CYCLE MULTIPLEXING FOR HIGHLY MULTIPLEXED QUANTITATIVE PCR

CROSS-REFERENCE TO RELATED APPLICATION AND INCORPORATION OF SEQUENCE LISTING

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 63/138,307, filed January 15, 2021, which is incorporated by reference herein in its entirety. This application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on January 13, 2022, is named P35047WO00_SL.txt, and is 1,945 bytes in size measured in Microsoft Windows®.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

[0002] This invention was made with United States government support under Grant No. R01CA203964 awarded by the National Institutes of Health. The United States government has certain rights in the invention.

FIELD

[0003] The present disclosure relates to the field of molecular biology. More particularly, it relates to methods and compositions useful for the detection, amplification, and quantification of nucleic acid molecules.

INCORPORATION OF SEQUENCE LISTING

[0004] Table 1 provides nucleic acid sequences used in this application.

Table 1. Nucleic acid sequences BACKGROUND

[0005] Multiplex quantitative polymerase chain reaction (multiplex qPCR) is a widely accepted molecular diagnostic technique for detecting and quantitating multiple different DNA target sequences. In multiplex qPCR, a single DNA sample comprising all, some, or none, of a set of DNA targets is mixed with a set of PCR primers, a set of fluorescent probes, a DNA polymerase, and a buffer and reagents required for DNA polymerase function.

[0006] In many embodiments of multiplex qPCR, the DNA polymerase used has a 5' to 3' exonuclease activity, and the DNA probes are TaqMan™ probes functionalized with a fluorophore at one end of the probe, and a fluorescence quencher at the other end of the probe. During the course of PCR amplification, PCR amplicons corresponding to the DNA target sequences will increase in concentration if the DNA target was initially present in the sample. As the PCR amplicons accumulate, the amplicons bind to the TaqMan™ probes and cause the hydrolysis of the phosphodiester backbone on the probes. The binding results in the delocalization of the fluorophore from the fluorescence quencher, which causes in increased solution fluorescence.

[0007] In multiplex qPCR, typically, TaqMan™ probes corresponding to different DNA targets will be functionalized with spectrally distinct fluorophores, so that the detection of a specific fluorescence signal corresponds to the presence of a specific DNA target. Due to physical and chemical property limitations, there are only a finite number of fluorophores that can be spectrally distinguished; depending on the exact fluorophores and instruments used, this limit is between 5 and 10 fluorophores.

[0008] For applications in oncology and in infectious disease syndromic testing, there may be more than 20 relevant DNA target sequences. Although it is possible in some cases to separate a DNA sample into multiple aliquots, with each aliquot undergoing a different multiplex qPCR reaction to detect a different set of DNA targets, in practice the extra labor involved and higher sample volumes requirements render this an undesirable solution. Methods for increasing the number of DNA targets that can be detected above the number of spectrally distinct fluorophores are thus in demand.

SUMMARY

[0009] In one aspect, this disclosure provides a mixture or kit comprising: (a) optionally, a DNA polymerase enzyme comprising 5' to 3' exonuclease activity; (b) optionally, reagents and a buffer required for the DNA polymerase enzyme to function; (c) optionally, at least one DNA template molecule; (d) a First Forward Primer (FP1) comprising a sequence complementary to a First Region of the at least one DNA template molecule; (e) a First Probe (TP1) comprising a sequence complementary or homologous to a Second Region of the at least one DNA templated molecule; (1) a First Reverse Primer (RP1) comprising a sequence homologous to a Third Region of the at least one DNA template molecule; (g) a Second Forward Primer (FP2) comprising a sequence complementary to a Fourth Region of the at least one DNA template molecule; (h) a Second Blocker (B2) comprising a sequence partially complementary to the Fourth Region of the at least one DNA template molecule and partially complementary to a Template Subsequence positioned 5' to the Fourth Region of the at least one DNA template molecule; (i) a Second Probe (TP2) comprising a sequence complementary or homologous to a Fifth Region of the at least one DNA template molecule; and (j) a Second Reverse Primer (RP2) comprising a sequence homologous to a Sixth Region of the at least one DNA template molecule, where the First Region, the Second Region, the Third Region, the Fourth Region, the Fifth Region, and the Sixth Region are non-overlapping; where the First Region, the Second Region, the Third Region, the Fourth Region, the Fifth Region, and the Sixth Region each comprise between 12 and 50 nucleotides; where between 0 and 5000 nucleotides are positioned between any two Regions; where the TP1 comprises a fluorophore (TP1 fluorophore) and a fluorescence-quenching moiety; where the TP2 comprises a fluorophore (TP2 fluorophore) and a fluorescence-quenching moiety; and where the TP1 fluorophore and the TP2 fluorophore are spectrally distinct from each other.

[0010] In one aspect, this disclosure provides a mixture or kit comprising: (a) optionally, a DNA polymerase enzyme comprising 5' to 3' exonuclease activity; (b) optionally, reagents and a buffer required for the DNA polymerase enzyme to function; (c) optionally, at least one DNA template molecule; (d) a First Forward Primer (FP1) comprising a sequence complementary to a Third Region of the at least one DNA template molecule; (e) a First Probe (TP1) comprising a sequence complementary or homologous to a Second Region of the at least one DNA template molecule; (1) a First Reverse Primer (RP1) comprising a sequence homologous to a First Region of the at least one DNA template molecule; (g) a Second Forward Primer (FP2) comprising a sequence complementary to a Fifth Region of the at least one DNA template molecule; (h) a Second Blocker (B2) comprising a sequence partially complementary to the Fifth Region of the at least one DNA template molecule and partially complementary to a subsequence of the at least one DNA template molecule positioned 5' to the Fifth Region of the at least one DNA template molecule; and (i) a Second Probe (TP2) comprising a sequence complementary or homologous to a Fourth Region of the at least one DNA template molecule; where the at least one DNA template molecule comprises, in order from 5' to 3', the First Region, the Second Region, the Third Region, the Fourth Region, and the Fifth Region; where the First Region, the Second Region, the Third Region, the Fourth Region, and the Fifth Region are non-overlapping; where the First Region, the Second Region, the Third Region, the Fourth Region, and the Fifth Region each comprise between 12 and 50 nucleotides; where the distance between the 5'-most nucleotide of the First Region and the 3'-most nucleotide of the Fifth Region is between 100 and 3000 nucleotides; where the TP1 comprises a fluorophore (TP1 fluorophore) and a fluorescence-quenching moiety; where the TP2 comprises a fluorophore (TP2 fluorophore) and a fluorescence-quenching moiety; and where the TP1 fluorophore and the TP2 fluorophore are spectrally distinct from each other.

[0011] In one aspect, this disclosure provides a method for generating fluorescence signal in an aqueous solution, the method comprising: (a) mixing a sample comprising a DNA template molecule with: (i) a DNA polymerase enzyme comprising 5' to 3' exonuclease activity; (ii) reagents and a buffer required for the DNA polymerase enzyme to function; (iii) a First Forward Primer (FP1) comprising a sequence complementary to a First Region of the DNA template molecule; (iv) a First Probe (TP1) comprising a sequence complementary or homologous to a Second Region of the DNA template molecule; (v) a First Reverse Primer (RP1) comprising a sequence homologous to a Third Region of the DNA template molecule; (vi) a Second Forward Primer (FP2) comprising a sequence complementary to a Fourth Region of the DNA template molecule; (vii) a Second Blocker (B2) comprising a sequence partially complementary to the Fourth Region of the DNA template molecule and partially complementary to a subsequence of the DNA template molecule positioned 5' to the Fourth Region of the DNA template molecule; (viii) a Second Probe (TP2) comprising a sequence complementary or homologous to a Fifth Region of the DNA template molecule; and (ix) a Second Reverse Primer (RP2) comprising a sequence homologous to a Sixth Region of the DNA template molecule to generate a solution; where the First Region, the Second Region, the Third Region, the Fourth Region, the Fifth Region, and the Sixth region are non-overlapping; where the First Region, the Second Region, the Third Region, the Fourth Region, the Fifth Region, and the Sixth Region each comprise between 12 and 50 nucleotides; where between 0 and 5000 nucleotides are positioned between any two Regions; where the TP1 comprises a fluorophore (TP1 fluorophore) and a fluorescence-quenching moiety; where the TP2 comprises a fluorophore (TP2 fluorophore) and a fluorescence-quenching moiety; and where the TP1 fluorophore and the TP2 fluorophore are spectrally distinct from each other; and (b) subjecting the sample from step (a) to at least 7 rounds of thermal cycling, where, optionally, each round comprises alternating between a temperature of equal to or greater than 78°C for between 1 second and 30 minutes and a temperature of less than 75°C for between 1 second and 4 hours.

[0012] In one aspect, this disclosure provides a method for generating fluorescence signal in an aqueous solution, the method comprising: (a) mixing a sample comprising a DNA template molecule with: (i) a DNA polymerase enzyme comprising 5' to 3' exonuclease activity; (ii) reagents and a buffer required for the DNA polymerase enzyme to function; (iii) a First Forward Primer (FP1) comprising a sequence complementary to a Third Region of the DNA template molecule; (iv) a First Probe (TP1) comprising a sequence complementary or homologous to a Second Region of the DNA template molecule; (v) a First Reverse Primer (RP1) comprising a sequence homologous to a First Region of the DNA template molecule; (vi) a Second Forward Primer (FP2) comprising a sequence complementary to a Fifth Region of the DNA template molecule; (vii) a Second Blocker (B2) comprising a sequence partially complementary to the Fifth Region of the DNA template molecule and partially complementary to a subsequence of the DNA template molecule positioned 5' to the Fifth Region of the DNA template molecule; and (viii) a Second Probe (TP2) comprising a sequence complementary or homologous to a Fourth Region of the DNA template molecule to generate a solution; where the at least one DNA template molecule comprises, in order from 5' to 3', the First Region, the Second Region, the Third Region, the Fourth Region, and the Fifth Region; where the First Region, the Second Region, the Third Region, the Fourth Region, and the Fifth Region are non-overlapping; where the First Region, the Second Region, the Third Region, the Fourth Region, and the Fifth Region each comprise between 12 and 50 nucleotides; where the distance between the 5'-most nucleotide of the First Region and the 3'-most nucleotide of the Fifth Region is between 100 and 3000 nucleotides; where the TP1 comprises a fluorophore (TP1 fluorophore) and a fluorescence-quenching moiety; where the TP2 comprises a fluorophore (TP2 fluorophore) and a fluorescence-quenching moiety; and where the TP1 fluorophore and the TP2 fluorophore are spectrally distinct from each other; and (b) subjecting the sample from step (a) to at least 7 rounds of thermal cycling, where, optionally, each round comprises alternating between a temperature of equal to or greater than 78°C for between 1 second and 30 minutes and a temperature of less than 75°C for between 1 second and 4 hours.

[0013] In one aspect, this disclosure provides a method for detecting a DNA template sequence in a sample, the method comprising: (a) mixing the sample with: (i) a DNA polymerase enzyme comprising 5' to 3' exonuclease activity; (ii) reagents and a buffer required for the DNA polymerase enzyme to function; (iii) a First Forward Primer (FP1) comprising a sequence complementary to a First Region of the DNA template molecule; (iv) a First Probe (TP1) comprising a sequence complementary or homologous to a Second Region of the DNA template molecule; (v) a First Reverse Primer (RP1) comprising a sequence homologous to a Third Region of the DNA template molecule; (vi) a Second Forward Primer (FP2) comprising a sequence complementary to a Fourth Region of the DNA template molecule; (vii) a Second Blocker (B2) comprising a sequence partially complementary to the Fourth Region of the DNA template molecule and partially complementary to a subsequence of the DNA template molecule positioned 5' to the Fourth Region of the DNA template molecule; (viii) a Second Probe (TP2) comprising a sequence complementary or homologous to a Fifth Region of the DNA template molecule; and (ix) a Second Reverse Primer (RP2) comprising a sequence homologous to a Sixth Region of the DNA template molecule to generate a solution; where the First Region, the Second Region, the Third Region, the Fourth Region, the Fifth Region, and the Sixth region are non-overlapping; where the First Region, the Second Region, the Third Region, the Fourth Region, the Fifth Region, and the Sixth Region each comprise between 12 and 50 nucleotides; where between 0 and 5000 nucleotides are positioned between any two Regions; where the TP1 comprises a fluorophore (TP1 fluorophore) and a fluorescence-quenching moiety; where the TP2 comprises a fluorophore (TP2 fluorophore) and a fluorescencequenching moiety; and where the TP1 fluorophore and the TP2 fluorophore are spectrally distinct from each other; (b) subjecting the solution from step (a) to at least 7 rounds of thermal cycling, where, optionally, each round comprises alternating between a temperature of equal to or greater than 78°C for between 1 second and 30 minutes and a temperature of less than 75°C for between 1 second and 4 hours; (c) measuring fluorescence values of the solution after each round of thermal cycling in step (b) in the fluorescence channels corresponding to the TP1 and the TP2; (d) determining a cycle threshold (Ct) value for the TP1 (Ctl) and the TP2 (Ct2); and (e) making a positive call for the presence of the DNA template in the sample if the Ctl is less than 35 cycles, Ct2 is less than 50 cycles, and the value of [Ct2 - Ctl] is between 1 cycle and 12 cycles.

[0014] In one aspect, this disclosure provides a method for detecting a DNA template sequence in a sample, the method comprising: (a) mixing the sample with: (i) a DNA polymerase enzyme comprising 5' to 3' exonuclease activity; (ii) reagents and a buffer required for the DNA polymerase enzyme to function; (iii) a First Forward Primer (FP1) comprising a sequence complementary to a Third Region of the DNA template molecule; (iv) a First Probe (TP1) comprising a sequence complementary or homologous to a Second Region of the DNA template molecule; (v) a First Reverse Primer (RP1) comprising a sequence homologous to a First Region of the DNA template molecule; (vi) a Second Forward Primer (FP2) comprising a sequence complementary to a Fifth Region of the DNA template molecule; (vii) a Second Blocker (B2) comprising a sequence partially complementary to the Fifth Region of the DNA template molecule and partially complementary to a subsequence of the DNA template molecule positioned 5' to the Fifth Region of the DNA template molecule; and (viii) a Second Probe (TP2) comprising a sequence complementary or homologous to a Fourth Region of the DNA template molecule to generate a solution; where the at least one DNA template molecule comprises, in order from 5' to 3', the First Region, the Second Region, the Third Region, the Fourth Region, and the Fifth Region; where the First Region, the Second Region, the Third Region, the Fourth Region, and the Fifth Region are non-overlapping; where the First Region, the Second Region, the Third Region, the Fourth Region, and the Fifth Region each comprise between 12 and 50 nucleotides; where the distance between the 5'-most nucleotide of the First Region and the 3'-most nucleotide of the Fifth Region is between 100 and 3000 nucleotides; where the TP1 comprises a fluorophore (TP1 fluorophore) and a fluorescence-quenching moiety; where the TP2 comprises a fluorophore (TP2 fluorophore) and a fluorescencequenching moiety; and where the TP1 fluorophore and the TP2 fluorophore are spectrally distinct from each other; and (b) subjecting the solution from step (a) to at least 7 rounds of thermal cycling, where, optionally, each round comprises alternating between a temperature of equal to or greater than 78°C for between 1 second and 30 minutes and a temperature of less than 75°C for between 1 second and 4 hours; (c) measuring fluorescence values of the solution after each round of thermal cycling in step (b) in the fluorescence channels corresponding to the TP1 and the TP2; (d) determining a cycle threshold (Ct) value for the TP1 (Ctl) and the TP2 (Ct2); and (e) making a positive call for the presence of the DNA template in the sample if the Ctl is less than 35 cycles, Ct2 is less than 50 cycles, and the value of [Ct2 - Ctl] is between 1 cycle and 12 cycles.

[0015] In one aspect, this disclosure provides a method for detection of multiple DNA targets in an analyte, the method comprising: (a) mixing the analyte with: (i) a DNA polymerase enzyme comprising 5' to 3' exonuclease activity; (ii) reagents and a buffer required for the DNA polymerase enzyme to function; (iii) a plurality of PCR primers; and (iv) a plurality of probes comprising a fluorophore and a fluorescence-quenching moiety to generate a solution; (b) subjecting the solution from step (a) to at least 7 rounds of thermal cycling, where, optionally, each round comprises alternating between a temperature of equal to or greater than 78°C for between 1 second and 30 minutes and a temperature of less than 75°C for between 1 second and 4 hours; (c) measuring fluorescence values of the solution after each round of thermal cycling in step (b) in at least two fluorescence channels; (d) determining a cycle threshold (Ct) value for each of the at least two fluorescence channels; (e) determining the temporal order of fluorescence signal increases; and (1) mapping the order of fluorescence signal increases to the presence, absence, or quantity of the multiple DNA targets.

[0016] In one aspect, this disclosure provides a mixture or kit comprising: (a) optionally, a DNA polymerase enzyme comprising 5' to 3' exonuclease activity; (b) optionally, reagents and a buffer required for the DNA polymerase enzyme to function; (c) optionally, a DNA template molecule; (d) a First Forward Primer (FP1) comprising a sequence complementary to a First Region of the DNA template molecule; (e) a First Probe (TP1) comprising a sequence complementary or homologous to a Second Region of the DNA template molecule; (1) a First Reverse Primer (RP1) comprising a sequence homologous to a Third Region of the DNA template molecule; (g) a Second Forward Primer (FP2) comprising a sequence complementary to a Fourth Region of the DNA template molecule, where the FP2 comprises one or more nucleotides at or near its 3' end that are not complementary to the DNA template molecule; (h) a Second Probe (TP2) comprising a sequence complementary or homologous to a Fifth Region of the DNA template molecule; and (i) a Second Reverse Primer (RP2) comprising a sequence homologous to a Sixth Region of the DNA template molecule; where the First Region, the Second Region, the Third Region, the Fourth Region, the Fifth Region, and the Sixth Region are non-overlapping; where the First Region, the Second Region, the Third Region, the Fourth Region, the Fifth Region, and the Sixth Region each comprise between 12 and 50 nucleotides; where between 0 and 5000 nucleotides are positioned between any two Regions; where the TP1 comprises a fluorophore (TP1 fluorophore) and a fluorescence-quenching moiety; where the TP2 comprises a fluorophore (TP2 fluorophore) and a fluorescence-quenching moiety; and where the TP1 fluorophore and the TP2 fluorophore are spectrally distinct from each other.

[0017] In one aspect, this disclosure provides a mixture or kit comprising: (a) optionally, a DNA polymerase enzyme comprising 5' to 3' exonuclease activity; (b) optionally, reagents and a buffer required for the DNA polymerase enzyme to function; (c) optionally, a DNA template molecule; (d) a First Forward Primer (FP1) comprising a sequence complementary to a Third Region of the DNA template molecule; (e) a First Probe (TP1) comprising a sequence complementary or homologous to a Second Region of the DNA template molecule; (1) a First Reverse Primer (RP1) comprising a sequence homologous to a First Region of the DNA template molecule; (g) a Second Forward Primer (FP2) comprising a sequence complementary to a Fifth Region of the DNA template molecule, where the FP2 comprises one or more nucleotides at or near its 3' end that are not complementary to the DNA template molecule; and (h) a Second Probe (TP2) comprising a sequence complementary or homologous to a Fourth Region of the DNA template molecule; where the at least one DNA template molecule comprises, in order from 5' to 3', the First Region, the Second Region, the Third Region, the Fourth Region, and the Fifth Region; where the First Region, the Second Region, the Third Region, the Fourth Region, and the Fifth Region are non-overlapping; where the First Region, the Second Region, the Third Region, the Fourth Region, and the Fifth Region each comprise between 12 and 50 nucleotides; where the distance between the 5'-most nucleotide of the First Region and the 3'-most nucleotide of the Fifth Region is between 100 and 3000 nucleotides; where the TP1 comprises a fluorophore (TP1 fluorophore) and a fluorescence-quenching moiety; where the TP2 comprises a fluorophore (TP2 fluorophore) and a fluorescence-quenching moiety; and where the TP1 fluorophore and the TP2 fluorophore are spectrally distinct from each other. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 depicts selected components and reagents of a cycle multiplexing system. The Template has six non-overlapping Regions from 5' to 3' (numbered 1, 2, 3, 4, 5, and 6). The black arrow on the left side of the Template, First Reverse Primer (RP1) and Second Reverse Primer (RP2), and the right side of the First Forward Primer (FP1) and Second Forward Primer (FP2) denote the 3' ends of the oligonucleotides. The FP1, First Probe (TP1), FP2, and Second Probe (TP2), are mostly or fully reverse complementary to the First Region, the Second Region, the Fourth Region, and the Fifth Region on the Template respectively. The RP1 and RP2 are mostly or fully homologous to the Third Region and the Sixth Region respectively. The Second Blocker (B2) is partially reverse complementary to the Fourth Region and partially reverse complementary to the region right next to 5' of the Fourth Region. The TP1 and TP2 have two distinct 5' fluorophore modifications. The system also includes a DNA polymerase.

[0019] Figure 2 depicts two embodiments of a cycle multiplexing system. In the top panel, the First Forward Primer (FP1), Second Forward Primer (FP2) and Second Probe (TP2) are mostly or fully reverse complementary to the First Region, the Fourth Region, and the Fifth Region on the Template respectively. The First Probe (TP1), First Reverse Primer (RP1) and Second Reverse Primer (RP2) are mostly or fully homologous to the Second Region, the Third Region and the Sixth Region respectively. The TP1 has a 3' fluorophore modification and the TP2 has a 5' fluorophore modification. In the bottom panel, the Template has six non-overlapping regions from 5' to 3': the Third Region (3), the Second Region (2), the First Region (1), the Sixth Region (6), the Fifth Region (5), and the Fourth Region (4). The FP1, TP1, and FP2 are mostly or fully reverse complementary to the First Region, the Second Region, and the Fourth Region on the Template respectively. The TP2 and RP2 are mostly or fully homologous to the Fifth Region and the Sixth Region. Both embodiments include a DNA polymerase.

[0020] Figure 3 depicts a schematic of a cycle multiplexing system. In a cycle multiplexing system with two TaqMan™ probes (TP1 and TP2), in the first stage of qPCR, the First Forward Primer (FP1) and First Reverse Primer (RP1) will function as a normal pair of primers and efficiently produce Amplicon 1. The Second Forward Primer (FP2) and Second Reverse Primer (RP2) cannot efficiently amplify Amplicon 2 because part of the Template sequence that binds to the FP2 is also the subsequence that binds to the Second Blocker (B2). Thus, the presence of B2 results in decreased efficiency of FP2 and RP2, causing a delay in Amplicon 2 amplification and fluorescence generation. As the reaction proceeds, both primer pairs could reach the amplification plateau stage in the final stage of qPCR.

[0021] Figure 4 depicts an example of cycle multiplexing. In a cycle multiplexing system having two TaqMan™ probes with distinct fluorophore modifications, one fluorescent signal would fluoresce normally, while the other would fluoresce relatively late in qPCR. Different color permutations are be used to indicate the presence of different templates. For example, if there is a 4-fluorescence-channel instrument available, the number of targets that can be detected simultaneously is up to 4 x (4-1) = 12.

[0022] Figure 5 depicts an example of two-level cycle multiplexing. In a cycle multiplexing system with three TaqMan™ probes (TP1, TP2, and TP3), a First Forward Primer (FP1) and a First Reverse Primer (RP1) will function as a standard primer pair to produce an amplicon and generate early fluorescence. The primer pair of a Second Forward Primer (FP2) and a Second Reverse Primer (RP2) with a Second Blocker (B2) has less primer efficiency in amplification, resulting in late fluorescence. Another primer pair of Third Forward Primer (FP3) and Third Reverse Primer (RP3) functions much less efficiently due to a much stronger Third Blocker (B3), thus producing a very late fluorescence. When using a two-level cycle multiplexing system, if there is a 4- fluorescence-channel instrument available, the number of targets that can be detected simultaneously is up to 4 x (4-1) x (4-2) = 24. Note that one-level and two-level cycle multiplexing can be used simultaneously.

[0023] Figure 6 depicts an experimental demonstration of a cycle multiplexing system. qPCR was applied to aNA18537 human genomic DNA template using the PowerUp™ SYBR™ Green DNA Polymerase Master Mix. As the schematic design of the experiment shown in the top panel, the 5' end of the First Probe (TP1) comprises a Cy5 fluorophore, and the 5' end of the Second Probe (TP2) comprises a ROX fluorophore. The two sets of primers (FP1 and RP1; FP2 and RP2) are separated by 12 nucleotides (nt). The amplicon produced by FP1 and RP1 is 121 nt in length, and the amplicon produced by FP2 and RP2 is 98 nucleotides in length. Experimental results are shown in the bottom panel. The consistent cycle threshold (Ct) value of the Cy5 curves among all the graphs under different conditions reveals that the efficiency of the primer pair is not influenced by other oligonucleotides inside the system. The Ct value of the ROX channel increases as the concentration of the Second Blocker (B2) increases, as does the ACt between the ROX channel and the Cy5 channel, demonstrating the success of the cycle multiplexing system.

[0024] Figure 7 depicts selected reagents and components of a cycle multiplexing system. In the top panel, the Template has five non-overlapping regions from 5' to 3' (1, 2, 3, 4, 5). The First Forward Primer (FP1), First Probe (TP1), Second Forward Primer (FP2), and Second Probe (TP2), are mostly or fully reverse complementary to the Third Region, the Second Region, the Fifth Region, and the Fourth Region on the Template, respectively. The First Reverse Primer (RP1) is mostly or fully homologous to the First Region. The Second Blocker (B2) is partially reverse complementary to the Fifth Region and partially reverse complementary to the region adjacent to the 5' end of the Fifth Region. The TP1 and TP2 each have a distinct 5' fluorophore modification. In the bottom panel, the Template has seven non-overlapping regions from 5' to 3' (1, 2, 3, 4, 5, 6, 7). The FP1, TP1, FP2, TP2, Third Forward Primer (FP3) and Third Probe (TP3) are mostly or fully reverse complementary to the Third Region, the Second Region, the Fifth Region, the Fourth Region, the Seventh Region and the Sixth Region on the Template, respectively. The RP1 is mostly or fully homologous to the First Region. The B2 and Third Blocker (B3) are partially reverse complementary to the Fifth Region and the Seventh Region, and partially reverse complementary to the region adjacent to the 5' end of the Fifth Region and the Seventh Region, respectively. The TP1, TP2, and TP3 each have 3' fluorophore modifications. Both panels also include a DNA polymerase.

[0025] Figure 8 depicts an alternative embodiment of cycle multiplexing using a 3' mismatched primer for the Second Forward Primer (FP2). A mismatch between FP2 and the Template at or near the 3' end of FP2 significantly reduces PCR amplification efficiency, resulting in cycle threshold (Ct) delay. The number of mismatches, nucleotide identities of the mismatches, and the proximity of the mismatches to the 3' end of FP2 can impact the quantitative amount of Ct delay. More mismatches, mismatches with larger AAG° values (such as C-C mismatches), and mismatches closer to the 3' end of FP2 are expected to generate larger Ct delays.

DETAILED DESCRIPTION

[0026] Unless defined otherwise, all technical and scientific terms used have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Where a term is provided in the singular, the inventors also contemplate aspects of the disclosure described by the plural of that term. Where there are discrepancies in terms and definitions used in references that are incorporated by reference, the terms used in this application shall have the definitions given herein. Other technical terms used have their ordinary meaning in the art in which they are used, as exemplified by various art-specific dictionaries, for example, “The American Heritage® Science Dictionary” (Editors of the American Heritage Dictionaries, 2011, Houghton Mifflin Harcourt, Boston and New York), the “McGraw-Hill Dictionary of Scientific and Technical Terms” (6th edition, 2002, McGraw-Hill, New York), or the “Oxford Dictionary of Biology” (6th edition, 2008, Oxford University Press, Oxford and New York).

[0027] Any references cited herein, including, e.g., all patents, published patent applications, and non-patent publications, are incorporated herein by reference in their entirety.

[0028] Any composition provided herein is specifically envisioned for use with any applicable method provided herein.

[0029] When a grouping of alternatives is presented, any and all combinations of the members that make up that grouping of alternatives is specifically envisioned. For example, if an item is selected from a group consisting of A, B, C, and D, the inventors specifically envision each alternative individually (e.g., A alone, B alone, etc.), as well as combinations such as A, B, and D; A and C; B and C; etc.

[0030] When a range of numbers is provided herein, the range is understood to inclusive of the edges of the range as well as any number between the defined edges of the range. For example, “between 1 and 10” includes any number between 1 and 10, as well as the number 1 and the number 10.

[0031] As used herein, the singular form “a,” “an,” and “the” include plural references unless the context clearly dictates otherwise. For example, the term “a compound” or “at least one compound” may include a plurality of compounds, including mixtures thereof. As used herein, the term “plurality” refers to any number greater than one.

[0032] As used herein, the term “plurality” means at least two. In an aspect, a method, kit, or mixture provided herein comprises a plurality of primers. In an aspect, a method, kit, or mixture provided herein comprises a plurality of probes. In an aspect, a method, kit or mixture provided herein comprises a plurality of blockers. [0033] In an aspect, a plurality of primers comprises at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 sets of forward and reverse primers. In an aspect, a plurality of probes comprises at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 probes. In an aspect, a plurality of blockers comprises at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 probes.

[0034] In an aspect, a primer, probe, or blocker is 100% complementary to a DNA template molecule. In an aspect, a primer, probe, or blocker is at least 99.5% complementary to a DNA template molecule. In an aspect, a primer, probe, or blocker is at least 99% complementary to a DNA template molecule. In an aspect, a primer, probe, or blocker is at least 98% complementary to a DNA template molecule. In an aspect, a primer, probe, or blocker is at least 97% complementary to a DNA template molecule. In an aspect, a primer, probe, or blocker is at least 96% complementary to a DNA template molecule. In an aspect, a primer, probe, or blocker is at least 95% complementary to a DNA template molecule. In an aspect, a primer, probe, or blocker is at least 90% complementary to a DNA template molecule. In an aspect, a primer, probe, or blocker is at least 85% complementary to a DNA template molecule.

[0035] Unless otherwise described, it will be understood that a “complementary” or “homologous” primer, probe, or blocker can comprise 0, 1, or 2 mismatches between the primer, probe, or blocker and a DNA template molecule to which it hybridizes.

[0036] As used herein, a “partially complementary” sequence refers to a primer, probe, or blocker that is between 85% and 99.9%, between 90% and 99.9%, between 91% and 99.9%, between 92% and 99.9%, between 93% and 99.9%, between 94% and 99.9%, between 95% and 99.9%, between 96% and 99.9%, between 97% and 99.9%, between 98% and 99.9%, between 99% and 99.9%, or between 99.5% and 99.9% complementary to a DNA template molecule.

[0037] In an aspect, a partially complementary sequence comprises one mismatch between a primer, probe, or blocker and a DNA template molecule. In an aspect, a partially complementary sequence comprises two mismatches between a primer, probe, or blocker and a DNA template molecule. In an aspect, a partially complementary sequence comprises three mismatches between a primer, probe, or blocker and a DNA template molecule. In an aspect, a partially complementary sequence comprises four mismatches between a primer, probe, or blocker and a DNA template molecule. In an aspect, a partially complementary sequence comprises five mismatches between a primer, probe, or blocker and a DNA template molecule. In an aspect, a partially complementary sequence comprises six mismatches between a primer, probe, or blocker and a DNA template molecule. In an aspect, a partially complementary sequence comprises seven mismatches between a primer, probe, or blocker and a DNA template molecule. In an aspect, a partially complementary sequence comprises eight mismatches between a primer, probe, or blocker and a DNA template molecule. In an aspect, a partially complementary sequence comprises nine mismatches between a primer, probe, or blocker and a DNA template molecule. In an aspect, a partially complementary sequence comprises ten mismatches between a primer, probe, or blocker and a DNA template molecule.

[0038] In an aspect, a probe, primer, or blocker is complementary, or partially complementary, to a single Region of a DNA template molecule. In an aspect, a probe, primer, or blocker is complementary, or partially complementary, to two or more Regions of a DNA template molecule. In an aspect, a probe, primer, or blocker is complementary, or partially complementary, to three or more Regions of a DNA template molecule. In an aspect, a probe, primer, or blocker is complementary, or partially complementary, to four or more Regions of a DNA template molecule.

[0039] As used herein, a “mismatch” refers to an alignment of two sequences that pairs two uncomplimentary nucleotides. Non-limiting examples of mismatches include G-A, G-T, G-U, G-G, C-A, C-T, C-U, C-C, A-A, T-T, and T-U. Conversely, “matched” alignments of nucleotides refer to complimentary pairs such as G-C, A-T, and A-U.

[0040] In an aspect, a primer, probe, or blocker is complementary (either completely or partially) or homologous to a DNA template molecule. As used herein, “homologous” refers to sequences that are identical, or similar to a DNA template molecule. A “similar” sequence comprises fewer than 5 mismatches between the primer, probe, or blocker and a DNA template.

[0041] For optimal alignment of sequences to calculate their percent complementarity, various pair-wise or multiple sequence alignment algorithms and programs are known in the art, such as ClustalW or Basic Local Alignment Search Tool® (BLAST™), etc., that can be used to compare the sequence complementarity or identity between two or more nucleotide sequences. Although other alignment and comparison methods are known in the art, the alignment and percent identity between two sequences (including the percent identity ranges described above) can be as determined by the ClustalW algorithm, see, e.g. , Chenna et al. , “Multiple sequence alignment with the Clustal series of programs,” Nucleic Acids Research 31 : 3497-3500 (2003); Thompson et al. , “Clustal W: Improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice,” Nucleic Acids Research 22: 4673-4680 (1994); Larkin MA et al., “Clustal W and Clustal X version 2.0,” Bioinformatics 23: 2947-48 (2007); and Altschul et al. "Basic local alignment search tool." J. Mol. Biol. 215:403-410 (1990), the entire contents and disclosures of which are incorporated herein by reference.

[0042] The terms “percent identity” or “percent identical” as used herein in reference to two or more nucleotide or amino acid sequences is calculated by (i) comparing two optimally aligned sequences (nucleotide or amino acid) over a window of comparison (the “alignable” region or regions), (ii) determining the number of positions at which the identical nucleic acid base (for nucleotide sequences) or amino acid residue (for proteins and polypeptides) occurs in both sequences to yield the number of matched positions, (iii) dividing the number of matched positions by the total number of positions in the window of comparison, and then (iv) multiplying this quotient by 100% to yield the percent identity. If the “percent identity” is being calculated in relation to a reference sequence without a particular comparison window being specified, then the percent identity is determined by dividing the number of matched positions over the region of alignment by the total length of the reference sequence. Accordingly, for purposes of the present application, when two sequences (query and subject) are optimally aligned (with allowance for gaps in their alignment), the “percent identity” for the query sequence is equal to the number of identical positions between the two sequences divided by the total number of positions in the query sequence over its length (or a comparison window), which is then multiplied by 100%.

[0043] The terms “percent complementarity” or “percent complementary” as used herein in reference to two nucleotide sequences refers to the percentage of nucleotides of a query sequence that optimally base-pair or hybridize to nucleotides a subject sequence when the query and subject sequences are linearly arranged and optimally base paired without secondary folding structures, such as loops, stems or hairpins. Such a percent complementarity can be between two DNA strands, two RNA strands, or a DNA strand and a RNA strand. The “percent complementarity” can be calculated by (i) optimally base-pairing or hybridizing the two nucleotide sequences in a linear and fully extended arrangement (i.e., without folding or secondary structures) over a window of comparison, (ii) determining the number of positions that base-pair between the two sequences over the window of comparison to yield the number of complementary positions, (iii) dividing the number of complementary positions by the total number of positions in the window of comparison, and (iv) multiplying this quotient by 100% to yield the percent complementarity of the two sequences. Optimal base pairing of two sequences can be determined based on the known pairings of complementary nucleotide bases, such as guanine (G)-cytosine (C), adenine (A)- thymine (T), and A-uracil (U), through hydrogen binding. If the “percent complementarity” is being calculated in relation to a reference sequence without specifying a particular comparison window, then the percent identity is determined by dividing the number of complementary positions between the two linear sequences by the total length of the reference sequence. Thus, for purposes of the present application, when two sequences (query and subject) are optimally base-paired (with allowance for mismatches or non-base-paired nucleotides), the “percent complementarity” for the query sequence is equal to the number of base-paired positions between the two sequences divided by the total number of positions in the query sequence over its length, which is then multiplied by 100%.

[0044] As a non-limiting example, the complement of the sequence 5'-ATGC-3' is 3'- TACG-5', and the reverse complement of 5'-ATGC-3' is 5'-GCAT-3'. Notably, the complement and reverse complement sequences are identical to each other when viewed in the 5' to 3' direction.

[0045] As used herein, a “primer” or “PCR primer” refers to a chemically synthesized single-stranded oligonucleotide which is designed to anneal to a specific site on a DNA template molecule. Without being limiting, a primer is used in PCR to initiate DNA synthesis. In an aspect, a primer is a DNA molecule. In an aspect, a primer is an RNA molecule. In an aspect, a primer comprises between 6 and 70 nucleotides. In an aspect, a primer comprises between 10 and 50 nucleotides. In an aspect, a primer comprises between 15 and 30 nucleotides. In an aspect, a primer comprises between 18 and 25 nucleotides. In an aspect, a primer comprises at least 6 nucleotides. In an aspect, a primer comprises at least 10 nucleotides. In an aspect, a primer comprises at least 15 nucleotides. In an aspect, a primer comprises at least 20 nucleotides. In an aspect, a primer is a forward primer. In an aspect, a primer is a reverse primer.

[0046] As used herein, “specific” or “specifically” when used in the context of a primer specific for a target nucleic acid refers to a level of complementarity between the primer and the target such that there exists an annealing temperature at which the primer will anneal to and mediate amplification of the target nucleic acid and will not anneal to or mediate amplification of non-target sequences present in a sample.

[0047] As used herein, a “forward primer” hybridizes to the anti-sense strand of dsDNA, and a “reverse primer” hybridizes to the sense strand of dsDNA. In an aspect, a forward primer comprises DNA. In an aspect, a reverse primer comprises DNA. In an aspect, a forward primer comprises RNA. In an aspect, a reverse primer comprises RNA. As used herein, a “set” of forward and reverse primers refers to a pair of forward and reverse primers that can produce an amplicon during PCR.

[0048] In an aspect, a primer comprises a sequence at least 80% identical or complementary to a DNA template molecule. In an aspect, a primer comprises a sequence at least 85% identical or complementary to a DNA template molecule. In an aspect, a primer comprises a sequence at least 90% identical or complementary to a DNA template molecule. In an aspect, a primer comprises a sequence at least 95% identical or complementary to a DNA template molecule. In an aspect, a primer comprises a sequence at least 99% identical or complementary to a DNA template molecule. In an aspect, a primer comprises a sequence 100% identical or complementary to a DNA template molecule.

[0049] The polymerase chain reaction (PCR) is a molecular biology technique that allows one to generate multiple copies of a targeted region of DNA. Copies of DNA made via PCR are termed “amplicons.” In an aspect, PCR comprises thermal cycling.

[0050] As used herein, “thermal cycling” refers to a controlled set of timed temperature changes. One “cycle” or “round” of thermal cycling comprises at least two stages. The first stage of a cycle or round comprises a first temperature maintained for a desired amount of time, and the second stage of a cycle or round comprises a second temperature maintained for a desired amount of time. In an aspect, a cycle or round further comprises a third stage comprising a third temperature maintained for a desired amount of time. In an aspect, a cycle or round further comprises a fourth stage comprising a fourth temperature maintained for a desired amount of time. Often, thermal cycling comprises repeating the same cycle or round several times.

[0051] In an aspect, a method comprises at least 1 cycle of thermal cycling In an aspect, a method comprises at least 3 cycles of thermal cycling. In an aspect, a method comprises at least 5 cycles of thermal cycling. In an aspect, a method comprises at least 7 cycles of thermal cycling. In an aspect, a method comprises at least 10 cycles of thermal cycling. In an aspect, a method comprises at least 12 cycles of thermal cycling. In an aspect, a method comprises at least 15 cycles of thermal cycling. In an aspect, a method comprises at least 18 cycles of thermal cycling. In an aspect, a method comprises at least 20 cycles of thermal cycling. In an aspect, a method comprises at least 25 cycles of thermal cycling. In an aspect, a method comprises at least 30 cycles of thermal cycling. In an aspect, a method comprises at least 35 cycles of thermal cycling. In an aspect, a method comprises at least 40 cycles of thermal cycling. In an aspect, a method comprises at least 45 cycles of thermal cycling. In an aspect, a method comprises at least 50 cycles of thermal cycling.

[0052] In an aspect, a method comprises between 2 cycles and 60 cycles of thermal cycling. In an aspect, a method comprises between 5 cycles and 60 cycles of thermal cycling. In an aspect, a method comprises between 7 cycles and 60 cycles of thermal cycling. In an aspect, a method comprises between 10 cycles and 60 cycles of thermal cycling. In an aspect, a method comprises between 12 cycles and 60 cycles of thermal cycling. In an aspect, a method comprises between 15 cycles and 60 cycles of thermal cycling. In an aspect, a method comprises between 18 cycles and 60 cycles of thermal cycling. In an aspect, a method comprises between 20 cycles and 60 cycles of thermal cycling. In an aspect, a method comprises between 25 cycles and 60 cycles of thermal cycling. In an aspect, a method comprises between 30 cycles and 60 cycles of thermal cycling. In an aspect, a method comprises between 35 cycles and 60 cycles of thermal cycling. In an aspect, a method comprises between 40 cycles and 60 cycles of thermal cycling. In an aspect, a method comprises between 50 cycles and 60 cycles of thermal cycling. In an aspect, a method comprises between 5 cycles and 50 cycles of thermal cycling. In an aspect, a method comprises between 5 cycles and 45 cycles of thermal cycling. In an aspect, a method comprises between 5 cycles and 30 cycles of thermal cycling. In an aspect, a method comprises between 7 cycles and 50 cycles of thermal cycling. In an aspect, a method comprises between 7 cycles and 45 cycles of thermal cycling.

[0053] In an aspect, a stage or round of thermal cycling comprises a temperature of greater than or equal to 40°C. In an aspect, a stage or round of thermal cycling comprises a temperature of greater than or equal to 50°C. In an aspect, a stage or round of thermal cycling comprises a temperature of greater than or equal to 60°C. In an aspect, a stage or round of thermal cycling comprises a temperature of greater than or equal to 65°C. In an aspect, a stage or round of thermal cycling comprises a temperature of greater than or equal to 70°C. In an aspect, a stage or round of thermal cycling comprises a temperature of greater than or equal to 75°C. In an aspect, a stage or round of thermal cycling comprises a temperature of greater than or equal to 76°C. In an aspect, a stage or round of thermal cycling comprises a temperature of greater than or equal to 77°C. In an aspect, a stage or round of thermal cycling comprises a temperature of greater than or equal to 78°C. In an aspect, a stage or round of thermal cycling comprises a temperature of greater than or equal to 79°C. In an aspect, a stage or round of thermal cycling comprises a temperature of greater than or equal to 80°C.

[0054] In an aspect, a stage or round of thermal cycling comprises a temperature of less than or equal to 40°C. In an aspect, a stage or round of thermal cycling comprises a temperature of less than or equal to 50°C. In an aspect, a stage or round of thermal cycling comprises a temperature of less than or equal to 60°C. In an aspect, a stage or round of thermal cycling comprises a temperature of less than or equal to 65°C. In an aspect, a stage or round of thermal cycling comprises a temperature of less than or equal to 70°C. In an aspect, a stage or round of thermal cycling comprises a temperature of less than or equal to 75°C. In an aspect, a stage or round of thermal cycling comprises a temperature of less than or equal to 76°C. In an aspect, a stage or round of thermal cycling comprises a temperature of less than or equal to 77°C. In an aspect, a stage or round of thermal cycling comprises a temperature of less than or equal to 78°C. In an aspect, a stage or round of thermal cycling comprises a temperature of less than or equal to 79°C. In an aspect, a stage or round of thermal cycling comprises a temperature of less than or equal to 80°C.

[0055] In an aspect, a stage or round of thermal cycling lasts for between 1 second and 6 hours. In an aspect, a stage or round of thermal cycling lasts for between 1 second and 5 hours. In an aspect, a stage or round of thermal cycling lasts for between 1 second and 4 hours. In an aspect, a stage or round of thermal cycling lasts for between 1 second and 3 hours. In an aspect, a stage or round of thermal cycling lasts for between 1 second and 2 hours. In an aspect, a stage or round of thermal cycling lasts for between 1 second and 1.5 hours. In an aspect, a stage or round of thermal cycling lasts for between 1 second and 1 hours. In an aspect, a stage or round of thermal cycling lasts for between 1 second and 50 minutes. In an aspect, a stage or round of thermal cycling lasts for between 1 second and 45 minutes. In an aspect, a stage or round of thermal cycling lasts for between 1 second and 30 minutes. In an aspect, a stage or round of thermal cycling lasts for between 1 second and 20 minutes. In an aspect, a stage or round of thermal cycling lasts for between 1 second and 15 minutes. In an aspect, a stage or round of thermal cycling lasts for between 1 second and 10 minutes. In an aspect, a stage or round of thermal cycling lasts for between 1 second and 7.5 minutes. In an aspect, a stage or round of thermal cycling lasts for between 1 second and 5 minutes. In an aspect, a stage or round of thermal cycling lasts for between 1 second and 4 minutes. In an aspect, a stage or round of thermal cycling lasts for between 1 second and 3 minutes. In an aspect, a stage or round of thermal cycling lasts for between 1 second and 2 minutes. In an aspect, a stage or round of thermal cycling lasts for between 1 second and 1 minute. In an aspect, a stage or round of thermal cycling lasts for between 1 second and 50 seconds. In an aspect, a stage or round of thermal cycling lasts for between 1 second and 40 seconds. In an aspect, a stage or round of thermal cycling lasts for between 1 second and 30 seconds. In an aspect, a stage or round of thermal cycling lasts for between 1 second and 20 seconds. In an aspect, a stage or round of thermal cycling lasts for between 1 second and 10 seconds.

[0056] In an aspect, PCR is quantitative PCR (qPCR). qPCR is used to quantify nucleic acid molecules in a given sample. qPCR can be performed using a fluorescent dye that binds to dsDNA. During each cycle of thermal cycling the dye binds to the newly formed dsDNA, which produces more fluorescence that can be measured. The fluorescence signal increases proportionally to the amount of replicated DNA, enabling quantification.

[0057] Probes (e.g. TaqMan™ probes) can also be used in qPCR in place of a fluorescent dye. Probes often comprise both a fluorophore and a quencher. Fluorescence resonance energy transfer prevents the emission of the fluorophore when the fluorophore and quencher are both present on the probe. During PCR, the probe, which binds between the two primers on the targeted region of DNA, is hydrolyzed during primer extension, allowing an amplification-dependent increase in fluorescence. Therefore, the measured fluorescence is proportional to the amount of the probe target sequence in a sample. However, this type of system will not work with DNA polymerases that lack 5' to 3' exonuclease activity.

[0058] The use of fluorophores during qPCR enables the quantification of a targeted DNA molecule. Either the absolute amount of a target DNA molecule, or the relative amount of a target molecule between samples, can be measured. A measurement called the threshold cycle (Ct) is often used to measure the relative concentration of a targeted DNA molecule in a qPCR sample. As used herein, “Ct” refers to the number of thermal cycles required for a fluorescent signal to cross the threshold (e.g., background fluorescence level) during qPCR. Ct levels are inversely proportional to the amount of target nucleic acid in a sample (e.g, the lower the Ct level, the greater the amount of target DNA molecule in the sample). Fluorescence levels can be detected by any suitable instrument used in the art. A non-limiting example of an instrument suitable for detecting qPCR fluorescence is a Bio-Rad CFX96 qPCR instrument.

[0059] In an aspect, an instrument for detecting qPCR fluorescence can detect fluorescence in at least two fluorescence channels. In an aspect, an instrument for detecting qPCR fluorescence can detect fluorescence in at least three fluorescence channels. In an aspect, an instrument for detecting qPCR fluorescence can detect fluorescence in at least four fluorescence channels.

[0060] In an aspect, a method provided herein comprises measuring fluorescence in at least two fluorescence channels. In an aspect, a method provided herein comprises measuring fluorescence in at least three fluorescence channels. In an aspect, a method provided herein comprises measuring fluorescence in at least four fluorescence channels.

[0061] In an aspect, a composition or method provided herein comprises at least one DNA polymerase enzyme. In an aspect, a composition or method provided herein comprises a DNA polymerase enzyme. In an aspect, a composition or method provided herein comprises at least one DNA polymerase enzyme comprising 5' to 3' exonuclease activity. In an aspect, a composition or method provided herein comprises a DNA polymerase enzyme comprising 5' to 3' exonuclease activity.

[0062] As used herein, a “DNA polymerase enzyme” refers to an enzyme that is capable of catalyzing the synthesis of a DNA molecule from nucleoside triphosphates. DNA polymerases add a nucleotide to the 3' end of a DNA strand one nucleotide at a time, creating an antiparallel DNA strand as compared to a template DNA strand. DNA polymerases are unable to begin a new DNA molecule de novo; they require a primer to which it can add a first new nucleotide.

[0063] Some DNA polymerase enzymes, such as Taq, possess 5' to 3' exonuclease activity. This 5' to 3' exonuclease activity allows the polymerase to remove primers at the 5' ends of newly synthesized DNA so that polymerase activity can fill in gaps. In an aspect, a DNA polymerase enzyme comprising 5' to 3' exonuclease activity is a Taq DNA polymerase enzyme. In an aspect, a DNA polymerase enzyme comprising 5' to 3' exonuclease activity is a Bst DNA polymerase enzyme. In an aspect, a DNA polymerase enzyme comprising 5' to 3' exonuclease activity is a DNA polymerase 1 enzyme. In an aspect, a DNA polymerase enzyme comprising 5' to 3' exonuclease activity is selected from the group consisting of a Taq DNA polymerase enzyme, a Bst DNA polymerase enzyme, and a DNA polymerase 1 enzyme.

[0064] In an aspect, a composition or method provided herein comprises reagents and a buffer required for a DNA polymerase enzyme to function. As used herein, a “reagent” refers to any substance or compound added to a mixture to cause a chemical reaction or to test if a chemical reaction occurs. In an aspect, a reagent comprises a component selected from the group consisting of magnesium, at least one dNTP, phosphatase, betaine, dimethyl sulfoxide (DMSO), and tetramethyl ammonium chloride (TMAC).

[0065] In an aspect, a composition or method provided herein comprises a DNA template molecule. In an aspect, a composition or method provided herein comprises at least one DNA template molecule. As used herein, a “DNA template molecule” refers to a DNA molecule that is bound by primers to produce amplicons during PCR. DNA template molecules can also be bound by blockers and probes.

[0066] A DNA template molecule can be subdivided into discrete “Regions.” In an aspect, a DNA template molecule comprises a First Region. In an aspect, a DNA template molecule comprises a First Region and a Second Region. In an aspect, a DNA template molecule comprises a First Region, a Second Region, and a Third Region. In an aspect, a DNA template molecule comprises a First Region, a Second Region, a Third Region, and a Fourth Region. In an aspect, a DNA template molecule comprises a First Region, a Second Region, a Third Region, a Fourth Region, and a Fifth Region. In an aspect, a DNA template molecule comprises a First Region, a Second Region, a Third Region, a Fourth Region, a Fifth Region, and a Sixth Region. In an aspect, a DNA template molecule comprises a First Region, a Second Region, a Third Region, a Fourth Region, a Fifth Region, a Sixth Region, and a Seventh Region. In an aspect, a DNA template molecule comprises a First Region, a Second Region, a Third Region, a Fourth Region, a Fifth Region, a Sixth Region, a Seventh Region, and an Eighth Region. In an aspect, a DNA template molecule comprises a First Region, a Second Region, a Third Region, a Fourth Region, a Fifth Region, a Sixth Region, a Seventh Region, an Eighth Region, and a Ninth Region. In an aspect, a DNA template molecule comprises up to Nth Regions, where N is any positive whole number.

[0067] In an aspect, a primer is complementary or partially complementary to a Region of a DNA template molecule. In an aspect, a forward primer is complementary or partially complementary to a Region of a DNA template molecule. In an aspect, a reverse primer is complementary or partially complementary to a Region of a DNA template molecule. In an aspect, a probe is complementary, partially complementary, or homologous to a Region of a DNA template molecule. In an aspect, a blocker is complementary or partially complementary to a Region of a DNA template molecule.

[0068] In an aspect, a primer and a probe hybridize to different Region(s) of a DNA template molecule. In an aspect, a primer and a probe hybridize to the same Region(s) of a DNA template molecule. In an aspect, a primer and a blocker hybridize to different Region(s) of a DNA template molecule. In an aspect, a primer and a blocker hybridize to the same Region(s) of a DNA template molecule. In an aspect, a probe and a blocker hybridize to different Region(s) of a DNA template molecule. In an aspect, a probe and a blocker hybridize to the same Region(s) of a DNA template molecule.

[0069] In an aspect, a DNA template molecule comprises non-overlapping Regions (e.g., no two Regions share any nucleotide positions). In an aspect, a DNA template molecule comprises overlapping Regions (e.g, at least two Regions share at least one nucleotide position). In an aspect, a DNA template molecule comprises both nonoverlapping Regions and overlapping Regions.

[0070] In an aspect, a Region comprises between 10 and 100 nucleotides. In an aspect, a Region comprises between 10 and 90 nucleotides. In an aspect, a Region comprises between 10 and 80 nucleotides. In an aspect, a Region comprises between 10 and 70 nucleotides. In an aspect, a Region comprises between 10 and 60 nucleotides. In an aspect, a Region comprises between 10 and 50 nucleotides. In an aspect, a Region comprises between 10 and 40 nucleotides. In an aspect, a Region comprises between 10 and 30 nucleotides. In an aspect, a Region comprises between 10 and 20 nucleotides.

[0071] In an aspect, a Region comprises between 12 and 100 nucleotides. In an aspect, a Region comprises between 12 and 90 nucleotides. In an aspect, a Region comprises between 12 and 80 nucleotides. In an aspect, a Region comprises between 12 and 70 nucleotides. In an aspect, a Region comprises between 12 and 60 nucleotides. In an aspect, a Region comprises between 12 and 50 nucleotides. In an aspect, a Region comprises between 12 and 40 nucleotides. In an aspect, a Region comprises between 12 and 30 nucleotides. In an aspect, a Region comprises between 12 and 20 nucleotides.

[0072] In an aspect, a Region comprises between 15 and 100 nucleotides. In an aspect, a Region comprises between 15 and 90 nucleotides. In an aspect, a Region comprises between 15 and 80 nucleotides. In an aspect, a Region comprises between 15 and 70 nucleotides. In an aspect, a Region comprises between 15 and 60 nucleotides. In an aspect, a Region comprises between 15 and 50 nucleotides. In an aspect, a Region comprises between 15 and 40 nucleotides. In an aspect, a Region comprises between 15 and 30 nucleotides. In an aspect, a Region comprises between 15 and 20 nucleotides. [0073] In an aspect, a Region comprises at least 10 nucleotides. In an aspect, a Region comprises at least 11 nucleotides. In an aspect, a Region comprises at least 12 nucleotides. In an aspect, a Region comprises at least 13 nucleotides. In an aspect, a Region comprises at least 14 nucleotides. In an aspect, a Region comprises at least 15 nucleotides. In an aspect, a Region comprises at least 20 nucleotides. In an aspect, a Region comprises at least 25 nucleotides. In an aspect, a Region comprises at least 30 nucleotides. In an aspect, a Region comprises at least 35 nucleotides. In an aspect, a Region comprises at least 40 nucleotides. In an aspect, a Region comprises at least 45 nucleotides. In an aspect, a Region comprises at least 50 nucleotides. In an aspect, a Region comprises at least 60 nucleotides.

[0074] In an aspect, between 0 and 5000 nucleotides are positioned between two adjacent Regions. In an aspect, between 0 and 4000 nucleotides are positioned between two adjacent Regions. In an aspect, between 0 and 3000 nucleotides are positioned between two adjacent Regions. In an aspect, between 0 and 2000 nucleotides are positioned between two adjacent Regions. In an aspect, between 0 and 1000 nucleotides are positioned between two adjacent Regions. In an aspect, between 0 and 750 nucleotides are positioned between two adjacent Regions. In an aspect, between 0 and 500 nucleotides are positioned between two adjacent Regions. In an aspect, between 0 and 250 nucleotides are positioned between two adjacent Regions. In an aspect, between 0 and 100 nucleotides are positioned between two adjacent Regions. In an aspect, between 0 and 50 nucleotides are positioned between two adjacent Regions.

[0075] In an aspect, between 0 and 5000 nucleotides are positioned between any two Regions of a DNA template molecule. In an aspect, between 0 and 4000 nucleotides are positioned between any two Regions of a DNA template molecule. In an aspect, between 0 and 3000 nucleotides are positioned between any two Regions of a DNA template molecule. In an aspect, between 0 and 2000 nucleotides are positioned between any two Regions of a DNA template molecule. In an aspect, between 0 and 1000 nucleotides are positioned between any two Regions of a DNA template molecule. In an aspect, between 0 and 750 nucleotides are positioned between any two Regions of a DNA template molecule. In an aspect, between 0 and 500 nucleotides are positioned between any two Regions of a DNA template molecule. In an aspect, between 0 and 250 nucleotides are positioned between any two Regions of a DNA template molecule. In an aspect, between 0 and 100 nucleotides are positioned between any two Regions of a DNA template molecule. In an aspect, between 0 and 50 nucleotides are positioned between any two Regions of a DNA template molecule.

[0076] In an aspect, two or more Regions can comprise a Group of Regions. In an aspect, a Sixth Region, a Fifth Region, and a Fourth Region comprise Group 1, and a Third Region, a Second Region, and a First Region comprise Group 2. In an aspect, Group 1 is positioned 5' to Group 2 on a DNA template molecule. In an aspect, Group 1 is positioned 3' to Group 2 on a DNA template molecule. In an aspect, a Seventh Region, an Eighth Region, and a Ninth Region comprise Group 3. In an aspect, a DNA template molecule comprises, in order from 5' to 3', Group 1, Group 2, Group 3. In an aspect, a DNA template molecule comprises, in order from 5' to 3', Group 1, Group 3, Group 2. In an aspect, a DNA template molecule comprises, in order from 5' to 3', Group 2, Group 1, Group 3. In an aspect, a DNA template molecule comprises, in order from 5' to 3', Group 2, Group 3, Group 1. In an aspect, a DNA template molecule comprises, in order from 5' to 3', Group 3, Group 2, Group 1. In an aspect, a DNA template molecule comprises, in order from 5' to 3', Group 3, Group 1, Group 2.

[0077] In an aspect, the number of nucleotides positioned between a Fourth Region and a Third Region is between 0 and 5000 nucleotides, between 0 and 2000 nucleotides, between 0 and 1500 nucleotides, between 0 and 1000 nucleotides, or between 0 and 800 nucleotides. In an aspect, the number of nucleotides positioned between a Fourth Region and a Third Region is between 0 and 4000 nucleotides. In an aspect, the number of nucleotides positioned between a Fourth Region and a Third Region is between 0 and 3000 nucleotides. In an aspect, the number of nucleotides positioned between a Fourth Region and a Third Region is between 0 and 2500 nucleotides. In an aspect, the number of nucleotides positioned between a Fourth Region and a Third Region is between 0 and 900 nucleotides. In an aspect, the number of nucleotides positioned between a Fourth Region and a Third Region is between 0 and 500 nucleotides.

[0078] In an aspect, the number of nucleotides positioned between a Sixth Region and a First Region is between 0 and 5000 nucleotides, between 0 and 2000 nucleotides, between 0 and 1500 nucleotides, between 0 and 1000 nucleotides, or between 0 and 800 nucleotides. In an aspect, the number of nucleotides positioned between a Sixth Region and a First Region is between 0 and 4000 nucleotides. In an aspect, the number of nucleotides positioned between a Sixth Region and a First Region is between 0 and 3000 nucleotides. In an aspect, the number of nucleotides positioned between a Sixth Region and a First Region is between 0 and 2500 nucleotides. In an aspect, the number of nucleotides positioned between a Sixth Region and a First Region is between 0 and 900 nucleotides. In an aspect, the number of nucleotides positioned between a Sixth Region and a First Region is between 0 and 500 nucleotides.

[0079] In an aspect, a mixture or method provided herein comprises a probe. As used herein, a “probe” refers to a single-stranded nucleic acid molecule used to detect the presence of a complementary nucleic acid sequence (e.g, a target sequence) via hybridization. In an aspect, a probe is a DNA probe. In an aspect, a probe is an RNA probe.

[0080] In an aspect, a probe comprises between 6 and 70 nucleotides. In an aspect, a probe comprises between 10 and 50 nucleotides. In an aspect, a probe comprises between 15 and 30 nucleotides. In an aspect, a probe comprises between 18 and 25 nucleotides. In an aspect, a probe comprises at least 6 nucleotides. In an aspect, a probe comprises at least 10 nucleotides. In an aspect, a probe comprises at least 15 nucleotides. In an aspect, a probe comprises at least 20 nucleotides.

[0081] In an aspect, a probe is a TaqMan™ probe. In an aspect, a probe is a molecular beacon.

[0082] In an aspect, a probe comprises a fluorophore. As used herein, a “fluorophore” refers to any fluorescent compound that can re-emit light upon light excitation. In an aspect, a probe comprises a fluorophore on its 5' end. In an aspect, a probe comprises a fluorophore on its 3' end. In an aspect, a probe comprises a fluorophore between its 5' end and 3' end.

[0083] In an aspect, a fluorophore is a peptide or protein. In an aspect, a fluorophore is a small organic compound. In an aspect, a fluorophore is a synthetic oligomer or polymer. In an aspect, a fluorophore is a multi-component system. In an aspect, a fluorophore is selected from the group consisting of a peptide or protein, a small organic compound, a synthetic oligomer or polymer, and a multi-component system. In an aspect, a fluorophore is an organic dye.

[0084] Non-limiting examples of peptide or protein fluorophores included green fluorescence protein (GFP), yellow fluorescence protein (YFP), and red fluorescence protein (RFP). [0085] Non-limiting examples of small organic compound fluorophores include xanthene derivatives (e.g, fluorescein, rhodamine, Oregon green, eosin, Texas red), cyanine derivatives (e.g. cyanine, indocarbocyanine, merocyanine), squaraine derivatives, squararine rotaxane derivatives, naphthalene derivatives, coumarin derivatives, oxadiazole derivatives, anthracene derivatives, pyrene derivatives (e.g , Cascade blue), oxazine derivatives (e.g , Nile red, Nile blue, cresyl violet), acridine derivatives (e.g, proflavine, acridine orange, acridine yellow), arylmethine derivatives (e.g. auramine, crystal violet, malachite green), tetrapyrrole derivatives, and dipyrromethene derivatives.

[0086] In an aspect, a fluorophore is selected from the group consisting of Cy3™, FAM, VIC, HEX, Cy5™, Cy5.5, Quasar 705, ROX, TET, Texas Red, or another organic dye.

[0087] In an aspect, a fluorophore is selected from the group consisting of a Freedom™ dye, an ATTO™ dye, an Alexa Fluor® dye, a LI-COR IRDye®, and a Rhodamine dye. Freedom™ dyes, ATTO™ dyes, Alexa Fluor® dyes, LI-COR IRDyes®, and Rhodamine dyes are obtainable from Integrated DNA Technologies (Coralville, Iowa). See, for example, idtdna[dot]com/site/Catalog/Modifications/Category/3.

[0088] In an aspect, two or more fluorophores are spectrally distinct. As used herein, “spectrally distinct,” when used in reference to two or more fluorophores, refers to the ability to emit light in different wavelengths such that detected light can be unambiguously assigned to a given fluorophore. As anon-limiting example, Cy5 (emits at 670 nm) and ROX (emits at 605 nm) are spectrally distinct fluorophores. In an aspect, a plurality of probes comprises proves that each comprise a spectrally distinct fluorophore.

[0089] In an aspect, a probe comprises a fluorescence-quenching moiety. As used herein, a “fluorescence-quenching moiety” or “quencher” refers to any substance that absorbs the excitation energy from a fluorophore, thereby reducing or eliminating the fluorescence intensity of the fluorophore. Quenchers can be used to decrease the fluorescence intensity of a given substance, such as a fluorophore. In an aspect, a quencher dissipates the light energy from a fluorophore as heat. In an aspect, a quencher is a dye that lacks native fluorescence.

[0090] In an aspect, a quencher is selected from the group consisting of MGB, Black Hole Quencher, Iowa Black® RQ, and Iowa Black® FQ. [0091] In an aspect, a probe comprises a fluorescence-quenching moiety on its 5' end. In an aspect, a probe comprises a fluorescence-quenching moiety on its 3' end. In an aspect, a probe comprises a fluorescence-quenching moiety between its 5' end and 3' end.

[0092] In an aspect, a plurality of probes each comprise a different fluorescencequenching moiety. In an aspect, a plurality of probes each comprise the same fluorescence-quenching moiety.

[0093] In an aspect, a mixture or method provided herein comprises a blocker. As used herein a “blocker” refers to an oligonucleotide that is designed to selectively bind to a DNA template molecule to retard the amplification of a target sequence. In an aspect, a blocker is a DNA molecule. In an aspect, a blocker is an RNA molecule. In another aspect, a blocker comprises at least one continuous strand of from about 12 to about 100 nucleotides in length which strand preferably anneals to a to-be-blocked allele sequence relative to a non-blocked allele sequence, and further comprising a functional group or nucleotide sequence at its 3’ end that prevents enzymatic extension during an amplification process such as polymerase chain reaction.

[0094] In an aspect, a blocker comprises between 11 and 100 nucleotides. In an aspect, a blocker comprises between 11 and 90 nucleotides. In an aspect, a blocker comprises between 11 and 80 nucleotides. In an aspect, a blocker comprises between 11 and 70 nucleotides. In an aspect, a blocker comprises between 11 and 60 nucleotides. In an aspect, a blocker comprises between 11 and 50 nucleotides. In an aspect, a blocker comprises between 11 and 40 nucleotides. In an aspect, a blocker comprises between 11 and 30 nucleotides. In an aspect, a blocker comprises between 11 and 20 nucleotides.

[0095] In an aspect, a blocker comprises at least 11 nucleotides. In an aspect, a blocker comprises at least 12 nucleotides. In an aspect, a blocker comprises at least 15 nucleotides. In an aspect, a blocker comprises at least 20 nucleotides. In an aspect, a blocker comprises at least 25 nucleotides. In an aspect, a blocker comprises at least 30 nucleotides. In an aspect, a blocker comprises at least 40 nucleotides. In an aspect, a blocker comprises at least 50 nucleotides. In an aspect, a blocker comprises at least 60 nucleotides. In an aspect, a blocker comprises at least 70 nucleotides. In an aspect, a blocker comprises at least 80 nucleotides. In an aspect, a blocker comprises at least 90 nucleotides. In an aspect, a blocker comprises at least 100 nucleotides.

[0096] In an aspect, a blocker comprises a chemical functionalization that prevents DNA polymerase extension. In an aspect, a blocker comprises a chemical functionalization that prevents DNA polymerase extension on its 3' end. In an aspect, a chemical functionalization comprises a 3-carbon spacer. In an aspect, a chemical functionalization comprises an inverted nucleotide. In an aspect, a chemical functionalization comprises a minor groove binder. In an aspect, a chemical functionalization comprises a dideoxynucleotide. In an aspect, a chemical functionalization is selected from the group consisting of a 3-carbon spacer, an inverted nucleotide, and a minor groove binder.

[0097] Typically, a blocker and a primer have partially overlapping sequences and thus they compete to bind to a given target site. The region of overlap between the blocker and primer sequences is referred to as an “overlapping subsequence.” An “overlapping subsequence” comprises a nucleotide sequence of at least 3 nucleotides of a primer sequence that is homologous with the blocker sequence.

[0098] In an aspect, a blocker and a primer comprise an overlapping sequence. In an aspect, a blocker and a forward primer comprise an overlapping sequence. In an aspect, a blocker and a reverse primer comprise an overlapping sequence. In an aspect, an overlapping sequence is positioned on the 3' end of a primer and the 5' end of a blocker.

[0099] When a primer and a blocker comprise an overlapping subsequence, the primer also has a “non-overlapping subsequence,” which refers to the portion of the primer sequence that does not overlap with the blocker sequence.

[0100] In an aspect, between 3 and 25 nucleotides of the 5' end of a blocker sequence are complementary to the same Region as a forward primer. In an aspect, between 3 and 20 nucleotides of the 5' end of a blocker sequence are complementary to the same Region as a forward primer. In an aspect, between 3 and 18 nucleotides of the 5' end of a blocker sequence are complementary to the same Region as a forward primer. In an aspect, between 3 and 15 nucleotides of the 5' end of a blocker sequence are complementary to the same Region as a forward primer. In an aspect, between 3 and 12 nucleotides of the 5' end of a blocker sequence are complementary to the same Region as a forward primer. In an aspect, between 3 and 10 nucleotides of the 5' end of a blocker sequence are complementary to the same Region as a forward primer. As a non-limiting example for this paragraph, if a forward primer binds to a Fourth Region, then at least a portion of the 5' end of the blocker would also be complementary to the Fourth Region.

[0101] In an aspect, between 8 and 100 nucleotides of the 3' end of a blocker are not complementary to the same Region as a forward primer. In an aspect, between 8 and 90 nucleotides of the 3' end of a blocker are not complementary to the same Region as a forward primer. In an aspect, between 8 and 80 nucleotides of the 3' end of a blocker are not complementary to the same Region as a forward primer. In an aspect, between 8 and 70 nucleotides of the 3' end of a blocker are not complementary to the same Region as a forward primer. In an aspect, between 8 and 60 nucleotides of the 3' end of a blocker are not complementary to the same Region as a forward primer. In an aspect, between 8 and 50 nucleotides of the 3' end of a blocker are not complementary to the same Region as a forward primer. In an aspect, between 8 and 40 nucleotides of the 3' end of a blocker are not complementary to the same Region as a forward primer. In an aspect, between 8 and 30 nucleotides of the 3' end of a blocker are not complementary to the same Region as a forward primer. In an aspect, between 8 and 20 nucleotides of the 3' end of a blocker are not complementary to the same Region as a forward primer. In an aspect, between 8 and 10 nucleotides of the 3' end of a blocker are not complementary to the same Region as a forward primer. As anon-limiting example for this paragraph, if a forward primer binds to a Fourth Region, then at least a portion of the 3' end of the blocker would not be complementary to the Fourth Region.

[0102] In an aspect, the overlapping subsequence of a blocker is capable of hybridizing to a DNA template molecule. In an aspect, a blocker comprises a sequence that is not complementary to a DNA template molecule and is termed a “non-compl ementary region.” In an aspect, a non-complementary region of a blocker forms at least one hairpin structure. In an aspect, a non-complementary region of a blocker comprises between 2 and 70 nucleotides at the 3' end of the blocker that are not complementary to a sequence adjacent to the 5' end of a Region to which the blocker can hybridize. In an aspect, a non-complementary region of a blocker comprises between 2 and 60 nucleotides at the 3' end of the blocker that are not complementary to a sequence adjacent to the 5' end of a Region to which the blocker can hybridize. In an aspect, a non-complementary region of a blocker comprises between 2 and 50 nucleotides at the 3' end of the blocker that are not complementary to a sequence adjacent to the 5' end of a Region to which the blocker can hybridize. In an aspect, a non-complementary region of a blocker comprises between 2 and 40 nucleotides at the 3' end of the blocker that are not complementary to a sequence adjacent to the 5' end of a Region to which the blocker can hybridize. In an aspect, anon-complementary region of a blocker comprises between 2 and 30 nucleotides at the 3' end of the blocker that are not complementary to a sequence adjacent to the 5' end of a Region to which the blocker can hybridize. In an aspect, a non-complementary region of a blocker comprises between 2 and 20 nucleotides at the 3' end of the blocker that are not complementary to a sequence adjacent to the 5' end of a Region to which the blocker can hybridize. In an aspect, a non-complementary region of a blocker comprises between 2 and 10 nucleotides at the 3' end of the blocker that are not complementary to a sequence adjacent to the 5' end of a Region to which the blocker can hybridize. As a non-limiting example for this paragraph, if a blocker is complementary, in part, to a Fourth Region, then its non- complementary region would be not complementary to a sequence adjacent to the 5' end of the Fourth Region.

[0103] In an aspect, this disclosure provides a mixture or kit comprising: (a) a DNA polymerase enzyme comprising 5' to 3' exonuclease activity; (b) reagents and a buffer required for the DNA polymerase enzyme to function; (c) at least one DNA template molecule; (d) a First Forward Primer (FP1) comprising a sequence complementary to a First Region of the at least one DNA template molecule; (e) a First Probe (TP1) comprising a sequence complementary or homologous to a Second Region of the at least one DNA templated molecule; (f) a First Reverse Primer (RP1) comprising a sequence homologous to a Third Region of the at least one DNA template molecule; (g) a Second Forward Primer (FP2) comprising a sequence complementary to a Fourth Region of the at least one DNA template molecule; (h) a Second Blocker (B2) comprising a sequence partially complementary to the Fourth Region of the at least one DNA template molecule and partially complementary to a Template Subsequence positioned 5' to the Fourth Region of the at least one DNA template molecule; (i) a Second Probe (TP2) comprising a sequence complementary or homologous to a Fifth Region of the at least one DNA template molecule; and (j) a Second Reverse Primer (RP2) comprising a sequence homologous to a Sixth Region of the at least one DNA template molecule, where the First Region, the Second Region, the Third Region, the Fourth Region, the Fifth Region, and the Sixth Region are non-overlapping; where the First Region, the Second Region, the Third Region, the Fourth Region, the Fifth Region, and the Sixth Region each comprise between 12 and 50 nucleotides; where between 0 and 5000 nucleotides are positioned between any two Regions; where the TP1 comprises a fluorophore (TP1 fluorophore) and a fluorescence-quenching moiety; where the TP2 comprises a fluorophore (TP2 fluorophore) and a fluorescencequenching moiety; and where the TP1 fluorophore and the TP2 fluorophore are spectrally distinct from each other. In an aspect, a mixture further comprises (k) a Third Forward Primer (FP3) comprising a sequence complementary to a Seventh Region of the at least one DNA template; (1) a Third Blocker (B3) comprising a sequence partially complementary to the Seventh Region of the at least one DNA template and partially complementary to a Template Subsequence positioned 5' to the Seventh Region of the at least one DNA template molecule; (m) a Third Probe (TP3) comprising a sequence complementary or homologous to an Eighth Region of the at least one DNA template molecule; and (n) a Third Reverse Primer (RP3) comprising a sequence homologous to a Ninth Region of the at least DNA template molecule; where the Seventh, Eighth, and Ninth Regions are non-overlapping subsequences of the at least one DNA template molecule; where the Seventh, Eighth, and Ninth Regions each comprise between 12 and 50 nucleotides; where between 0 and 5000 nucleotides are positioned between any two Regions; where the TP3 comprises a fluorophore (TP3 fluorophore) and a fluorescence-quenching moiety; and where the TP3 fluorophore is spectrally distinct from the TP 1 fluorophore and the TP2 fluorophore.

[0104] In an aspect, between 3 and 25 nucleotides at the 5' end of a blocker are complementary to a Fourth Region. In an aspect, between 3 and 20 nucleotides at the 5' end of a blocker are complementary to a Fourth Region. In an aspect, between 3 and 18 nucleotides at the 5' end of a blocker are complementary to a Fourth Region. In an aspect, between 3 and 15 nucleotides at the 5' end of a blocker are complementary to a Fourth Region. In an aspect, between 3 and 12 nucleotides at the 5' end of a blocker are complementary to Fourth Fifth Region. In an aspect, between 3 and 10 nucleotides at the 5' end of a blocker are complementary to a Fourth Region.

[0105] In an aspect, between 8 and 100 nucleotides at the 3' end of a blocker are not complementary to a Fourth Region. In an aspect, between 8 and 90 nucleotides at the 3' end of a blocker are not complementary to a Fourth Region. In an aspect, between 8 and 80 nucleotides at the 3' end of a blocker are not complementary to a Fourth Region. In an aspect, between 8 and 70 nucleotides at the 3' end of a blocker are not complementary to a Fourth Region. In an aspect, between 8 and 60 nucleotides at the 3' end of a blocker are not complementary to a Fourth Region. In an aspect, between 8 and 50 nucleotides at the 3' end of a blocker are not complementary to a Fourth Region. In an aspect, between 8 and 40 nucleotides at the 3' end of a blocker are not complementary to a Fourth Region. In an aspect, between 8 and 30 nucleotides at the 3' end of a blocker are not complementary to a Fourth Region. In an aspect, between 8 and 20 nucleotides at the 3' end of a blocker are not complementary to a Fourth Region. In an aspect, between 8 and 15 nucleotides at the 3' end of a blocker are not complementary to a Fourth Region.

[0106] In an aspect, between 2 and 70 nucleotides at the 3' end of a blocker (the noncompl ementary region) are not complementary to the a sequence adjacent to the 5' end of a Fourth Region. In an aspect, between 2 and 60 nucleotides at the 3' end of a blocker (the non-compl ementary region) are not complementary to the a sequence adjacent to the 5' end of a Fourth Region. In an aspect, between 2 and 50 nucleotides at the 3' end of a blocker (the non-complementary region) are not complementary to the a sequence adjacent to the 5' end of a Fourth Region. In an aspect, between 2 and 40 nucleotides at the 3' end of a blocker (the non-complementary region) are not complementary to the a sequence adjacent to the 5' end of a Fourth Region. In an aspect, between 2 and 30 nucleotides at the 3' end of a blocker (the non-complementary region) are not complementary to the a sequence adj acent to the 5 ' end of a Fourth Region. In an aspect, between 2 and 20 nucleotides at the 3' end of a blocker (the non-complementary region) are not complementary to the a sequence adjacent to the 5' end of a Fourth Region. In an aspect, between 2 and 10 nucleotides at the 3' end of a blocker (the non- complementary region) are not complementary to the a sequence adjacent to the 5' end of a Fourth Region.

[0107] In an aspect, any mixture or kit provided herein comprises a plurality of Second Forward Primers, Second Blockers, Second Probes, and Second Reverse Primers, wherein at least two Second Probes comprise spectrally distinct fluorophores. In an aspect, any mixture or kit provided herein comprises a plurality of Third Forward Primers, Third Blockers, Third Probes, and Third Reverse Primers, wherein at least two Third Probes comprise spectrally distinct fluorophores.

[0108] In an aspect, this disclosure provides a mixture or kit comprising: (a) a DNA polymerase enzyme comprising 5' to 3' exonuclease activity; (b) reagents and a buffer required for the DNA polymerase enzyme to function; (c) at least one DNA template molecule; (d) a First Forward Primer (FP1) comprising a sequence complementary to a Third Region of the at least one DNA template molecule; (e) a First Probe (TP1) comprising a sequence complementary or homologous to a Second Region of the at least one DNA template molecule; (I) a First Reverse Primer (RP1) comprising a sequence homologous to a First Region of the at least one DNA template molecule; (g) a Second Forward Primer (FP2) comprising a sequence complementary to a Fifth Region of the at least one DNA template molecule; (h) a Second Blocker (B2) comprising a sequence partially complementary to the Fifth Region of the at least one DNA template molecule and partially complementary to a subsequence of the at least one DNA template molecule positioned 5' to the Fifth Region of the at least one DNA template molecule; and (i) a Second Probe (TP2) comprising a sequence complementary or homologous to a Fourth Region of the at least one DNA template molecule; where the at least one DNA template molecule comprises, in order from 5' to 3', the First Region, the Second Region, the Third Region, the Fourth Region, and the Fifth Region; where the First Region, the Second Region, the Third Region, the Fourth Region, and the Fifth Region are non-overlapping; where the First Region, the Second Region, the Third Region, the Fourth Region, and the Fifth Region each comprise between 12 and 50 nucleotides; where the distance between the 5'-most nucleotide of the First Region and the 3'-most nucleotide of the Fifth Region is between 100 and 3000 nucleotides; where the TP1 comprises a fluorophore (TP1 fluorophore) and a fluorescence-quenching moiety; where the TP2 comprises a fluorophore (TP2 fluorophore) and a fluorescence-quenching moiety; and where the TP1 fluorophore and the TP2 fluorophore are spectrally distinct from each other. In an aspect, a mixture further comprises (j) a Third Forward Primer (FP3) comprising a sequence complementary to a Seventh Region of the at least one DNA template molecule; (k) a third Blocker (B3) comprising a sequence partially complementary to the Seventh Region of the at least one DNA template molecule and partially complementary to a subsequence of the at least one DNA template molecule positioned 5' to the Seventh Region of the at least one DNA template molecule; and (1) a Third Probe (TP3) comprising a sequence complementary or homologous to a Sixth Region of the at least one DNA template molecule; where the Sixth Region and the Seventh Region are nonoverlapping; where the Sixth Region and the Seventh Region each comprise between 12 and 50 nucleotides; where the Sixth Region and the Seventh Region are positioned 3’ to the Fifth Region; where the TP3 comprises a fluorophore (TP3 fluorophore) and a fluorescence-quenching moiety; and where the TP3 fluorophore is spectrally distinct from the TP 1 fluorophore and the TP2 fluorophore.

[0109] In an aspect, the distance between the 5'-most nucleotide of a First Region and the 3 '-most nucleotide of a Fifth Region is between 100 and 3000 nucleotides. In an aspect, the distance between the 5'-most nucleotide of a First Region and the 3'-most nucleotide of a Fifth Region is between 100 and 2000 nucleotides. In an aspect, the distance between the 5'-most nucleotide of a First Region and the 3'-most nucleotide of a Fifth Region is between 100 and 1500 nucleotides. In an aspect, the distance between the 5 '-most nucleotide of a First Region and the 3 '-most nucleotide of a Fifth Region is between 100 and 1000 nucleotides. In an aspect, the distance between the 5'-most nucleotide of a First Region and the 3'-most nucleotide of a Fifth Region is between 100 and 800 nucleotides. In an aspect, the distance between the 5'-most nucleotide of a First Region and the 3'-most nucleotide of a Fifth Region is between 100 and 500 nucleotides. In an aspect, the distance between the 5'-most nucleotide of a First Region and the 3'-most nucleotide of a Fifth Region is between 100 and 300 nucleotides.

[0110] In an aspect, between 3 and 25 nucleotides at the 5' end of a blocker are complementary to a Fifth Region. In an aspect, between 3 and 20 nucleotides at the 5' end of a blocker are complementary to a Fifth Region. In an aspect, between 3 and 18 nucleotides at the 5' end of a blocker are complementary to a Fifth Region. In an aspect, between 3 and 15 nucleotides at the 5' end of a blocker are complementary to a Fifth Region. In an aspect, between 3 and 12 nucleotides at the 5' end of a blocker are complementary to a Fifth Region. In an aspect, between 3 and 10 nucleotides at the 5' end of a blocker are complementary to a Fifth Region.

[0111] In an aspect, between 8 and 100 nucleotides at the 3' end of a blocker are not complementary to a Fifth Region. In an aspect, between 8 and 90 nucleotides at the 3' end of a blocker are not complementary to a Fifth Region. In an aspect, between 8 and 80 nucleotides at the 3' end of a blocker are not complementary to a Fifth Region. In an aspect, between 8 and 70 nucleotides at the 3' end of a blocker are not complementary to a Fifth Region. In an aspect, between 8 and 60 nucleotides at the 3' end of a blocker are not complementary to a Fifth Region. In an aspect, between 8 and 50 nucleotides at the 3' end of a blocker are not complementary to a Fifth Region. In an aspect, between 8 and 40 nucleotides at the 3' end of a blocker are not complementary to a Fifth Region. In an aspect, between 8 and 30 nucleotides at the 3' end of a blocker are not complementary to a Fifth Region. In an aspect, between 8 and 20 nucleotides at the 3' end of a blocker are not complementary to a Fifth Region. In an aspect, between 8 and 15 nucleotides at the 3' end of a blocker are not complementary to a Fifth Region.

[0112] In an aspect, between 2 and 70 nucleotides at the 3' end of a blocker (the noncompl ementary region) are not complementary to the a sequence adjacent to the 5' end of a Fifth Region. In an aspect, between 2 and 60 nucleotides at the 3' end of a blocker (the non-compl ementary region) are not complementary to the a sequence adjacent to the 5' end of a Fifth Region. In an aspect, between 2 and 50 nucleotides at the 3' end of a blocker (the non-complementary region) are not complementary to the a sequence adjacent to the 5' end of a Fifth Region. In an aspect, between 2 and 40 nucleotides at the 3' end of a blocker (the non-complementary region) are not complementary to the a sequence adjacent to the 5' end of a Fifth Region. In an aspect, between 2 and 30 nucleotides at the 3' end of a blocker (the non-complementary region) are not complementary to the a sequence adjacent to the 5' end of a Fifth Region. In an aspect, between 2 and 20 nucleotides at the 3' end of a blocker (the non-complementary region) are not complementary to the a sequence adjacent to the 5' end of a Fifth Region. In an aspect, between 2 and 10 nucleotides at the 3' end of a blocker (the non-complementary region) are not complementary to the a sequence adjacent to the 5' end of a Fifth Region.

[0113] In one aspect, this disclosure provides a method for generating fluorescence signal in an aqueous solution, the method comprising: (a) mixing a sample comprising a DNA template molecule with: (i) a DNA polymerase enzyme comprising 5' to 3' exonuclease activity; (ii) reagents and a buffer required for the DNA polymerase enzyme to function; (iii) a First Forward Primer (FP1) comprising a sequence complementary to a First Region of the DNA template molecule; (iv) a First Probe (TP1) comprising a sequence complementary or homologous to a Second Region of the DNA template molecule; (v) a First Reverse Primer (RP1) comprising a sequence homologous to a Third Region of the DNA template molecule; (vi) a Second Forward Primer (FP2) comprising a sequence complementary to a Fourth Region of the DNA template molecule; (vii) a Second Blocker (B2) comprising a sequence partially complementary to the Fourth Region of the DNA template molecule and partially complementary to a subsequence of the DNA template molecule positioned 5' to the Fourth Region of the DNA template molecule; (viii) a Second Probe (TP2) comprising a sequence complementary or homologous to a Fifth Region of the DNA template molecule; and (ix) a Second Reverse Primer (RP2) comprising a sequence homologous to a Sixth Region of the DNA template molecule to generate a solution; where the First Region, the Second Region, the Third Region, the Fourth Region, the Fifth Region, and the Sixth region are non-overlapping; where the First Region, the Second Region, the Third Region, the Fourth Region, the Fifth Region, and the Sixth Region each comprise between 12 and 50 nucleotides; where between 0 and 5000 nucleotides are positioned between any two Regions; where the TP1 comprises a fluorophore (TP1 fluorophore) and a fluorescence-quenching moiety; where the TP2 comprises a fluorophore (TP2 fluorophore) and a fluorescence-quenching moiety; and where the TP1 fluorophore and the TP2 fluorophore are spectrally distinct from each other; and (b) subjecting the sample from step (a) to at least 7 rounds of thermal cycling, where, optionally, each round comprises alternating between a temperature of equal to or greater than 78°C for between 1 second and 30 minutes and a temperature of less than 75°C for between 1 second and 4 hours. In an aspect, a solution further comprises (x) a Third Forward Primer (FP3) comprising a sequence complementary to a Seventh Region of the at least one DNA template; (xi) a Third Blocker (B3) comprising a sequence partially complementary to the Seventh Region of the at least one DNA template and partially complementary to a Template Subsequence positioned 5' to the Seventh Region of the at least one DNA template molecule; (xii) a Third Probe (TP3) comprising a sequence complementary or homologous to an Eighth Region of the at least one DNA template molecule; and (xiii) a Third Reverse Primer (RP3) comprising a sequence homologous to a Ninth Region of the at least DNA template molecule; where the Seventh, Eighth, and Ninth Regions are non-overlapping subsequences of the at least one DNA template molecule; where the Seventh, Eighth, and Ninth Regions each comprise between 12 and 50 nucleotides; where between 0 and 5000 nucleotides are positioned between any two Regions; where the TP3 comprises a fluorophore (TP3 fluorophore) and a fluorescence-quenching moiety; and where the TP3 fluorophore is spectrally distinct from the TP 1 fluorophore and the TP2 fluorophore.

[0114] In an aspect, any solution provided herein comprises a plurality of First Forward Primers, First Blockers, First Probes, and First Reverse Primers, wherein at least two First Probes comprise spectrally distinct fluorophores. In an aspect, any solution provided herein comprises a plurality of Second Forward Primers, Second Blockers, Second Probes, and Second Reverse Primers, wherein at least two Second Probes comprise spectrally distinct fluorophores. In an aspect, any solution provided herein comprises a plurality of Third Forward Primers, Third Blockers, Third Probes, and Third Reverse Primers, wherein at least two Third Probes comprise spectrally distinct fluorophores.

[0115] In one aspect, this disclosure provides a method for generating fluorescence signal in an aqueous solution, the method comprising: (a) mixing a sample comprising a DNA template molecule with: (i) a DNA polymerase enzyme comprising 5' to 3' exonuclease activity; (ii) reagents and a buffer required for the DNA polymerase enzyme to function; (iii) a First Forward Primer (FP1) comprising a sequence complementary to a Third Region of the DNA template molecule; (iv) a First Probe (TP1) comprising a sequence complementary or homologous to a Second Region of the DNA template molecule; (v) a First Reverse Primer (RP1) comprising a sequence homologous to a First Region of the DNA template molecule; (vi) a Second Forward Primer (FP2) comprising a sequence complementary to a Fifth Region of the DNA template molecule; (vii) a Second Blocker (B2) comprising a sequence partially complementary to the Fifth Region of the DNA template molecule and partially complementary to a subsequence of the DNA template molecule positioned 5' to the Fifth Region of the DNA template molecule; and (viii) a Second Probe (TP2) comprising a sequence complementary or homologous to a Fourth Region of the DNA template molecule to generate a solution; where the at least one DNA template molecule comprises, in order from 5' to 3', the First Region, the Second Region, the Third Region, the Fourth Region, and the Fifth Region; where the First Region, the Second Region, the Third Region, the Fourth Region, and the Fifth Region are non-overlapping; where the First Region, the Second Region, the Third Region, the Fourth Region, and the Fifth Region each comprise between 12 and 50 nucleotides; where the distance between the 5'-most nucleotide of the First Region and the 3'-most nucleotide of the Fifth Region is between 100 and 3000 nucleotides; where the TP1 comprises a fluorophore (TP1 fluorophore) and a fluorescence-quenching moiety; where the TP2 comprises a fluorophore (TP2 fluorophore) and a fluorescence-quenching moiety; and where the TP1 fluorophore and the TP2 fluorophore are spectrally distinct from each other; and (b) subjecting the sample from step (a) to at least 7 rounds of thermal cycling, where, optionally, each round comprises alternating between a temperature of equal to or greater than 78°C for between 1 second and 30 minutes and a temperature of less than 75°C for between 1 second and 4 hours. In an aspect, a solution further comprises (ix) a Third Forward Primer (FP3) comprising a sequence complementary to a Seventh Region of the at least one DNA template molecule; (x) a third Blocker (B3) comprising a sequence partially complementary to the Seventh Region of the at least one DNA template molecule and partially complementary to a subsequence of the at least one DNA template molecule positioned 5' to the Seventh Region of the at least one DNA template molecule; and (xi) a Third Probe (TP3) comprising a sequence complementary or homologous to a Sixth Region of the at least one DNA template molecule; where the Sixth Region and the Seventh Region are non-overlapping; where the Sixth Region and the Seventh Region each comprise between 12 and 50 nucleotides; where the Sixth Region and the Seventh Region are positioned 3' to the Fifth Region; where the TP3 comprises a fluorophore (TP3 fluorophore) and a fluorescence-quenching moiety; and where the TP3 fluorophore is spectrally distinct from the TP1 fluorophore and the TP2 fluorophore.

[0116] In one aspect, this disclosure provides a method for detecting a DNA template sequence in a sample, the method comprising: (a) mixing the sample with: (i) a DNA polymerase enzyme comprising 5' to 3' exonuclease activity; (ii) reagents and a buffer required for the DNA polymerase enzyme to function; (iii) a First Forward Primer (FP1) comprising a sequence complementary to a First Region of the DNA template molecule; (iv) a First Probe (TP1) comprising a sequence complementary or homologous to a Second Region of the DNA template molecule; (v) a First Reverse Primer (RP1) comprising a sequence homologous to a Third Region of the DNA template molecule; (vi) a Second Forward Primer (FP2) comprising a sequence complementary to a Fourth Region of the DNA template molecule; (vii) a Second Blocker (B2) comprising a sequence partially complementary to the Fourth Region of the DNA template molecule and partially complementary to a subsequence of the DNA template molecule positioned 5' to the Fourth Region of the DNA template molecule; (viii) a Second Probe (TP2) comprising a sequence complementary or homologous to a Fifth Region of the DNA template molecule; and (ix) a Second Reverse Primer (RP2) comprising a sequence homologous to a Sixth Region of the DNA template molecule to generate a solution; where the First Region, the Second Region, the Third Region, the Fourth Region, the Fifth Region, and the Sixth region are non-overlapping; where the First Region, the Second Region, the Third Region, the Fourth Region, the Fifth Region, and the Sixth Region each comprise between 12 and 50 nucleotides; where between 0 and 5000 nucleotides are positioned between any two Regions; where the TP1 comprises a fluorophore (TP1 fluorophore) and a fluorescence-quenching moiety; where the TP2 comprises a fluorophore (TP2 fluorophore) and a fluorescencequenching moiety; and where the TP1 fluorophore and the TP2 fluorophore are spectrally distinct from each other; (b) subjecting the solution from step (a) to at least 7 rounds of thermal cycling, where, optionally, each round comprises alternating between a temperature of equal to or greater than 78°C for between 1 second and 30 minutes and a temperature of less than 75°C for between 1 second and 4 hours; (c) measuring fluorescence values of the solution after each round of thermal cycling in step (b) in the fluorescence channels corresponding to the TP1 and the TP2; (d) determining a cycle threshold (Ct) value for the TP1 (Ctl) and the TP2 (Ct2); and (e) making a positive call for the presence of the DNA template in the sample if the Ctl is less than 35 cycles, Ct2 is less than 50 cycles, and the value of [Ct2 - Ctl] is between 1 cycle and 12 cycles. In an aspect, a solution further comprises (x) a Third Forward Primer (FP3) comprising a sequence complementary to a Seventh Region of the at least one DNA template; (xi) a Third Blocker (B3) comprising a sequence partially complementary to the Seventh Region of the at least one DNA template and partially complementary to a Template Subsequence positioned 5' to the Seventh Region of the at least one DNA template molecule; (xii) a Third Probe (TP3) comprising a sequence complementary or homologous to an Eighth Region of the at least one DNA template molecule; and (xiii) a Third Reverse Primer (RP3) comprising a sequence homologous to a Ninth Region of the at least DNA template molecule; where the Seventh, Eighth, and Ninth Regions are non-overlapping subsequences of the at least one DNA template molecule; where the Seventh, Eighth, and Ninth Regions each comprise between 12 and 50 nucleotides; where between 0 and 5000 nucleotides are positioned between any two Regions; where the TP3 comprises a fluorophore (TP3 fluorophore) and a fluorescence-quenching moiety; and where the TP3 fluorophore is spectrally distinct from the TP 1 fluorophore and the TP2 fluorophore.

[0117] In one aspect, this disclosure provides a method for detecting a DNA template sequence in a sample, the method comprising: (a) mixing the sample with: (i) a DNA polymerase enzyme comprising 5' to 3' exonuclease activity; (ii) reagents and a buffer required for the DNA polymerase enzyme to function; (iii) a First Forward Primer (FP1) comprising a sequence complementary to a Third Region of the DNA template molecule; (iv) a First Probe (TP1) comprising a sequence complementary or homologous to a Second Region of the DNA template molecule; (v) a First Reverse Primer (RP1) comprising a sequence homologous to a First Region of the DNA template molecule; (vi) a Second Forward Primer (FP2) comprising a sequence complementary to a Fifth Region of the DNA template molecule; (vii) a Second Blocker (B2) comprising a sequence partially complementary to the Fifth Region of the DNA template molecule and partially complementary to a subsequence of the DNA template molecule positioned 5' to the Fifth Region of the DNA template molecule; and (viii) a Second Probe (TP2) comprising a sequence complementary or homologous to a Fourth Region of the DNA template molecule to generate a solution; where the at least one DNA template molecule comprises, in order from 5' to 3', the First Region, the Second Region, the Third Region, the Fourth Region, and the Fifth Region; where the First Region, the Second Region, the Third Region, the Fourth Region, and the Fifth Region are non-overlapping; where the First Region, the Second Region, the Third Region, the Fourth Region, and the Fifth Region each comprise between 12 and 50 nucleotides; where the distance between the 5'-most nucleotide of the First Region and the 3'-most nucleotide of the Fifth Region is between 100 and 3000 nucleotides; where the TP1 comprises a fluorophore (TP1 fluorophore) and a fluorescence-quenching moiety; where the TP2 comprises a fluorophore (TP2 fluorophore) and a fluorescencequenching moiety; and where the TP1 fluorophore and the TP2 fluorophore are spectrally distinct from each other; and (b) subjecting the solution from step (a) to at least 7 rounds of thermal cycling, where, optionally, each round comprises alternating between a temperature of equal to or greater than 78°C for between 1 second and 30 minutes and a temperature of less than 75°C for between 1 second and 4 hours; (c) measuring fluorescence values of the solution after each round of thermal cycling in step (b) in the fluorescence channels corresponding to the TP1 and the TP2; (d) determining a cycle threshold (Ct) value for the TP1 (Ctl) and the TP2 (Ct2); and (e) making a positive call for the presence of the DNA template in the sample if the Ctl is less than 35 cycles, Ct2 is less than 50 cycles, and the value of [Ct2 - Ctl] is between 1 cycle and 12 cycles. In an aspect, a solution further comprises (ix) a Third Forward Primer (FP3) comprising a sequence complementary to a Seventh Region of the at least one DNA template molecule; (x) a third Blocker (B3) comprising a sequence partially complementary to the Seventh Region of the at least one DNA template molecule and partially complementary to a subsequence of the at least one DNA template molecule positioned 5' to the Seventh Region of the at least one DNA template molecule; and (xi) a Third Probe (TP3) comprising a sequence complementary or homologous to a Sixth Region of the at least one DNA template molecule; where the Sixth Region and the Seventh Region are non-overlapping; where the Sixth Region and the Seventh Region each comprise between 12 and 50 nucleotides; where the Sixth Region and the Seventh Region are positioned 3’ to the Fifth Region; where the TP3 comprises a fluorophore (TP3 fluorophore) and a fluorescence-quenching moiety; and where the TP3 fluorophore is spectrally distinct from the TP1 fluorophore and the TP2 fluorophore.

[0118] In one aspect, this disclosure provides a mixture or kit comprising: (a) a DNA polymerase enzyme comprising 5' to 3' exonuclease activity; (b) reagents and a buffer required for the DNA polymerase enzyme to function; (c) a DNA template molecule; (d) a First Forward Primer (FP1) comprising a sequence complementary to a First Region of the DNA template molecule; (e) a First Probe (TP1) comprising a sequence complementary or homologous to a Second Region of the DNA template molecule; (f) a First Reverse Primer (RP1) comprising a sequence homologous to a Third Region of the DNA template molecule; (g) a Second Forward Primer (FP2) comprising a sequence complementary to a Fourth Region of the DNA template molecule, where the FP2 comprises one or more nucleotides at or near its 3' end that are not complementary to the DNA template molecule; (h) a Second Probe (TP2) comprising a sequence complementary or homologous to a Fifth Region of the DNA template molecule; and (i) a Second Reverse Primer (RP2) comprising a sequence homologous to a Sixth Region of the DNA template molecule; where the First Region, the Second Region, the Third Region, the Fourth Region, the Fifth Region, and the Sixth Region are nonoverlapping; where the First Region, the Second Region, the Third Region, the Fourth Region, the Fifth Region, and the Sixth Region each comprise between 12 and 50 nucleotides; where between 0 and 5000 nucleotides are positioned between any two Regions; where the TP1 comprises a fluorophore (TP1 fluorophore) and a fluorescencequenching moiety; where the TP2 comprises a fluorophore (TP2 fluorophore) and a fluorescence-quenching moiety; and where the TP1 fluorophore and the TP2 fluorophore are spectrally distinct from each other.

[0119] In one aspect, this disclosure provides a mixture or kit comprising: (a) a DNA polymerase enzyme comprising 5' to 3' exonuclease activity; (b) reagents and a buffer required for the DNA polymerase enzyme to function; (c) a DNA template molecule; (d) a First Forward Primer (FP1) comprising a sequence complementary to a Third Region of the DNA template molecule; (e) a First Probe (TP1) comprising a sequence complementary or homologous to a Second Region of the DNA template molecule; (f) a First Reverse Primer (RP1) comprising a sequence homologous to a First Region of the DNA template molecule; (g) a Second Forward Primer (FP2) comprising a sequence complementary to a Fifth Region of the DNA template molecule, where the FP2 comprises one or more nucleotides at or near its 3' end that are not complementary to the DNA template molecule; and (h) a Second Probe (TP2) comprising a sequence complementary or homologous to a Fourth Region of the DNA template molecule; where the at least one DNA template molecule comprises, in order from 5' to 3', the First Region, the Second Region, the Third Region, the Fourth Region, and the Fifth Region; where the First Region, the Second Region, the Third Region, the Fourth Region, and the Fifth Region are non-overlapping; where the First Region, the Second Region, the Third Region, the Fourth Region, and the Fifth Region each comprise between 12 and 50 nucleotides; where the distance between the 5'-most nucleotide of the First Region and the 3'-most nucleotide of the Fifth Region is between 100 and 3000 nucleotides; where the TP1 comprises a fluorophore (TP1 fluorophore) and a fluorescence-quenching moiety; where the TP2 comprises a fluorophore (TP2 fluorophore) and a fluorescence-quenching moiety; and where the TP1 fluorophore and the TP2 fluorophore are spectrally distinct from each other.

[0120] In one aspect, this disclosure provides a method for detection of multiple DNA targets in an analyte, the method comprising: (a) mixing the analyte with: (i) a DNA polymerase enzyme comprising 5' to 3' exonuclease activity; (ii) reagents and a buffer required for the DNA polymerase enzyme to function; (iii) a plurality of PCR primers; and (iv) a plurality of probes comprising a fluorophore and a fluorescence-quenching moiety to generate a solution; (b) subjecting the solution from step (a) to at least 7 rounds of thermal cycling, where, optionally, each round comprises alternating between a temperature of equal to or greater than 78°C for between 1 second and 30 minutes and a temperature of less than 75°C for between 1 second and 4 hours; (c) measuring fluorescence values of the solution after each round of thermal cycling in step (b) in at least two fluorescence channels; (d) determining a cycle threshold (Ct) value for each of the at least two fluorescence channels; (e) determining the temporal order of fluorescence signal increases; and (1) mapping the order of fluorescence signal increases to the presence, absence, or quantity of the multiple DNA targets.

[0121] If no qPCR fluorescence is observed for a DNA target, it can be said to be absent in a sample. If qPCR fluorescence is observed for a DNA target, it can be said to present in a sample. As used herein, a “DNA target” refers to a nucleic acid sequence between a forward and reverse primer that is desired to be amplified via PCR.

[0122] In an aspect, the term “multiple DNA targets” comprises at least 2 targets, at least 3 targets, at least 4 targets, at least 5 targets, at least 6 targets, at least 7 targets, at least 8 targets, at least 9 targets, at least 10 targets, at least 11 targets, at least 12 targets, at least 13 targets, at least 14 targets, at least 15 targets, at least 18 targets, at least 20 targets, at least 25 targets, at least 30 targets, at least 35 targets, at least 40 targets, at least 45 targets, at least 50 targets, at least 55 targets, at least 60 targets, or at least 64 targets. [0123] In an aspect, a DNA target is an animal genome DNA target. In an aspect, a DNA target is a plant genome DNA target. In an aspect, a DNA target is a bacterial genome DNA target. In an aspect, a DNA target is a fungal genome DNA target. In an aspect, a DNA target is a viral genome DNA target. In an aspect, a DNA target is a human genome DNA target.

[0124] Samples (also referred to interchangeably with the term “analyte”) can be obtained from any organism, including animals, plants, bacteria, fungi, and viruses. In an aspect, a sample is obtained from blood. In an aspect, a sample is obtained from saliva. In an aspect, a sample is obtained from urine. In an aspect, a sample is obtained from feces. In an aspect, a sample is obtained from an organ. In an aspect, a sample is obtained from a tissue. In an aspect, a sample is obtained from a single cell. In an aspect, a sample is obtained from multiple cells. In an aspect, a sample is obtained from a tumor. In an aspect, a sample is obtained from a benign tumor. In an aspect, a sample is obtained from a malignant tumor.

[0125] The following non-limiting embodiments are specifically envisioned:

1. A mixture or kit comprising:

(a) optionally, a DNA polymerase enzyme comprising 5' to 3' exonuclease activity;

(b) optionally, reagents and a buffer required for the DNA polymerase enzyme to function;

(c) optionally, at least one DNA template molecule;

(d) a First Forward Primer (FP1) comprising a sequence complementary to a First Region of the at least one DNA template molecule;

(e) a First Probe (TP1) comprising a sequence complementary or homologous to a Second Region of the at least one DNA templated molecule;

(I) a First Reverse Primer (RP1) comprising a sequence homologous to a Third Region of the at least one DNA template molecule;

(g) a Second Forward Primer (FP2) comprising a sequence complementary to a Fourth Region of the at least one DNA template molecule;

(h) a Second Blocker (B2) comprising a sequence partially complementary to the Fourth Region of the at least one DNA template molecule and partially complementary to a Template Subsequence positioned 5' to the Fourth Region of the at least one DNA template molecule;

(i) a Second Probe (TP2) comprising a sequence complementary or homologous to a Fifth Region of the at least one DNA template molecule; and (j) a Second Reverse Primer (RP2) comprising a sequence homologous to a Sixth Region of the at least one DNA template molecule. wherein the First Region, the Second Region, the Third Region, the Fourth Region, the Fifth Region, and the Sixth Region are non-overlapping; wherein the First Region, the Second Region, the Third Region, the Fourth Region, the Fifth Region, and the Sixth Region each comprise between 12 and 50 nucleotides; wherein between 0 and 5000 nucleotides are positioned between any two Regions; wherein the TP1 comprises a fluorophore (TP1 fluorophore) and a fluorescencequenching moiety; wherein the TP2 comprises a fluorophore (TP2 fluorophore) and a fluorescencequenching moiety; and wherein the TP1 fluorophore and the TP2 fluorophore are spectrally distinct from each other.

2. The mixture or kit of embodiment 1, wherein the Sixth Region, the Fifth Region, and the Fourth Region comprise Group 1, wherein the Third Region, the Second Region, and the First Region comprise Group 2, and wherein Group 1 is positioned 5' to Group 2 on the at least one DNA template molecule.

3. The mixture or kit of embodiment 1 or 2, wherein the number of nucleotides positioned between the Fourth Region and the Third Region is between 0 and 5000 nucleotides, between 0 and 2000 nucleotides, between 0 and 1500 nucleotides, between 0 and 1000 nucleotides, or between 0 and 800 nucleotides.

4. The mixture or kit of any one of embodiments 1-3, wherein the Sixth Region, the Fifth Region, and the Fourth Region comprise Group 1, wherein the Third Region, the Second Region, and the First Region comprise Group 2, and wherein Group 1 is positioned 3' to Group 2 on the at least one DNA template molecule.

5. The mixture or kit of embodiment 4, wherein the number of nucleotides positioned between the Sixth Region and the First Region is between 0 and 5000 nucleotides, between 0 and 2000 nucleotides, between 0 and 1500 nucleotides, between 0 and 1000 nucleotides, or between 0 and 800 nucleotides.

6. The mixture or kit of any one of embodiments 1-5, wherein between 3 and 25 nucleotides at the 5' end of the B2 are complementary to the Fourth Region.

7. The mixture or kit of any one of embodiments 1-6, wherein between 8 and 100 nucleotides at the 3' end of the B2 are not complementary to the Fourth Region. The mixture or kit of embodiment 7, wherein between 2 and 70 nucleotides at the 3' end of the B2 (the B2 non-compl ementary region) are not complementary to the a sequence adjacent to the 5' end of the Fourth Region. The mixture or kit of embodiment 8, wherein the B2 non-complementary region forms at least one hairpin structure. The mixture or kit of embodiment 6, wherein the 3' end of the B2 comprises a chemical functionalization that prevents DNA polymerase extension. The mixture or kit of embodiment 10, wherein the chemical functionalization is selected from the group consisting of a 3-carbon spacer, an inverted nucleotide, and a minor groove binder. The mixture or kit of any one of embodiments 1-11, wherein the TP1 fluorophore and the TP2 fluorophore are selected from the group consisting of Cy3, FAM, VIC, HEX, Cy5, Cy5.5, Quasar 705, ROX, TET, Texas Red, or another organic dye. The mixture or kit of any one of embodiments 1-12, wherein the TP1 fluorescencequenching moiety and the TP2 fluorescence-quenching moiety are selected from the group consisting of MGB, Black Hole Quencher, Iowa Black RQ, and Iowa Black FQ. The mixture or kit of any one of embodiments 1-13, wherein the mixture or kit further comprises:

(k) a Third Forward Primer (FP3) comprising a sequence complementary to a Seventh Region of the at least one DNA template;

(l) a Third Blocker (B3) comprising a sequence partially complementary to the Seventh Region of the at least one DNA template and partially complementary to a Template Subsequence positioned 5' to the Seventh Region of the at least one DNA template molecule ;

(m)a Third Probe (TP3) comprising a sequence complementary or homologous to an Eighth Region of the at least one DNA template molecule; and

(n) a Third Reverse Primer (RP3) comprising a sequence homologous to a Ninth Region of the at least DNA template molecule; wherein the Seventh, Eighth, and Ninth Regions are non-overlapping subsequences of the at least one DNA template molecule; wherein the Seventh, Eighth, and Ninth Regions each comprise between 12 and 50 nucleotides; wherein between 0 and 5000 nucleotides are positioned between any two Regions; wherein the TP3 comprises a fluorophore (TP3 fluorophore) and a fluorescencequenching moiety; and wherein the TP3 fluorophore is spectrally distinct from the TP1 fluorophore and the TP2 fluorophore.

15. The mixture or kit of any one of embodiments 1-14, wherein the mixture or kit comprises a plurality of Second Forward Primers, Second Blockers, Second Probes, and Second Reverse Primers, wherein at least two Second Probes comprise spectrally distinct fluorophores.

16. The mixture or kit of any one of embodiments 1-15, wherein the mixture or kit comprises a plurality of Third Forward Primers, Third Blockers, Third Probes, and Third Reverse Primers, wherein at least two Third Probes comprise spectrally distinct fluorophores.

17. A mixture or kit comprising:

(a) optionally, a DNA polymerase enzyme comprising 5' to 3' exonuclease activity;

(b) optionally, reagents and a buffer required for the DNA polymerase enzyme to function;

(c) optionally, at least one DNA template molecule;

(d) a First Forward Primer (FP1) comprising a sequence complementary to a Third Region of the at least one DNA template molecule;

(e) a First Probe (TP1) comprising a sequence complementary or homologous to a Second Region of the at least one DNA template molecule;

(1) a First Reverse Primer (RP1) comprising a sequence homologous to a First Region of the at least one DNA template molecule;

(g) a Second Forward Primer (FP2) comprising a sequence complementary to a Fifth Region of the at least one DNA template molecule;

(h) a Second Blocker (B2) comprising a sequence partially complementary to the Fifth Region of the at least one DNA template molecule and partially complementary to a subsequence of the at least one DNA template molecule positioned 5' to the Fifth Region of the at least one DNA template molecule;

(i) a Second Probe (TP2) comprising a sequence complementary or homologous to a Fourth Region of the at least one DNA template molecule; wherein the at least one DNA template molecule comprises, in order from 5' to 3', the First Region, the Second Region, the Third Region, the Fourth Region, and the Fifth Region; wherein the First Region, the Second Region, the Third Region, the Fourth Region, and the Fifth Region are non-overlapping; wherein the First Region, the Second Region, the Third Region, the Fourth Region, and the Fifth Region each comprise between 12 and 50 nucleotides; wherein the distance between the 5 '-most nucleotide of the First Region and the 3'- most nucleotide of the Fifth Region is between 100 and 3000 nucleotides; wherein the TP1 comprises a fluorophore (TP1 fluorophore) and a fluorescencequenching moiety; wherein the TP2 comprises a fluorophore (TP2 fluorophore) and a fluorescencequenching moiety; and wherein the TP1 fluorophore and the TP2 fluorophore are spectrally distinct from each other.

18. The mixture or kit of embodiment 17, wherein the distance between the 5'-most nucleotide of the First Region and the 3'-most nucleotide of the Fifth Region is between 100 and 3000 nucleotides, between 100 and 2000 nucleotides, between 100 and 1500 nucleotides, between 100 and 1000 nucleotides, between 100 and 800 nucleotides, between 100 and 500 nucleotides, or between 100 and 300 nucleotides.

19. The mixture or kit of embodiment 17 or 18, wherein between 3 and 25 nucleotides at the 5' end of the B2 are complementary to the Fifth Region.

20. The mixture or kit of any one of embodiments 17-19, wherein between 8 and 100 nucleotides at the 3' end of the B2 are not complementary to the Fifth Region.

21. The mixture or kit of embodiment 20, wherein between 2 and 70 nucleotides at the 3' end of the B2 (the B2 non-complementary region) are not complementary to the sequence adjacent to the 5' end of the Fifth Region.

22. The mixture or kit of embodiment 21, wherein the B2 non-complementary region forms at least one hairpin structure.

23. The mixture or kit of embodiment 20, wherein the B2 comprises a chemical functionalization that prevents DNA polymerase extension.

24. The mixture or kit of embodiment 23, wherein the chemical functionalization is selected from the group consisting of a 3-carbon spacer, an inverted nucleotide, and a minor groove binder.

25. The mixture or kit of any one of embodiments 17-24, wherein the TP1 fluorophore and the TP2 fluorophore are selected from the group consisting of Cy3, FAM, VIC, HEX, Cy5, Cy5.5, Quasar 705, ROX, TET, Texas Red, or another organic dye. The mixture or kit of any one of embodiments 17-25, wherein the TP1 fluorescencequenching moiety and the TP2 fluorescence-quenching moiety are selected from the group consisting of MGB, Black Hole Quencher, Iowa Black RQ, and Iowa Black FQ. The mixture or kit of any one of embodiments 17-26, wherein the mixture or kit further comprises:

(j) a Third Forward Primer (FP3) comprising a sequence complementary to a Seventh Region of the at least one DNA template molecule;

(k) a third Blocker (B3) comprising a sequence partially complementary to the Seventh Region of the at least one DNA template molecule and partially complementary to a subsequence of the at least one DNA template molecule positioned 5' to the Seventh Region of the at least one DNA template molecule;

(l) a Third Probe (TP3) comprising a sequence complementary or homologous to a Sixth Region of the at least one DNA template molecule; wherein the Sixth Region and the Seventh Region are non-overlapping; wherein the Sixth Region and the Seventh Region each comprise between 12 and 50 nucleotides; wherein the Sixth Region and the Seventh Region are positioned 3' to the Fifth Region; wherein the TP3 comprises a fluorophore (TP3 fluorophore) and a fluorescencequenching moiety; and wherein the TP3 fluorophore is spectrally distinct from the TP1 fluorophore and the TP2 fluorophore. The mixture or kit of any one of embodiments 17-27, wherein the mixture or kit comprises a plurality of Second Forward Primers, Second Blockers, and Second Probes, wherein at least two Second Probes comprise spectrally distinct fluorophores. The mixture or kit of any one of embodiments 17-28, wherein the mixture or kit comprises a plurality of Third Forward Primers, Third Blockers, and Third Probes, wherein at least two Third Probes comprise spectrally distinct fluorophores. A method for generating fluorescence signal in an aqueous solution, the method comprising:

(a) mixing a sample comprising a DNA template molecule with:

(i) a DNA polymerase enzyme comprising 5' to 3' exonuclease activity;

(ii) reagents and a buffer required for the DNA polymerase enzyme to function; (iii) a First Forward Primer (FP1) comprising a sequence complementary to a First Region of the DNA template molecule;

(iv) a First Probe (TP1) comprising a sequence complementary or homologous to a Second Region of the DNA template molecule;

(v) a First Reverse Primer (RP1) comprising a sequence homologous to a Third Region of the DNA template molecule;

(vi) a Second Forward Primer (FP2) comprising a sequence complementary to a Fourth Region of the DNA template molecule;

(vii) a Second Blocker (B2) comprising a sequence partially complementary to the Fourth Region of the DNA template molecule and partially complementary to a subsequence of the DNA template molecule positioned 5' to the Fourth Region of the DNA template molecule;

(viii) a Second Probe (TP2) comprising a sequence complementary or homologous to a Fifth Region of the DNA template molecule; and

(ix) a Second Reverse Primer (RP2) comprising a sequence homologous to a Sixth Region of the DNA template molecule to generate a solution; wherein the First Region, the Second Region, the Third Region, the Fourth Region, the Fifth Region, and the Sixth region are non-overlapping; wherein the First Region, the Second Region, the Third Region, the Fourth Region, the Fifth Region, and the Sixth Region each comprise between 12 and 50 nucleotides; wherein between 0 and 5000 nucleotides are positioned between any two Regions; wherein the TP 1 comprises a fluorophore (TP 1 fluorophore) and a fluorescencequenching moiety; wherein the TP2 comprises a fluorophore (TP2 fluorophore) and a fluorescencequenching moiety; and wherein the TP1 fluorophore and the TP2 fluorophore are spectrally distinct from each other; and

(b) subjecting the sample from step (a) to at least 7 rounds of thermal cycling, wherein, optionally, each round comprises alternating between a temperature of equal to or greater than 78°C for between 1 second and 30 minutes and a temperature of less than 75°C for between 1 second and 4 hours. 31. The method of embodiment 30, wherein the Sixth Region, the Fifth Region, and the Fourth Region comprise Group 1, wherein the Third Region, the Second Region, and the First Region comprise Group 2, and wherein Group 1 is positioned 5' to Group 2 on the DNA template molecule of interest.

32. The method of embodiment 30 or 31, wherein the number of nucleotides positioned between the Fourth Region and the Third Region is between 0 and 5000 nucleotides, between 0 and 2000 nucleotides, between 0 and 1500 nucleotides, between 0 and 1000 nucleotides, or between 0 and 800 nucleotides.

33. The method of any one of embodiments 30-32, wherein the Sixth Region, the Fifth Region, and the Fourth Region comprise Group 1, wherein the Third Region, the Second Region, and the First Region comprise Group 2, and wherein Group 1 is positioned 3' to Group 1 on the DNA template molecule of interest.

34. The method of any one of embodiments 31-33, wherein the number of nucleotides positioned between the Sixth Region and the First Region is between 0 and 5000 nucleotides, between 0 nucleotides and 2000 nucleotides, between 0 nucleotides and 1500 nucleotides, between 0 nucleotides and 1000 nucleotides, or between 0 nucleotides and 800 nucleotides.

35. The method of any one of embodiments 30-34, wherein between 3 nucleotides and 25 nucleotides at the 5' end of the B2 are complementary to the Fourth Region.

36. The method of any one of embodiments 30-35, wherein between 8 nucleotides and 100 nucleotides at the 3' end of the B2 are not complementary to the Fourth Region.

37. The method of embodiment 36, wherein between 2 and 70 nucleotides at the 3' end of the B2 (the B2 non-complementary region) are not complementary to the sequence adjacent to the 5' end of the Fourth Region.

38. The method of embodiment 37, wherein the B2 non-complementary region forms at least one hairpin structure.

39. The method of embodiment 35, wherein the 3' end of the B2 comprises a chemical functionalization that prevents DNA polymerase extension.

40. The method of embodiment 39, wherein the chemical functionalization is selected from the group consisting of a 3-carbon spacer, an inverted nucleotide, and a minor groove binder.

41. The method of any one of embodiments 30-40, wherein the TP1 fluorophore and the TP2 fluorophore are selected from the group consisting of Cy3, FAM, VIC, HEX, Cy5, Cy5.5, Quasar 705, ROX, TET, Texas Red, or another organic dye. 42. The method of any one of embodiments 30-41, wherein the TP1 fluorescencequenching moiety and the TP2 fluorescence-quenching moiety are selected from the group consisting of MGB, Black Hole Quencher, Iowa Black RQ, and Iowa Black FQ.

43. The method of any one of embodiments 30-42, wherein the solution further comprises:

(x) a Third Forward Primer (FP3) comprising a sequence complementary to a Seventh Region of the DNA template of interest;

(xi) a Third Blocker (B3) comprising a sequence partially complementary to the Seventh Region of the DNA template of interest and partially complementary to a Template Subsequence positioned 5' to the Seventh Region of the DNA template molecule of interest ;

(xii) a Third Probe (TP3) comprising a sequence complementary or homologous to an Eighth Region of the DNA template molecule of interest; and

(xiii) a Third Reverse Primer (RP3) comprising a sequence homologous to a Ninth Region of the DNA template molecule of interest; wherein the Seventh, Eighth, and Ninth Regions are non-overlapping subsequences of the DNA template molecule of interest; wherein the Seventh, Eighth, and Ninth Regions each comprise between 12 nucleotides and 50 nucleotides; wherein between 0 nucleotides and 5000 nucleotides are positioned between any two Regions; wherein the TP3 comprises a fluorophore (TP3 fluorophore) and a fluorescencequenching moiety; and wherein the TP3 fluorophore is spectrally distinct from the TP1 fluorophore and the TP2 fluorophore.

44. The method of any one of embodiments 30-43, wherein the solution comprises a plurality of Second Forward Primers, Second Blockers, Second Probes, and Second Reverse Primers, wherein at least two Second Probes comprise spectrally distinct fluor ophores.

45. The method of any one of embodiments 30-44, wherein the solution comprises a plurality of Third Forward Primers, Third Blockers, Third Probes, and Third Reverse Primers, wherein at least two Third Probes comprise spectrally distinct fluorophores.

46. A method for generating fluorescence signal in an aqueous solution, the method comprising: (a) mixing a sample comprising a DNA template molecule with:

(i) a DNA polymerase enzyme comprising 5' to 3' exonuclease activity;

(ii) reagents and a buffer required for the DNA polymerase enzyme to function;

(iii) a First Forward Primer (FP1) comprising a sequence complementary to a Third Region of the DNA template molecule;

(iv) a First Probe (TP1) comprising a sequence complementary or homologous to a Second Region of the DNA template molecule;

(v) a First Reverse Primer (RP1) comprising a sequence homologous to a First Region of the DNA template molecule;

(vi) a Second Forward Primer (FP2) comprising a sequence complementary to a Fifth Region of the DNA template molecule;

(vii) a Second Blocker (B2) comprising a sequence partially complementary to the Fifth Region of the DNA template molecule and partially complementary to a subsequence of the DNA template molecule positioned 5' to the Fifth Region of the DNA template molecule; and

(viii) a Second Probe (TP2) comprising a sequence complementary or homologous to a Fourth Region of the DNA template molecule to generate a solution; wherein the DNA template molecule comprises, in order from 5' to 3', the First Region, the Second Region, the Third Region, the Fourth Region, and the Fifth Region; wherein the First Region, the Second Region, the Third Region, the Fourth Region, and the Fifth Region are non-overlapping; wherein the First Region, the Second Region, the Third Region, the Fourth Region, and the Fifth Region each comprise between 12 nucleotides and 50 nucleotides; wherein the distance between the 5 '-most nucleotide of the First Region and the 3'- most nucleotide of the Fifth Region is between 100 nucleotides and 3000 nucleotides; wherein the TP1 comprises a fluorophore (TP1 fluorophore) and a fluorescencequenching moiety; wherein the TP2 comprises a fluorophore (TP2 fluorophore) and a fluorescencequenching moiety; and wherein the TP1 fluorophore and the TP2 fluorophore are spectrally distinct from each other; and (b) subjecting the sample from step (a) to at least 7 rounds of thermal cycling, wherein, optionally, each round comprises alternating between a temperature of equal to or greater than 78°C for between 1 second and 30 minutes and a temperature of less than 75°C for between 1 second and 4 hours.

47. The method of embodiment 46, wherein the distance between the 5 '-most nucleotide of the First Region and the 3 '-most nucleotide of the Fifth Region is between 100 nucleotides and 3000 nucleotides, between 100 nucleotides and 2000 nucleotides, between 100 nucleotides and 1500 nucleotides, between 100 nucleotides and 1000 nucleotides, between 100 nucleotides and 800 nucleotides, between 100 nucleotides and 500 nucleotides, or between 100 nucleotides and 300 nucleotides.

48. The method of embodiment 46 or 47, wherein between 3 nucleotides and 25 nucleotides at the 5' end of the B2 are complementary to the Fifth Region.

49. The method of any one of embodiments 46-48, wherein between 8 nucleotides and 100 nucleotides at the 3' end of the B2 are not complementary to the Fifth Region.

50. The method of embodiment 49, wherein between 2 nucleotides and 70 nucleotides at the 3' end of the B2 (the B2 non-compl ementary region) are not complementary to the sequence adjacent to the 5' end of the Fifth Region.

51. The method of embodiment 50, wherein the B2 non-complementary region forms at least one hairpin structure.

52. The method of embodiment 49, wherein the B2 comprises a chemical functionalization that prevents DNA polymerase extension.

53. The method of embodiment 52, wherein the chemical functionalization is selected from the group consisting of a 3-carbon spacer, an inverted nucleotide, and a minor groove binder.

54. The method of any one of embodiments 46-53, wherein the TP1 fluorophore and the TP2 fluorophore are selected from the group consisting of Cy3, FAM, VIC, HEX, Cy5, Cy5.5, Quasar 705, ROX, TET, Texas Red, or another organic dye.

55. The method of any one of embodiments 46-54, wherein the TP1 fluorescencequenching moiety and the TP2 fluorescence-quenching moiety are selected from the group consisting of MGB, Black Hole Quencher, Iowa Black RQ, and Iowa Black FQ.

56. The method of any one of embodiments 46-55, wherein the solution further comprises:

(ix) a Third Forward Primer (FP3) comprising a sequence complementary to a Seventh Region of the DNA template molecule of interest; (x) a third Blocker (B3) comprising a sequence partially complementary to the Seventh Region of the DNA template molecule of interest and partially complementary to a subsequence of the DNA template molecule of interest positioned 5' to the Seventh Region of the DNA template molecule of interest; and

(xi) a Third Probe (TP3) comprising a sequence complementary or homologous to a Sixth Region of the DNA template molecule of interest; wherein the Sixth Region and the Seventh Region are non-overlapping; wherein the Sixth Region and the Seventh Region each comprise between 12 and 50 nucleotides; wherein the Sixth Region and the Seventh Region are positioned 3' to the Fifth Region; wherein the TP3 comprises a fluorophore (TP3 fluorophore) and a fluorescencequenching moiety; and wherein the TP3 fluorophore is spectrally distinct from the TP1 fluorophore and the TP2 fluorophore. The method of any one of claims 46-56, wherein the solution comprises a plurality of Second Forward Primers, Second Blockers, and Second Probes, wherein at least two Second Probes comprise spectrally distinct fluorophores. The method of any one of embodiments 46-57, wherein the solution comprises a plurality of Third Forward Primers, Third Blockers, and Third Probes, wherein at least two Third Probes comprise spectrally distinct fluorophores. A method for detecting a DNA template sequence in a sample, the method comprising:

(a) mixing the sample with:

(i) a DNA polymerase enzyme comprising 5' to 3' exonuclease activity;

(ii) reagents and a buffer required for the DNA polymerase enzyme to function;

(iii) a First Forward Primer (FP1) comprising a sequence complementary to a First Region of the DNA template molecule;

(iv) a First Probe (TP1) comprising a sequence complementary or homologous to a Second Region of the DNA template molecule;

(v) a First Reverse Primer (RP1) comprising a sequence homologous to a Third Region of the DNA template molecule; (vi) a Second Forward Primer (FP2) comprising a sequence complementary to a Fourth Region of the DNA template molecule;

(vii) a Second Blocker (B2) comprising a sequence partially complementary to the Fourth Region of the DNA template molecule and partially complementary to a subsequence of the DNA template molecule positioned 5' to the Fourth Region of the DNA template molecule;

(viii) a Second Probe (TP2) comprising a sequence complementary or homologous to a Fifth Region of the DNA template molecule; and

(ix) a Second Reverse Primer (RP2) comprising a sequence homologous to a Sixth Region of the DNA template molecule to generate a solution; wherein the First Region, the Second Region, the Third Region, the Fourth Region, the Fifth Region, and the Sixth region are non-overlapping; wherein the First Region, the Second Region, the Third Region, the Fourth Region, the Fifth Region, and the Sixth Region each comprise between 12 and 50 nucleotides; wherein between 0 and 5000 nucleotides are positioned between any two Regions; wherein the TP 1 comprises a fluorophore (TP 1 fluorophore) and a fluorescencequenching moiety; wherein the TP2 comprises a fluorophore (TP2 fluorophore) and a fluorescencequenching moiety; and wherein the TP1 fluorophore and the TP2 fluorophore are spectrally distinct from each other;

(b) subjecting the solution from step (a) to at least 7 rounds of thermal cycling, wherein, optionally, each round comprises alternating between a temperature of equal to or greater than 78°C for between 1 second and 30 minutes and a temperature of less than 75°C for between 1 second and 4 hours;

(c) measuring fluorescence values of the solution after each round of thermal cycling in step (b) in the fluorescence channels corresponding to the TP1 and the TP2;

(d) determining a cycle threshold (Ct) value for the TP1 (Ctl) and the TP2 (Ct2); and (e) making a positive call for the presence of the DNA template in the sample if the Ctl is less than 35 cycles, Ct2 is less than 50 cycles, and the value of [Ct2 - Ctl] is between 1 cycle and 12 cycles. The method of embodiment 59, wherein the solution further comprises:

(x) a Third Forward Primer (FP3) comprising a sequence complementary to a Seventh Region of the DNA template;

(xi) a Third Blocker (B3) comprising a sequence partially complementary to the Seventh Region of the DNA template and partially complementary to a Template Subsequence positioned 5' to the Seventh Region of the DNA template molecule ;

(xii) a Third Probe (TP3) comprising a sequence complementary or homologous to an Eighth Region of the DNA template molecule; and

(xiii) a Third Reverse Primer (RP3) comprising a sequence homologous to a Ninth Region of the DNA template molecule; wherein the Seventh, Eighth, and Ninth Regions are non-overlapping subsequences of the DNA template molecule; wherein the Seventh, Eighth, and Ninth Regions each comprise between 12 and 50 nucleotides; wherein between 0 and 5000 nucleotides are positioned between any two Regions; wherein the TP3 comprises a fluorophore (TP3 fluorophore) and a fluorescencequenching moiety; and wherein the TP3 fluorophore is spectrally distinct from the TP1 fluorophore and the TP2 fluorophore. The method of embodiment 59 or 60, wherein the solution comprises a plurality of First Forward Primers, First Blockers, First Probes, and First Reverse Primers, wherein at least two First Probes comprise spectrally distinct fluorophores. The method of any one of embodiments 59-61, wherein the solution comprises a plurality of Second Forward Primers, Second Blockers, Second Probes, and Second Reverse Primers, wherein at least two Second Probes comprise spectrally distinct fluorophores. The method of any one of embodiments 59-62, wherein the solution comprises a plurality of Third Forward Primers, Third Blockers, Third Probes, and Third Reverse Primers, wherein at least two Third Probes comprise spectrally distinct fluorophores. A method for detecting a DNA template sequence in a sample, the method comprising: (a) mixing the sample with:

(i) a DNA polymerase enzyme comprising 5' to 3' exonuclease activity;

(ii) reagents and a buffer required for the DNA polymerase enzyme to function;

(iii) a First Forward Primer (FP1) comprising a sequence complementary to a Third Region of the DNA template molecule;

(iv) a First Probe (TP1) comprising a sequence complementary or homologous to a Second Region of the DNA template molecule;

(v) a First Reverse Primer (RP1) comprising a sequence homologous to a First Region of the DNA template molecule;

(vi) a Second Forward Primer (FP2) comprising a sequence complementary to a Fifth Region of the DNA template molecule;

(vii) a Second Blocker (B2) comprising a sequence partially complementary to the Fifth Region of the DNA template molecule and partially complementary to a subsequence of the DNA template molecule positioned 5' to the Fifth Region of the DNA template molecule; and

(viii) a Second Probe (TP2) comprising a sequence complementary or homologous to a Fourth Region of the DNA template molecule to generate a solution; wherein the DNA template molecule comprises, in order from 5' to 3', the First Region, the Second Region, the Third Region, the Fourth Region, and the Fifth Region; wherein the First Region, the Second Region, the Third Region, the Fourth Region, and the Fifth Region are non-overlapping; wherein the First Region, the Second Region, the Third Region, the Fourth Region, and the Fifth Region each comprise between 12 and 50 nucleotides; wherein the distance between the 5 '-most nucleotide of the First Region and the 3'- most nucleotide of the Fifth Region is between 100 and 3000 nucleotides; wherein the TP1 comprises a fluorophore (TP1 fluorophore) and a fluorescencequenching moiety; wherein the TP2 comprises a fluorophore (TP2 fluorophore) and a fluorescencequenching moiety; and wherein the TP1 fluorophore and the TP2 fluorophore are spectrally distinct from each other; and (b) subjecting the solution from step (a) to at least 7 rounds of thermal cycling, wherein, optionally, each round comprises alternating between a temperature of equal to or greater than 78°C for between 1 second and 30 minutes and a temperature of less than 75°C for between 1 second and 4 hours;

(c) measuring fluorescence values of the solution after each round of thermal cycling in step (b) in the fluorescence channels corresponding to the TP1 and the TP2;

(d) determining a cycle threshold (Ct) value for the TP1 (Ctl) and the TP2 (Ct2); and

(e) making a positive call for the presence of the DNA template in the sample if the Ctl is less than 35 cycles, Ct2 is less than 50 cycles, and the value of [Ct2 - Ctl] is between 1 cycle and 12 cycles. The method of embodiment 64, wherein the solution further comprises:

(ix) a Third Forward Primer (FP3) comprising a sequence complementary to a Seventh Region of the DNA template molecule;

(x) a third Blocker (B3) comprising a sequence partially complementary to the Seventh Region of the DNA template molecule and partially complementary to a subsequence of the DNA template molecule positioned 5' to the Seventh Region of the DNA template molecule; and

(xi) a Third Probe (TP3) comprising a sequence complementary or homologous to a Sixth Region of the DNA template molecule; wherein the Sixth Region and the Seventh Region are non-overlapping; wherein the Sixth Region and the Seventh Region each comprise between 12 and 50 nucleotides; wherein the Sixth Region and the Seventh Region are positioned 3' to the Fifth Region; wherein the TP3 comprises a fluorophore (TP3 fluorophore) and a fluorescencequenching moiety; and wherein the TP3 fluorophore is spectrally distinct from the TP1 fluorophore and the TP2 fluorophore. The method of embodiment 64 or 65, wherein the solution comprises a plurality of First Forward Primers, First Blockers, First Probes, and First Reverse Primers, wherein at least two First Probes comprise spectrally distinct fluorophores. 67. The method of any one of embodiments 64-66, wherein the solution comprises a plurality of Second Forward Primers, Second Blockers, Second Probes, and Second Reverse Primers, wherein at least two Second Probes comprise spectrally distinct fluor ophores.

68. The method of any one of embodiments 64-67, wherein the solution comprises a plurality of Third Forward Primers, Third Blockers, Third Probes, and Third Reverse Primers, wherein at least two Third Probes comprise spectrally distinct fluorophores.

69. A method for detection of multiple DNA targets in an analyte, the method comprising:

(a) mixing the analyte with:

(i) a DNA polymerase enzyme comprising 5' to 3' exonuclease activity;

(ii) reagents and a buffer required for the DNA polymerase enzyme to function;

(iii) a plurality of PCR primers; and

(iv) a plurality of probes comprising a fluorophore and a fluorescencequenching moiety to generate a solution;

(b) subjecting the solution from step (a) to at least 7 rounds of thermal cycling, wherein, optionally, each round comprises alternating between a temperature of equal to or greater than 78°C for between 1 second and 30 minutes and a temperature of less than 75°C for between 1 second and 4 hours;

(c) measuring fluorescence values of the solution after each round of thermal cycling in step (b) in at least two fluorescence channels;

(d) determining a cycle threshold (Ct) value for each of the at least two fluorescence channels;

(e) determining the temporal order of fluorescence signal increases; and

(1) mapping the order of fluorescence signal increases to the presence, absence, or quantity of the multiple DNA targets.

70. The method of embodiment 69, wherein the plurality of probes each comprise a spectrally distinct fluorophore.

71. The method of embodiment 69 or 70, wherein the plurality of probes each comprise a different fluorescence-quenching moiety.

72. The method of embodiment 69 or 70, wherein the plurality of probes each comprise the same fluorescence-quenching moiety. 73. The method of any one of embodiments 69-72, wherein the fluorophore is selected from the group consisting of Cy3, FAM, VIC, HEX, Cy5, Cy5.5, Quasar 705, ROX, TET, Texas Red, or another organic dye.

74. The method of any one of embodiments 69-73, wherein the fluorescence-quenching moiety is selected from the group consisting of MGB, Black Hole Quencher, Iowa Black RQ, and Iowa Black FQ.

75. The method of any one of embodiments 69-74, wherein the plurality of PCR primers comprises at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 sets of forward and reverse primers.

76. The method of any one of embodiments 69-75, wherein the plurality of probes comprises at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 probes.

77. The method of any one of embodiments 69-76, wherein the solution further comprises a plurality of Blockers.

78. The method of embodiment 77, wherein the plurality of Blockers comprises at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 Blockers.

79. The method of any one of embodiments 69-78, wherein the multiple DNA targets comprises at least 3 targets, at least 4 targets, at least 5 targets, at least 6 targets, at least 7 targets, at least 8 targets, at least 9 targets, at least 10 targets, at least 11 targets, at least 12 targets, at least 13 targets, at least 14 targets, at least 15 targets, at least 18 targets, at least 20 targets, at least 25 targets, at least 30 targets, at least 35 targets, at least 40 targets, at least 45 targets, at least 50 targets, at least 55 targets, at least 60 targets, or at least 64 targets.

80. The method of any one of embodiments 69-79, wherein the multiple DNA targets comprise DNA targets from an animal genome, a plant genome, a bacterial genome, a fungal genome, or a viral genome.

81. The method of embodiment 80, wherein the animal genome is a human genome.

82. The method of any one of embodiments 69-81, wherein step (c) comprises measuring fluorescence in at least three fluorescence channels.

83. The method of any one of embodiments 30-82, wherein the method comprises at least 10 cycles, at least 12 cycles, at least 15 cycles, at least 18 cycles, at least 20 cycles, at least 25 cycles, at least 30 cycles, at least 35 cycles, at least 40 cycles, at least 45 cycles, or at least 50 cycles of thermal cycling. 84. The method of any one of embodiments 30-83, wherein the DNA polymerase enzyme is selected from the group consisting of a Taq DNA polymerase enzyme, a Best DNA polymerase, and DNA polymerase 1.

85. A mixture or kit comprising:

(a) optionally, a DNA polymerase enzyme comprising 5' to 3' exonuclease activity;

(b) optionally, reagents and a buffer required for the DNA polymerase enzyme to function;

(c) optionally, a DNA template molecule;

(d) a First Forward Primer (FP1) comprising a sequence complementary to a First Region of the DNA template molecule;

(e) a First Probe (TP1) comprising a sequence complementary or homologous to a Second Region of the DNA template molecule;

(1) a First Reverse Primer (RP1) comprising a sequence homologous to a Third Region of the DNA template molecule;

(g) a Second Forward Primer (FP2) comprising a sequence complementary to a Fourth Region of the DNA template molecule, wherein the FP2 comprises one or more nucleotides at or near its 3' end that are not complementary to the DNA template molecule;

(h) a Second Probe (TP2) comprising a sequence complementary or homologous to a Fifth Region of the DNA template molecule; and

(i) a Second Reverse Primer (RP2) comprising a sequence homologous to a Sixth Region of the DNA template molecule; wherein the First Region, the Second Region, the Third Region, the Fourth Region, the Fifth Region, and the Sixth Region are non-overlapping; wherein the First Region, the Second Region, the Third Region, the Fourth Region, the Fifth Region, and the Sixth Region each comprise between 12 and 50 nucleotides; wherein between 0 and 5000 nucleotides are positioned between any two Regions; wherein the TP1 comprises a fluorophore (TP1 fluorophore) and a fluorescencequenching moiety; wherein the TP2 comprises a fluorophore (TP2 fluorophore) and a fluorescencequenching moiety; and wherein the TP1 fluorophore and the TP2 fluorophore are spectrally distinct from each other.

86. A mixture or kit comprising: (a) optionally, a DNA polymerase enzyme comprising 5' to 3' exonuclease activity;

(b) optionally, reagents and a buffer required for the DNA polymerase enzyme to function;

(c) optionally, a DNA template molecule;

(d) a First Forward Primer (FP1) comprising a sequence complementary to a Third Region of the DNA template molecule;

(e) a First Probe (TP1) comprising a sequence complementary or homologous to a Second Region of the DNA template molecule;

(1) a First Reverse Primer (RP1) comprising a sequence homologous to a First Region of the DNA template molecule;

(g) a Second Forward Primer (FP2) comprising a sequence complementary to a Fifth Region of the DNA template molecule, wherein the FP2 comprises one or more nucleotides at or near its 3' end that are not complementary to the DNA template molecule; and

(h) a Second Probe (TP2) comprising a sequence complementary or homologous to a Fourth Region of the DNA template molecule; wherein the at least one DNA template molecule comprises, in order from 5' to 3', the First Region, the Second Region, the Third Region, the Fourth Region, and the Fifth Region; wherein the First Region, the Second Region, the Third Region, the Fourth Region, and the Fifth Region are non-overlapping; wherein the First Region, the Second Region, the Third Region, the Fourth Region, and the Fifth Region each comprise between 12 and 50 nucleotides; wherein the distance between the 5 '-most nucleotide of the First Region and the 3'- most nucleotide of the Fifth Region is between 100 and 3000 nucleotides; wherein the TP1 comprises a fluorophore (TP1 fluorophore) and a fluorescencequenching moiety; wherein the TP2 comprises a fluorophore (TP2 fluorophore) and a fluorescencequenching moiety; and wherein the TP1 fluorophore and the TP2 fluorophore are spectrally distinct from each other.

[0126] Having now generally described the disclosure, the same will be more readily understood through reference to the following examples that are provided by way of illustration, and are not intended to be limiting of the present disclosure, unless specified.

EXAMPLES

Example 1. Color combinations

[0127] In applications where certain combinations of DNA targets are unlikely to be simultaneously present in the same sample, it is possible to detect more DNA targets than the number of distinct fluorescence color channels through the use of combinations of fluorescent probes. For example, in syndromic testing for infectious pathogens, most patients are affected with at most one pathogen out of a list of common pathogens.

[0128] Through the use of four fluorescence color channels A, B, C, and D, it is possible to identify up to 4 + 6 + 4 + l = 2 ^{(4 )} = 15 different pathogens, assuming that no more than one pathogen is present in any sample. An example mapping of fluorescence observed vs. DNA target is shown in Table 2.

Table 2. Mapping of fluorescence observed vs. DNA target

[0129] A sigmoidal qPCR trace indicative of a positive result in two distinct fluorescence color channels A and B could indicate a distinctive DNA target #5. This can be implemented through using two distinct TaqMan™ probes for DNA target #5, with one TaqMan™ probe having the A fluorophore and the other TaqMan™ probe having the B fluorophore.

[0130] Even in cases where multiple DNA targets may be present in solution, it may be possible to exceed the number of fluorescence color channels as long as not all possible combinations of pathogens are likely be to present. For example, using four fluorescence color channels, it may is possible to detect six different DNA targets as long as at most one of target #1, #2, and #3 are present, and at most one of target #4, #5, and #6 are present in a sample. See Table 3.

Table 3. Mapping of fluorescence observed vs. DNA target Example 2. Cycle multiplexing

[0131] Cycle multiplexing allows one to increase the number of distinct reportable DNA targets through the use of color permutations. Cycle multiplexing is based on the rational design of multiplex qPCR reactions so that when a given DNA target is present, a first fluorescence channel shows a fluorescence increase at an earlier cycle, and then a second fluorescence channel shows fluorescence increases at a later cycle. Thus, whereas in color combinations (see Example 1), fluorescence increases in A and B channels (written as AB) can indicate a third target DNA sequence, with color multiplexing, the permutation A>B (fluorescence increases in A first, then B) can represent a different DNA target than the permutation B>A.

[0132] Using 4 color channels, cycle multiplexing can allow identification of up to 4+4x3+4x3x2+4x3x2xl = 64 distinct DNA targets, assuming that the sample has at most 1 of the DNA targets shown in Table 4.

Table 4. Color permutations

[0133] In the notation in Table 4, A>B>C>D indicates that A fluorescence increases first, followed by B, followed by C, followed by D. In some embodiments, the number of PCR cycles between the rise of one fluorescence signal and the rise of the subsequence fluorescence signal can be between 3 and 5, so that it can be easy to determine the order of fluorescence signals. Cycle multiplexing thus allows the encoding of 64 distinct signals using four color channels, a 4-fold increase over color combinations (see Example 1). This increased number of distinct signals can likewise be used for applications where certain sets of different DNA targets may co-exist in solution.

Example 3. Blocker displacement amplification

[0134] One non-limiting example embodiment of cycle multiplexing is to use Blocker Displacement Amplification (BDA) to engineer Ct delays for different amplicons on the same DNA target. In BDA, a rationally designed Blocker oligonucleotide competes with a forward primer in binding to the template, and results in a delay in PCR amplification, resulting in larger Ct values than a PCR reaction without the Blocker. The exact value of the Ct delay can be modulated via the sequence of the Blocker and via the concentration of the Blocker. This feature allows multiple different amplicons to engineered to give Ct values differing by a desired number of cycles. The ability to rationally design Ct delay values across a wide range of possibilities is also helpful for two-level and three-level cycle multiplexing, in which the order of three or four different fluorescence signals are used to determine the DNA target present in a sample. Examples of BDA were previously described in U.S. Patent Application Publication No. 20170067090 and WO 2019/164885, which are both incorporated by reference herein in their entireties.

[0135] A First Forward Primer (FP 1) and a Second Forward Primer (FP2) are designed that exhibits significant reverse complementarity to the corresponding Template, a Second Blocker (B2) that is partially overlapped with the 3' region of the Second Forward Primer and mostly or fully reverse complementary to the Template (see Figure 1). A First Probe (TP1) and a Second Probe (TP2) that fall into the region between a First Reverse Primer (RP1) that exhibits significant sequence similarity to the corresponding Template.

[0136] In some embodiments, the system also has a Second Reverse Primer (RP2) that is mostly or fully homologous to the Template. In some embodiments, the system has a Third Forward Primer (FP3), a Third Blocker (B3) and a Third TaqMan™ Probe (TP3). In some embodiments, the system has a Third Forward Primer (FP3), a Third Blocker (B3), a Third TaqMan™ Probe (TP3) and a Third Reverse Primer (RP3). Example 4. Variants

[0137] There are many obvious variants apparent to one of ordinary skill in the art. For example, a Probe can bind to either the (+) strand or the (-) strand of the DNA template. For example, a Probe can be functionalized with the fluorophore at the 5' end, the 3' end, or in the middle. The quencher can likewise be functionalized at the 5' end, the 3' end, or in the middle. The primers can be designed to be perfectly or imperfectly complementary to the intended DNA target sequences. The primers may have 5' overhangs that can serve as adapters for subsequent re-analysis such as using sequencing. The primers or probes may contain DNA nucleoside modifications such as non-natural nucleotides such as inosine, 5'-nitroindole, PEG spacers, iso-C, or iso- G. The primers or probes may contain DNA backbone modifications such as phosphorothioate, locked nucleic acid, 2'-O-methyl, or 5 '-2' DNA linkage. The reaction buffer may include dUTP in addition to the standard dATP, dTTP, dCTP, and dGTP to prevent carry-over contamination. The reaction buffer may include crowding agents to improve PCR amplification of high G/C content DNA sequences.

Example 5. Cycle Multiplex PCR

[0138] qPCR was applied to aNA18537 human genomic DNA template (20 ng; Coriell Cell Repositories) using the PowerUp™ SYBR™ Green DNA Polymerase Master Mix (ThermoFisher). Figure 6 provides a schematic design of the experiment in the top panel. The 5' end of the First Probe (TP1 ; SEQ ID NO: 7) comprises a Cy5 fluorophore, and the 5' end of the Second Probe (TP2; SEQ ID NO: 4) comprises a ROX fluorophore. The two sets of primers (FP1 (SEQ ID NO: 5) and RP1 (SEQ ID NO: 6); FP2 (SEQ ID NO: 1) and RP2 (SEQ ID NO: 2)) are separated by 12 nucleotides. The amplicon produced by FP1 and RP1 is 121 nt in length, and the amplicon produced by FP2 and RP2 is 98 nucleotides in length. Experimental results are shown in the bottom panel of Figure 6. The consistent cycle threshold (Ct) value of the Cy5 curves among all the graphs under different conditions reveals that the efficiency of the primer pair is not influenced by other oligonucleotides inside the system. The Ct value of the ROX channel increases as the concentration of the Second Blocker (B2; SEQ ID NO: 3) increases, as does the ACt between the ROX channel and the Cy5 channel, demonstrating the success of the cycle multiplexing system. See Figure 6. [0139] Thermal cycling and fluorescence measurement were performed using a BioRad CFX96 qPCR instrument. The thermal cycling protocol is as follows: 1 cycle of 95°C for 3 minutes; then 50 cycles of (95°C for 10 seconds, 60°C for 30 seconds). Fluorescent signals are collected under 60 °C.