Reagents, mixtures, kits, and methods for amplifying nucleic acids

Abstract

To provide simplified and affordable means for use in detecting low abundance target polynucleotides.SOLUTION: This disclosure relates to reagents, mixtures, kits and methods for use in detecting low-frequency target polynucleotides, such as rare allelic variants.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 62 / 719,074, filed August 16, 2018, the disclosure of which is incorporated herein by reference in its entirety.

[0002] The present disclosure relates to reagents, mixtures, kits, and methods for use in detecting target polynucleotides, particularly mutant or low abundance target polynucleotides. [Background technology]

[0003] Cancer detection and treatment have advanced in recent years. Much of this progress is due to our growing understanding of the molecular basis of cancer and the detailed molecular mechanisms used by cancer cells to evade the immune system. Accordingly, biomarkers have been identified that help identify patients who will likely respond to specific cancer treatments. For example, biomarkers found on tumor cells, such as programmed death-ligand 1 (PD-L1), identify patients who will likely respond to immunotherapies such as anti-PD-1 antibodies. Furthermore, biomarkers include specific mutations in specific genes. For example, detecting mutations in a specific kinase can help identify patients who are much more likely to respond to kinase inhibitor cancer therapeutics. Then, as the cancer mutates further and overcomes blockade of the first kinase, the detection of new mutations that confer the cancer the ability to overcome this first kinase can be used to identify subsequent treatment options that may be effective against this type of cancer.

[0004] The ability to detect mutations in cancer requires improved methods for detecting low-abundance alleles.Due to the heterogeneity of cancerous cells that constitute tumors, detecting low-abundance alleles is important when identifying the genetic defects underlying tumors.Furthermore, due to the presence of relatively small amounts of circulating tumor DNA compared with the circulating DNA from normal cells, detecting low-abundance alleles is important in analyzing the circulating DNA of cancer patients.In both contexts, the ability to better detect such low-abundance alleles should lead to improved detection and improved targeted treatment of cancer patients.Currently available systems are either unable to detect target polynucleotides (e.g., including mutant nucleic acid sequences) at very low copy numbers (e.g., less than 10 copies and / or less than 0.1% in a 30 ng test nucleic acid sample), or are very expensive, time-consuming, and complicated.Therefore, there is a need in the art for simplified and affordable reagents, mixtures, kits, and methods for use in detecting low-abundance target polynucleotides. Summary of the Invention

[0005] Reagents, mixtures, kits, and methods for use in detecting low-abundance target polynucleotides are provided herein. In some embodiments, the present disclosure provides a method for detecting low-abundance target polynucleotides, comprising: a) a first oligonucleotide configured to hybridize to a first sequence in a first target polynucleotide strand (such as a first strand in a double-stranded polynucleotide), the first sequence in the first target polynucleotide strand having a target variant nucleotide, the first oligonucleotide further having a nucleotide residue at its 3' end that is positioned to hybridize to the target variant nucleotide; and b) a second oligonucleotide configured to hybridize to a sequence complementary to a second sequence in the first target polynucleotide strand (e.g., a portion of the second strand that is complementary to the first strand). The present invention relates to a mixture comprising: (a) a second oligonucleotide having a sequence configured to hybridize to a first target polynucleotide strand, wherein the second sequence of the first target polynucleotide strand is located 5' upstream from the first sequence of the first target polynucleotide strand; and (b) a third oligonucleotide having a sequence configured to hybridize to a third sequence of the first target polynucleotide strand, wherein the third sequence of the first target polynucleotide strand at least partially overlaps with the first target polynucleotide strand and the first sequence of the target variant nucleotide. In some embodiments, additional oligonucleotides and / or sets of oligonucleotides are also provided. In some embodiments, the third oligonucleotide is detectable. Reagents combined to provide such mixtures are also contemplated herein, as are kits and methods that include and / or use the same. Further details regarding aspects and embodiments of the present disclosure are provided throughout this patent application. The sections and section headers are not intended to limit the methods, compositions, and kits or the combinations of functional elements therein. [Brief explanation of the drawings]

[0006] [Figure 1]Exemplary first oligonucleotides (e.g., target sequence-specific primers ("TSPs")), second oligonucleotides (e.g., locus-specific primers ("LSPs")), and third oligonucleotides (e.g., target site-specific probes) aligned with exemplary target polynucleotides. [Figures 2A-2F] Exemplary amplification of target polynucleotides using exemplary target sequence-specific primers (TSPs) and locus-specific primers (LSPs) for detection of KRAS G12R(34G>C) target polynucleotides with (Figures 2A, 2B, and 2C) and without (Figures 2D, 2E, and 2F) enrichment cycles. A sample of wild-type DNA (10 ng) or a spiked sample (10 ng of wild-type DNA spiked with 0.1% allelic variant DNA (e.g., mutant DNA) (control DNA from CEPH individual 1347-02, Thermo Fisher Scientific Cat. No. 403062, hereafter referred to as "CEPH")) was combined with 300 nM of each primer, 250 nM of probe, 1 mM dNTPs, 39 mM Tris pH 8, 2.55 mM MgCl, 30 mM KCl, 16 mM (NH)SO, 0.1 mg / ml BSA, 7% glycerol, and 0.085 U / µL Platinum Taq to form a reaction mixture. A 10 µl aliquot of the mixture was then plated into four replicate wells of a 96-well plate. qPCR reactions were performed in each well on a QuantStudio 5 F96 (Thermo Fisher Scientific, Waltham, MA) with (Figures 2A, 2B, and 2C) or without (Figures 2D, 2E, and 2F) an enrichment phase. [Figures 3A-3F]3A, 3B, and 3C) and without enrichment cycles (FIGS. 3D, 3E, and 3F), using exemplary target sequence-specific primers (TSPs) and locus-specific primers (LSPs) for the KRAS G12A (35G>C) target polynucleotide. A sample of wild-type DNA (10 ng) or a spiked sample (10 ng of wild-type (CEPH) DNA spiked with 0.1% allelic variant DNA (e.g., mutant DNA)) was combined with 300 nM of each primer, 250 nM of probe, 1 mM dNTPs, 39 mM Tris pH 8, 2.55 mM MgCl, 30 mM KCl, 16 mM (NH)SO, 0.1 mg / ml BSA, 7% glycerol, and 0.085 U / μL Platinum Taq to form a reaction mixture. Next, 10 μl aliquots of the mixture were seeded into four replicate wells of a 96-well plate. qPCR reactions were performed in each well on a QuantStudio 5 F96 (Thermo Fisher Scientific, Waltham, MA). PCR reactions were performed with (Figures 3A, 3B, and 3C) or without (Figures 3D, 3E, and 3F) an enrichment phase. [Figures 4A-4F]Wild-type (CEPH) DNA and 0.1% of the following allelic variant (e.g., mutant) KRAS DNA: KRAS G12R (34G>C, Horizon Discovery Ltd. Cat. No. HD287) (Figure 4A), KRAS G12A (35G>C, Horizon Discovery Ltd. Cat. No. HD265) (Figure 4B), KRAS G12S (34G>A, Horizon Discovery Ltd. Cat. No. HD288) (Figure 4C), KRAS G12C (34G>T, Horizon Discovery Ltd. Cat. No. HD269) (Figure 4D), KRAS G12D (35G>A, Horizon Discovery Ltd. Cat. No. HD272) (Figure 4E), or KRAS G12V (35G>T, Horizon Discovery Ltd. Cat. No. HD273) (Figure 4F). Discrimination of wild-type (CEPH) DNA spiked individually with CEPH (Cat. No. HD289) (Figure 4F). PCR conditions were as described above for Figures 2 and 3, with enrichment. [Figure 5A-5B] In Figure 5A, differentiation between mutant KRAS G13D ("G13D") target polynucleotides and wild-type (CEPH) nucleic acids was observed in qPCR reactions containing up to 60 mM KCl (potassium chloride) and up to 30 mM (NH4)2SO4 (ammonium sulfate). In Figure 5B, differentiation between mutant KRAS G13D target polynucleotides and wild-type (CEPH) nucleic acids was not observed in qPCR reactions lacking KCl and (NH4)2SO4 (ammonium sulfate). Wild-type DNA was spiked with 0.2% mutant KRAS G13D target polynucleotides in both reactions. [Figures 6A-6D]Effect of potassium chloride and ammonium sulfate on target polynucleotide amplification and detection. A 20 μL reaction mixture was prepared containing 10 ng of wild-type (CEPH) DNA, 300 nM of each primer (TSP and LSP), 250 nM of probe, 1 mM dNTP, 0.085 U / μL Platinum Taq, 2.55 mM MgCl, 45 nM ROX passive reference, 39 mM Tris, pH 8, and 7% glycerol. Potassium chloride and ammonium sulfate were titrated into the reaction mixture as described below. Figure 6A: No KCl or ammonium sulfate (no differentiation); Figure 6B: 30 mM ammonium sulfate, no potassium chloride (some differentiation); Figure 6C: 30 mM KCl and 30 mM ammonium sulfate (sufficient differentiation); and Figure 6D: 60 mM KCl and 30 mM ammonium sulfate (inhibited reaction). Reactions with the G13D mutant spike contained 20 pg of KRAS G13D reference standard DNA (Horizon Discovery Ltd., Cat. No. HD290) spiked into wild-type (CEPH) DNA at 0.2%. Reactions were amplified on a QuantStudio 7 instrument using the following thermal cycling protocol: 95°C (3 min), 19 cycles of 95°C (3 s) / 64°C (20 s) (enrichment phase), followed by 40 cycles of 95°C (3 s) / 60°C (20 s) (amplification and detection phase). [Figures 7A-7B] Using qPCR conditions as described above in Figure 6 (with 0.15% mutant DNA spiked into wild-type DNA), but with intermediate concentrations of KCl and ammonium sulfate as indicated, Figure 7A contained 45 mM KCl and 30 mM ammonium sulfate. Figure 7B contained 45 mM KCl and 22 mM ammonium sulfate. [Figure 8]Effect of distance of target variant nucleotide from the end of a third oligonucleotide (e.g., target site-specific probe). Experiments were performed in 20 μL reactions containing 1 mM dNTPs, 45 mM KCl, 22 mM ammonium sulfate, 0.085 U / μL Platinum Taq, 2.55 mM MgCl2, 45 nM ROX passive reference, 39 mM Tris pH 8, and 7% glycerol, 300 nM of each primer (TSP and LSP), 250 nM of one of probes 1, 2, or 3, 10 ng of wild-type (CEPH) DNA, and 10 pg of EGFR L858R reference standard DNA (Horizon Discovery Ltd., Cat. No. HD254). The data shown demonstrate that effective amplification and real-time detection were achieved with target variant nucleotides located 3 (probe 1), 4 (probe 2), or 5 (probe 3) nucleotides from the 3' end of the probe. [Figures 9A-9F]Titration and differentiation of mutant target DNA spiked into wild-type DNA. These experiments were performed in 20 μL reactions containing 10 ng of wild-type (CEPH) DNA, 1 mM dNTPs, 45 mM KCl, 22 mM ammonium sulfate, 0.085 U / μL Platinum Taq, 2.55 mM MgCl, 45 nM ROX passive reference, 39 mM Tris pH 8, and 7% glycerol. A 50 fM solution of the artificial mutant template containing the indicated point mutation was first diluted to 3000 copies / μL, then to 250 copies / μL, followed by two-fold dilutions to 2 copies / μL. Reactions were amplified on a QuantStudio 7 instrument using the following thermal cycling protocol: 95°C (3 min), 19 cycles of 95°C (3 s) / 64°C (20 s) (enrichment phase), followed by 40 cycles of 95°C (3 s) / 60°C (20 s). Experiments were performed using the following amounts and copies of primers and target polynucleotides: Figure 9A contained 300 nM of each primer and 250 copies of target polynucleotide; Figure 9B contained 300 nM of each primer and 16 copies of target polynucleotide; Figure 9C contained 300 nM of each primer and 2 copies of target polynucleotide; Figure 9D contained 450 nM of each primer and 250 copies of target polynucleotide; Figure 9E contained 450 nM of each primer and 16 copies of target polynucleotide; and Figure 9F contained 450 nM of each primer and 2 copies of target polynucleotide. [Figure 10]Titration of mutant DNA into wild-type DNA. These experiments were performed as described for Figures 9A-9F using 20 μL reactions containing 10 ng of wild-type (CEPH) DNA, 1 mM dNTPs, 45 mM KCl, 22 mM ammonium sulfate, 0.085 U / μL Platinum Taq, 2.55 mM MgCl, 45 nM ROX passive reference, 39 mM Tris pH 8, and 7% glycerol. A 50 fM solution of the artificial mutant template was first diluted to 3000 copies / μL, then to 250 copies / μL, followed by two-fold dilutions to 2 copies / μL. Reactions were amplified on a QuantStudio 7 instrument using the following thermal cycling protocol: 95°C (3 min), 19 cycles of 95°C (3 s) / 64°C (20 s) (enrichment phase), followed by 40 cycles of 95°C (3 s) / 60°C (20 s). The amount of each primer and copies of variant allele (e.g., mutant) polynucleotide spiked into the reaction were as indicated. The left y-axis shows the Cq of FAM. The right axis shows the delta Cq of FAM-VIC. [Figure 11]The indicated dilutions of the target polynucleotide (NRAS Q61R) were spiked into wild-type DNA at 3,000, 300, 30, and 3 copies. For these experiments, a 20-µL reaction mixture was prepared containing 300 nM of each primer (TSP and LSP), 250 nM of probe, 10 ng of wild-type (CEPH) DNA, and the indicated amount of mutant DNA (Horizon Discovery Ltd., Cat. No. HD574) at <1, 3, 30, 300, or 3,000 copies, 1 mM dNTPs, 45 mM KCl, 22 mM ammonium sulfate, 0.085 U / µL Platinum Taq, 2.55 mM MgCl, 45 nM ROX passive reference, 39 mM Tris, pH 8, and 7% glycerol. Data were exported in Excel format, Cq values ​​for replicate reactions were averaged, and the delta average Cq of the target under each condition was determined and plotted (data not shown). Delta Cq was used as the quantification method. The thermal cycling conditions for the QuantStudio 7 were 95°C (3 min), enrichment phase—19 cycles of 95°C (3 s) / 64°C (20 s), amplification—40 cycles of 95°C (3 s) / 60°C (20 s). "ntc" = no template control. [Figure 12]Dilutions of the indicated copy numbers (2, 4, 8, 16, 31, 62, 125, or 250 copies) of the target polynucleotide (NRAS Q61K) were spiked into wild-type DNA. For these experiments, a 20-µL reaction mixture was prepared containing 300 nM of each primer (TSP and LSP), 250 nM of probe, 10 ng of wild-type (CEPH) DNA, and the indicated amount of mutant (Horizon Discovery Ltd., Cat. No. HD351) (2, 4, 8, 16, 31, 62.5, 125, or 250 copies), 1 mM dNTPs, 45 mM KCl, 22 mM ammonium sulfate, 0.085 U / µL Platinum Taq, 2.55 mM MgCl, 45 nM ROX passive reference, 39 mM Tris, pH 8, and 7% glycerol. Data were exported in Excel format, Cq values ​​for replicate reactions were averaged, and the delta average Cq of the target under each condition was determined and plotted (data not shown). Delta Cq was used as the quantification method. Thermal cycling conditions for the Quant Studio 7 were 95°C (3 min), 19 cycles of 95°C (3 s) / 64°C (20 s), and 40 cycles of 95°C (3 s) / 60°C (20 s). [Figures 13A-13C]Three copies of the target polynucleotide (KRAS G12R) were diluted into 20 ng (0.05% spike-in, FIG. 13A), 10 ng (0.1% spike-in, FIG. 13B), and 5 ng (0.2% spike-in, FIG. 13C) wild-type DNA. These experiments were performed in a reaction mixture containing 300 nM KRAS forward and reverse primers, 100 nM RPPH1 forward and reverse primers, 250 nM KRAS-FAM probe, 150 nM RPPH1-VIC probe, 1 mM dNTPs, 45 mM KCl, 22 mM ammonium sulfate, 0.085 U / uL Platinum Taq, 2.55 mM MgCl, 45 nM ROX passive reference, 39 mM Tris pH 8, 7% glycerol. Ten pg of KRAS G12R reference standard DNA (Horizon Discovery Ltd., Cat. No. HD287) and the indicated amount of wild-type (CEPH) DNA were prepared. Reactions were run in a QuantStudio 5 using the following thermal cycling protocol: 95°C (2 min), 19 cycles of 95°C (1 s) / 64°C (20 s) (enrichment), followed by 40 cycles of 95°C (1 s) / 60°C (20 s). [Figure 14] Use of the reverse KRAS G12D (35G>A) primer in the amplification of target polynucleotides. These experiments were performed in a 20 μL reaction mixture containing 10 ng of wild-type (CEPH) DNA, 300 nM of the indicated KRAS forward and reverse primers, 250 nM of KRAS probe, and a 10 pg spike of mutant KRAS (G12D or G12S, as indicated) reference standard DNA (Horizon Discovery Ltd., Cat. No. HD272 or HD288, respectively). 1 mM dNTPs, 45 mM KCl, 22 mM ammonium sulfate, 0.085 U / μL Platinum Taq, 2.55 mM MgCl, 45 nM ROX passive reference, 39 mM Tris, pH 8, and 7% glycerol were used. Thermal cycling conditions were 95°C (3 min), 19 cycles of 95°C (3 s), 64°C (20 s) (enrichment), followed by 40 cycles of 95°C (3 s) / 60°C (20 s). [Figure 15]Use of the reverse KRAS G12S(34G>A) primer in the amplification of a target polynucleotide performed as described for Figure 14, but using instead the reverse primer for KRAS G12S(34G>A). [Figures 16A-16F]Amplification of target polynucleotides in the presence of wild-type DNA (0.1% mutant target polynucleotides). Figure 16A. EGFR20 T790M (2369C>T, Horizon Discovery Ltd., Cat. No. HD258) target polynucleotide. Figure 16B. EGFR19 (del746-750, Horizon Discovery Ltd., Cat. No. HD251) target polynucleotide. Figure 16C. NRAS G12D (35G>A, Horizon Discovery Ltd., Cat. No. HD745) target polynucleotide. Figure 16D. BRAF V600E (1799T>A, Horizon Discovery Ltd., Cat. No. HD238) target polynucleotide. Figure 16E. NRAS G13D (38G>A, Horizon Discovery Ltd., Cat. No. HD745) target polynucleotide. Figure 16F. EGFR L861Q (2582T>A, Horizon Discovery Ltd., Cat. No. HD257) target polynucleotide. Each experiment was performed in a 20 uL reaction mixture containing 10 ng of wild-type (CEPH) DNA, 300 nM each of first and second oligonucleotides (e.g., target sequence-specific primer (TSP) and locus-specific primer (LSP)), 250 nM of probe, and utilized a 10 pg spike of the corresponding mutant DNA listed above (Horizon Discovery Ltd., Reference Standards). 1 mM dNTPs, 45 mM KCl, 22 mM ammonium sulfate, 0.085 U / uL Platinum Taq, 2.55 mM MgCl, 45 nM ROX passive reference, 39 mM Tris, pH 8, and 7% glycerol were prepared. Data were exported in Excel format, Cq values ​​for replicate reactions were averaged, and the delta average Cq of the target under each condition was determined and plotted (data not shown). Delta Cq was used as the quantification method. The thermal cycling conditions used were 95°C (3 min), 19 cycles of 95°C (3 s) / 64°C (20 s) (enrichment), followed by 40 cycles of 95°C (3 s) / 60°C (20 s). [Figures 17A-17B]Amplification of target polynucleotides using probes of different lengths. Figure 17A. NRAS Q61L (182A>T), 16- and 21-nucleotide probes. Figure 17B. NRAS Q61H (183A>T), 15- and 20-nucleotide probes. These experiments were performed in 20 μL reaction mixtures containing 300 nM of each primer, 250 nM of the corresponding probe, 10 ng of wild-type (CEPH) DNA, a 20 pg mutant spike of NRAS Q61L or Q61H reference standard DNA (Horizon Discovery Ltd., Cat. No. HD412 or HD303, respectively), 1 mM dNTPs, 45 mM KCl, 22 mM ammonium sulfate, 0.085 U / μL Platinum Taq, 2.55 mM MgCl, 45 nM ROX passive reference, 39 mM Tris, pH 8, and 7% glycerol. Reactions were run on a QuantStudio 5 using the following thermal protocol: 95°C (2 min), 19 cycles of 95°C (1 s) / 64°C (20 s) (enrichment), then 40 cycles of 95°C (1 s) / 60°C (20 s). [Figures 18A-18C]Amplification of target polynucleotides (0.1% mutant target polynucleotides) in the presence of wild-type (CEPH) DNA. Figure 18A. ESR1 E380Q (1138G>C) target polynucleotide. Figure 18B. PIK3CA H1047R (3140A>G) target polynucleotide. Figure 18C. TP53 H179Q (537T>A) target polynucleotide. Each experiment was performed in a 20 μL reaction mixture containing 10 ng of wild-type (CEPH) DNA, 300 nM each of the first and second oligonucleotides (e.g., target sequence-specific primer (TSP) and locus-specific primer (LSP) for each of the indicated mutant and RPPH1 control targets), 250 nM of probe for each of the indicated mutant and RPPH1 control targets, and utilized a 10 pg spike of the corresponding mutant DNA listed above. The buffer was prepared in 1 mM dNTPs, 45 mM KCl, 22 mM ammonium sulfate, 0.085 U / μL Platinum Taq, 2.55 mM MgCl, 45 nM ROX passive reference, 39 mM Tris, pH 8, and 7% glycerol. Data were exported in Excel format, Cq values ​​for replicate reactions were averaged, and the delta-average Cq for the target in each reaction condition was determined and plotted (data not shown). The delta Cq between the RPPH1 control target and each individual mutant target was used as the quantification method. The thermal cycling conditions used were 95°C (3 min), 19 cycles of 95°C (3 s) / 64°C (20 s) (enrichment), followed by 40 cycles of 95°C (3 s) / 60°C (20 s). [Figures 19A-19B]Amplification of target polynucleotides (0.1% mutant target polynucleotides) in the presence of wild-type (CEPH) DNA. Figure 19A. TP53 Y220C (659A>G) target polynucleotide. Figure 19B. TP53 R249M (746G>T) target polynucleotide. Each experiment was performed in a 20 μL reaction mixture containing 10 ng of wild-type (CEPH) DNA, 300 nM each of the first and second oligonucleotides (e.g., target sequence-specific primer (TSP) and locus-specific primer (LSP) for each of the indicated mutant and RPPH1 control targets), 250 nM of probe for each of the indicated mutant and RPPH1 control targets, and utilized a 10 pg spike of the corresponding mutant DNA listed above. The buffer was prepared in 1 mM dNTPs, 45 mM KCl, 22 mM ammonium sulfate, 0.085 U / μL Platinum Taq, 2.55 mM MgCl, 45 nM ROX passive reference, 39 mM Tris, pH 8, and 7% glycerol. Data were exported in Excel format, Cq values ​​for replicate reactions were averaged, and the delta-average Cq for the target in each reaction condition was determined and plotted (data not shown). The delta Cq between the RPPH1 control target and each individual mutant target was used as the quantification method. The thermal cycling conditions used were 95°C (3 min), 19 cycles of 95°C (3 s) / 64°C (20 s) (enrichment), followed by 40 cycles of 95°C (3 s) / 60°C (20 s). [Figure 20] Exemplary wild-type EGFR and BRAF sequences. Bolded nucleotides represent bases that are mutated in the mutant polynucleotides described elsewhere herein. [Figure 21] Exemplary wild-type KRAS and NRAS sequences. Bolded nucleotides represent bases that are mutated in mutant polynucleotides described elsewhere herein (e.g., KRAS c.34G, c.35G, c.38G).

[0007] definition As used herein, the term "minor allele" or "minor allelic variant" refers to a target polynucleotide that is present at a lower level in a sample compared to alternative allelic variants (e.g., "abundant alleles" such as "major alleles" and / or "wild-type alleles"). For example, a minor allelic variant may be found at a frequency of less than 1 / 10, 1 / 100, 1 / 1,000, 1 / 10,000, 1 / 100,000, 1 / 1,000,000, 1 / 10,000,000, 1 / 100,000,000, or 1 / 1,000,000,000 compared to another allelic variant for a given single nucleotide polymorphism (SNP) or gene (e.g., having a minor allele frequency ("MAF")). Alternatively, the rare allelic variant can be, for example, less than 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 50, 75, 100, 250, 500, 750, 1,000, 2,500, 5,000, 7,500, 10,000, 25,000, 50,000, 75,000, 100,000, 250,000, 500,000, 750,000, or 1,000,000 copies per 1, 10, 100, 1,000 microliters of sample or reaction volume. In some embodiments, an allele that is present at a frequency of 1 in 1,000 copies or less relative to another allelic variant of a given SNP or gene may be referred to herein as a "rare allele," "rare allelic variant," "low abundance allele," or "low abundance allelic variant." One of skill in the art will understand that minor alleles or minor allelic variants other than those explicitly defined herein are applicable to the present disclosure.

[0008] As used herein, the term "abundant allele" refers to a target polynucleotide that is present at a higher level in a sample compared to alternative allelic variants.Amplete alleles can also be referred to as "major alleles" and / or "wild-type alleles".For example, abundant alleles can be found at a frequency of more than 10x, 100x, 1,000x, 10,000x, 100,000x, 1,000,000x, 10,000,000x, 100,000,000x, or 1,000,000,000x compared to the allelic variants and / or major alleles (or wild-type alleles) for a given SNP or gene. Alternatively, an abundant allelic variant can be present at, for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 50, 75, 100, 250, 500, 750, 1,000, 2,500, 5,000, 7,500, 10,000, 25,000, 50,000, 75,000, 100,000, 250,000, 500,000, 750,000, or greater than 1,000,000 copies per 1, 10, 100, or 1,000 microliters of sample or reaction volume. Those skilled in the art will understand that abundant alleles other than those expressly defined herein are applicable to the present disclosure. DETAILED DESCRIPTION OF THE INVENTION

[0009] The present disclosure relates to reagents, mixtures, kits, and methods for use in detecting one or more target polynucleotides (alternatively, which may be referred to herein as target polynucleotides and / or target polynucleotide sequences), such as low abundance (or "rare") target polynucleotides, comprising at least one target variant nucleotide (e.g., a mutated genetic variant and / or a minor / specific allelic variant) using at least a first oligonucleotide that functions as a target sequence-specific primer (sometimes abbreviated as "TSP") having specificity for at least one target variant nucleotide, at least one second oligonucleotide (sometimes referred to as a locus-specific primer ("LSP")) that functions as a primer having specificity for the target polynucleotide but not the target variant nucleotide, and at least a third oligonucleotide that functions as a target site-specific probe having binding specificity for a target polynucleotide sequence comprising the at least one target variant nucleotide, and which may include a detectable property (e.g., a detectable label). In some exemplary embodiments, the present disclosure relates to reagents, kits, and methods that use first, second, and third oligonucleotides in amplification reactions to amplify low-abundance target polynucleotides from within a sample (e.g., a mixture) containing abundant alternative nucleic acid sequences (e.g., non-mutated, "wild-type," or major allelic variants). In some embodiments, the low-abundance target polynucleotides can be identified by detecting a change in a detectable property of a detectable oligonucleotide (e.g., a target site-specific probe such as a third oligonucleotide). Each mixture containing first, second, and third oligonucleotides typically contains only one type of third oligonucleotide (e.g., a target site-specific probe) that has binding specificity for a nucleic acid sequence that contains or is complementary to a particular target polynucleotide sequence that contains (or is complementary to) a particular target variant nucleotide.However, in some embodiments, the mixture can include different target sequence-specific primers, locus-specific primers, and / or target site-specific probes (e.g., multiplex reactions, etc.). In some embodiments, oligonucleotides (i.e., primers and probes), and / or mixtures thereof, can be used to detect low abundance, e.g., 3 copies or less, of one or more low-abundance target polynucleotide(s) (e.g., rare target polynucleotides) in the presence of more abundant "wild-type" nucleic acids (e.g., non-mutated nucleic acids, or nucleic acids representing major alleles (e.g., "major alleles" or "major allele variants"). In illustrative examples, the mixture can include, for example, about 10 pg of low-abundance (or "rare") target polynucleotides and about 10 ng of genomic DNA, or about 0.1% of the low-abundance (or "rare") target polynucleotides. Other embodiments, variations, etc. are contemplated herein and will be understood by those skilled in the art from this disclosure.

[0010] Target variant nucleotide A target variant nucleotide is a nucleotide residue within a target polynucleotide sequence that varies between different versions of a nucleic acid sequence (e.g., a gene and / or coding sequence corresponding to a particular mutant and / or allele). In some embodiments, a target variant nucleotide is said to "correspond to," be associated with, and / or be found within an allele (i.e., an allelic variant), which, for purposes of this disclosure, represents a difference in DNA sequence between two or more variants of a particular gene, which may be found within or associated with the coding sequence of the gene or the non-coding sequence of the gene. A target variant nucleotide can correspond to a major or minor allele, and such a minor allele may be found at a frequency that qualifies it as a low-abundance or rare allele. In some embodiments, the target variant nucleotide is part of a larger allelic sequence difference. In humans, the presence of a particular allele within an individual's genome can result in, for example, a change in eye color, but may also be associated with or correlated to a particular disease state. In non-mammals, such as plants, the presence of a particular allele in the genome can be used, for example, to identify a particular plant species, subtype, and / or genotype. Target variant nucleotides can also be present in target polynucleotides as a result of genetic, stochastic (i.e., random), or other mutations. Exemplary mutations containing target variant nucleotides can include nucleic acid sequences containing point or other mutations resulting, for example, from the substitution, insertion, or deletion of a "normal" nucleotide for another, resulting in an abnormal condition (e.g., disease). In some embodiments, target variant nucleotides can correspond to allelic variants that exist in a population of nucleic acid sequences at frequencies of less than 1 / 10, 1 / 100, 1 / 1,000, 1 / 10,000, 1 / 100,000, 1 / 1,000,000, 1 / 10,000,000, 1 / 100,000,000, or 1 / 1,000,000,000, and any fractional range therebetween.For example, in some embodiments, the target variant nucleotide corresponds to the identity of a minor allele sequence having a population frequency of less than about any of 1%, 0.1%, 0.01%, 0.001%, or 0.0001% of the sample nucleic acid population (i.e., "population frequency" is referred to herein in preference to the "sample population" because if the sample contains the minor allele, it may be 50% prevalent in heterozygous individuals). In certain embodiments, the target allele is a rare allele or a low-abundance allele.

[0011] In some embodiments, the target variant nucleotide may correspond to and / or occur at a single nucleotide polymorphism ("SNP"). SNPs are heritable single base pair variations that occur throughout the genome of an organism. SNPs comprise the most common form of genetic variation, with some estimates suggesting that there are over 10 million SNPs in a given human genome. SNP genotyping plays a central role in characterizing individuals and populations, studying disease traits in humans and other organisms, and identifying genes responsible for advantageous crop traits. Thus, SNPs represent a common form of genetic variation between organisms, in which a particular nucleotide is found in the genome of an individual organism that differs from that found at the corresponding position in another individual organism. SNPs can be junctional SNPs (located outside a gene without an effect on protein production or function), coding SNPs (i.e., located within the coding region of a gene), or non-coding SNPs (i.e., located within the regulatory sequence of a gene). In the human genome, SNPs typically occur at a frequency of about 1 in every 300 nucleotides (i.e., there may be about 10 million SNPs across the human genome), and some are associated with disease (e.g., disease-associated SNPs). SNPs are also associated with expression quantitative trait loci (eQTLs), some of which may be cell-type specific. Disease-associated SNPs are particularly relevant to the present disclosure. SNPs are also found in non-human organisms, such as plants. In some embodiments, the target variant nucleotide may correspond to and / or occur in any such SNP.

[0012] In some embodiments, the target variant nucleotide can correspond to a point mutation, which can also be considered herein as an allelic variant. In humans, exemplary diseases that can result from one or more point mutations that can serve as target variant nucleotides include, but are not limited to, cystic fibrosis (caused by F508 mutation), cancer (e.g., tumor suppressor genes or certain cancer-related kinases), neurofibromatosis (neurofibromin 1 or 2 mutation), sickle cell anemia (β-globin mutation), Tay-Sachs disease (HEXA mutation), and color blindness (e.g., X-chromosome mutation). Exemplary mutations associated with cancer include, but are not limited to, mutations in Ras (e.g., KRAS (e.g., at codon 12 and / or codon 13) or and / or NRAS mutation), EGFR, Kit, pTEN, TP53 (also known as p53), PIK3CA, AKT1, and / or ESR1 (such as those listed in Table 1 and Table 2 and described in more detail below). The target variant nucleotide may correspond to, match, or be related to any such mutation. Thus, in some embodiments, a mutant allele may comprise the target variant nucleotide. In some embodiments, the mutant allele may be either a single purine-to-purine or pyrimidine-to-pyrimidine point or base mutation at the target variant nucleotide. In some embodiments, the mutant allele may be a stochastic mutation. In some embodiments, the target variant nucleotide may have an identity corresponding to a major allele sequence or a minor allele sequence. The methods provided herein can be used to detect, and optionally quantitate, major and minor alleles. Thus, the identity of the target variant nucleotide can be used with the oligonucleotides, mixtures, and methods disclosed herein to detect, differentiate, and optionally quantitate major and / or minor alleles.Similarly, the identity of the target variant nucleotide can be used in conjunction with the oligonucleotides, mixtures, and methods disclosed herein to detect, differentiate, and optionally, quantify target polynucleotides associated with inherited or acquired diseases and / or disorders. [Table 1] [Table 2]

[0013] First oligonucleotide (target sequence-specific primer, "TSP") In certain embodiments provided herein, the first oligonucleotide (e.g., a target sequence-specific primer "TSP") typically corresponds to, is hybridizable to (e.g., configured to hybridize to), or includes the complement of a target variant nucleotide at its terminal nucleotide or within three nucleotides of the terminal nucleotide. Although the target variant nucleotide is typically identified via a single variant nucleotide, it will be understood by those skilled in the art that this target variant nucleotide is typically present within a longer sequence of nucleotides (e.g., within a target polynucleotide, such as an allele or mutant gene), sometimes referred to herein as the first sequence in the first target polynucleotide. Thus, the first oligonucleotide (e.g., a TSP) corresponds to, is hybridizable to (e.g., configured to hybridize to), or includes a nucleotide sequence complementary to the target polynucleotide strand, and the first oligonucleotide includes, but is not limited to, a nucleotide complementary to the target variant nucleotide sequence, and is typically terminated by it. The target polynucleotide strand can be either strand of a double-stranded nucleic acid. Thus, the complementarity of a first oligonucleotide (e.g., TSP) to a target polynucleotide sequence is determined in part by the binding specificity defined by the first oligonucleotide (e.g., TSP) as a whole, but in particular by the target variant nucleotide (or its complement) present at the end of the first oligonucleotide (e.g., TSP). Whether the first oligonucleotide (e.g., TSP) is a forward or reverse primer (e.g., in an amplification reaction), the target variant nucleotide is located at the 3' end of the first oligonucleotide (e.g., TSP).Thus, in some embodiments, a first oligonucleotide is configured to hybridize to a first sequence in a first target polynucleotide strand, the first sequence in the first target polynucleotide having a target variant nucleotide, and the first oligonucleotide further having a nucleotide at its 3' end positioned to hybridize to the target variant nucleotide. In some embodiments, the first oligonucleotide (e.g., TSP) can contain 10-30 nucleotides (e.g., any of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides), or in exemplary embodiments, 12-30 nucleotides, such as 15-22 nucleotides. As will be appreciated by those skilled in the art, other forms and / or versions of the first oligonucleotide (e.g., TSP) are also contemplated herein.

[0014] Second oligonucleotide (Locus-specific primer, "LSP") The second oligonucleotide (e.g., a locus-specific primer "LSP") typically exhibits binding specificity for a target polynucleotide (i.e., a target polynucleotide), but not at a target variant nucleotide (or its complement) position. The second oligonucleotide (e.g., LSP) and the first oligonucleotide (e.g., TSP) typically, but not necessarily, bind to different strands of a double-stranded target polynucleotide sequence. Thus, the second oligonucleotide (e.g., LSP) typically has binding specificity for a nucleotide sequence, sometimes referred to as the second sequence of the first polynucleotide strand, upstream or downstream of the first oligonucleotide (e.g., TSP). For example, if the target polynucleotide is a double-stranded nucleic acid, the second oligonucleotide (e.g., LSP) is typically complementary to a nucleotide sequence located 3', or in illustrative examples, 5', and thereon, of the strand to which the first oligonucleotide (e.g., TSP) binds, and in exemplary embodiments is configured to hybridize to a sequence that is identical or significantly identical thereto (i.e., the second oligonucleotide (e.g., LSP)) binds to the strand complementary to that to which the first oligonucleotide (e.g., TSP) hybridizes, but at a different site or location than that to which the first oligonucleotide (e.g., TSP) binds). The second oligonucleotide (e.g., LSP) and the third oligonucleotide (e.g., target site-specific probe) typically, although not necessarily, have binding specificity for different nucleotide sequences on the same strand of a double-stranded target polynucleotide sequence. Thus, in some embodiments, the second oligonucleotide has a sequence configured to hybridize to a sequence complementary to a second sequence of the first target polynucleotide strand, the second sequence of the first target polynucleotide strand being located 5' upstream from the first sequence of the first target polynucleotide strand.In some embodiments, the second oligonucleotide (e.g., LSP) can contain 10-30 nucleotides (e.g., any of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides), or in exemplary embodiments, 12-30, or 15-25 nucleotides, such as 16-24 nucleotides. As will be appreciated by those of skill in the art, other forms and / or versions of the second oligonucleotide (e.g., LSP) are also contemplated herein.

[0015] Third oligonucleotide (target site-specific probe) The third oligonucleotide (e.g., a target site-specific probe) also has binding specificity at a target variant nucleotide position or at a position corresponding to or complementary to a sequence containing the target variant nucleotide, and is typically configured to hybridize to a sequence complementary to a third sequence of the first target polynucleotide strand, with the target variant nucleotide being 1 to 8 nucleotides from the 5' end of the third oligonucleotide. However, typically, the third oligonucleotide has a nucleotide identical to the target variant nucleotide, but typically not at the terminal nucleotide of the third oligonucleotide. Thus, in some embodiments, the nucleotide identical to the target variant nucleotide in the third oligonucleotide is instead positioned near the terminal nucleotide, but not at the terminal nucleotide. For example, the target variant nucleotide is typically positioned at least 2 and / or within 3 to 6 nucleotides of the terminal nucleotide of the third oligonucleotide (e.g., a target site-specific probe). Thus, in some embodiments, the target variant nucleotide in a target site-specific probe (e.g., a third oligonucleotide) is at least two nucleotides and / or within 2, 3, 4, 5, or 6 nucleotides from its 3' end. In some exemplary embodiments, the target variant nucleotide in a target site-specific probe (e.g., a third oligonucleotide) is near its center, e.g., within 1 to 7 nucleotide residues (i.e., 1, 2, 3, 4, 5, 6, or 7 nucleotide residues) from the 3' or 5' end of the target site-specific probe (e.g., a third oligonucleotide). In some embodiments, the target variant nucleotide is located within 3 to 5 nucleotide residues from the 3' or 5' end of the target site-specific probe (e.g., a third oligonucleotide). In some preferred embodiments, the target variant nucleotide is located within 3 to 5 nucleotide residues from the 3' end of the target site-specific probe (e.g., a third oligonucleotide).In some preferred embodiments, the target variant nucleotide is positioned within 3 to 5 nucleotide residues from the 5' end of the target site-specific probe (e.g., the third oligonucleotide). In some embodiments, the target variant nucleotide in the target site-specific probe is positioned about 1 to 3 nucleotide residues from the nucleotide(s) at its intermediate position(s). In some embodiments, the target variant nucleotide in the target site-specific probe (e.g., the third oligonucleotide) is at least two nucleotides from its 3' end. The target site-specific probe (e.g., the third oligonucleotide) also typically includes a nucleotide sequence that overlaps with (and is typically complementary to) the first sequence of the first target polynucleotide to which the first oligonucleotide (e.g., TSP) is configured to hybridize, and also includes a sequence (e.g., 2 to 7 nucleotides in some embodiments) that does not overlap with (and is not complementary to) the first sequence of the first target polynucleotide. In some embodiments, the number of overlapping nucleotide residues between the target site-specific probe and the first sequence of the first target polynucleotide to which the first oligonucleotide (e.g., TSP) is configured to hybridize is 2 to 7 (i.e., 2, 3, 4, 5, 6, or 7 nucleotide residues). In some preferred embodiments, the number of overlapping nucleotide residues between the target site-specific probe and the first sequence of the first target polynucleotide to which the first oligonucleotide (e.g., TSP) is configured to hybridize is 3 to 5 (i.e., 3, 4, or 5 nucleotide residues). In some preferred embodiments, the number of overlapping nucleotide residues between the target site-specific probe and the first sequence of the first target polynucleotide to which the first oligonucleotide (e.g., TSP) is configured to hybridize is 3 nucleotides (e.g., 3 bases).In some embodiments, the target site-specific probe (e.g., a third oligonucleotide) can be designed according to the methods and principles described in U.S. Patent No. 6,727,356 (the disclosure of which is incorporated herein by reference in its entirety) and / or can be a hydrolysis or TaqMan® probe (Thermo-Fisher, Foster City, CA). The target site-specific probe (e.g., a third oligonucleotide) and the first oligonucleotide (e.g., a TSP) are typically, but not necessarily, hybridizable to different strands of a double-stranded target polynucleotide sequence. In some embodiments, the target site-specific probe (e.g., a third oligonucleotide) is detectable (e.g., comprises a detectable property such as a detectable label) and has a sequence configured to hybridize to a sequence complementary to the third sequence of the first target polynucleotide strand, wherein the third sequence of the first target polynucleotide strand at least partially overlaps with the first sequence of the first target polynucleotide strand and the target variant nucleotide. As will be appreciated by one of skill in the art, other forms and / or versions of the third oligonucleotide are also contemplated herein.

[0016] In some embodiments, the target site-specific probe (e.g., the third oligonucleotide) can be 12 to 40 nucleotides in length (e.g., any of 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 nucleotides), or in exemplary embodiments, 10 to 25 nucleotides in length, such as 11 to 23 nucleotides. In some embodiments, the melting temperature (T m ) is the T of the first oligonucleotide (e.g., TSP) m In some embodiments, the T of the target site-specific probe (e.g., third oligonucleotide) is at least 5° C. and no more than 25° C. higher than mis the T of the first oligonucleotide (e.g., TSP) m In some embodiments, the T of the first oligonucleotide is at least 8° C. and no more than 12° C. higher than m is the T of the second oligonucleotide (e.g., LSP) m In some embodiments, the T of the first oligonucleotide (e.g., TSP) is within 5°C of m is 45°C to about 60°C, about 48°C to about 58°C, or 48°C to 58°C, and the T of the second oligonucleotide (e.g., LSP) m are between 45°C and about 60°C, but within about 5°C of each other. As used herein, the T m refers to the temperature (in degrees Celsius) at which 50% of the oligonucleotides in a population of single-stranded oligonucleotides are hybridized to their complementary sequences and 50% of the oligonucleotides in the population are not hybridized to their complementary sequences. m can be determined empirically by melting curves or by using other methods or formulas well known in the art (e.g., as described by Maniatis, T., et al. in Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY: 1982) and elsewhere in the art).

[0017] The target site-specific probe (e.g., the third oligonucleotide) also optionally, and in some embodiments preferably, comprises at least one detectable property, such as may be provided by at least one detectable label (e.g., a fluorescent label), that provides a detectable signal upon amplification of the target polynucleotide sequence. Such a detectable label may be, and preferably is, located on the first terminal nucleotide (e.g., the 5'- or 3'-terminal base or nucleotide residue) of the target site-specific probe (e.g., the third oligonucleotide), but is typically not located on the same nucleotide residue as another moiety, such as a minor groove binder (MGB). In some embodiments, the detectable label is located on the first terminal nucleotide of the target site-specific probe. In some embodiments, the detectable label is located on the 5'-terminal nucleotide of the target site-specific probe. In other embodiments, the detectable label is located internally but near the 5'-end of the probe (e.g., within the 5' portion of the probe). A change in the detectable property of the target site-specific probe (e.g., the third oligonucleotide) during amplification of the target polynucleotide sequence typically indicates that a low-abundance target polynucleotide sequence is present in the sample (e.g., tissue sample) being assayed. Typically, but not necessarily, neither the first oligonucleotide (e.g., TSP) nor the second oligonucleotide (e.g., LSP) contains a detectable property, such as may be provided by a detectable label. The detectable property may be provided to the target site-specific probe (e.g., the third oligonucleotide) via one or more detectable labels.Suitable, non-limiting, and exemplary detectable labels include, for example, DNA binding dyes, reporter dyes, fluorescent probes, 6-carboxyfluorescein (FAM™), tetrachlorofluorescin (TET™), 6-carboxy-4′,5′-dichloro-2′,7′-dimethoxyfluorescein, succinimidyl ester (JOE™), VIC™, sulfonate derivatives of fluorescein dyes containing SO3 in place of the carboxylate group, among others. Fluorescein phosphoramidite forms, CY5 phosphoramidite forms, non-FRET labels, ferrocene reagents, ABY™, NED™, and JUN™, Fluor® 488, AlexaFluor® 532, AlexaFluor® 546, AlexaFluor® 594, AlexaFluor® 647, AlexaFluor® 660, TYE™ 563, TYE™ 665, and TYE™ 705 are included. In some embodiments, the detectable label on the third oligonucleotide (e.g., the target site-specific probe) is on its first terminal nucleotide residue. In some embodiments, the target site-specific probe can be a hydrolysis probe. Thus, in certain embodiments, the third oligonucleotide is cleaved by the exonuclease activity of a polymerase during nucleic acid synthesis or polymerization (e.g., when the enzyme extends the primer into the probe region), releasing and detecting the fluorescently labeled nucleotide or nucleotide fragment. Also contemplated herein are multiplex assays in which two or more (e.g., two, three, four, five or more) of such detectable labels may be present in a single reaction (e.g., mixture). In some embodiments, the mixture may also include at least one passive reference dye (e.g., ROX™, Mustang Purple™).

[0018] The target site-specific probe (e.g., a third oligonucleotide) may also contain a quencher moiety capable of quenching a signal from a detectable label (e.g., a fluorescent label) prior to amplification of the target polynucleotide. Such a quencher moiety is typically attached to a nucleotide at a position where it can quench a signal from the detectable label. Thus, the quencher and label can preferably be positioned any length of nucleotide apart (and any length from the end of the probe) unless the two are constrained by their positions relative to each other, and can be in close proximity to each other such that the signal from the detectable label is quenched (e.g., suppressed) when the probe is not hybridized to a complementary strand. For example, in some embodiments, the detectable label is attached to the target site-specific probe at its 5' end and the quencher moiety is attached to the target site-specific probe at its 3' end. In some embodiments, the quencher moiety is on or near the 3' end of the third oligonucleotide, and the quencher moiety is on or near the 5' end. In some embodiments, the quencher moiety is on the second terminal nucleotide of the target site-specific probe (e.g., the third oligonucleotide), such as when the detectable label is on its first terminal nucleotide. Thus, in some embodiments, the quenching moiety can quench the signal from the detectable label. Suitable non-limiting exemplary quenchers include, for example, tetramethylrhodamine (TAMRA), non-fluorescent quencher (NFQ), black hole quencher, Iowa Black, QSY, QSY7, QSY21, NFQ, dabcyl, and / or dabcyl sulfonate / carboxylate quencher, among others. In some exemplary embodiments, where the third oligonucleotide comprises both a detectable label and a quenching moiety, the third oligonucleotide is a hydrolysis probe.

[0019] In some embodiments, the target site-specific probe (e.g., the third oligonucleotide) may be non-extendable, to which point it may contain a non-extendable blocker moiety such as a dideoxynucleotide (e.g., 2'3'-ddX) (X can be C, A, G, or T), a spacer such as a three-carbon linker (C3), an inverted dT, a modified non-extendable primer blocker (NEBP, AS-NEBP-PCR (Wang, et al. al. J. Mol. Diagn. 15(1):62-9 (2013)), or minor groove binder (MGB, which can be referred to as an "MGB," "MGB group," "MGB compound," or "MBG moiety"). In exemplary embodiments, the non-extendable blocker moiety is located at the 3'-terminal nucleotide residue of the third oligonucleotide. Thus, in some embodiments, the non-extendable blocker moiety can be an MGB. Oligonucleotides bound to an MGB moiety form highly stable duplexes with single- and double-stranded DNA targets, thereby allowing shorter probes to be used in hybridization-based assays. Compared to unmodified oligonucleotides or probes, MGB probes have higher melting temperatures (Tm) and increased specificity, especially when mismatches are located near the MGB region of the hybridized duplex. (See, e.g., Kutyavin, et al. Nucleic Acids Research, 2000, Vol. 28, No. 2: 655-661.) This means that MGB probes are significantly shorter than conventional probes, offering better sequence discrimination and the flexibility to accommodate more targets. Generally speaking, MGBs have a crescent-shaped three-dimensional structure and a strong preference for AT (adenine and thymine)-rich regions of the B-form of double-stranded DNA. Nevertheless, MGB compounds that would exhibit a preference for CG (cytosine and guanine)-rich regions may also be useful as described herein. Some MGBs have a 10 3 M -1MGBs can bind within the minor groove of double-stranded DNA with an association constant equal to or greater than 100 kJ / s. Such binding can be detected by well-established spectrophotometric methods, such as ultraviolet (UV), nuclear magnetic resonance (NMR) spectroscopy, and / or gel electrophoresis. NMR spectroscopy, which utilizes a shift in the UV spectrum upon binding of minor groove binder molecules, and the "Nuclear Overhauser" (NOSEY) effect, are particularly well-known and useful techniques for this purpose. Gel electrophoresis detects binding of MGBs to double-stranded DNA or fragments thereof, due to the change in the mobility of double-stranded DNA after such binding. A variety of suitable minor groove binders have been described in the literature (see, e.g., Kutyavin, et al., U.S. Pat. No. 5,801,155; Wemmer, DE, and Dervan, PB, Current Opinion in Structural Biology, 7:355-361 (1997); Walker, WL, Kopka, JL and Goodsell, DS, Biopolymers, 44:323-334 (1997); Zimmer, C. & Wahnert, U., Prog. Biophys. Molec. Bio. 47:31-112 (1986); and / or Reddy, BSP, Dondhi, SM, and Lown, JW, Pharmacol. Therap., 84:1-111 (1999)). A preferred MGB according to the present disclosure is DPI3. Methods for synthesizing and / or sources of such MGBs are also well known in the art. (See, e.g., U.S. Pat. Nos. 5,801,155, 6,492,346, 6,084,102, and 6,727,356, the disclosures of which are incorporated by reference in their entireties.) When attached to the 3' end of an oligonucleotide, an MGB group can function as a non-extendable blocker moiety. In some embodiments, a third oligonucleotide (e.g., a target site-specific probe) may contain an MGB moiety at its 3' and / or 5' end.In some embodiments, the MGB is located at the 3'-end (e.g., the 3'-terminal nucleotide residue) or the second or third nucleotide from the 3'-terminal nucleotide of the target site-specific probe (e.g., the third oligonucleotide). In some embodiments, the MGB moiety can be covalently linked to a quencher moiety. Thus, in some embodiments, the target site-specific probe (e.g., the third oligonucleotide) is typically detectable (e.g., comprises one or more detectable labels), comprises a quencher, and may also comprise a blocking moiety such as an MGB. As discussed above, when the target site-specific probe (e.g., the third oligonucleotide) comprises one or more detectable labels and also comprises a quencher, the detectable labels and quencher are typically located at opposite ends of the probe.

[0020] An embodiment of a mixture of at least one first oligonucleotide (e.g., TSP, first primer), at least one second oligonucleotide (e.g., LSP, second primer), and at least one third oligonucleotide (e.g., detectable target site-specific probe, first probe) in the context of a double-stranded target polynucleotide (comprising a "forward" and "reverse" strand) is shown in Figure 1. In this illustration, the first oligonucleotide (e.g., TSP) is a "forward primer" complementary to the forward strand of the target polynucleotide and contains at its 3' end a complement to a target variant nucleotide (e.g., "T") present in the top strand. The third oligonucleotide (e.g., target site-specific probe) shown in Figure 1 is complementary to the corresponding reverse strand of the target polynucleotide, which contains a sequence including the target variant nucleotide (i.e., has identity to a portion of the forward strand of the target polynucleotide). In some exemplary embodiments, the target site-specific probe also contains nucleotide residues that are complementary to the sequence to which the first oligonucleotide (e.g., TSP) binds, overlapping with and non-overlapping with the sequence to which the first oligonucleotide (e.g., TSP) binds. For example, the embodiment shown in FIG. 1 includes a target site-specific probe having a 3' sequence (or portion) that overlaps with the sequence of the target polynucleotide to which the target-specific primer (TSP) binds (i.e., the target site) and a 5' sequence (or portion) that does not overlap with the target site. In some embodiments, such as when the TSP is complementary to the "reverse" strand of the target polynucleotide, the target site-specific probe (e.g., the first oligonucleotide) may contain an overlapping sequence at its 5' end. In some embodiments, for example, the overlapping sequence may span two, three, four, five, six, or seven nucleotide residues. Also shown is a second oligonucleotide (e.g., LSP) that is complementary to the reverse strand of the target polynucleotide 5' of the target variant nucleotide (relative to the forward strand in FIG. 1). The target site-specific probe (eg, the third oligonucleotide) shown in Figure 1 also comprises a detectable label ("R") at its 5' end and a quencher ("Q") at its 3' end.Thus, this combination of a first oligonucleotide (e.g., TSP, forward primer) and a second oligonucleotide (e.g., LSP, reverse primer) can be used to amplify a target polynucleotide, and amplification of the target polynucleotide is determined by detecting the release of a detectable label from the target site-specific probe (e.g., a third oligonucleotide, the first probe, which in some embodiments can be a hydrolysis probe such as a TaqMan™ probe). The first, second, and third oligonucleotides, or various combinations thereof (e.g., first and second oligonucleotides, first and third oligonucleotides, second and third oligonucleotides) can be combined in the same or different amounts (e.g., equimolar or non-equimolar) to provide mixture(s) for use in practicing the methods described herein.

[0021] Thus, in some embodiments, the present disclosure provides compositions and / or mixtures, as well as kits containing and methods for using the same, comprising: a) a first oligonucleotide complementary to a first sequence in a first target polynucleotide strand, wherein the first sequence comprises a target variant nucleotide, and wherein the first oligonucleotide comprises a nucleotide at its 3' end that is complementary to the target variant nucleotide; b) a second oligonucleotide containing a sequence configured to hybridize to a sequence complementary to (e.g., having identity to) a second sequence located upstream or downstream of the first sequence on the first target polynucleotide strand; and c) an (optionally detectable) third oligonucleotide (e.g., comprising a detectable label, and optionally a quencher and / or non-extendable blocker moiety) having a sequence configured to hybridize to a sequence complementary to a third sequence in the first target polynucleotide strand, wherein the third sequence shares identity with and at least partially overlaps with the first sequence and comprises the target variant nucleotide. In some embodiments, the first and second oligonucleotides are extendible. In some embodiments, the first and second oligonucleotides are primers. In some embodiments, the third oligonucleotide may be non-extendible, may be a probe (e.g., a target site-specific probe) that may include a detectable label (e.g., as described above) and / or a quencher moiety (e.g., as described above), may include a minor groove binder (MGB) moiety (e.g., as described above), and / or is preferably distinguishable from any other oligonucleotides in the mixture that function as probes (e.g., second, third, or fourth oligonucleotide probes, if present). In some embodiments, the first, second, and / or third oligonucleotides comprise 10-40 nucleotides. In some embodiments, the present disclosure provides kits that may include methods for their use, as well as instructions for using and / or storing such reagents and / or for practicing such methods.

[0022] Also in some embodiments, the present disclosure provides a hybridization method comprising: a) a first oligonucleotide configured to hybridize to a first sequence in a first target polynucleotide strand, the first sequence having a target variant nucleotide, the first oligonucleotide further having a nucleotide residue at its 3' end that is complementary to the target variant nucleotide; b) a second oligonucleotide having a sequence configured to hybridize to a second sequence in the first target polynucleotide strand, the second sequence being located upstream or downstream from the first sequence on the first target polynucleotide strand; and c) a second oligonucleotide (e.g., optionally a detectable and a (optionally detectable) third oligonucleotide (e.g., a target site-specific probe) having a sequence configured to hybridize to a third sequence of a first target polynucleotide strand, the third sequence of the first target polynucleotide strand at least partially overlapping with the first sequence of the first target polynucleotide strand and comprising a target variant nucleotide, as well as methods of using the same, and kits which may include instructions for using and / or storing such reagents and / or for practicing such methods.

[0023] In some embodiments, the disclosure provides a hybridization method comprising: a) a first oligonucleotide configured to hybridize to a first sequence (A) present in a first target polynucleotide strand, the first sequence comprising a target variant nucleotide residue ("first variant nucleotide"), the first oligonucleotide further having a nucleotide at its 3' end positioned to hybridize to the first variant nucleotide; and b) a second oligonucleotide configured to hybridize to a second sequence (B), the second sequence being complementary to a third sequence (C). a) a second oligonucleotide, wherein a third sequence (C) is present in the first target polynucleotide strand, and the third sequence (C) is located 5' upstream from the first sequence (A) of the first target polynucleotide strand; and b) a third oligonucleotide configured to hybridize to a fourth sequence (D) complementary to a fifth sequence (E), wherein the fifth sequence is present in the first target polynucleotide strand, the fifth sequence (E) at least partially overlaps with the first sequence (A) in the first target polynucleotide strand, and the third oligonucleotide comprises a first target variant nucleotide. In some embodiments, the present disclosure provides a kit that can include methods for its use, as well as instructions for using and / or storing such reagents and / or for performing such methods. In some embodiments, the first and second oligonucleotides are extendable. In some embodiments, the first and second oligonucleotides are primers. In some embodiments, the third oligonucleotide may be non-extendable, may be a probe (e.g., a target site-specific probe) that may include a detectable label (e.g., as described above) and / or a quencher moiety (e.g., as described above), may include a minor groove binder (MGB) moiety (e.g., as described above), and / or is preferably distinguishable from any other oligonucleotides in the mixture that function as probes (e.g., second, third, or fourth oligonucleotide probes, if present).In some embodiments, the first, second, and / or third oligonucleotides comprise 10 to 40 nucleotides. In some embodiments, the disclosure provides kits that can include methods for their use, as well as instructions for using and / or storing such reagents and / or for practicing such methods.

[0024] Fourth, fifth, and / or sixth oligonucleotides In some embodiments, the present disclosure provides a method for hybridizing a polynucleotide comprising: a) a fourth oligonucleotide having a sequence configured to hybridize to a first sequence in a second target polynucleotide strand, wherein the first sequence in the second target polynucleotide comprises a second target variant nucleotide, and wherein the fourth oligonucleotide further comprises a nucleotide at its 3' end positioned to hybridize to the second target variant nucleotide; b) a fifth oligonucleotide comprising a sequence configured to hybridize to a sequence complementary to a second sequence in the second target polynucleotide strand, wherein the second sequence in the second target polynucleotide strand is located 3' upstream from the first sequence on the second target polynucleotide strand; and c) a sixth oligonucleotide configured to hybridize to a sequence complementary to a third sequence in the second target polynucleotide strand. and a sixth oligonucleotide (in some embodiments, including those described above having the first, second, and third oligonucleotides, and in some embodiments, excluding those described above having such first, second, and third oligonucleotides), wherein the third sequence of the second target polynucleotide strand at least partially overlaps with the first sequence on the second target polynucleotide strand and comprises a second target variant nucleotide (e.g., functioning as a probe, e.g., by including a detectable label and, optionally, a quencher and / or a non-extendable blocker moiety), and the third sequence of the second target polynucleotide strand at least partially overlaps with the first sequence on the second target polynucleotide strand and comprises the second target variant nucleotide. In some embodiments, the fourth and fifth oligonucleotides are extendible. In some embodiments, the fourth and fifth oligonucleotides are primers.In some embodiments, the sixth oligonucleotide may be non-extendable, may be a probe (e.g., a target site-specific probe) that may include a detectable label (e.g., as described above) and / or a quencher moiety (e.g., as described above), may include a minor groove binder (MGB) moiety (e.g., as described above), and / or is preferably distinguishable from any other oligonucleotide in the mixture that functions as a probe (e.g., the third oligonucleotide, if present). In some embodiments, the fourth, fifth, and / or sixth oligonucleotides comprise 10-40 nucleotides. In some embodiments, the present disclosure provides kits that may include methods for their use, as well as instructions for using and / or storing such reagents and / or for practicing such methods.

[0025] In some embodiments, the disclosure provides compositions and / or mixtures (in some embodiments, including those described above having first, second, and third oligonucleotides, and in some embodiments, excluding those described above having such first, second, and third oligonucleotides) comprising: a) a fourth oligonucleotide comprising a sequence configured to hybridize to a sequence complementary to the first oligonucleotide, wherein the fourth oligonucleotide is configured to substantially hybridize to the first sequence and comprises a nucleotide at its 3' end that is different from the complement of the target variant nucleotide; and b) a fifth oligonucleotide (optionally detectable) configured to hybridize to a sequence complementary to the third oligonucleotide, wherein the fifth oligonucleotide comprises a nucleotide at a position corresponding to the same or non-complementary target variant nucleotide. In some embodiments, the fifth oligonucleotide may be non-extendable, may be a probe (e.g., a target site-specific probe), may contain a detectable label (e.g., as described above), a quencher moiety (e.g., as described above), and / or a minor groove binder (MGB) moiety (e.g., as described above), and may be distinguishable by its detectable property (e.g., a detectable label, and optionally a quencher and / or non-extendable blocker moiety) from any other oligonucleotide in the mixture that functions as a probe (e.g., the third oligonucleotide, if present). In some embodiments, the fourth, fifth, and / or sixth oligonucleotides comprise 10-40 nucleotides. In some embodiments, the present disclosure provides kits that may include methods for their use, as well as instructions for using and / or storing such reagents and / or for performing such methods.

[0026] Oligonucleotide Types In some embodiments, oligonucleotides disclosed herein, particularly those that function as probes (e.g., third oligonucleotides, target site-specific probes), can contain one or more modified bases in addition to the naturally occurring bases adenine, cytosine, guanine, thymine, and uracil. In some embodiments, the modified base(s) reduce the T between matched and mismatched target sequences. mThis can increase the difference in base cleavage and / or reduce mismatch priming efficiency, thereby improving not only assay specificity but also selectivity. Modified bases can differ from naturally occurring bases by the addition or deletion of one or more functional groups, differences in the heterocyclic ring structure (i.e., substitution of a carbon with a heteroatom or vice versa), and / or the attachment of one or more linker arm structures to the base. Such modified base(s) can include, for example, 8-aza-7-deaza-dA (ppA), 8-aza-7-deaza-dG (ppG), locked nucleic acid (LNA), or 2'-O,4'-C-ethylene nucleic acid (ENA) bases. Other examples of modified bases include, but are not limited to, the general classes of base analogs: 7-deazapurines and their derivatives, and pyrazolopyrimidines and their derivatives (e.g., as described in PCT WO 90 / 14353). When present in an oligonucleotide, these base analogs can enhance hybridization and improve mismatch discrimination. All tautomeric forms of naturally occurring bases, modified bases, and base analogs may be included. Modified internucleotide linkages may also be present in the oligonucleotides described herein. Such modified linkages include, but are not limited to, peptide, phosphate, phosphodiester, phosphotriester, alkyl phosphate, alkane phosphonate, thiophosphate, phosphorothioate, phosphorodithioate, methyl phosphonate, phosphoramidate, substituted phosphoramidate, etc. Some further modifications of bases, sugars, and / or internucleotide linkages that are compatible with their use in oligonucleotides that function as probes and / or primers will be apparent to those skilled in the art. In addition, in some embodiments, nucleotide units incorporated into oligonucleotides that act as probes (e.g., the third and / or sixth oligonucleotides, target site-specific probes), such as those containing MGB moieties, may have a cross-linking function (alkylating agent) covalently bound to one or more of the bases through a linking arm.Similarly, modified sugars or sugar analogs can be present on one or more of the nucleotide subunits of the oligonucleotides disclosed herein. Sugar modifications include, but are not limited to, attachment of substituents to the 2', 3', and / or 4' carbon atoms of the sugar, different epimeric forms of the sugar, differences in the alpha or beta configuration of the glycosidic linkage, and other anomeric variations. Sugar moieties include, but are not limited to, pentose, deoxypentose, hexose, deoxyhexose, ribose, deoxyribose, glucose, arabinose, pentofuranose, xylose, lyxose, and cyclopentyl. In some embodiments, the sugar or glycoside moiety of some embodiments of oligonucleotides acting as probes (e.g., the third and / or sixth oligonucleotides, target site-specific probes), e.g., those containing an MGB moiety, can include deoxyribose, ribose, 2-fluororibose, 2-O alkyl, or alkenylribose, where the alkyl group can have 1 to 6 carbons and the alkenyl group can have 2 to 6 carbons. In some embodiments, in naturally occurring nucleotides and the modifications and analogs described herein, the deoxyribose or ribose moiety can form a furanose ring, and purine bases can be linked to the sugar moiety through position 9, pyrimidines through position I, and pyrazolopyrimidines through position I. Also, in some embodiments, particularly in oligonucleotides acting as probes (e.g., third and / or sixth oligonucleotides, target site-specific probes), the nucleotide units of the oligonucleotide can be interconnected by a "phosphate" backbone and / or can include phosphorothioates and methylphosphonates in addition to "natural" phosphodiester bonds, as is well known in the art. As will be appreciated by those skilled in the art, other types of oligonucleotides or modified bases are also contemplated herein.

[0027] Oligonucleotide Set In some embodiments, the present disclosure provides compositions and / or mixtures comprising a plurality of oligonucleotide sets, each oligonucleotide set comprising: a) a first oligonucleotide (e.g., a TSP) that corresponds to, is hybridizable to (e.g., configured to hybridize to), or comprises a nucleotide sequence complementary to a target polynucleotide strand, where the first oligonucleotide comprises, but is typically terminated by, a nucleotide complementary to the target variant nucleotide sequence, and the first oligonucleotide further comprises a nucleotide at its 3' end that is positioned to hybridize to the target variant nucleotide; and b) a second oligonucleotide that is configured to hybridize to a sequence that is complementary to the second sequence of the target polynucleotide strand. The set of oligonucleotides typically includes: a) a second oligonucleotide (e.g., an LSP) comprising a sequence configured as follows ...Thus, in some embodiments, the compositions and / or mixtures disclosed herein may comprise a plurality of oligonucleotide sets, each oligonucleotide set comprising: a) a first oligonucleotide (e.g., TSP) configured to hybridize to a first sequence in a target polynucleotide strand, wherein the first sequence comprises a target variant nucleotide, and the first oligonucleotide further comprises a nucleotide at its 3' end positioned to hybridize to the target variant nucleotide; and b) a second oligonucleotide (e.g., LS) comprising a sequence configured to hybridize to a second sequence in the target polynucleotide strand that is complementary to the second sequence. P) a second oligonucleotide, wherein the second sequence is located 5' upstream from the first sequence; and c) a third oligonucleotide (e.g., a target site-specific probe) comprising both a detectable label and a sequence configured to hybridize to a sequence complementary to a third sequence of a target polynucleotide strand, wherein the third sequence overlaps the first sequence and comprises a target variant nucleotide (or its complement), wherein the first oligonucleotides of each set are configured to hybridize to different first sequences, the third oligonucleotides of each set have identity to different third sequences, and the third oligonucleotides of each set comprise different distinguishable detectable labels. Thus, in some embodiments, each set of oligonucleotides comprises a first oligonucleotide (e.g., the TSP described above), a second oligonucleotide (e.g., the LSP described above), and a detectable third oligonucleotide (e.g., the target site-specific probe described above), and each third oligonucleotide of each set comprises a different detectable characteristic so that amplification of a first target polynucleotide sequence to which a first third oligonucleotide of the set binds can be distinguished from amplification of any other target polynucleotide sequence to which any other third oligonucleotide of another set binds. As will be understood by those skilled in the art, other conditions are also contemplated by the present disclosure.

[0028] PCR reaction mixture In certain embodiments, the compositions and / or mixtures provided herein comprise, in addition to a first oligonucleotide (e.g., a TSP), a second oligonucleotide (e.g., an LSP), and a third oligonucleotide (e.g., a target site-specific probe), or set of oligonucleotides, provided herein, one or more components known in the art as components of a PCR reaction mixture. In certain embodiments, the first oligonucleotide (e.g., TSP), second oligonucleotide (e.g., LSP), and third oligonucleotide (e.g., target site-specific probe) are each present at the same or different concentrations, which may be 0.05 uM to 1 uM, and in exemplary embodiments, 0.15 uM to 1 uM (e.g., about 250 nM, about 300 nM, about 400 nM, 450 nM, about 500 nM, about 550 nM, about 600 nM, about 650 nM, about 700 nM, about 750 nM, about 800 nM, about 850 nM, or about 900 nM, and in preferred embodiments, about 300 nM or about 450 nM. In exemplary embodiments, the first (e.g., The first (e.g., TSP) and second (e.g., LSP) oligonucleotides can each be present at a concentration of about 0.15 uM to about 0.45 uM, preferably about 0.15 uM, 0.30 uM, or 0.45 uM, and the third oligonucleotide (e.g., target site-specific probe) can be present at a concentration of about 0.25 uM. In an exemplary embodiment, such a mixture contains components that result in amplification of the target when subjected to PCR thermal cycling conditions. Such PCR reaction mixture components include dNTPs, one or more PCR buffers, one or more thermostable polymerases, and Mg at concentrations that allow for PCR or that are used in such reaction mixtures. 2+ For example, Mg 2+The nucleotides may be present at 0.5-4 mM (preferably 2.55 mM), e.g., as MgSO or MgCl, and dNTPs may be present at equal concentrations of 0.1-5 mM each, e.g., 2 mM each, or preferably 1 mM, for all four nucleotides. Furthermore, in exemplary embodiments, such reaction mixtures include a target polynucleotide capable of serving as a template polynucleotide in a PCR reaction. In some reaction mixtures provided herein, the target polynucleotide is present at 0.1-1 μg / ul or 0.01-1 ng / nl. The thermostable polymerase(s) may be present at a known activity for PCR, e.g., 0.01 units to 0.1 units per ul of reaction mixture. PCR buffers are known in the art and can be used at 0.5-2x, e.g., 1x, concentrations. Additionally, additional components, such as potassium phosphate and ammonium sulfate, may be present, as discussed in more detail herein. Additional components such as deoxynucleoside triphosphates (dNTPs), albumin such as bovine serum albumin (BSA, e.g., 10-100 μg / ml), buffer (e.g., Tris-HCl, pH about 8-9.5), gelatin (such as fish and / or human gelatin, e.g., 0.01%), formamide (e.g., 1.25-10%), glycerol (e.g., 5-20%), polyethylene glycol (e.g., 5-15%), non-ionic detergent(s) (e.g., Tween 20, Triton X-100, e.g., 0.05-1%), NNN-trimethylglycine (betaine, e.g., 1-3 M), dimethyl sulfoxide (DMSO, e.g., 1-10%), tetramethylammonium chloride (TMAC), and / or betaine, and / or combinations thereof, among other components, may also be present in the reaction mixtures provided herein.

[0029] Potassium phosphate and ammonium sulfate The inventors / applicants have surprisingly found that additional components, when included in the reactions and / or methods described herein in effective amounts, increase the efficiency of target polynucleotide sequence amplification. Thus, in some embodiments, a mixture can include such additional components to increase the efficiency of target polynucleotide sequence amplification. In some embodiments, such additional components include an effective amount of potassium chloride and / or ammonium sulfate. Potassium chloride (KCl) and / or ammonium sulfate ((NH4)2SO4) are included in an amplification reaction in an "effective amount," i.e., an amount of potassium chloride and / or ammonium sulfate that improves amplification of a target variant nucleotide relative to a more abundant nucleotide at a target nucleotide position in an amplification reaction (e.g., as shown in the Examples) when compared to an amplification reaction that does not contain that amount of potassium chloride and / or ammonium sulfate. Thus, a mixture can include a concentration of potassium chloride and / or ammonium sulfate that improves the discrimination of a target polynucleotide from a more abundant wild-type nucleic acid, as determined by Cq after an amplification reaction, compared to Cq after an amplification reaction lacking that concentration of potassium chloride and / or ammonium sulfate. In some embodiments, the mixture contains effective concentrations of potassium chloride and ammonium sulfate, each of which improves the discrimination of mutant target polynucleotides from wild-type nucleic acids as determined by Cq after amplification, compared to Cq after amplification without the combination. For example, the effective concentration of potassium chloride can be at least 20 mM to 80 mM, 30 mM to 80 mM, 40 mM to 70 mM, at least 40 mM to less than 70 mM, less than 70 mM, 60 mM, 40 mM to 48 mM, 45 mM, or 10 mM to 40 mM. Also, for example, the effective concentration of ammonium sulfate can be at least 20 mM, 20 mM to 35 mM, at least 20 mM to less than 35 mM, less than 35 mM, 20 mM to 25 mM, 20 mM to 24 mM, 22 mM, 10 mM to 20 mM, or 15 mM.As shown in the Examples herein, particularly effective concentrations of potassium chloride and ammonium sulfate in combination can be 60 mM and 15 mM, respectively, or 30 mM and 16 mM, respectively, or preferably 45 mM and 22 mM, respectively. Other concentrations of each of potassium chloride and ammonium sulfate are also contemplated and can be used, as can be determined by one of skill in the art (e.g., by determining whether differentiation of a target polynucleotide (e.g., a mutant or variant nucleic acid) from a more abundant target polynucleotide (e.g., a wild-type nucleic acid) in the methods described herein is improved in the presence of a particular concentration compared to other concentrations).

[0030] polymerase The mixtures disclosed herein can also include at least one polymerase (e.g., a DNA polymerase) and at least one nucleotide source (e.g., dNTPs). The polymerase can be a DNA polymerase with 5' to 3' exonuclease activity. In some embodiments, the polymerase can be a "thermostable polymerase," which refers to an enzyme that is thermostable, thermotolerant, and / or not irreversibly inactivated when subjected to high temperatures for the time required to destabilize single-stranded nucleic acids or denature double-stranded nucleic acids during amplification (e.g., not irreversibly denatured at about 90 to about 100°C, as typically required for amplification (e.g., in polymerase chain reaction (PCR))), and that catalyzes the polymerization of deoxyribonucleotides to form primer extension products complementary to target polynucleotide strands. Thermostable polymerases can be obtained, for example, from various commercially available thermophilic bacteria (e.g., from the American Type Culture Collection, Rockville, Md.) using methods well known to those skilled in the art (see, e.g., U.S. Patent No. 6,245,533). Bacterial cells can be grown according to standard microbiological techniques using media and incubation conditions suitable for growing active cultures of the particular species, as known to those skilled in the art (see, e.g., Brock, T.D., and Freeze, H., J. Bacteriol. 98(1):289-297 (1969); Oshima, T., and Imahori, K., Int. J. Syst. Bacteriol. 24(1):102-112 (1974)).Suitable sources of thermostable polymerases include, for example, the thermophilic bacteria Thermus aquaticus, Thermus thermophilus, Thermococcus litoralis, Pyrococcus furiosus, Pyrococcus woosii, and other species of the genus Pyrococcus, Bacillus stearothermophilus, Sulfolobus acidocaldarius, Thermoplasma acidophilum, Thermus flavus, Thermus ruber, Thermus brockianus, Thermotoga neapolitana, Thermotoga maritima, and other species of the genus Thermotoga, and Methanobacterium thermoautotrophicum, as well as mutants of each of these species. Exemplary thermostable polymerases can include, but are not limited to, any of SuperScript, Platinum, TaqMan, MicroAmp, AmpliTaq, and / or fusion polymerases.Exemplary polymerases include Taq™ DNA polymerase, AmpliTaq DNA polymerase, AmpliTaq™ Gold DNA polymerase, DreamTaq™ DNA polymerase, a recombinant, modified form of the Thermus aquaticus DNA polymerase gene expressed in E. coli (Thermo Fisher Scientific), iTaq™ (Bio-Rad), Platinum Taq DNA Polymerase High Fidelity, Platinum™ II Taq™ Hot-Start DNA Polymerase, Platinum SuperFi DNA Polymerase, AccuPrime Taq™ DNA Polymerase High Fidelity, Tne DNA polymerase, Tma DNA polymerase, Phire Hot Start II DNA polymerase, Phusion U Hot Start DNA Polymerase, Phusion Hot Start II High-Fidelity DNA Polymerase, iProof High Fidelity DNA Polymerase (Bio-Rad), HotStart Taq Polymerase (Qiagen), e.g., a chemically modified polymerase that blocks its activity at a specific temperature, such as room temperature, and / or mutants, derivatives, and / or fragments thereof. In some embodiments, oligonucleotides or aptamers may also be used as hot start agents, and / or the hot start function may result from chemical modifications to a polymerase that block its activity at a specific temperature (e.g., room temperature) (e.g., TaqGold, FlashTaq, Hot-Start Taq).In some embodiments, the hot start component can be one or more antibodies directed against (i.e., have binding specificity for) the thermostable polymerase in the mixture (e.g., available from Thermo Fisher Scientific in Platinum™ II Hot-Start Green PCR Master Mix, DreamTaq™ Hot Start Green PCR Master Mix, Phusion U Green Muliplex PCR Master Mix, Phire Green Hot Start II Master Mix, or AmpliTaq® Gold 360 Master Mix (Thermo Fisher Scientific)). In some embodiments, a dual hot start mechanism can be used. For example, a first hot start component, such as an oligonucleotide, can be used as a hot start agent in combination with a second hot start component, such as one or more antibodies. In some embodiments, the first and second hot start components of a dual hot start mechanism can be the same type or different (oligo-based, antibody-based, chemical-based, etc.). In some embodiments, the first and second hot-start components of the dual hot-start mechanism can be inhibitory to the same polymerase (e.g., a dual hot-start mechanism using an inhibitory antibody directed against Taq DNA polymerase and an inhibitory oligonucleotide specific to Taq DNA polymerase). In some embodiments, the polymerase can be a fusion or chimeric polymerase, which refers to an enzyme or polymerase composed of different domains or sequences derived from different sources. For example, a fusion polymerase can include a polymerase domain, such as a Thermus aquaticus (Taq) polymerase domain, fused to a DNA-binding domain, such as a single-stranded or double-stranded DNA-binding protein domain. Fusion or chimeric polymerases can be obtained, for example, using methods well known to those skilled in the art (see, e.g., U.S. Pat. No. 8,828,700), the disclosure of which is incorporated by reference in its entirety.In some embodiments, such fusion or chimeric polymerases are thermostable. In some embodiments, the mixture is a mixture that is a master mix and / or a reaction mixture (e.g., TaqPath™ ProAmp™ Master Mix (Applied Biosystems™), TaqPath™ ProAmp™ Multiplex Master Mix (Applied Biosystems™), TaqMan™ PreAmp Master Mix (Applied Biosystems™), TaqMan™ Universal Master Mix II with UNG (Applied Biosystems™), TaqMan™ Universal PCR Master Mix II (no UNG) (Applied Biosystems™), TaqMan™ Gene Expression Master Mix II with UNG (Applied Biosystems™), EXPRESS qPCR Supermix, universal (Invitrogen), TaqMan™ Fast Advanced Master Mix (Applied Biosystems™), TaqMan™ Multiplex Master Mix (Applied Biosystems™), TaqMan™ Multiplex Master Mix (Applied Biosystems™), TaqMan™ Universal Master Mix II with UNG (Applied Biosystems™), TaqMan™ Multiplex Master Mix ... Biosystems™), TaqMan™ PreAmp Master Mix Kit (Applied Biosystems™), TaqMan™ Universal PCR Master Mix, no AmpErase™ UNG (Applied Biosystems™), PowerUp SYBR Green Master Mix (Applied Biosystems™), or FlashTaq HotStart 2X MeanGreen Master Mix (Empirical Biosciences). In some embodiments, the mixture can further include one or more of at least one detergent, glycerol, and at least one reference dye (e.g., ROX™, Mustang Purple™).In some embodiments, the reaction mixture can further include amplicons that include a target polynucleotide sequence (e.g., a first sequence) of a target polynucleotide strand. In some embodiments, the mixture does not include amplicons that include a sequence (e.g., of a major allelic variant) of a second polynucleotide strand.

[0031] Target Polynucleotide In some embodiments, the mixtures disclosed herein include a nucleic acid sample suspected of containing a target polynucleotide strand (e.g., a target polynucleotide sequence or target polynucleotide). The target polynucleotide sequence (e.g., a target polynucleotide) can be any suitable single-stranded, double-stranded, or otherwise configured polynucleotide to which a target sequence-specific primer (e.g., a first oligonucleotide, TSP), a locus-specific primer (e.g., a second oligonucleotide, LSP), and a target site-specific probe (e.g., a third oligonucleotide) can bind and support its amplification. In some embodiments, the nucleic acid sample can be deoxyribonucleic acid (DNA), such as genomic DNA (gDNA) or complementary DNA (cDNA). Thus, in some embodiments, the mixtures disclosed herein can include a single-stranded target polynucleotide comprising a target polynucleotide strand, and / or a double-stranded target polynucleotide comprising a target polynucleotide strand and a target complement polynucleotide strand, where the target complement polynucleotide strand is substantially complementary to the target polynucleotide strand. In some embodiments, the double-stranded target polynucleotide comprises a target polynucleotide strand and a target complement polynucleotide strand, wherein the target complement polynucleotide strand is substantially complementary to the target polynucleotide strand; the double-stranded target polynucleotide comprises a variant polynucleotide strand and a variant complement polynucleotide strand, wherein the variant polynucleotide strand has identity or is substantially identical to the target polynucleotide strand and comprises nucleotides that differ from the target polynucleotide strand at the target variant nucleotide; and the variant complement polynucleotide strand is substantially complementary to the variant polynucleotide strand.

[0032] As described above, a target polynucleotide sequence can include target variant nucleotides (i.e., allelic variants, such as those represented by SNPs and / or mutations) that "correspond" to, are hybridizable with, are associated with, and / or can be found within an allele. Such SNPs or mutations can include, but are not limited to, those found in, for example, EGFR (epidermal growth factor receptor) (e.g., Figure 20), KRAS (e.g., at codon 12 and / or codon 13), or NRAS mutations (e.g., Figure 21), Kit, pTEN TP53, ESR1, PIK2CA, TSC1, MDM2, ERBB2, SMAD4, and FGFR2 genes (including those listed in Tables 1 and 2). For example, as shown in the examples herein, the mixtures and methods of the present disclosure may be used to produce the following exemplary, non-limiting mutations: a guanosine to adenosine (G>A (GGT>GAT)) mutation, which encodes a glycine to aspartic acid (G12D) substitution at amino acid 12 of the translated protein; a guanosine to thymidine (G>T (GGT>GTT)) mutation, which encodes a glycine to valine (G12V) substitution at amino acid 12 of the translated protein; a guanosine to thymidine (G>T (GGT>TGT)) mutation, which encodes a glycine to cysteine ​​(G12C) substitution at amino acid 12 of the translated protein; a glycine to serine (G>T (GGT>TGT)) mutation, which encodes a glycine to aspartic acid (G12D) substitution at amino acid 12 of the translated protein; The present invention can identify and / or quantitate KRAS having any of the following mutations: a guanosine to adenosine (G>A (GGT>AGT)) mutation encoding a glycine to alanine (G12A) substitution at amino acid 12 of the translated protein; a guanosine to cytosine (G>C (GGT>GCT)) mutation encoding a glycine to arginine (G12R) substitution at amino acid 12 of the translated protein; and / or a guanosine to adenosine (G>A (GGC>GAC)) mutation encoding a glycine to aspartic acid (G13D) substitution at amino acid 13 of the translated protein.As will be appreciated by those of skill in the art, other KRAS mutations as described above, and other KRAS mutations beyond those listed in Table 2, are also contemplated and may also be suitable for analysis and / or detection using the reagents and methods described herein.

[0033] As also shown in the examples herein, the compositions and / or mixtures and methods of the present disclosure may be used to produce the following exemplary, non-limiting mutations: a guanine to adenosine (G>A) mutation encoding a glycine to aspartic acid mutation at amino acid 12 (G12D) of the translated protein; a guanosine to adenosine (G>A) mutation encoding a glycine to aspartic acid mutation at amino acid 13 (G13D) of the translated protein; an adenosine to thymidine (A>T) mutation encoding a glutamine to lysine (G>T) mutation at amino acid 61 of the translated protein; NRAS having any of the following mutations can be identified and / or quantified: an adenosine to guanosine (A>G) mutation encoding a glutamine to arginine mutation at amino acid 61 (Q61R) of the translated protein; an adenosine to thymidine (A>T) mutation encoding a glutamine to histidine mutation at amino acid 61 (Q61H) of the translated protein; and / or an adenosine to thymidine (A>T) mutation encoding a glutamine to leucine mutation at amino acid 61 (Q61L) of the translated protein. As will be appreciated by those skilled in the art, other NRAS mutations as described above, and other NRAS mutations beyond those listed in Table 2, are also contemplated and may also be suitable for analysis and / or detection using the reagents and methods described herein.

[0034] As also shown in the examples herein, the compositions and / or mixtures and methods of the present disclosure can be used to identify and / or quantitate EGFRs having any of the following exemplary, non-limiting mutations: a cytosine to thymidine (C>T) mutation, which encodes a threonine to methionine mutation at amino acid 790 of the translated protein (EGFR20), a deletion of nucleotides 746-750 of the wild-type coding sequence (EGFR19), and / or a thymidine to adenosine (T>A) mutation, which encodes a leucine to glutamic acid mutation at amino acid 861 (L861Q) of the translated protein. As will be understood by those of skill in the art, other EGFR mutations, as described above, and other EGFR mutations beyond those listed in Table 2, are also contemplated and may also be suitable for analysis and / or detection using the reagents and methods described herein.

[0035] As also shown in the examples herein, the compositions and / or mixtures and methods of the present disclosure can be used to identify and / or quantify, by way of non-limiting example, BRAF having a thymidine to adenosine mutation, which encodes a valine to glutamic acid mutation at amino acid 600 (V600E) of the translated protein. As will be appreciated by those skilled in the art, other BRAF mutations, as described above, and other BRAF mutations beyond those listed in Table 2, are also contemplated and may also be suitable for analysis and / or detection using the reagents and methods described herein.

[0036] As also shown in the examples herein, the compositions and / or mixtures and methods of the present disclosure may be used to identify ESR1 having any of the following exemplary, non-limiting mutations: a guanine to cytosine (G>C) mutation encoding a glutamic acid to glutamine mutation at amino acid 380 (E380Q) of the translated protein; PIK3CA having any of the following exemplary, non-limiting mutations: an adenosine to guanine (A>G) mutation encoding a histidine to arginine mutation at amino acid 1047 (H1047R) of the translated protein; or a PIK3CA having any of the following exemplary, non-limiting mutations: an adenosine to guanine (A>G) mutation encoding a histidine to arginine mutation at amino acid 273 (R273H) of the translated protein. ), a thymidine to adenosine (T>A) mutation encoding a histidine to glutamine mutation at amino acid 179 (H179Q) of the translated protein, an adenosine to guanine (A>G) mutation encoding a tyrosine to cysteine ​​mutation at amino acid 220 (Y220C) of the translated protein, or a guanine to thymidine (G>T) mutation encoding an arginine to methionine mutation at amino acid 179 (R249M) of the translated protein. As will be appreciated by those skilled in the art, other ESR1, PIK3CA, and TP53 mutations as described above, as well as other ESR1, PIK3CA, and TP53 mutations beyond those listed in Table 1 and / or Table 2, are also contemplated and may also be suitable for analysis and / or detection using the reagents and methods described herein.

[0037] Samples in which target polynucleotides may be present include, for example, tissues, cells, and / or fluids (e.g., circulating, dried, reconstituted) from mammalian or non-mammalian organisms (e.g., plants, viruses, bacteriophages, bacteria, fungi, and / or other organisms, including, but not limited to, tissues, cells, and / or fluids. In some embodiments, the sample may be or be derived from, for example, mammalian saliva, buccal epithelial cells, buccal tissue, lymph, cerebrospinal fluid, skin, hair, blood, plasma, urine, feces, semen, tumor samples (e.g., cancer cells), cultured cells, or cultured tumor cells. The target polynucleotide may be DNA in genomic form, or it may be cloned in a plasmid, bacteriophage, bacterial artificial chromosome (BAC), yeast artificial chromosome (YAC), and / or other vector. Other types of samples may also be useful in the methods described herein, for example, as may be relevant to diagnostic or forensic assays.

[0038] Freeze drying In some embodiments, individual types of oligonucleotides and / or mixtures thereof can include additional components suitable for lyophilization and / or can be lyophilized and / or otherwise stabilized (e.g., prepared as lyophilized (e.g., frozen, primary drying, secondary drying) or evaporated compositions) and thus can include components or be treated to provide such stabilization. Mixtures can be prepared as compositions stable for about 2 years at -20°C (e.g., dried or in a solution of water or TE buffer (10 mM Tris, pH 7.5-8, 1 mM EDTA)), about 1 year at 4°C (e.g., dried or in a solution of water or TE buffer), about 3-6 months at room temperature (e.g., dried or in a solution of water or TE buffer), and / or about 1-2 months at temperatures above room temperature (e.g., dried or in a solution of water or TE buffer). The kits described below may also include a buffer for reconstitution of lyophilized or otherwise stabilized oligonucleotides and / or mixtures (e.g., water (e.g., sterile, nuclease-free water), or a weak buffer such as TE or Tris (10 mM Tris-HCl, pH 8.0)).

[0039] method Polymerase chain reaction (PCR) generally refers to the cycling polymerase-mediated exponential amplification of nucleic acids using primers hybridized to complementary strands of a target polynucleotide, typically performed using a thermostable enzyme and / or a thermal cycler device designed to perform such a reaction, as described, for example, in Innis et al., PCR Protocols: A Guide to Methods and Applications, Academic Press (1990). Suitable PCRs include, but are not limited to, real-time PCR (e.g., quantitative PCR (qPCR)), nested PCR, multiplex PCR, end-point PCR, digital PCR (dPCR), drop-dPCR, isothermal PCR, touchdown PCR, co-amplification at lower denaturation temperature (COLD) PCR, and / or isothermal PCR. In a preferred embodiment, the PCR can be real-time PCR (e.g., quantitative PCR (qPCR)). Those skilled in the art will appreciate that the melting temperature ("T") of an oligonucleotide can be used to determine the temperature at which the PCR is performed. m It is understood that the T of an oligonucleotide can significantly affect PCR performance. m refers to the temperature (typically in degrees Celsius) at which 50% of the polynucleotides in a population of single-stranded oligonucleotides hybridize to their complementary sequences and 50% of the polynucleotides in the population do not hybridize to their complementary sequences. As will be understood by those skilled in the art, the T of an oligonucleotide (e.g., a primer) m Tm can be empirically determined by melting curves. Tm may depend on the length of the primer, the percentage of GC content, the molecular weight, and its decay coefficient. In some cases, T m can be calculated using formulas and / or calculators well known in the art (e.g., Maniatis, T., et al., Molecular cloning: a laboratory manual / Cold Spring Harbor Laboratory, Cold Spring Harbor, NY: 1982, Thermo-Fisher's T m(See Calculator, TaqPipe, PrimerExpress.) A skilled artisan will also appreciate that the T m The appropriate annealing temperature can be determined using the thermodynamic method of Allawi and SantaLucia, understanding that PCR specificity and yield may also depend on primer concentration and the polymerase used. Specific calculations may need to be performed based on which polymerase is used. For example, the modified Allawi and SantaLucia thermodynamic method provides the T for reactions with Platinum SuperFi, Phusion, and Phire DNA polymerases. m and can be used to calculate the annealing temperature (Biochemistry, 36(34):10581-94(1997)). In some embodiments, the annealing temperature for PCR is determined by the lowest T m Slightly higher (e.g., within 5–10 degrees) than the T of both oligonucleotides m In some embodiments, the T of the first oligonucleotide may be higher than m is the T of the second oligonucleotide m In some embodiments, the T of the first oligonucleotide is within 5°C of m The T of the second oligonucleotide can be 48-58 °C. m In some embodiments, the T of the third oligonucleotide may be 48 to 58°C. m is the T of the first oligonucleotide m In some embodiments, the T of the third oligonucleotide may be at least 5° C. and no more than 25° C. higher than m is the T of the first oligonucleotide m In some embodiments, the annealing temperature for PCR using primers (e.g., first and second oligonucleotides) disclosed herein may be at least 8°C and no more than 12°C higher than the higher T of a particular primer pair. m For example, the T of the first oligonucleotide may be within 5 to 10°C. m is the T of the second oligonucleotidem If the annealing temperature is within 5 °C of the higher calculated T of the oligonucleotide, m In some embodiments, for example, the T of the first oligonucleotide may be within 5°C of m can be 48-58 °C, and the T of the second oligonucleotide m For example, if the T can be 48 to 58°C, the annealing temperature can be 48°C or higher than 58°C (e.g., the higher T m (within about 5°C of the initial temperature). As will be appreciated by one of skill in the art, other conditions for PCR are also contemplated by the present disclosure.

[0040] In some embodiments, the PCR reaction can include an "enrichment phase" or "enrichment cycle(s)" in which low-abundance nucleic acids (i.e., target polynucleotides or target polynucleotide molecules) are amplified in preference to more abundant nucleic acids (e.g., major alleles, wild-type nucleic acids), by subjecting the mixture to one cycle of 95°C for 2 minutes, 15-20 cycles of 95°C for 1-3 seconds and 64°C for 20 seconds (enrichment phase), and 40 cycles of 95°C for 1 second and 60°C for 20 seconds (amplification and detection phase). Without being limited by theory, it is possible that the elevated temperature in the enrichment phase favors annealing of sequence-specific primers to low-abundance target polynucleotides that form perfect matches when bound to the target sequence-specific primers, compared to annealing of target sequence-specific primers to abundant nucleic acids that contain a single-base mismatch (e.g., a single-base mismatch) when bound to the target sequence-specific primers. In some embodiments, the qPCR reaction was performed without an enrichment phase, and the mixture was subjected to 1-10 minutes, e.g., 1 cycle of 95°C for 2 minutes and 40 cycles of 95°C for 1 second and 60°C for 20 seconds (amplification and detection phase). In some embodiments, enrichment is performed by measuring the calculated melting temperature (T m) can be performed using PCR involving 15-25 cycles at temperatures 12-16°C higher than the calculated primer T. m The approach described here uses a high temperature in the enrichment phase to favor annealing and amplification of target polynucleotides (i.e., low-abundance or rare nucleic acid molecules) at the expense of abundant target polynucleotide sequences (e.g., major alleles, wild-type nucleic acids). A TSP is a perfect match of the (rare) target polynucleotide sequence but contains a single-base mismatch with the more abundant target polynucleotide (e.g., major allele, wild-type nucleic acid). This mismatch, combined with a higher annealing / extension temperature, allows amplification of low-abundance polynucleotide species (i.e., low-abundance and / or rare target polynucleotide molecules). The annealing / extension temperature is typically 3°C to 8°C lower than the enrichment temperature. If the enrichment and annealing / extension temperatures are equivalent, the distinction between rare and abundant targets will be lost. In some embodiments, qPCR can be performed with serial dilutions (e.g., 5-log dilutions) using a minimum of three replicates, especially for samples expected to contain low copy numbers of target polynucleotides (for statistical analysis of results). As will be appreciated by one of skill in the art, other conditions for PCR are also contemplated by the present disclosure.

[0041] In some embodiments, the present disclosure provides a method for detecting a target polynucleotide containing a target variant nucleotide by forming a reaction mixture of a test nucleic acid sample potentially containing the target polynucleotide sequence and a mixture of a first oligonucleotide (e.g., TSP), a second oligonucleotide (e.g., LSP), and a third oligonucleotide (e.g., target site-specific probe), performing an amplification reaction using at least the first and second oligonucleotides as primers to generate an amplicon, and detecting the amplicon by detecting a change in a detectable property of the third oligonucleotide (e.g., target site-specific probe), wherein detection of the amplicon indicates the presence of the target polynucleotide in the test nucleic acid sample. In some embodiments, the test nucleic acid sample comprises a mixture of nucleic acids containing target polynucleotides that contain or correspond to the target variant nucleotides and wild-type nucleic acids that do not contain or correspond to the target variant nucleotides. In some embodiments, as described above and shown in the examples, the method enriches (e.g., increases) the number of target polynucleotides in a test nucleic acid sample relative to more abundant (e.g., wild-type) nucleic acid polynucleotides, e.g., by performing an amplification reaction under conditions different from those used to amplify the target polynucleotide sequence for detection (i.e., by increasing, e.g., the calculated melting temperature (T) of the first oligonucleotide). m ) at a temperature 12-16°C higher than the T m and performing 15-60, 20-50, 30-50, 35-45, 38-42, or 40 cycles at about 5°C to 25°C and 3-8°C lower than the temperature at which enrichment was performed).

[0042] The amplification resulting from PCR typically occurs at a threshold cycle (C t ), is quantified by measuring a relative measure of the concentration of amplified nucleic acid in the sample, which is the intersection point between the amplification curve and the threshold line. tThe data is presented in an amplification plot showing the variation of log(ΔRn) with PCR cycle number. Rn is the fluorescence of the reporter dye divided by the fluorescence of the passive reference dye, i.e., Rn is the reporter signal normalized to the reporter dye fluorescence signal. Rn is plotted against PCR cycle number. ΔRn is Rn minus the baseline fluorescence value (e.g., background FAM fluorescence) and may vary depending on the amplification conditions (e.g., the type of reporter dye used and / or the type of master mix used). Exemplary reporter dyes (or passive reference dyes) include, but are not limited to, ROX™ or Mustang Purple™. C t The value increases as the amount of template decreases. In some embodiments, such as those shown in the examples herein, multiple qPCR reactions (e.g., two or more) can be performed with the same mixture (e.g., a control and a test reaction), resulting in two C values ​​that are distinct from each other on an amplification plot. t yields the value of delta C t (ΔC t ) In some embodiments, ΔC t can be, for example, at least about 8 (e.g., Example 1 herein has a ΔC of 9.0 to 16.3). t (Values ​​are shown). Data generated from the amplification reactions can be exported in Excel format, the Ct (also called Cq) values ​​of the replicate reactions are averaged, and the delta average Cq of the labeled target polynucleotide (e.g., FAM and VIC targets) in each condition is determined and plotted. Thus, the delta Cq can be used to quantify the starting amount of the target polynucleotide or target variant polynucleotide in a relative or absolute manner (e.g., as shown in the examples and figures of the present disclosure).

[0043] In some embodiments, the compositions and / or mixtures disclosed herein may contain ribonucleic acid (RNA), which can serve as a starting material in reverse transcriptase PCR (RT-PCR), for example, using the compositions and / or mixtures and methods disclosed herein. In such embodiments, the compositions and / or mixtures may include a reverse transcriptase (RT) and related components. In some embodiments, the RT-PCR may be a one-step procedure using one or more target sequence-specific primers (e.g., TSPs or first oligonucleotides), one or more locus-specific primers (e.g., LSPs or second oligonucleotides), and one or more target site-specific probes (e.g., third oligonucleotides). Suitable exemplary RTs may include, for example, SuperScript Reverse Transcriptase (Thermo Fisher Scientific), SuperScript IV Reverse Transcriptase (Thermo Fisher Scientific), or Maxima Reverse Transcriptase (Thermo Fisher Scientific). The compositions and / or mixtures may also include any other components necessary to perform such reactions, such as may be found in SuperScript IV VILO Master Mix (Thermo Fisher Scientific) or any other suitable master mix (including those described above).

[0044] Devices have been developed that can perform thermal cycling reactions with compositions containing fluorescent indicators that emit a light beam of a specific wavelength, read the intensity of the fluorescent dye, and display the intensity of the fluorescence after each cycle. Devices comprising a thermal cycler, a light beam emitter, and a fluorescent signal detector are described, for example, in U.S. Pat. Nos. 5,928,907, 6,015,674, 6,174,670, and 6,814,934, and include, among others, the Prism® 7700 Sequence Detection System (Thermo Fisher Scientific), the ABI GeneAmp® 5700 Sequence Detection System (Thermo Fisher Scientific), the ABI GeneAmp® 7300 Sequence Detection System (Thermo Fisher Scientific), the ABI GeneAmp® 7500 Sequence Detection System (Thermo Fisher Scientific), the StepOne™ Real-Time PCR System (Thermo Fisher Scientific), the ABI GeneAmp® 7900 Sequence Detection System (Thermo Fisher Scientific), the QuantStudio 12K Flex Real-Time PCR System (Thermo Fisher Scientific), the QuantStudio 7 ... Fisher Scientific), QuantStudio 6 Flex Real-Time PCR System (Thermo Fisher Scientific), QuantStudio 5 Flex Real-Time PCR System (Thermo Fisher Scientific), QuantStudio 3 Flex Real-Time PCR System (Thermo Fisher Scientific), ViiA Real-Time PCR System (ThermoExamples of suitable thermal cyclers include, but are not limited to, Bio-Rad Thermostats (Fisher Scientific), C1000 Touch™ Thermal Cycler (Bio-Rad), S1000™ Thermal Cycler (Bio-Rad), T100™ Thermal Cycler (Bio-Rad), CFX96 Touch™ (Bio-Rad), CFX384 Touch™ (Bio-Rad), CFX Connect™ (Bio-Rad), and Rotor-Gene Q (Qiagen), and systems (e.g., software) therefor. As known to those skilled in the art, these systems can be used to simultaneously analyze multiple samples (e.g., 96-well or 384-well systems) and / or multiple detectable labels (e.g., multiplex assays), and are suitable for use with the mixtures and methods described herein. For example, these devices can include multiple channels for detecting different detectable labels (e.g., two channels for detecting green and yellow, four channels for detecting green, yellow, orange, and red, five channels for detecting green, yellow, orange, red, and crimson, six channels for detecting blue, green, yellow, orange, red, and crimson, etc.). Many of these systems are automated and / or require software (e.g., Applied Biosystems QuantStudio™ (Thermo Fisher Scientific), CFX Maestro Software (Bio-Rad), CFX Automation System).It is also well known that PCR systems may rely on and / or use various PCR equipment (e.g., ELISA Kits ...

[0045] In some embodiments, the methods disclosed herein can detect target variant nucleotides indicative of mutations, even when a nucleic acid sample contains only about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 to about 10, about 10 to 15, about 15 to 20, about 20 to 25, about 26 to 50, about 50 to 75, or about 75 to 100 copies of any target polynucleotide (e.g., in the presence of a much larger number of more abundant polynucleotides). For example, the major allele(s) and / or wild-type polynucleotide may comprise greater than 99% of the polynucleotides, and / or the target polynucleotide comprises, for example, about 2%, 1%, 0.1%, 0.01%, 0.001%, or 0.0001% of the sample polynucleotide population (e.g., test sample). Target polynucleotides can be detected within such sample polynucleotide populations using the methods described herein. In some embodiments, a method for detecting and / or quantifying low-abundance (e.g., rare) allelic variants comprising a target variant nucleotide in a pool or mixed sample containing other alleles. In some embodiments, the target variant nucleotide comprises a purine base, and the wild-type nucleotide at the target variant nucleotide position comprises a different purine base; the target variant nucleotide comprises a pyrimidine base, and the wild-type nucleotide at the target variant nucleotide position comprises a different pyrimidine base. In some embodiments, a target site-specific probe (e.g., a third oligonucleotide) is located at the TSP (e.g., the first oligonucleotide) at the TSP position. m T 6 to 20°C (optimally 8 to 12°C) higher than m and an amplification reaction (e.g., qPCR) is performed to identify the TSP T mThe amplification reaction is performed at an annealing temperature within 5°C of the target polynucleotide. In some embodiments, the test nucleic acid sample is derived from mammalian or non-mammalian animal tissue or cells, or plant tissue or cells. In some embodiments, detection of the amplicon indicates the presence of cancer cells in the tissue from which the test nucleic acid sample is derived. In some embodiments, the target polynucleotide comprises at least one mutation in Ras, EFGR, Kit, pTEN, and / or p53, and / or at least one KRAS or NRAS mutation. In some embodiments, the amplification reaction performed in these methods is, relies on, or includes polymerase chain reaction (PCR), including but not limited to real-time PCR. For example, in the specific example below, a target variant nucleotide present within an allelic variant KRAS DNA (which may be referred to elsewhere herein as a target polynucleotide) is identified, a forward or reverse primer (TSP) is designed to bind to the target variant nucleotide by including a nucleotide at its 3' end that is complementary to the target variant nucleotide of the allelic variant KRAS DNA being assayed, a labeled TaqMan probe is designed to include the target variant nucleotide, and the target polynucleotide is amplified and detected (e.g., KRAS G12C and C of wild-type (WT) DNA). t Figure 4D, which distinguishes between values ​​of C and C based on the amount of target polynucleotide in the sample. t (See Figure 10, which shows the variation in values.) As will be appreciated by those skilled in the art, other embodiments are contemplated.

[0046] In certain embodiments, the methods disclosed herein may be used to detect a target polynucleotide sequence (e.g., a first allelic variant) that is present in a sample at a frequency of less than 1 / 10, 1 / 100, 1 / 1,000, 1 / 10,000, 1 / 100,000, 1 / 1,000,000, 1 / 10,000,000, 1 / 100,000,000, or 1 / 1,000,000,000, and any fractional ranges therebetween, of the wild-type nucleic acid sequence (e.g., a second allelic variant) for a given nucleic acid sequence (e.g., a SNP or gene). In some embodiments, the methods disclosed herein can be used to detect a target polynucleotide sequence (e.g., a first allelic variant) present at less than 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 50, 75, 100, 250, 500, 750, 1,000, 2,500, 5,000, 7,500, 10,000, 25,000, 50,000, 75,000, 100,000, 250,000, 500,000, 750,000, or 1,000,000 copies per 1, 10 ... In some embodiments, the second allelic variant is a wild-type nucleic acid sequence. In some embodiments, the method can include detecting one target polynucleotide sequence (e.g., a first allelic variant, mutant) in a background of at least 1,000 to 1,000,000, e.g., about 1,000 to 10,000, about 10,000 to 100,000, or about 100,000 to 1,000,000 wild-type polynucleotides, or any fractional range therebetween. In some embodiments, the method can provide high sensitivity and efficiency at least comparable to TaqMan®-based assays.In some embodiments, comparison of a first amplicon (e.g., representing a target polynucleotide sequence) and a second amplicon (e.g., representing a wild-type nucleic acid sequence) using the methods disclosed herein can provide an improvement in specificity of 100-1,000,000-fold difference, e.g., about 100-1,000-fold, about 1,000-10,000-fold, about 10,000-100,000-fold, or about 100,000-1,000,000-fold, or any fractional range therebetween. In some embodiments, the size of the amplicon ranges from about 60 to 120 nucleotides in length.

[0047] In some embodiments, the oligonucleotides, mixtures, compositions, methods, and / or kits comprising or related thereto (e.g., as described herein) can be used for genotyping tetra-, tri-, and diallelic SNPs. In some embodiments, the compositions, methods, and / or kits can be used for DNA typing from mixed DNA samples for quality control (QC) and human specific assays, cell line QC for cellular contamination, allele expression analysis, virus typing / rare pathogen detection, mutation detection from pooled samples, detection of circulating tumor cells in the blood, and / or prenatal diagnosis. In some embodiments, the oligonucleotides, mixtures, compositions, methods, and / or kits comprising or related thereto (e.g., as described herein) can be used to detect tumor cells in the blood for early cancer diagnosis. In some embodiments, the compositions, methods, and / or kits can be used for the detection and validation of cancer- or disease-associated genetic or somatic mutations. In some embodiments, the oligonucleotides, mixtures, compositions, methods, and / or kits comprising or related thereto (e.g., as described herein) may be compatible with various instruments, such as, for example, SDS software-based instruments from Applied Biosystems (Foster City, Calif.). Other uses and applications of the oligonucleotides, mixtures, compositions, methods, and / or kits comprising or related thereto disclosed (e.g., as described herein) are also contemplated by the present disclosure, as will be understood by those of skill in the art.

[0048] kit The present disclosure also provides kits for using the oligonucleotides, mixtures, and compositions disclosed herein and / or for carrying out the methods described herein. In some embodiments, the present disclosure provides kits for quantifying a target polynucleotide sequence (e.g., a first allelic variant, a mutant) in a sample containing a high-abundance nucleic acid sequence (e.g., a second allelic variant, a wild-type nucleic acid sequence), the kit comprising: (a) a target sequence-specific oligonucleotide (e.g., a first oligonucleotide or a first allele-specific primer) capable of hybridizing to a target variant nucleotide in a target polynucleotide; (b) a locus-specific oligonucleotide (e.g., a second oligonucleotide or a second allele-specific primer); and (c) a probe (e.g., a third oligonucleotide) capable of hybridizing to a nucleotide corresponding to the target variant nucleotide in the target polynucleotide sequence, typically detectable (e.g., comprising a detectable label) and comprising a quencher moiety and / or an MGB moiety. Optionally, other reaction components, such as a polymerase and / or dNTPs, and / or Mg 2+Cofactors such as may also be included in the kit. In some embodiments, the kit may include reagents and the like necessary for performing RT-PCR (e.g., as described herein), where one or more RTs may be contained in the same or separate container as the DNA polymerase(s) that may be included in the kit. In some embodiments, the kit may include two or more containers containing such components, either independently dispensed in such containers or contained together in any combination in one or another container. In some embodiments, such a kit may include a first container containing a mixture provided herein (or any component thereof) and a second container containing a control nucleic acid sample comprising a first target polynucleotide strand. Thus, in some embodiments, the kit includes a first container (e.g., a first oligonucleotide, a second oligonucleotide, and a third oligonucleotide) containing (a) a target sequence-specific oligonucleotide (e.g., a first oligonucleotide or a first allele-specific primer) capable of hybridizing to a target variant nucleotide in a target polynucleotide, (b) a locus-specific oligonucleotide (e.g., a second oligonucleotide or a second allele-specific primer), and (c) a probe (e.g., a third oligonucleotide) that is typically detectable (e.g., includes a detectable label) and capable of hybridizing to a nucleotide corresponding to the target variant nucleotide in the target polynucleotide sequence that includes a quencher moiety and / or an MGB moiety, as well as a second container and / or kit containing a control nucleic acid sample comprising the first target polynucleotide strand. In some embodiments, the oligonucleotides and / or other components included in the kit are lyophilized or otherwise stabilized for storage and / or shipment and can be reconstituted as desired by the user. Instructions for use can also be included.

[0049] Illustrative Embodiments Thus, in some embodiments, the present disclosure provides a mixture comprising: a) a first oligonucleotide configured to hybridize to a first sequence in a first target polynucleotide strand, wherein the first sequence comprises a target variant nucleotide, and wherein the first oligonucleotide further has a nucleotide residue at its 3' end positioned to hybridize to the target variant nucleotide; b) a second oligonucleotide having a sequence configured to hybridize to a sequence complementary to a second sequence in the first target polynucleotide strand, wherein the second sequence in the first target polynucleotide strand is located 5' upstream from the first sequence in the first target polynucleotide strand; and c) a third oligonucleotide having a sequence configured to hybridize to a sequence complementary to a third sequence in the first target polynucleotide strand, wherein the third sequence in the first target polynucleotide strand at least partially overlaps with the first sequence in the first target polynucleotide strand, and wherein the third sequence comprises the target variant nucleotide.In some embodiments, the disclosure provides a hybridization method comprising: a) a first oligonucleotide configured to hybridize to a first sequence (A) in a first target polynucleotide strand, the first sequence comprising a target variant nucleotide ("first variant nucleotide"), the first oligonucleotide further having a nucleotide at its 3' end positioned to hybridize to the first variant nucleotide; and b) a second oligonucleotide configured to hybridize to a second sequence (B), the second sequence being complementary to a third sequence (C), the third sequence being present in the first target polynucleotide strand, the third sequence (C) being and (c) a third oligonucleotide configured to hybridize to a fourth sequence (D) complementary to a fifth sequence (E), the fifth sequence being present in the first target polynucleotide strand, the fifth sequence (E) at least partially overlapping the first sequence (A) in the first target polynucleotide strand, and the third oligonucleotide comprising a first target variant nucleotide. Methods for using the same and kits that may include instructions for using and / or storing such reagents and / or performing such methods are provided. An exemplary embodiment is shown in FIG. 1. In some embodiments, the first target polynucleotide strand is a single-stranded polynucleotide molecule comprising the first target polynucleotide strand. In some embodiments, the sequences A, E, and C described herein are located within a single-stranded B polynucleotide molecule on the first target polynucleotide strand.

[0050] In some embodiments, at least one additional set of oligonucleotides suitable for amplifying and detecting a second target polynucleotide may be included. The oligonucleotides of this at least one additional set of oligonucleotides correspond in function, but not nucleotide sequence, to those used to amplify and detect the first target polynucleotide as described above. For example, the at least one additional set of oligonucleotides may include: a) a first oligonucleotide configured to hybridize to a first sequence (F) present in the second target polynucleotide strand, the first sequence comprising a target variant nucleotide ("second variant nucleotide"), the first oligonucleotide further having a nucleotide at its 3' end positioned to hybridize to the second variant nucleotide; and b) a second oligonucleotide configured to hybridize to a second sequence (G), the second sequence (G) comprising: The hybridization set may include: a) a second oligonucleotide complementary to a third sequence (H), the third sequence being present in a second target polynucleotide strand and located 5' upstream from a first sequence (F) in the second target polynucleotide strand; and b) a third oligonucleotide configured to hybridize to a fourth sequence (I) complementary to a fifth sequence (J), the fifth sequence being present in the second target polynucleotide strand, the fifth sequence (J) at least partially overlapping with the first sequence (F) in the second target polynucleotide strand, and comprising a second target variant nucleotide. Additional sets of oligonucleotides arranged as described herein may also be included. In some embodiments, the first, second, and / or third oligonucleotides comprise 10-30 nucleotides. In some embodiments, the first and second oligonucleotides are extendable. In some embodiments, the first and second oligonucleotides are primers. In some embodiments, the target variant nucleotides in the third oligonucleotide are at least two nucleotides from the 3' or 5' end of the third oligonucleotide.In some embodiments, the third oligonucleotide can comprise 3 to 6 consecutive nucleotides of the first sequence. In some embodiments, the third oligonucleotide further comprises a sequence of nucleotides of the first target polynucleotide strand that does not overlap with the sequence of nucleotides of the first sequence. In some embodiments, the third oligonucleotide can be non-extendable and / or can be a probe. In some embodiments, the third oligonucleotide comprises a minor groove binder (MGB) moiety that can be located at the 3'-terminal nucleotide of the third oligonucleotide. In some embodiments, the third oligonucleotide can be a hydrolysis probe. In some embodiments, the T of the first oligonucleotide. m is the T of the second oligonucleotide m In some embodiments, the T of the first oligonucleotide is within 5°C of m The T of the second oligonucleotide can be 45-60°C. m In some embodiments, the T of the third oligonucleotide may be 45 to 60°C. m is the T of the first oligonucleotide m In some embodiments, the T of the third oligonucleotide may be at least 5° C. and no more than 25° C. higher than m is the T of the first oligonucleotide m may be at least 8°C and up to 12°C higher than

[0051] In some embodiments, the third oligonucleotide comprises a detectable label, such as a fluorescent label, which may be on the first terminal nucleotide of the third oligonucleotide. In some embodiments, the detectable label is a DNA binding dye, a reporter dye, a fluorescent probe, 6-carboxyfluorescein (FAM™), tetrachlorofluorescein (TET™), 6-carboxy-4',5'-dichloro-2',7'-dimethoxyfluorescein, succinimidyl ester (JOE™), VIC™, sulfonate derivatives of fluorescein dyes containing SO3 instead of the carboxylate group, phosphoramidite forms of fluorescein, phosphoramidite forms of CY5, or the like. The quencher label may be selected from the group consisting of sulfamidite-type, non-FRET labels, ferrocene reagents, ABY™, NED™, JUN™, Fluor® 488, AlexaFluor® 532, AlexaFluor® 546, AlexaFluor® 594, AlexaFluor® 647, AlexaFluor® 660, TYE™ 563, TYE™ 665, TYE™ 705, and combinations thereof. In some embodiments, the third oligonucleotide may further comprise a quencher moiety (e.g., capable of quenching a signal from the detectable label) on the second terminal nucleotide of the third oligonucleotide. In some embodiments, the quencher label may be selected from the group consisting of tetramethylrhodamine (TAMRA), non-fluorescent quencher (NFQ), Black Hole Quencher, Iowa Black, QSY, QSY7, QSY21, NFQ, dabcyl, and / or dabcyl sulfonate / carboxylate quencher. In some embodiments, the first terminal nucleotide is the 5' terminal nucleotide. In some embodiments, the second terminal nucleotide is the 3' terminal nucleotide.

[0052] In some embodiments, the composition and / or mixture does not include a fourth oligonucleotide. In some embodiments, the mixture does not include a fourth oligonucleotide comprising a detectable label. In some embodiments, the mixture does not include a fourth oligonucleotide that binds to a target polynucleotide strand. In some embodiments, the mixture does not include a fourth oligonucleotide comprising a detectable label that binds to a target polynucleotide strand. In some embodiments, the third oligonucleotide is the only oligonucleotide in the mixture that has a detectable label and a sequence configured to hybridize to the sequence of a target polynucleotide strand.

[0053] In some embodiments, the present disclosure provides compositions and / or mixtures comprising at least one single-stranded target polynucleotide comprising a target polynucleotide strand. In some embodiments, the mixture comprises at least one double-stranded target polynucleotide comprising a target polynucleotide strand and a target complementary polynucleotide strand, wherein the target complementary polynucleotide strand is substantially complementary to the target polynucleotide strand. In some embodiments, the mixture comprises at least one double-stranded target polynucleotide comprising a target polynucleotide strand and a target complementary polynucleotide strand, wherein the target complementary polynucleotide strand is substantially complementary to the target polynucleotide strand, and the double-stranded target polynucleotide comprises a variant polynucleotide strand and a variant complementary polynucleotide strand, wherein the variant polynucleotide strand has identity or is substantially identical to the target polynucleotide strand, and the target variant nucleotide comprises a different nucleotide from the target polynucleotide strand, and the variant complementary polynucleotide strand is substantially complementary to the variant polynucleotide strand. In some embodiments, the mutant allele may comprise a targeted variant nucleotide, may have an identity corresponding to a major or minor allele sequence (e.g., an allele with a population frequency of less than 1%), may occur at a single nucleotide polymorphism, such as, but not limited to, an allele of EGFR, KRAS, NRAS, BRAF PIK3CA, AKT1, ESR1, TP53, and / or may be a stochastic mutation. In some embodiments, the mutant allele is either a purine-to-purine or pyrimidine-to-pyrimidine single point mutation at the targeted variant nucleotide.

[0054] In some embodiments, the compositions and / or mixtures comprise: a) a fourth oligonucleotide configured to hybridize to a first sequence in a second target polynucleotide strand, wherein the first sequence in the second target polynucleotide comprises a second target variant nucleotide, and wherein the fourth oligonucleotide further comprises a nucleotide at its 3' end positioned to hybridize to the second target variant nucleotide; and b) a fifth oligonucleotide comprising a sequence configured to hybridize to a sequence complementary to a second sequence in the second target polynucleotide strand. The oligonucleotide may further comprise: a fifth oligonucleotide, wherein the second sequence of the second target polynucleotide strand is located 3' downstream from the first sequence on the second target polynucleotide strand; and c) a sixth oligonucleotide (optionally detectable) comprising both a detectable label and a sequence configured to hybridize to a sequence complementary to the third sequence of the second target polynucleotide strand, wherein the third sequence of the second target polynucleotide strand at least partially overlaps with the first sequence on the second target polynucleotide strand and comprises a second target variant nucleotide. In some embodiments, the fourth, fifth, and / or sixth oligonucleotides may comprise 10-40 nucleotides, with the fourth and fifth oligonucleotides preferably comprising 10-30 nucleotides. In some embodiments, the fourth and fifth oligonucleotides may be extendable and / or may be primers. In some embodiments, the target variant nucleotide in the sixth oligonucleotide is at least two nucleotides from the 3' or 5' end of the sixth oligonucleotide. In some embodiments, the sixth oligonucleotide can include a detectable label (e.g., a fluorescent label), which can be on the first terminal nucleotide of the sixth oligonucleotide. The sixth oligonucleotide can further include a quencher moiety capable of quenching a signal from the detectable label, which can be on the second terminal nucleotide of the sixth oligonucleotide.In some embodiments, the sixth oligonucleotide may be non-extendable and / or may be a probe. In some embodiments, the sixth oligonucleotide comprises a minor groove binder (MGB) moiety, which may be on the 3'-terminal nucleotide of the sixth oligonucleotide.

[0055] In some embodiments, the composition and / or mixture may further comprise: a) a fourth oligonucleotide comprising a sequence configured to hybridize to a sequence complementary to the first oligonucleotide, wherein the fourth oligonucleotide is configured to substantially hybridize to the first sequence and comprises a nucleotide at its 3' end that is different from the complement of the target variant nucleotide; and b) a fifth oligonucleotide (optionally detectable) configured to hybridize to a sequence complementary to the third oligonucleotide, wherein the fifth oligonucleotide comprises a nucleotide that is different at the position of the target variant nucleotide. In some embodiments, the fourth and fifth oligonucleotides comprise 10-40 nucleotides, with the fourth oligonucleotide preferably being 10-30 nucleotides. In some embodiments, the fourth oligonucleotide may be extendable and / or may be a primer. In some embodiments, the target variant nucleotide in the fifth oligonucleotide may be at least two nucleotides from the 3' or 5' end of the fifth oligonucleotide. In some embodiments, the fifth oligonucleotide may include a detectable label (e.g., a fluorescent label) on the first terminal nucleotide, which may be distinguishable from the detectable label on the third oligonucleotide. The fifth oligonucleotide may further include a quencher moiety that can quench the signal from the detectable label on the second terminal nucleotide of the third oligonucleotide. In some embodiments, the fifth oligonucleotide is non-extendable and / or is a probe. In some embodiments, the fifth oligonucleotide includes a minor groove binder (MGB) moiety on its 3' terminal nucleotide.

[0056] In some embodiments, the present disclosure provides compositions and / or mixtures comprising a plurality of oligonucleotide sets, each oligonucleotide set comprising: a) a first oligonucleotide configured to hybridize to a first sequence in a target polynucleotide strand, wherein the first sequence comprises a target variant nucleotide, and the first oligonucleotide further comprises a nucleotide residue at its 3' end positioned to hybridize to the target variant nucleotide; and b) a second oligonucleotide comprising a sequence configured to hybridize to a sequence that is complementary to a second sequence within the first target polynucleotide strand, , wherein the second sequence is located 5' upstream from the first sequence; and c) detectable third oligonucleotides comprising a sequence configured to hybridize to a sequence complementary to a third sequence of the target polynucleotide strand, wherein the third sequence at least partially overlaps with the first sequence and comprises a target variant nucleotide, wherein the first oligonucleotides of each set are configured to hybridize to different first sequences, the third oligonucleotides of each set share sequence similarity with the different third sequences, and the third oligonucleotides of each set comprise a different distinguishable detectable label.

[0057] In some embodiments, the present disclosure provides compositions and / or mixtures comprising a plurality of oligonucleotide sets, each oligonucleotide set comprising: a) a first oligonucleotide configured to hybridize to a first sequence in a first target polynucleotide strand, wherein the first sequence comprises a target variant nucleotide, the first oligonucleotide further having a nucleotide residue at its 3' end positioned to hybridize to the target variant nucleotide; and b) a second oligonucleotide having a sequence configured to hybridize to a sequence complementary to a second sequence within the first target polynucleotide strand, wherein the second sequence in the first target nucleotide strand is complementary to the first target nucleotide. and c) a second oligonucleotide located 5' upstream from the first sequence of the nucleotide strand; and c) a third oligonucleotide having a sequence configured to hybridize to a sequence complementary to a third sequence within the first target polynucleotide strand, wherein the third sequence of the first target nucleotide strand at least partially overlaps with the first sequence of the first target nucleotide strand and comprises a target variant nucleotide, wherein the first oligonucleotides of each set are configured to hybridize to different first sequences, the third oligonucleotides of each set share sequence similarity with the different third sequences, and / or the third oligonucleotides of each set comprise different distinguishable detectable labels.

[0058] In some embodiments, the present disclosure provides compositions and / or mixtures comprising a plurality of oligonucleotide sets, each oligonucleotide set comprising: a) a first oligonucleotide configured to hybridize to a first sequence (A) present in a first target polynucleotide strand, wherein the first sequence comprises a target variant nucleotide ("first variant nucleotide"), the first oligonucleotide further having a nucleotide at its 3' end positioned to hybridize to the first variant nucleotide; and b) a second oligonucleotide configured to hybridize to a second sequence (B), wherein the second sequence is complementary to a third sequence (C), the third sequence present in the first target polynucleotide strand and the third a second oligonucleotide, wherein sequence (C) is located 5' upstream from the first sequence (A) of the first target polynucleotide strand; and c) a third oligonucleotide configured to hybridize to a fourth sequence (D) complementary to a fifth sequence (E), wherein the fifth sequence is present in the first target polynucleotide strand, the fifth sequence (E) at least partially overlaps with the first sequence (A) in the first target polynucleotide strand and comprises a first target variant nucleotide, wherein the first oligonucleotides of each set are configured to hybridize to different first sequences, and the third oligonucleotides of each set share sequence similarity with different third sequences and / or the third oligonucleotides of each set comprise different distinguishable detectable labels.

[0059] In some embodiments, the compositions and / or mixtures of the present disclosure can include additional components. For example, the mixture can further include about 10 mM to about 80 mM potassium chloride and / or about 10 mM to about 40 mM ammonium sulfate. In some embodiments, the mixture can include potassium chloride at a concentration of 30 mM to 80 mM and ammonium sulfate at a concentration of 10 mM to 40 mM. In some embodiments, the mixture can include about 45 mM potassium chloride and about 22 mM ammonium sulfate. In some embodiments, the mixture can further include a polymerase, which may be thermostable, and can further include a hot start component, such as an antibody, oligonucleotide, and / or aptamer directed against the thermostable polymerase. In some embodiments, the mixture can further include a nucleotide source. In some embodiments, the mixture is a reaction mixture. In some other embodiments, the mixture is a storage mixture. In some embodiments, the mixture can include a nucleic acid sample suspected of containing a target polynucleotide strand. In some embodiments, the mixture can include and / or be a master mix. In some embodiments, the mixture can further include an amplicon comprising a first sequence of the target polynucleotide strand. In some embodiments, the mixture does not include an amplicon comprising a sequence of a second polynucleotide strand. In some embodiments, the present disclosure provides a mixture comprising one or more of at least one detergent, glycerol, at least one reference dye, bovine serum albumin, and / or gelatin. In some embodiments, the mixture can be lyophilized.

[0060] In some embodiments, the present disclosure provides a kit comprising a first container containing one or more mixtures described herein and a second container containing a control nucleic acid sample comprising a first target polynucleotide strand that shares sequence similarity with the first target polynucleotide strand. In some embodiments, the control nucleic acid sample can contain only a portion (e.g., a heterologous sequence) of the target polynucleotide, and in some embodiments, the control nucleic acid sample contains the entire first target polynucleotide strand.

[0061] The present disclosure also provides a method for detecting a target polynucleotide containing a target variant nucleotide using the reagents (e.g., oligonucleotides) described herein. In some embodiments, the method can include: a) forming a reaction mixture of a test nucleic acid sample and one or more of the mixtures described herein; b) performing an amplification reaction using at least a first and a second oligonucleotide as primers to generate an amplicon of the target polynucleotide sequence of the target polynucleotide, if present in the sample; and c) detecting the amplicon by detecting a change in the detectable property of the third oligonucleotide, wherein detecting the amplicon in step c) indicates that the target polynucleotide is present in the test nucleic acid sample. In some embodiments, the target polynucleotide can be identified in a sample containing a mixture of nucleic acids containing a target polynucleotide (e.g., a rare / low-abundance nucleic acid) and a non-target polynucleotide (e.g., a high-abundance nucleic acid) that does not contain the target variant nucleotide.

[0062] In some embodiments, the present disclosure provides a method for detecting a target polynucleotide molecule comprising a target variant nucleotide in a test polynucleotide sample, the method comprising: a) forming a reaction mixture of the test polynucleotide sample and a mixture of first, second, and third oligonucleotides described herein (in some embodiments, also comprising a fourth, fifth, and / or sixth oligonucleotide described herein); b) performing an amplification reaction using at least the first and second oligonucleotides as primers to generate an amplicon of the target polynucleotide sequence of the target polynucleotide molecule, if present in the test polynucleotide sample; and c) detecting the amplicon generated in step b) by detecting a change in a detectable property of the third oligonucleotide, wherein detecting the amplicon in step c) indicates the presence of the target polynucleotide molecule in the test polynucleotide sample. In some embodiments of these methods, the target polynucleotide molecule is detected in a test polynucleotide sample comprising a mixture of polynucleotide molecules, the mixture comprising a polynucleotide molecule comprising a first variant form of a target variant nucleotide (a "first variant target polynucleotide molecule") and a polynucleotide molecule comprising a second variant form of the target variant nucleotide (a "second variant target polynucleotide molecule"). In some embodiments of these methods, the test polynucleotide sample comprises a polynucleotide strand that does not comprise a target polynucleotide sequence (a "non-target polynucleotide molecule"). In some embodiments of these methods, the test sample comprises more non-target polynucleotide molecules than target polynucleotide molecules. In some embodiments of these methods, the target polynucleotide molecule is a rare allele or a mutant polynucleotide sequence. In some embodiments of these methods, the non-target polynucleotide molecule is a major allele or a wild-type polynucleotide sequence.In some embodiments of these methods, the test sample contains less than 2%, 1%, 0.1%, 0.01%, or 0.001% of target polynucleotide molecules compared to non-target polynucleotide molecules. In some embodiments of these methods, the test sample contains less than 2%, 1%, 0.1%, 0.01%, or 0.001% of second variant target polynucleotide molecules compared to first variant target polynucleotide molecules. In some embodiments of these methods, the first variant target polynucleotide molecule and / or the second variant target polynucleotide molecule are mutant polynucleotide sequences. In some embodiments of these methods, the first variant target polynucleotide molecule and / or the second variant target polynucleotide molecule are wild-type polynucleotide sequences.

[0063] In some embodiments, the methods further comprise enriching the number of first variant polynucleotide molecules in the polynucleotide test sample relative to the second variant polynucleotide molecules prior to steps a)-c) (the "enrichment" step). In some embodiments of these methods, the enrichment step comprises an amplification reaction under conditions different from those used in steps a)-c). In some embodiments, the enrichment step increases (enriches) the number of first variant polynucleotide molecules in the polynucleotide test sample relative to the second variant polynucleotide molecules by at least 2-fold, 4-fold, 6-fold, 8-fold, or 10-fold. For example, in ... M In some embodiments, steps a) through c) are performed using PCR comprising 15-25 cycles at a temperature 12-16 degrees higher than the T of the target site-specific probe (e.g., third oligonucleotide) at a temperature 4-6 degrees lower than the temperature at which enrichment is performed. mIn some embodiments of these methods, the test polynucleotide sample is derived from mammalian or non-mammalian animal tissue or cells, or plant tissue or cells. In some embodiments of these methods, the sample is selected from the group consisting of saliva, buccal tissue, skin, hair, blood, plasma, urine, feces, semen, and a tumor sample. In some embodiments of these methods, the polynucleotide test sample is derived from cancer cells. In some embodiments of these methods, detection of the amplicon indicates the presence of cancer cells in the tissue from which the test polynucleotide sample is derived. In some embodiments of these methods, the target polynucleotide comprises at least one mutation in EFGR (e.g., FIG. 20), Ras (e.g., at least one KRAS or NRAS mutation (e.g., FIG. 21)), Kit, pTEN, and / or p53, and / or at least one KRAS or NRAS mutation, and / or at least one mutation listed in Table 1 and / or Table 2. In some embodiments of these methods, the amplification reaction is a polymerase chain reaction (PCR), such as real-time PCR. In some embodiments of these methods, the third oligonucleotide has a Tm 6-20°C (optimally 8-12°C) higher than the Tm of the first oligonucleotide, and PCR is performed at an annealing temperature within 5°C of the Tm of the first oligonucleotide. In some embodiments, these methods are performed using a kit comprising a first container containing at least the first oligonucleotide, the second oligonucleotide, and the third oligonucleotide, and a second container containing a control polynucleotide sample comprising the first target polynucleotide strand. In some embodiments, the method detects a target variant nucleotide indicative of a mutation, and the polynucleotide sample comprises 1-10 copies of the target polynucleotide.In some embodiments of these methods, the target variant nucleotide comprises a purine base and the corresponding major allele or wild-type nucleotide at the target variant nucleotide position comprises a different purine base, or the target variant nucleotide comprises a pyrimidine base and the corresponding major allele or wild-type nucleotide at the target variant nucleotide position comprises a different pyrimidine base.

[0064] In some embodiments, the method can further include enriching the number of target polynucleotides in the sample relative to wild-type nucleic acids prior to steps a)-c). In some embodiments, this enrichment process can include an amplification reaction that includes different conditions as used in steps a)-c). For example, in some embodiments, the enrichment step can include enriching the number of target polynucleotides in the sample relative to wild-type nucleic acids based on the calculated melting temperature (T m In some embodiments, steps a) to c) may be performed using a polymerase chain reaction comprising 15 to 25 cycles at a temperature 12 to 16° C. higher than the T of the third oligonucleotide. mThe amplification method can include 40 cycles at a temperature close to the enrichment temperature and 4-6 degrees lower than the temperature at which enrichment is performed (e.g., "close to" can mean 4-6 degrees lower than the temperature used in the enrichment process). In some embodiments, the test nucleic acid sample is derived from mammalian or non-mammalian animal tissue or cells, or plant tissue or cells (e.g., saliva, buccal tissue, skin, hair, blood, plasma, urine, feces, semen, and tumor samples (e.g., cancer cells)). In some embodiments, detection of amplicons indicates the presence of cancer cells in the tissue from which the test nucleic acid sample is derived. In some embodiments, the target polynucleotide comprises one or more of EFGR (e.g., FIG. 20), Ras (e.g., at least one KRAS or NRAS mutation (e.g., FIG. 21)), BRAF, Kit, pTEN, ESR1, and / or p53; and / or one or more of the mutations listed in Table 1 and / or Table 2. In some embodiments, the amplification reaction is a polymerase chain reaction (PCR), such as, but not limited to, real-time PCR or quantitative PCR (qPCR). In some embodiments, for example, the third oligonucleotide is a T m T 6 to 20°C (optimally 8 to 12°C) higher than m and PCR can include a first oligonucleotide T m The annealing temperature may be within 5°C of the annealing temperature.

[0065] In some embodiments, the method can be performed using a kit including a first container containing at least a first oligonucleotide, a second oligonucleotide, and a third oligonucleotide, and a second container containing a control nucleic acid sample containing a first target polynucleotide strand. In some embodiments, the method can detect a target variant nucleotide indicative of a mutation, wherein the nucleic acid sample contains 1 to 10 copies of the target polynucleotide sequence from a background of a much larger number of wild-type nucleic acid sequences. In some embodiments, the target variant nucleotide contains a purine base, and the corresponding wild-type nucleotide at the target variant nucleotide position contains a different purine base, and / or contains a pyrimidine base, and the corresponding wild-type nucleotide at the target variant nucleotide position contains a different pyrimidine base. As will be appreciated by those skilled in the art, other methods are also contemplated by the present disclosure.

[0066] General information The terms "about," "approximately," and the like, when preceding a list of numerical values ​​or ranges, refer independently to each individual value in that list or range, as if the term were immediately preceding each individual value in that list or range. These terms mean that the value to which the same refers is exactly, nearly, or similar to that.

[0067] As used herein, subject or host refers to an individual.Subject can include domestic animals such as cats and dogs, livestock (e.g., cows, horses, pigs, sheep, and goats), laboratory animals (e.g., mice, rabbits, rats, guinea pigs), and birds.In one aspect, subject is a mammal such as a primate or human.

[0068] "Any" or "optionally" means that the subsequently described event or circumstance may or may not occur, and the description includes instances in which the event or circumstance occurs and instances in which it does not occur. For example, the phrase "optionally, the composition may include a combination" means that the composition may include a combination of different polynucleotides or may not include a combination, such that the description includes both a combination and the absence of a combination (i.e., individual members of a combination).

[0069] Ranges may be expressed herein as from about one particular value and / or to about another particular value. When such a range is expressed, another aspect includes from the one particular value and / or to the other particular value. Similarly, when values ​​are expressed as approximations, by using the antecedent "about" or "approximately," it will be understood that the particular value forms another aspect. It will further be understood that the endpoints of each range are significant both in relation to the other endpoint and independently of the other endpoint. A range (e.g., 90-100%) is meant to include the range itself and each individual value within that range, as if each value were individually recited.

[0070] The terms "combined" or "in combination" or "in conjunction with" can refer to the physical combination of agents administered together, or the use of two or more agents in a regimen (e.g., administered separately, physically, and / or in time) to treat, prevent, and / or ameliorate a particular disease.

[0071] The terms treatment, prevention, and / or amelioration, or their derivatives, when used herein in connection with a given treatment for a given condition (e.g., prevention of cancer infection by HIV), are meant to convey that a treated patient will not develop clinically observable levels of the condition at all, or will develop the condition at a slower and / or less severe level than if the treatment had not been administered. These terms are not limited to situations in which the patient does not experience any aspect of the condition at all. For example, a treatment is said to have prevented a condition if it is administered during a patient's exposure to a stimulus that would be expected to result in a given manifestation of the condition, resulting in the patient experiencing fewer and / or milder symptoms of the condition than would otherwise be expected. For example, a treatment can "prevent" an infection by resulting in the patient exhibiting only mild, overt symptoms of the infection, and does not imply that there must have been no invasion of any cells by the infectious microorganism.

[0072] Similarly, "reduce," "reducing," and "reduction," as used herein in connection with the prevention, treatment, and / or amelioration of a given condition by a particular treatment, typically refer to a subject developing an infection more slowly or to a lesser extent compared to a control or basal level of infection without treatment. As a result of a reduced risk of infection, a patient may exhibit only mild overt symptoms of infection or delayed symptoms of infection, and does not mean that there must have been no invasion of any cells by the infectious microorganism.

[0073] All references cited within this disclosure are incorporated herein by reference in their entirety. Certain embodiments are further described in the following examples. These embodiments are provided by way of example only and are not intended to limit the scope of the claims in any way. [Example]

[0074] The reagents and assay designs disclosed herein exemplify the detection of fewer than 10 copies (e.g., as few as 1-3 copies) of a target nucleotide sequence (e.g., a mutant, rare, or low-abundance target nucleotide sequence) from a background of non-target nucleotide sequences (e.g., "wild-type," common, or higher abundance nucleotide sequences). As discussed above, these methods can be useful for detecting such rare (e.g., low-abundance) target nucleic acids in a variety of assays, including, but not limited to, cell-free DNA cancer-related assays (e.g., for early diagnosis and / or detection of recurrence), single nucleotide polymorphism (SNP) determination assays, forensic-related assays, and / or agricultural-related assays.

[0075] As shown in these examples, quantitative polymerase chain reaction (qPCR) reactions were performed to detect, identify, and / or quantify low-abundance nucleic acid sequences (e.g., representing mutant genes or allelic variants) in the presence of more abundant wild-type nucleic acid sequences (e.g., representing non-mutated genes or major alleles) using exemplary primer and probe assay designs such as those shown in Figure 1. Each qPCR reaction utilized a mixture of at least one forward primer (e.g., a first oligonucleotide), at least one reverse primer (e.g., a second oligonucleotide), and at least one probe (e.g., a third oligonucleotide or TaqMan probe, which may be a 5' nuclease), as exemplified but not limited to in the examples described below, used to amplify target polynucleotides comprising first and second target polynucleotide strands. One of the primers (i.e., either the forward or reverse primer, the first oligonucleotide) had binding specificity for a first nucleotide sequence (sometimes referred to as the "first sequence") containing at least one target variant nucleotide (e.g., a target site that may represent a mutant gene or a low-abundance allelic variant) in the first target polynucleotide strand. The target variant nucleotide corresponded to the terminal nucleotide of the primer (i.e., meaning that it was hybridizable to it and was the same as or complementary to it), so that the binding specificity of the primer was primarily determined by the target variant nucleotide. Thus, the first oligonucleotide, either the forward or reverse primer, was designed to bind to the first nucleotide sequence and contain a nucleotide complementary to the target variant nucleotide (e.g., a nucleotide corresponding to a mutant gene or an allelic variant) at its 3' end. In some embodiments, the nucleotide complementary to the target variant nucleotide at the 3' end of the first oligonucleotide may be within two nucleotides (n+2, n+1, n) of the actual 3'-terminal nucleotide (n) of the first oligonucleotide.

[0076] A second oligonucleotide (i.e., another primer; forward or reverse primer) and a third oligonucleotide (e.g., a target site-specific probe such as a TaqMan™ probe or hydrolysis probe), each having a sequence that shares sequence identity with the second and third sequences of the first polynucleotide strand, respectively (i.e., the second and third sequences are typically complementary to the second polynucleotide strand of the double-stranded target polynucleotide). The second oligonucleotide (e.g., the second primer) in the mixture is designed to share identity with the first target polynucleotide strand, but is located upstream or downstream of the first sequence to which the first oligonucleotide (e.g., the first primer) is hybridized, and does not overlap. The third sequence to which the third oligonucleotide (e.g., the TaqMan™ probe) is hybridized is designed to at least partially overlap with the first sequence and contain a target variant nucleotide. In some exemplary embodiments, the third oligonucleotide (e.g., TaqMan™ probe) also contained additional nucleotides identical to the first target polynucleotide strand that did not overlap with the first sequence bound by the first oligonucleotide or first primer. In some exemplary embodiments, the third oligonucleotide (e.g., target site-specific probe such as TaqMan™ probe) used in these experiments contained an oligonucleotide having a fluorophore (e.g., 6-FAM) covalently attached to its 5' end, a quencher (non-fluorescent quencher, sometimes referred to as "NFQ"), and a minor groove binder ("MGB") covalently attached to its 3' end. Without being limited by theory, it is believed that the minor groove binder attached to the 3' end of the oligonucleotide renders it non-extendible by Taq polymerase. Generally, the third oligonucleotide (e.g., TaqMan™ probe) is selected to have a melting temperature (T) that is 6-20°C (optimally 8-12°C) higher than the melting temperature of the first oligonucleotide (e.g., first primer). M) and PCR was performed using standard techniques (e.g., T of a third oligonucleotide (e.g., a target site-specific probe such as a TaqMan probe) M (40 cycles at an annealing / extension temperature close to 0.05°C). For example, in an exemplary assay for KRAS G12R, the T m (as calculated by Primer Express) was 52.4°C, and the T of the second oligonucleotide m is 50.8 °C, and the T of the third oligonucleotide m In another example, in an assay for KRAS G12C, the T m is 50.2 °C, and the T of the second oligonucleotide m is 51.6 °C, and the T of the third oligonucleotide m In yet another example for KRAS G12A, the Tm of the first oligonucleotide was 51.4°C, the Tm of the second oligonucleotide was 51.6°C, and the Tm of the third oligonucleotide was 61°C.

[0077] In certain embodiments, the qPCR reaction included an "enrichment cycle" or "enrichment phase" to amplify low-abundance nucleic acids preferentially over high-abundance (e.g., wild-type) nucleic acids. The mixture was subjected to one cycle of 95°C for 2 minutes, 15-20 cycles of 95°C for 1 second and 64°C for 20 seconds (enrichment phase), and 40 cycles of 95°C for 1 second and 60°C for 20 seconds (amplification and detection phase). In some embodiments, the qPCR reaction was performed without an enrichment phase. The mixture was subjected to one cycle of 95°C for 2 minutes and 40 cycles of 95°C for 1 second and 60°C for 20 seconds (amplification and detection phase). If the reaction included a VIC-labeled RPPH1 assay, the data generated from the amplification reaction was exported to Excel format, the Cq values ​​of the replicate reactions were averaged, and the delta average Cq of the FAM and VIC targets for each condition was determined and plotted. The delta Cq was used for the quantification method.

[0078] In certain illustrative examples below, for example, a target variant nucleotide is present in allelically variant DNA (which may be referred to elsewhere herein as the target nucleic acid) such as KRAS, BRAF, or EGFR, a forward or reverse primer (a first oligonucleotide) is designed to bind to the target variant nucleotide by including a nucleotide at its 3' end that is complementary to the target variant nucleotide of the DNA being analyzed, such as the allelic variant KRAS, BRAF, or EGFR, a labeled third oligonucleotide (which may be a TaqMan probe) is designed to include the target variant nucleotide, and the target nucleic acid is amplified and detected. Additional details of the various assays performed in these illustrative examples are provided below.

[0079] Example 1 Distinguishing between wild-type and mutant sequences A. qPCR Assay For qPCR analysis, samples of wild-type DNA (control DNA from CEPH individual 1347-02 (“CEPH”), Thermo Fisher Scientific catalog number 403062) were spiked with or without 0.1% target allele variant KRAS DNA (KRAS Reference Standards DNA purchased from Horizon Discovery Ltd, catalog numbers: HD287 for KRAS G12R and HD264 for KRAS G12A). A wild-type DNA sample (10 ng) or a spiked sample (10 ng of wild-type (CEPH) DNA containing an allelic variant spike (e.g., mutant sample)) was added to 300 nM of each primer (forward and reverse, first and second oligonucleotides), 250 nM of probe (third oligonucleotide), 1 mM dNTPs, 39 mM Tris pH 8, 2.55 mM MgCl, 30 mM KCl, 16 mM (NH)SO, 0.1 mg / mL BSA, 7% glycerol, and 0.085 U / uL Platinum Taq to form the reaction mixture, and 10 μL aliquots of the mixture were plated in four replicate wells of a 96-well plate using a QuantStudio 5 F96 (Thermo Fisher Scientific). qPCR reactions were performed on a PCR PCR system (Scientific, Waltham, MA). For qPCR reactions performed without an enrichment phase, the mixture was subjected to one cycle of 95°C for 2 minutes and 40 cycles of 95°C for 1 second, followed by 58–62°C for 20 seconds (amplification and detection phase). For qPCR reactions performed with an enrichment phase, the mixture was subjected to one cycle of 95°C for 2 minutes, 19 cycles of 95°C for 3 seconds and 64°C for 20 seconds (enrichment), and 40 cycles of 95°C for 1 second, followed by 58–62°C for 20 seconds (amplification and detection phase). Primers, probes, and target nucleic acids utilized in these assays were designed as described above. Data were exported to Excel, and Cq values ​​for replicate reactions were averaged to determine and plot the delta average Cq of the target. Delta Cq was used for quantification.

[0080] B. Experimental Results As described above, qPCR reactions with an assay design in which the reaction contained a target site-specific probe and two primers, one of which was a first oligonucleotide (e.g., a target site-specific primer) and the other a second oligonucleotide (e.g., a locus-specific primer), differentially amplified low-abundance target nucleic acids in the presence of highly similarly abundant nucleic acids (Figures 2-4). Notably, the assay design was able to discriminate between samples with and without spiked allelic variant KRAS DNA with or without an amplification enrichment phase (results from an experiment including an enrichment phase shown in Figures 2A-C and 3A-C). Under these reaction conditions and assay, an amplification curve indicating successful amplification was observed only when low-abundance allelic variant KRAS DNA (10 ng of wild-type (CEPH) DNA and 10 pg of target sequence (0.1% or 3 copies)) was present in the reaction mixture (i.e., not when only wild-type DNA (10 ng of wild-type (CEPH) DNA) was present) (Figures 2A-F and 3A-F).

[0081] Reactions that included an enrichment phase had lower Ct values, indicating that the enrichment phase preferentially, if not exclusively, amplifies low-abundance allelic variant DNA, thus further improving performance using this assay design (Figures 2A-F and 3A-F). Meanwhile, the background fluorescence of some samples containing only wild-type KRAS DNA increased with the inclusion of the enrichment phase. These background qPCR curves were readily distinguishable from typical qPCR curves, as seen when low-abundance allelic variants are present (e.g., Figures 2A-C and 3A). Without being limited by theory, it is believed that the high temperature in the enrichment phase favors annealing of target sequence-specific primers to low-abundance target nucleic acids that form perfect matches when bound to the target sequence-specific primer, compared to annealing of target sequence-specific primers to abundant nucleic acids that contain a single-base mismatch when bound to the target sequence-specific primer. This subsequently results in preferential amplification of low-abundance target nucleic acids over abundant nucleic acids.

[0082] In subsequent experiments, a similar assay design was used (Horizon Discovery Ltd.) to distinguish wild-type DNA spiked with 0.1% of the following allelic variant KRAS DNA from wild-type DNA spiked with 0.1% of the following allelic variants: 34 G>C (Figure 4A), 35 G>C (Figure 4B), 34 G>A (Figure 4C), 34 G>T (Figure 4D), 35 G>A (Figure 4E), and 35 G>T (Figure 4F). qPCR reactions composed of a first oligonucleotide (e.g., target sequence-specific primer), a second oligonucleotide (e.g., locus-specific primer), and a third oligonucleotide (e.g., target site-specific probe) had lower Ct values ​​in samples containing wild-type KRAS DNA spiked with 0.1% of the indicated allelic variant KRAS DNA than in samples containing only wild-type KRAS DNA. Table 3 shows the average Ct values ​​with and without spiked allelic variant KRAS DNA from these qPCR reactions, and the difference between the two Ct values ​​(dCt) is shown. All primer / probe combinations showed effective discrimination between samples containing low-abundance KRAS allele DNA and wild-type KRAS DNA, with five of the six allelic variants having dCt values ​​of 9 or greater. [Table 3]

[0083] Additional experiments using assay designs and cycling conditions similar to those disclosed in this example were also performed on a variety of other samples, detecting 1 to 3 copies of allelic variant DNA in a background of wild-type target nucleic acid (data not shown).

[0084] Example 2 Potassium chloride and ammonium sulfate The effects of potassium chloride and ammonium sulfate concentrations on the detection of low-abundance target nucleic acids were also tested. As shown in this illustrative example, fewer than 10 copies of a target nucleic acid differing from an abundant nucleic acid by a single base change were detected using the primer and probe assay design shown in Figure 1 and / or described in more detail below, and amplification mixtures containing optimized concentrations of both KCl and (NH4)2SO4. When the single base change is in the same structural family (purine to purine or pyrimidine to pyrimidine), discrimination of a low-abundance target nucleic acid in the presence of a highly similar abundant nucleic acid is particularly challenging. In these examples, potassium chloride was demonstrated to improve amplification, while ammonium sulfate improved discrimination of a low-abundance target nucleic acid in the presence of a highly similar abundant nucleic acid. The concentrations of both potassium chloride and ammonium sulfate were optimized for amplification of low-abundance target nucleic acids and suppression of amplification of abundant targets. As generally described above, the third oligonucleotide (e.g., TaqMan probe) in these assays was designed to bind to a nucleotide spanning the target variant nucleotide (e.g., allelic variant) of the target nucleic acid. In addition to a third oligonucleotide (e.g., a TaqMan probe), either a forward or reverse primer is designed to contain a nucleotide at its 3' end that is complementary to the target variant nucleotide on the target nucleic acid. As described above, the primer containing a nucleotide at its 3' end that is complementary to the target variant is a first oligonucleotide (e.g., a target sequence-specific primer (TSP)). The other primer (i.e., a second oligonucleotide) that has binding specificity for the target nucleic acid does not contain a nucleotide at its 3' end that is complementary to the target variant nucleotide described herein and may be referred to as a locus-specific primer (LSP).

[0085] A. Effect of potassium chloride and ammonium sulfate on amplification In these experiments, the following mixture was used: 0.3 μM forward [or reverse] primer (e.g., first oligonucleotide (e.g., target sequence-specific primer (TSP)), 0.3 μM reverse [or forward] primer (e.g., second oligonucleotide (e.g., locus-specific primer (LSP)), 0.25 μM detection probe (e.g., third oligonucleotide (e.g., target site-specific probe)), 1 mM dNTPs, 2.55 mM MgCl2, 39 mM Tris pH 8, 0.085 U / uL Platinum A 20 μL reaction mixture was made containing Taq and amplification reagent with 0.1 mg / mL BSA; zero, 30 mM, or 60 mM KCl (only zero and 60 mM data shown); and zero, 15 mM, or 30 mM (NH4)2SO4 (only zero and 30 mM data shown), and 10 ng of wild-type DNA (with or without 0.2% (4 copies) of allelic variant DNA spiked in, as shown in Figures 5 and 6). qPCR was performed in a QuantStudio 7 with the following cycles: 1 cycle at 95°C for 3 minutes, 19 cycles at 95°C for 3 seconds / 64°C for 20 seconds (enrichment phase), and 40 cycles at 95°C for 3 seconds / 60°C for 20 seconds (amplification and detection phase). These experiments and the resulting data are further described below.

[0086] We observed that qPCR reactions containing up to 60 mM KCl and up to 30 mM (NH4)2SO4, and the primer and probe assay designs described in Figure 1 and further above, discriminated between samples containing only wild-type DNA and wild-type DNA spiked with 0.2% allelic variant DNA, as indicated by lower Ct values ​​(Figure 5A, reactions performed in the presence of 60 mM KCl and 30 mM (NH4)2SO4). In contrast, qPCR reactions without either salt showed similar Ct values ​​for reactions containing wild-type DNA and wild-type DNA spiked with 0.2% allelic variant DNA (Figure 5B, reactions performed in the absence of KCl and (NH4)2SO4). Thus, including effective amounts of KCl and (NH)SO in a qPCR master mix, along with the primer and probe assay design provided in FIG. 1 (e.g., as exemplified herein above), provided an effective method for detecting as little as 0.2% (e.g., 4 copies) of a target nucleic acid in a sample containing abundant (e.g., wild-type) nucleic acids that differ from the target nucleic acid by only a single nucleotide (i.e., the target variant nucleotide).

[0087] Additional experiments were performed to determine the range of concentrations that provided discrimination between low-copy number target nucleic acids and wild-type nucleic acids. A 20 μL reaction mixture was prepared containing 10 ng of wild-type (CEPH) DNA (Thermo Fisher Scientific catalog number 403062), 300 nM of each primer (forward and reverse, first and second oligonucleotides, TSP and LSP), 250 nM probe (third oligonucleotide), 1 mM dNTPs, 0.085 U / μL Platinum Taq, 2.55 mM MgCl2, 45 nM ROX passive reference, 39 mM Tris, pH 8, and 7% glycerol. Potassium chloride and ammonium sulfate were titrated into the reaction mixture as shown below: Figure 6A: no KCl or ammonium sulfate; Figure 6B: 30 mM ammonium sulfate, no potassium chloride; Figure 6C: 30 mM KCl and 30 mM ammonium sulfate; and Figure 6D: 60 mM KCl and 30 mM ammonium sulfate. Reactions with the G13D mutant spike contained 20 pg of KRAS G13D Reference Standard DNA (Horizon Discovery Ltd., catalog number HD290). Reactions were amplified on a QuantStudio7 instrument using the following thermal protocol: 95°C (3 min), 19 cycles of 95°C (3 s) / 64°C (20 s) (enrichment phase), then 40 cycles of 95°C (3 s) / 60°C (20 s) (amplification and detection phase). As shown in Figure 6C, the conditions used in the exemplary reactions (30 mM KCl and 30 mM ammonium sulfate) showed clear discrimination between target and wild-type nucleic acids, whereas omission of both KCl and ammonium sulfate (Figure 6A) did not. Thus, in these experiments, "effective amounts" (amounts that provide discrimination between target and wild-type nucleic acids) of KCl and ammonium sulfate were found to be up to about 60 mM and up to about 30 mM, respectively.

[0088] The effects of the two salts were further investigated in experiments using KCl and ammonium sulfate concentrations, testing concentrations between the initial high concentration (60 mM KCl, 30 mM ammonium sulfate) and the initial low concentration (30 mM KCl, 15 mM ammonium sulfate). As Figure 6C demonstrates, the effect of the two salts when used at higher concentrations provides discrimination but also shows delayed Cq values. Therefore, intermediate concentrations were tested to determine whether either could provide acceptable discrimination and lower Cq values ​​for low-abundance targets. In Figure 7A, the concentrations of the two salts were 45 mM (KCl) and 30 mM (ammonium sulfate), while in Figure 7B, the concentration of ammonium sulfate was reduced to 22 mM (KCl held at 45 mM). Reactions performed in the presence of 45 mM KCl and 22 mM (NH4)2SO4 (Figure 7B) reduced the Cq of the mutants while maintaining clear separation of the mutants from the wild-type polynucleotide. The average Cq for wells with mutant spikes decreased from 35.1 (Figure 7A, for reactions using 30 mM ammonium sulfate) to 28.0 (Figure 7B, for reactions using 22 mM ammonium sulfate), while maintaining separation from wells containing only wild-type. Based on these experiments, the use of 45 mM KCl and 22 mM ammonium sulfate was selected as optimal for further studies using similar conditions.

[0089] Example 3 Effect of target variant nucleotide distance from the 3' end of the probe The effect of the distance of the target variable nucleotide from either end of the first oligonucleotide (e.g., target sequence-specific primer (TSP)) and with different numbers of overhanging bases that do not bind to the target nucleic acid was evaluated. Experiments were performed in 20 μL reactions containing 1 mM dNTP, 45 mM KCl, 22 mM ammonium sulfate, 0.085 U / μL Platinum Taq, 2.55 mM MgCl2, 45 nM ROX passive reference, 39 mM Tris pH 8 and 7% glycerol, 300 nM of each primer (TSP and LSP), 250 nM of one of probes 1, 2, or 3, 10 ng of wild-type (CEPH) DNA (Thermo Fisher Scientific Catalog No. 403962), and 10 pg of EGFR L858R Reference Standard DNA (Horizon Discovery Ltd., Catalog No. HD254). The data shown in Figure 8 demonstrate that effective amplification and real-time detection was achieved with target variant nucleotides located more than two bases from the end of the probe (i.e., at 3, 4, or 5 nucleotides from the 3' end for probes 1, 2, and 3, respectively). Increasing the distance of the target variant nucleotide 5 bases from the 3' end (probe 3) compared to 3 bases from the 3' end (probe 1) resulted in a decrease in the cycle number at which the change in fluorescence of the 6-FAM reporter dye on the probe divided by the passive reference dye present in the reaction exceeded a cutoff of 0.1.

[0090] Example 4 Titration of mutant DNA into wild-type DNA A. Detection of target nucleic acid using different primer concentrations These experiments were performed in 20 uL reactions containing 10 ng of wild-type (CEPH) DNA (Thermo Fisher Scientific catalog number 403062) and the indicated amount of mutant (Horizon Discover Ltd., catalog number HD574 (NRAS Q61R) (spiked-in), 1 mM dNTPs, 45 mM KCl, 22 mM ammonium sulfate, 0.085 U / μL Platinum Taq, 2.55 mM MgCl, 45 nM ROX passive reference, 39 mM Tris pH 8, and 7% glycerol. Reactions indicated with "300 nM primers" contained 10 ng of wild-type (CEPH) DNA (Thermo Fisher Scientific catalog number 403062) and the indicated amount of mutant and control target (NRAS Reactions labeled "450 nM primers" contained 300 nM forward and reverse primers (TSP and LSP) for both the mutant and control targets, while reactions labeled "450 nM primers" contained 450 nM forward and reverse primers (TSP and LSP) for both the mutant and control targets. In all cases, the FAM-labeled (mutant) and VIC-labeled (RPPH1) probe concentrations were 250 nM. The Q61R mutant target was added to the reactions at the indicated concentrations (e.g., 250 copies, 125 copies, 62.5 copies, 31 copies, 16 copies, 8 copies, 4 copies, and 2 copies). A 50 fM solution of the mutant template (30,000 copies / uL) was diluted to 3,000 copies / uL and then to 250 copies / uL. From there, two-fold dilutions were performed down to 2 copies / uL. Reactions were amplified on a QuantStudio7 instrument using the following thermal protocol: 95°C (3 min), 19 cycles of 95°C (3 s) / 64°C (20 s), then 40 cycles of 95°C (3 s) / 60°C (20 s). Data were exported to Excel, and the Cq values ​​of the replicate reactions were averaged to determine and plot the delta average Cq of the target for each condition (Figure 10). The delta Cq was used for quantification.As shown in Figures 9 and 10 , including either 300 nM or 450 nM primers in combination with a lower number of target nucleic acids (i.e., 2 or 16 copies) demonstrated discrimination between the endogenous control target, RPPH1, and the mutant target (e.g., Figures 9C, 9E, and 9F).

[0091] B. Detection of target nucleic acid at different copy numbers For these experiments, a 20 uL reaction mixture was prepared containing 300 nM of each primer (TSP and LSP), 250 nM of probe, 10 ng of wild-type (CEPH) DNA (Thermo Fisher Scientific catalog number 403062), and the indicated amount and type of mutant (Horizon Discover Ltd., catalog number HD574 (NRAS Q61R) or HD351 (NRAS Q61K) (spiked-in), 1 mM dNTPs, 45 mM KCl, 22 mM ammonium sulfate, 0.085 U / μL Platinum Taq, 2.55 mM MgCl, 45 nM ROX passive reference, 39 mM Tris pH 8, and 7% glycerol. Data were exported in Excel format, and Cq values ​​of replicate reactions were averaged. The delta average Cq of the target in each condition was determined and plotted (data not shown). Delta Cq was used as the quantification method. Quant Studio The thermal cycling conditions on 7 were 95°C (3 min), 19 cycles of 95°C (3 sec) / 64°C (20 sec), and 40 cycles of 95°C (3 sec) / 60°C (20 sec). As shown in Figure 11 (NRAS Q61R) and Figure 12 (NRAS Q61K), as few as 3 and 4 copies of the target nucleic acid could be detected using the system described herein.

[0092] C. Reaction with different amounts of wild-type DNA To perform these experiments, reaction mixtures containing 300 nM KRAS forward and reverse (TSP and LSP) primers, 150 nM RPPH1 forward and reverse primers, 250 nM KRAS FAM probe, 150 nM RPPH1 VIC probe, 1 mM dNTPs, 45 mM KCl, 22 mM ammonium sulfate, 0.085 U / µL Platinum Taq, 2.55 mM MgCl2, 45 nM ROX passive reference, 39 mM Tris pH 8, and 7% glycerol were prepared. 10 pg of KRAS G12R Reference Standard DNA (Horizon Discovery Ltd., catalog no. HD287) and the indicated amount of wild-type (CEPH) DNA were added to each reaction. Reactions were run on a QuantStudio 5 using the following thermal protocol: 95°C (2 min), 19 cycles of 95°C (1 sec) / 64°C (20 sec), then 40 cycles of 95°C (1 sec) / 60°C (20 sec). As shown in Figures 13A-C, mutant (e.g., lower abundance) target nucleic acids could be detected in a background of 5, 10, and 20 ng of wild-type (e.g., higher abundance) DNA.

[0093] Example 5 Target site at the 3' end of the reverse primer To perform these experiments, a 20 μL reaction mixture containing 10 ng of CEPH DNA (Thermo Fisher Scientific catalog number 403062), 300 nM of each primer (TSP and LSP), 250 nM of probe, 10 pg of mutant spike of KRAS G12D or G12S Reference Standard DNA (Horizon Discovery Ltd., catalog number HD272 or HD288, respectively) was utilized as indicated, 1 mM dNTPs, 45 mM KCl, 22 mM ammonium sulfate, 0.085 U / μL Platinum Taq, 2.55 mM MgCl, 45 nM ROX passive reference, 39 mM Tris pH 8, and 7% glycerol was prepared. The thermal cycling conditions were 95°C (3 min), 95°C (3 s), 64°C (20 s) for 19 cycles, followed by 40 cycles of 95°C (3 s) / 60°C (20 s). As shown in Figure 14 (KRAS G12D) and Figure 15 (KRAS G12S), the reverse primer can be a target-specific primer (TSP) (e.g., used for hybridization to either strand of a double-stranded polynucleotide. The target site can be located at the 3' end of either the forward or reverse TSP primer). The system described herein functions well, demonstrating discrimination between low-abundance (e.g., mutant) and high-abundance (e.g., wild-type) targets.

[0094] Example 6 Additional genes and rare variants To perform these experiments, a 20 μL reaction mixture containing 10 ng of wild-type (CEPH) DNA (Thermo Fisher Scientific catalog number 403062), 300 nM each of first and second oligonucleotides (e.g., target sequence-specific primer (TSP) and locus-specific primer (LSP)), 250 nM of probe (third oligonucleotide, target site-specific probe), 10 pg of the corresponding mutant DNA spike (e.g., mutant EGFR, BRAF, KRAS as shown in Figures 16A-16F; see Table 2) (Horizon Discovery Ltd., Reference Standards catalog number as provided elsewhere herein) was used for the mutant samples, 1 mM dNTPs, 45 mM KCl, 22 mM ammonium sulfate, 0.085 U / μL Platinum Taq, 2.55 mM MgCl, 45 nM ROX passive reference, 39 mM Tris pH 8, and 7% glycerol was prepared. The data were exported in Excel format, the Cq values ​​of the replicate reactions were averaged, and the delta average Cq of the target under each condition was determined and plotted (data not shown). Delta Cq was used as the quantification method. The thermal cycling conditions used were 95°C (3 min), 19 cycles of 95°C (3 s) / 64°C (20 s), followed by 40 cycles of 95°C (3 s) / 60°C (20 s). As shown in Figures 16A-16F, each of the various target mutant nucleic acids tested, such as those listed in Table 2, was clearly distinguished from the wild-type nucleic acid.

[0095] Example 7 Reaction with probes of different lengths To perform these experiments, a 20 μL reaction mixture was prepared containing 300 nM of each primer, 250 nM of the appropriate probe, 10 ng of wild-type (CEPH) DNA (Thermo Fisher Scientific catalog number 403062), 20 pg of mutant spike of NRAS Q61L or Q61H Reference Standard DNA as indicated (Horizon Discovery Ltd., catalog number HD412 or HD303, respectively), 1 mM dNTPs, 45 mM KCl, 22 mM ammonium sulfate, 0.085 U / μL Platinum Taq, 2.55 mM MgCl, 45 nM ROX passive reference, 39 mM Tris pH 8, and 7% glycerol. Reactions were run on a QuantStudio 5 using the following thermal protocol: 95°C (2 min), 19 cycles of 95°C (1 sec) / 64°C (20 sec), then 40 cycles of 95°C (1 sec) / 60°C (20 sec). As shown in Figure 17, varying probe length by up to 5 nucleotides had little effect on the ability to detect low-abundance targets in these experiments, although in some other cases, probe length was observed to decrease Cq values ​​(data not shown). Figure 17A demonstrates the detection of NRAS Q61L (182A>T) using two different probes with lengths of either 16 or 21 nucleotides. Figure 17B demonstrates the detection of NRAS Q61H (183A>T) using two different probes with lengths of either 15 or 20 nucleotides.

[0096] Example 8 Various mutant targets tested When target reference material was not commercially available, an artificial double-stranded DNA template was used. A 300-bp artificial dsDNA fragment with the indicated mutation was ordered from Thermo Fisher Scientific (GeneArt Strings DNA Fragments, catalog number 815010DE). The dsDNA fragment was diluted to 50 fM (30,000 copies / uL) based on the assay information provided with each dsDNA product. Subsequent dilutions were made as needed for addition to individual qPCR reactions. Each 20-uL reaction also contained 10 ng of wild-type (CEPH) DNA. In addition to wild-type DNA (10 ng of CEPH) and artificial template, 20 μL reactions were prepared containing 300 nM each of first and second oligonucleotides (e.g., target sequence-specific primer (TSP) and locus-specific primer (LSP) for either the indicated mutant target or RPPH1 control target), 250 nM differentially labeled probe for either the indicated mutant target or RPPH1 control target, 1.3 mM dNTPs, 45 mM KCl, 22 mM ammonium sulfate, 0.175 U / μL Platinum Taq, 2.85 mM MgCl, 45 nM ROX passive reference, 40 mM Tris, pH 8, and 7% glycerol. For duplicate reactions containing probes for both mutant and control targets, data were exported to Excel format, Cq values ​​for the duplicate reactions were averaged, and the delta-average Cq for the target in each condition was determined and plotted (data not shown). The delta Cq between the indicated mutant target and the RPPH1 target was used as the quantification method. The thermal cycling conditions used were 95°C (3 min), 19 cycles of 95°C (3 s) / 64°C (20 s), followed by 40 cycles of 95°C (3 s) / 60°C (20 s).

[0097] As shown in Figures 18A-18C and Figures 19A and 19B, numerous artificial mutant targets, such as those listed in Table 2, were detected using the methods described herein.

[0098] The foregoing examples illustrate various aspects of the present invention and the practice of the methods of the present invention. The examples are not intended to provide an exhaustive description of the many different embodiments of the present invention. Thus, while the foregoing invention has been described in some detail by way of illustration and example for clarity of understanding, those skilled in the art will readily appreciate that many changes and modifications can be made thereto without departing from the spirit or scope of the following section and appended claims.

[0099] Additional embodiments may follow the numbered paragraphs below. 1. a) a first oligonucleotide configured to hybridize to a first sequence in a first target polynucleotide strand, the first sequence comprising a target variant nucleotide, the first oligonucleotide further having a nucleotide residue at its 3' end positioned to hybridize to the target variant nucleotide; b) a second oligonucleotide having a sequence configured to hybridize to a sequence complementary to a second sequence in the first target polynucleotide strand, wherein the second sequence of the first target polynucleotide strand is located 5' upstream from the first sequence of the first target polynucleotide strand. 2. c) a third oligonucleotide having a sequence configured to hybridize to a sequence complementary to a third sequence in the first target polynucleotide strand, wherein the third sequence of the first target polynucleotide strand at least partially overlaps with the first sequence of the first target polynucleotide strand, and the third sequence comprises a target variant nucleotide. 3. a) a first oligonucleotide configured to hybridize to a first sequence (A) present in a first target polynucleotide strand, the first sequence comprising a target variant nucleotide ("first variant nucleotide"), the first oligonucleotide further having a nucleotide residue at its 3' end positioned to hybridize to the first variant nucleotide; b) a second oligonucleotide configured to hybridize to a second sequence (B), wherein the second sequence is complementary to a third sequence (C), wherein the third sequence is present within the first target polynucleotide strand, and the third sequence (C) is located 5' upstream from the first sequence (A) of the first target polynucleotide strand. 4. c) the mixture of paragraph 2, further comprising a third oligonucleotide configured to hybridize to a fourth sequence (D) complementary to a fifth sequence (E), wherein the fifth sequence is present in the first target polynucleotide strand, the fifth sequence (E) at least partially overlaps with the first sequence (A) in the first target polynucleotide strand, and the third oligonucleotide comprises a first target variant nucleotide. 5. The mixture of paragraph 2 or 4, wherein the target variant nucleotide in the third oligonucleotide is at least two nucleotides from the 3' or 5' end of the third oligonucleotide. 6. The mixture of any one of paragraphs 2, 4, and 5, wherein the third oligonucleotide comprises a detectable label. 7. The mixture according to paragraph 6, wherein the detectable label is a fluorescent label. 8. The mixture of paragraph 6 or 7, wherein the detectable label is on the first terminal nucleotide. 9. The mixture of any one of paragraphs 6 to 8, wherein the third oligonucleotide further comprises a quenching moiety. 10. The mixture of paragraph 9, wherein the quenching moiety is on the second terminal nucleotide of the third oligonucleotide. 11. The mixture of paragraph 9 or 10, wherein the quenching moiety is capable of quenching a signal from a detectable label. 12. The mixture according to paragraph 10, wherein the first terminal nucleotide is the 5' terminal nucleotide. 13. The mixture of paragraph 10, wherein the second terminal nucleotide is the 3' terminal nucleotide. 14. The mixture of paragraph 2 or 4, wherein the mixture comprises a single-stranded polynucleotide molecule comprising a first target polynucleotide strand. 15. The mixture of item 4, wherein sequences A, E, and C are located within a single-stranded polynucleotide molecule on the first target polynucleotide strand. 16. The mixture of paragraph 2 or 4, wherein the mixture comprises double-stranded polynucleotide molecules comprising a first target polynucleotide strand and a first target complement polynucleotide strand, and the first target complement polynucleotide strand is substantially complementary to the first target polynucleotide strand. 17. The mixture of paragraph 4, wherein the mixture comprises double-stranded polynucleotide molecules comprising a first target polynucleotide strand and a first target complement polynucleotide strand, wherein the first target complement polynucleotide strand is substantially complementary to the first target polynucleotide strand, and wherein sequences D and B are located within the double-stranded polynucleotide molecule on the target complement polynucleotide strand, and sequences A, E, and C are located within the double-stranded polynucleotide molecule on the target polynucleotide strand. 18. The mixture of paragraph 2 or 4, wherein the mixture comprises double-stranded polynucleotide molecules comprising a first target polynucleotide strand and a first target complement polynucleotide strand, wherein the first target complement polynucleotide strand is substantially complementary to the first target polynucleotide strand, the double-stranded polynucleotide molecules comprise a variant polynucleotide strand and a variant complement polynucleotide strand, wherein the variant polynucleotide strand is substantially identical to the target polynucleotide strand and comprises a nucleotide that differs from the target polynucleotide strand at the target variant nucleotide, and the variant complement polynucleotide strand is substantially complementary to the variant polynucleotide strand. 19. The mixture according to item 2 or 4, wherein the third oligonucleotide comprises 3 to 6 consecutive nucleotides of the first sequence. 20. The mixture of paragraph 2 or 4, wherein the third oligonucleotide further comprises a sequence of nucleotides of the first target polynucleotide strand that does not overlap with the sequence of nucleotides of the first sequence. 21. The mixture of paragraph 2 or 4, wherein the mixture does not contain a fourth oligonucleotide. 22. The mixture of paragraph 2 or 4, wherein the mixture does not include a fourth oligonucleotide comprising a detectable label. 23. The mixture of paragraph 2 or 4, wherein the mixture does not contain a fourth oligonucleotide that binds to the first target polynucleotide strand. 24. The mixture of paragraph 2 or 4, wherein the mixture does not contain a fourth oligonucleotide having a detectable label and a sequence that binds to the first target polynucleotide strand. 25. The mixture of paragraph 2 or 4, wherein the third oligonucleotide is the only oligonucleotide in the mixture having a detectable label and a sequence configured to hybridize to a sequence of a target polynucleotide strand. 26. The mixture according to item 2 or 4, wherein the mixture further contains 30 mM to 80 mM potassium chloride. 27. The mixture according to item 26, wherein the concentration of potassium chloride is at least 40 mM. 28. The mixture described in item 26, wherein the potassium chloride concentration is less than 70 mM. 29. The mixture of item 26, wherein the concentration of potassium chloride is at least 40 mM and less than 70 mM. 30. The mixture according to item 2 or 4, wherein the mixture further contains 10 mM to 40 mM ammonium sulfate. 31. The mixture according to item 30, wherein the concentration of ammonium sulfate is at least 20 mM. 32. The mixture according to item 30, wherein the concentration of ammonium sulfate is less than 35 mM. 33. The mixture of paragraph 30, wherein the concentration of ammonium sulfate is at least 20 mM and less than 35 mM. 34. The mixture according to item 30, wherein the concentration of ammonium sulfate is 20 to 25 mM. 35. The mixture is a) a potassium chloride concentration of 30 mM to 80 mM; b) The mixture according to item 2 or 4, further comprising an ammonium sulfate concentration of 10 mM to 40 mM. 36. The mixture according to item 35, wherein the concentration of potassium chloride is 40 mM to 70 mM and the concentration of ammonium sulfate is 20 mM to 35 mM. 37. The mixture according to item 35, wherein the concentration of potassium chloride is 40 mM to 48 mM and the concentration of ammonium sulfate is 20 to 24 mM. 38. The mixture according to item 35, wherein the concentration of potassium chloride is 45 mM and the concentration of ammonium sulfate is 22 mM. 39. The detectable label is a DNA binding dye, a reporter dye, a fluorescent probe, 6-carboxyfluorescein (FAM™), tetrachlorofluorescin (TET™), 6-carboxy-4',5'-dichloro-2',7'-dimethoxyfluorescein, succinimidyl ester (JOE™), VIC™, a sulfonate derivative of a fluorescein dye containing SO3 instead of the carboxylate group, a phosphoramidite form of fluorescein, CY5 Item 7. The mixture of item 6, wherein the fluorophore is selected from the group consisting of a phosphoramidite form of fluorophore, a non-FRET label, a ferrocene reagent, ABY™, NED™, JUN™, Fluor™ 488, AlexaFluor™ 532, AlexaFluor™ 546, AlexaFluor™ 594, AlexaFluor™ 647, AlexaFluor™ 660, TYE™ 563, TYE™ 665, TYE™ 705, and combinations thereof. 40. The mixture of any one of paragraphs 9 to 11, wherein the quenching moiety is selected from the group consisting of tetramethylrhodamine (TAMRA), non-fluorescent quenchers (NFQs), black hole quenchers, Iowa black quenchers, QSY quenchers, QSY7 quenchers, QSY21 quenchers, dabcyl and / or dabcyl sulfonate / carboxylate quenchers. 41. T of the third oligonucleotide m is the T of the first oligonucleotide m Item 5. The mixture according to item 2 or 4, wherein the temperature is at least 5°C and not more than 25°C higher than the temperature of the mixture according to item 3. 42. T of the third oligonucleotide m is the T of the first oligonucleotide m 42. The mixture according to claim 41, wherein the temperature is at least 8°C and not more than 12°C higher than the reference temperature. 43. T of the first oligonucleotide m is the T of the second oligonucleotide m Item 5. The mixture according to item 2 or 4, wherein the temperature is within 5°C of 44. T of the first oligonucleotide m is 45 to 60°C, and the T of the second oligonucleotide m Item 44. The mixture according to any one of items 42 and 43, wherein the temperature is 45 to 60°C. 45. The mixture of any one of paragraphs 1 to 4, wherein the first and second oligonucleotides are extendable. 46. ​​The mixture according to any one of paragraphs 1 to 4, wherein the first and second oligonucleotides are primers. 47. The mixture of paragraph 2 or 4, wherein the third oligonucleotide is non-extendable. 48. The mixture of paragraph 2 or 4, wherein the third oligonucleotide is a probe. 49. The mixture of paragraph 2 or 4, wherein the first, second, and / or third oligonucleotide comprises 10 to 40 nucleotides. 50. The mixture of paragraph 2 or 4, wherein the third oligonucleotide comprises a blocking moiety. 51. The mixture of paragraph 50, wherein the blocking moiety is a minor groove binding agent (MGB) moiety. 52. The mixture according to paragraph 51, wherein the MGB portion is located at the 3' end and / or at the 3' portion of the third oligonucleotide. 53. The mixture is a) a fourth oligonucleotide configured to hybridize to a first sequence in a second target polynucleotide strand, wherein the first sequence in the second target polynucleotide comprises a second target variant nucleotide, and the fourth oligonucleotide further comprises a nucleotide residue at its 3' end positioned to hybridize to the second target variant nucleotide; b) a fifth oligonucleotide having a sequence configured to hybridize to a sequence complementary to a second sequence in a second target polynucleotide strand, wherein the second sequence of the second target polynucleotide strand is located 5' upstream from the first sequence on the second target polynucleotide strand; and c) a sixth oligonucleotide having a sequence configured to hybridize to a sequence complementary to a third sequence in the second target polynucleotide strand, wherein the third sequence of the second target polynucleotide strand at least partially overlaps with the first sequence on the second target polynucleotide strand and comprises a second target variant nucleotide. 54. The mixture is a) a fourth oligonucleotide configured to hybridize to a first sequence (F) in a second target polynucleotide strand, wherein the first sequence in the second target polynucleotide comprises a second target variant nucleotide ("second variant nucleotide"), and wherein the fourth oligonucleotide further has a nucleotide residue at its 3' end that is positioned to hybridize to the second target variant nucleotide; b) a fifth oligonucleotide configured to hybridize to a second sequence (G) in a second target polynucleotide strand, wherein the second sequence of the second target polynucleotide strand is complementary to a third sequence (H) of the second target polynucleotide strand, the third sequence of the target polynucleotide strand being present within the second target polynucleotide strand, and the third sequence (H) being located 5' upstream from the first sequence (F) of the second target polynucleotide strand; c) a sixth oligonucleotide configured to hybridize to a fourth sequence (I) of the second target polynucleotide strand, wherein the fourth sequence (I) is complementary to a fifth sequence (J) of the second target polynucleotide strand, the fifth sequence (J) being present in the second target polynucleotide strand, the fifth sequence (J) at least partially overlapping with the first sequence (F) in the second target polynucleotide strand, and comprising a second target variant nucleotide. 55. The mixture of paragraph 53 or 54, wherein the target variant nucleotide in the sixth oligonucleotide is at least two nucleotides from the 3' or 5' end of the sixth oligonucleotide. 56. The mixture of paragraph 53, 54, or 55, wherein the sixth oligonucleotide comprises a detectable label. 57. The mixture of paragraph 56, wherein the detectable label of the sixth oligonucleotide is a fluorescent label. 58. The mixture of paragraph 56 or 57, wherein the detectable label of the sixth oligonucleotide is on the first terminal nucleotide. 59. The mixture of any one of paragraphs 53, 54, or 56, wherein the sixth oligonucleotide further comprises a quenching moiety. 60. The mixture of paragraph 59, wherein the quenching moiety of the sixth oligonucleotide is on the second terminal nucleotide of the sixth oligonucleotide. 61. The mixture of paragraph 59 or 60, wherein the quenching moiety of the sixth oligonucleotide is capable of quenching a signal from the detectable label of the sixth oligonucleotide. 62. The mixture of paragraph 53 or 54, wherein the fourth and fifth oligonucleotides are extendable. 63. The mixture of paragraph 53 or 54, wherein the fourth and fifth oligonucleotides are primers. 64. The mixture of paragraph 53 or 54, wherein the sixth oligonucleotide is non-extendable. 65. The mixture of paragraph 53 or 54, wherein the sixth oligonucleotide is a probe. 66. The mixture of paragraph 53 or 54, wherein the fourth, fifth, and / or sixth oligonucleotides comprise 10 to 30 nucleotides. 67. The mixture of paragraph 53 or 54, wherein the sixth oligonucleotide comprises a blocking moiety. 68. The mixture of paragraph 67, wherein the blocking moiety of the sixth oligonucleotide is a minor groove binder (MGB) moiety. 69. The mixture of paragraph 68, wherein the MGB portion of the sixth oligonucleotide is located at the 3' end and / or 3' portion of the sixth oligonucleotide. 70. The mixture is a) a fourth oligonucleotide having a sequence configured to hybridize to a sequence complementary to the first oligonucleotide, wherein the fourth oligonucleotide is configured to hybridize substantially to the first sequence and includes a nucleotide at its 3' end that differs from the complement of the target variant nucleotide; b) a fifth oligonucleotide having a sequence configured to hybridize to a sequence complementary to the third oligonucleotide, wherein the fifth oligonucleotide comprises a different nucleotide at the position of the target variant nucleotide. 71. The mixture of paragraph 70, wherein the different nucleotides are at least two nucleotides from the 3' end. 72. The mixture of paragraph 70 or 71, wherein the fifth oligonucleotide comprises a detectable label. 73. The mixture of paragraph 72, wherein the detectable label of the fifth oligonucleotide is a fluorescent label. 74. The mixture of paragraph 72 or 73, wherein the detectable label of the fifth oligonucleotide is on the first terminal nucleotide of the fifth oligonucleotide. 74. The mixture of any one of paragraphs 72 to 74, wherein the detectable label on the fifth oligonucleotide is distinguishable from the detectable label on the third oligonucleotide. 75. The mixture of any one of paragraphs 72 to 74, wherein the fifth oligonucleotide further comprises a quenching moiety. 76. The mixture of paragraph 75, wherein the quenching moiety of the fifth oligonucleotide is on the second terminal nucleotide of the fifth oligonucleotide. 77. The mixture of paragraph 75 or 76, wherein the quenching moiety of the fifth oligonucleotide is capable of quenching a signal from the detectable label of the fifth oligonucleotide. 78. The mixture of paragraph 70, wherein the fourth oligonucleotide is extendible. 79. The mixture of paragraph 70, wherein the fourth oligonucleotide is a primer. 80. The mixture of paragraph 70, wherein the fifth oligonucleotide is non-extendable. 81. The mixture of paragraph 70, wherein the fifth oligonucleotide is a probe. 82. The mixture according to paragraph 70, wherein the fourth and fifth oligonucleotides contain 10 to 40 nucleotides. 83. The mixture of paragraph 70, wherein the fifth oligonucleotide comprises a blocking moiety. 84. The mixture of paragraph 83, wherein the blocking moiety of the fifth oligonucleotide is a minor groove binder (MGB) moiety. 85. The mixture of paragraph 84, wherein the MGB portion of the fifth oligonucleotide is located at the 3' end and / or 3' portion of the fifth oligonucleotide. 86. A mixture comprising: A mixture comprising a plurality of oligonucleotide sets, each oligonucleotide set comprising: a) a first oligonucleotide configured to hybridize to a first sequence in a first target polynucleotide strand, the first sequence comprising a target variant nucleotide, the first oligonucleotide further having a nucleotide residue at its 3' end positioned to hybridize to the target variant nucleotide; b) a second oligonucleotide having a sequence configured to hybridize to a sequence complementary to a second sequence in the first target polynucleotide strand, wherein the second sequence of the first target polynucleotide strand is located 5' upstream from the first sequence of the first target polynucleotide strand; A mixture wherein the first oligonucleotides of each set are configured to hybridize to a different first sequence. 87. Each oligonucleotide set is: c) a third oligonucleotide having a sequence configured to hybridize to a sequence complementary to a third sequence in the first target polynucleotide strand, wherein the third sequence of the first target polynucleotide strand at least partially overlaps with the first sequence of the first target polynucleotide strand, and the third sequence comprises a target variant nucleotide; the third oligonucleotides of each set share sequence similarity with a different third sequence; 87. The mixture of paragraph 86, wherein the third oligonucleotide of each set comprises a different distinguishable detectable label. 88. A mixture comprising a plurality of oligonucleotide sets, each oligonucleotide set comprising: a) a first oligonucleotide configured to hybridize to a first sequence (A) present in a first target polynucleotide strand, the first sequence comprising a target variant nucleotide ("first variant nucleotide"), the first oligonucleotide further having a nucleotide residue at its 3' end positioned to hybridize to the first variant nucleotide; b) a second oligonucleotide configured to hybridize to a second sequence (B), wherein the second sequence is complementary to a third sequence (C), wherein the third sequence is present within the first target polynucleotide strand, and the third sequence (C) is located 5' upstream from the first sequence (A) of the first target polynucleotide strand; A mixture wherein the first oligonucleotides of each set are configured to hybridize to a different first sequence. 89. Each oligonucleotide set is c) a third oligonucleotide configured to hybridize to a fourth sequence (D) complementary to a fifth sequence (E), wherein the fifth sequence is present in the first target polynucleotide strand, the fifth sequence (E) at least partially overlaps with the first sequence (A) in the first target polynucleotide strand and comprises a first target variant nucleotide; the third oligonucleotides of each set share sequence similarity with a different third sequence; 89. The mixture of paragraph 88, wherein the third oligonucleotide of each set comprises a different distinguishable detectable label. 90. The mixture of any one of paragraphs 1 to 89, wherein the mixture further comprises a polymerase. 91. The mixture of paragraph 90, wherein the polymerase is thermostable. 92. The mixture of paragraph 91, wherein the mixture further comprises a hot start component. 93. The mixture of paragraph 92, wherein the hot-start component comprises an antibody, an oligonucleotide, an aptamer directed against a thermostable polymerase, and / or a chemical modification of the polymerase. 94. The mixture of any one of paragraphs 1 to 93, wherein the mixture further comprises a source of nucleotides. 95. The mixture of any one of paragraphs 1 to 94, wherein the third oligonucleotide is a hydrolysis probe. 96. The mixture of any one of paragraphs 1 to 95, further comprising a nucleic acid sample suspected of containing the first target polynucleotide strand. 97. The mixture of any one of paragraphs 1 to 96, wherein the target variant nucleotide is in a mutant allele. 98. The mixture of paragraph 97, wherein the mutant allele is either a purine-to-purine single point mutation or a pyrimidine-to-pyrimidine single point mutation at the target variant nucleotide. 99. The mixture of paragraph 97 or 98, wherein the mutant allele is an allele of the KRAS oncogene. 100. The mixture of paragraph 97, wherein the mutant allele is a stochastic mutation. 101. The mixture of any one of paragraphs 1 to 100, wherein the target variant nucleotide has an identity corresponding to a major allelic variant or a minor allelic variant. 102. The mixture of paragraph 101, wherein the target variant nucleotide has the identity of a minor allele variant having a minor allele frequency (MAF) of less than 1%. 103. The mixture of paragraph 102, wherein the target variant nucleotide has a minor allelic variant identity with a minor allele frequency (MAF) of less than 0.1%, less than 0.01%, or less than 0.001%. 104. The mixture of any one of paragraphs 1 to 103, wherein the target variant nucleotide occurs at a single nucleotide polymorphism position. 105. The mixture of any one of paragraphs 1 to 95, wherein the mixture is a master mix. 106. The mixture according to paragraph 105, wherein the master mix is ​​stable at 4-8°C for up to 12 months. 107. The mixture according to any one of paragraphs 1 to 104, wherein the mixture is a reaction mixture. 108. The mixture of paragraph 107, wherein the reaction mixture further comprises an amplicon comprising a first sequence of the first target polynucleotide strand. 109. The mixture of paragraph 108, wherein the reaction mixture does not contain amplicons comprising the sequence of any other different (e.g., second) polynucleotide strand. 110. A mixture of: a) at least one cleaning agent; b) glycerol, and c) The mixture according to any one of paragraphs 1 to 109, further comprising one or more of at least one reference dye. 111. The mixture according to any one of paragraphs 1 to 110, wherein the mixture further comprises bovine serum albumin and / or gelatin. 112. The mixture according to any one of paragraphs 1 to 106, wherein the mixture is freeze-dried. 113. A kit comprising: a first container containing a mixture described in any preceding paragraph; and a second container containing a control polynucleotide sample comprising polynucleotide molecules that share sequence similarity with a first target polynucleotide strand. 114. The kit of paragraph 113, wherein the control polynucleotide sample comprises the entire first target polynucleotide strand. 115. The kit of paragraph 113, wherein the control polynucleotide sample shares no sequence similarity with the first target polynucleotide strand at the target variant nucleotide. 116. A method for detecting a target polynucleotide molecule comprising a target variant nucleotide in a test polynucleotide sample, the method comprising: a) forming a reaction mixture of a test polynucleotide sample and the mixture described in item 2 or 4; b) performing an amplification reaction using at least the first and second oligonucleotides as primers to generate an amplicon of the target polynucleotide sequence of the target polynucleotide molecule, if present in the test polynucleotide sample; c) detecting the amplicons produced in step b) by detecting a change in the detectable property of the third oligonucleotide; The method wherein detecting the amplicon in step c) indicates the presence of the target polynucleotide molecule in the test polynucleotide sample. 117. The method of paragraph 116, wherein the target polynucleotide molecule is detected in a test polynucleotide sample comprising a mixture of polynucleotide molecules, the mixture comprising a polynucleotide molecule comprising a first variant form of the target variant nucleotide ("first variant target polynucleotide molecule"), and a polynucleotide molecule comprising a second variant form of the target variant nucleotide ("second variant target polynucleotide molecule"). 118. The method of paragraph 116 or 117, wherein the test polynucleotide sample comprises polynucleotide strands that do not contain target polynucleotide sequences ("non-target polynucleotide molecules"). 119. The method of paragraph 118, wherein the test sample contains more non-target polynucleotide molecules than target polynucleotide molecules. 120. The method of any one of paragraphs 116 to 119, wherein the target polynucleotide molecule is a mutant polynucleotide sequence. 121. The method of any one of paragraphs 118 to 120, wherein the non-target polynucleotide molecule is a major allele or a wild-type polynucleotide sequence. 122. The method of any one of paragraphs 118 to 121, wherein the test sample contains less than 1%, less than 0.1%, less than 0.01%, or less than 0.001% of target polynucleotide molecules compared to non-target polynucleotide molecules. 123. The method of any one of paragraphs 117-119, wherein the test sample contains less than 1%, less than 0.1%, less than 0.01%, or less than 0.001% of the second variant target polynucleotide molecules compared to the first variant target polynucleotide molecules. 124. The method of any one of paragraphs 117-119, wherein the first variant target polynucleotide molecule and / or the second variant target polynucleotide molecule is a mutant polynucleotide sequence. 125. The method of any one of paragraphs 117-119, wherein the first variant target polynucleotide molecule and / or the second variant target polynucleotide molecule is a wild-type polynucleotide sequence. 126. The method of paragraphs 117-125, further comprising enriching the number of first variant polynucleotide molecules in the polynucleotide test sample relative to the second variant polynucleotide molecules prior to steps a)-c). 127. The method of paragraph 126, wherein enriching comprises an amplification reaction comprising different conditions compared to those used in any of steps a) to c). 128. The method of paragraph 126 or 127, wherein the number of first variant polynucleotide molecules in the polynucleotide test sample is enriched by at least 2-fold, 4-fold, 6-fold, 8-fold, or 10-fold compared to the second variant polynucleotide molecules. 129. Enrichment is performed by determining whether the calculated melting temperature (T M 129. The method of any one of paragraphs 126 to 128, wherein the method is carried out using a polymerase chain reaction comprising 15 to 25 cycles at a temperature 12 to 16 degrees higher than the temperature of the reaction mixture. 130. Step b) is a step of converting the T M 130. The method of any one of paragraphs 126 to 129, wherein the method is carried out using 35 to 40 cycles at a temperature close to the temperature at which enrichment is carried out and 4 to 6 degrees lower than the temperature at which enrichment is carried out. 131. The method of any one of paragraphs 116-130, wherein the test polynucleotide sample is derived from mammalian or non-mammalian animal tissue or cells, or plant tissue or cells. 132. The method of paragraph 131, wherein the sample is selected from the group consisting of saliva, buccal tissue, skin, hair, blood, plasma, urine, feces, semen, and a tumor sample. 133. The method of paragraph 131 or 132, wherein the polynucleotide test sample is derived from a cancer cell. 134. The method of any one of paragraphs 131-133, wherein detection of the amplicon indicates the presence of cancer cells in the tissue from which the test polynucleotide sample is derived. 135. The method of any one of paragraphs 116 to 134, wherein the target polynucleotide comprises at least one mutation in Ras, EFGR, Kit, pTEN, and / or p53, and / or at least one KRAS or NRAS mutation. 136. The method of any one of clauses 116 to 135, wherein the amplification reaction is a polymerase chain reaction (PCR). 137. The method of clause 136, wherein the PCR is real-time PCR or quantitative PCR (qPCR). 138. The method of paragraph 136 or 137, wherein the third oligonucleotide has a Tm that is 8-12°C higher than the Tm of the first oligonucleotide, and PCR is performed at an annealing temperature that is within 5°C of the Tm of the first oligonucleotide. 139. The method of any one of paragraphs 116 to 138, wherein the method is carried out using a kit comprising a first container containing at least a first oligonucleotide, a second oligonucleotide, and a third oligonucleotide, and a second container containing a control polynucleotide sample comprising the first target polynucleotide strand. 140. The method of any one of paragraphs 116-139, wherein the method detects a target variant nucleotide indicative of a mutation, and the test polynucleotide sample comprises 1 to 10 copies of the target polynucleotide. 141. The method of any one of paragraphs 116 to 140, wherein the target variant nucleotide comprises a purine base and the corresponding wild-type nucleotide at the target variant nucleotide position comprises a different purine base. 142. The method of any one of paragraphs 116 to 140, wherein the target variant nucleotide comprises a pyrimidine base and the corresponding wild-type nucleotide at the target variant nucleotide position comprises a different pyrimidine base. 143. A method for detecting a target polynucleotide molecule comprising a target variant nucleotide in a test polynucleotide sample, the method comprising: a) forming a reaction mixture of a test polynucleotide sample and a mixture according to any one of paragraphs 1 to 94, 104, 105, and 109 to 111; b) performing an amplification reaction using at least the first and second oligonucleotides as primers to generate an amplicon of the target polynucleotide sequence of the target polynucleotide molecule, if present in the test polynucleotide sample; c) detecting the amplicons produced in step b) by detecting a change in the detectable property of the third oligonucleotide; The method wherein detecting the amplicon in step c) indicates the presence of the target polynucleotide molecule in the test polynucleotide sample. 144. A method for detecting at least two different target polynucleotide molecules in a test polynucleotide sample, each of which contains a target variant nucleotide, the method comprising: a) forming a reaction mixture of a test polynucleotide sample and the mixture according to paragraph 53 or 54; b) performing an amplification reaction using the first and second oligonucleotides as primers to generate an amplicon of the first target polynucleotide sequence of the first target polynucleotide molecule, if present in the test polynucleotide sample; c) performing an amplification reaction using the fourth and fifth oligonucleotides as primers to generate a second amplicon of a second target polynucleotide sequence of a second target polynucleotide molecule, if present in the test polynucleotide sample; d) detecting the amplicons produced in steps b) and c) by detecting a change in the detectable property of the third and sixth oligonucleotides; The method wherein detecting the amplicon in step d) indicates the presence of the first and / or second target polynucleotide molecule within the test polynucleotide sample. 145. The method of paragraph 144, wherein the detectable property of the third and sixth oligonucleotides is a fluorescent label. 146. The method of paragraph 145, wherein the fluorescent label on each of the third and sixth oligonucleotides is different. 147. The mixture of any one of paragraphs 1 to 112, wherein the mixture is used to detect one or more mutations listed in Table 1 and / or Table 2. 148. The kit of any one of paragraphs 113-115, wherein the kit is used for the detection of one or more mutations listed in Table 1 and / or Table 2. 149. The method of any one of paragraphs 116-146, wherein the method is used to detect one or more mutations listed in Table 1 and / or Table 2. Another aspect of the present invention may be as follows. [1] a) a first oligonucleotide configured to hybridize to a first sequence in a first target polynucleotide strand, the first sequence comprising a target variant nucleotide, the first oligonucleotide further having a nucleotide residue at its 3' end positioned to hybridize to the target variant nucleotide; b) a second oligonucleotide having a sequence configured to hybridize to a sequence complementary to a second sequence in the first target polynucleotide strand, wherein the second sequence of the first target polynucleotide strand is located 5' upstream from the first sequence of the first target polynucleotide strand. [2] The mixture described in [1], further comprising: c) a third oligonucleotide having a sequence configured to hybridize to a sequence complementary to a third sequence within the first target polynucleotide strand, wherein the third sequence of the first target polynucleotide strand at least partially overlaps with the first sequence of the first target polynucleotide strand, and the third sequence comprises the target variant nucleotide. [3] a) a first oligonucleotide configured to hybridize to a first sequence (A) present in a first target polynucleotide strand, wherein the first sequence comprises a target variant nucleotide ("first variant nucleotide"), and the first oligonucleotide further has a nucleotide residue at its 3' end positioned to hybridize to the first variant nucleotide; b) a second oligonucleotide configured to hybridize to a second sequence (B), wherein the second sequence is complementary to a third sequence (C), the third sequence being present within the first target polynucleotide strand, and the third sequence (C) being located 5' upstream of the first sequence (A) of the first target polynucleotide strand. [4] The mixture described in [2], further comprising: c) a third oligonucleotide configured to hybridize to a fourth sequence (D) complementary to a fifth sequence (E), wherein the fifth sequence is present in the first target polynucleotide strand, the fifth sequence (E) at least partially overlaps with the first sequence (A) in the first target polynucleotide strand, and the third oligonucleotide further comprises the first target variant nucleotide. [5] The mixture described in [2] or [4], wherein the target variant nucleotide in the third oligonucleotide is at least two nucleotides from the 3' end or 5' end of the third oligonucleotide. [6] The mixture described in any one of [2], [4], and [5], wherein the third oligonucleotide comprises a detectable label. [7] The mixture described in [6], wherein the detectable label is a fluorescent label. [8] The mixture described in [6] or [7], wherein the detectable label is on the first terminal nucleotide. [9] The mixture described in any one of [6] to [8] above, wherein the third oligonucleotide further comprises a quenching moiety.

[10] The mixture described in [9], wherein the quenching moiety is on the second terminal nucleotide of the third oligonucleotide.

[11] The mixture described in [9] or

[10] , wherein the quenching moiety is capable of quenching a signal from the detectable label.

[12] The mixture described in

[10] , wherein the first terminal nucleotide is a 5' terminal nucleotide.

[13] The mixture described in

[10] , wherein the second terminal nucleotide is a 3' terminal nucleotide.

[14] The mixture described in [2] or [4], wherein the mixture contains a single-stranded polynucleotide molecule comprising the first target polynucleotide strand.

[15] The mixture described in [4], wherein sequences A, E, and C are located within a single-stranded polynucleotide molecule on the first target polynucleotide strand.

[16] The mixture described in [2] or [4], wherein the mixture contains double-stranded polynucleotide molecules comprising the first target polynucleotide strand and a first target complementary polynucleotide strand, and the first target complementary polynucleotide strand is substantially complementary to the first target polynucleotide strand.

[17] The mixture described in [4], wherein the mixture contains double-stranded polynucleotide molecules comprising the first target polynucleotide strand and a first target complementary polynucleotide strand, the first target complementary polynucleotide strand being substantially complementary to the first target polynucleotide strand, sequences D and B being located within the double-stranded polynucleotide molecule on the target complementary polynucleotide strand, and sequences A, E, and C being located within the double-stranded polynucleotide molecule on the target polynucleotide strand.

[18] The mixture of [2] or [4], wherein the mixture comprises double-stranded polynucleotide molecules comprising the first target polynucleotide strand and a first target complement polynucleotide strand, wherein the first target complement polynucleotide strand is substantially complementary to the first target polynucleotide strand, the double-stranded polynucleotide molecules comprise a variant polynucleotide strand and a variant complement polynucleotide strand, wherein the variant polynucleotide strand is substantially identical to the target polynucleotide strand and comprises a nucleotide that differs from the target polynucleotide strand at the target variant nucleotide, and the variant complement polynucleotide strand is substantially complementary to the variant polynucleotide strand.

[19] The mixture described in [2] or [4], wherein the third oligonucleotide contains 3 to 6 consecutive nucleotides of the first sequence.

[20] The mixture described in [2] or [4], wherein the third oligonucleotide further comprises a sequence of nucleotides of the first target polynucleotide strand that does not overlap with the sequence of nucleotides of the first sequence.

[21] The mixture described in [2] or [4], wherein the mixture does not contain a fourth oligonucleotide.

[22] The mixture described in [2] or [4], wherein the mixture does not contain a fourth oligonucleotide comprising a detectable label.

[23] The mixture described in [2] or [4], wherein the mixture does not contain a fourth oligonucleotide that binds to the first target polynucleotide strand.

[24] The mixture described in [2] or [4], wherein the mixture does not contain a fourth oligonucleotide having a detectable label and a sequence that binds to the first target polynucleotide strand.

[25] The mixture described in [2] or [4], wherein the third oligonucleotide is the only oligonucleotide in the mixture having a detectable label and a sequence configured to hybridize to the sequence of the target polynucleotide strand.

[26] The mixture according to [2] or [4], further comprising 30 mM to 80 mM potassium chloride.

[27] The mixture described in

[26] above, wherein the concentration of potassium chloride is at least 40 mM.

[28] The mixture described in

[26] above, wherein the potassium chloride concentration is less than 70 mM.

[29] The mixture described in

[26] above, wherein the concentration of potassium chloride is at least 40 mM and less than 70 mM.

[30] The mixture according to [2] or [4], further comprising 10 mM to 40 mM ammonium sulfate.

[31] The mixture according to

[30] , wherein the concentration of ammonium sulfate is at least 20 mM.

[32] The mixture described in

[30] above, wherein the concentration of ammonium sulfate is less than 35 mM.

[33] The mixture according to

[30] , wherein the concentration of ammonium sulfate is at least 20 mM and less than 35 mM.

[34] The mixture according to

[30] , wherein the concentration of ammonium sulfate is 20 to 25 mM.

[35] The mixture is a) a potassium chloride concentration of 30 mM to 80 mM; b) the mixture according to [2] or [4], further comprising an ammonium sulfate concentration of 10 mM to 40 mM.

[36] The mixture according to

[35] , wherein the concentration of potassium chloride is 40 mM to 70 mM and the concentration of ammonium sulfate is 20 mM to 35 mM.

[37] The mixture according to

[35] , wherein the concentration of potassium chloride is 40 mM to 48 mM and the concentration of ammonium sulfate is 20 mM to 24 mM.

[38] The mixture described in

[35] above, wherein the concentration of potassium chloride is 45 mM and the concentration of ammonium sulfate is 22 mM.

[39] The detectable label is a DNA binding dye, a reporter dye, a fluorescent probe, 6-carboxyfluorescein (FAM™), tetrachlorofluorescin (TET™), 6-carboxy-4',5'-dichloro-2',7'-dimethoxyfluorescein, succinimidyl ester (JOE™), VIC™, SO instead of a carboxylate group, or the like. 3 7. The mixture of claim 6, wherein the fluorescein dye is selected from the group consisting of a sulfonate derivative of a fluorescein dye, a phosphoramidite form of fluorescein, a phosphoramidite form of CY5, a non-FRET label, a ferrocene reagent, ABY™, NED™, JUN™, Fluor™ 488, AlexaFluor™ 532, AlexaFluor™ 546, AlexaFluor™ 594, AlexaFluor™ 647, AlexaFluor™ 660, TYE™ 563, TYE™ 665, TYE™ 705, and combinations thereof.

[40] The mixture according to any one of [9] to

[11] , wherein the quenching moiety is selected from the group consisting of tetramethylrhodamine (TAMRA), non-fluorescent quenchers (NFQ), black hole quenchers, Iowa black quenchers, QSY quenchers, QSY7 quenchers, QSY21 quenchers, dabcyl and / or dabcyl sulfonate / carboxylate quenchers.

[41] T of the third oligonucleotide m is T of the first oligonucleotide m The mixture according to [2] or [4], wherein the temperature is at least 5°C and 25°C higher than the reference temperature.

[42] T of the third oligonucleotide m is T of the first oligonucleotide m The mixture according to

[41] , wherein the temperature is at least 8°C and 12°C higher than the reference temperature.

[43] T of the first oligonucleotide m is T of the second oligonucleotide m The mixture according to [2] or [4], wherein the temperature is within 5°C of

[44] T of the first oligonucleotide m is 45 to 60°C, and the T of the second oligonucleotide m The mixture according to any one of

[42] and

[43] , wherein the temperature is 45 to 60°C.

[45] The mixture described in any one of [1] to [4] above, wherein the first and second oligonucleotides are extendable.

[46] The mixture described in any one of [1] to [4] above, wherein the first and second oligonucleotides are primers.

[47] The mixture described in [2] or [4], wherein the third oligonucleotide is non-extendable.

[48] ​​The mixture described in [2] or [4], wherein the third oligonucleotide is a probe.

[49] The mixture described in [2] or [4], wherein the first, second, and / or third oligonucleotides contain 10 to 40 nucleotides.

[50] The mixture described in [2] or [4], wherein the third oligonucleotide comprises a blocking moiety.

[51] The mixture described in

[50] , wherein the blocking moiety is a minor groove binding moiety (MGB).

[52] The mixture described in

[51] , wherein the MGB portion is located at the 3' end and / or 3' portion of the third oligonucleotide.

[53] The mixture a) a fourth oligonucleotide configured to hybridize to a first sequence in a second target polynucleotide strand, wherein the first sequence in the second target polynucleotide comprises a second target variant nucleotide, and the fourth oligonucleotide further comprises a nucleotide residue at its 3' end positioned to hybridize to the second target variant nucleotide; b) a fifth oligonucleotide having a sequence configured to hybridize to a sequence complementary to a second sequence in the second target polynucleotide strand, wherein the second sequence of the second target polynucleotide strand is located 5' upstream from the first sequence on the second target polynucleotide strand; and c) a sixth oligonucleotide having a sequence configured to hybridize to a sequence complementary to a third sequence within the second target polynucleotide strand, wherein the third sequence of the second target polynucleotide strand at least partially overlaps with the first sequence on the second target polynucleotide strand and comprises the second target variant nucleotide.

[54] The mixture a) a fourth oligonucleotide configured to hybridize to a first sequence (F) in a second target polynucleotide strand, wherein the first sequence in the second target polynucleotide comprises a second target variant nucleotide ("second variant nucleotide"), and the fourth oligonucleotide further has a nucleotide residue at its 3' end positioned to hybridize to the second target variant nucleotide; b) a fifth oligonucleotide configured to hybridize to a second sequence (G) in the second target polynucleotide strand, wherein the second sequence of the second target polynucleotide strand is complementary to a third sequence (H) of the second target polynucleotide strand, the third sequence of the target polynucleotide strand being present within the second target polynucleotide strand, and the third sequence (H) being located 5' upstream from the first sequence (F) of the second target polynucleotide strand; c) a sixth oligonucleotide configured to hybridize to a fourth sequence (I) of the second target polynucleotide strand, wherein the fourth sequence (I) is complementary to the fifth sequence (J) of the second target polynucleotide strand, the fifth sequence (J) being present in the second target polynucleotide strand, the fifth sequence (J) at least partially overlapping with the first sequence (F) in the second target polynucleotide strand, and comprising the second target variant nucleotide.

[55] The mixture described in

[53] or

[54] , wherein the target variant nucleotide in the sixth oligonucleotide is at least two nucleotides from the 3' end or 5' end of the sixth oligonucleotide.

[56] The mixture described in

[53] ,

[54] , or

[55] , wherein the sixth oligonucleotide comprises a detectable label.

[57] The mixture described in

[56] , wherein the detectable label of the sixth oligonucleotide is a fluorescent label.

[58] The mixture described in

[56] or

[57] , wherein the detectable label of the sixth oligonucleotide is on the first terminal nucleotide.

[59] The mixture described in any one of

[53] ,

[54] , or

[56] , wherein the sixth oligonucleotide further comprises a quenching moiety.

[60] The mixture described in

[59] , wherein the quenching portion of the sixth oligonucleotide is located on the second terminal nucleotide of the sixth oligonucleotide.

[61] The mixture described in

[59] or

[60] , wherein the quenching portion of the sixth oligonucleotide is capable of quenching a signal from the detectable label of the sixth oligonucleotide.

[62] The mixture described in

[53] or

[54] , wherein the fourth and fifth oligonucleotides are extendable.

[63] The mixture described in

[53] or

[54] , wherein the fourth and fifth oligonucleotides are primers.

[64] The mixture described in

[53] or

[54] , wherein the sixth oligonucleotide is non-extendable.

[65] The mixture described in

[53] or

[54] , wherein the sixth oligonucleotide is a probe.

[66] The mixture described in

[53] or

[54] , wherein the fourth, fifth, and / or sixth oligonucleotides contain 10 to 30 nucleotides.

[67] The mixture described in

[53] or

[54] , wherein the sixth oligonucleotide comprises a blocking portion.

[68] The mixture described in

[67] , wherein the blocking portion of the sixth oligonucleotide is a minor groove binder (MGB) portion.

[69] The mixture described in

[68] , wherein the MGB portion of the sixth oligonucleotide is located at the 3' end and / or 3' portion of the sixth oligonucleotide.

[70] The mixture, a) a fourth oligonucleotide having a sequence configured to hybridize to a sequence complementary to the first oligonucleotide, wherein the fourth oligonucleotide is configured to hybridize substantially to the first sequence and includes a nucleotide at its 3' end that differs from the complement of the target variant nucleotide; b) a fifth oligonucleotide having a sequence configured to hybridize to a sequence complementary to the third oligonucleotide, wherein the fifth oligonucleotide comprises a different nucleotide at the position of the target variant nucleotide.

[71] The mixture described in

[70] , wherein the different nucleotides are at least two nucleotides from the 3' end.

[72] The mixture described in

[70] or

[71] , wherein the fifth oligonucleotide comprises a detectable label.

[73] The mixture described in

[72] , wherein the detectable label of the fifth oligonucleotide is a fluorescent label. 〔74〕

[74] The mixture described in

[72] or

[73] , wherein the detectable label of the fifth oligonucleotide is on the first terminal nucleotide of the fifth oligonucleotide.

[74] The mixture described in any one of

[72] to

[74] , wherein the detectable label on the fifth oligonucleotide is distinguishable from the detectable label on the third oligonucleotide.

[75] The mixture described in any one of

[72] to

[74] , wherein the fifth oligonucleotide further comprises a quenching moiety.

[76] The mixture described in

[75] , wherein the quenching portion of the fifth oligonucleotide is located on the second terminal nucleotide of the fifth oligonucleotide.

[77] The mixture described in

[75] or

[76] , wherein the quenching moiety of the fifth oligonucleotide is capable of quenching a signal from the detectable label of the fifth oligonucleotide.

[78] The mixture described in

[70] , wherein the fourth oligonucleotide is extendible.

[79] The mixture described in

[70] , wherein the fourth oligonucleotide is a primer.

[80] The mixture described in

[70] , wherein the fifth oligonucleotide is non-extendable.

[81] The mixture described in

[70] , wherein the fifth oligonucleotide is a probe.

[82] The mixture described in

[70] , wherein the fourth and fifth oligonucleotides contain 10 to 40 nucleotides.

[83] The mixture described in

[70] , wherein the fifth oligonucleotide comprises a blocking moiety.

[84] The mixture described in

[83] , wherein the blocking portion of the fifth oligonucleotide is a minor groove binder (MGB) portion.

[85] The mixture described in

[84] , wherein the MGB portion of the fifth oligonucleotide is located at the 3' end and / or 3' portion of the fifth oligonucleotide.

[86] A mixture comprising a plurality of oligonucleotide sets, wherein each oligonucleotide set is: a) a first oligonucleotide configured to hybridize to a first sequence in a first target polynucleotide strand, the first sequence comprising a target variant nucleotide, the first oligonucleotide further having a nucleotide residue at its 3' end positioned to hybridize to the target variant nucleotide; b) a second oligonucleotide having a sequence configured to hybridize to a sequence complementary to a second sequence in the first target polynucleotide strand, wherein the second sequence of the first target polynucleotide strand is located 5' upstream from the first sequence of the first target polynucleotide strand; A mixture wherein each set of said first oligonucleotides is configured to hybridize to a different first sequence.

[87] Each oligonucleotide set is c) a third oligonucleotide having a sequence configured to hybridize to a sequence complementary to a third sequence in the first target polynucleotide strand, wherein the third sequence of the first target polynucleotide strand at least partially overlaps with the first sequence of the first target polynucleotide strand, and the third sequence comprises the target variant nucleotide; the third oligonucleotides of each set share sequence similarity with a different third sequence; The mixture of claim 86, wherein the third oligonucleotides of each set contain different, distinguishable detectable labels.

[88] A mixture comprising a plurality of oligonucleotide sets, wherein each oligonucleotide set is: a) a first oligonucleotide configured to hybridize to a first sequence (A) present in a first target polynucleotide strand, said first sequence comprising a target variant nucleotide ("first variant nucleotide"), said first oligonucleotide further having a nucleotide residue at its 3' end positioned to hybridize to said first variant nucleotide; b) a second oligonucleotide configured to hybridize to a second sequence (B), wherein the second sequence is complementary to a third sequence (C), the third sequence being present within the first target polynucleotide strand, and the third sequence (C) being located 5' upstream of the first sequence (A) of the first target polynucleotide strand; A mixture wherein each set of said first oligonucleotides is configured to hybridize to a different first sequence.

[89] Each oligonucleotide set is c) a third oligonucleotide configured to hybridize to a fourth sequence (D) complementary to a fifth sequence (E), wherein the fifth sequence is present in the first target polynucleotide strand, the fifth sequence (E) at least partially overlaps with the first sequence (A) in the first target polynucleotide strand, and the third oligonucleotide further comprises the first target variant nucleotide; the third oligonucleotides of each set share sequence similarity with a different third sequence; The mixture of claim 88, wherein the third oligonucleotides of each set contain different, distinguishable detectable labels.

[90] The mixture described in any one of [1] to

[89] , further comprising a polymerase.

[91] The mixture described in

[90] , wherein the polymerase is thermostable.

[92] The mixture described in

[91] , wherein the mixture further comprises a hot start component.

[93] The mixture described in

[92] , wherein the hot start component comprises an antibody, an oligonucleotide, an aptamer directed against a thermostable polymerase, and / or a chemical modification of the polymerase.

[94] The mixture described in any one of [1] to

[93] above, further comprising a nucleotide source.

[95] The mixture described in any one of [1] to

[94] above, wherein the third oligonucleotide is a hydrolysis probe.

[96] The mixture described in any one of [1] to

[95] above, further comprising a nucleic acid sample suspected of containing the first target polynucleotide strand.

[97] The mixture described in any one of [1] to

[96] , wherein the target variant nucleotide is in a mutant allele.

[98] The mixture described in

[97] , wherein the mutant allele is either a purine to purine single point mutation or a pyrimidine to pyrimidine single point mutation in the target variant nucleotide.

[99] The mixture described in

[97] or

[98] , wherein the mutant allele is an allele of the KRAS oncogene.

[100] The mixture described in

[97] , wherein the mutant allele is a stochastic mutation.

[101] The mixture described in any one of [1] to

[100] , wherein the target variant nucleotide has an identity corresponding to a major allelic variant or a minor allelic variant.

[102] The mixture described in

[101] , wherein the target variant nucleotide has the identity of a minor allele variant having a minor allele frequency (MAF) of less than 1%.

[103] The mixture described in

[102] , wherein the target variant nucleotide has the identity of a minor allele variant having a minor allele frequency (MAF) of less than 0.1%, less than 0.01%, or less than 0.001%.

[104] The mixture described in any one of [1] to

[103] , wherein the target variant nucleotide occurs at a single nucleotide polymorphism position.

[105] The mixture described in any one of [1] to

[95] above, wherein the mixture is a master mix.

[106] The mixture described in

[105] , wherein the master mix is ​​stable at 4 to 8°C for up to 12 months.

[107] The mixture according to any one of [1] to

[104] above, wherein the mixture is a reaction mixture.

[108] The mixture described in

[107] , wherein the reaction mixture further comprises an amplicon comprising the first sequence of the first target polynucleotide strand.

[109] The mixture described in

[108] , wherein the reaction mixture does not contain amplicons containing the sequence of any other different (e.g., second) polynucleotide strand.

[110] The mixture comprises: a) at least one cleaning agent; b) glycerol, and c) The mixture according to any one of [1] to

[109] above, further comprising one or more of at least one reference dye.

[111] The mixture described in any one of [1] to

[110] above, further comprising bovine serum albumin and / or gelatin.

[112] The mixture described in any one of [1] to

[106] above, wherein the mixture is freeze-dried.

[113] A kit comprising: a first container containing the mixture described in any preceding embodiment; and a second container containing a control polynucleotide sample comprising a polynucleotide molecule that shares sequence similarity with the first target polynucleotide strand.

[114] The kit described in

[113] , wherein the control polynucleotide sample contains the entire first target polynucleotide strand.

[115] The kit described in

[113] , wherein the control polynucleotide sample does not share sequence similarity with the first target polynucleotide strand at the target variant nucleotide.

[116] A method for detecting a target polynucleotide molecule containing a target variant nucleotide in a test polynucleotide sample, the method comprising: a) forming a reaction mixture of a test polynucleotide sample and the mixture described in [2] or [4]; b) performing an amplification reaction using at least the first and second oligonucleotides as primers to generate an amplicon of the target polynucleotide sequence of the target polynucleotide molecule, if present in the test polynucleotide sample; c) detecting the amplicon produced in step b) by detecting a change in a detectable property of the third oligonucleotide; wherein detecting the amplicon in step c) indicates the presence of the target polynucleotide molecule in the test polynucleotide sample.

[117] The method of

[116] , wherein the target polynucleotide molecule is detected in a test polynucleotide sample comprising a mixture of polynucleotide molecules, the mixture comprising a polynucleotide molecule comprising a first variant form of the target variant nucleotide (a "first variant target polynucleotide molecule"), and a polynucleotide molecule comprising a second variant form of the target variant nucleotide (a "second variant target polynucleotide molecule").

[118] The method of

[116] or

[117] , wherein the test polynucleotide sample contains polynucleotide strands that do not contain the target polynucleotide sequence ("non-target polynucleotide molecules").

[119] The method of

[118] , wherein the test sample contains more non-target polynucleotide molecules than target polynucleotide molecules.

[120] The method according to any one of

[116] to

[119] above, wherein the target polynucleotide molecule is a mutant polynucleotide sequence.

[121] The method according to any one of

[118] to

[120] , wherein the non-target polynucleotide molecule is a major allele or a wild-type polynucleotide sequence.

[122] The method described in any one of

[118] to

[121] , wherein the test sample contains less than 1%, less than 0.1%, less than 0.01%, or less than 0.001% of target polynucleotide molecules compared to non-target polynucleotide molecules.

[123] The method of any one of

[117] to

[119] , wherein the test sample contains less than 1%, less than 0.1%, less than 0.01%, or less than 0.001% of second variant target polynucleotide molecules compared to the first variant target polynucleotide molecules.

[124] The method of any one of

[117] to

[119] , wherein the first variant target polynucleotide molecule and / or the second variant target polynucleotide molecule is a mutant polynucleotide sequence.

[125] The method described in any one of

[117] to

[119] , wherein the first variant target polynucleotide molecule and / or the second variant target polynucleotide molecule is a wild-type polynucleotide sequence.

[126] The method of any one of

[117] to

[125] , further comprising enriching the number of first variant polynucleotide molecules in the polynucleotide test sample compared to the second variant polynucleotide molecules prior to steps a) to c).

[127] The method according to

[126] , wherein the enriching comprises an amplification reaction comprising different conditions compared to those used in any of steps a) to c).

[128] The method of

[126] or

[127] , wherein the number of first variant polynucleotide molecules in the polynucleotide test sample is enriched by at least 2-fold, 4-fold, 6-fold, 8-fold, or 10-fold compared to the number of second variant polynucleotide molecules.

[129] The enrichment step is performed by increasing the calculated melting temperature (T M The method according to any one of

[126] to

[128] , wherein the method is carried out using a polymerase chain reaction comprising 15 to 25 cycles at a temperature 12 to 16 degrees higher than that of the PCR product.

[130] Step b) is a step of converting the T M The method according to any one of

[126] to

[129] , wherein the method is carried out using 35 to 40 cycles at a temperature close to the temperature at which the enrichment is carried out and 4 to 6 degrees lower than the temperature at which the enrichment is carried out.

[131] The method according to any one of

[116] to

[130] , wherein the test polynucleotide sample is derived from mammalian or non-mammalian animal tissue or cells, or plant tissue or cells.

[132] The method of

[131] , wherein the sample is selected from the group consisting of saliva, buccal tissue, skin, hair, blood, plasma, urine, feces, semen, and a tumor sample.

[133] The method according to

[131] or

[132] , wherein the polynucleotide test sample is derived from a cancer cell.

[134] The method according to any one of

[131] to

[133] above, wherein detection of the amplicon indicates the presence of cancer cells in the tissue from which the test polynucleotide sample is derived.

[135] The method of any one of

[116] to

[134] , wherein the target polynucleotide contains at least one mutation in Ras, EFGR, Kit, pTEN, and / or p53, and / or at least one KRAS or NRAS mutation.

[136] The method according to any one of

[116] to

[135] above, wherein the amplification reaction is a polymerase chain reaction (PCR).

[137] The method according to

[136] , wherein the PCR is real-time PCR or quantitative PCR (qPCR).

[138] The method according to

[136] or

[137] , wherein the third oligonucleotide has a Tm that is 8 to 12°C higher than the Tm of the first oligonucleotide, and the PCR is performed at an annealing temperature that is within 5°C of the Tm of the first oligonucleotide.

[139] The method of any one of

[116] to

[138] , wherein the method is carried out using a kit comprising a first container containing at least the first oligonucleotide, the second oligonucleotide, and the third oligonucleotide, and a second container containing a control polynucleotide sample containing the first target polynucleotide strand.

[140] A method according to any one of

[116] to

[139] , wherein the method detects a target variant nucleotide that indicates a mutation, and the test polynucleotide sample contains 1 to 10 copies of the target polynucleotide.

[141] The method of any one of

[116] to

[140] , wherein the target variant nucleotide contains a purine base and the corresponding wild-type nucleotide at the target variant nucleotide position contains a different purine base.

[142] The method of any one of

[116] to

[140] , wherein the target variant nucleotide contains a pyrimidine base and the corresponding wild-type nucleotide at the target variant nucleotide position contains a different pyrimidine base.

[143] A method for detecting a target polynucleotide molecule containing a target variant nucleotide in a test polynucleotide sample, the method comprising: a) forming a reaction mixture of a test polynucleotide sample and a mixture according to any one of [1] to

[94] ,

[104] ,

[105] , and 109 to 111; b) performing an amplification reaction using at least the first and second oligonucleotides as primers to generate an amplicon of the target polynucleotide sequence of the target polynucleotide molecule, if present in the test polynucleotide sample; c) detecting the amplicon produced in step b) by detecting a change in a detectable property of the third oligonucleotide; wherein detecting the amplicon in step c) indicates the presence of the target polynucleotide molecule in the test polynucleotide sample.

[144] A method for detecting at least two different target polynucleotide molecules in a test polynucleotide sample, each of which contains a target variant nucleotide, the method comprising: a) forming a reaction mixture of a test polynucleotide sample and the mixture described in

[53] or

[54] ; b) performing an amplification reaction using the first and second oligonucleotides as primers to generate an amplicon of a first target polynucleotide sequence of a first target polynucleotide molecule, if present in the test polynucleotide sample; c) performing an amplification reaction using the fourth and fifth oligonucleotides as primers to generate a second amplicon of a second target polynucleotide sequence of a second target polynucleotide molecule, if present in the test polynucleotide sample; d) detecting the amplicons produced in steps b) and c) by detecting a change in a detectable property of the third and sixth oligonucleotides; wherein detecting the amplicon in step d) indicates the presence of the first and / or second target polynucleotide molecule within the test polynucleotide sample.

[145] The method of

[144] , wherein the detectable property of the third and sixth oligonucleotides is a fluorescent label.

[146] The method described in

[145] , wherein the fluorescent labels on each of the third and sixth oligonucleotides are different.

[147] The mixture described in any one of [1] to

[112] above, wherein the mixture is used to detect one or more mutations listed in Table 1 and / or Table 2.

[148] The kit according to any one of

[113] to

[115] , wherein the kit is used for detecting one or more mutations listed in Table 1 and / or Table 2.

[149] The method of any one of

[116] to

[146] above, wherein the method is used to detect one or more mutations listed in Table 1 and / or Table 2.

Claims

1. A mixture comprising a plurality of oligonucleotide sets, about 20 mM to about 80 mM potassium chloride, and about 10 mM to about 40 mM ammonium sulfate, Each oligonucleotide set is a) a first oligonucleotide configured to hybridize to a first sequence in a first target polynucleotide strand, the first sequence comprising a target variant nucleotide, the first oligonucleotide further having a nucleotide residue at its 3' end positioned to hybridize to the target variant nucleotide; b) a second oligonucleotide having a sequence configured to hybridize to a sequence complementary to a second sequence in the first target polynucleotide strand, wherein the second sequence of the first target polynucleotide strand is located 5' upstream from the first sequence of the first target polynucleotide strand; c) a third oligonucleotide having a sequence configured to hybridize to a sequence complementary to a third sequence in said first target polynucleotide strand, wherein said third sequence of said first target polynucleotide strand at least partially overlaps with said first sequence of said first target polynucleotide strand, and said third sequence comprises said target variant nucleotide.

2. The mixture described in claim 1, wherein the target variant nucleotides in the third oligonucleotide are at least two nucleotides from the 3' end or 5' end of the third oligonucleotide.

3. The mixture described in claim 1, wherein the third oligonucleotide comprises a detectable label.

4. The mixture described in claim 3, wherein the detectable label is a fluorescent label.

5. The mixture described in claim 3, wherein the detectable label is on the first terminal nucleotide.

6. The mixture described in claim 3, wherein the third oligonucleotide further comprises a quenching moiety.

7. The mixture described in claim 6, wherein the quenching moiety is on the second terminal nucleotide of the third oligonucleotide.

8. The mixture described in claim 6, wherein the quenching moiety is capable of quenching a signal from the detectable label.

9. The mixture of claim 7, wherein the first terminal nucleotide is a 5' terminal nucleotide.

10. The mixture of claim 7, wherein the second terminal nucleotide is a 3' terminal nucleotide.

11. The mixture described in claim 1, wherein the mixture contains a single-stranded polynucleotide molecule comprising the first target polynucleotide strand.

12. The mixture described in claim 1, wherein the mixture comprises a double-stranded polynucleotide molecule comprising the first target polynucleotide strand and a first target complementary polynucleotide strand, and the first target complementary polynucleotide strand is substantially complementary to the first target polynucleotide strand.

13. The mixture of claim 1, wherein the mixture comprises double-stranded polynucleotide molecules comprising the first target polynucleotide strand and a first target complement polynucleotide strand, wherein the first target complement polynucleotide strand is substantially complementary to the first target polynucleotide strand, the double-stranded polynucleotide molecules comprise a variant polynucleotide strand and a variant complement polynucleotide strand, wherein the variant polynucleotide strand is substantially identical to the target polynucleotide strand and comprises a nucleotide that differs from the target polynucleotide strand in the target variant nucleotide, and the variant complement polynucleotide strand is substantially complementary to the variant polynucleotide strand.

14. The mixture described in claim 1, wherein the third oligonucleotide comprises 3 to 6 consecutive nucleotides of the first sequence.

15. The mixture described in claim 1, wherein the third oligonucleotide further comprises a sequence of nucleotides of the first target polynucleotide strand that does not overlap with a sequence of nucleotides of the first sequence.

16. A kit comprising: a first container containing a mixture described in any one of claims 1 to 15; and a second container containing a control polynucleotide sample comprising a polynucleotide molecule that shares sequence similarity with the first target polynucleotide strand.

17. The mixture of claim 1, wherein the third oligonucleotide of each oligonucleotide set comprises a different detectable label.

Images