.g., a haloacetyl moiety) is covalently linked to a second bioconjugate reactive group (e.g., a sulfhydryl group). In embodiments, a first bioconjugate reactive group (e.g., a pyridyl moiety) is covalently linked to a second bioconjugate reactive group (e.g., a sulfhydryl group). In embodiments, a first bioconjugate reactive group (e.g., a -N-hydroxysuccinimide moiety) is covalently linked to a second bioconjugate reactive group (e.g., an amine). In embodiments, a first bioconjugate reactive group (e.g., a maleimide moiety) is covalently linked to a second bioconjugate reactive group (e.g., a sulfhydryl group). In embodiments, a first bioconjugate reactive group (e.g., a -sulfo-N-hydroxysuccinimide moiety) is covalently linked to a second bioconjugate reactive group (e.g., an amine).

[0091] Useful bioconjugation reactive moieties for bioconjugation chemistry herein include, for example: (a) carboxyl groups and their various derivatives, including, but not limited to, N-hydroxysuccinimide esters, N-hydroxybenzotriazole esters, acyl halides, acyl imidazoles, thioesters, p-nitrophenyl esters, alkyl, alkenyl, alkynyl, and aromatic esters; (b) hydroxyl groups that can be converted into esters, ethers, aldehydes, and the like; (c) alkyl halides, where the halide can be later replaced by a nucleophilic group, such as, for example, an amine, a carboxylate anion, a thiol anion, a carbon anion, or an alkoxyl ion, resulting in a new group covalently attached at the site of the halogen atom; (d) dienophiles that are capable of participating in a Diels-Alder reaction. (e) aldehyde or ketone groups, allowing for subsequent derivatization by formation of carbonyl derivatives, such as, for example, imines, hydrazones, semicarbazones or oximes, or by mechanisms such as Grignard addition or alkyllithium addition; (f) sulfonyl halide groups for subsequent reaction with amines, for example to form sulfonamides; (g) thiol groups, which can be converted to disulfides, reacted with acyl halides, or bonded to metals such as gold, or reacted with maleimides; (h) amines or sulfhydryl groups (e.g., as present in cysteine), which can, for example, be acylated, alkylated or oxidized; (i) olefins, which can undergo, for example, cycloadditions, acylations, Michael additions, etc. (j) epoxides, which can react with, for example, amines and hydroxyl compounds; (k) phosphoramidites and other standard functional groups used in nucleic acid synthesis; (l) metal silicon oxide bonding; (m) metal bonding to reactive phosphorus groups (e.g., phosphines) to form, for example, phosphodiester bonds; (n) coupling of azides to alkynes using copper-catalyzed cycloaddition click chemistry; and (o) biotin conjugates can react with avidin or streptavidin to form avidin-biotin complexes or streptavidin-biotin complexes.

[0092] The bioconjugate reactive groups may be selected so that they do not participate in or interfere with the chemical stability of the conjugates described herein. Alternatively, reactive functional groups may be protected from participating in cross-linking reactions by the presence of protecting groups. In embodiments, the bioconjugate comprises a molecular entity derived from an unsaturated bond (such as maleimide) and a sulfhydryl reaction.

[0093] "Analog," "analogue," or "derivative" is used according to its plain ordinary meaning in chemistry and biology and refers to a chemical compound that is structurally similar to another compound, the so-called "reference" compound, but differs in composition, e.g., the replacement of one atom by an atom of a different element, or the presence of a specific functional group, or the replacement of one functional group by another, or the absolute stereochemistry of one or more chiral centers of the reference compound. Thus, an analog is a compound that is similar or equivalent to the reference compound in function and appearance, but differs from the reference compound in structure or origin.

[0094] As used herein, the term "a" or "an" means one or more. In addition, as used herein, the phrase "substituted" means that the specified group can be substituted with one or more of any or all of the specified substituents. For example, when a group such as an alkyl or heteroaryl group is "substituted with unsubstituted C1-C 20 alkyl or unsubstituted 2 to 20 membered heteroalkyl”, the group may contain one or more unsubstituted C1-C 20 alkyl and / or one or more unsubstituted 2 to 20 membered heteroalkyl groups.

[0095] Furthermore, when a moiety is substituted with an R substituent, the group may be referred to as "R-substituted". When a moiety is R-substituted, the moiety is substituted with at least one R substituent, and each R substituent is optionally different. In cases where a particular R group is present in the description of a chemical genus such as Formula (I), a Roman letter symbol may be used to distinguish each occurrence of that particular R group. For example, when multiple R 13 When a substituent is present, each R 13 Substituents can be divided into R 13.A , R 13.B , R 13.C , R 13.D etc., where each R 13.A , R 13.B , R 13.C , R 13.D Waiting in R 13 When an R moiety, group or substituent as disclosed herein is connected by a single bond, and the R moiety, group or substituent is an oxo, one of ordinary skill in the art will immediately recognize that according to the normal rules of chemical valence, the oxo is connected by a double bond.

[0096] The description of the compounds of the present disclosure is limited by the principles of chemical bonding known to those skilled in the art. Thus, when a group may be substituted by one or more of a number of substituents, such substitutions are selected so as to conform to the principles of chemical bonding and to give compounds that are not inherently unstable and / or are known to those of ordinary skill in the art to be unstable under ambient conditions (e.g., aqueous, neutral, and several known physiological conditions). For example, heterocycloalkyl or heteroaryl groups are connected to the rest of the molecule via ring heteroatoms, thereby avoiding inherently unstable compounds, according to the principles of chemical bonding known to those skilled in the art.

[0097] "Nucleic acid" refers to nucleotides (e.g., deoxyribonucleotides or ribonucleotides) and polymers thereof, or their complements, in single-stranded, double-stranded or multi-stranded forms; or nucleosides (e.g., deoxyribonucleosides or ribonucleosides). In embodiments, "nucleic acid" does not include nucleosides. The terms "polynucleotide," "oligonucleotide," "oligonucleotide," etc. refer to linear nucleotide sequences in the usual and customary sense. The length of an oligonucleotide is generally about 5, 6, 7, 8, 9, 10, 12, 15, 25, 30, 40, 50 or more nucleotides, up to about 100 nucleotides in length. Nucleic acids and polynucleotides are polymers of any length, including longer lengths, for example, 200, 300, 500, 1000, 2000, 3000, 5000, 7000, 10,000, etc. In certain embodiments, the nucleic acids herein comprise phosphodiester bonds. In other embodiments, nucleic acid analogs are included, which may have alternative backbones, including, for example, phosphoramidate, phosphorothioate, phosphorodithioate or O-methylphosphoamidite bonds; and peptide nucleic acid backbones and linkages. Other similar nucleic acids include those with positive backbones; non-ionic backbones and non-ribose backbones. Nucleic acids containing one or more carbocyclic sugars are also included in a definition of nucleic acids. Modification of the ribose-phosphate backbone can be for a variety of reasons, such as increasing the stability and half-life of such molecules in a physiological environment or as a probe on a biochip. A mixture of naturally occurring nucleic acids and analogs can be prepared; alternatively, a mixture of different nucleic acid analogs can be prepared, as well as a mixture of naturally occurring nucleic acids and analogs. As described herein, the residue of a nucleic acid is a monomer (e.g., nucleotide) of a nucleic acid. The term "nucleoside" refers to a glycosylamine including a nucleobase and a pentose (ribose or deoxyribose) in the usual and customary sense. Non-limiting examples of nucleosides include cytidine, uridine, adenosine, guanosine, thymidine and inosine. Nucleosides can be modified on bases and / or sugars. The term "nucleotide" refers to a single unit of a polynucleotide, i.e., a monomer, in the usual and customary sense. A nucleotide may be a ribonucleotide, a deoxyribonucleotide, or a modified form thereof. Examples of polynucleotides encompassed herein include single-stranded and double-stranded DNA, single-stranded and double-stranded RNA, and hybrid molecules having a mixture of single-stranded and double-stranded DNA and RNA. Examples of nucleic acids, for example, polynucleotides contemplated herein, include any type of RNA, such as mRNA, siRNA, miRNA, and guide RNA, as well as any type of DNA, genomic DNA, plasmid DNA, and small circular DNA, and any fragments thereof. In the context of polynucleotides, the term "duplex" refers to a double strand in the usual and customary sense. Nucleic acids may be straight or branched. For example, a nucleic acid may be a straight chain of nucleotides, or a nucleic acid may be branched, for example, so that the nucleic acid comprises an arm or branch of one or more nucleotides. Optionally, branched nucleic acids are repeatedly branched to form higher-order structures, such as dendritic polymers, etc. "Nucleic acid moiety" as used herein is a monovalent form of a nucleic acid.In an embodiment, the nucleic acid moiety is linked to the 3' or 5' position of a nucleotide or nucleoside.

[0098] Nucleic acids, including, for example, nucleic acids having a phosphorothioate backbone, can include one or more reactive moieties. As used herein, the term reactive moiety includes any group capable of reacting with another molecule, such as a nucleic acid or a polypeptide, through covalent, non-covalent, or other interactions. For example, a nucleic acid can include an amino acid reactive moiety that reacts with an amino acid on a protein or polypeptide through covalent, non-covalent, or other interactions.

[0099] As used herein, "nucleotide" refers to a nucleoside-5'-phosphate (e.g., polyphosphate) compound or a structural analog thereof, which can be incorporated by a nucleic acid polymerase (e.g., partially incorporated as a nucleoside-5'-monophosphate or a derivative thereof) to extend a growing nucleic acid chain (e.g., a primer). Nucleotide may include a base, such as adenine (A), cytosine (C), guanine (G), thymine (T), uracil (U) or their analogs, and may include 1, 2, 3, 4, 5, 6, 7, 8 or more phosphates in a phosphate group. Nucleotide may be modified at one or more of a base, sugar or phosphate group. Nucleotide may be connected with a label or tag ("labeled nucleotide" or "labeled nucleotide"). In an embodiment, the nucleotide is a deoxyribonucleotide. In an embodiment, the nucleotide is a ribonucleotide. In an embodiment, the nucleotide includes 3 phosphate groups (e.g., a triphosphate group).

[0100] The term also includes nucleic acids containing known nucleotide analogs or modified backbone residues or linkages, and the nucleic acids are synthetic, naturally occurring, and non-naturally occurring, with binding properties similar to reference nucleic acids, and the mode of metabolism is similar to reference nucleotides. The example of such analogs includes, but is not limited to, phosphodiester derivatives, including, for example, phosphoramidates, diaminophosphoroesters, thiophosphates (also referred to as thiophosphates with oxygen in double-bond sulfur-substituted phosphates), dithiophosphates, phosphonocarboxylic acids, phosphonocarboxylates, phosphonoacetic acid, phosphonoformic acid, methylphosphonates, borophosphonates, or O-methylphosphoramidate linkages (see Eckstein, Oligonucleotides and Analogues: A Practical Approach, Oxford University Press, Oxford University Press) and modifications to nucleotide bases, such as 5-methylcytidine or pseudouridine; and peptide nucleic acid backbones and linkages. Other similar nucleic acids include those with positive backbones; non-ionic backbones, modified sugars, and non-ribose backbones (e.g., phosphorodiamidate morpholino oligomers or locked nucleic acids (LNA) known in the art). Nucleic acids containing one or more carbocyclic sugars are also included in a definition of nucleic acids. Modification of the ribose-phosphate backbone can be for a variety of reasons, such as increasing the stability and half-life of such molecules in a physiological environment or as probes on a biochip. Mixtures of naturally occurring nucleic acids and analogs can be prepared; alternatively, mixtures of different nucleic acid analogs can be prepared, as well as mixtures of naturally occurring nucleic acids and analogs. In an embodiment, the internucleotide linkage in DNA is a phosphodiester, a phosphodiester derivative, or a combination of the two.

[0101] "Nucleoside" is similar to nucleotide in structure, but lacks the phosphate moiety present in nucleotide. The example of nucleoside analog is a nucleoside analog in which the label is connected to the base and there is no phosphate group connected to the sugar molecule. "Nucleoside" as used herein refers to a glycosyl compound consisting of a core base and a 5-membered ring sugar (e.g., ribose or deoxyribose). Nucleoside can include a base, such as adenine (A), cytosine (C), guanine (G), thymine (T), uracil (U) or their analogs. Nucleoside can be modified at base and / or sugar. In an embodiment, a nucleoside is a deoxyribonucleoside. In an embodiment, a nucleoside is a ribonucleoside.

[0102] As used herein, the term "complementary" or "substantially complementary" refers to the hybridization, base pairing or duplex formation between nucleotides or nucleic acids. For example, when nucleotides (e.g., RNA or DNA) or nucleotide sequences can base pair with corresponding homologous nucleotides or homologous nucleotide sequences, there is complementarity between the two chains of a double-stranded DNA molecule or between the primer binding sites on an oligonucleotide primer and a single-stranded nucleic acid. As described herein and well known in the art, the complementary (matching) nucleotide of adenosine (A) is thymidine (T), and the complementary (matching) nucleotide of guanosine (G) is cytosine (C). Therefore, the complement may include a nucleotide sequence that is base-paired with the corresponding complementary nucleotides of the second nucleic acid sequence. The nucleotides of the complement may partially or completely match the nucleotides of the second nucleic acid sequence. When the nucleotides of the complement fully match each nucleotide of the second nucleic acid sequence, the complement forms a base pair with each nucleotide of the second nucleic acid sequence. When the nucleotides of the complement partially match the nucleotides of the second nucleic acid sequence, only some of the nucleotides of the complement in the complement form a base pair with the nucleotides of the second nucleic acid sequence. Examples of complements include coding sequences and non-coding sequences, wherein the non-coding sequence contains nucleotides that are complementary to the coding sequence and thus forms the complement of the coding sequence. Additional examples of complements are sense and antisense sequences, wherein the sense sequence contains complementary nucleotides to the antisense sequence and thus forms the complement of the antisense sequence. "Double" means that at least two fully or partially complementary oligonucleotides and / or polynucleotides undergo Watson-Crick type base pairing in all or most of their nucleotides, thereby forming a stable complex.

[0103] As described herein, the complementarity of sequences can be partial, where only some of the nucleic acids in the nucleic acids match according to base pairing, or complete, where all nucleic acids match according to base pairing. Thus, two sequences that are complementary to each other can have a specified percentage of nucleotides that are complementary to each other (e.g., about 60%, preferably 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher complementarity in a specific region). In an embodiment, when two sequences are fully complementary, they are complementary and have 100% complementarity.

[0104] As used herein, the term "polymerase" refers to any naturally occurring or non-natural enzyme or other catalyst capable of catalyzing a polymerization reaction, such as the polymerization of nucleotide monomers to form nucleic acid polymers. Exemplary types of polymerases that can be used in the compositions and methods of the present disclosure include nucleic acid polymerases, such as DNA polymerases, RNA polymerases that rely on DNA or RNA, and reverse transcriptases. In some cases, the DNA polymerase is 9N polymerase or a variant thereof, Escherichia coli (E. Coli) DNA polymerase I, bacteriophage T4 DNA polymerase, sequencing enzyme, Taq DNA polymerase, DNA polymerase from Bacillus stearothermophilus, Bst 2.0 DNA polymerase, 9N polymerase, 9N polymerase (exo-) A485L / Y409V, Phi29 DNA polymerase ( DNA polymerase, T7 DNA polymerase, DNA polymerase II, DNA polymerase III holoenzyme, DNA polymerase IV, DNA polymerase V, VentR DNA polymerase, TherminatorTMII DNA polymerase, TherminatorTMIII DNA polymerase or TherminatorTMIX DNA polymerase. As used herein, the terms "DNA polymerase" and "nucleic acid polymerase" are used according to their ordinary common sense and refer to enzymes capable of synthesizing nucleic acid molecules from nucleotides (e.g., deoxyribonucleotides). Typically, DNA polymerases add nucleotides to the 3' end of a DNA chain, one nucleotide at a time. In embodiments, the DNA polymerase is Pol I DNA polymerase, Pol II DNA polymerase, Pol III DNA polymerase, Pol IV DNA polymerase, Pol V DNA polymerase, Pol β DNA polymerase, Pol μ DNA polymerase, Pol λ DNA polymerase, Pol σ DNA polymerase, Pol α DNA polymerase, Pol δ DNA polymerase, Pol ε DNA polymerase, Pol η DNA polymerase, Pol ι DNA polymerase, Pol κ DNA polymerase, Pol ζ DNA polymerase, Pol γ DNA polymerase, Pol θ DNA polymerase, Pol υ DNA polymerase, or a thermophilic nucleic acid polymerase (e.g., thermopolymerase γ, 9N polymerase (exo-), thermopolymerase II, thermopolymerase III, or thermopolymerase IX). In embodiments, the DNA polymerase is a modified archaeal DNA polymerase. In embodiments, the polymerase is a reverse transcriptase. In an embodiment, the polymerase is a mutant Pichia abyssi polymerase (e.g., a mutant Pichia abyssi polymerase as described in WO 2018 / 148723 or WO 2020 / 056044). As used herein, the term "thermophilic nucleic acid polymerase" refers to a family of DNA polymerases (e.g., 9NTM) and mutants thereof, derived from DNA polymerases originally isolated from hyperthermophilic archaea Thermococcus sp. 9 degrees N-7, found in hydrothermal vents (Eastern Pacific Rise) at this latitude (Southworth MW, et al. Proceedings of the National Academy of Sciences (PNAS) 1996; 93(11): 5281-5285). Thermophilic nucleic acid polymerases are members of the B DNA polymerases.

[0105] As used herein, the term "exonuclease activity" is used according to its common meaning in the art, and refers to the removal of nucleotides from nucleic acid by DNA polymerase. For example, during polymerization, nucleotides are added to the 3' end of the primer chain. Sometimes DNA polymerase incorporates incorrect nucleotides at the 3'-OH end of the primer chain, wherein the incorrect nucleotides cannot form hydrogen bonds with the corresponding bases in the template chain. Due to the 3' to 5' exonuclease activity of DNA polymerase, the nucleotides added by such errors are removed from the primer. When referring to 3'-5' exonuclease activity, it is understood that DNA polymerase promotes hydrolysis reaction, which destroys the phosphodiester bond at the 3' end of the polynucleotide chain to excise nucleotides. In an embodiment, 3'-5' exonuclease activity refers to the continuous removal of nucleotides in single-stranded DNA in the 3'→5' direction, releasing deoxyribonucleoside 5'-monophosphates one by one.

[0106] The terms "determine," "calculate," and "estimate" are used synonymously herein. These terms are not intended to imply a precise level of measurement accuracy. Thus, where values ​​are "determined," "calculated," or "estimated" using the embodiments described herein, it should be understood that such values ​​may include some degree of inherent error due to factors such as instrument tolerances, rounding, chemical reaction variability, and other inherent measurement imperfections known and understood by those skilled in the art.

[0107] For any given element of a component of a described embodiment, any possible alternatives listed for that element or component may generally be used alone or in combination with one another, unless implicitly or explicitly stated otherwise.

[0108] In addition, unless otherwise indicated, the numbers used in the specification and claims to express quantity, composition, distance or other measurement should be understood as optionally modified by the term "about" or its synonyms. When the terms "about", "approximately", "substantially", etc. are used in conjunction with the amount, value or condition, they may mean an amount, value or condition that is less than 20%, less than 10%, less than 5%, less than 1%, less than 0.1% or less than 0.01% away from the amount, value or condition. At the very least, and without attempting to limit the application of the doctrine of equivalents to the scope of the claims, each numerical parameter should be interpreted in accordance with the number of significant figures reported and by applying ordinary rounding techniques.

[0109] Any headings and sub-headings used herein are for organizational purposes only and are not meant to be used to limit the scope of the description or the claims.

[0110] It should also be noted that, as used in this specification and the appended claims, the singular forms "a," "an," and "the" do not include plural referents unless the context clearly dictates otherwise. Thus, for example, an embodiment referring to a singular referent (e.g., "a widget") may also include two or more such referents.

[0111] It should also be understood that the embodiments described herein may also include characteristics and / or features (e.g., ingredients, components, members, elements, parts and / or portions) described in one or more separate embodiments, and are not necessarily strictly limited to the features explicitly described for that particular embodiment. Therefore, the various features of a given embodiment may be combined with other embodiments of the present disclosure and / or incorporated into other embodiments of the present disclosure. Therefore, certain features are disclosed with reference to a specific embodiment of the present disclosure, which should not be considered as limiting the application of the features to or incorporation into the specific embodiment. On the contrary, it should be understood that other embodiments may also include such features.

[0112] Overview of multiplexing using probes with spectrally similar labels

[0113] Figure 1A The emission spectra of various fluorescent dyes that can be used in nucleic acid detection assays are shown. As described above, some multiplexed assays assign each dye to a separate target and then determine the presence and / or amount of each target by measuring the fluorescent signal, for example, in separate detection channels that each correspond to the emission wavelength of the corresponding dye. Figure 1B As shown, in some cases, there can be a large amount of overlap in the emission spectra of the dyes. For example, Figure 1B Dye 1 and Dye 2 in FIG. 1 show substantially overlapping emission spectra and can be determined by Figure 1B Although multiplex dyes are generally selected with the goal of minimizing spectral overlap, when using existing technologies, the finite nature of emission spectra effectively limits the number of individual dyes that can be combined in the same multiplex assay, and therefore limits the number of different targets that can be detected and / or measured.

[0114] Embodiments described herein solve one or more of the aforementioned problems by providing multiple detectable signals, each of which is associated with a different assay target or target group, having a similar emission spectrum, such as, for example, corresponding to detection in the same detection channel. Multiple detectable signals can be resolved and analyzed individually, thereby allowing detection and / or quantification of each target. By allowing the use of the same one or more dyes with spectral similarity to measure multiple targets, the disclosed embodiments can beneficially increase the "diversity" (i.e., the number of targets that can be detected and quantified) of multiple assays, without relying on additional dyes and the problem of increased spectral overlap that accompanies them. For example, according to various aspects of the present disclosure, a public detection channel can be used to detect dyes with spectral similarity but intended for different target analytes. Similarly, embodiments described herein can beneficially reduce the number of separate dyes required in multiple assays without reducing the diversity of the assay. In addition, various embodiments can allow the use of the same dye as a marker for different target nucleic acids in multiple assays, including using the same dye for different targets simultaneously during the reaction. In addition, various embodiments can allow the detection of the same dye in the same detection channel.

[0115] Figure 2A It is a schematic overview of the method for detecting multiple target nucleic acids by providing different first probe types and second probe types, changing reaction mixture conditions, and measuring the resulting total signal under each set of conditions, utilizing a detectable label with spectral similarity to detect multiple target nucleic acids. As shown in the figure, the first probe 202 is designed to specifically interact ("combine") with the first target 206. The first probe 202 includes a first label 210 that can produce a first label signal 214. The second probe 204 is designed to specifically interact with a second target 208 that is different from the first target 206. The second probe 204 includes a second label 212 that can produce a second label signal 216.

[0116] In some embodiments, the first label 210 and the second label 212 are the same. For example, the first label 210 and the second label 212 may include the same fluorescent dye. In some embodiments, the first label 210 and the second label 212 may be different, but still designed to produce substantially the same signal (e.g., having spectral similarity). For example, the first label 210 and the second label 212 may include a dye that is chemically different but functions to emit a fluorescent signal with a similar wavelength. In some embodiments, the first label signal 214 and the second label signal 216 are measured using the same detection channel (e.g., including a filter arrangement) in a detection instrument.

[0117] The first probe 202 and the second probe 204 can be provided in the same reaction mixture, and are allowed to specifically interact with any first target 206 and the second target 208 in the reaction mixture, respectively. As shown, the reaction mixture is subjected to at least two groups of different reaction conditions. The first probe 202 is designed so that during both the first group of conditions 218 and the second group of conditions 220, the first label 210 produces a first label signal 214, and its degree is related to the amount of the specific interaction between the first probe 202 and the first target 206 (e.g., proportional). On the contrary, the second probe 204 is designed so that the second label 212 produces a second label signal 216 during the second group of conditions 220 rather than during the first group of conditions 218, and its degree is related to the amount of the specific interaction between the second probe 204 and the second target 208 (e.g., proportional). In other words, under first set of conditions 218, first label signal 214 increases due to the specific interaction of first probe 202 with first target 206, but second label signal 216 is not emitted (increased) due to the specific interaction of second probe 204 with second target 208. Under second set of conditions 220, second label signal 216 increases due to the specific interaction of second probe 204 with second target 208, while first label signal 214 also further increases or remains at a level that is at least somewhat increased compared to first set of conditions 218.

[0118] During the first set of conditions 218, the second marker 212 will not generate a "substantial signal (e.g., fluorescence)", and therefore the second marker signal 216 will not be substantially different from the background (i.e., baseline) level of the emission signal (e.g., fluorescence) in the reaction mixture. That is, although there may be a certain non-zero level of signal generated by the second marker 212 during the first set of conditions 218, the second marker signal 216 will generally remain below the threshold that separates the background signal from the meaningful signal. As described above, this threshold can vary according to the specific test protocol and application requirements.

[0119] In at least some embodiments, when both first target 206 and second target 208 are present in the reaction mixture, second marker signal 216 will differ to a greater extent between first set of conditions 218 and second set of conditions 220 than first marker signal 214 will differ to a greater extent between first set of conditions 218 and second set of conditions 220. Thus, while first marker signal 214 may differ somewhat between first set of conditions 218 and second set of conditions 220, the difference will generally be less than the difference in second marker signal 216 between first set of conditions 218 and second set of conditions 220.

[0120] Various embodiments of the present disclosure utilize the differences in the way that the first marker signal 214 and the second marker signal 216 respond to different sets of conditions so that the detected first marker signal 214 and the second marker signal 216 can be resolved (separated), even if, for example, they are detected in the same detection channel. As shown, the total signal during the first set of conditions 218 ("first total signal") is measured, and the total signal during the second set of conditions 220 ("second total signal") is measured. Fluorescence (or emission) signal data representing the first total signal is sometimes referred to as "first fluorescence signal data" or "first emission signal data" in this article, and fluorescence signal data representing the second total signal is sometimes referred to as "second fluorescence signal data" or "composite fluorescence signal data" or "second emission signal data" or "composite emission signal data" in this article. As used herein, the first and second in this context are not necessarily used to represent the temporal order of detection or conditions, although such a temporal order may occur.

[0121] During first set of conditions 218, the total signal will be substantially equal to first marker signal 214. That is, the first total signal will be primarily composed of first marker signal 214, with negligible contribution from second marker signal 216. During second set of conditions 220, the total signal will include a combination of first marker signal 214 and second marker signal 216. Thus, first marker signal 214 and second marker signal 216 may be resolved separately based on the first total signal and the second total signal. For example, first marker signal 214 may be determined based on the first total signal, and second marker signal 216 may be resolved by subtracting the first total signal from the second total signal.

[0122] In some embodiments, first marker signal 214 is directly equal to first total signal. In other embodiments, first marker signal 214 is determined as a function of first total signal. In some embodiments, the function is a linear function (although nonlinear functions may be used in some implementations). For example, as described above, first marker signal 214 may be slightly different between first set of conditions 218 and second set of conditions 220 even when the amount of first target 206 does not change. In some applications, first marker signal 214 under second set of conditions 220 may better correspond to a standard curve that equates first marker signal 214 to the amount of first target 206. Therefore, estimating first marker signal 214 as a function of the first total signal, rather than directly equal to the first total signal, may make the calculated first marker signal 214 closer to the signal to be measured under second set of conditions 220 (i.e., second marker signal 216 without any interference).

[0123] In some embodiments, the function for converting the first total signal into the first label signal 214 is determined by comparing the first label signal 214 under the first set of conditions 218 with the first label signal 214 under the second set of conditions 220 without any second probe interacting with the second target. The first label signal 214 under the first set of conditions 218 and the second set of conditions 220 is usually related to each other according to a linear function. In other embodiments, it can be related using a nonlinear function. When a linear function is used, a multiplier factor (e.g., a correction factor) can be used to convert the first total signal into the first label signal 214. Once such a linear function is determined, it can be used in subsequent determinations without the need for additional comparisons of the first label signal 214 under the first set of conditions 218 and under the second set of conditions 220 without the second probe having the second target. In some embodiments, the function for converting the first total signal into the first label signal can be nonlinear. In some embodiments, the function / correlation is determined on the stage of the thermal cycle or between thermal cycles with the same number of expected cleavage probes. For example, even if different signals are detected in the same detection channel, this method can also be used to resolve the different signals.

[0124] As described in more detail below, the first probe 202 and the second probe 204 have different mechanisms of action that achieve different signal responses to the first set of conditions 218 and the second set of conditions 220 according to the probe type. Advantageously, the ability to resolve separate signals associated with each different probe type does not need to rely on properties, such as the different melting temperatures of the probes. Therefore, although the first probe 202 and the second probe 204 may have different melting temperatures, this is not a prerequisite for allowing their associated label signals to be effectively resolved. For example, in some embodiments, the melting temperature (T) of the first probe 202 m ) (generally defined as the temperature at which 50% of the strands are in double-stranded form and 50% are single-stranded) and the T of the second probe 204 m Difference is within about 8 ℃, or about 6 ℃, or about 4 ℃, or about 2 ℃. In some embodiments, under the first set of conditions or the second set of conditions, both probes are combined (e.g., hybridized) to their corresponding targets. Optionally, under the first set of conditions or the second set of conditions, both probes are not substantially combined (e.g., hybridized) with their corresponding targets. In an example, under the first set of conditions, the first probe and the second probe are substantially combined (e.g., hybridized) to their corresponding targets, and under the second set of conditions, the first probe and the second probe are not substantially combined (e.g., hybridized) to their corresponding targets. In another example, in an example, under the first set of conditions, the first probe and the second probe are substantially combined (e.g., hybridized) to their corresponding targets, and under the second set of conditions, the first probe and the second probe are substantially combined (e.g., hybridized) to their corresponding targets.

[0125] Figure 2B 20 is a graph showing that when the reaction mixture is cycled between the first set of reaction conditions 218 and the second set of reaction conditions 220 and when both the first target 206 and the second target 208 are present in the reaction mixture, Figure 2A 218. A graph of the signal response over time of the method outlined in . The cycle of conditions may include, for example, different conditions for various stages associated with thermal cycling in a nucleic acid amplification reaction (such as, for example, PCR). In such a reaction, a first set of reaction conditions 218 may correspond to supporting a denaturation stage, and a second set of reaction conditions 220 may correspond to supporting an annealing and / or extension stage ("annealing / extension stage") of the thermal cycle. Thus, in various embodiments, the first set of reaction conditions 218 includes a first temperature or a first temperature range, and the second set of reaction conditions 220 includes a second temperature or a second temperature range (lower than the first temperature).

[0126] As shown, under the second set of reaction conditions 220, the first label signal 214 and the second label signal 216 are both increased. Under the first set of reaction conditions 218, the first label signal 214 remains substantially the same as when the previous cycle ends (although as mentioned above, it may change slightly), and the second label signal 216 drops to a level similar to the baseline signal level of the second label signal 216, which can be substantially constant in multiple amplification cycles. In other words, during the first set of reaction conditions, the second label signal 216 shows a baseline signal higher than the background signal level. In some cases, the second label signal may show a baseline signal level that changes at different stages of the amplification cycle, but is still enough to be distinguished from the level under the second set of reaction conditions and is lower than the level under the second set of reaction conditions. This may be due to the different states of the probe and the proximity of the quencher to the label.

[0127] As shown, first marker signal 214 and second marker signal 216 cumulatively increase at each successive occurrence of second set of conditions 220. This is a result of additional specific interactions in the reaction mixture between first probe 202 and first target 206 and additional specific interactions in the reaction mixture between second probe 204 and second target 208. However, as first marker signal 214 moves from the end of one cycle to the beginning of another cycle (i.e., when moving from second set of conditions 220 at the end of a cycle to first set of conditions 218 at the beginning of a subsequent cycle), second marker signal 216 returns to a level close to the baseline at the beginning of each cycle (i.e., at each occurrence of first set of conditions 218).

[0128] Although some embodiments described herein can utilize multiplexing in channels (detection in the same channel), the disclosure is not limited thereto. In addition, the disclosure also contemplates the combination of multiplexing between channels and multiplexing in channels with multiplexing between channels, to further increase the diversity of the assay. For example, assays can be designed with a variety of different dyes (and therefore with a variety of different detection channels), wherein two or more different channels each include a variety of detectable signals, which can be resolved using the techniques and methods described herein.

[0129] Cleavable and non-cleavable probes

[0130] In some embodiments, the first probe (e.g., first probe 202) is a "cleavable" probe. The first probe can be designed so that as a result of hybridization of the first probe to the first target (e.g., first target 206), the first label (e.g., first label 210) is separated from the first probe (and, for example, released from the corresponding quencher). Therefore, once released, the first label continues to contribute to the total signal in the reaction mixture. The first probe can be a TaqMan probe, for example, which undergoes cleavage due to the 5' to 3' exonuclease activity of a DNA polymerase during extension of the target molecule to which the probe hybridizes. TaqMan probes are described in U.S. Pat. Nos. 4,889,818, 5,079,352, 5,210,015, 5,436,134, 5,487,972, 5,658,751, 5,210,015, 5,487,972, 5,538,848, 5,618,711, 5,677,152, 5,723,591, 5,773,25 8, 5,789,224, 5,801,155, 5,804,375, 5,876,930, 5,994,056, 6,030,787, 6,084,102, 6,127,155, 6,171,785, 6,214,979, 6,258,569, 6,814,934, 6,821,727, 7,141,377, and 7,445,900, all of which are hereby incorporated by reference.

[0131] In some embodiments, the second probe (e.g., second probe 204) is a "non-cleavable" probe. The second label (e.g., second label 212) of the non-cleavable probe is intended to remain associated with the probe throughout the assay and to change the level of the generated signal (e.g., second label signal 216) based on the probe configuration rather than the release of the label. The second probe can be an extendible fluorescent ("EF") probe, for example, which quenches the label when in a single-stranded configuration, but allows the signal when incorporated into a double-stranded molecule.

[0132] In some embodiments, the second probe (e.g., the second probe 204) is a compound or a salt thereof as described below, and in a patent application entitled “Multiplex Dye Compounds” filed concurrently with the U.S. Patent and Trademark Office on June 29, 2023, the entire contents of which are incorporated herein by reference.

[0133] Figure 3A Shown during the annealing stage, extension stage and the denaturation stage of the PCR reaction thermal cycle, the activity of cleavable probe 302 and non-cleavable probe 312, this cleavable probe can be TaqMan probe in various embodiments, and this non-cleavable probe can be EF probe in various embodiments.As shown in the figure, during the annealing stage, TaqMan probe 302 and its corresponding target nucleic acid amplicon 304 (as used herein, target nucleic acid amplicon can refer to the single strand of target double-stranded nucleic acid, and should be understood by the context when describing the PCR reaction with reference) hybridization.During the extension of the primer 303 hybridized with the target nucleic acid amplicon 304 upstream of probe 302, the 5' to 3' exonuclease activity of DNA polymerase is cracked by TaqMan probe mark 306 from the remainder of probe 302, thereby it is separated from corresponding TaqMan probe quencher 309.This causes the corresponding increase of fluorescent signal.During the denaturation period, mark 306 keeps free in the reaction mixture solution, and therefore continues to contribute to the total fluorescent signal.

[0134] EF probe 312 includes EF probe label 316 and EF probe quencher 319, which remain close to each other when probe 312 is in a single-stranded configuration. Therefore, when the EF probe is in a single-stranded configuration, the fluorescence signal from label 316 remains substantially quenched. During the annealing phase and the extension phase, EF probe 312 hybridizes with its corresponding target template amplicon 314 and extends to form an extended probe amplicon 313. The extension of the target template 314 then forms a complement 315 of the extended probe amplicon 313. The resulting double-stranded amplicon 317 forces the label 316 to move away from the quencher 319, reaching a distance sufficient to allow fluorescence emission. During denaturation, the extended probe amplicon 313 is separated from its complement 315. When returning to the single-stranded configuration, the label 316 and quencher 318 are brought back into proximity, and the fluorescence is quenched again.

[0135] Figure 3B302 and EF probe 312 over time during thermal cycling of the amplification process. The temperature of the thermal cycle can vary depending on the needs of the particular application. For example, the denaturation phase can be performed at a temperature in the range of about 80°C to about 100°C, such as about 85°C to about 95°C, or such as about 90°C to about 95°C. The annealing / extension phase can be performed at a lower temperature, such as in the range of about 40°C to about 75°C, such as about 50°C to about 70°C, such as about 55°C to about 65°C. In some embodiments, the first set of reaction conditions (e.g., reference Figure 2A The first set of conditions discussed above (218) corresponds to the denaturation stage 318, while the second set of reaction conditions (e.g., Figure 2A The second set of conditions discussed (220) corresponds to the annealing / extension phase (320).

[0136] Although various embodiments circulate between denaturation stage 318 and combined annealing / extension stage 320 (i.e., the amplification process circulates between two target temperatures), other embodiments may include separate annealing and extension steps. In such embodiments, temperature and possible other reaction conditions may vary between the annealing step and the extension step. For example, the extension step may be carried out at a temperature higher than the annealing temperature. In some embodiments, for at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% or at least 95% of the circulation of the amplification process, the amplification process circulates between at least two target temperatures.

[0137] Figure 3B 3. It is shown that the fluorescent signal associated with the TaqMan probe 302 increases during the extension phase 320 and then remains at a similar level during the denaturation phase 318 of the next cycle, while the fluorescent signal associated with the EF probe 304 increases during the extension phase 320, but decreases to the baseline signal level associated with the EF probe 304 once the subsequent denaturation phase 318 reaches the target denaturation temperature. It will be appreciated by those of ordinary skill in the art that Figure 3B The cycles N, N+1, N+2 may be started at different stages, however, in which case the comparison of the above signal levels may be shifted.

[0138] In some embodiments, the first set of reaction conditions (e.g., denaturation conditions 318) includes a first measurement temperature at which the first label signal is measured, and the second set of reaction conditions (e.g., annealing / extension conditions 320) includes a second, different measurement temperature at which the first label signal and the second label signal are measured. In some embodiments, the first measurement temperature and the second measurement temperature differ by at least about 10°C or more, about 15°C or more, about 20°C or more, about 25°C or more, or about 30°C or more. The first measurement temperature can be, for example, a target denaturation temperature in the range of about 80°C to about 100°C, for example, about 85°C to about 95°C, or, for example, about 90°C to about 95°C, and the second measurement temperature can be, for example, a target annealing / extension temperature in the range of about 40°C to about 75°C, for example, about 50°C to about 70°C, for example, about 55°C to about 65°C.

[0139] EF probe template formation

[0140] Figure 4A The process of using a tailed primer (also referred to herein as a tailed primer) 422 that is specific to a nucleic acid target 424 to form a target template 414 to which an EF probe 412 can hybridize is shown. The tailed primer 422 includes a tail 426 and a target specific portion 428. Figure 4B An example of a more detailed view of a tailed primer 422 as a forward primer, a target-specific primer 423 paired with the tailed primer 422 as a reverse primer, and the EF probe 412 is shown.

[0141] As shown, in the first stage, the target specific portion 428 hybridizes to the target 424. Extension of the target specific portion 428 forms a tailed amplicon 425. Primer 423, which is paired with the tailed primer 422, enables extension of the complement of the tailed amplicon 425. It is this complement that forms the target template 414. As shown, the target template 414 includes a tail complement portion 427.

[0142] In the second stage, the EF probe 412 is hybridized to the target template 414, and amplification can continue, as Figure 3A As shown. Figure 4A As shown, EF probe 412 includes a probe tail 417 that has substantial homology to tail 426 and is therefore complementary to tail complement portion 427 of target template 414. Extension of probe 412 and target template 414 forms a double-stranded amplicon 419. In the third stage, primer 423, shown here as paired with tailed primer 422, can also be used as primer 423 paired with EF probe 412 to enable formation of double-stranded amplicon 419, as shown in FIG. Figure 3A shown.

[0143] like Figure 4BAs shown, tail 426 may be formed with the 5' end of tail primer 422. EF probe 412 may include a stem-loop portion, wherein stem portion 410 is on either side of loop portion 411, configured to form a stem-loop structure when EF probe 412 is single-stranded. For example, label 416 may be located on one side of the stem-loop portion, and quencher 418 may be located on the opposite side of the stem-loop portion, such that when the stem-loop structure is formed, label 416 and quencher 418 are in close proximity, but when EF probe 412 is constrained to a more linear configuration (e.g., when incorporating a double-stranded amplicon), the label and quencher are spaced further apart.

[0144] In the illustrated embodiment, the label 416 is located at or near the 5' end of the EF probe 412, and the quencher 418 is located at the 3' end of the label 416. In other embodiments, the positions of the label 416 and the quencher 418 can be reversed. Preferably, as shown, the stem-loop portion is disposed at the 5' end of the probe tail 417, so that the stem-loop portion remains at the end of the amplicon generated by the extension of the EF probe 412, so that the stem-loop structure formation (when single-stranded) is less likely to be impaired.

[0145] In some embodiments, an EF probe includes a non-stem-loop portion that separates a label (located at or near the 5' end of the EF probe) from a quencher located at or near the 3' end of the EF probe.

[0146] In addition to or as an alternative to the EF probes described herein, some embodiments may include other labeled oligonucleotides that produce increased fluorescence when incorporated into a double-stranded amplicon (relative to when in a single-stranded state), such as, for example, during the extension phase and / or annealing phase of a PCR process. For example, LUX TM The primer includes an internal fluorophore that is quenched by a hairpin structure located 5' of the fluorophore. As with the EF probe, when incorporated into a double-stranded amplicon, the LUX TM The primers provide increased fluorescence, and the hairpin structure is linearized. In addition, any primer or probe described herein may include one or more locked nucleic acids (LNA) known in the art.

[0147] In some embodiments, tailed primer 422 and corresponding (tailless) primer 423 are provided at different concentrations. For example, primer 423 can be provided at a higher concentration than tailed primer 422. For example, primer 423 can be provided at a concentration of about 2X (2 times) to about 30X (30 times) the concentration of tailed primer 422, or about 5X to about 25X the concentration of tailed primer 422, or about 10X to about 20X the concentration of tailed primer 422. Because primer 423 can play a role in (1) driving the formation of target template 414 (e.g., Figure 4A ) and (2) driving the formation of complement 415 of extended probe amplicon 413 (as shown Figure 3A), so providing it at a higher concentration than the corresponding tailed primer 422 can beneficially balance the reaction and help drive the overall reaction efficiency.

[0148] In some embodiments, EF probe 412 is provided at a concentration that is different from the concentration of tailed primer 422 and / or the concentration of primer 423. For example, EF probe 412 can be provided at a concentration that is greater than the concentration of tailed primer 422, and less than the concentration of primer 423. In some embodiments, EF probe 412 is provided at a concentration that is about 2X to about 20X the concentration of tailed primer 422, or about 3X to about 15X the concentration of tailed primer 422. As described above, providing a relatively high concentration of primer 423 helps drive the overall efficiency of the reaction. Providing EF probe 412 at a higher concentration than tailed primer 422, but not necessarily higher than primer 423, pushes more of the associated amplification toward EF probe 412, but still allows primer 423 to be the primary driver of reaction efficiency.

[0149] In addition to or as an alternative to using a "universal" EF probe with a probe tail 417, other embodiments include and / or utilize EF probes having a target-specific portion other than the probe tail 417. Such EF probes can hybridize directly to a target template nucleic acid, such as Figure 4A shown, and therefore does not need to follow Figure 4A The two-stage process shown is used to generate a target template 414 having a tail complement portion 427. In such an embodiment, the probe tail 417 of the EF probe 412 is replaced with a target-specific portion that hybridizes directly to the target 424. The process is otherwise similar to Figure 3A That is, after the EF probe is extended, a subsequent round of annealing / extension will extend the complementary strands to form a double-stranded amplicon that separates the fluorophore and quencher to allow the generation of a fluorescent signal.

[0150] Figure 4C A three-stage thermal cycling method is shown that can be used during an amplification process involving a non-cleavable probe (eg, an EF probe) and an optional cleavable probe (eg, a TaqMan probe). Figure 4C The amplification process shown can be used in combination with any other method disclosed herein. The amplification process shown includes a first stage with a first target annealing / extension temperature, a second stage with a second different annealing / extension temperature, and a third stage with a third annealing / extension temperature. In this embodiment, the third annealing / extension temperature is the same as the first annealing / extension temperature. Other embodiments may include a third annealing / extension temperature that is different from both the first annealing / extension temperature and the second annealing / extension temperature.

[0151] Thus, the amplification process shown includes a denaturation temperature that varies throughout the amplification process and a plurality of different annealing / extension temperatures. Such an amplification process advantageously provides an initial stage (stage 1) in which the target template 414 is initially formed, an intermediate stage (stage 2) in which the interaction between the EF probe 412 and the target template 414 increases to form the initially extended probe amplicon 413, and a later stage (stage 3) in which further amplification involving the probe amplicon 413 and 415 can proceed.

[0152] As shown in the figure, the first annealing / extension temperature may be higher than the second annealing / extension temperature. The first series of denaturation steps and annealing / extension steps (in stage 1) are circulated more times than the second series of denaturation steps and annealing / extension steps (in stage 2). The third series of denaturation steps and annealing / extension steps (in stage 3) may be circulated more times than the first series of denaturation steps and annealing / extension steps. The denaturation temperature of each stage may be the same or different. As a non-limiting example, the denaturation temperature of each stage may be higher than 80°C, but different from each other. For example, the temperature in the early stage may be higher than the temperature in the subsequent stage. In an embodiment, the denaturation temperature in the first stage may be about 95°C, and the denaturation temperature in the second stage may be about 85°C.

[0153] Thus, stages 1 and 2 serve as pre-loading stages that primarily generate target template 414 (in stage 1), followed by providing a lower annealing / extension temperature (in stage 2) for at least one cycle to allow for increased interaction between EF probe 412 and target template 414. Thereafter, multiple amplification cycles may be performed at a third annealing / extension temperature to drive amplification of probe amplicons 413 and 415 primarily involving EF probe 412, primer 423, and / or extensions thereof. Thus, most amplification cycles are typically performed during stage 3.

[0154] Figure 4C The various specific temperatures, times, and temperature change rates depicted in the are exemplary only and should not be construed as limiting the scope of the present disclosure and claims. Other specific temperatures, times, and temperature change rates may be employed and will be understood from the rest of the present disclosure.

[0155] Embodiments are not limited to three-stage thermal cycling methods. During amplification involving a non-cleavable probe (e.g., EF probe) and an optional cleavable probe (e.g., TaqMan probe), a one-stage thermal cycling method (wherein the annealing / extension temperature and the denaturation temperature do not vary from cycle to cycle) or a two-stage thermal cycling method (wherein one of the annealing / extension temperature or the denaturation temperature varies from cycle to cycle) may be utilized.

[0156] Digital PCR Implementation

[0157] PCR is a method for in vitro enzymatic synthesis of specific DNA sequences using two oligonucleotide primers that hybridize to opposite strands and flank a region of interest in a target DNA. A series of repeated reaction phases involving template denaturation, primer annealing, and extension of the annealed primers by a DNA polymerase results in the exponential accumulation of specific fragments, the ends of which are defined by the primers. PCR can selectively enrich specific DNA sequences by several orders of magnitude. As described above, the annealing phase and the extension phase can be different phases under different reaction conditions, or can occur under the same channel set reaction conditions.

[0158] Digital polymerase chain reaction (dPCR) is a specific application of PCR that can be used to directly quantify the target nucleic acid in a sample. In dPCR, the reaction mixture is partitioned into many small reaction volumes (also referred to as separators) so that the target nucleic acid is in some but not all reaction volumes. The reaction volume is subjected to thermal cycling, and the proportion of the "positive" reaction volume that produces a signal indicating the presence of the target (e.g., an emission signal from a detectable label, such as fluorescence) is determined. Quantitative Poisson-based applications use the number of negative / non-reactive reaction volumes and assume a Poisson distribution to determine the number of initial copies distributed in all reaction volumes.

[0159] The features and principles of other amplification processes described herein are also generally applicable to dPCR processes. Therefore, although the description of this section provides a more detailed disclosure of dPCR embodiments, the disclosure provided elsewhere herein is also applicable to dPCR embodiments. Embodiments including dPCR can utilize various separation mechanisms or devices known in the art or that may be developed in the future. For example, some conventional dPCR systems utilize multiple droplets encapsulated by an oil phase to form multiple separators / reaction volumes. Other embodiments may utilize microchamber arrays. An example of such a system is the QuantStudio Absolute Q system available from Thermo Fisher Scientific, which uses a microfluidic array plate to divide / separate samples and generate reaction volumes. Those of ordinary skill in the art are familiar with various types of systems for separating samples into small reaction volumes, subjecting those reaction volumes to PCR, and detecting emission signals from reaction volumes.

[0160] In some dPCR embodiments, the reaction mixture is fully formed before being separated into multiple reaction volumes. In alternative embodiments, one or more components of the reaction mixture can be preloaded onto or into the reaction volume. For example, probes and / or primers can be applied to the walls of the microchamber, and then the sample and / or other components of the reaction mixture are added to the microchamber to form multiple reaction mixtures in each reaction volume.

[0161] Figure 5Ais a schematic overview of a method for detecting multiple target nucleic acids using a PCR process and probes carrying detectable labels with spectral similarity. The PCR process can include real-time / quantitative PCR (qPCR), which typically monitors amplification during the reaction, dPCR, which typically includes an endpoint measurement to count and determine the number of "positive" partitions, and / or endpoint PCR. Therefore, Figure 5A It is shown that under both the first set of conditions 518 (e.g., denaturation conditions, such as about 95° C.) and the second set of conditions 520 (e.g., annealing / extension conditions, such as about 65° C.), the first probe 502 associated with the first label 510 and configured to specifically interact with the first target 506 will be shown as positive (+) at the end of the reaction due to the emission of the first label signal 514. On the other hand, due to the absence of an emission signal from the second label signal 516, the second probe 504 associated with the second label 512 and configured to specifically interact with the second target 508 will be shown as negative (-) under the first set of conditions 518, but will be shown as positive (+) under the second set of conditions 520 due to the emission signal from the second label signal 516.

[0162] This is similar to Figure 2A The scheme shown, and Figure 5A It is shown that the same principle can be applied to endpoint measurements such as in dPCR.As described in other embodiments, the first probe 502 can be a cleavable probe, such as a TaqMan probe, and the second probe 504 can be a non-cleavable probe, such as an EF probe.

[0163] Figure 5B How the signal of dPCR reaction volume (for example, at the end point cycle of PCR) is shown to be active and change according to whether the first probe 502, the second probe 504 or both are active in the reaction volume during the reaction. As shown in the figure, if only the first probe 502 provides signal 514, then under the first set of conditions 518 and the second set of conditions 520, the reaction volume will be positive (+). If only the second probe 504 provides signal 516, then the reaction volume will be negative (-) under the first set of conditions 518 and positive (+) under the second set of conditions 520. If the first probe 502 and the second probe 504 both provide signal, then the reaction volume is positive (+) under the first set of conditions 518, and is highly positive (++) (due to the contribution of two label signals 514, 516 and high emission signal is produced) under the second set of conditions 520. Of course, if both probe types do not provide signal, then under any set of conditions 518, 520, the reaction volume will be negative (-).

[0164] The total count of reaction volumes that are positive for the first probe 502 (and therefore estimated to be positive for the first target 506) is determined by counting the number of reaction volumes that are positive (+) or highly positive (++) under the two sets of conditions 518, 520. The total count of reaction volumes that are positive for the second probe 504 (and therefore estimated to be positive for the second target 508) is determined by counting (i) the number of reaction volumes that are positive (+) under the second set of conditions 520 but negative (-) under the first set of conditions 518, and adding it to (ii) the number of reaction volumes that are highly positive (++) under the second set of conditions 520. These reaction volume counts can be calculated or estimated by plotting the signal under the first set of conditions 518 at the PCR endpoint cycle with the signal under the second set of conditions 520 at the endpoint cycle and identifying clusters. For example, see Figure 8 The concentration of the first target 506 and the second target 508 in the sample can then be estimated using standard dPCR techniques.

[0165] Thus, a method for determining the presence and / or amount of multiple targets using the multiplexing technology described herein in a dPCR application may include: preparing a reaction mixture comprising a first probe type (e.g., a TaqMan probe) and a second probe type (e.g., an EF probe) designed to specifically interact with a first nucleic acid target and a second nucleic acid target, respectively; loading / partitioning the sample into a plurality of reaction volumes; measuring the signal of the reaction volume under a first set of reaction conditions (e.g., denaturation conditions, such as about 95° C.) during an endpoint cycle of PCR; measuring the signal of the reaction volume under a second set of reaction conditions (e.g., annealing / extension conditions, such as about 65° C.) during an endpoint cycle of PCR; classifying the reaction volumes according to the signal characteristics measured at the endpoint signal measurement; determining or estimating the count of each probe type based on the classified reaction volumes (i.e., the count of the reaction volume in which the first probe type is active and the count of the reaction volume in which the second probe type is active); and determining or estimating the presence and / or amount of the first nucleic acid target and the second nucleic acid target based on the counts.

[0166] End-point PCR implementation

[0167] The analysis techniques according to various embodiments may also be used when performing a traditional endpoint PCR process, where the sample is subjected to bulk PCR (or a larger reaction volume that is not intended to capture single or no DNA molecules using Poisson statistics), and as will be familiar to those of ordinary skill in the art. In such a PCR process, the measurement of the signals from the two different probe types may occur at the end cycle of the PCR and under different reaction conditions (such as, for example, denaturation conditions and annealing and / or extension conditions as described herein), similar to the methods described above for the dPCR process. However, the detected marker signal will follow Figure 2A The signal as outlined in the above, wherein the signal thus indicates the presence or absence of the corresponding first target and the second target. Figure 3B , the measured endpoint cycle signals under two different reaction conditions can therefore result in different signal levels schematically shown at cycle N+2 (analogously to the endpoint cycle). By comparing the different signal levels obtained, using Figure 2A The algorithms set forth in and further described above can determine the presence or absence of a first target nucleic acid and a second target nucleic acid using detectable labels having overlapping emission signal spectra and detectable in the same detection channel.

[0168] Thus, the methods for determining the presence or absence of multiple targets using the intra-channel multiplexing features described herein can also be performed using endpoint PCR (i.e., using a volume or larger reaction volume whose size is not intended to rely on Poisson statistics and capturing a single molecule of a target nucleic acid in the reaction volume, as is familiar to those of ordinary skill in the art). The methods employed in endpoint PCR applications include; preparing a reaction mixture comprising a first probe type (e.g., a TaqMan probe) and a second probe type (e.g., an EF probe), the first probe type and the second probe type being configured to specifically interact with a first nucleic acid target and a second nucleic acid target, respectively; subjecting the reaction mixture to an amplification reaction (e.g., PCR); measuring an endpoint cycle signal of the reaction mixture under a first set of reaction conditions (e.g., denaturation conditions, such as about 95°C); and measuring an endpoint signal of the reaction volume under a second set of reaction conditions (e.g., annealing / extension conditions, such as about 65°C) (it is worth noting that for measurements at the endpoint, the second set of The measurement under the reaction conditions (annealing / extension) will occur before the measurement under the first set of reaction conditions (denaturation); by measuring a first total emission (e.g., fluorescence) signal including any first emission (e.g., fluorescence) signal (if any) during the first set of reaction conditions, measuring a second total emission (e.g., fluorescence) signal including any first emission (e.g., fluorescence) signal (if any) during the second set of reaction conditions, and estimating the first emission (e.g., fluorescence) signal and / or the second emission (e.g., fluorescence) signal based on the first total emission (e.g., fluorescence) signal and the second total emission (e.g., fluorescence) signal.

[0169] Compound

[0170] In embodiments, the second probe or the non-cleavable probe is a compound having the formula:

[0171]

[0172] Where Q A is the quencher part,

[0173] B is a divalent nucleobase,

[0174] L 1 is a divalent linker,

[0175] R 2 is hydrogen or –OR 2A ,

[0176] R 3 Yes – OR 3A or –OP(NR 3B R 3C )-OR3A ,

[0177] R 4 is hydrogen or unsubstituted methyl, or R 2 and R 4 The substituents are linked to form a substituted or unsubstituted heterocycloalkyl (e.g., 3 to 8 members, 3 to 6 members, 4 to 6 members, 4 to 5 members, or 5 to 6 members),

[0178] R 5 Yes – OR 5A ,and

[0179] R 2A , R 3A , R 3B , R 3C and R 5A are independently hydrogen, -CCl3, -CBr3, -CF3, -CI3, -CHCl2, -CHBr2, -CHF2, -CHI2, -CH2Cl, -CH2Br, -CH2F, -CH2I, -CN, -OH, -NH2, -COOH, -CONH2, -OCCl3, -OCF3, -OCBr3, -OCI3, -OCHCl2, -OCHBr2, -OCHI2, -OCHF2, -OCH2Cl, -OCH2Br, -OCH2 I, -OCH2F, substituted or unsubstituted alkyl (e.g., C1-C8, C1-C6, C1-C4, or C1-C2), substituted or unsubstituted heteroalkyl (e.g., 2-8, 2-6, 4-6, 2-3, or 4-5 members), substituted or unsubstituted cycloalkyl (e.g., C3-C8, C3-C6, C4-C6, or C5-C6), substituted or unsubstituted heterocycloalkyl (e.g., 3-8, 3-6, 4-6, 4-5, or 5-6 members), substituted or unsubstituted aryl (e.g., C6-C 10 or phenyl), or a substituted or unsubstituted heteroaryl (e.g., 5- to 10-membered, 5- to 9-membered, or 5- to 6-membered).

[0180] In an embodiment, the quencher moiety is a monovalent form of QSY7.

[0181] In embodiments, the quencher moiety is Unit price form.

[0182] In embodiments, the quencher moiety is

[0183]

[0184] In embodiments, the quencher moiety is a monovalent form of QSY21.

[0185] In embodiments, the quencher moiety is Unit price form.

[0186] In embodiments, the quencher moiety is

[0187] In an embodiment, the quencher moiety is a monovalent form of QSY9.

[0188] In embodiments, the quencher moiety is Unit price form.

[0189] In embodiments, the quencher moiety is

[0190] In embodiments, the quencher moiety is a monovalent form of BHQ1.

[0191] In embodiments, the quencher moiety is Unit price form.

[0192] In embodiments, the quencher moiety is

[0193] In embodiments, the quencher moiety is a monovalent form of BHQ2.

[0194] In embodiments, the quencher moiety is Unit price form.

[0195] In embodiments, the quencher moiety is

[0196] In embodiments, the quencher moiety is a monovalent form of BHQ3.

[0197] In embodiments, the quencher moiety is

[0198] Unit price form.

[0199] In embodiments, the quencher moiety is

[0200]

[0201] In an embodiment, the quencher moiety is a monovalent form of Dabcyl.

[0202] In embodiments, the quencher moiety is

[0203] Unit price form.

[0204] In embodiments, the quencher moiety is

[0205]

[0206] In an embodiment, the quencher moiety is a monovalent form of Dabsyl.

[0207] In embodiments, the quencher moiety is

[0208]

[0209] In embodiments, the quencher moiety is a monovalent form of Eclipse.

[0210] In embodiments, the quencher moiety is

[0211]

[0212] In embodiments, the quencher moiety is

[0213]

[0214] In embodiments, the quencher moiety is a monovalent form of BBQ-650.

[0215] In embodiments, the quencher moiety is

[0216]

[0217] In embodiments, the quencher moiety is a monovalent form of Iowa Black RQ.

[0218] In embodiments, the quencher moiety is Iowa Black FQ in monovalent form.

[0219] In embodiments, the quencher moieties described above are all interchangeable. In embodiments, the quencher moieties can be in formula (I), (IA), (II), (III), (IV), (V), (VI), (VI-1), (VI-2), (VI-3), (VI-4), (VI-5), (VII), (VII-1), (VII-2), (VII-3), (VII-4), (VII-5), (VIII), (VIII-1), (VIII-2), (VIII-3), (VIII-4), (VIII-5), (IX), (IX-1), (IX-2), (IX-3), (IX-4), (IX-5), (X), (XI), (XII), (IX-2), (IX-3), (IX-4), (IX-5), (XII), (IX-1 ... XII), (XIII), (XIV), (XV), (XV-1), (XV-2), (XV-3), (XV-4), (XV-5), (XVI), (XVI-1), (XVI-2), (XVI-3), (XVI-4), (XVI-5), (XVII), (XVII-1), (XVII-2), (XVII-3), (XVII-4), (XVII-5), (XVIII), (XVIII-1), (XVIII-2), (XVIII-3), (XVIII-4), (XVIII-5), (XIX) and embodiments thereof are substituted.

[0220] In embodiments, the second probe or the non-cleavable probe is a compound having the formula:

[0221]

[0222] in

[0223] B is a divalent nucleobase,

[0224] L 1 is a divalent linker,

[0225] R 2 is hydrogen or –OR 2A ,

[0226] R 3 Yes – OR 3A or –OP(NR 3B R 3C )-OR 3A ,

[0227] R 4 is hydrogen or unsubstituted methyl, or R 2 and R 4 The substituents are linked to form a substituted or unsubstituted heterocycloalkyl (e.g., 3 to 8 members, 3 to 6 members, 4 to 6 members, 4 to 5 members, or 5 to 6 members),

[0228] R 5 Yes – OR 5A ,

[0229] R 1 and R 10 are independently hydrogen, -CCl3, -CBr3, -CF3, -CI3, -CHCl2, -CHBr2, -CHF2, -CHI2, -CH2Cl, -CH2Br, -CH2F, -CH2I, -CN, -OH, -NH2, -COOH, -CONH2, -OCCl3, -OCF3, -OCBr3, -OCI3, -OCHCl2, -OCHBr2, -OCHI2, -OCHF2, -OCH2Cl, -OCH2Br, -OCHI, -OCH2F, substituted or unsubstituted alkyl (e.g., C1-C8, C1-C6, C1-C4 or C1-C2), or substituted or unsubstituted heteroalkyl (e.g., 2-8, 2-6, 4-6, 2-3 or 4-5 members), and

[0230] R 6 , R 7 , R 8 and R 9 independently hydrogen, halogen, -CCl3, -CBr3, -CF3, -CI3, -CH2Cl, -CH2Br, -CH2F, -CH2I, -CHCl2, -CHBr2, -CHF2, -CHI2, -CN, -OH, -NH2, -COOH, -CONH2, -NO2, -SH, -SO3R A , -SO2NH2, -NHNH2, -ONH2, -NHC(O)NH2, -NHSO2H, -NHC(O)H, -NHC(O)OH, -NHOH, -OCCl3, -OCBr3, -OCF3, -OCI3, -OCH2Cl, -OCH2Br, -OCH2F, -OCH2I, -OCHCl2, -OCHBr2, -OCHF2, -OCHI2, -SF5, -N3, substituted or unsubstituted C1-C8, C1-C6, C1-C4 or C1-C2), substituted or unsubstituted heteroalkyl (e.g., 2-8, 2-6, 4-6, 2-3 or 4-5 members), substituted or unsubstituted cycloalkyl (e.g., C3-C8, C3-C6, C4-C6 or C5-C6), substituted or unsubstituted heterocycloalkyl (e.g., 3-8, 3-6, 4-6, 4-5 or 5-6 members), substituted or unsubstituted aryl (e.g., C6-C 10 or phenyl), or a substituted or unsubstituted heteroaryl (e.g., 5- to 10-membered, 5- to 9-membered, or 5- to 6-membered).

[0231] In an embodiment, R 1 and R 6 They may be linked to form a substituted or unsubstituted heterocycloalkyl group (e.g., 3-8, 3-6, 4-6, 4-5, or 5-6 membered) or a substituted or unsubstituted heteroaryl group (e.g., 5-10, 5-9, or 5-6 membered).

[0232] In an embodiment, R 8 and R 10 They may be linked to form a substituted or unsubstituted heterocycloalkyl group (e.g., 3-8, 3-6, 4-6, 4-5, or 5-6 membered) or a substituted or unsubstituted heteroaryl group (e.g., 5-10, 5-9, or 5-6 membered).

[0233] In an embodiment, R 2A , R 3A , R 3B , R 3C and R 5A and R A can be independently hydrogen, -CCl 3 , -CBr3, -CF3, -CI3, -CHCl2, -CHBr2, -CHF2, -CHI2, -CH2Cl, -CH2Br, -CH2F, -CH2I, -CN, -OH, -NH2, -COOH, - CONH2, -OCCl3, -OCF3, -OCBr3, -OCI3, -OCHCl2, -OCHBr2, -OCHI2, -OCHF2, -OCH2Cl, -OCH2Br, -OCH2I, -OCH 2F, substituted or unsubstituted alkyl (e.g., C1-C8, C1-C6, C1-C4, or C1-C2), substituted or unsubstituted heteroalkyl (e.g., 2 to 8 members, 2 to 6 members, 4 to 6 members, 2 to 3 members, or 4 to 5 members), substituted or unsubstituted cycloalkyl (e.g., C3-C8, C3-C6, C4-C6, or C5-C6), substituted or unsubstituted heterocycloalkyl (e.g., 3 to 8 members, 3 to 6 members, 4 to 6 members, 4 to 5 members, or 5 to 6 members), substituted or unsubstituted aryl (e.g., C6-C 10 or phenyl), or a substituted or unsubstituted heteroaryl (e.g., 5- to 10-membered, 5- to 9-membered, or 5- to 6-membered).

[0234] One of ordinary skill in the art will appreciate that the compound may exist as a neutral species with a counter ion.

[0235] In an embodiment, the compound has the formula:

[0236]

[0237] B.L 1 , R2 , R 3 and R 5 As described herein, included in the embodiments. - is a counterion. In an embodiment, A - It is Cl - In an embodiment, A - It's F3CC(O)O - In an embodiment, A - It is the acetate or bromide.

[0238] In an embodiment, the compound has the formula:

[0239]

[0240] B.L 1 , R 2 , R 3 and R 5 As described herein, included in the embodiments.

[0241] In an embodiment, the compound has the formula:

[0242]

[0243] B.L 1 , R 2 , R 3 and R 5 As described herein, included in the embodiments.

[0244] In an embodiment, the compound has the formula:

[0245]

[0246] B.L 1 , R 3 and R 5 As described herein, included in the embodiments.

[0247] In an embodiment, the compound has the formula:

[0248]

[0249] B.L 1 , R 3 and R 5 As described herein, included in the embodiments.

[0250] In an embodiment, B is a divalent cytosine or a derivative thereof, a divalent guanine or a derivative thereof, a divalent adenine or a derivative thereof, a divalent thymine or a derivative thereof, a divalent uracil or a derivative thereof, a divalent hypoxanthine or a derivative thereof, a divalent xanthine or a derivative thereof, a divalent 7-methylguanine or a derivative thereof, a divalent 5,6-dihydrouracil or a derivative thereof, a divalent 5-methylcytosine or a derivative thereof, or a divalent 5-hydroxymethylcytosine or a derivative thereof. In an embodiment, B is a divalent cytosine or a derivative thereof, a divalent guanine or a derivative thereof, a divalent adenine or a derivative thereof, a divalent thymine or a derivative thereof, or a divalent uracil or a derivative thereof. In an embodiment, B is a divalent cytosine or a derivative thereof. In an embodiment, B is a divalent guanine or a derivative thereof. In an embodiment, B is a divalent adenine or a derivative thereof. In an embodiment, B is a divalent thymine or a derivative thereof. In an embodiment, B is a divalent uracil or a derivative thereof.

[0251] In an embodiment, the compound has the formula:

[0252]

[0253] L 1 , R 2 , R 3 and R 5 As described herein, included in the embodiments.

[0254] In an embodiment, the compound has the formula:

[0255]

[0256] L 1 , R 2 , R 3 and R 5 As described herein, included in the embodiments.

[0257] In an embodiment, the compound has the formula:

[0258]

[0259] L 1 , R 2 , R 3 and R 5 As described herein, included in the embodiments.

[0260] In an embodiment, the compound has the formula:

[0261]

[0262] L 1 , R 2 , R3 and R 5 As described herein, included in the embodiments.

[0263] In an embodiment, the compound has the formula:

[0264]

[0265] L 1 , R 2 , R 3 and R 5 As described herein, included in the embodiments.

[0266] In an embodiment, the compound has the formula:

[0267]

[0268] L 1 , R 2 , R 3 and R 5 As described herein, included in the embodiments.

[0269] In an embodiment, the compound has the formula:

[0270]

[0271] L 1 , R 2 , R3 and R 5 As described herein, included in the embodiments.

[0272] In an embodiment, the compound has the formula:

[0273] L 1 , R 2 , R 3 and R 5 As described herein, included in the embodiments.

[0274] In an embodiment, the compound has the formula:

[0275] L 1 , R 2 , R 3 and R 5 As described herein, included in the embodiments.

[0276] In an embodiment, the compound has the formula:

[0277] L 1 , R 2 , R3 and R 5 As described herein, included in the embodiments.

[0278] In an embodiment, the compound has the formula:

[0279] L 1 , R 2 , R 3 and R 5 As described herein, included in the embodiments.

[0280] In an embodiment, the compound has the formula:

[0281] L 1 , R 2 , R 3 and R 5 As described herein, included in the embodiments.

[0282] In an embodiment, the compound has the formula:

[0283] L 1 , R 3 and R 5 As described herein, included in the embodiments.

[0284] In an embodiment, the compound has the formula:

[0285] L 1 , R 3 and R 5 As described herein, included in the embodiments.

[0286] In an embodiment, the compound has the formula:

[0287] L 1 , R 3 and R 5 As described herein, included in the embodiments.

[0288] In an embodiment, the compound has the formula:

[0289] L 1 , R 3 and R 5 As described herein, included in the embodiments.

[0290] In an embodiment, the compound has the formula:

[0291] L1 , R 3 and R 5 As described herein, included in the embodiments.

[0292] In an embodiment, the compound has the formula:

[0293] L 1 , R 3 and R 5 As described herein, included in the embodiments.

[0294] In an embodiment, the compound has the formula:

[0295] L 1 , R 3 and R 5 As described herein, included in the embodiments.

[0296] In an embodiment, the compound has the formula:

[0297] L 1 , R 3 and R 5 As described herein, included in the embodiments.

[0298] In an embodiment, the compound has the formula:

[0299] L 1 , R 3 and R 5 As described herein, included in the embodiments.

[0300] In an embodiment, the compound has the formula:

[0301] L 1 , R 3 and R 5 As described herein, included in the embodiments.

[0302] In an embodiment, the compound has the formula:

[0303] L 1 , R 3 and R 5 As described herein, included in the embodiments.

[0304] In an embodiment, the compound has the formula:

[0305] L 1 , R3 and R 5 As described herein, included in the embodiments.

[0306] In an embodiment, L 1 It is a divalent linker comprising 4 to 30 atoms.

[0307] In an embodiment, L 1 YesL 101 -L 102 -L 103 -L 104 -L 105 .

[0308] L 101 , L 102 , L 103 , L 104 and L 105 The alkylene groups are independently a bond, -NH-, -O-, -S-, -S(O)-, -S(O)2-, -C(O)-, -C(O)NH-, -NHC(O)-, -NHC(O)NH-, -C(O)O-, -OC(O)-, substituted or unsubstituted alkylene (e.g., C1-C8, C1-C6, C1-C4, or C1-C2), substituted or unsubstituted heteroalkylene (e.g., 2 to 8 members, 2 to 6 members, 4 to 6 members, 2 to 3 members, or 4 to 5 members), substituted or unsubstituted cycloalkylene (e.g., C3-C8, C3-C6, C4-C6, or C5-C6), substituted or unsubstituted heterocycloalkylene (e.g., 3 to 8 members, 3 to 6 members, 4 to 6 members, 4 to 5 members, or 5 to 6 members), substituted or unsubstituted arylene (e.g., C6-C 10 or phenylene), or a substituted or unsubstituted heteroarylene (e.g., 5- to 10-membered, 5- to 9-membered, or 5- to 6-membered).

[0309] In an embodiment, substituted L 101 (e.g., substituted alkylene, substituted heteroalkylene, substituted cycloalkylene, substituted heterocycloalkylene, substituted arylene and / or substituted heteroarylene) is substituted with at least one substituent, size-restricted substituent or lower substituent; wherein if the substituted L 101 is substituted with a plurality of groups selected from substituents, size-restricted substituents and lower substituents; each substituent, size-restricted substituent and / or lower substituent may be optionally different. In an embodiment, when L 101 When substituted, it is substituted with at least one substituent. In an embodiment, when L 101 When substituted, it is substituted with at least one size-restricted substituent. In an embodiment, when L 101 When substituted, it is substituted with at least one lower substituent.

[0310] In an embodiment, L 101 is a bond. In an embodiment, L 101 is -NH-. In an embodiment, L 101 is -O-. In an embodiment, L 101 is -S-. In an embodiment, L 101 is -S(O)-. In an embodiment, L 101 is -S(O)2-. In an embodiment, L 101 is -C(O)-. In an embodiment, L 101 is -C(O)NH-. In an embodiment, L 101 is -NHC(O)-. In an embodiment, L 101 is -NHC(O)NH-. In an embodiment, L 101 is -C(O)O-. In an embodiment, L 101 is -OC(O)-. In an embodiment, L 101 is a substituted or unsubstituted C1-C4 alkylene group. 101 is a substituted or unsubstituted 2- to 6-membered heteroalkylene group.

[0311] In an embodiment, substituted L 102 (e.g., substituted alkylene, substituted heteroalkylene, substituted cycloalkylene, substituted heterocycloalkylene, substituted arylene and / or substituted heteroarylene) is substituted with at least one substituent, size-restricted substituent or lower substituent; wherein if the substituted L 102 is substituted with a plurality of groups selected from substituents, size-restricted substituents and lower substituents; each substituent, size-restricted substituent and / or lower substituent may be optionally different. In an embodiment, when L 102 When substituted, it is substituted with at least one substituent. In an embodiment, when L 102 When substituted, it is substituted with at least one size-restricted substituent. In an embodiment, when L 102 When substituted, it is substituted with at least one lower substituent.

[0312] In an embodiment, L102 is a bond. In an embodiment, L 102 is -NH-. In an embodiment, L102 is -O-. In an embodiment, L 102 is -S-. In an embodiment, L 102 is -S(O)-. In an embodiment, L 102 is -S(O)2-. In an embodiment, L 102 is -C(O)-. In an embodiment, L 102is -C(O)NH-. In an embodiment, L 102 is -NHC(O)-. In an embodiment, L 102 is -NHC(O)NH-. In an embodiment, L 102 is -C(O)O-. In an embodiment, L 102 is -OC(O)-. In an embodiment, L 102 is a substituted or unsubstituted C1-C4 alkylene group. 102 is a substituted or unsubstituted 2 to 6 membered heteroalkylene. 102 is an unsubstituted 3 to 8 membered heterocycloalkyl. 102 is unsubstituted piperidinyl. In an embodiment, L 102 yes

[0313] In an embodiment, substituted L 103 (e.g., substituted alkylene, substituted heteroalkylene, substituted cycloalkylene, substituted heterocycloalkylene, substituted arylene and / or substituted heteroarylene) is substituted with at least one substituent, size-restricted substituent or lower substituent; wherein if the substituted L 103 is substituted with a plurality of groups selected from substituents, size-restricted substituents and lower substituents; each substituent, size-restricted substituent and / or lower substituent may be optionally different. In an embodiment, when L 103 When substituted, it is substituted with at least one substituent. In an embodiment, when L 103 When substituted, it is substituted with at least one size-restricted substituent. In an embodiment, when L 103 When substituted, it is substituted with at least one lower substituent.

[0314] In an embodiment, L 103 is a bond. In an embodiment, L 103 is -NH-. In an embodiment, L103 is -O-. In an embodiment, L 103 is -S-. In an embodiment, L 103 is -S(O)-. In an embodiment, L 103 is -S(O)2-. In an embodiment, L 103 is -C(O)-. In an embodiment, L 103 is -C(O)NH-. In an embodiment, L 103 is -NHC(O)-. In an embodiment, L 103 is -NHC(O)NH-. In an embodiment, L 103 is -C(O)O-. In an embodiment, L 103is -OC(O)-. In an embodiment, L 103 is a substituted or unsubstituted C1-C4 alkylene group. 103 is a substituted or unsubstituted 2- to 6-membered heteroalkylene group.

[0315] In an embodiment, substituted L 104 (e.g., substituted alkylene, substituted heteroalkylene, substituted cycloalkylene, substituted heterocycloalkylene, substituted arylene and / or substituted heteroarylene) is substituted with at least one substituent, size-restricted substituent or lower substituent; wherein if the substituted L 104 is substituted with a plurality of groups selected from substituents, size-restricted substituents and lower substituents; each substituent, size-restricted substituent and / or lower substituent may be optionally different. In an embodiment, when L 104 When substituted, it is substituted with at least one substituent. In an embodiment, when L 104 When substituted, it is substituted with at least one size-restricted substituent. In an embodiment, when L 104 When substituted, it is substituted with at least one lower substituent.

[0316] In an embodiment, L 104 is a bond. In an embodiment, L 104 is -NH-. In an embodiment, L 104 is -O-. In an embodiment, L 104 is -S-. In an embodiment, L 104 is -S(O)-. In an embodiment, L 104 is -S(O)2-. In an embodiment, L 104 is -C(O)-. In an embodiment, L 104 is -C(O)NH-. In an embodiment, L 104 is -NHC(O)-. In an embodiment, L 104 is -NHC(O)NH-. In an embodiment, L 104 is -C(O)O-. In an embodiment, L 104 is -OC(O)-. In an embodiment, L 104 is an unsubstituted C1-C 10 In an embodiment, L 104 is an unsubstituted methylene. In an embodiment, L 104 is unsubstituted ethylene. In an embodiment, L 104 is unsubstituted propylene. In an embodiment, L 104 is unsubstituted n-propylene. In an embodiment, L 104 is unsubstituted butylene. In an embodiment, L104 is unsubstituted n-butene. In an embodiment, L 104 is unsubstituted pentylene. In an embodiment, L 104 is unsubstituted n-pentyl. In an embodiment, L 104 is an unsubstituted hexamethylene group. In an embodiment, L 104 is an unsubstituted n-hexyl group. In an embodiment, L 104 is unsubstituted heptenyl. In an embodiment, L 104 is unsubstituted n-heptenyl. In an embodiment, L 104 is unsubstituted octenyl. In an embodiment, L 104 is unsubstituted n-octenyl. In an embodiment, L 104 is an unsubstituted C2-C6 alkynylene. In an embodiment, L 104 is an unsubstituted ethynyl. In an embodiment, L 104 is unsubstituted propynyl. In an embodiment, L 104 is an unsubstituted butynyl group. In an embodiment, L 104 is an unsubstituted pentynyl. In an embodiment, L 104 is unsubstituted hexynyl. In an embodiment, L 104 yes In an embodiment, L 104 is a substituted or unsubstituted 2 to 6 membered heteroalkylene. 104 is an unsubstituted 2 to 6 membered heteroalkylene. 104 yes In an embodiment, L 104 yes wherein n104 is an integer from 1 to 10. In an embodiment, n104 is 1. In an embodiment, n104 is 2. In an embodiment, n104 is 3. In an embodiment, n104 is 4. In an embodiment, n104 is 5. In an embodiment, n104 is 6. In an embodiment, n104 is 7. In an embodiment, n104 is 8. In an embodiment, n104 is 9. In an embodiment, n104 is 10. In an embodiment, L 104 is a substituted or unsubstituted phenylene. In an embodiment, L 104 is unsubstituted phenylene. In an embodiment, L 104 yes

[0317] In an embodiment, substituted L 105(e.g., substituted alkylene, substituted heteroalkylene, substituted cycloalkylene, substituted heterocycloalkylene, substituted arylene and / or substituted heteroarylene) is substituted with at least one substituent, size-restricted substituent or lower substituent; wherein if the substituted L 105 is substituted with a plurality of groups selected from substituents, size-restricted substituents and lower substituents; each substituent, size-restricted substituent and / or lower substituent may be optionally different. In an embodiment, when L 105 When substituted, it is substituted with at least one substituent. In an embodiment, when L 105 When substituted, it is substituted with at least one size-restricted substituent. In an embodiment, when L 105 When substituted, it is substituted with at least one lower substituent.

[0318] In an embodiment, L 105 is a bond. In an embodiment, L 105 is -NH-. In an embodiment, L 105 is -O-. In an embodiment, L 105 is -S-. In an embodiment, L 105 is -S(O)-. In an embodiment, L 105 is -S(O)2-. In an embodiment, L 105 is -C(O)-. In an embodiment, L 105 is -C(O)NH-. In an embodiment, L 105 is -NHC(O)-. In an embodiment, L 105 is -NHC(O)NH-. In an embodiment, L 105 is -C(O)O-. In an embodiment, L 105 is -OC(O)-. In an embodiment, L 105 is an unsubstituted C1-C 10 In an embodiment, L105 is a substituted or unsubstituted C1-C4 alkylene. In an embodiment, L 105 is an unsubstituted methylene. In an embodiment, L 105 is unsubstituted ethylene. In an embodiment, L 105 is unsubstituted propylene. In an embodiment, L 105 is unsubstituted n-propylene. In an embodiment, L 105 is unsubstituted butylene. In an embodiment, L 105 is unsubstituted n-butene. In an embodiment, L 105 is unsubstituted pentylene. In an embodiment, L 105 is unsubstituted n-pentyl. In an embodiment, L 105is an unsubstituted hexamethylene group. In an embodiment, L 105 is an unsubstituted n-hexyl group. In an embodiment, L 105 is unsubstituted heptenyl. In an embodiment, L 105 is unsubstituted n-heptenyl. In an embodiment, L 105 is unsubstituted octenyl. In an embodiment, L 105 is unsubstituted n-octenyl. In an embodiment, L 105 is an unsubstituted C2-C6 alkynylene. In an embodiment, L 105 is an unsubstituted ethynyl. In an embodiment, L 105 is unsubstituted propynyl. In an embodiment, L 105 is an unsubstituted butynyl group. In an embodiment, L 105 is an unsubstituted pentynyl. In an embodiment, L 105 is unsubstituted hexynyl. In an embodiment, L 105 yes In an embodiment, L 105 is a substituted 2 to 8 membered heteroalkylene. 105 is an oxo-substituted 2 to 8 membered heteroalkylene. 105 is an oxo-substituted 2- to 8-membered heteroalkenylene. 105 yes In an embodiment, L 105 yes In an embodiment, L 105 yes In an embodiment, L 105 is a substituted or unsubstituted 2 to 8 membered heteroalkynyl. 105 yes In an embodiment, L 105 yes wherein n105 is an integer from 1 to 10. In an embodiment, n105 is 1. In an embodiment, n105 is 2. In an embodiment, n105 is 3. In an embodiment, n105 is 4. In an embodiment, n105 is 5. In an embodiment, n105 is 6. In an embodiment, n105 is 7. In an embodiment, n105 is 8. In an embodiment, n105 is 9. In an embodiment, n105 is 10. In an embodiment, L 105 is an unsubstituted 5- to 10-membered heteroarylene. In an embodiment, L 105 is an unsubstituted triazolidine. In an embodiment, L 105 yes

[0319] In an embodiment, L 1yes In an embodiment, L 1 yes

[0320] In an embodiment, L 1 yes In an embodiment, L 1 yes In an embodiment, L 1 yes In an embodiment, L 1 yes In an embodiment, L 1 yes In an embodiment, L 1 yes In an embodiment, L 1 yes In an embodiment, L 1 yes

[0321] In an embodiment, substituted R1 (e.g., substituted alkyl and / or substituted heteroalkyl) is substituted with at least one substituent, a substituent of limited size, or a lower substituent; wherein if substituted R1 1 is substituted with a plurality of groups selected from substituents, size-restricted substituents and lower substituents; each substituent, size-restricted substituent and / or lower substituent may be optionally different. In an embodiment, when R 1 When substituted, it is substituted with at least one substituent. In an embodiment, when R 1 When substituted, it is substituted with at least one substituent of limited size. In an embodiment, when R 1 When substituted, it is substituted with at least one lower substituent.

[0322] In an embodiment, R 1 is hydrogen. In an embodiment, R 1 is an unsubstituted C1-C4 alkyl. In an embodiment, R 1 is unsubstituted methyl. In an embodiment, R 1 is an unsubstituted ethyl group. In an embodiment, R 1 is unsubstituted propyl. In an embodiment, R 1 is unsubstituted n-propyl. In an embodiment, R 1 is unsubstituted isopropyl. In an embodiment, R 1 is unsubstituted butyl. In an embodiment, R 1 is unsubstituted n-butyl. In an embodiment, R 1 is unsubstituted isobutyl. In an embodiment, R 1 is an unsubstituted tert-butyl group.

[0323] In an embodiment, R 2 is hydrogen or -OH. In an embodiment, R 2 is hydrogen. In an embodiment, R 2 Yes – OR 2A In an embodiment, R 2 It is -OH.

[0324] In an embodiment, substituted R 2A (e.g., substituted alkyl, substituted heteroalkyl, substituted cycloalkyl, substituted heterocycloalkyl, substituted aryl and / or substituted heteroaryl) is substituted with at least one substituent, size-restricted substituent or lower substituent; wherein if the substituted R 2A is substituted with a plurality of groups selected from substituents, size-restricted substituents and lower substituents; each substituent, size-restricted substituent and / or lower substituent may be optionally different. In an embodiment, when R 2A When substituted, it is substituted with at least one substituent. In an embodiment, when R 2A When substituted, it is substituted with at least one substituent of limited size. In an embodiment, when R 2A When substituted, it is substituted with at least one lower substituent.

[0325] In an embodiment, R 2A is hydrogen. In an embodiment, R 2A is an unsubstituted C1-C4 alkyl. In an embodiment, R 2A is unsubstituted methyl. In an embodiment, R 2A is an unsubstituted ethyl group. In an embodiment, R 2A is unsubstituted propyl. In an embodiment, R 2A is unsubstituted n-propyl. In an embodiment, R 2A is unsubstituted isopropyl. In an embodiment, R 2A is unsubstituted butyl. In an embodiment, R 2A is unsubstituted n-butyl. In an embodiment, R 2A is unsubstituted isobutyl. In an embodiment, R 2A is an unsubstituted tert-butyl group.

[0326] In an embodiment, R 3 Yes – OR 3A In an embodiment, R 3 is –OH. In an embodiment, R 3 Yes – OP (NR 3B R 3C )-OR 3A In an embodiment, R 3yes

[0327] In an embodiment, substituted R 3A (e.g., substituted alkyl, substituted heteroalkyl, substituted cycloalkyl, substituted heterocycloalkyl, substituted aryl and / or substituted heteroaryl) is substituted with at least one substituent, size-restricted substituent or lower substituent; wherein if the substituted R 3A is substituted with a plurality of groups selected from substituents, size-restricted substituents and lower substituents; each substituent, size-restricted substituent and / or lower substituent may be optionally different. In an embodiment, when R 3A When substituted, it is substituted with at least one substituent. In an embodiment, when R 3A When substituted, it is substituted with at least one substituent of limited size. In an embodiment, when R 3A When substituted, it is substituted with at least one lower substituent.

[0328] In an embodiment, R 3A is hydrogen. In an embodiment, R 3A is an unsubstituted C1-C4 alkyl. In an embodiment, R 3A is unsubstituted methyl. In an embodiment, R 3A is an unsubstituted ethyl group. In an embodiment, R 3A is unsubstituted propyl. In an embodiment, R 3A is unsubstituted n-propyl. In an embodiment, R 3A is unsubstituted isopropyl. In an embodiment, R 3A is unsubstituted butyl. In an embodiment, R 3A is unsubstituted n-butyl. In an embodiment, R 3A is unsubstituted isobutyl. In an embodiment, R 3A is unsubstituted tert-butyl. In an embodiment, R 3A is a substituted C1-C4 alkyl. 3A is a cyano-substituted C1-C4 alkyl group. 3A yes

[0329] In an embodiment, substituted R 3B (e.g., substituted alkyl, substituted heteroalkyl, substituted cycloalkyl, substituted heterocycloalkyl, substituted aryl and / or substituted heteroaryl) is substituted with at least one substituent, size-restricted substituent or lower substituent; wherein if the substituted R 3Bis substituted with a plurality of groups selected from substituents, size-restricted substituents and lower substituents; each substituent, size-restricted substituent and / or lower substituent may be optionally different. In an embodiment, when R 3B When substituted, it is substituted with at least one substituent. In an embodiment, when R 3B When substituted, it is substituted with at least one substituent of limited size. In an embodiment, when R 3B When substituted, it is substituted with at least one lower substituent.

[0330] In an embodiment, R 3B is hydrogen. In an embodiment, R 3B is an unsubstituted C1-C4 alkyl. In an embodiment, R 3B is unsubstituted methyl. In an embodiment, R 3B is an unsubstituted ethyl group. In an embodiment, R 3B is unsubstituted propyl. In an embodiment, R 3B is unsubstituted n-propyl. In an embodiment, R 3B is unsubstituted isopropyl. In an embodiment, R 3B is unsubstituted butyl. In an embodiment, R 3B is unsubstituted n-butyl. In an embodiment, R 3B is unsubstituted isobutyl. In an embodiment, R 3B is an unsubstituted tert-butyl group.

[0331] In an embodiment, substituted R 3C (e.g., substituted alkyl, substituted heteroalkyl, substituted cycloalkyl, substituted heterocycloalkyl, substituted aryl and / or substituted heteroaryl) is substituted with at least one substituent, size-restricted substituent or lower substituent; wherein if the substituted R 3C is substituted with a plurality of groups selected from substituents, size-restricted substituents and lower substituents; each substituent, size-restricted substituent and / or lower substituent may be optionally different. In an embodiment, when R 3C When substituted, it is substituted with at least one substituent. In an embodiment, when R 3C When substituted, it is substituted with at least one substituent of limited size. In an embodiment, when R 3C When substituted, it is substituted with at least one lower substituent.

[0332] In an embodiment, R 3C is hydrogen. In an embodiment, R 3C is an unsubstituted C1-C4 alkyl. In an embodiment, R 3C is unsubstituted methyl. In an embodiment, R 3Cis an unsubstituted ethyl group. In an embodiment, R 3C is unsubstituted propyl. In an embodiment, R 3C is unsubstituted n-propyl. In an embodiment, R 3C is unsubstituted isopropyl. In an embodiment, R 3C is unsubstituted butyl. In an embodiment, R 3C is unsubstituted n-butyl. In an embodiment, R 3C is unsubstituted isobutyl. In an embodiment, R 3C is an unsubstituted tert-butyl group.

[0333] In an embodiment, R 4 is hydrogen. In an embodiment, R 4 is an unsubstituted methyl group.

[0334] In an embodiment, when R 2 and R 4 The substituted ring formed when the substituents are connected (e.g., a substituted heterocycloalkyl) is substituted with at least one substituent, a size-restricted substituent, or a lower substituent; wherein when R 2 and R 4 The substituted ring formed when the substituents are connected is substituted with a plurality of groups selected from substituents, size-restricted substituents, and lower substituents; each substituent, size-restricted substituent, and / or lower substituent may be optionally different. In an embodiment, when R 2 and R 4 When the substituted ring formed when the substituents are attached is substituted, it is substituted with at least one substituent. 2 and R 4 When the substituted ring formed when the substituents are attached is substituted, it is substituted with at least one substituent of limited size. 2 and R 4 When a substituted ring formed when substituents are linked is substituted, it is substituted with at least one lower substituent.

[0335] In an embodiment, R 2 and R 4 The substituents are linked to form a substituted or unsubstituted 3 to 8 membered heterocycloalkyl. 2 and R 4 The substituents are linked to form a substituted or unsubstituted tetrahydrofuranyl group.

[0336] In an embodiment, R 5 It is –OH.

[0337] In an embodiment, substituted R 5A(e.g., substituted alkyl, substituted heteroalkyl, substituted cycloalkyl, substituted heterocycloalkyl, substituted aryl and / or substituted heteroaryl) is substituted with at least one substituent, size-restricted substituent or lower substituent; wherein if the substituted R 5A is substituted with a plurality of groups selected from substituents, size-restricted substituents and lower substituents; each substituent, size-restricted substituent and / or lower substituent may be optionally different. In an embodiment, when R 5A When substituted, it is substituted with at least one substituent. In an embodiment, when R 5A When substituted, it is substituted with at least one substituent of limited size. In an embodiment, when R 5A When substituted, it is substituted with at least one lower substituent.

[0338] In an embodiment, R 5A is hydrogen or substituted C1-C6 alkyl. 5A is hydrogen. In an embodiment, R 5A is an unsubstituted C1-C4 alkyl. In an embodiment, R 5A is unsubstituted methyl. In an embodiment, R 5A is an unsubstituted ethyl group. In an embodiment, R 5A is unsubstituted propyl. In an embodiment, R 5A is unsubstituted n-propyl. In an embodiment, R 5A is unsubstituted isopropyl. In an embodiment, R 5A is unsubstituted butyl. In an embodiment, R 5A is unsubstituted n-butyl. In an embodiment, R 5A is unsubstituted isobutyl. In an embodiment, R 5A is unsubstituted tert-butyl. In an embodiment, R 5A is a substituted C1-C6 alkyl. 5A is dimethoxytrityl. In an embodiment, R 5A yes

[0339] In an embodiment, substituted R 6 (e.g., substituted alkyl, substituted heteroalkyl, substituted cycloalkyl, substituted heterocycloalkyl, substituted aryl and / or substituted heteroaryl) is substituted with at least one substituent, size-restricted substituent or lower substituent; wherein if the substituted R 6 is substituted with a plurality of groups selected from substituents, size-restricted substituents and lower substituents; each substituent, size-restricted substituent and / or lower substituent may be optionally different. In an embodiment, when R 6When substituted, it is substituted with at least one substituent. In an embodiment, when R 6 When substituted, it is substituted with at least one substituent of limited size. In an embodiment, when R 6 When substituted, it is substituted with at least one lower substituent. In an embodiment, R 6 is hydrogen. In an embodiment, R 6 is halogen. In an embodiment, R 6 is –F. In the embodiment, R 6 is –Cl. In an embodiment, R 6 is -Br. In an embodiment, R6 is -I. In an embodiment, R 6 is -CCl3. In an embodiment, R 6 is -CBr3. In an embodiment, R 6 is -CF3. In an embodiment, R 6 is -CI3. In an embodiment, R 6 is -CH2Cl. In an embodiment, R 6 is -CH2Br. In an embodiment, R 6 is -CH2F. In an embodiment, R 6 is -CH2I. In an embodiment, R 6 is -CHCl2. In an embodiment, R 6 is -CHBr2. In an embodiment, R 6 is -CHF2. In an embodiment, R 6 is -CH2. In an embodiment, R 6 is -CN. In an embodiment, R 6 is –OH. In an embodiment, R 6 is -NH2. In an embodiment, R 6 is -COOH. In an embodiment, R 6 is -CONH2. In an embodiment, R 6 is -NO2. In an embodiment, R 6 is -SH. In an embodiment, R 6 Yes-SO3R A In an embodiment, R 6 is -SO3H. In an embodiment, R 6 is -SO2NH2. In an embodiment, R 6 is -NHNH2. In an embodiment, R 6 is -ONH2. In an embodiment, R 6 is -NHC(O)NH2. In an embodiment, R 6 is -NHSO2H. In an embodiment, R6 is -NHC(O)H. In an embodiment, R 6 is -NHC(O)OH. In an embodiment, R 6 is NHOH. In an embodiment, R 6 is -OCCl3. In an embodiment, R 6 is -OCBr3. In an embodiment, R 6 is -OCF3. In an embodiment, R 6 is -OCI3. In an embodiment, R 6 is -OCH2Cl. In an embodiment, R 6 is -OCH2Br. In an embodiment, R 6 is -OCH2F. In an embodiment, R 6 is -OCH2I. In an embodiment, R 6 is -OCHCl2. In an embodiment, R 6 is -OCHBr2. In an embodiment, R 6 is -OCHF2. In an embodiment, R 6 is -OCHI2. In an embodiment, R 6 is -SF5. In an embodiment, R 6 is N3. In an embodiment, R 6 is an unsubstituted C1-C4 alkyl. In an embodiment, R 6 is unsubstituted methyl. In an embodiment, R 6 is an unsubstituted ethyl group. In an embodiment, R 6 is unsubstituted propyl. In an embodiment, R 6 is unsubstituted n-propyl. In an embodiment, R 6 is unsubstituted isopropyl. In an embodiment, R 6 is unsubstituted butyl. In an embodiment, R 6 is unsubstituted n-butyl. In an embodiment, R 6 is unsubstituted isobutyl. In an embodiment, R 6 is unsubstituted tert-butyl. In an embodiment, R 6 is an unsubstituted 2 to 6 membered heteroalkyl. 6 is unsubstituted methoxy. In an embodiment, R 6 is an unsubstituted ethoxy group. In an embodiment, R 6 is unsubstituted propoxy. In an embodiment, R 6 is unsubstituted n-propoxy. In an embodiment, R 6 is unsubstituted isopropoxy. In an embodiment, R 6 It is an unsubstituted butoxy group.

[0340] In an embodiment, substituted R 7 (e.g., substituted alkyl, substituted heteroalkyl, substituted cycloalkyl, substituted heterocycloalkyl, substituted aryl and / or substituted heteroaryl) is substituted with at least one substituent, size-restricted substituent or lower substituent; wherein if the substituted R 7 is substituted with a plurality of groups selected from substituents, size-restricted substituents and lower substituents; each substituent, size-restricted substituent and / or lower substituent may be optionally different. In an embodiment, when R 7 When substituted, it is substituted with at least one substituent. In an embodiment, when R 7 When substituted, it is substituted with at least one substituent of limited size. In an embodiment, when R 7 When substituted, it is substituted with at least one lower substituent.

[0341] In an embodiment, R 7 is hydrogen. In an embodiment, R 7 is halogen. In an embodiment, R 7 is –f. In the embodiment, R 7 is –Cl. In an embodiment, R 7 is -Br. In an embodiment, R 7 is -I. In an embodiment, R 7 is -CCl3. In an embodiment, R 7 is -CBr3. In an embodiment, R 7 is -CF3. In an embodiment, R 7 is -CI3. In an embodiment, R 7 is -CH2Cl. In an embodiment, R 7 is -CH2Br. In an embodiment, R 7 is -CH2F. In an embodiment, R 7 is -CH2I. In an embodiment, R 7 is -CHCl2. In an embodiment, R 7 is -CHBr2. In an embodiment, R 7 is -CHF2. In an embodiment, R 7 is -CHI2. In an embodiment, R 7 is -CN. In an embodiment, R 7 is –OH. In an embodiment, R 7 is -NH2. In an embodiment, R 7 is -COOH. In an embodiment, R 7 is -CONH2. In an embodiment, R 7is -NO2. In an embodiment, R 7 is -SH. In an embodiment, R 7 Yes-SO3R A In an embodiment, R 7 is -SO3H. In an embodiment, R 7 is -SO2NH2. In an embodiment, R 7 is -NHNH2. In an embodiment, R 7 is -ONH2. In an embodiment, R 7 is -NHC(O)NH2. In an embodiment, R 7 is -NHSO2H. In an embodiment, R 7 is -NHC(O)H. In an embodiment, R 7 is -NHC(O)OH. In an embodiment, R 7 is -NHOH. In an embodiment, R 7 is -OCCl3. In an embodiment, R 7 is -OCBr3. In an embodiment, R 7 is -OCF3. In an embodiment, R 7 is -OCI3. In an embodiment, R 7 is -OCH2Cl. In an embodiment, R 7 is -OCH2Br. In an embodiment, R 7 is -OCH2F. In an embodiment, R 7 is -OCH2I. In an embodiment, R 7 is -OCHCl2. In an embodiment, R 7 is -OCHBr2. In an embodiment, R 7 is -OCHF2. In an embodiment, R 7 is -OCHI2. In an embodiment, R 7 is -SF5. In an embodiment, R 7 is -N3. In an embodiment, R 7 is an unsubstituted C1-C4 alkyl. In an embodiment, R 7 is unsubstituted methyl. In an embodiment, R 7 is an unsubstituted ethyl group. In an embodiment, R 7 is unsubstituted propyl. In an embodiment, R 7 is unsubstituted n-propyl. In an embodiment, R 7 is unsubstituted isopropyl. In an embodiment, R 7 is unsubstituted butyl. In an embodiment, R 7 is unsubstituted n-butyl. In an embodiment, R7 is unsubstituted isobutyl. In an embodiment, R 7 is unsubstituted tert-butyl. In an embodiment, R 7 is an unsubstituted 2 to 6 membered heteroalkyl. 7 is unsubstituted methoxy. In an embodiment, R 7 is an unsubstituted ethoxy group. In an embodiment, R 7 is unsubstituted propoxy. In an embodiment, R 7 is unsubstituted n-propoxy. In an embodiment, R 7 is unsubstituted isopropoxy. In an embodiment, R 7 It is an unsubstituted butoxy group.

[0342] In an embodiment, substituted R 8 (e.g., substituted alkyl, substituted heteroalkyl, substituted cycloalkyl, substituted heterocycloalkyl, substituted aryl and / or substituted heteroaryl) is substituted with at least one substituent, size-restricted substituent or lower substituent; wherein if the substituted R 8 is substituted with a plurality of groups selected from substituents, size-restricted substituents and lower substituents; each substituent, size-restricted substituent and / or lower substituent may be optionally different. In an embodiment, when R 8 When substituted, it is substituted with at least one substituent. In an embodiment, when R 8 When substituted, it is substituted with at least one substituent of limited size. In an embodiment, when R 8 When substituted, it is substituted with at least one lower substituent.

[0343] In an embodiment, R 8 is hydrogen. In an embodiment, R 8 is halogen. In an embodiment, R 8 is –f. In the embodiment, R 8 is –Cl. In an embodiment, R 8 is -Br. In an embodiment, R 8 is -I. In an embodiment, R 8 is -CCl3. In an embodiment, R 8 is -CBr3. In an embodiment, R 8 is -CF3. In an embodiment, R 8 is -CI3. In an embodiment, R 8 is -CH2Cl. In an embodiment, R 8 is -CH2Br. In an embodiment, R 8 is -CH2F. In an embodiment, R 8 is -CH2I. In an embodiment, R8 is -CHCl2. In an embodiment, R 8 is -CHBr2. In an embodiment, R 8 is -CHF2. In an embodiment, R 8 is -CHI2. In an embodiment, R 8 is -CN. In an embodiment, R 8 is –OH. In an embodiment, R 8 is -NH2. In an embodiment, R 8 is -COOH. In an embodiment, R 8 is -CONH2. In an embodiment, R 8 is -NO2. In an embodiment, R 8 is -SH. In an embodiment, R 8 Yes-SO3R A In an embodiment, R 8 is -SO3H. In an embodiment, R 8 is -SO2NH2. In an embodiment, R 8 is -NHNH2. In an embodiment, R 8 is -ONH2. In an embodiment, R 8 is -NHC(O)NH2. In an embodiment, R 8 is -NHSO2H. In an embodiment, R 8 is -NHC(O)H. In an embodiment, R 8 is -NHC(O)OH. In an embodiment, R 8 is -NHOH. In an embodiment, R 8 is -OCCl3. In an embodiment, R 8 is -OCBr3. In an embodiment, R 8 is -OCF3. In an embodiment, R 8 is -OCI3. In an embodiment, R 8 is -OCH2Cl. In an embodiment, R 8 is -OCH2Br. In an embodiment, R 8 is -OCH2F. In an embodiment, R 8 is -OCH2I. In an embodiment, R 8 is -OCHCl2. In an embodiment, R 8 is -OCHBr2. In an embodiment, R 8 is -OCHF2. In an embodiment, R 8 is -OCHI2. In an embodiment, R 8 is -SF5. In an embodiment, R 8is -N3. In an embodiment, R 8 is an unsubstituted C1-C4 alkyl. In an embodiment, R 8 is unsubstituted methyl. In an embodiment, R 8 is an unsubstituted ethyl group. In an embodiment, R 8 is unsubstituted propyl. In an embodiment, R 8 is unsubstituted n-propyl. In an embodiment, R 8 is unsubstituted isopropyl. In an embodiment, R 8 is unsubstituted butyl. In an embodiment, R 8 is unsubstituted n-butyl. In an embodiment, R 8 is unsubstituted isobutyl. In an embodiment, R 8 is unsubstituted tert-butyl. In an embodiment, R 8 is an unsubstituted 2 to 6 membered heteroalkyl. 8 is unsubstituted methoxy. In an embodiment, R 8 is an unsubstituted ethoxy group. In an embodiment, R 8 is unsubstituted propoxy. In an embodiment, R 8 is unsubstituted n-propoxy. In an embodiment, R 8 is unsubstituted isopropoxy. In an embodiment, R 8 It is an unsubstituted butoxy group.

[0344] In an embodiment, substituted R 9 (e.g., substituted alkyl, substituted heteroalkyl, substituted cycloalkyl, substituted heterocycloalkyl, substituted aryl and / or substituted heteroaryl) is substituted with at least one substituent, size-restricted substituent or lower substituent; wherein if the substituted R 9 is substituted with a plurality of groups selected from substituents, size-restricted substituents and lower substituents; each substituent, size-restricted substituent and / or lower substituent may be optionally different. In an embodiment, when R 9 When substituted, it is substituted with at least one substituent. In an embodiment, when R 9 When substituted, it is substituted with at least one substituent of limited size. In an embodiment, when R 9 When substituted, it is substituted with at least one lower substituent.

[0345] In an embodiment, R 9 is hydrogen. In an embodiment, R 9 is halogen. In an embodiment, R 9 is –F. In the embodiment, R 9 is –Cl. In an embodiment, R 9is -Br. In an embodiment, R 9 is -I. In an embodiment, R 9 is -CCl3. In an embodiment, R 9 is -CBr3. In an embodiment, R 9 is -CF3. In an embodiment, R 9 is -CI3. In an embodiment, R 9 is -CH2Cl. In an embodiment, R 9 is -CH2Br. In an embodiment, R 9 is -CH2F. In an embodiment, R 9 is -CH2I. In an embodiment, R 9 is -CHCl2. In an embodiment, R 9 is -CHBr2. In an embodiment, R 9 is -CHF2. In an embodiment, R 9 is -CHI2. In an embodiment, R 9 is -CN. In an embodiment, R 9 is –OH. In an embodiment, R 9 is -NH2. In an embodiment, R 9 is -COOH. In an embodiment, R 9 is -CONH2. In an embodiment, R 9 is -NO2. In an embodiment, R 9 is -SH. In an embodiment, R 9 Yes-SO3R A In an embodiment, R 9 is -SO3H. In an embodiment, R 9 is -SO2NH2. In an embodiment, R 9 is -NHNH2. In an embodiment, R 9 is -ONH2. In an embodiment, R 9 is -NHC(O)NH2. In an embodiment, R 9 is -NHSO2H. In an embodiment, R 9 is -NHC(O)H. In an embodiment, R 9 is -NHC(O)OH. In an embodiment, R 9 is -NHOH. In an embodiment, R 9 is -OCCl3. In an embodiment, R 9 is -OCBr3. In an embodiment, R 9 is -OCF3. In an embodiment, R 9 is -OCI3. In an embodiment, R 9is -OCH2Cl. In an embodiment, R 9 is -OCH2Br. In an embodiment, R 9 is -OCH2F. In an embodiment, R 9 is -OCH2I. In an embodiment, R 9 is -OCHCl2. In an embodiment, R 9 is -OCHBr2. In an embodiment, R 9 is -OCHF2. In an embodiment, R 9 is -OCHI2. In an embodiment, R 9 is -SF5. In an embodiment, R 9 is -N3. In an embodiment, R 9 is an unsubstituted C1-C4 alkyl. In an embodiment, R 9 is unsubstituted methyl. In an embodiment, R 9 is an unsubstituted ethyl group. In an embodiment, R 9 is unsubstituted propyl. In an embodiment, R 9 is unsubstituted n-propyl. In an embodiment, R 9 is unsubstituted isopropyl. In an embodiment, R 9 is unsubstituted butyl. In an embodiment, R 9 is unsubstituted n-butyl. In an embodiment, R 9 is unsubstituted isobutyl. In an embodiment, R 9 is unsubstituted tert-butyl. In an embodiment, R 9 is an unsubstituted 2 to 6 membered heteroalkyl. 9 is unsubstituted methoxy. In an embodiment, R 9 is an unsubstituted ethoxy group. In an embodiment, R 9 is unsubstituted propoxy. In an embodiment, R 9 is unsubstituted n-propoxy. In an embodiment, R 9 is unsubstituted isopropoxy. In an embodiment, R 9 It is an unsubstituted butoxy group.

[0346] In an embodiment, substituted R A (e.g., substituted alkyl, substituted heteroalkyl, substituted cycloalkyl, substituted heterocycloalkyl, substituted aryl and / or substituted heteroaryl) is substituted with at least one substituent, size-restricted substituent or lower substituent; wherein if the substituted R A is substituted with a plurality of groups selected from substituents, size-restricted substituents and lower substituents; each substituent, size-restricted substituent and / or lower substituent may be optionally different. In an embodiment, when RA When substituted, it is substituted with at least one substituent. In an embodiment, when R A When substituted, it is substituted with at least one substituent of limited size. In an embodiment, when R A When substituted, it is substituted with at least one lower substituent.

[0347] In an embodiment, R A is hydrogen. In an embodiment, R A is an unsubstituted C1-C4 alkyl. In an embodiment, R A is unsubstituted methyl. In an embodiment, R A is an unsubstituted ethyl group. In an embodiment, R A is unsubstituted propyl. In an embodiment, R A is unsubstituted n-propyl. In an embodiment, R A is unsubstituted isopropyl. In an embodiment, R A is unsubstituted butyl. In an embodiment, R A is unsubstituted n-butyl. In an embodiment, R A is unsubstituted isobutyl. In an embodiment, R A is an unsubstituted tert-butyl group.

[0348] In an embodiment, substituted R 10 (e.g., substituted alkyl and / or substituted heteroalkyl) is substituted with at least one substituent, a size-restricted substituent, or a lower substituent; wherein if the substituted R 10 is substituted with a plurality of groups selected from substituents, size-restricted substituents and lower substituents; each substituent, size-restricted substituent and / or lower substituent may be optionally different. In an embodiment, when R 10 When substituted, it is substituted with at least one substituent. In an embodiment, when R 10 When substituted, it is substituted with at least one substituent of limited size. In an embodiment, when R 10 When substituted, it is substituted with at least one lower substituent.

[0349] In an embodiment, R 10 is hydrogen. In an embodiment, R 10 is an unsubstituted C1-C4 alkyl. In an embodiment, R 10 is unsubstituted methyl. In an embodiment, R 10 is an unsubstituted ethyl group. In an embodiment, R 10 is unsubstituted propyl. In an embodiment, R 10 is unsubstituted n-propyl. In an embodiment, R 10 is unsubstituted isopropyl. In an embodiment, R10 is unsubstituted butyl. In an embodiment, R 10 is unsubstituted n-butyl. In an embodiment, R 10 is unsubstituted isobutyl. In an embodiment, R 10 is an unsubstituted tert-butyl group.

[0350] In an embodiment, when R 1 and R 6 The substituted ring formed when the substituents are connected (e.g., substituted heterocycloalkyl and / or substituted heteroaryl) is substituted with at least one substituent, a size-restricted substituent, or a lower substituent; wherein when R 1 and R 6 The substituted ring formed when the substituents are connected is substituted with a plurality of groups selected from substituents, size-restricted substituents, and lower substituents; each substituent, size-restricted substituent, and / or lower substituent may be optionally different. In an embodiment, when R 1 and R 6 When the substituted ring formed when the substituents are attached is substituted, it is substituted with at least one substituent. 1 and R 6 When the substituted ring formed when the substituents are attached is substituted, it is substituted with at least one substituent of limited size. 1 and R 6 When a substituted ring formed when substituents are linked is substituted, it is substituted with at least one lower substituent.

[0351] In an embodiment, R 1 and R 6 The substituents are linked to form a substituted or unsubstituted 3 to 8 membered heterocycloalkyl. 1 and R 6 The substituents are linked to form a substituted or unsubstituted pyrrolidinyl group. 1 and R 6 The substituents are linked to form an unsubstituted pyrrolidinyl group. 1 and R 6 The substituents are linked to form a substituted or unsubstituted 5- to 6-membered heteroaryl group.

[0352] In an embodiment, when R 8 and R 10 The substituted ring formed when the substituents are connected (e.g., substituted heterocycloalkyl and / or substituted heteroaryl) is substituted with at least one substituent, a size-restricted substituent, or a lower substituent; wherein when R 8 and R 10The substituted ring formed when the substituents are connected is substituted with a plurality of groups selected from substituents, size-restricted substituents, and lower substituents; each substituent, size-restricted substituent, and / or lower substituent may be optionally different. In an embodiment, when R 8 and R 10 When the substituted ring formed when the substituents are attached is substituted, it is substituted with at least one substituent. 8 and R 10 When the substituted ring formed when the substituents are attached is substituted, it is substituted with at least one substituent of limited size. 8 and R 10 When a substituted ring formed when substituents are linked is substituted, it is substituted with at least one lower substituent.

[0353] In an embodiment, R 8 and R 10 The substituents are linked to form a substituted or unsubstituted 3 to 8 membered heterocycloalkyl. 8 and R 10 The substituents are linked to form a substituted or unsubstituted pyrrolidinyl group. 8 and R 10 The substituents are linked to form an unsubstituted pyrrolidinyl group. 8 and R 10 The substituents are linked to form a substituted or unsubstituted 5- to 6-membered heteroaryl group.

[0354] In an embodiment, the compound has the formula:

[0355]

[0356] In an embodiment, the compound has the formula:

[0357]

[0358] In an embodiment, the compound has the formula:

[0359]

[0360] In an embodiment, the compound has the formula:

[0361]

[0362] In an embodiment, the compound has the formula:

[0363]

[0364] In an embodiment, the compound has the formula:

[0365]

[0366] In an embodiment, the compound has the formula:

[0367]

[0368] In an embodiment, the compound has the formula:

[0369]

[0370] In an embodiment, the compound has the formula:

[0371]

[0372] In an embodiment, the compound has the formula:

[0373]

[0374] In an embodiment, the compound has the formula:

[0375]

[0376] In an embodiment, the compound has the formula:

[0377]

[0378] In an embodiment, the compound has the formula:

[0379]

[0380] In an embodiment, the compound has the formula:

[0381]

[0382] In an embodiment, the compound has the formula:

[0383]

[0384] In an embodiment, the compound has the formula:

[0385]

[0386] In an embodiment, the compound has the formula:

[0387]

[0388] In an embodiment, the compound has the formula:

[0389]

[0390] In an embodiment, the compound has the formula:

[0391]

[0392] In an embodiment, the compound has the formula:

[0393]

[0394] In an embodiment, the compound has the formula:

[0395]

[0396] In an embodiment, the compound has the formula:

[0397]

[0398] In an embodiment, the compound has the formula:

[0399]

[0400] In an embodiment, the compound has the formula:

[0401]

[0402] In an embodiment, the compound has the formula:

[0403]

[0404] In an embodiment, the compound has the formula:

[0405]

[0406] In an embodiment, the compound has the formula:

[0407]

[0408] In an embodiment, the compound has the formula:

[0409]

[0410] In an embodiment, the compound has the formula:

[0411]

[0412] In an embodiment, the compound has the formula:

[0413]

[0414] In an embodiment, the compound has the formula:

[0415]

[0416] In an embodiment, the compound has the formula:

[0417]

[0418] In embodiments, the compound is a compound as described herein, including in the embodiments. In embodiments, the compound is a compound as described herein (eg, in the Examples section, Figures, Tables, or Claims).

[0419] Additional Marker / Dye Details

[0420] Exemplary non-limiting detectable labels that can be used in the embodiments described herein include, for example:

[0421] Fluorescein (e.g., 5-carboxy-2,7-dichlorofluorescein, 5-carboxyfluorescein (5-FAM), 6-JOE, 6-carboxyfluorescein (6-FAM), VIC, FITC, 6-carboxy-4',5'-dichloro-2',7'-dimethoxyfluorescein (JOE)), 5 and 6-carboxy-1,4-dichloro-2',7'-dichlorofluorescein (TET), 5 and 6-carboxy-1,4-dichloro-2',4',5',7'-tetrachlorofluorescein, HEX, PET, NED, Oregon Green (e.g., 488, 500, 514));

[0422] Pyrenes (e.g., Cascade Blue; AlexaFluor405);

[0423] coumarins (e.g., Pacific Blue, Atto 425, Alexa Fluor 350, Alexa Fluor 430);

[0424] Cyanine dyes (e.g., Cy dyes such as Cy3, Cy3.18, Cy3.5, Cy5, Cy5.18, Cy5.5, Cy7);

[0425] Rhodamines (e.g., 110, 123, B, B 200, BB, BG, B Extra, 5 and 6-carboxytetramethylrhodamine (5-TAMRA, 6-TAMRA), 5 and 6-carboxyrhodamine 6G, Lissamine, Lissamine Rhodamine B, Rhod-2, ROX (6-carboxy-X-rhodamine), 5 and 6-ROX (carboxy-X-rhodamine), Sulforhodamine B can C, Sulforhodamine G Extra, 5 and 6TAMRA (6-carboxytetramethyl-rhodamine), (TRITC), ABY, JUN, LIZ, RAD, RXJ, Texas Red; and Texas Red-X);

[0426] ALEXA FLUOR fluorophores (this is a broad class that includes many dye types, such as cyanines) (e.g., Alexa 350, 405, 430, 488, 500, 514, 532, 546, 555, 568, 594, 610, 633, 635, 647, 660, 676, 680, 700, 750);

[0427] FRET donor / acceptor pairs (e.g., fluorescein / fluorescein, fluorescein / rhodamine, fluorescein / cyanine, rhodamine / cyanine, fluorescein / Alexa Fluor, Alexa Fluor / rhodamine); and other types of dyes known to those skilled in the art.

[0428] Fluorophore labels can be associated with quenchers, such as dark fluorescence quenchers (DFQ), black hole quenchers (BHQ), Iowa Black, QSY7, QSY21 quenchers, Dabsyl and Dabcel sulfonate / carboxylate quenchers, and MGB-NFQ quenchers. For example, fluorophore labels can also include sulfonate derivatives of fluorescein dyes with SO3 replacing the carboxylate group, phosphoramidite forms of fluorescein, and / or phosphoramidite forms of Cy5.

[0429] Additional Amplification Details

[0430] Amplification products produced using one or more embodiments described herein can be produced, detected and / or analyzed on any suitable platform. In some embodiments, the nucleic acid target can be single-stranded, double-stranded, or any other nucleic acid molecule of any size or conformation. Amplification processes described herein may include PCR (see, e.g., U.S. Patent No. 4,683,202). In some embodiments, PCR is real-time or quantitative PCR (qPCR). In some embodiments, PCR is endpoint PCR. In some embodiments, PCR is digital PCR (dPCR).

[0431] In some embodiments, the amplification process includes reverse transcription PCR (RT-PCR). The disclosed method may include, for example, subjecting the target nucleic acid to a reverse transcription reaction prior to amplification by PCR. In some embodiments, the amplification process includes a one-step RT-PCR (e.g., in a single container or reaction volume), wherein one or more reverse transcriptases are used in combination with one or more DNA polymerases.

[0432] Optionally, certain qPCR assays can be plated in individual wells of a single array or multi-well plate, such as a TaqMan Array Card (see, e.g., Thermo Fisher Scientific, Waltham, MA; catalog numbers 4346800 and 4342265) or a MicroAmp multi-well (e.g., 96-well, 384-well) reaction plate (see, e.g., Thermo Fisher Scientific, Waltham, MA; catalog numbers 4346906, 4366932, 4306737, 4326659, and N8010560). Optionally, different qPCR assays present in different wells of an array or plate can be dried or freeze-dried in situ, and the array or plate can be stored or shipped prior to use. In some embodiments, the concepts described herein can be used in in situ hybridization applications that are not necessarily associated with PCR.

[0433] Other amplification methods, such as, for example, loop-mediated isothermal amplification ("LAMP") and other isothermal methods can also be used in the assay embodiments described herein.

[0434] The components described herein for realizing multiplexing using a probe with a spectrally detectable label can be provided in a kit together with one or more additional components to realize the amplification process. Such components can include, for example, dNTPs, DNA polymerase, amplification buffer / reagents, master mix components known in the art, and other components known in the art for realizing or assisting nucleic acid amplification.

[0435] Computer system implementation

[0436] In some embodiments, at least a portion of the methods described herein may be implemented using one or more computer systems. In some cases, the techniques discussed herein are represented by computer executable instructions that may be stored on one or more hardware storage devices. Computer executable instructions may be executable by one or more processors to implement (or configure a system to implement) the disclosed techniques. In some embodiments, the system may be configured to send computer executable instructions to a remote device to configure the remote device to perform the disclosed techniques.

[0437] In an exemplary embodiment, a computer system includes one or more processors and a memory storing one or more instructions that, when executed by the one or more processors, cause the one or more processors to perform the following process: at multiple time points during one or more cycles of an amplification process, obtain emission (e.g., fluorescent) signal data associated with a composite emission (e.g., fluorescent) signal from at least a first probe type comprising a first detectable label (e.g., fluorophore) and a second probe type comprising a second detectable label (e.g., fluorophore), the second detectable label having a spectral similarity to the first detectable label (e.g., fluorophore) and / or producing the same or substantially the same signal, the first probe type and the second probe type differing in thermal and / or temporal characteristics; and during the one or more cycles of the amplification process, determine emission signal data associated with the emission signal of a given probe type from at least the first probe type and the second probe type based at least in part on the emission signal data associated with the composite emission signal and the thermal and / or temporal characteristics of one or more of the at least the first probe type and the second probe type.

[0438] In some embodiments, using emission signal data associated with a composite emission signal and first emission signal data as input for generating emission signal data associated with an emission signal from a given probe type includes: generating transformed first emission signal data by applying a transformation (e.g., linear) to the first emission signal data; and modifying the emission signal data associated with the composite emission signal with the transformed first emission signal data to generate emission signal data associated with an emission signal from a given probe type.

[0439] In some embodiments, when executed by one or more processors, the one or more instructions further cause the one or more processors to perform the following processes: quantifying a first target associated with a first probe type based at least on the first emission signal data; and quantifying a second target associated with a second probe type based at least on the generated emission signal data associated with the emission signal from a given probe type.

[0440] Some embodiments include one or more computer-readable media storing one or more instructions that, when executed by one or more processors of at least one computing device, cause the one or more processors to perform the foregoing processes or other computer-implemented processes as described herein.

[0441] Systems for implementing the disclosed embodiments may include various components, such as, as non-limiting examples, processors, memories, sensors, I / O systems, communication systems, and the like. A processor may include one or more groups of electronic circuits that include any number of logic units, registers, and / or control units to facilitate the execution of computer-readable instructions (e.g., instructions that form a computer program). Such computer-readable instructions may be stored in a storage device. The storage device may include physical system memory and may be volatile, non-volatile, or some combination thereof. In addition, the storage device may include a local storage device, a remote storage device (e.g., accessible via a communication system or otherwise), or some combination thereof.

[0442] In addition, the system may include or communicate with an I / O system. The I / O system may include any type of input or output device, such as, by way of non-limiting example, a touch screen, a mouse, a keyboard, a controller, a speaker, and / or others, but is not limited thereto. For example, the I / O system may include a display system, which may include any number of display panels, optics, laser scanning display components, and / or other components.

[0443] The disclosed embodiments may also include physical and other computer-readable media for carrying or storing computer-executable instructions and / or data structures. Such computer-readable media may be any available media that can be accessed by a general-purpose or special-purpose computer system. Computer storage media (also referred to as "hardware storage devices") are computer-readable hardware storage devices based on RAM, flash memory, phase change memory ("PCM") or other types of memory, such as RAM, ROM, EEPROM, CD-ROM, solid-state drive ("SSD"), or other optical disk storage, magnetic disk storage or other magnetic storage devices, or any other medium that can be used to store required program code tools in hardware in the form of computer-executable instructions, data or data structures and can be accessed by a general-purpose or special-purpose computer.

[0444] Those skilled in the art will appreciate that various embodiments are practiced in a network computing environment with many types of computer system configurations, including personal computers, desktop computers, laptop computers, message processors, handheld devices, multiprocessor systems, microprocessor-based or programmable consumer electronics, network PCs, minicomputers, mainframe computers, mobile phones, PDAs, pagers, routers, switches, wearable devices, etc. The embodiments may also be implemented in a distributed system environment, where multiple computer systems (e.g., local and remote systems) linked by a network (by hardwired data links, wireless data links, or by a combination of hardwired and wireless data links) perform tasks. In a distributed system environment, program modules may be located in local and / or remote memory storage devices.

[0445] Alternatively or additionally, the functions described herein may be performed at least in part by one or more hardware logic components. For example, but not limited to, illustrative types of hardware logic components that may be used include field programmable gate arrays (FPGAs), application specific integrated circuits (ASICs), application specific standard products (ASSPs), systems on chips (SOCs), complex programmable logic devices (CPLDs), central processing units (CPUs), graphics processing units (GPUs), and / or other components.

[0446] As used herein, the terms "executable module," "executable component," "component," "module," or "engine" may refer to a hardware processing unit or a software object, routine, or method that can be executed on one or more computer systems. The various components, modules, engines, and services described herein may be implemented as objects or processors (e.g., as separate threads) that execute on one or more computer systems.

[0447] Example

[0448] Fig. 6A Shown is the change in fluorescence signal as a function of cycle number measured at the annealing / extension temperature (65° C. in this example) and the denaturation temperature (95° C. in this example) using the TaqMan probe and EF probe combination.

[0449] FIG. 6B to FIG. 6D Results of a qPCR duplex assay are shown, measuring the fluorescence signal in the FAM detection channel, where the TaqMan probe and the EF probe were designed to produce fluorescence signals that are spectrally similar (e.g., detectable in the same detection channel) ( Figure 6B ) or are not spectrally similar (e.g., detectable in different detection channels) ( Figure 6C and Fig.6D ).exist Figure 6B In the assay shown, both the TaqMan probe and the EF probe were labeled with FAM. Figure 6C In the assay shown, the TaqMan probe was labeled with ABY and the EF probe was labeled with FAM. Fig.6D In the assays shown, the TaqMan probe was labeled with FAM and the EF probe was labeled with ABY. The reaction mixture composition, template DNA concentration and amplification conditions were kept constant in each assay.

[0450] exist Figure 6BIn, the top row shows the change of the FAM channel fluorescence signal measured at the denaturation temperature (95°C in this embodiment) with the cycle number. It is expected that the signal includes the fluorescence (those that have been cleaved from the probe) mainly produced by the TaqMan probe label. The second row shows the change of the fluorescence signal measured at the denaturation temperature (95°C in this embodiment) with the cycle number, and is modified by a linear function, which relates the 95°C measured values ​​of the TaqMan probe to the 65°C measured values. It is expected that the signal includes the fluorescence produced by the TaqMan probe label, but does not include the significant fluorescence from the EF probe label. The third row shows the change of the fluorescence signal measured at the annealing / extension temperature (65°C in this embodiment) with the cycle number. It is expected that the signal includes the fluorescence produced by the TaqMan probe label (those that have been cleaved from the probe) and the EF probe label (those that have been incorporated into the double-stranded amplicon). The bottom row shows the resolved fluorescence signal determined by subtracting the second row signal from the third row signal. The signal is expected to be used to estimate the fluorescence produced by the EF probe label, separated from the fluorescence produced by the TaqMan probe label.

[0451] exist Figure 6C The top, second, third, and bottom rows represent Figure 6B The same signal measurement type in the assay, where the TaqMan probe is labeled with ABY and the EF probe is labeled with FAM. As shown in the top and second rows, at the denaturation temperature, the EF probe label (FAM) produces insignificant (essentially baseline level) fluorescence in the FAM channel. Because the TaqMan and EF probes are labeled differently in this assay, the bottom row shows the resolved signal of the EF probe label, which essentially matches the EF probe signal at the annealing / extension temperature in the FAM channel (third row).

[0452] exist Fig.6D The top, second, third, and bottom rows represent Figure 6B The same signal measurement type in the assay, but the TaqMan probe is labeled with FAM and the EF probe is labeled with ABY. The first row of fluorescent signals is mainly produced by the TaqMan probe labeling (those that have been cleaved from the probe). The second row shows the change in the fluorescent signal measured at the denaturation temperature (95°C in this embodiment) with the number of cycles, and is modified by a linear function that relates the 95°C measurement value of the TaqMan probe to the 65 measurement value. Because the TaqMan and EF probes are labeled differently in this assay, the derived TaqMan signal in the FAM channel (second row) essentially matches the signal measured at the annealing / extension temperature in the FAM channel (third row), while the resolved signal of the EF probe label in the FAM channel (bottom row) is essentially zero.

[0453] Although FIG. 6B to FIG. 6D Results of qPCR assays are shown, but the same principles shown can be extended to end-point PCR assays with similar results. Specifically, a correction factor can be applied to the signal measured under a set of denaturation conditions (e.g., denaturation temperature); the derived signal can be subtracted from the measured signal under a set of extension conditions (e.g., extension temperature) to obtain the derived signal from the second probe (non-cleavable), and the measured signal from the denaturation condition set represents the signal from the first probe (cleavable probe).

[0454] Fig. 6E The baseline-adjusted resolved EF-related fluorescence signals (ΔRn) were compared ( Figure 6B ) and the baseline-adjusted EF-related fluorescence signal (ΔRn) ( Figure 6C The bottom row of ), which represents the direct measurement of the EF probe labeled fluorescence in the FAM channel. The results show that the resolved signal is closely related to the measured signal. Therefore, the results show that the fluorescence signals of different probe types in the same detection channel can be resolved separately.

[0455] Fig. 6F The baseline adjustment (ΔRn) ( Figure 6B The second row of the TaqMan fluorescence signal derived after baseline adjustment (ΔRn) in the FAM channel ( Fig.6D The results show that when the EF probe is similarly labeled ( Figure 6B ) or labeled with different dyes ( Fig.6D ) were closely correlated with the TaqMan signals derived from separate assays.

[0456] Figure 7 Results of another assay test are shown, which included 5 different detection channels / dyes, four detection channels with corresponding TaqMan probes and EF probes (each channel having a different dye common to both the TaqMan and EF probes in that channel), and one channel with only the TaqMan probe (in Figure 7 The results show that the fluorescence signals of different probe types can be determined independently and that 9-plex reactions can be efficiently performed using 5 detection channels.

[0457] Figure 8 is a graph comparing the endpoint signals after a dPCR process at reaction volumes at 65°C and 95°C. As shown, the signals fall into identifiable clusters. Clustering can be estimated using clustering analysis algorithms known in the art. Figure 8In the above, the "EF" cluster represents those partitions that provide signals at the annealing / extension temperature but have limited signals at the denaturation temperature, the "T" cluster represents those partitions that provide signals at both the annealing / extension temperature and the denaturation temperature, and the "T+EF" cluster represents those partitions that provide signals at the denaturation temperature and enhance the signals at the annealing / extension temperature. The total counts of the partitions where the TaqMan probe generates signals are equal to the counts of cluster T plus the counts of cluster T+EF, and the total counts of the partitions where the EF probe generates signals are equal to the counts of cluster EF plus the counts of cluster T+EF. The concentrations of the first target and the second target in the sample can then be estimated using standard dPCR techniques.

[0458] Figure 8 The cluster analysis technique of is one embodiment of a technique for analyzing signals obtained during dPCR. For other data analysis techniques that can be used to parse signal data obtained during multiplex dPCR using detectable labels with spectral similarity according to the present disclosure.

[0459] The present disclosure includes, but is not limited to, the embodiments represented by the following clauses.

[0460] Clause 1: A method for detecting nucleic acid in a sample, the method comprising:

[0461] (i) providing a reaction mixture, the reaction mixture comprising

[0462] at least a portion of the sample,

[0463] a first probe detectably labeled with a first label configured to produce a first emission signal,

[0464] a second probe detectably labeled with a second label configured to produce a second emission signal,

[0465] wherein the first probe and the second probe have different sequences, and

[0466] wherein the first label and the second label are identical and / or produce substantially identical emissions;

[0467] (ii) subjecting the reaction mixture to an amplification process comprising a first set of reaction conditions and a second set of reaction conditions, the first set of reaction conditions being different from the second set of reaction conditions; and

[0468] (iii) determining the presence of the first nucleic acid target and / or the second nucleic acid target in the sample by the following steps:

[0469] measuring an emission signal during the first set of reaction conditions, the emission signal during the first set of reaction conditions being associated with a specific interaction, or lack of interaction, between the first probe and the first nucleic acid target,

[0470] measuring an emission signal during the second set of reaction conditions, the emission signal during the second set of reaction conditions being associated with a specific interaction, or lack thereof, between the first probe and the first nucleic acid target and being associated with a specific interaction, or lack thereof, between the second probe and the second nucleic acid target, and

[0471] The presence and / or amount of each of the first nucleic acid target and the second nucleic acid target is estimated.

[0472] Clause 2. A method for detecting nucleic acid in a sample, the method comprising:

[0473] (i) providing a reaction mixture, the reaction mixture comprising:

[0474] at least a portion of the sample,

[0475] a first probe detectably labeled with a first label configured to generate a first emission signal indicative of the presence or absence of a first nucleic acid target,

[0476] a second probe detectably labeled with a second label configured to generate a second emission signal indicative of the presence or absence of a second nucleic acid target,

[0477] wherein the first probe and the second probe have different sequences, and

[0478] wherein the first marker and the second marker are identical and / or produce substantially identical emission signals;

[0479] (ii) subjecting the reaction mixture to an amplification process comprising a first set of reaction conditions and a second set of reaction conditions, the first set of reaction conditions being different from the second set of reaction conditions; and

[0480] (iii) determining any presence and / or amount of the first nucleic acid target and / or the second nucleic acid target in the reaction mixture by the following steps:

[0481] measuring a first total emission signal during the first set of reaction conditions, the first total emission signal including any first emission signal, if present, and any second emission signal, if present,

[0482] measuring a second total emission signal during the second set of reaction conditions, the second total emission signal including any first emission signal, if present, and any second emission signal, if present, and

[0483] The first transmit signal and / or the second transmit signal are estimated based on the first total transmit signal and the second total transmit signal.

[0484] Item 3: A method according to item 1 or 2, wherein the first emission signal and the second emission signal are a first fluorescent signal and a second fluorescent signal, and wherein both the first probe and the second probe are excited at the same wavelength and / or both the first probe and the second probe are excited during detection of their corresponding first fluorescent signals and second fluorescent signals.

[0485] Clause 4: A method according to any one of clauses 1 to 3, wherein the emission signal is a fluorescent signal, and wherein:

[0486] Measuring the fluorescent signal during the first set of reaction conditions includes measuring a combined signal including the first fluorescent signal and the second fluorescent signal during the first set of reaction conditions to obtain a first total fluorescent signal;

[0487] Measuring the fluorescent signal during the second set of reaction conditions includes measuring a combined signal including the first fluorescent signal and the second fluorescent signal during the second set of reaction conditions to obtain a second total fluorescent signal; and

[0488] Estimating the presence and / or amount of each of the first nucleic acid target and the second nucleic acid target includes estimating the first fluorescent signal and / or the second fluorescent signal based on the first total fluorescent signal and the second total fluorescent signal.

[0489] Item 5: A method according to Item 4, wherein the degree of difference between the second fluorescent signal between the first set of reaction conditions and the second set of reaction conditions is greater than the degree of difference between the first fluorescent signal between the first set of reaction conditions and the second set of reaction conditions.

[0490] Clause 6: A method according to any one of clauses 4 to 5, wherein

[0491] The first total fluorescence value includes (i) fluorescence from a first label that is free and unquenched in the reaction mixture and is emitted due to cleavage of the first label after hybridization of the first probe to the first amplicon, and (ii) background fluorescence of the second label, and

[0492] The second total fluorescence value is based on (i) fluorescence from the first label, which is free and unquenched in the reaction mixture and is emitted due to cleavage of the first label after hybridization of the first probe to the first amplicon, and (ii) fluorescence from the second label, which is above the background fluorescence of the second label and is emitted due to hybridization of the second probe to the second amplicon.

[0493] Clause 7: The method according to any one of clauses 4 to 6, further comprising:

[0494] calculating the amount of the first nucleic acid target based on the first fluorescent signal; and

[0495] The amount of the second nucleic acid target is calculated based on the second fluorescent signal.

[0496] Clause 8: The method according to any one of clauses 4 to 7, wherein the first fluorescent signal is above background levels during both the first set of reaction conditions and the second set of reaction conditions, indicating the presence of the first nucleic acid target in the reaction mixture.

[0497] Clause 9: A method according to any one of clauses 4 to 8, wherein the second fluorescent signal is above background levels during the second set of reaction conditions but not during the first set of reaction conditions, indicating the presence of the second nucleic acid target in the reaction mixture.

[0498] Clause 10: A method according to any one of clauses 5 to 8, wherein the first set of reaction conditions includes a first measurement temperature for measuring the first fluorescence signal, and the second set of reaction conditions includes a second measurement temperature for measuring the second fluorescence signal, the second measurement temperature being different from the first measurement temperature.

[0499] Clause 11: The method of clause 10, wherein the first measured temperature and the second measured temperature differ by at least about 10°C or more, about 15°C or more, about 20°C or more, about 25°C or more, or about 30°C or more.

[0500] Clause 12: The method of clause 10 or clause 11, wherein at least one of the first measured temperature or the second measured temperature is a denaturation temperature at which the DNA in the reaction mixture denatures, such as in the range of about 80°C or above.

[0501] Clause 13: The method according to any one of clauses 1 to 12, wherein the reaction mixture is subjected to a plurality of amplification cycles during the amplification process, each of the amplification cycles comprising the first set of reaction conditions and the second set of reaction conditions.

[0502] Clause 14: A method according to any one of clauses 1 to 13, wherein the amplification process comprises thermal cycling.

[0503] Clause 15: The method of clause 14, wherein subjecting the reaction mixture to the first set of reaction conditions comprises thermally cycling the reaction mixture at a first temperature sufficient to cause denaturation of the first amplicon and the second amplicon.

[0504] Item 16: A method according to Item 15, wherein subjecting the reaction mixture to the second set of reaction conditions includes thermally cycling the reaction mixture at a second temperature sufficient to cause the first nucleic acid target and the second nucleic acid target to anneal and / or extend to form the first amplicon and the second amplicon, respectively, and the second temperature is lower than the first temperature.

[0505] Clause 17: The method according to any one of clauses 1 to 16, wherein the first probe is a cleavable probe.

[0506] Clause 18: The method of Clause 17, wherein the first emission signal increases as the cleavable probe is cleaved during the annealing / extension phase.

[0507] Item 19: A method according to item 17 or item 18, wherein the first probe comprises a fluorophore and a quencher, and wherein the first probe is configured such that fluorescence from the fluorophore is quenched by the quencher until the probe is cleaved during the annealing / extension phase of the amplification process.

[0508] Clause 20: The method according to Clause 19, wherein the first probe is a TaqMan probe.

[0509] Clause 21: The method according to any one of clauses 1 to 20, wherein the second probe is a non-cleavable probe.

[0510] Clause 22: The method according to clause 21, wherein the second probe comprises a stem-loop portion configured to form a stem-loop structure when the second probe is single-stranded.

[0511] Item 23: A method according to Item 21 or Item 22, wherein the second probe comprises a fluorophore and a quencher separated from each other, so that when the second probe is single-stranded, the fluorophore is quenched, and when the second probe is incorporated into a double-stranded amplicon, the fluorophore is not quenched.

[0512] Clause 24: The method of clause 23, wherein the fluorophore is located at or near the 5' end of the second probe and the quencher is 3' to the fluorophore.

[0513] Clause 25: The method of clause 23 or clause 24, wherein both the fluorophore and the quencher are disposed at or near the stem-loop portion of the second probe.

[0514] Item 26: A method according to any one of items 1 to 25, wherein the reaction mixture further comprises: a first primer pair complementary to a first nucleic acid target of the nucleic acid or its complement, the first nucleic acid target being configured to produce a first amplicon with which the first probe can hybridize; and a second primer pair complementary to a second nucleic acid target of the nucleic acid or its complement, the second nucleic acid target being configured to produce a second amplicon with which the second probe can hybridize.

[0515] Clause 27: The method of clause 26, wherein the second primer pair comprises a tailed primer.

[0516] Clause 28: The method according to clause 27, wherein the tail forms the 5' end of the primer carrying the tail.

[0517] Clause 29: The method according to clause 27 or clause 28, wherein the second probe is capable of hybridizing to the tail or its complement.

[0518] Clause 30: The method according to any one of clauses 20 to 29, wherein the amplification process utilizes a series of thermal cycling stages comprising at least three different target temperatures.

[0519] Clause 31: The method of clause 30, wherein the amplification process comprises a denaturation temperature and a plurality of different annealing / extension temperatures that vary throughout the amplification process.

[0520] Item 32: A method according to Item 31, wherein a first series of denaturation phases and annealing / extension phases are performed at a first annealing / extension temperature, and wherein a second series of denaturation phases and annealing / extension phases are performed at a second annealing / extension temperature different from the first annealing / extension temperature.

[0521] Clause 33: The method of clause 32, wherein the first annealing / extension temperature is higher than the second annealing / extension temperature.

[0522] Clause 34: A method according to clause 32 or clause 33, wherein the first series of denaturation phases and annealing / extension phases is cycled more times than the second series of denaturation phases and annealing / extension phases.

[0523] Clause 35: The method according to any one of clauses 32 to 34, wherein the amplification process further comprises a third series of denaturation steps and annealing / extension steps performed using a third annealing / extension temperature.

[0524] Clause 36: The method of clause 35, wherein the third annealing / extension temperature is the same as the first annealing / extension temperature.

[0525] Clause 37: A method according to clause 35 or clause 36, wherein the third series of denaturation phases and annealing / extension phases is cycled more times than the first series of denaturation phases and annealing / extension phases.

[0526] Clause 38: The method according to any one of clauses 32 to 37, wherein for each series of the denaturation stages, the denaturation temperature is the same.

[0527] Clause 39: The method of any one of clauses 2738, wherein the second primer pair further comprises a tailless primer, and wherein the concentration of the primer with the tail in the reaction mixture is different from the concentration of the tailless primer in the reaction mixture.

[0528] Clause 40: The method of clause 39, wherein the concentration of the tailless primer is greater than the concentration of the primer with the tail.

[0529] Item 41: A method according to Item 40, wherein the concentration of the tailless primer is about 2X to about 30X the concentration of the primer with the tail, or about 5X to about 25X the concentration of the primer with the tail, or about 10X to about 20X the concentration of the primer with the tail.

[0530] Clause 42: The method according to any one of clauses 39 to 41, wherein the second probe is provided at a concentration different from the concentration of the primer with the tail and the concentration of the tailless primer.

[0531] Clause 43: The method of clause 42, wherein the second probe is provided at a concentration greater than the concentration of the primer with the tail.

[0532] Clause 44: The method according to clause 42 or clause 43, wherein the second probe is provided at a concentration lower than the concentration of the tailless primer.

[0533] Clause 45: The method of any one of clauses 42 to 44, wherein the second probe is provided at a concentration of about 2X to about 10X the concentration of the primer with the tail, or about 3X to about 7.5X the concentration of the primer with the tail.

[0534] Clause 46: A method according to any one of clauses 1 to 45, wherein the melting temperature (T m ) and the T of the second probe m Within about 8°C, or about 6°C, or about 4°C, or about 2°C of each other.

[0535] Clause 47: The method of any one of clauses 1 to 48, wherein for a plurality of cycles of the amplification process, the amplification process is cycled between at least two target temperatures.

[0536] Item 48: A method according to item 47, wherein for at least 5% of the cycles of the amplification process, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% or at least 95%, the amplification process cycles between at least two target temperatures.

[0537] Clause 49: The method according to any one of clauses 1 to 48, wherein the method further comprises dividing the reaction mixture into a plurality of reaction volumes, and wherein the amplification process is a digital PCR (dPCR) process.

[0538] Clause 50: A method according to clause 49, wherein measuring the emission signal during the first set of reaction conditions includes measuring the emission signal at or near completion of subjecting the reaction mixture to the first set of reaction conditions to obtain a first endpoint measurement, and wherein measuring the emission signal during the second set of reaction conditions includes measuring the emission signal at or near completion of subjecting the reaction mixture to the second set of reaction conditions to obtain a second endpoint measurement.

[0539] Clause 51: The method of clause 50, wherein estimating the presence and / or amount of each of the first nucleic acid target and the second nucleic acid target comprises:

[0540] classifying the plurality of reaction volumes according to the emission signal measured at the first endpoint metric and according to the emission signal measured at the second endpoint metric; and

[0541] Based on the classification, a count of the plurality of reaction volumes in which the first probe shows activity and a count of the plurality of reaction volumes in which the second probe shows activity are determined.

[0542] Clause 52: A method according to any one of clauses 1 to 48, wherein measuring the emission signal during the first set of reaction conditions includes measuring the emission signal during a denaturation phase of an endpoint cycle of the amplification process, and wherein measuring the emission signal during the second set of reaction conditions includes measuring the emission signal during an annealing and / or extension state of an endpoint cycle of the amplification process.

[0543] Clause 53: The method according to any one of clauses 1 to 48 or 52, wherein the amplification process is an endpoint PCR process.

[0544] Clause 54: A method for detecting nucleic acid in a sample, the method comprising:

[0545] A reaction mixture is provided, the reaction mixture comprising:

[0546] a primer pair that is complementary to the nucleic acid target or its complement for producing an amplicon, and

[0547] a non-cleavable probe configured to hybridize to the amplicon, the non-cleavable probe comprising a detectable label configured to provide an emission signal corresponding to the amount of amplicon produced,

[0548] subjecting the reaction mixture to an amplification process to produce the amplicon, wherein the label produces emission during the amplification process without cleaving the non-cleavable probe, and wherein the amplification process utilizes a series of thermal cycling stages including at least three different target temperatures; and

[0549] The emission signal from the non-cleavable probe is measured.

[0550] Clause 55: The method of clause 54, further comprising quantifying the amount of the nucleic acid target based on the measured emission signal.

[0551] Clause 56: The method according to clause 54 or clause 55, wherein the non-cleavable probe comprises a stem-loop portion, which is capable of forming a stem-loop structure when the non-cleavable probe is single-stranded.

[0552] Clause 57: A method according to any one of clauses 54 to 56, wherein the uncleavable probe comprises a fluorophore and a quencher, and the fluorophore and the quencher are spaced such that when the uncleavable probe is single-stranded, the fluorophore is quenched, but when the probe is incorporated into a double-stranded amplicon, the fluorophore is enabled.

[0553] Clause 58: The method of clause 57, wherein the fluorophore is located at or near the 5' end of the probe and the quencher is 3' to the fluorophore.

[0554] Clause 59: The method of clause 57 or clause 58, wherein both the fluorophore and the quencher are at or near the stem-loop portion of the probe.

[0555] Clause 60: The method according to any one of clauses 54 to 59, wherein the primer pair comprises a tailed primer.

[0556] Clause 61: The method according to clause 60, wherein the tail forms the 5' end of the primer carrying the tail.

[0557] Clause 62: The method of clause 60 or clause 61, wherein the non-cleavable probe is configured to hybridize to the tail or its complement.

[0558] Clause 63: The method according to clause 62, wherein the 3' portion of the non-cleavable probe is configured to hybridize to the tail or its complement.

[0559] Clause 64: The method according to any one of clauses 54 to 63, wherein the amplification process comprises a denaturation temperature and a plurality of different annealing / extension temperatures that vary throughout the amplification process.

[0560] Item 65: A method according to Item 64, wherein a first series of denaturation phases and annealing / extension phases are performed at a first annealing / extension temperature, and wherein a second series of denaturation phases and annealing / extension phases are performed at a second annealing / extension temperature different from the first annealing / extension temperature.

[0561] Clause 66: The method of clause 65, wherein the first annealing / extension temperature is higher than the second annealing / extension temperature.

[0562] Clause 67: A method according to clause 65 or clause 66, wherein the first series of denaturation phases and annealing / extension phases is cycled more times than the second series of denaturation phases and annealing / extension phases.

[0563] Clause 68: A method according to any one of clauses 65 to 67, wherein the amplification process further comprises a third series of denaturation phases and annealing / extension phases using a third annealing / extension temperature.

[0564] Clause 69: The method of clause 68, wherein the third annealing / extension temperature is the same as the first annealing / extension temperature.

[0565] Clause 70: A method according to clause 68 or clause 69, wherein the third series of denaturation phases and annealing / extension phases is cycled more times than the first series of denaturation phases and annealing / extension phases.

[0566] Clause 71: The method according to any one of clauses 65 to 70, wherein for each series of the denaturation stages, the denaturation temperature is the same.

[0567] Clause 72: The method according to any one of clauses 65 to 71, wherein the primer pair further comprises a tailless primer, and wherein the concentration of the primer with the tail in the reaction mixture is different from the concentration of the tailless primer.

[0568] Clause 73: The method according to clause 72, wherein said tailless primer is provided at a higher concentration than said primer with said tail.

[0569] Item 74: A method according to Item 73, wherein the tailless primer is provided at a concentration of about 2X to about 30X the concentration of the primer with the tail, or at a concentration of about 5X to about 25X the concentration of the primer with the tail, or at a concentration of about 10X to about 20X the concentration of the primer with the tail.

[0570] Clause 75: The method according to any one of clauses 74 to 74, wherein the concentration of the non-cleavable probe in the reaction mixture is different from the concentration of the primer with the tail and the concentration of the tailless primer in the reaction mixture.

[0571] Clause 76: The method according to clause 75, wherein the concentration of the non-cleavable probe in the reaction mixture is greater than the concentration of the primer with the tail in the reaction mixture.

[0572] Clause 77: The method according to clause 75 or clause 76, wherein the non-cleavable probe is provided at a concentration lower than the concentration of the tailless primer.

[0573] Clause 78: The method of any one of clauses 75 to 77, wherein the non-cleavable probe is provided at a concentration of about 2X to about 10X the concentration of the tailless primer, or about 3X to about 7.5X the concentration of the tailless primer.

[0574] Clause 79: A method according to any one of clauses 54 or 55,

[0575] The reaction mixture further comprises:

[0576] a primer pair that is complementary to the second nucleic acid target or its complement for producing a second amplicon, and

[0577] a cleavable probe configured to hybridize to the second amplicon, the cleavable probe comprising a detectable label configured to provide an emission signal corresponding to the amount of the second amplicon produced,

[0578] wherein said subjecting said reaction mixture to said amplification process produces a second amplicon, wherein said detectable label of said cleavable probe produces emission due to cleavage of said cleavable probe during said amplification process; and

[0579] Wherein the method further comprises measuring the emission signal from the cleavable probe.

[0580] Clause 80: The method of clause 81, further comprising quantifying the amount of the second nucleic acid target based on the measured emission signal.

[0581] Clause 81: A method for detecting nucleic acid in a sample, the method comprising:

[0582] A reaction mixture is provided, the reaction mixture comprising:

[0583] A primer pair targeting a nucleic acid target for producing an amplicon, the primer pair comprising a primer with the tail and a primer without a tail provided at different concentrations, and

[0584] a detectably labeled, non-cleavable probe configured to hybridize to the amplicon and configured to produce a fluorescent signal corresponding to the amount of amplicon produced,

[0585] subjecting the reaction mixture to an amplification process to produce the amplicons, wherein the non-cleavable probe produces emission without being cleaved during the amplification process; and

[0586] The emission signal from the non-cleavable probe is measured.

[0587] Clause 82: The method according to clause 81, wherein the tailless primer is provided at a higher concentration than the primer with the tail.

[0588] Item 83: A method according to item 82, wherein the tailless primer is provided at a concentration of about 2X to about 30X the concentration of the primer with the tail, or at a concentration of about 5X to about 25X the concentration of the primer with the tail, or at a concentration of about 10X to about 20X the concentration of the primer with the tail.

[0589] Clause 84: The method according to any one of clauses 80 to 83, wherein the non-cleavable probe is provided at a concentration different from the concentration of the primer with the tail and the concentration of the tailless primer.

[0590] Clause 85: The method of clause 84, wherein the non-cleavable probe is provided at a concentration greater than the concentration of the primer with the tail.

[0591] Clause 86: A method according to clause 84 or clause 84, wherein the non-cleavable probe is provided at a concentration lower than the concentration of the tailless primer.

[0592] Clause 87: The method of any one of clauses 84 to 86, wherein the non-cleavable probe is provided at a concentration of about 2X to about 10X the concentration of the tailless primer, or about 3X to about 7.5X the concentration of the tailless primer.

[0593] Clause 88: A method according to clause 81,

[0594] The reaction mixture further comprises:

[0595] a primer pair targeting a second nucleic acid target different from the nucleic acid target and used to produce a second amplicon, the primer pair comprising a primer with the tail and a tailless primer provided at different concentrations, and

[0596] a detectably labeled, cleavable probe configured to hybridize to the second amplicon and configured to generate an emission signal corresponding to the amount of the second amplicon produced;

[0597] wherein subjecting the reaction mixture to an amplification process produces the second amplicon, wherein the cleavable probe produces an emission signal other than due to cleavage during the amplification process; and

[0598] Wherein the method further comprises measuring the emission signal from the cleavable probe.

[0599] Clause 89: A method of detecting the presence or amount of a first target and / or a second target in a reaction mixture, the method comprising:

[0600] including a first probe and a second probe in the reaction mixture, wherein the first probe is capable of specifically interacting with a first target and comprises a first label capable of producing a first detectable signal, and the second probe is capable of specifically interacting with a second target and comprises a second label capable of producing a second detectable signal;

[0601] allowing the first probe and the second probe to specifically interact with any first target and second target in the reaction mixture, respectively;

[0602] measuring a first total signal through an optical filter under a first set of conditions, wherein the first total signal includes the first detectable signal and the second detectable signal from the first label and the second label, and wherein under the first set of conditions, the first detectable signal increases due to the specific interaction of the first probe with the first target, but the second detectable signal does not increase due to the specific interaction of the second probe with the second target;

[0603] measuring a second total signal through the same filter under a second set of conditions, wherein the second total signal includes the first detectable signal and the second detectable signal from the first label and the second label, and wherein under the second set of conditions, the second detectable signal is increased due to the specific interaction of the second probe with the second target; and

[0604] By estimating the first detectable signal and the second detectable signal based on both the first total signal and the second total signal, the presence or amount of the first target and / or the second target is assessed.

[0605] Clause 90: The method of clause 89, wherein the first label and the second label are the same and / or produce substantially the same fluorescence.

[0606] Clause 91: A method according to clause 89 or clause 90, wherein the degree of difference of the second fluorescent signal between the first set of conditions and the second set of conditions is greater than the degree of difference of the first fluorescent signal between the first set of conditions and the second set of conditions.

[0607] Clause 92: The method according to any one of clauses 89 to 91, wherein the first probe is a cleavable probe.

[0608] Clause 93: The method of clause 92, wherein an increase in the first detectable signal indicates that the cleavable probe is cleaved.

[0609] Clause 94: The method of clause 92 or clause 93, wherein the first probe comprises a fluorophore and a quencher, and wherein the first probe is configured such that fluorescence from the fluorophore is quenched by the quencher until the probe is cleaved.

[0610] Clause 95: The method of clause 94, wherein the first probe is a TaqMan probe.

[0611] Clause 96: The method according to any one of clauses 89 to 95, wherein the second probe is a non-cleavable probe.

[0612] Clause 97: The method according to clause 96, wherein the second probe comprises a stem-loop portion, and when the second probe is single-stranded, the stem-loop portion is capable of forming a stem-loop structure.

[0613] Item 98: A method according to item 96 or item 97, wherein the second label of the second probe is a fluorophore, and wherein the second probe further comprises a quencher, and the quencher is spaced such that when the second probe is single-stranded, the fluorophore is quenched, but when the second probe is incorporated into a double-stranded nucleic acid, the fluorophore is enabled.

[0614] Clause 99: The method of clause 98, wherein the fluorophore is located at or near the 5' end of the second probe and the quencher is 3' to the fluorophore.

[0615] Clause 100: The method of clause 98 or clause 99, wherein both the fluorophore and the quencher are disposed at or near the stem-loop portion of the second probe.

[0616] Clause 101: A method according to any one of clauses 89 to 100, wherein the melting temperature (T m ) and the T of the second probe m Within about 8°C, or about 6°C, or about 4°C, or about 2°C of each other.

[0617] Clause 102: The method of any of clauses 89 to 101, wherein the first set of conditions comprises a first measurement temperature at which the first fluorescent signal is measured, and the second set of conditions comprises a second, different measurement temperature at which the second fluorescent signal is measured.

[0618] Clause 103: The method of clause 102, wherein the first measured temperature and the second measured temperature differ by at least about 10°C or more, about 15°C or more, about 20°C or more, about 25°C or more, or about 30°C or more.

[0619] Clause 104: The method of clause 102 or clause 103, wherein at least one of the first measured temperature or the second measured temperature is a denaturation temperature at which the DNA in the reaction mixture denatures, such as about 90°C or above.

[0620] Clause 105: The method of any one of clauses 89 to 104, further comprising thermally cycling the reaction mixture between two target temperatures for a plurality of cycles.

[0621] Item 106: A method according to item 105, wherein the thermal cycle cycles between two target temperatures for at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% of the time.

[0622] Clause 107: The method of any one of clauses 54 to 106, wherein measuring the signal occurs at an endpoint thermal cycle of the amplification process.

[0623] Clause 108: A method according to any one of clauses 1, 2 or 86, wherein the first probe is configured to produce a cumulative signal across different stages of a cycle of an amplification process and the second probe is configured to produce a transient signal during different stages of a cycle of an amplification process.

[0624] Clause 109: The method according to any one of clauses 1 to 108, wherein the second probe is a compound having the formula:

[0625] or a salt thereof, wherein

[0626] Q is an internal quencher moiety having the formula:

[0627]

[0628] B is a divalent nucleobase;

[0629] L 1 It is a divalent linker;

[0630] L 5 is a divalent oligonucleotide linker comprising 4 to 40 nucleotides;

[0631] L 50 is a bond, -NH-, -O-, -S-, -S(O)-, -S(O)2-, -C(O)-, -C(O)NH-, -NHC(O)-, -NHC(O)NH-, -C(O)O-, -OC(O)-, substituted or unsubstituted alkylene, substituted or unsubstituted heteroalkylene, substituted or unsubstituted cycloalkylene, substituted or unsubstituted heterocycloalkylene, substituted or unsubstituted arylene, or substituted or unsubstituted heteroarylene;

[0632] R 50 is the second label or the detectable label;

[0633] R 30 Yes – OR30A ;

[0634] R 30A is a monovalent oligonucleotide moiety;

[0635] R 2 is hydrogen or –OR 2A ;

[0636] R 4 is hydrogen or unsubstituted methyl, or R 2 and R 4 The substituents are linked to form a substituted or unsubstituted heterocycloalkyl group;

[0637] R 1 and R 10 are independently hydrogen, -CCl3, -CBr3, -CF3, -CI3, -CHCl2, -CHBr2, -CHF2, -CHI2, -CH2Cl, -CH2Br, -CH2F, -CH2I, -CN, -OH, -NH2, -COOH, -CONH2, -OCCl3, -OCF3, -OCBr3, -OCI3, -OCHCl2, -OCHBr2, -OCHI2, -OCHF2, -OCH2Cl, -OCH2Br, -OCH2I, -OCH2F, substituted or unsubstituted alkyl, or substituted or unsubstituted heteroalkyl;

[0638] R 6 , R 7 , R 8 and R 9 independently hydrogen, halogen, -CCl3, -CBr3, -CF3, -CI3, -CH2Cl, -CH2Br, -CH2F, -CH2I, -CHCl2, -CHBr2, -CHF2, -CHI2, -CN, -OH, -NH2, -COOH, -CONH2, -NO2, -SH, -SO3R A , -SO2NH2, -NHNH2, -ONH2, -NHC(O)NH2, -NHSO2H, -NHC(O)H, -NHC(O)OH, -NHOH, -OCCl3, -OCBr3, -OCF3, -OCI3, -OCH2Cl, -OCH2Br, -OCH2F, -OCHI, -OCHCl2, -OCHBr2, -OCHF2, -OCHI2, -SF5, -N3, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl;

[0639] R 1 and R 6Capable of connecting to form a substituted or unsubstituted heterocycloalkyl group or a substituted or unsubstituted heteroaryl group;

[0640] R 8 and R 10 capable of being linked to form a substituted or unsubstituted heterocycloalkyl group or a substituted or unsubstituted heteroaryl group; and

[0641] R 2A and R A and -CHCl, -CHBr, -CHF, -CHI, -CHCl, -CHBr, -CHF, -CHI, -CN, -OH, -NH, -COOH, -CONH, -OCCl, -OCF, -OCBr, -OCI, -OCHCl, -OCHBr, -OCHI, -OCHF, -OCHCl, -OCHBr, -OCHI, -OCHF, -OCHCl, -OCHBr, -OCHI, -OCHF, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl.

[0642] Clause 110: The method according to clause 109, wherein when the second probe is single-stranded, L 5 Forming a stem-loop structure.

[0643] Clause 111: The method according to clause 109, wherein R 50 is the fluorophore, and Q and R 50 are spaced apart from each other so that when the second probe is single-stranded, R 50 is quenched, and when the second probe is incorporated into the double-stranded amplicon, it is not quenched.

[0644] Clause 112: The method according to clause 111, wherein Q and R 50 Both are arranged at or near the stem-loop portion of the second probe.

[0645] Clause 113: The method according to clause 109, wherein L 5 Contains 11 to 30 nucleotides.

[0646] Clause 114: The method according to clause 109, wherein L 5 Contains 19 to 23 nucleotides.

[0647] Clause 115: The method according to clause 109, wherein L 5 Contains 4 to 14 nucleotides.

[0648] Clause 116: The method according to clause 109, wherein L 5 Contains 6 to 12 nucleotides.

[0649] Clause 117: The method according to clause 109, wherein the nucleotides are DNA nucleotides.

[0650] Clause 118: The method according to clause 109, wherein the nucleotide is an RNA molecule.

[0651] Clause 119: The method according to clause 109, wherein the compound has the formula:

[0652]

[0653] Clause 120: The method according to clause 109, wherein the compound has the formula:

[0654]

[0655] Clause 121: The method according to clause 109, wherein the compound has the formula:

[0656]

[0657] Clause 122: The method according to clause 109, wherein the compound has the formula:

[0658]

[0659] Item 123: A method according to Item 109, wherein B is divalent cytosine or a derivative thereof, divalent guanine or a derivative thereof, divalent adenine or a derivative thereof, divalent thymine or a derivative thereof, divalent uracil or a derivative thereof, divalent hypoxanthine or a derivative thereof, divalent xanthine or a derivative thereof, divalent 7-methylguanine or a derivative thereof, divalent 5,6-dihydrouracil or a derivative thereof, divalent 5-methylcytosine or a derivative thereof, or a divalent 5-hydroxymethylcytosine or a derivative thereof.

[0660] Clause 124: The method according to clause 109, wherein B is divalent cytosine or a derivative thereof, divalent guanine or a derivative thereof, divalent adenine or a derivative thereof, divalent thymine or a derivative thereof, or divalent uracil or a derivative thereof.

[0661] Clause 125: The method according to clause 109, wherein the compound has the formula:

[0662]

[0663] Clause 126: The method according to clause 109, wherein the compound has the formula:

[0664]

[0665] Clause 127: The method according to clause 109, wherein the compound has the formula:

[0666]

[0667] Clause 128: The method according to clause 109, wherein the compound has the formula:

[0668]

[0669] Clause 129: A method according to clause 109, wherein L 1 YesL 101 -L 102 -L 103 -L 104 -L 105 ;and

[0670] L 101 , L 102 , L 103 , L 104 and L 105 and -NH-, -O-, -S-, -S(O)-, -S(O)2-, -C(O)-, -C(O)NH-, -NHC(O)-, -NHC(O)NH-, -C(O)O-, -OC(O)-, substituted or unsubstituted alkylene, substituted or unsubstituted heteroalkylene, substituted or unsubstituted cycloalkylene, substituted or unsubstituted heterocycloalkylene, substituted or unsubstituted arylene, or substituted or unsubstituted heteroarylene.

[0671] Clause 130: A method according to clause 129, wherein L 101 It is -S(O)2-.

[0672] Clause 131: A method according to clause 129, wherein L 102 is an unsubstituted 3- to 8-membered heterocycloalkyl group.

[0673] Clause 132: A method according to clause 129, wherein L 102 is an unsubstituted piperidinyl.

[0674] Clause 133: A method according to clause 129, wherein L 102 yes

[0675] Clause 134: A method according to clause 129, wherein L 103 It is –C(O)NH-.

[0676] Clause 135: A method according to clause 129, wherein L 104 is an unsubstituted C1-C10 alkylene group, an unsubstituted 2- to 6-membered heteroalkylene group, or an unsubstituted phenylene group.

[0677] Clause 136: A method according to clause 129, wherein L 104 is unsubstituted n-hexylene,

[0678] Clause 137: A method according to clause 129, wherein L 105 is an unsubstituted C1-C 10 alkylene, substituted or unsubstituted 2- to 8-membered heteroalkylene, or unsubstituted 5- to 10-membered heteroarylene.

[0679] Clause 138: A method according to clause 129, wherein L 105 yes

[0680]

[0681] Clause 139: A method according to clause 129, wherein L 1 yes

[0682]

[0683] Clause 140: The method according to clause 109, wherein L 50 is a substituted 2- to 10-membered heteroalkylene.

[0684] Clause 141: A method according to clause 109, wherein L 50 yes

[0685] Clause 142: A method according to clause 109, wherein R 50 It is the fluorescent part.

[0686] Clause 143: The method according to clause 142, wherein R 50 It is a monovalent form of FAM, a monovalent form of VIC, a monovalent form of ABY, a monovalent form of JUN, a monovalent form of AF647, a monovalent form of Cy5, a monovalent form of AF676, or a monovalent form of Cy5.5.

[0687] Clause 144: The method according to clause 109, wherein R 2 It is hydrogen or -OH.

[0688] Clause 145: The method according to clause 109, wherein R2 is hydrogen.

[0689] Clause 146: A method according to any one of clauses 109 to 145, wherein R 30 It is -OH.

[0690] Clause 147: A method according to any one of clauses 109 to 145, wherein R 30 yes

[0691]

[0692] Clause 148: A method according to any one of clauses 109 to 145, wherein the 3' blocking moiety is a monovalent form of dideoxycytidine (3'ddC), a monovalent form of dideoxyadenosine (ddA), 3' reverse dT, a 3' amino modifier, a monovalent form of QSY7, a monovalent form of QSY21, a monovalent form of QSY9, a monovalent form of BHQ1, a monovalent form of BHQ2, a monovalent form of BHQ3, a monovalent form of Dabcyl, a monovalent form of Dabsyl, a monovalent form of Eclipse, a monovalent form of BBQ-650, a monovalent form of Iowa Black RQ, a monovalent form of Iowa Black FQ,

[0693] Clause 149: A composition for detecting nucleic acid in a sample, the composition comprising:

[0694] a first probe detectably labeled with a first label configured to produce a first emission signal,

[0695] a second probe detectably labeled with a second label configured to produce a second emission signal,

[0696] wherein the first probe and the second probe have different sequences, and

[0697] wherein the first label and the second label are identical and / or produce substantially identical emissions

[0698] wherein under a first set of conditions, the first label produces a first emission signal that is increased due to a specific interaction of the first probe with a first nucleic acid target, and the second label produces a second emission signal that is not increased due to a specific interaction of the second probe with a second nucleic acid target; and

[0699] Wherein under a second set of conditions different from the first set of conditions, the second emission signal increases due to the specific interaction of the second probe with the second nucleic acid target.

[0700] Clause 150: The composition of clause 149, wherein the first probe is a cleavable probe.

[0701] Clause 151: A composition according to any of clauses 149 and 150, wherein the first probe comprises a fluorophore and a quencher, and wherein the first probe is configured such that fluorescence from the fluorophore is quenched by the quencher until the probe is cleaved during the annealing / extension phase of the amplification process.

[0702] Clause 152: The composition according to any one of clauses 149 to 151, wherein the first probe is a TaqMan probe.

[0703] Clause 153: A composition according to any one of clauses 149 to 152, wherein the second probe is a non-cleavable probe.

[0704] Clause 154: The composition according to any one of clauses 149 to 152, wherein the second probe comprises a stem-loop portion configured to form a stem-loop structure when the second probe is single-stranded.

[0705] Clause 155: A composition according to any of clauses 153 or 154, wherein the second probe comprises a fluorophore and a quencher spaced apart from each other such that the fluorophore is quenched when the second probe is single-stranded and is not quenched when the second probe is incorporated into a double-stranded amplicon.

[0706] Clause 156: The composition of clause 155, wherein the fluorophore is located at or near the 5' end of the second probe and the quencher is 3' to the fluorophore.

[0707] Clause 157: The composition of any of clauses 155 or 156, wherein both the fluorophore and the quencher are disposed at or near the stem-loop portion of the second probe.

[0708] Item 158: A composition according to any one of items 149 to 157, wherein the reaction mixture further comprises: a first primer pair complementary to a first nucleic acid target of the nucleic acid or its complement, the first nucleic acid target being configured to produce a first amplicon with which the first probe can hybridize; and a second primer pair complementary to a second nucleic acid target of the nucleic acid or its complement, the second nucleic acid target being configured to produce a second amplicon with which the second probe can hybridize.

[0709] Clause 159: The composition of clause 158, wherein the second primer pair comprises a tailed primer.

[0710] Clause 160: The composition of clause 159, wherein the tail forms the 5' end of the primer carrying the tail.

[0711] Clause 161: The composition according to any of clauses 159 or 160, wherein the second probe is capable of hybridizing to the tail or its complement.

[0712] Clause 162: The composition of any one of clauses 159 to 161, wherein the second primer pair further comprises a tailless primer, and wherein the concentration of the primer with the tail in the reaction mixture is different from the concentration of the tailless primer in the reaction mixture.

[0713] Clause 163: The composition of clause 162, wherein the concentration of the tailless primer is greater than the concentration of the primer with the tail.

[0714] Clause 164: A composition according to clause 163, wherein the concentration of the tailless primer is about 2X to about 30X the concentration of the primer with the tail, or about 5X to about 25X the concentration of the primer with the tail, or about 10X to about 20X the concentration of the primer with the tail.

[0715] Clause 165: The composition of any one of clauses 162 to 164, wherein the second probe is provided at a concentration different from the concentration of the primer with the tail and the concentration of the tailless primer.

[0716] Clause 166: The composition of clause 165, wherein the second probe is provided at a concentration greater than the concentration of the primer with the tail.

[0717] Clause 167: The composition according to any of clauses 165 or 166, wherein the second probe is provided at a concentration lower than the concentration of the tailless primer.

[0718] Clause 168: The composition of any one of clauses 165 to 167, wherein the second probe is provided at a concentration of about 2X to about 10X the concentration of the primer with the tail, or about 3X to about 7.5X the concentration of the primer with the tail.

[0719] Clause 169: A composition comprising:

[0720] a primer pair that is complementary to the nucleic acid target or its complement for producing an amplicon, and

[0721] a non-cleavable probe configured to hybridize to the amplicon, the non-cleavable probe comprising a detectable label configured to provide an emission signal corresponding to the amount of amplicon produced,

[0722] Wherein the detectable label produces emission during an amplification process without cleaving the non-cleavable probe, the amplification process comprising a series of thermal cycling stages comprising at least two different target temperatures.

[0723] Clause 170: The composition of clause 169, wherein the composition is a reaction mixture.

[0724] Clause 171: A composition according to clause 169 or 170, wherein the non-cleavable probe comprises a stem-loop portion which is capable of forming a stem-loop structure when the non-cleavable probe is single-stranded.

[0725] Clause 172: A composition according to any one of clauses 159 to 170, wherein the uncleavable probe comprises a fluorophore and a quencher, wherein the fluorophore and the quencher are spaced such that when the uncleavable probe is single-stranded, the fluorophore is quenched, but when the probe is incorporated into a double-stranded amplicon, the fluorophore is enabled.

[0726] Clause 173: The composition of clause 172, wherein the fluorophore is located at or near the 5' end of the probe and the quencher is 3' to the fluorophore.

[0727] Clause 174: The composition of clause 172, wherein both the fluorophore and the quencher are at or near the stem-loop portion of the probe.

[0728] Clause 175: The composition of any one of clauses 169 to 174, wherein the primer pair comprises a tailed primer.

[0729] Clause 176: The composition according to clause 175, wherein the tail forms the 5' end of the primer carrying the tail.

[0730] Clause 177: The composition of clause 175 or clause 176, wherein the non-cleavable probe is configured to hybridize to the tail or its complement.

[0731] Clause 178: The composition of clause 177, wherein the 3' portion of the non-cleavable probe is configured to hybridize to the tail or its complement.

[0732] Clause 179: The composition of any of Clause 169, wherein the primer pair comprises a tailed primer and a tailless primer provided at different concentrations.

[0733] Clause 180: The composition of clause 179, wherein the tailless primer is provided at a higher concentration than the primer with the tail.

[0734] Clause 181: A composition according to clause 180, wherein the tailless primer is provided at a concentration of about 2X to about 30X the concentration of the primer with the tail, or at a concentration of about 5X to about 25X the concentration of the primer with the tail, or at a concentration of about 10X to about 20X the concentration of the primer with the tail.

[0735] Clause 182: The composition of clause 179, wherein the concentration of the non-cleavable probe in the reaction mixture is greater than the concentration of the primer with the tail in the reaction mixture.

[0736] Clause 183: The composition of clause 179, wherein the non-cleavable probe is provided at a concentration lower than the concentration of the tailless primer.

[0737] Clause 184: The composition of any of Clause 183, wherein the non-cleavable probe is provided at a concentration of about 2X to about 10X the concentration of the tailless primer, or about 3X to about 7.5X the concentration of the tailless primer.

[0738] Clause 185: A composition according to any one of clauses 169 to 184,

[0739] The reaction mixture further comprises:

[0740] a primer pair that is complementary to the second nucleic acid target or its complement for producing a second amplicon, and

[0741] a cleavable probe configured to hybridize to the second amplicon, the cleavable probe comprising a detectable label configured to provide an emission signal corresponding to the amount of the second amplicon produced,

[0742] wherein subjecting the reaction mixture to the amplification process produces a second amplicon, wherein the detectable label of the cleavable probe produces emission due to cleavage of the cleavable probe during the amplification process; and

[0743] Wherein the method further comprises measuring the emission signal from the cleavable probe.

[0744] Clause 186: A kit comprising the composition according to any one of clauses 149 to 185.

[0745] Those skilled in the art will appreciate that various other modifications may be made to the structures, arrangements, methods, materials, etc. without departing from the scope and operating principles of the present disclosure. For example, although various embodiments describe methods for detecting two different targets using a first probe and a second probe having spectral similarity, the method may include other numbers of targets and probes to increase the diversity of the overall amplification and detection assay.

[0746] Those of ordinary skill in the art will understand how any feature or operation disclosed herein may be combined with any one or combination of other features and operations disclosed herein. In addition, content or features in any one figure may be used in combination with any content or features used in any other figure. In this regard, content disclosed in any one figure is not mutually exclusive, but may be combined with content from any other figure.

[0747] The described embodiments are to be considered in all respects only as illustrative and not restrictive. All changes coming within the meaning and equivalency range of the claims are intended to be embraced within their scope.

Claims

1. A method for detecting nucleic acid in a sample, the method comprising: (i) providing a reaction mixture, the reaction mixture comprising at least a portion of the sample, a first probe detectably labeled with a first label configured to produce a first emission signal, a second probe detectably labeled with a second label configured to produce a second emission signal, wherein the first probe and the second probe have different sequences, and wherein the first label and the second label are identical and / or produce substantially identical emissions; (ii) subjecting the reaction mixture to an amplification process comprising a first set of reaction conditions and a second set of reaction conditions, the first set of reaction conditions being different from the second set of reaction conditions; as well as (iii) determining the presence of the first nucleic acid target and / or the second nucleic acid target in the sample by the following steps: measuring an emission signal during the first set of reaction conditions, the emission signal during the first set of reaction conditions being associated with a specific interaction, or lack of interaction, between the first probe and the first nucleic acid target, measuring an emission signal during the second set of reaction conditions, the emission signal during the second set of reaction conditions being associated with a specific interaction, or lack thereof, between the first probe and the first nucleic acid target and being associated with a specific interaction, or lack thereof, between the second probe and the second nucleic acid target, and The presence and / or amount of each of the first nucleic acid target and the second nucleic acid target is estimated.

2. A method for detecting nucleic acid in a sample, the method comprising: (i) providing a reaction mixture, the reaction mixture comprising: at least a portion of the sample, a first probe detectably labeled with a first label configured to generate a first emission signal indicative of the presence or absence of a first nucleic acid target, a second probe detectably labeled with a second label configured to generate a second emission signal indicative of the presence or absence of a second nucleic acid target, wherein the first probe and the second probe have different sequences, and wherein the first label and the second label are identical and / or produce substantially identical emission signals; (ii) subjecting the reaction mixture to an amplification process comprising a first set of reaction conditions and a second set of reaction conditions, the first set of reaction conditions being different from the second set of reaction conditions; and (iii) determining any presence and / or amount of the first nucleic acid target and / or the second nucleic acid target in the reaction mixture by the following steps: measuring a first total emission signal during the first set of reaction conditions, the first total emission signal including any first emission signal, if present, and any second emission signal, if present, measuring a second total emission signal during the second set of reaction conditions, the second total emission signal including any first emission signal, if present, and any second emission signal, if present, and The first transmit signal and / or the second transmit signal are estimated based on the first total transmit signal and the second total transmit signal.

3. The method according to claim 1 or 2, wherein the first emission signal and the second emission signal are a first fluorescent signal and a second fluorescent signal, and wherein both the first probe and the second probe are excited at the same wavelength and / or both the first probe and the second probe are excited during detection of their corresponding first fluorescent signals and second fluorescent signals.

4. The method according to any one of claims 1 to 3, wherein the emission signal is a fluorescent signal, and wherein: Measuring the fluorescent signal during the first set of reaction conditions includes measuring a combined signal including the first fluorescent signal and the second fluorescent signal during the first set of reaction conditions to obtain a first total fluorescent signal; Measuring the fluorescent signal during the second set of reaction conditions includes measuring a combined signal including the first fluorescent signal and the second fluorescent signal during the second set of reaction conditions to obtain a second total fluorescent signal; as well as Estimating the presence and / or amount of each of the first nucleic acid target and the second nucleic acid target includes estimating the first fluorescent signal and / or the second fluorescent signal based on the first total fluorescent signal and the second total fluorescent signal.

5. The method of claim 4, wherein the degree of difference between the second fluorescent signal between the first set of reaction conditions and the second set of reaction conditions is greater than the degree of difference between the first fluorescent signal between the first set of reaction conditions and the second set of reaction conditions.

6. The method according to any one of claims 4 to 5, wherein The first total fluorescence value includes (i) fluorescence from a first label that is free and unquenched in the reaction mixture and is emitted due to cleavage of the first label after hybridization of the first probe to the first amplicon, and (ii) background fluorescence of the second label, and The second total fluorescence value is based on (i) fluorescence from the first label, which is free and unquenched in the reaction mixture and is emitted due to cleavage of the first label after hybridization of the first probe to the first amplicon, and (ii) fluorescence from the second label, which is above the background fluorescence of the second label and is emitted due to hybridization of the second probe to the second amplicon.

7. The method according to any one of claims 4 to 6, further comprising: calculating the amount of the first nucleic acid target based on the first fluorescent signal; as well as The amount of the second nucleic acid target is calculated based on the second fluorescent signal.

8. The method of any one of claims 4 to 7, wherein the first fluorescent signal is above background levels during both the first set of reaction conditions and the second set of reaction conditions, indicating the presence of the first nucleic acid target in the reaction mixture.

9. The method of any one of claims 4 to 8, wherein the second fluorescent signal is above background levels during the second set of reaction conditions but not during the first set of reaction conditions, indicating the presence of the second nucleic acid target in the reaction mixture.

10. The method according to any one of claims 5 to 8, wherein the first set of reaction conditions includes a first measurement temperature for measuring the first fluorescence signal, and the second set of reaction conditions includes a second measurement temperature for measuring the second fluorescence signal, the second measurement temperature being different from the first measurement temperature.

11. The method of claim 10, wherein the first measured temperature and the second measured temperature differ by at least about 10°C or more, about 15°C or more, about 20°C or more, about 25°C or more, or about 30°C or more.

12. The method of claim 10 or claim 11, wherein at least one of the first measured temperature or the second measured temperature is a denaturation temperature at which the DNA in the reaction mixture denatures, such as in the range of about 80°C or above.

13. The method according to any one of claims 1 to 12, wherein the reaction mixture is subjected to a plurality of amplification cycles during the amplification process, each of the amplification cycles comprising the first set of reaction conditions and the second set of reaction conditions.

14. The method according to any one of claims 1 to 13, wherein the amplification process comprises thermal cycling.

15. The method of claim 14, wherein subjecting the reaction mixture to the first set of reaction conditions comprises thermally cycling the reaction mixture at a first temperature sufficient to cause denaturation of the first amplicon and the second amplicon.

16. The method of claim 15, wherein subjecting the reaction mixture to the second set of reaction conditions comprises thermally cycling the reaction mixture at a second temperature sufficient to cause annealing and / or extension of the first nucleic acid target and the second nucleic acid target to form the first amplicon and the second amplicon, respectively, the second temperature being lower than the first temperature.

17. The method according to any one of claims 1 to 16, wherein the first probe is a cleavable probe.

18. The method of claim 17, wherein the first emission signal increases as the cleavable probe is cleaved during the annealing / extension phase.

19. A method according to claim 17 or claim 18, wherein the first probe comprises a fluorophore and a quencher, and wherein the first probe is configured such that fluorescence from the fluorophore is quenched by the quencher until the probe is cleaved during the annealing / extension phase of the amplification process.

20. The method of claim 19, wherein the first probe is a TaqMan probe.

21. The method of any one of claims 1 to 20, wherein the second probe is a non-cleavable probe.

22. The method of claim 21, wherein the second probe comprises a stem-loop portion configured to form a stem-loop structure when the second probe is single-stranded.

23. A method according to claim 21 or claim 22, wherein the second probe comprises a fluorophore and a quencher spaced apart from each other such that the fluorophore is quenched when the second probe is single-stranded and is not quenched when the second probe is incorporated into a double-stranded amplicon.

24. The method of claim 23, wherein the fluorophore is located at or near the 5' end of the second probe and the quencher is 3' to the fluorophore.

25. The method of claim 23 or claim 24, wherein both the fluorophore and the quencher are disposed at or near the stem-loop portion of the second probe.

26. A method according to any one of claims 1 to 25, wherein the reaction mixture further comprises: a first primer pair complementary to a first nucleic acid target of the nucleic acid or its complement, the first nucleic acid target being configured to produce a first amplicon with which the first probe can hybridize; and a second primer pair complementary to a second nucleic acid target of the nucleic acid or its complement, the second nucleic acid target being configured to produce a second amplicon with which the second probe can hybridize.

27. The method of claim 26, wherein the second primer pair comprises a tailed primer.

28. The method of claim 27, wherein the tail forms the 5' end of the primer carrying the tail.

29. A method according to claim 27 or claim 28, wherein the second probe is capable of hybridising to the tail or its complement.

30. The method of any one of claims 20 to 29, wherein the amplification process utilizes a series of thermal cycling stages comprising at least three different target temperatures.

31. The method of claim 30, wherein the amplification process comprises a denaturation temperature and a plurality of different annealing / extension temperatures that vary throughout the amplification process.

32. A method according to claim 31, wherein a first series of denaturation phases and annealing / extension phases are performed at a first annealing / extension temperature, and wherein a second series of denaturation phases and annealing / extension phases are performed at a second annealing / extension temperature different from the first annealing / extension temperature.

33. The method of claim 32, wherein the first annealing / extension temperature is higher than the second annealing / extension temperature.

34. A method according to claim 32 or claim 33, wherein the first series of denaturation phases and annealing / extension phases is cycled more times than the second series of denaturation phases and annealing / extension phases.

35. The method according to any one of claims 32 to 34, wherein the amplification process further comprises a third series of denaturation steps and annealing / extension steps performed using a third annealing / extension temperature.

36. The method of claim 35, wherein the third annealing / extension temperature is the same as the first annealing / extension temperature.

37. A method according to claim 35 or claim 36, wherein the third series of denaturation phases and annealing / extension phases is cycled more times than the first series of denaturation phases and annealing / extension phases.

38. A method according to any one of claims 32 to 37, wherein for each series of the denaturation stages, the denaturation temperature is the same.

39. The method of any one of claims 2738, wherein the second primer pair further comprises a tailless primer, and wherein the concentration of the primer with the tail in the reaction mixture is different from the concentration of the tailless primer in the reaction mixture.

40. The method of claim 39, wherein the concentration of the tailless primer is greater than the concentration of the primer with the tail.

41. The method of claim 40, wherein the concentration of the tailless primer is about 2X to about 30X the concentration of the primer with the tail, or about 5X to about 25X the concentration of the primer with the tail, or about 10X to about 20X the concentration of the primer with the tail.

42. The method of any one of claims 39 to 41, wherein the second probe is provided at a concentration that is different from the concentration of the primer with the tail and the concentration of the tailless primer.

43. The method of claim 42, wherein the second probe is provided at a concentration greater than the concentration of the primer with the tail.

44. The method of claim 42 or claim 43, wherein the second probe is provided at a concentration lower than the concentration of the tailless primer.

45. The method of any one of claims 42 to 44, wherein the second probe is provided at a concentration of about 2X to about 10X the concentration of the primer with the tail, or about 3X to about 7.5X the concentration of the primer with the tail.

46. ​​according to the method described in any one of claims 1 to 45, wherein the melting temperature (T m ) and the T of the second probe m Within about 8°C, or about 6°C, or about 4°C, or about 2°C of each other.

47. The method of any one of claims 1 to 48, wherein for multiple cycles of the amplification process, the amplification process is cycled between at least two target temperatures.

48. A method according to claim 47, wherein the amplification process cycles between at least two target temperatures for at least 5% of the cycles of the amplification process, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% or at least 95%.

49. The method of any one of claims 1 to 48, wherein the method further comprises dividing the reaction mixture into a plurality of reaction volumes, and wherein the amplification process is a digital PCR (dPCR) process.

50. The method of claim 49, wherein measuring the emission signal during the first set of reaction conditions comprises measuring the emission signal at or near completion of subjecting the reaction mixture to the first set of reaction conditions to obtain a first endpoint measurement, and wherein measuring the emission signal during the second set of reaction conditions comprises measuring the emission signal at or near completion of subjecting the reaction mixture to the second set of reaction conditions to obtain a second endpoint measurement.

51. The method of claim 50, wherein estimating the presence and / or amount of each of the first nucleic acid target and the second nucleic acid target comprises: classifying the plurality of reaction volumes according to the emission signal measured at the first endpoint metric and according to the emission signal measured at the second endpoint metric; as well as Based on the classification, a count of the plurality of reaction volumes in which the first probe shows activity and a count of the plurality of reaction volumes in which the second probe shows activity are determined.

52. A method according to any one of claims 1 to 48, wherein measuring the emission signal during the first set of reaction conditions includes measuring the emission signal during a denaturation phase of an endpoint cycle of the amplification process, and wherein measuring the emission signal during the second set of reaction conditions includes measuring the emission signal during an annealing and / or extension state of an endpoint cycle of the amplification process.

53. The method of any one of claims 1 to 48 or 52, wherein the amplification process is an endpoint PCR process.

54. A method for detecting nucleic acid in a sample, the method comprising: A reaction mixture is provided, the reaction mixture comprising: a primer pair that is complementary to the nucleic acid target or its complement for producing an amplicon, and a non-cleavable probe configured to hybridize to the amplicon, the non-cleavable probe comprising a detectable label configured to provide an emission signal corresponding to the amount of amplicon produced, subjecting the reaction mixture to an amplification process to produce the amplicon, wherein the label produces emission during the amplification process without cleaving the non-cleavable probe, and wherein the amplification process utilizes a series of thermal cycling stages including at least three different target temperatures; as well as The emission signal from the non-cleavable probe is measured.

55. The method of claim 54, further comprising quantifying the amount of the nucleic acid target based on the measured emission signal.

56. The method of claim 54 or claim 55, wherein the non-cleavable probe comprises a stem-loop portion that is capable of forming a stem-loop structure when the non-cleavable probe is single-stranded.

57. The method of any one of claims 54 to 56, wherein the non-cleavable probe comprises a fluorophore and a quencher, wherein the fluorophore and the quencher are spaced such that when the non-cleavable probe is single-stranded, the fluorophore is quenched, but when the probe is incorporated into a double-stranded amplicon, the fluorophore is enabled.

58. The method of claim 57, wherein the fluorophore is located at or near the 5' end of the probe and the quencher is 3' to the fluorophore.

59. The method of claim 57 or claim 58, wherein both the fluorophore and the quencher are at or near the stem-loop portion of the probe.

60. The method of any one of claims 54 to 59, wherein the primer pair comprises a tailed primer.

61. The method of claim 60, wherein the tail forms the 5' end of the primer carrying the tail.

62. The method of claim 60 or claim 61, wherein the non-cleavable probe is configured to hybridize to the tail or its complement.

63. The method of claim 62, wherein the 3' portion of the non-cleavable probe is configured to hybridize to the tail or its complement.

64. The method of any one of claims 54 to 63, wherein the amplification process comprises a denaturation temperature and a plurality of different annealing / extension temperatures that vary throughout the amplification process.

65. A method according to claim 64, wherein a first series of denaturation phases and annealing / extension phases are performed at a first annealing / extension temperature, and wherein a second series of denaturation phases and annealing / extension phases are performed at a second annealing / extension temperature different from the first annealing / extension temperature.

66. The method of claim 65, wherein the first annealing / extension temperature is higher than the second annealing / extension temperature.

67. A method according to claim 65 or claim 66, wherein the first series of denaturation phases and annealing / extension phases is cycled more times than the second series of denaturation phases and annealing / extension phases.

68. The method of any one of claims 65 to 67, wherein the amplification process further comprises a third series of denaturation phases and annealing / extension phases performed using a third annealing / extension temperature.

69. The method of claim 68, wherein the third annealing / extension temperature is the same as the first annealing / extension temperature.

70. The method of claim 68 or claim 69, wherein the third series of denaturation phases and annealing / extension phases is cycled more times than the first series of denaturation phases and annealing / extension phases.

71. A method according to any one of claims 65 to 70, wherein the denaturation temperature is the same for each series of the denaturation stages.

72. The method of any one of claims 65 to 71, wherein the primer pair further comprises a tailless primer, and wherein the concentration of the primer with the tail in the reaction mixture is different from the concentration of the tailless primer.

73. The method of claim 72, wherein the tailless primer is provided at a higher concentration than the primer with the tail.

74. The method of claim 73, wherein the tailless primer is provided at a concentration of about 2X to about 30X the concentration of the primer with the tail, or at about 5X to about 25X the concentration of the primer with the tail, or at a concentration of about 10X to about 20X the concentration of the primer with the tail.

75. The method of any one of claims 74 to 74, wherein the concentration of the non-cleavable probe in the reaction mixture is different from the concentration of the primer with the tail and the concentration of the tailless primer in the reaction mixture.

76. The method of claim 75, wherein the concentration of the non-cleavable probe in the reaction mixture is greater than the concentration of the primer with the tail in the reaction mixture.

77. The method of claim 75 or claim 76, wherein the non-cleavable probe is provided at a concentration lower than the concentration of the tailless primer.

78. The method of any one of claims 75 to 77, wherein the non-cleavable probe is provided at a concentration of about 2X to about 10X the concentration of the tailless primer, or about 3X to about 7.5X the concentration of the tailless primer.

79. The method according to any one of claims 54 or 55, The reaction mixture further comprises: a primer pair that is complementary to the second nucleic acid target or its complement for producing a second amplicon, and a cleavable probe configured to hybridize to the second amplicon, the cleavable probe comprising a detectable label configured to provide an emission signal corresponding to the amount of the second amplicon produced, wherein said subjecting said reaction mixture to said amplification process produces a second amplicon, wherein said detectable label of said cleavable probe produces emission due to cleavage of said cleavable probe during said amplification process; and Wherein the method further comprises measuring the emission signal from the cleavable probe.

80. The method of claim 81, further comprising quantifying the amount of the second nucleic acid target based on the measured emission signal.

81. A method for detecting nucleic acid in a sample, the method comprising: A reaction mixture is provided, the reaction mixture comprising: A primer pair targeting a nucleic acid target for producing an amplicon, the primer pair comprising a primer with the tail and a primer without a tail provided at different concentrations, and a detectably labeled, non-cleavable probe configured to hybridize to the amplicon and configured to produce a fluorescent signal corresponding to the amount of amplicon produced, subjecting the reaction mixture to an amplification process to produce the amplicons, wherein the non-cleavable probe produces emission without being cleaved during the amplification process; and The emission signal from the non-cleavable probe is measured.

82. The method of claim 81, wherein the tailless primer is provided at a higher concentration than the primer with the tail.

83. The method of claim 82, wherein the tailless primer is provided at a concentration of about 2X to about 30X the concentration of the primer with the tail, or at about 5X to about 25X the concentration of the primer with the tail, or at a concentration of about 10X to about 20X the concentration of the primer with the tail.

84. The method of any one of claims 80 to 83, wherein the non-cleavable probe is provided at a concentration that is different from the concentration of the primer with the tail and the concentration of the tailless primer.

85. The method of claim 84, wherein the non-cleavable probe is provided at a concentration greater than the concentration of the primer with the tail.

86. The method of claim 84 or claim 84, wherein the non-cleavable probe is provided at a concentration lower than the concentration of the tailless primer.

87. The method of any one of claims 84 to 86, wherein the non-cleavable probe is provided at a concentration of about 2X to about 10X the concentration of the tailless primer, or about 3X to about 7.5X the concentration of the tailless primer.

88. The method according to claim 81, The reaction mixture further comprises: a primer pair targeting a second nucleic acid target different from the nucleic acid target and used to produce a second amplicon, the primer pair comprising a primer with the tail and a tailless primer provided at different concentrations, and a detectably labeled, cleavable probe configured to hybridize to the second amplicon and configured to generate an emission signal corresponding to the amount of the second amplicon produced; wherein subjecting the reaction mixture to an amplification process produces the second amplicon, wherein the cleavable probe produces an emission signal other than due to cleavage during the amplification process; and Wherein the method further comprises measuring the emission signal from the cleavable probe.

89. A method for detecting the presence or amount of a first target and / or a second target in a reaction mixture, the method comprising: including a first probe and a second probe in the reaction mixture, wherein the first probe is capable of specifically interacting with a first target and comprises a first label capable of producing a first detectable signal, and the second probe is capable of specifically interacting with a second target and comprises a second label capable of producing a second detectable signal; allowing the first probe and the second probe to specifically interact with any first target and second target in the reaction mixture, respectively; measuring a first total signal through an optical filter under a first set of conditions, wherein the first total signal includes the first detectable signal and the second detectable signal from the first label and the second label, and wherein under the first set of conditions, the first detectable signal increases due to the specific interaction of the first probe with the first target, but the second detectable signal does not increase due to the specific interaction of the second probe with the second target; measuring a second total signal through the same filter under a second set of conditions, wherein the second total signal includes the first detectable signal and the second detectable signal from the first label and the second label, and wherein under the second set of conditions, the second detectable signal is increased due to the specific interaction of the second probe with the second target; as well as By estimating the first detectable signal and the second detectable signal based on both the first total signal and the second total signal, the presence or amount of the first target and / or the second target is assessed.

90. The method of claim 89, wherein the first label and the second label are the same and / or produce substantially the same fluorescence.

91. A method according to claim 89 or claim 90, wherein the degree of difference in the second fluorescent signal between the first set of conditions and the second set of conditions is greater than the degree of difference in the first fluorescent signal between the first set of conditions and the second set of conditions.

92. The method of any one of claims 89 to 91, wherein the first probe is a cleavable probe.

93. The method of claim 92, wherein the increase in the first detectable signal indicates that the cleavable probe is cleaved.

94. A method according to claim 92 or claim 93, wherein the first probe comprises a fluorophore and a quencher, and wherein the first probe is configured such that fluorescence from the fluorophore is quenched by the quencher until the probe is cleaved.

95. The method of claim 94, wherein the first probe is a TaqMan probe.

96. The method of any one of claims 89 to 95, wherein the second probe is a non-cleavable probe.

97. The method of claim 96, wherein the second probe comprises a stem-loop portion that is capable of forming a stem-loop structure when the second probe is single-stranded.

98. A method according to claim 96 or claim 97, wherein the second label of the second probe is a fluorophore, and wherein the second probe further comprises a quencher, and the quencher is spaced such that when the second probe is single-stranded, the fluorophore is quenched, but when the second probe is incorporated into a double-stranded nucleic acid, the fluorophore is enabled.

99. The method of claim 98, wherein the fluorophore is located at or near the 5' end of the second probe and the quencher is 3' to the fluorophore.

100. The method of claim 98 or claim 99, wherein both the fluorophore and the quencher are disposed at or near the stem-loop portion of the second probe.

101. according to the method described in any one of claims 89 to 100, wherein the melting temperature (T m ) and the T of the second probe m Within about 8°C, or about 6°C, or about 4°C, or about 2°C of each other.

102. The method of any one of claims 89 to 101, wherein the first set of conditions comprises a first measurement temperature at which the first fluorescent signal is measured, and the second set of conditions comprises a second, different measurement temperature at which the second fluorescent signal is measured.

103. The method of claim 102, wherein the first measured temperature and the second measured temperature differ by at least about 10°C or more, about 15°C or more, about 20°C or more, about 25°C or more, or about 30°C or more.

104. The method of claim 102 or claim 103, wherein at least one of the first measured temperature or the second measured temperature is a denaturation temperature at which the DNA in the reaction mixture denatures, such as about 90°C or above.

105. The method of any one of claims 89 to 104, further comprising thermally cycling the reaction mixture between two target temperatures for a plurality of cycles.

106. The method of claim 105, wherein the thermal cycle cycles between two target temperatures for at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% of the cycle.

107. The method of any one of claims 54 to 106, wherein measuring the signal occurs at an endpoint thermal cycle of the amplification process.

108. A method according to any one of claims 1, 2 or 86, wherein the first probe is configured to produce a cumulative signal across different stages of a cycle of an amplification process, and the second probe is configured to produce a transient signal during different stages of a cycle of an amplification process.

109. The method of any one of claims 1 to 108, wherein the second probe is a compound having the formula: or a salt thereof, wherein Q is an internal quencher moiety having the formula: B is a divalent nucleobase; L 1 It is a divalent linker; L 5 is a divalent oligonucleotide linker comprising 4 to 40 nucleotides; L 50 is a bond, -NH-, -O-, -S-, -S(O)-, -S(O)2-, -C(O)-, -C(O)NH-, -NHC(O)-, -NHC(O)NH-, -C(O)O-, -OC(O)-, substituted or unsubstituted alkylene, substituted or unsubstituted heteroalkylene, substituted or unsubstituted cycloalkylene, substituted or unsubstituted heterocycloalkylene, substituted or unsubstituted arylene, or substituted or unsubstituted heteroarylene; R 50 is the second label or the detectable label; R 30 is – OR 30A ; R 30A is a monovalent oligonucleotide moiety; R 2 is hydrogen or –OR 2A ; R 4 is hydrogen or unsubstituted methyl, or R 2 and R 4 The substituents are linked to form a substituted or unsubstituted heterocycloalkyl group; R 1 and R 10 are independently hydrogen, -CCl3, -CBr3, -CF3, -CI3, -CHCl2, -CHBr2, -CHF2, -CHI2, -CH2Cl, -CH2Br, -CH2F, -CH2I, -CN, -OH, -NH2, -COOH, -CONH2, -OCCl3, -OCF3, -OCBr3, -OCI3, -OCHCl2, -OCHBr2, -OCHI2, -OCHF2, -OCH2Cl, -OCH2Br, -OCH2I, -OCH2F, substituted or unsubstituted alkyl, or substituted or unsubstituted heteroalkyl; R 6 , R 7 , R 8 and R 9 independently hydrogen, halogen, -CCl3, -CBr3, -CF3, -CI3, -CH2Cl, -CH2Br, -CH2F, -CH2I, -CHCl2, -CHBr2, -CHF2, -CHI2, -CN, -OH, -NH2, -COOH, -CONH2, -NO2, -SH, -SO3R A , -SO2NH2, -NHNH2, -ONH2, -NHC(O)NH2, -NHSO2H, -NHC(O)H, -NHC(O)OH, -NHOH, -OCCl3, -OCBr3, -OCF3, -OCI3, -OCH2Cl, -OCH2Br, -OCH2F, -OCHI, -OCHCl2, -OCHBr2, -OCHF2, -OCHI2, -SF5, -N3, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl; R 1 and R 6 Capable of connecting to form a substituted or unsubstituted heterocycloalkyl group or a substituted or unsubstituted heteroaryl group; R 8 and R 10 capable of being linked to form a substituted or unsubstituted heterocycloalkyl group or a substituted or unsubstituted heteroaryl group; and R 2A and R A and -CHCl, -CHBr, -CHF, -CHI, -CHCl, -CHBr, -CHF, -CHI, -CN, -OH, -NH, -COOH, -CONH, -OCCl, -OCF, -OCBr, -OCI, -OCHCl, -OCHBr, -OCHI, -OCHF, -OCHCl, -OCHBr, -OCHI, -OCHF, -OCHCl, -OCHBr, -OCHI, -OCHF, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl.

110. The method of claim 109, wherein when the second probe is single-stranded, L 5 Forming a stem-loop structure.

111. The method of claim 109, wherein R 50 is the fluorophore, and Q and R 50 are spaced apart from each other so that when the second probe is single-stranded, R 50 is quenched, and when the second probe is incorporated into the double-stranded amplicon, it is not quenched.

112. The method of claim 111, wherein Q and R 50 Both are arranged at or near the stem-loop portion of the second probe.

113. The method of claim 109, wherein L 5 Contains 11 to 30 nucleotides.

114. The method of claim 109, wherein L 5 Contains 19 to 23 nucleotides.

115. The method of claim 109, wherein L 5 Contains 4 to 14 nucleotides.

116. The method of claim 109, wherein L 5 Contains 6 to 12 nucleotides.

117. The method of claim 109, wherein the nucleotides are DNA nucleotides.

118. The method of claim 109, wherein the nucleotide is an RNA molecule.

119. The method of claim 109, wherein the compound has the formula:

120. The method of claim 109, wherein the compound has the formula:

121. The method of claim 109, wherein the compound has the formula:

122. The method of claim 109, wherein the compound has the formula:

123. The method of claim 109, wherein B is a divalent cytosine or a derivative thereof, a divalent guanine or a derivative thereof, a divalent adenine or a derivative thereof, a divalent thymine or a derivative thereof, a divalent uracil or a derivative thereof, a divalent hypoxanthine or a derivative thereof, a divalent xanthine or a derivative thereof, a divalent 7-methylguanine or a derivative thereof, a divalent 5,6-dihydrouracil or a derivative thereof, a divalent 5-methylcytosine or a derivative thereof, or a divalent 5-hydroxymethylcytosine or a derivative thereof.

124. The method of claim 109, wherein B is a divalent cytosine or a derivative thereof, a divalent guanine or a derivative thereof, a divalent adenine or a derivative thereof, a divalent thymine or a derivative thereof, or a divalent uracil or a derivative thereof.

125. The method of claim 109, wherein the compound has the formula:

126. The method of claim 109, wherein the compound has the formula:

127. The method of claim 109, wherein the compound has the formula:

128. The method of claim 109, wherein the compound has the formula:

129. The method of claim 109, wherein L 1 YesL 101 -L 102 -L 103 -L 104 -L 105 ;and L 101 , L 102 , L 103 , L 104 and L 105 are independently a bond, -NH-, -O-, -S-, -S(O)-, -S(O)2-, -C(O)-, -C(O)NH-, -NHC(O)-, -NHC(O)NH-, - C(O)O-, -OC(O)-, substituted or unsubstituted alkylene, substituted or unsubstituted heteroalkylene, substituted or unsubstituted cycloalkylene, substituted or unsubstituted heterocycloalkylene, substituted or unsubstituted arylene, or substituted or unsubstituted heteroarylene.

130. The method of claim 129, wherein L 101 It is -S(O)2-.

131. The method of claim 129, wherein L 102 is an unsubstituted 3- to 8-membered heterocycloalkyl group.

132. The method of claim 129, wherein L 102 is an unsubstituted piperidinyl.

133. The method of claim 129, wherein L 102 yes 134. The method of claim 129, wherein L 103 It is –C(O)NH-.

135. The method of claim 129, wherein L 104 is an unsubstituted C1-C10 alkylene group, an unsubstituted 2- to 6-membered heteroalkylene group, or an unsubstituted phenylene group.

136. The method of claim 129, wherein L 104 is an unsubstituted n-hexylene, 137. The method of claim 129, wherein L 105 is an unsubstituted C1-C 10 alkylene, substituted or unsubstituted 2- to 8-membered heteroalkylene, or unsubstituted 5- to 10-membered heteroarylene.

138. The method of claim 129, wherein L 105 yes 139. The method of claim 129, wherein L 1 yes or 140. The method of claim 109, wherein L 50 is a substituted 2- to 10-membered heteroalkylene.

141. The method of claim 109, wherein L 50 yes 142. The method of claim 109, wherein R 50 It is the fluorescent part.

143. The method of claim 142, wherein R 50 It is a monovalent form of FAM, a monovalent form of VIC, a monovalent form of ABY, a monovalent form of JUN, a monovalent form of AF647, a monovalent form of Cy5, a monovalent form of AF676, or a monovalent form of Cy5.

5.

144. The method of claim 109, wherein R 2 It is hydrogen or -OH.

145. The method of claim 109, wherein R2 is hydrogen.

146. The method of any one of claims 109 to 145, wherein R 30 It is -OH.

147. The method of any one of claims 109 to 145, wherein R 30 for 148. The method of any one of claims 109 to 145, wherein the 3' blocking moiety is a monovalent form of dideoxycytidine (3'ddC), a monovalent form of dideoxyadenosine (ddA), a 3' inverted dT, a 3' amino modifier, a monovalent form of QSY7, a monovalent form of QSY21, a monovalent form of QSY9, a monovalent form of BHQ1, a monovalent form of BHQ2, a monovalent form of BHQ3, a monovalent form of Dabcyl, a monovalent form of Dabsyl, a monovalent form of Eclipse, a monovalent form of BBQ-650, a monovalent form of Iowa Black RQ, a monovalent form of Iowa Black FQ, 149. A composition for detecting nucleic acid in a sample, the composition comprising: a first probe detectably labeled with a first label configured to produce a first emission signal, a second probe detectably labeled with a second label configured to produce a second emission signal, wherein the first probe and the second probe have different sequences, and wherein the first label and the second label are identical and / or produce substantially identical emissions wherein under a first set of conditions, the first label produces a first emission signal that is increased due to a specific interaction of the first probe with a first nucleic acid target, and the second label produces a second emission signal that is not increased due to a specific interaction of the second probe with a second nucleic acid target; and Wherein under a second set of conditions different from the first set of conditions, the second emission signal increases due to the specific interaction of the second probe with the second nucleic acid target.

150. The composition of claim 149, wherein the first probe is a cleavable probe.

151. A composition according to any one of claims 149 and 150, wherein the first probe comprises a fluorophore and a quencher, and wherein the first probe is configured such that fluorescence from the fluorophore is quenched by the quencher until the probe is cleaved during the annealing / extension phase of the amplification process.

152. The composition of any one of claims 149 to 151, wherein the first probe is a TaqMan probe.

153. The composition of any one of claims 149 to 152, wherein the second probe is a non-cleavable probe.

154. The composition of any one of claims 149 to 152, wherein the second probe comprises a stem-loop portion configured to form a stem-loop structure when the second probe is single-stranded.

155. A composition according to any one of claims 153 or 154, wherein the second probe comprises a fluorophore and a quencher spaced apart from each other such that the fluorophore is quenched when the second probe is single-stranded and the fluorophore is not quenched when the second probe is incorporated into a double-stranded amplicon.

156. The composition of claim 155, wherein the fluorophore is located at or near the 5' end of the second probe and the quencher is 3' to the fluorophore.

157. A composition according to any one of claims 155 or claim 156, wherein both the fluorophore and the quencher are disposed at or near the stem-loop portion of the second probe.

158. A composition according to any one of claims 149 to 157, wherein the reaction mixture further comprises: a first primer pair complementary to a first nucleic acid target of the nucleic acid or its complement, the first nucleic acid target being configured to produce a first amplicon with which the first probe can hybridize; and a second primer pair complementary to a second nucleic acid target of the nucleic acid or its complement, the second nucleic acid target being configured to produce a second amplicon with which the second probe can hybridize.

159. The composition of claim 158, wherein the second primer pair comprises a tailed primer.

160. The composition of claim 159, wherein the tail forms the 5' end of the primer carrying the tail.

161. The composition of any one of claims 159 or 160, wherein the second probe is capable of hybridizing to the tail or its complement.

162. The composition of any one of claims 159 to 161, wherein the second primer pair further comprises a tailless primer, and wherein the concentration of the primer with the tail in the reaction mixture is different from the concentration of the tailless primer in the reaction mixture.

163. The composition of claim 162, wherein the concentration of the tailless primer is greater than the concentration of the primer with the tail.

164. The composition of claim 163, wherein the concentration of the tailless primer is about 2X to about 30X the concentration of the primer with the tail, or about 5X to about 25X the concentration of the primer with the tail, or about 10X to about 20X the concentration of the primer with the tail.

165. The composition of any one of claims 162 to 164, wherein the second probe is provided at a concentration different from the concentration of the primer with the tail and the concentration of the tailless primer.

166. The composition of claim 165, wherein the second probe is provided at a concentration greater than the concentration of the primer with the tail.

167. The composition of any one of claims 165 or 166, wherein the second probe is provided at a concentration lower than the concentration of the tailless primer.

168. The composition of any one of claims 165 to 167, wherein the second probe is provided at a concentration of about 2X to about 10X the concentration of the primer with the tail, or about 3X to about 7.5X the concentration of the primer with the tail.

169. A composition comprising: a primer pair that is complementary to the nucleic acid target or its complement for producing an amplicon, and a non-cleavable probe configured to hybridize to the amplicon, the non-cleavable probe comprising a detectable label configured to provide an emission signal corresponding to the amount of amplicon produced, Wherein the detectable label produces emission during an amplification process without cleaving the non-cleavable probe, the amplification process comprising a series of thermal cycling stages comprising at least two different target temperatures.

170. The composition of claim 169, wherein the composition is a reaction mixture.

171. A composition according to claim 169 or claim 170, wherein the non-cleavable probe comprises a stem-loop portion, which is capable of forming a stem-loop structure when the non-cleavable probe is single-stranded.

172. A composition according to any one of claims 159 to 170, wherein the non-cleavable probe comprises a fluorophore and a quencher, and the fluorophore and the quencher are spaced such that when the non-cleavable probe is single-stranded, the fluorophore is quenched, but when the probe is incorporated into a double-stranded amplicon, the fluorophore is enabled.

173. The composition of claim 172, wherein the fluorophore is located at or near the 5' end of the probe and the quencher is 3' to the fluorophore.

174. The composition of claim 172, wherein both the fluorophore and the quencher are at or near the stem-loop portion of the probe.

175. The composition of any one of claims 169 to 174, wherein the primer pair comprises a tailed primer.

176. The composition of claim 175, wherein the tail forms the 5' end of the primer carrying the tail.

177. A composition according to claim 175 or claim 176, wherein the non-cleavable probe is configured to hybridize to the tail or its complement.

178. The composition of claim 177, wherein the 3' portion of the non-cleavable probe is configured to hybridize to the tail or its complement.

179. The composition of any one of claims 169, wherein the primer pairs comprise tailed primers and tailless primers provided at different concentrations.

180. The composition of claim 179, wherein the tailless primer is provided at a higher concentration than the primer with the tail.

181. The composition of claim 180, wherein the tailless primer is provided at a concentration of about 2X to about 30X the concentration of the primer with the tail, or about 5X to about 25X the concentration of the primer with the tail, or about 10X to about 20X the concentration of the primer with the tail.

182. The composition of claim 179, wherein the concentration of the non-cleavable probe in the reaction mixture is greater than the concentration of the primer with the tail in the reaction mixture.

183. The composition of claim 179, wherein the non-cleavable probe is provided at a concentration lower than the concentration of the tailless primer.

184. The composition of any one of claims 183, wherein the non-cleavable probe is provided at a concentration of about 2X to about 10X the concentration of the tailless primer, or about 3X to about 7.5X the concentration of the tailless primer.

185. A composition according to any one of claims 169 to 184, The reaction mixture further comprises: a primer pair that is complementary to the second nucleic acid target or its complement for producing a second amplicon, and a cleavable probe configured to hybridize to the second amplicon, the cleavable probe comprising a detectable label configured to provide an emission signal corresponding to the amount of the second amplicon produced, wherein subjecting the reaction mixture to the amplification process produces a second amplicon, wherein the detectable label of the cleavable probe produces emission due to cleavage of the cleavable probe during the amplification process; and Wherein the method further comprises measuring the emission signal from the cleavable probe.

186. A kit comprising the composition of any one of claims 149 to 185.

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