Use of variant-signaling primers for the detection of genetic rearrangements and modifications

Abstract

Disclosed are variant-signaling primers for primer-dependent nucleic acid amplification methods. Also disclosed are multiplex assay methods, related reagent kits, and oligonucleotides for such methods.

Description

[0001] USE OF VARIANT-SIGNALING PRIMERS FOR THE DETECTION OF GENETIC REARRANGEMENTS AND MODIFICATIONS

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims the benefit under 35 U.S.C. §119(e) of the earlier filing date of U.S. Provisional Patent No. 63 / 568,792, filed on March 22, 2024 and U.S. Provisional Patent No. 63 / 566,523, filed on March 18, 2024, which are hereby incorporated by reference in their entirety.

[0004] REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

[0005] The contents of the electronic sequence listing (096747.00495 SeqList.xml; Size: 12,788 bytes; and Date of Creation: March 13, 2025) is herein incorporated by reference in its entirety.

[0006] FIELD OF THE INVENTION

[0007] This invention relates to nucleic acid amplification and detection assays, for example, PCR amplification and detection methods, and to primers, reaction mixtures and kits for such methods.

[0008] BACKGROUND OF THE INVENTION

[0009] It has been a long-sought medical goal to be able to detect at a very early stage extremely rare mutations whose presence in a clinical sample are useful for diagnosing cancer, determining prognosis, and indicating the choice of effective therapy. The detection and quantitative assessment of relevant somatic mutations have multiple uses, including: (i) the detection of cancer at a treatable stage in patients who inherit genes that make cancer more likely; (ii) the detection of mutations in benign cancer cells that indicate that they may now metastasize; (iii) measurement of the abundance of cancer cells during treatment; and (iv) the detection of drug-resistant cancer cells that may arise during treatment, so that therapy can be adjusted. A further goal is to develop methods that enable multiplex assays that can simultaneously measure the abundance of different genomic alterations such as single nucleotide variants (SNVs), indels, structural variants (SVs), and somatic copy-number aberrations.

[0010] Integrating diverse genomic alterations, such as structural variants (SVs), single nucleotide variants (SNVs), small insertions and deletions (indels), and copy number variations (CNVs), into cancer diagnostics provides a comprehensive understanding of the tumor’s genomic landscape. This multi-faceted approach not only increases the number of actionable markers per tumor, thereby enhancing diagnostic sensitivity, but also amplifies the prognostic and predictive value of genetic signatures, ultimately improving patient outcomes in cancer care.

[0011] Spurring on these efforts is the realization that cancer cells, no matter where in the body they are located, divide frequently, and undergo apoptosis and necrosis, resulting in cancer cells liberating genomic DNA fragments that can be found in a patient's blood plasma. “Liquid biopsies”, which analyze tumor-derived materials such as DNA, RNA, or proteins in a cancer patient’s blood plasma, can detect and measure rare mutations associated with diagnosis, prognosis, and treatment options. This minimally invasive approach enables the identification of cancer-related changes, often at an early stage. Circulating tumor DNA (ctDNA) serves as a potent biomarker for molecular profiling, guiding targeted therapy, detecting minimal residual disease (MRD), and assessing treatment response.

[0012] Other examples of "liquid biopsies" or bodily fluids apart from blood, include urine, saliva, cerebrospinal fluid, pleural fluid, and ascites fluid. They offer a minimally invasive or non- invasive approach to monitor disease progression.

[0013] The challenge facing assay designers is to find a means of selectively detecting and quantitating these biomarkers in plasma DNA, especially in early-stage cancer patients (< 0.01% variant allele fraction), despite the presence of abundant related wild-type DNA sequence fragments originating from normal cells throughout the body, and despite the fact that different relevant mutations, often occur in the same or adjacent codons. The success of "next-generation" sequencing for the detection of rare mutant sequence fragments in plasma DNA, though complex and costly, has illustrated the value of this approach.

[0014] Molecular diagnostic assays based on the exponential amplification of nucleic acid target sequences, such as polymerase chain reactions, are inexpensive and sufficiently sensitive to generate signals sometimes from as few as a single molecule. Conventionally, specificity is obtained by making a primer sufficiently long so that under the amplification reaction conditions, primarily during the primer-annealing step, the primer only hybridizes with its perfect complement on the single stranded DNA / RNA target. For distinguishing between or among target sequences, allele-specific hybridization probes such as molecular beacon probes are commonly used (Marras SAE et al. Multiplex detection of single-nucleotide variations using molecular beacons. Genetic Analysis: Biomolecular Engineering. 1999, 14, 151-156). If the sequence being investigated is an allele, such as a single-nucleotide polymorphism (SNP) that is present in a mixture with another allele, for example, a wild-type (WT) variant, distinguishing by use of a probe has a practical detection limit of about 3% (allele selectivity of not less than about 300 target allele molecules in the presence of 10,000 molecules of the alternate allele) due to the tendency of amplification of the prevalent allele to overwhelm amplification of the less abundant allele.

[0015] Researchers have turned to modifying amplification primers to improve the selectivity of amplification assays. A primer that is highly allele-selective enables the exponential amplification of a mutant DNA sequence while simultaneously suppressing amplification of a far more abundant wild-type sequence that is present, even when the difference between them is an SNP. Shortening a conventional amplification primer may improve allele selectivity, but this comes at the cost of specificity, making it less effective for analyzing mixtures of alleles. Other modifications of primers have been developed to improve their selectivity while retaining specificity. One such approach is ARMS (“amplification refractory mutation system”). An ARMS primer has a 3'- terminal nucleotide that is complementary to the sequence in the mutant being investigated, but that is mismatched to another allele or alleles. ARMS relies on the refractory nature of certain DNA polymerases, that is, a tendency not to extend a primer-target hybrid having such a mismatch. ARMS has been demonstrated to be useful for determining zygosity (homozygous WT, heterozygous, or homozygous mutant (MUT)), but it has a practical detection limit for other uses of about 1% (not less than about 100 target allele molecules in the presence of 10,000 molecules of the alternate allele) (Newton CR et al. Analysis of any point mutation in DNA. The amplification refractory mutation system (ARMS). Nucleic Acids Research. 1989 17, 2503-2516).

[0016] Other approaches seek to increase the likelihood that a primer specific for a mutant sequence will hybridize to the mutant sequence and lead to its amplification, while minimizing the chance that it will hybridize to the corresponding wild-type sequence, thereby suppressing the amplification of the wild-type. Several primer designs have in common that they possess a priming sequence that is perfectly complementary to a mutant target and contain an internal interrogating nucleotide that is mismatched with the corresponding wild-type sequence. Such designs include dual priming oligonucleotide (DPO) primers (Chun J-Y et al. Dual priming oligonucleotide system for the multiplex detection of respiratory viruses and SNP genotyping of CYP2C19 gene. Nucleic Acids Research. 2007 35, e40); MyT primers, Makarov V and Chupreta, United States Patent Publication 2011 / 0129832 Al; hairpin primers (U.S, Patent No. 6,277,607); and PlexPrime primers (Tan LY et al. Superior multiplexing capacity of PlexPrimers enables sensitive and specific detection of SNPs and clustered mutations in qPCR. PLoS ONE. 2017 12, eOl 70087). The length of their priming sequence is chosen so that, under annealing conditions, perfectly complementary mutant hybrids are likely to form, and generate amplicons, while mismatched wild-type hybrids are much less likely to form, and are therefore much less likely to lead to the generation of amplicons.

[0017] PCR using allele-specific primers is highly selective and offers a valuable approach for detecting mutations in cancer samples, but comes at the cost of poor sensitivity. Some of these technologies, due to the presence of unnatural nucleotides in their sequence, are not sensitive enough to detect extremely rare mutants or have not demonstrated utility for quantitative determinations in multiplex real-time PCR assays.

[0018] One type of allele-discriminating primers are known as SuperSelective primers (see WO2021 / 067527; and U.S. Patent Nos. 9,909,159; 10,815,512; 11,111,515; and 11,542,547).

[0019] SuperSelective primers exhibit remarkable sensitivity, capable of detecting SNVs even when present in low abundance relative to the wild-type allele (Vargas DY et al. Multiplex realtime PCR assays that measure the abundance of extremely rare mutations associated with cancer. PLoS ONE. 2016 11, e0156546; Vargas DY et al. Suppression of wild-type amplification by selectivity enhancing agents in PCR assays that utilize SuperSelective primers for the detection of rare somatic mutations. Journal of Molecular Diagnostics. 2018 20, 415-427; and Kramer FR and Vargas DY. SuperSelective primer pairs for sensitive detection of rare somatic mutations. Nature Scientific Reports. 2021 11, Article 22384). SuperSelective primers have proven effective in multiplex reactions, especially when testing clinical samples containing a limited number of mutant targets. They facilitate the precise identification of mutations within such samples (Vargas DY et al. Multiplex SuperSelective PCR assays for the detection and quantitation of rare somatic mutations in liquid biopsies. Journal of Molecular Diagnostics. 2022 24, 189-204).

[0020] Allele-specific primers are designed for known target sequences that can affect their effectiveness in regions with high sequence variability. Flexibility and mutation tolerance are crucial attributes for primers to accommodate sequence variations, and the amplification of different variants present in the same or adjacent codons.

[0021] It is important to analyze an extensive number of biomarkers, encompassing SNVs, SVs, indels, and CNVs, to ensure clinicians achieve greater sensitivity and specificity in cancer diagnosis. Analyzing a wide range of biomarkers facilitates early detection and monitoring, as well as leads to better treatment and survival outcomes. Tndels are genomic variations where small segments of DNA are either inserted or deleted from the genome, and they present significant challenges in detection.

[0022] Primer design is further complicated by complexity within certain regions of the human genome where some of the genetic rearrangements occur. Assay specificity is impacted by high GC content, which increases the probability of the formation of stable secondary structures like hairpins and stem-loop structures, while repetitive sequences increase the risk of nonspecific annealing and amplification, ultimately affecting assay specificity. Conversely, AT-rich regions can impact amplification efficiency and sensitivity due to low annealing probability.

[0023] Despite employing advanced bioinformatics tools to mitigate the aforementioned challenges, there remains a strong demand for innovative techniques to enable comprehensive detection of structural variations.

[0024] SUMMARY OF THE INVENTION

[0025] This disclosure provides variant-signaling primers for primer-dependent nucleic acid amplification methods. In one aspect, provided is an amplification and detection method that is capable of detecting as few as five copies of at least one rare mutant DNA target sequence in a mixture containing, for each mutant target sequence, 10,000 copies of a closely related wild-type DNA target sequence, comprising:

[0026] (a) repeatedly cycling a reaction mixture in a primer-dependent amplification reaction having a primer-annealing temperature, said reaction mixture including said at least one mutant target sequence or its closely related wild-type target sequence or both, a DNA polymerase, other reagents needed for amplification, and for each mutant target sequence a primer pair that includes a variant-signaling primer comprising, in the 5' to 3' direction the following three contiguous DNA sequences: an anchor sequence that hybridizes with the mutant target sequence and with its closely related wild-type target sequence during primer annealing; a bridge sequence at least four nucleotides long that does not hybridize to either the mutant target sequence or its closely related wild-type target sequence during primer annealing; and a foot sequence that is at least five nucleotides long, perfectly complementary to the mutant sequence and mismatched to its wild-type sequence by at least three or more nucleotides, wherein: (i) if the anchor sequence and the foot sequence of the primer are hybridized to the mutant target sequence, there is in the target sequence an intervening sequence that does not hybridize to the primer's bridge sequence during primer-annealing, and the bridge sequence and the intervening sequence, together create a bubble in the hybrid having a circumference of at least twelve nucleotides, wherein the intervening sequence is at least four nucleotides long,

[0027] (ii) the circumference of the bubble and the length of the foot sequence in combination result in a weak foot / mutant-target- sequence hybrid that makes copying the mutant target sequence unlikely,

[0028] (iii) the bridge sequence and the foot sequence do not together prime non-target sequences in the mixture, and

[0029] (iv) the probability that during said cycling a variant-signaling primer / wild-type target sequence hybrid will be extended is at least 10,000 times lower than the probability that during said cycling a variant-signaling primer / mutant target sequence hybrid will be extended, as evidenced by a AC-rof at least 13.3 cycles; and

[0030] (b) detecting amplified product or products with a dsDNA binding dye, or for each variantsignaling primer a fluorescent hybridization probe that signals upon hybridization to the amplification product of the primer, or for each variant-signaling primer a quenched, fluorescently labeled oligonucleotide hairpin at the primer's 5' end that fluoresces only when incorporated in or hybridized to the primer's amplified product, wherein copying is “unlikely” means that, if the reaction were begun with a reaction mixture containing 106copies of the at least one mutant DNA target sequence, no wild-type sequence and the variant-signaling primer, and separately with a reaction mixture containing 106copies of the at least one mutant DNA target sequence, no wild-type sequence and a corresponding conventional primer in place of the variant-signaling primer, the threshold cycle (CT) of the reaction utilizing the variant-signaling primer would be delayed by at least three cycles compared to the CT of the reaction utilizing the conventional primer.

[0031] In some embodiments: i. the bridge sequence is at least 6 nucleotides long, the intervening sequence is at least 6 nucleotides long, and the bubble has a circumference of 28-52 nucleotides; or ii. for each of the at least one variant-signaling primers, the CT delay for a reaction begun with 106copies of the mutant DNA target sequence and the variant-signaling primer as compared to a reaction begun with 106copies of the mutant DNA target sequence and a conventional primer is at least 5 cycles.

[0032] In some embodiments, the bubble circumference is 28-44 nucleotides.

[0033] In one aspect, provided is a primer-dependent amplification and detection method that is capable of amplifying and detecting in a sample as few as five copies of at least one rare DNA intended target sequence (“rare target sequence”) in a mixture containing, for each rare target sequence, 10,000 copies of a closely related unintended target sequence (“closely related sequence” or “unintended target sequence”) that differs from the rare target sequence by at least three nucleotides, comprising:

[0034] (a) preparing a primer-dependent amplification reaction mixture that includes the sample, a DNA polymerase, deoxyribonucleoside triphosphates, an amplification buffer, homogeneous fluorescence detection means for detecting amplification products, and for each rare target sequence a pair of a first primer and a second primer that are specific for the rare target sequence but mismatched to the closely related sequence,

[0035] (b) repeatedly cycling the primer-dependent amplification reaction mixture by said primerdependent amplification method to amplify each rare target sequence present in the sample, and

[0036] (c) detecting that rare target sequence by measuring the intensity of fluorescence from the homogeneous fluorescence detection means; wherein (i) the first primer is an allele-discriminating variant-signaling primer comprising from the 5 ' end to the 3 ' end a first anchor sequence, a first bridge sequence, and a first foot sequence that is mismatched to the closely related sequence by at least three nucleotides, and (ii) the second primer is an allele-discriminating primer.

[0037] In some embodiments, each primer contains a 3 '-terminal interrogating nucleotide that is complementary to the rare target sequence, but mismatched to the unintended target sequence. In some embodiments, the at least one rare target sequence in the sample includes at least two different rare target sequences, and the homogeneous fluorescence detection means comprises at least two different homogeneous fluorescence detection probes for the at least two different rare target sequences respectively. In some embodiments, each different unintended target sequence differs from its corresponding rare target sequence by at least three base pairs, and both the first and the second primers are mismatched to said base pairs.

[0038] In one aspect, provided is an amplification and detection method, contingent upon the identity of the mismatch, that is capable of detecting as few as five copies of at least one rare mutant DNA target sequence in a mixture containing, for each mutant target sequence, 10,000 copies of a closely related wild-type DNA target sequence, comprising:

[0039] (a) repeatedly cycling a reaction mixture in a primer-dependent amplification reaction having a primer-annealing temperature, said reaction mixture including said at least one mutant target sequence or its closely related wild-type target sequence or both, a DNA polymerase, other reagents needed for amplification, and for each mutant target sequence a primer pair that includes a variant-signaling primer comprising, in the 5' to 3' direction the following three contiguous DNA sequences: an anchor sequence that hybridizes with the mutant target sequence and with its closely related wild-type target sequence during primer annealing; a bridge sequence at least four nucleotides long that does not hybridize to either the mutant target sequence or its closely related wild-type target sequence during primer annealing; and a foot sequence that is 5-14 nucleotides long, perfectly complementary to the mutant sequence and mismatched to its wild-type sequence by at least three or more nucleotides, wherein:

[0040] (i) if the anchor sequence and the foot sequence of the primer are hybridized either to the mutant target sequence or to its closely related wild-type target sequence, there is in the target sequence an intervening sequence at least eight nucleotides long that does not hybridize to the primer's bridge sequence during primer-annealing, and the bridge sequence and the intervening sequence, together create a bubble in the hybrid having a circumference of 28-52 nucleotides,

[0041] (ii) the circumference of the bubble and the length of the foot sequence in combination result in a weak foot / mutant-target-sequence hybrid that makes copying the intended target sequence unlikely as evidenced by a delay of at least five cycles in the threshold cycle (CT) of amplification of said at least one mutant target sequence using said variant-signaling primer as compared to using a conventional primer, (iii) the bridge sequence and the foot sequence do not together prime non-target sequences in the mixture, and

[0042] (iv) the probability that during said cycling a variant-signaling primer / wild-type-target- sequence hybrid will be extended is at least 10,000 times lower than the probability that during said cycling a variant-signaling primer / mutant-target-sequence hybrid will be extended, as evidenced by a ACrof at least 13.3 cycles; and

[0043] (b) detecting amplified product or products with a dsDNA binding dye, or for each variantsignaling primer a fluorescent hybridization probe that signals upon hybridization to the amplification product of the primer, or for each variant-signaling primer a quenched, fluorescently labeled oligonucleotide hairpin at the primer's 5' end that fluoresces only when incorporated in or hybridized to the primer's amplified product.

[0044] In some embodiments, the foot sequence of each variant-signaling primer is mismatched to its wild-type target at any one or each of the primer's 3' nucleotide, 3' penultimate nucleotide, and 3' antepenultimate nucleotide. In some embodiments, the at least one mutant target sequence is cDNA. In some embodiments, the variant-signaling primer for the mutant target sequence has a foot sequence that is 6-7 nucleotides long, wherein the bubble circumference is 28-44 nucleotides long, and wherein detection of amplified product is by SYBR Green or another dsDNA binding dye.

[0045] In one aspect, provided is a multiplex assay method for amplifying and detecting in a sample copies of each of at least two different closely related, intended rare mutant DNA target sequences in the presence of copies of a wild-type allele of the mutant DNA target sequences (“a related wild-type DNA target sequence”), where the mutant DNA target sequences differ from each other and from the wild-type DNA target sequence, comprising:

[0046] (a) preparing a non-symmetric primer-dependent amplification reaction mixture that includes the sample, a DNA polymerase, deoxyribonucleoside triphosphates, other reagents required for amplification, 10-70 mM tetramethylammonium chloride (TMAC), a distinguishably labeled homogeneous fluorescence detection probe that is specific for an amplification product of each rare mutant DNA target sequence, an excess concentration of a reverse primer for the closely related mutant target sequences, and for each intended rare mutant target sequence, a limiting concentration of a unique variant-signaling primer: wherein the sequence of each variant-signaling primer comprises, in the 5' to 3' direction, the following three contiguous DNA sequences that are copied by extension of the reverse primer: an anchor DNA sequence that is sufficiently long so that it is able to hybridize with the closely related mutant DNA target sequences and with the related wild-type target sequence during primer annealing; a unique bridge DNA sequence at least four nucleotides long that does not hybridize during primer annealing to the unique variant-signaling primer's intended DNA target sequence, to any other closely related mutant target DNA sequences, or to the related wild-type DNA target sequence during primer annealing; and a unique foot DNA sequence that is at least 5 nucleotides long to 14 nucleotides long and that is perfectly complementary to the intended DNA target sequence but mismatches each other mutant target sequence and the related wild-type DNA sequence by three or more nucleotides, wherein all of the following requirements are met:

[0047] (i) if the anchor DNA sequence and the foot DNA sequence of the variant-signaling primer are both hybridized to its intended target DNA sequences thereby creating a primer-target hybrid, the primer-target hybrid comprises in the 5' to 3' direction of the variant-signaling primer: an anchor-target hybrid, a bubble, and a foot-target hybrid, said bubble having a circumference of 24 to 40 nucleotides and being formed by an intervening DNA sequence in the target DNA sequence that is at least four nucleotides long and does not hybridize to the primer's bridge DNA sequence during primer annealing;

[0048] (ii) the bubble isolates the function of the foot-target hybrid from the function of the anchortarget hybrid;

[0049] (iii) the variant-signaling primer that has generated an amplicon strand has bridge and foot DNA sequences that are perfectly complementary to the amplicon strand's complementary strand; and

[0050] (b) repeatedly cycling the reaction mixture to amplify the closely related rare mutant target DNA sequences present in the sample and detecting the presence of those DNA sequences by measuring the intensity of fluorescence from each distinguishably labeled probe by real-time or end-point detection. In some embodiments, cycling is temperature cycling in a polymerase chain reaction (PCR) method. In some embodiments, a Ct value for one target DNA sequence represents the same number of starting templates as it does for any other target DNA sequence. In some embodiments: i. for at least one intended rare target sequence the variant-signaling primer includes a 5'-tag sequence that is not complementary to any target sequence, that is unique for each target sequence or target-sequence group that is to be separately identified, and whose complement in the amplicon strand initiated by the reverse primer is the target of the probe; or ii. for at least one intended rare target sequence the target of the probe is the complement of the bridge sequence in the amplicon strand initiated by the reverse primer; or iii. for at least one intended rare target sequence the target of the probe is the interprimer sequence in the amplicon that lies between where the primers bind.

[0051] In some embodiments, the foot DNA sequence mismatches the closely related wild-type DNA sequence by three or more nucleotides, and wherein at least one of which is the 3 '-terminal nucleotide. In some embodiments, cycling the reaction mixture is performed by an instrument, wherein the number of different target DNA sequences exceeds the number of colors the instrument can separately detect, and wherein multiple different probes are thermospecific hybridization probes having the same fluorophore but the hybrids that they form have different melting temperatures. In some embodiments, amplification and detection are a digital PCR method. In some embodiments, the probes are color-coded molecular beacon probes. In some embodiments, provided is a kit of reagents for detecting the amplification product described herein by a fluorescent detection reagent.

[0052] BRIEF DESCRIPTION OF THE FIGURES

[0053] FIG. 1 is a schematic representation of a method for identifying genetic rearrangements, such as insertions, inversions, and / or translocations. This approach utilizes a variant-signaling primer whose foot sequence is designed to anneal to one of the junctions in the mutant template where the inserted sequence joins the target gene sequence. The foot sequence of the variantsignaling primer overlaps this junction, which contains the "breakpoint" within the junction. In addition, a conventional primer anneals to the insertion within the complementary strand containing the insertion, enabling the exponential amplification of the mutant. FIG. 2 is a schematic representation of a method for identifying genetic rearrangements, such as insertions, inversions, and / or translocations. The only difference between this method and the method illustrated in FIG. 1 is that the foot sequence of the variant-signaling primers is designed to hybridize to a sequence within the insertion, instead of the sequence containing the breakpoint.

[0054] FIG. 3 is a schematic representation of a method for detecting deletions. The occurrence of a deletion brings together the two sequences that flank the deletion, resulting in a new sequence that is exclusively present in the mutant. The foot sequence of the variant-signaling primer is designed to hybridize to this junction, but cannot hybridize to the non-deleted wild-type sequence. A conventional primer is designed to bind to the complementary strand that is outside of the deletion. If the deletion has occurred, the hybridization of the foot sequence of the variantsignaling primer, in combination with the hybridization of the conventional primer, results in exponential amplification. However, if the deletion has not occurred, the foot sequence of the variant-signaling primer cannot bind to the template, and the conventional primer can only initiate the synthesis of linear copies of the template.

[0055] FIG. 4 is a schematic representation that illustrates a method for detecting indels and small translocations that are difficult to identify. The technique employs a pair of variant-signaling primers, with each primer's foot sequence designed to hybridize to one of the two junctions where a small insertion, for example, has occurred.

[0056] FIGS. 5A and 5B illustrate sensitive detection of APC structural variant mutant templates in the presence of 30,000 wild-type templates. FIG. 5A is a schematic representation of Example 1, of how two different variant-signaling primers can be used to detect the presence of an insertion in the APC (adenomatous polyposis coli) gene exon 15. FIG. 5B is a graph of the results of the real-time PCR assay depicted in FIG. 5A, in which each reaction mixture contained 30,000 copies of wild-type gDNA as the target, along with 500, 50, 5, or 0 copies of the APC mutant targets.

[0057] FIG. 6 is a schematic representation of a method for detecting relatively large deletions, where the immediate sequences surrounding both ends of the deletion are joined together, and the resulting junction closely resembles a corresponding region of the deleted sequence. Thus, resulting in a new junction whose sequence is similar to the undeleted wild-type target sequence. The variant-signaling primer's anchor sequence is designed to bind upstream and near to that breakpoint; and the variant-signaling primer's foot sequence is designed to bind downstream of that junction region. The substantial non-deleted sequence within the wild-type target becomes a significant component of the potential bubble formed with the variant-signaling primer, thereby either thermodynamically forming a foot-target hybrid that exists for either an extremely short time or not at all. As a consequence, amplification is prevented. On the other hand, this sequence is deleted in the mutant strand, causing the bubble to be small, thereby enabling the formation of the foot hybrid, leading to the exponential amplification of the mutant target.

[0058] FIGS. 7A and 7B display the results of the real-time PCR assays described in Example 2, for the detection of two distinct small insertions (indels) commonly found in EGFR Exon 20, wherein each reaction mixture contains as targets, 30,000 copies of wild-type genomic DNA plus 500, 50, 5, or 0 copies of the EGFR mutant targets. FIG. 7A is a graph that shows the detection of sequence EGFR D770_N771insG. FIG. 7B is a graph that shows the detection of sequence EGFR D770_N771ins SVD.

[0059] FIGS. 8A and 8B show the results of the real-time PCR assays described in Example 3, designed to detect two distinct deletions commonly found in BRCA2. One deletion is classified as an indel, while the other deletion is considered to be a structural variant, because of its large size, wherein each reaction mixture contains as targets, 30,000 copies of wild-type genomic DNA plus 500, 50, 5, or 0 copies of the BRCA2 mutant targets. FIG. 8A is a graph that shows the detection of sequence BRCA2c. 6174delT & UK2223. FIG. 8B is a graph that shows the detection of sequence B RCA 2c. 6174delT & UK1999.

[0060] DETAILED DESCRIPTION

[0061] This disclosure provides assays that address a critical unmet need of highly sensitive detection of genomic modifications. These assays are primer-dependent amplification and detection methods such as, Reverse Transcription Polymerase Chain Reaction (RT-PCR) and Polymerase Chain Reaction (PCR) amplification. Provided are multiplex assays that are capable of detecting in a sample containing genomic DNA fragments the presence of at least two closely related mutations of a selected wild-type sequence in the presence of an abundance of the related wild-type sequence. Reaction mixtures utilized in such methods include a primer for each mutant target sequence.

[0062] Also provided are variant-signaling primers for primer-dependent nucleic acid amplification methods, including particularly polymerase chain reaction methods, that are capable of distinguishing between a rare intended target (e.g. , a mutant DNA target) and a closely related sequence (e.g., a wild-type DNA target) that differ in their genetic rearrangements or genomic modifications, such as methylated DNA. Variant-signaling primers offer numerous advantageous such as enabling high sensitivity for detecting genomic modifications. The variant-signaling primers described herein address a long-felt need of enabling early detection of genomic modifications. Early detection of genomic modifications is important because such detection facilitates early disease diagnosis such as early detecting of cancer before symptoms are present. Additionally, variant-signaling primers can be used to detect rare bacteria, viruses, or bacteriophages in infectious disease samples. Moreover, variant-signaling primers can detect rare genetic rearrangements and modifications in both plants and animals.

[0063] Variant-signaling primers detect and amplify DNA sequences that differ from wild-type targets by three or more nucleotides, making them invaluable for identifying genomic modifications, such as methylated DNA. Detecting methylated DNA typically involves a preparatory chemical treatment, like bisulfite conversion, which converts unmethylated cytosines into uracil while preserving methylated cytosines. Variant-signaling primers can be designed to target these modified sequences with precision.

[0064] After bisulfite treatment, methylated and unmethylated DNA sequences differ at cytosine residues. The primer's "foot" region is designed to perfectly match the methylated sequence, ensuring selective amplification. This foot region also incorporates intentional mismatches with the unmethylated sequence at critical positions, minimizing non-specific binding and extension.

[0065] When used in amplification reactions, such as real-time PCR or dPCR platforms, variantsignaling primers are capable of detecting low-abundance methylated DNA within a background of unmethylated DNA. They also can selectively amplify DNA with other modifications or base substitutions. Their high specificity and sensitivity make them powerful tools in epigenetic biomarker detection, particularly methylation patterns linked to cancer. They are also well- suited for liquid biopsy applications, enabling non-invasive detection of methylated DNA in blood or other body fluids for cancer monitoring. (Jinrong et al. DNA methylation profiles in cancer: functions, therapy, and beyond. Cancer Biology & Medicine. Dec 2023).

[0066] Notably, other mutations within the same or adjacent codons may also trigger or avoid amplification by the assay-specific variant-signaling primer, contingent upon the binding site of the foot relative to the breakpoint. This becomes especially important in scenarios where distinguishing between various deletions within the sample is required. Primer dimers are reduced or improved if the foot of the variant-signaling primer is accommodated at different positions. Additionally, the variant-signaling primers described herein achieve better sensitivity if three or more nucleotides mismatch the wild-type target. Furthermore, the foot target sequence gains more flexibility when the foot can be accommodated at different positions based on the breakpoint or junction sequence. Thus, a strong 3’ domain sequence serves as a "foot clamp." Furthermore, purine-pyrimidine mismatches in the foot target hybrid formed with the wild-type sequence (especially terminal G:T mismatches) can be avoided, thereby significantly repressing the initiation of amplification when the variant-signaling primers are hybridized to the abundant wildtype sequences.

[0067] Variant-signaling primers are made up of three distinct functional segments. The function of efficiently binding to a gene of interest is assigned to a relatively long sequence segment (termed the “5’ domain,” the “anchor sequence” or the “anchor”). The 5’ domain hybridizes at varying distances 5' from the genomic rearrangement junction. At the 3 ' end of a variant-signaling primer lies a shorter segment (termed the “3’ domain,” the “foot sequence” or the “foot”), that hybridizes at varying distances related to the junction. The foot sequence can contain three or more nucleotide mismatches compared to the wild-type sequence and form a perfectly complementary hybrid with the altered or chimeric junction target sequence. Alternatively, the foot sequence can span the breakpoint or junction and anneal within the region inserted during the rearrangement. This ensures that the 3’ domain will not anneal in the absence of the mutation. In other words, the primer is only extended by DNA polymerase when the genetic rearrangement is present. These two distinct sequence domains are connected by a non-hybridizing segment of synthetic sequence (the middle domain or the bridge sequence), carefully designed to maintain the 5' and 3' hybridizing segments at an optimal distance from each other.

[0068] The middle domain (referred to as the “middle domain,” “bridge sequence,” or “bridge”) can bridge across the breakpoint or junction. The bridge is chosen so as to ensure that it does not form secondary structures and is not complementary to the “intervening sequence” in the template molecule that joins the target sequence for the anchor to the target sequence for the foot. Consequently, when the primer is hybridized to a template molecule, the bridge sequence in the primer and the intervening sequence in the template form a single-stranded “bubble” with the intervening sequence in the template molecule that separates the gene-identification function of the anchor hybrid from the mutant-identification function of the foot hybrid. In some embodiments, the variant-signaling primers comprise an anchor sequence that binds to the gene of interest in both the mutant and the wild type, and a foot sequence that possesses at least three nucleotides that are complementary to the mutant target sequence, but mismatch the closely related wild-type sequence.

[0069] As used herein, the term “breakpoint,” refers to the precise locations in the genome where genetic rearrangements occur, indicating sites where DNA strands are broken and then reattached in a different configuration.

[0070] Non-contiguous primers, such as variant-signaling primers, offer a more adaptable approach. Since a variant-signaling primer’ s foot is shorter than that of conventional primers, they enhance specificity. This targeted approach enables accurate amplification only at the desired junction and improves detection accuracy.

[0071] The variant-signaling primers described herein contain a foot sequence. The foot sequence is located at the 3' end of the primer and is complementary to the region of the mutant target. In some embodiments, the foot sequence is complementary to the mutant sequence and mismatched to its wild-type sequence by multiple nucleotide sequences such as 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 nucleotides. In one embodiment, the foot sequence is complementary to the mutant sequence and mismatched to its wild-type sequence by at least three nucleotides. The three or more nucleotides in the foot of the variant-signaling primers that are complementary to nucleotides in the mutant target sequence to which the foot binds need not be consecutive, nor do they need to be at the 3' end of the foot of the variant-signaling primer. In one embodiment, the foot sequence is completely mismatched to its wild-type sequence. In one embodiment, the foot sequence is complementary to the mutant and the wild-type sequence (for instance, in the case of relatively large deletions).

[0072] In one embodiment, the anchor sequence and the foot sequence of the primer are hybridized either to the mutant target sequence or to its closely related wild-type target sequence. In one embodiment, there is an intervening sequence present in the target that does not hybridize to the primer's bridge sequence during primer-annealing, and the bridge sequence and the intervening sequence together create a single-stranded bubble in the hybrid having a circumference of at least twelve nucleotides, wherein the intervening sequence is at least four nucleotides long. In one embodiment, the circumference of the bubble does not need to maintain the same size when formed with the mutant compared to the circumference of the bubble formed with the wild-type. In one embodiment, the bubble has a circumference of 12 to 15 nucleotides such as 12 nucleotides, 13 nucleotides, 14 nucleotides, or 15 nucleotides. In one embodiment, the bubble has a circumference of 24 to 40 nucleotides. In one embodiment, the bubble has a circumference of 28 to 44 nucleotides. In one embodiment, the bubble has a circumference of 28 to 52 nucleotides, such as 28, 29, 30, 31, 31, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 59, 50, 51, or 52 nucleotides.

[0073] In one embodiment, the circumference of the bubble is not limited to a particular size, as long as it remains single-stranded and separates the function of the anchor sequence from the function of the foot sequence.

[0074] In one embodiment, the variant-signaling primers described herein are used in a multiplex assay (the simultaneous measurement of different substances from a single sample). In these assays, the variant-signaling primers can have unique bridge sequences that enable probes (such as molecular beacon probes) to identify which primer was extended due to the presence of a specific mutation. Having different bridges for primers designed to detect mutations present in the same or nearby codons is crucial, as it ensures that the amplicon generated from the extension of the variant-signaling primer will differ in the newly incorporated bridge sequence. This approach maintains assay specificity, even when amplicons from multiple mutants are amplified in the same reaction, because only the correct primer will anneal to the target sequence. Alternatively, a 5'-tag sequence can serve a similar function, allowing molecular beacon probes to bind to the complement of the 5’-tag sequence in the generated amplicons during the amplification process. The presence of a unique bridge sequence or a 5 ’-tag sequence ensures that only the targeted DNA or mRNA isoform is amplified and labeled, facilitating the precise detection and analysis of genomic modifications, genetic rearrangements, or alternative splicing events.

[0075] In one embodiment, the bridge sequence is four nucleotides long. In one embodiment the bridge sequence is five nucleotides long.

[0076] In one embodiment, the variant-signaling primers described herein are designed to function as shown in FIG. 1. Specifically, if the mutation involves an insertion, the foot sequence of the variant-signaling primer is designed to anneal to one of the junction regions overlapping the breakpoint where the inserted sequence joins the original target sequence in the mutant template; and the anchor of that variant-signaling primer is designed to anneal to the target sequence near to that junction. The conventional primer is designed to anneal to the complementary strand within the inserted sequence, or to anneal downstream of the inserted sequence beyond the other junction.

[0077] In one embodiment, the variant-signaling primers described herein are designed to function as shown in FIG. 2. In this case, the foot sequence of the variant-signaling primers described herein is designed to anneal to a sequence within the insertion. The middle domain of each variantsignaling primer bridges the junction between the inserted sequence and the original target strand, but is designed to not hybridize to the bridged junction; and the anchor of that variant-signaling primer is designed to anneal to the target sequence near to that junction.

[0078] In one embodiment, the variant-signaling primers described herein are designed to function as shown in FIG. 3. In this case, the foot sequence of the variant-signaling primer is designed to anneal to the junction region overlapping the breakpoint where the two target sequences that surrounded the deleted sequence are joined together; whereas the anchor of that variant-signaling primer is designed to anneal to the target sequence near to that junction. In this embodiment, the conventional primer is designed to anneal to the complementary strand downstream of that breakpoint.

[0079] In one embodiment, the variant-signaling primers described herein are designed to function as shown in FIG. 4. In this case, where a pair of different variant-signaling primers described herein are utilized to detect small insertions, each primer annealing to a different complementary strand of the mutant target, the anchor of each variant-signaling primer anneals to a sequence within the original target strand close to the insertion; and the foot of each primer anneals to one of the junction regions overlapping the breakpoint where the inserted sequence joins the original target sequence in the mutant template.

[0080] In one embodiment (FIG. 5), the variant-signaling primers described herein are utilized to detect a large insertion (where the insertion harbors one or more distinct internal mutations), each primer annealing to a different complementary strand of the mutant target, the anchor of one variant-signaling primer is designed to anneal to the target sequence near to the junction of the inserted sequence and the original target sequence, while the foot of that variant-signaling primer is designed to anneal within the insertion. The other variant-signaling primer is designed to anneal to a sequence within the insertion, and its foot is designed to anneal to a portion of the insertion containing one or more of the distinctive mutations, thereby ensuring increased specificity. In one embodiment, if the mutation inside the inserted sequence is an SNV and the second primer is a SuperSelective primer. If more than two nucleotides differ, then the second primer is a variant-signaling primer.

[0081] In one embodiment, pairs of variant-signaling primers described herein are utilized to detect deletions, each primer in a pair annealing to a different complementary strand of the mutant target, and the anchor of each variant-signaling primer is designed to anneal to the original target strand close to the breakpoint, while their feet overlap the junction region, potentially with one or more complementary nucleotides shared between the feet of both primers.

[0082] In one embodiment, the anchor domain of a variant-signaling primer described herein is designed to anneal to an exon close to the junction neighboring the aberrant sequences, while the foot of the variant-signaling primer is designed to specifically anneal to the aberrant sequence that is unique to each splice variant. This occurs in instances involving intragenic rearrangements, which occur as the result of aberrant RNA splicing, where one exon is shared among a number of different splice variants, but the adjacent exon differs.

[0083] In one embodiment (FIG. 6), if the mutation involves a deletion (for example, at least 40 nucleotides in length) where the immediate sequences surrounding both ends of the deletion become joined together, and if in addition that junction closely resembles the corresponding region of the deleted sequence, resulting in a new junction whose sequence is similar to the undeleted wild-type target sequence, then the variant-signaling primer's anchor is designed to bind near to that breakpoint; and the primer's foot is designed to bind downstream of that junction region, with the middle domain of the variant-signaling primer designed to span the deleted sequence so that it anneals to a unique sequence in the wild-type target. Consequently, this bridge creates a smaller single-stranded bubble with the intervening sequence in the mutant target in which the deletion has occurred, and it forms a much larger single-stranded bubble with the intervening sequence (containing the potential deleted region) in the wild-type target in which the deletion has not occurred. Although both the 5’ and 3’ domains of the variant-signaling primer are designed to bind to both the wild-type target and to the mutant target, the thermodynamic characteristics related to the length of the bubble formed between the bridge and the intervening sequence in the mutant target favors the formation of hybrids between the foot and its corresponding mutant target, and that hybrid persists much longer than the persistence time of the highly improbable hybrid formed with the mismatched wild-type target. In contrast, a conventional primer is designed to bind to the complementary strand downstream from the foot-binding region, downstream of the breakpoint.

[0084] In one embodiment, a pair of different variant-signaling primers described herein are utilized to detect small insertions, each primer binding to a different complementary strand of the mutant target, the anchor of each variant-signaling primer anneals to a sequence within the original target strand close to the insertion; and the foot of each primer anneals to a sequence within the insertion. The bridge sequence of each variant-signaling primer bridges the junction between the inserted sequence and the original target strand, but is designed to not hybridize to the bridged junction.

[0085] Variant-signaling primers differ from SuperSelective primers not only in the number of nucleotides that mismatch to the wild-type target present in the foot, but also in the different schemes that can be used with such primers. Examples of their use include, but are not limited to:

[0086] 1) a foot that binds to the unique junction where an insertion combines with the original sequence;

[0087] 2) a foot that binds to a unique sequence within an insertion; 3) a foot that binds to the unique junction where a deletion has occurred; 4) a situation in which two variant-signaling primers are utilized, one of which binds to the unique junction at one end of an insertion, and the other of which binds to the unique junction at the other end of the insertion; and 5) a situation in which two variant-signaling primers are utilized, with the foot of each primer binding within a unique insertion, one at one end of the genetic rearrangement, and the other at the opposite end within the same genetic rearrangement.

[0088] As used herein, the term “genomic modifications” refers to changes involving three or more nucleotides in the genome that do not involve alterations to the DNA sequence itself, but that can still impact gene expression, regulation, and cellular functions. These modifications can be epigenetic, meaning they affect how genes are expressed without changing the underlying genetic code. Examples of such genomic modifications include, but are not limited to, methylated DNA.

[0089] As used herein, the term “genetic rearrangements” refers to structural DNA or RNA variants that are larger than single-nucleotide substitutions. Examples of such genetic rearrangements include, but are not limited to, small deletions, large deletions, insertions (e. ., from the same chromosome, a different chromosome, a virus, or a bacterium), translocations (often between non-homologous chromosomes), copy number alterations (CNAs), inversions, gene amplifications, and gene fusions. Genetic rearrangements can influence the biology of cancer in diverse ways. First, genetic rearrangements can disrupt gene organization, and perturb crucial cellular pathways, such as those governing cell division, and ultimately compromising gene function. Second, the accumulation of structural variations, encompassing SVs, indels, or CNVs, along with single nucleotide variants (SNVs), can contribute to genome instability by indirectly inducing DNA damage and affecting DNA repair mechanisms. This includes mutations affecting DNA damage sensors and checkpoint genes, such as the tumor suppressors TP53, CEIEK1, CEIEK2, BRCA1, and BRCA2.

[0090] In some embodiments, mutations are present and occur on the same chromosome (in cis), in which case the protein encoded by that gene on the mutant chromosome contains both mutations. Whereas in other embodiments, mutations occur on separate sister chromosomes (in trans).

[0091] Specific genetic modifications and rearrangements serve as key hallmarks of cancer subtypes, clinical outcomes, and responses to therapy. Some of these changes alter gene expression patterns and impact the efficacy of immunotherapy for patients. Early identification of these alterations could facilitate the implementation of tailored immunotherapy treatments targeting the molecular characteristics of individual tumors. Some may be passenger mutations with no direct functional impact, but can still serve as biomarkers for diagnostic purposes. The ultimate goal is the early detection of rare genetic modifications and rearrangements present in individuals before symptoms manifest, to significantly improve the likelihood of successful treatment and cure.

[0092] Primer-dependent amplification reactions useful in methods described herein may be any suitable exponential amplification method, including the polymerase chain reaction (PCR), the ligase chain reaction (LCR), the nicking enzyme amplification reaction (NEAR), strand-displacement amplification (SDA), nucleic acid sequence-based amplification (NASBA), transcription-mediated amplification (TMA), loop-mediated isothermal amplification (LAMP), and rolling circle amplification (RCA). Preferred methods utilize PCR. In non-symmetric PCR amplification methods, for example asymmetric PCR, one primer, the limiting primer, is present in a limiting amount so as to be exhausted prior to completion of amplification, after which linear amplification occurs, using the remaining primer, the excess primer. A non-symmetric PCR method useful in this disclosure is LATE-PCR (see, for example, European Patent No. EP 1,468,114) and Pierce KE et al. Linear-After-The-Exponential (LATE)-PCR: primer design criteria for high yields of specific single-stranded DNA and improved real-time detection. Proceedings of the National Academy of Sciences (PNAS). 2005 Jun 14;102(24):8609-14). In a non-symmetric amplification method according to this disclosure, the variant-signaling primer or, in the case of a variant-signaling primer pair, one of the variant-signaling primers used to detect a mutant, serves as the limiting primer. Preferred methods also include digital PCR (Vogelstein B et al. Digital PCR. Proceedings of the National Academy of Sciences (PNAS). 1999 Aug 3;96(16):9236-41), where it is desirable to detect amplicons from a single mutant template molecule that is present in reactions that contain related wild-type molecules. Carrying out variantsignaling primer assays in a digital PCR format can enhance assay sensitivity and provide a precise quantitative assessment of the target nucleic acid molecules present in the sample (Garcia-Murillas I et al. Assessment of Molecular Relapse Detection in Early-Stage Breast Cancer. JAMA Oncol. 2019 Oct l;5(10): 1473-1478).

[0093] In some embodiments, either variant-signaling forward primer or the conventional reverse primer can serve as the limiting primer, allowing for the addition of a 5' tag to either primer. In some embodiments, an interprimer-specific molecular beacon is designed to anneal to either the sense or antisense strand.

[0094] If the amplification reaction utilizes an RNA-dependent DNA polymerase (an example being NASBA), the amplification reaction is isothermal. Repeated rounds of synthesis of amplified product in these isothermal reactions are referred to as “cycles”, but they are not actually thermal cycles. For such amplifications, the “intended target sequence” and the “unintended target sequence” that are primed by a variant-signaling primer according to this disclosure are RNA sequences that occur in the original sample and in the amplification reaction mixture, where they are present with the DNA polymerase and the variant-signaling primer.

[0095] If the amplification reaction utilizes a DNA-dependent DNA polymerase (an example being PCR), an original sample may contain either DNA or RNA targets. For such amplifications, the “intended target sequence” and the “unintended target sequence” that are primed by a variantsignaling primer that is useful in methods of this disclosure are DNA sequences that either occur in an original sample or are made by reverse transcribing RNA sequences that occur in the original sample. If the variant-signaling primer is used for reverse transcription, the “intended target sequence” and the “unintended target sequence" are RNA as well as cDNA. If a separate, outside primer is used for reverse transcription, the “intended target sequence” and the “unintended target sequence” are cDNA. In either case, the “intended target sequence” and the “unintended target sequence” are nucleic acid sequences that are present in the amplification reaction mixture with the DNA polymerase and the variant-signaling primers. Primer-dependent amplification reactions comprise repeated thermal cycles of primer annealing, primer extension, and strand denaturation (strand melting). Primer annealing may be performed at a temperature below the primer-extension temperature (for example, three-temperature PCR), or primer annealing and primer extension may be performed at the same temperature (for example, two-temperature PCR). The overall thermal profile of the reaction may include repetitions of a particular cycle, or temperatures / times may be varied during one or more cycles. For example, once amplification has begun and the priming sequence of a variant-signaling primer is lengthened, a higher annealing temperature appropriate for the longer primer might be used to complete the amplification reaction. This concept is referred to as “temperature jump” and it is used to optimize the specificity and efficiency of the PCR reaction by adjusting the annealing temperature dynamically during cycling. Unlike traditional PCR methods that employ a fixed annealing temperature throughout, temperature-jump PCR utilizes a series of cycles with varying annealing temperatures. This approach ensures the robust amplification of the desired product while effectively reducing nonspecific amplification. Through the dynamic optimization of annealing temperatures, temperature-jump PCR significantly improves the accuracy and reliability of assay results.

[0096] Reagents required for the amplification reaction described herein include, but are not limited to tetramethylammonium chloride (TMAC) or other types of Hofmeister salts, quaternary ammonium compounds (QACs), or quaternary ammonium salts (e.g., betaine, tetramethylammonium chloride, Zizx-tetramethylammonium oxalate, etc.). Hofmeister salts or quaternary salts can be used at various concentrations ranging from 1-100 mM such as 1 mM, 10 mM, 20 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, 80 mM, 90 mM or 100 mM. Betaine can be used at concentrations such as (but not limited to) at least 100 mM, 150 mM, 200 mM, etc. Chemicals, such as tetramethylammonium chloride, that enhance the specificity of interaction between the foot sequence and the target sequence, and minimizes the binding of the foot sequence to the wild-type target.

[0097] In some embodiments, samples are enriched using a pre-amplification step. Each cycle of pre-amplification creates a number of new copies of each target sequence (mutant and wild-type) equal to the starting number. Pre-amplification is described in U.S. Patent No. 11,542,547, which is incorporated herein by reference in its entirety. In one aspect, provided is an amplification and detection method that is capable of detecting as few as five copies of at least one rare mutant DNA target sequence in a mixture containing, for each mutant target sequence, 10,000 copies of a closely related wild-type DNA target sequence, comprising:

[0098] (a) repeatedly cycling a reaction mixture in a primer-dependent amplification reaction having a primer-annealing temperature, said reaction mixture including said at least one mutant target sequence or its closely related wild-type target sequence or both, a DNA polymerase, other reagents needed for amplification, and for each mutant target sequence a primer pair that includes a variant-signaling primer comprising, in the 5' to 3' direction the following three contiguous DNA sequences: an anchor sequence that hybridizes with the mutant target sequence and with its closely related wild-type target sequence during primer annealing; a bridge sequence at least four nucleotides long that does not hybridize to either the mutant target sequence or its closely related wild-type target sequence during primer annealing; and a foot sequence that is at least five nucleotides long, perfectly complementary to the mutant sequence and mismatched to its wild-type sequence by at least three or more nucleotides, wherein:

[0099] (i) if the anchor sequence and the foot sequence of the primer are hybridized to the mutant target sequence, there is in the target sequence an intervening sequence that does not hybridize to the primer's bridge sequence during primer-annealing, and the bridge sequence and the intervening sequence, together create a bubble in the hybrid having a circumference of at least twelve nucleotides, wherein the intervening sequence is at least four nucleotides long,

[0100] (ii) the circumference of the bubble and the length of the foot sequence in combination result in a weak foot / mutant-target-sequence hybrid that makes copying the mutant target sequence unlikely,

[0101] (iii) the bridge sequence and the foot sequence do not together prime non-target sequences in the mixture, and

[0102] (iv) the probability that during said cycling a variant-signaling primer / wild-type target sequence hybrid will be extended is at least 10,000 times lower than the probability that during said cycling a variant-signaling primer / mutant target sequence hybrid will be extended, as evidenced by a ACrof at least 13.3 cycles; and

[0103] (b) detecting amplified product or products with a dsDNA binding dye, or for each variantsignaling primer a fluorescent hybridization probe that signals upon hybridization to the amplification product of the primer, or for each variant-signaling primer a quenched, fluorescently labeled oligonucleotide hairpin at the primer's 5' end that fluoresces only when incorporated in or hybridized to the primer's amplified product, wherein copying is “unlikely” means that, if the reaction were begun with a reaction mixture containing 106copies of the at least one mutant DNA target sequence, no wild-type sequence and the variant-signaling primer, and separately with a reaction mixture containing 106copies of the at least one mutant DNA target sequence, no wild-type sequence and a corresponding conventional primer in place of the variant-signaling primer, the threshold cycle (CT) of the reaction utilizing the variant-signaling primer would be delayed by at least three cycles compared to the CT of the reaction utilizing the conventional primer.

[0104] In some embodiments, when the variant-signaling primer's anchor and foot anneal to the mutant target, an intervening sequence in the target remains unbound by the bridge. This results in the formation of a single- stranded bubble, which occurs in cases of any genetic rearrangement, such as deletions or inversions.

[0105] In some embodiments: i. the bridge sequence is at least 6 nucleotides long, the intervening sequence is at least 6 nucleotides long, and the bubble has a circumference of 28-52 nucleotides; or ii. for each of the at least one variant-signaling primers, the CT delay for a reaction begun with 106copies of the mutant DNA target sequence and the variant-signaling primer as compared to a reaction begun with 106copies of the mutant DNA target sequence and a conventional primer is at least 5 cycles.

[0106] In some embodiments, the bubble circumference is 28-44 nucleotides. In some embodiments, each variant-signaling primer contains a unique functional moiety located 5' to the anchor sequence, said functional group not hybridizing either to the mutant target sequence or to the wild-type target sequence.

[0107] In one aspect, provided is a primer-dependent amplification and detection method that is capable of amplifying and detecting in a sample as few as five copies of at least one rare DNA intended target sequence (“rare target sequence”) in a mixture containing, for each rare target sequence, 10,000 copies of a closely related unintended target sequence (“closely related sequence” or “unintended target sequence”) that differs from the rare target sequence by at least three nucleotides, comprising:

[0108] (a) preparing a primer-dependent amplification reaction mixture that includes the sample, a DNA polymerase, deoxyribonucleoside triphosphates, an amplification buffer, homogeneous fluorescence detection means for detecting amplification products, and for each rare target sequence a pair of a first primer and a second primer that are specific for the rare target sequence but mismatched to the closely related sequence,

[0109] (b) repeatedly cycling the primer-dependent amplification reaction mixture by said primerdependent amplification method to amplify each rare target sequence present in the sample, and

[0110] (c) detecting that rare target sequence by measuring the intensity of fluorescence from the homogeneous fluorescence detection means; wherein (i) the first primer is an allele-discriminating variant-signaling primer comprising from the 5' end to the 3' end a first anchor sequence, a first bridge sequence, and a first foot sequence that is mismatched to the closely related sequence by at least three nucleotides, and (ii) the second primer is an allele-discriminating primer.

[0111] As used herein, the term “an allele-discriminating primer” is a nucleic acid (e.g., DNA) amplification primer that has an internal sequence, termed a “bridge sequence”, that is not sufficiently complementary to the target sequence to hybridize therewith under primer-annealing conditions and that is sandwiched between two target-complementary sequences that are termed an “anchor sequence” and a “foot sequence.” Examples of an allele-discriminating include, but are not limited to an ARMS primer, a SuperSelective primer, and a variant-signaling primer.

[0112] In some embodiments, each primer contains a 3 '-terminal interrogating nucleotide that is complementary to the rare target sequence, but mismatched to the unintended target sequence. In some embodiments, the at least one rare target sequence in the sample includes at least two different rare target sequences, and the homogeneous fluorescence detection means comprises at least two different homogeneous fluorescence detection probes for the at least two different rare target sequences respectively. In some embodiments, each different unintended target sequence differs from its corresponding rare target sequence by at least three base pairs, and both the first and the second primers are mismatched to said base pairs. In one embodiment, the probe is a shared-stem molecular beacon. In some embodiments, the homogeneous fluorescence method for detecting each rare target sequence comprises an interprimer-specific molecular beacon probe. In one embodiment, the amplification reaction is a symmetric PCR reaction. In some embodiments, each variant-signaling primer contains a unique functional moiety located 5' to the anchor sequence, said functional group not hybridizing either to the mutant target sequence or to the wildtype target sequence. In some embodiments, the at least one mutant target sequence comprises at least two mutant target sequences that are close to each other in an intended target, wherein there is a variant-signaling forward primer for each of said at least two mutant sequences and a common conventional reverse primer, wherein each variant-signaling primer has a different bridge sequence, and wherein amplified products from each variant-signaling primer are separately detected.

[0113] In one aspect, provided is an amplification and detection method, contingent upon the identity of the mismatch, that is capable of detecting as few as five copies of at least one rare mutant DNA target sequence in a mixture containing, for each mutant target sequence, 10,000 copies of a closely related wild-type DNA target sequence, comprising:

[0114] (a) repeatedly cycling a reaction mixture in a primer-dependent amplification reaction having a primer-annealing temperature, said reaction mixture including said at least one mutant target sequence or its closely related wild-type target sequence or both, a DNA polymerase, other reagents needed for amplification, and for each mutant target sequence a primer pair that includes a variant-signaling primer comprising, in the 5' to 3' direction the following three contiguous DNA sequences: an anchor sequence that hybridizes with the mutant target sequence and with its closely related wild-type target sequence during primer annealing; a bridge sequence at least four nucleotides long that does not hybridize to either the mutant target sequence or its closely related wild-type target sequence during primer annealing; and a foot sequence that is 5-14 nucleotides long, perfectly complementary to the mutant sequence and mismatched to its wild-type sequence by at least three or more nucleotides, wherein: (i) if the anchor sequence and the foot sequence of the primer are hybridized either to the mutant target sequence or to its closely related wild-type target sequence, there is in the target sequence an intervening sequence at least eight nucleotides long that does not hybridize to the primer's bridge sequence during primer-annealing, and the bridge sequence and the intervening sequence, together create a bubble in the hybrid having a circumference of 28-52 nucleotides,

[0115] (ii) the circumference of the bubble and the length of the foot sequence in combination result in a weak foot / mutant-target-sequence hybrid that makes copying the intended target sequence unlikely as evidenced by a delay of at least five cycles in the threshold cycle (CT) of amplification of said at least one mutant target sequence using said variant-signaling primer as compared to using a conventional primer,

[0116] (iii) the bridge sequence and the foot sequence do not together prime non-target sequences in the mixture, and

[0117] (iv) the probability that during said cycling a variant-signaling primer / wild-type-target- sequence hybrid will be extended is at least 10,000 times lower than the probability that during said cycling a variant-signaling primer / mutant-target-sequence hybrid will be extended, as evidenced by a ACrof at least 13.3 cycles; and

[0118] (b) detecting amplified product or products with a dsDNA binding dye, or for each variantsignaling primer a fluorescent hybridization probe that signals upon hybridization to the amplification product of the primer, or for each variant-signaling primer a quenched, fluorescently labeled oligonucleotide hairpin at the primer's 5' end that fluoresces only when incorporated in or hybridized to the primer's amplified product.

[0119] In some embodiments, the foot sequence of each variant-signaling primer is mismatched to its wild-type target at any one or each of the primer's 3' nucleotide, 3' penultimate nucleotide, and 3' antepenultimate nucleotide. In some embodiments, the at least one mutant target sequence is cDNA. In some embodiments, the variant-signaling primer for the mutant target sequence has a foot sequence that is 6-7 nucleotides long, wherein the bubble circumference is 28-44 nucleotides long, and wherein detection of amplified product is by SYBR Green or another dsDNA binding dye. In some embodiments, provided is a kit of reagents for detecting the amplification product described herein by a fluorescent detection reagent. The kit can further comprise instructions for use.

[0120] In a multiplex assay utilizing variant-signaling primers, each primer is designed to have a unique bridge sequence, which when bound to the target gene forms a single-stranded bubble with the intervening sequence in the template that separates the function of the anchor sequence from the function of the foot sequence. The complement of the unique bridge sequence can optionally serve as a molecular "bar code" that enables probes, such as molecular beacons, to identify which primer was extended due to the presence of a specific structural variation. Alternatively, incorporating a 5'-tag sequence into a variant-signaling primer can fulfill a similar barcoding function. This addition allows molecular beacons to bind specifically to the complementary sequence of this tag within the synthesized complementary amplicon during the amplification process. In some embodiments, each variant-signaling primer possesses a unique identifying bridge sequence, and in multiplex exponential amplification assays they simultaneously identify and quantitate different target sequences in the same sample.

[0121] Another method for detecting amplicons generated by the variant-signaling primers described herein involves the use of "thermospecific" hybridization probes, preferably molecular beacon probes, such as sloppy molecular beacon probes. Each variant-signaling primer will feature a distinctive "5'-tag sequence," with the concentration of each of these primers being limited. This ensures that after exponential amplification, there is an excess of complementary amplicon strands containing the complement of the primer's 5'-tag sequence, which then serves as a target for the hybridization of identifying molecular beacon probes. Following amplification, a post-amplification melt can be conducted, typically using a spectrofluorometric thermal cycler, allowing probes to be distinguished from one another based on the melting temperature (Tm) of the hybrid that it forms with its target sequence, and in combination with the identity of the color of the probe’s fluorescent signal.

[0122] Variant-signaling primers described herein can be used in a variety of amplicon detection methods. These methods include, but are not limited to the use of TaqMan probes, molecular beacon probes, and intercalating fluorescent dyes. In particular, fluorescent amplicon detection probes light up either the complement of a 5'-tag sequence on the variant-signaling primer that becomes incorporated into the 3'-end of the amplicon; the complement of the variant-signaling primer's bridge sequence; or the use of an "interprimer-specific molecular beacon" that lights up a portion of the amplicons that lies between where the primers bind.

[0123] Screening assays are multiplex assays in which it is expected that only one of many possible target sequences will be present in a sample. In one embodiment, the hybridization probes are used for in situ hybridization techniques, such as fluorescence in situ hybridization (FISH). Employing variant-signaling primers in FISH techniques offers a potent means to illuminate specific genetic alterations within DNA or RNA. This advancement holds promise for deepening the understanding of how these genetic variations influence disease susceptibility and progression. For example, variant-signaling primers that serve as fluorescent in situ hybridization probes light up mutant messenger RNAs (or cells possessing mutant chromosomes) in image-based techniques.

[0124] In these embodiments, one or more of the primers described herein are variant-signaling primers. The ability of variant-signaling primers to detect genomic modifications and rearrangements can be used to understand and address a spectrum of diseases, including but not limited to cancer, genetic disorders, developmental disorders, neurological disorders, and hematological disorders. Epigenetic therapies, such as DNA methyltransferase inhibitors (e.g., azacitidine, decitabine), are being developed to target some of these changes.

[0125] An effective approach for detecting splicing variants, as described herein, involves the utilization of variant- signaling primers that anneal precisely at the exon-exon junctions where alternative splicing events occur. Once a reverse transcription PCR (RT-PCR) assay is conducted using these primers, it enables accurate detection and analysis of the splicing variants. The methods described herein enable the targeting of precise regions of interest within the transcriptome. By strategically designing variant-signaling primers to span the splice junctions, the RT-PCR assay becomes highly selective, amplifying only the cDNA fragments corresponding to the alternative splicing variants under investigation.

[0126] As used herein, the term “alternative splicing” refers to a process in which different combinations of exons are included or excluded from mRNA transcripts. Variations in mRNA transcripts, originated from the same gene, can yield a different pattern of protein isoforms, each possessing distinct structural and functional attributes. These different proteins have the potential to impact crucial cellular processes such as proliferation, apoptosis, and metastasis that promote cancer cell growth (Kanayama M et al. AR Splicing Variants and Resistance to AR Targeting Agents. Cancers (Basel). 2021 May 23; 13(11):2563). Examples of alternative splicing include, but are not limited to, variable exon skipping and alternative 3' and 5' splicing site usage.

[0127] Genes susceptible to variable exon skipping, with significant implications for cancer, include, but are not limited to the TP53 gene, which encodes tumor protein 53, and which is associated with breast and colorectal cancers; and the epidermal growth factor receptor gene (EGFR), which is associated with non-small cell lung cancer and glioblastomas. Certain isoforms of these genes may confer resistance to targeted therapies, which emphasizes the importance of understanding and targeting these alternative splicing mechanisms in cancer treatment.

[0128] An example of a gene where alternative 3'- or 5'-splicing-site usage has been implicated in cancer is CD44. CD44 is expressed in a large number of mammalian cell types, and encodes a cell-surface glycoprotein whose isoforms are related to breast cancer, colorectal cancer, and pancreatic cancer. Also, alternative splicing of BRCA1 pre-mRNA can lead to the production of truncated isoforms, which lack functional domains, and can contribute to tumorigenesis associated with hereditary breast and ovarian cancers.

[0129] The diagnostic identification of these alternative splicing isoforms has significant implications in clinical diagnostics and in the choice of effective therapeutic responses (El Marabti E et al. The cancer spiiceosome: reprograming of alternative splicing in cancer. Frontiers in Molecular Biosciences. 2018 5:80; El Marabti E et al. Alternative splicing and cancer: a systematic review. Signal Transduction and Targeted Therapy. 2021 6, 78; Yan Y et al. RNA splicing alterations in lung cancer pathogenesis and therapy, Cancer Pathogenesis and Therapy. 2023 1 , 272-283; Fackenthal JD. Alternative mRNA splicing and promising therapies in cancer. Biomolecules. 2023 13, 561; and Bradley RK, Anczukow O. RNA splicing dysregulation and the hallmarks of cancer. Nature Reviews, Cancer. 2023 23, 135—155). Variant-signaling primers provide a sensitive strategy for detecting and investigating these alternative splicing events, and in facilitating the exploration of the diversity of mRNA isoforms that originate from a single gene.

[0130] Existing assays require knowledge of the exact mutant sequence that is present within which the single-nucleotide substitution occurs, whereas many genetic modifications and genetic rearrangements can occur at different positions. There also can be many different genetic rearrangements causing the same disease and it is not practical to have a large set of variantsignaling primers. These genetic rearrangements typically occur in positions within the gene that are near each other, and designing many primers to bind to adjacent regions could lead to competition among the primers, which may negatively affect the overall sensitivity of the multiplex assay ( . e. , it is desirable to have a setup where only one or two variant-signaling primers will enable the detection of a larger set of relevant genetic alterations).

[0131] A special case arises in hotspot regions, where various mutations may arise from the deletion of relatively large sequences within a gene. In such instances, the anchor sequence of the variant-signaling primer is designed to anneal to the conserved region upstream of the potential breakpoint, while the foot of the variant-signaling primer is designed to hybridize to a conserved region downstream of the potential breakpoint. Regardless of whether any of the mutant deletions have occurred, the deleted sequences (relative short intervening sequences) promote the formation of the hybrid between the foot and its target sequence, facilitating the amplification of any of the mutant targets. Conversely, the undeleted sequence in the wild-type target generates a substantial bubble between the bridge of the variant-signaling primer and the relatively long intervening sequence. This ensures that amplification occurs only if any deletions occurred, because when the circumference of the bubble is very large, the random Brownian motion of the assay fluid rapidly pulls the foot hybrid apart before a DNA polymerase is likely to encounter a transient foot-target hybrid.

[0132] Another special case occurs when two different sequences are inserted next to each other in a target gene, in which case there are two different breakpoints (each detected by a different variant-signaling primer, and each primer is bound to a different complementary strand). In this case, an exponentially amplified amplicon only occurs if both insertions have taken place.

[0133] Importantly, traditional methods, such as PCR using conventional primers, lack the capability to detect indels or small translocations resulting in either a single breakpoint (small deletions) or two distinct characteristic breakpoints located in close proximity (small insertions). However, employing variant-signaling primer pairs ensures exponential amplification solely when either the breakpoint or both targeted breakpoints (no matter their rarity) are present in the analyzed nucleic acid sample.

[0134] In one aspect, provided is a multiplex assay method for amplifying and detecting in a sample copies of each of at least two different closely related, intended rare mutant DNA target sequences in the presence of copies of a wild-type allele of the mutant DNA target sequences (“a related wild-type DNA target sequence”), where the mutant DNA target sequences differ from each other and from the wild-type DNA target sequence, comprising: (a) preparing a non-symmetric primer-dependent amplification reaction mixture that includes the sample, a DNA polymerase, deoxyribonucleoside triphosphates, other reagents required for amplification, 10-70 mM tetramethylammonium chloride (TMAC), a distinguishably labeled homogeneous fluorescence detection probe that is specific for an amplification product of each rare mutant DNA target sequence, an excess concentration of a reverse primer for the closely related mutant target sequences, and for each intended rare mutant target sequence, a limiting concentration of a unique variant-signaling primer: wherein the sequence of each variant-signaling primer comprises, in the 5' to 3' direction, the following three contiguous DNA sequences that are copied by extension of the reverse primer: an anchor DNA sequence that is sufficiently long so that it is able to hybridize with the closely related mutant DNA target sequences and with the related wild-type target sequence during primer annealing; a unique bridge DNA sequence at least four nucleotides long that does not hybridize during primer annealing to the unique variant-signaling primer's intended DNA target sequence, to any other closely related mutant target DNA sequences, or to the related wild-type DNA target sequence during primer annealing; and a unique foot DNA sequence that is at least 5 nucleotides long to 14 nucleotides long and that is perfectly complementary to the intended DNA target sequence but mismatches each other mutant target sequence and the related wild-type DNA sequence by three or more nucleotides, wherein all of the following requirements are met:

[0135] (i) if the anchor DNA sequence and the foot DNA sequence of the variant-signaling primer are both hybridized to its intended target DNA sequences thereby creating a primer-target hybrid, the primer-target hybrid comprises in the 5' to 3' direction of the variant-signaling primer: an anchor-target hybrid, a bubble, and a foot-target hybrid, said bubble having a circumference of 24 to 40 nucleotides and being formed by an intervening DNA sequence in the target DNA sequence that is at least four nucleotides long and does not hybridize to the primer's bridge DNA sequence during primer annealing;

[0136] (ii) the bubble isolates the foot-target hybrid from the anchor-target hybrid;

[0137] (iii) the variant-signaling primer that has generated an amplicon strand has bridge and foot DNA sequences that are perfectly complementary to the amplicon strand's complementary strand; and (b) repeatedly cycling the reaction mixture to amplify the closely related rare mutant target DNA sequences present in the sample and detecting the presence of those DNA sequences by measuring the intensity of fluorescence from each distinguishably labeled probe by real-time or end-point detection.

[0138] In some embodiments, cycling is temperature cycling in a polymerase chain reaction (PCR) method. In some embodiments, a Ct value for one target DNA sequence represents the same number of starting templates as it does for any other target DNA sequence. In some embodiments: i. for at least one intended rare target sequence the variant-signaling primer includes a 5 '-tag sequence that is not complementary to any target sequence, that is unique for each target sequence or target-sequence group that is to be separately identified, and whose complement in the amplicon strand initiated by the reverse primer is the target of the probe; or ii. for at least one intended rare target sequence the target of the probe is the complement of the bridge sequence in the amplicon strand initiated by the reverse primer; or iii. for at least one intended rare target sequence the target of the probe is the interprimer sequence in the amplicon that lies between where the primers bind.

[0139] In some embodiments, the foot DNA sequence mismatches the closely related wild-type DNA sequence by three or more nucleotides, and wherein at least one of which is the 3 '-terminal nucleotide. In some embodiments, cycling the reaction mixture is performed by an instrument, wherein the number of different target DNA sequences exceeds the number of colors the instrument can separately detect, and wherein multiple different probes are thermospecific hybridization probes having the same color fluorophore, but the hybrids that they form have different melting temperatures. In some embodiments, amplification and detection are a digital PCR method. In some embodiments, the probes are color-coded molecular beacon probes.

[0140] The methods disclosed herein facilitate the selection of the most suitable foot sequence among several that are complementary to the junction where the insertion or deletion occurs. This leads to optimal discrimination against the wild-type sequence and enhanced sensitivity towards the mutant variant.

[0141] It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims.

[0142] EXAMPLES

[0143] Example 1. Utilization of variant-signaling primers in real-time PCR assays for the detection of an insertion into Exon 15 of the APC ( (adenomatous polyposis coli) gene

[0144] Table 1 shows the sequences of the primers and molecular beacon used in the PCR assays of this example.

[0145] Table 1. Variant-signaling primers and molecular beacon sequences.

[0146] The 5 ’-tag sequence (where there is a tag) within the variant-signaling primer is underlined. The bridge nucleotides, demarcated from both the 5’ and 3’ domain nucleotides by dashes, are indicated by bold and italic letters. Within the 3' domain, nucleotides that do not match the corresponding nucleotides in the wild-type target, but align with those in the mutant target, are indicated with bold and underlined letters. A variant-signaling primer is referred to in such a format as, e.g., SEQ ID NO: 1, a "22-22-6 / 5-10" primer, referring to a 5 ’-tag sequence that is 22 nucleotides long, an anchor sequence that is 22 nucleotides long, a bridge sequence that is 6 nucleotides long, and occurs opposite an intervening sequence that is 5 nucleotides long, and finally, a foot sequence that is ten nucleotides long and that is not complementary to the corresponding nucleotides in the wild-type target, but that is complementary to the corresponding nucleotides in the mutant target. The molecular beacon probe is designed to hybridize to the complement of the 5’-tag sequence, with those nucleotides that form the singlestranded loop emphasized by underlining.

[0147] Variant-signaling primers comprising the sequences of SEQ ID NO: 1 and SEQ ID NO: 2 detect the presence of a 750-nucleotide long sequence that was inserted into Exon 15 of the APC (adenomatous polyposis coli) gene. The PC gene encodes a tumor suppressor that is found in chromosome 5q21-22, and it plays a critical role in regulating cell growth and division. Mutations in APC can lead to colorectal cancer. Individuals with these mutations are prone to developing polyps in their colon and rectum, which can progress to cancer if left untreated. Detecting structural modifications in the APC gene is important for understanding their role in the development of colorectal cancer, as these mutations contribute significantly to the initiation and progression of the disease, and lead to new treatments that are tailored to each person's specific genetic makeup. These treatments aim to improve patient care and outcomes.

[0148] The manner in which two different variant-signaling primers can be used to detect the presence of the insertion in the APC gene is shown in FIG. 5A, which is a modification of a diagram in Miki et al. (Miki Y et al. Disruption of the APC gene by a retrotransposal insertion of LI sequence in a colon cancer. Cancer Research. 1992 Feb l;52(3):643-5). As described by the authors, the inserted sequenced is composed of:

[0149] "The first fifth [nucleotides 1 to 150] of the insertion was almost identical [2-base pair difference] to the inverse complement of the LI consensus sequence (nucleotides 5697 to 5548). The central part of the insertion (nucleotides 151 to 531) was highly homologous to the 3' portion of the LI consensus sequence (nucleotides 5778 to 6161, more than 98% homology). The last fifth of the insertion (nucleotides 532 to 750) was composed of a polyadenylation signal and subsequent 180-base polyadenylate tract. Furthermore, duplication of an AT-rich 8 nucleotides (GAATAATG) of the APC sequence was observed at the target site without deletion of any APC sequence."

[0150] The anchor of SEQ ID NO: 1 anneals to the LI consensus sequence, and the foot of SEQ ID NO: 1 anneals to the inverse complement of the LI consensus sequence. The anchor of SEQ ID NO: 2 anneals upstream of the breakpoint between the APC gene and the LI consensus sequence, and the foot of SEQ ID NO: 2 anneals to the insertion. This targeted annealing strategy guarantees that SEQ ID NO: 1 exclusively binds to the rearranged LI sequence, which only occurs in this particular insertion. The distance between these two targeted breakpoints is only 150 nucleotides, enabling robust exponential amplification indicative of the presence of this specific structural variant.

[0151] A PCR assay was performed utilizing mutant plasmids and wild-type human genomic DNA containing the target gene sequence, in which the “anchor” sequence of one variant-signaling primer (which we will call the “forward” primer) binds to all (-) template strands (both the rare intended target and the abundant unintended target) in the sample.

[0152] In this study, the limiting forward primer contains a unique “5’-tag sequence.” When the forward allele-discriminating primer binds to a mutant (-) template and initiates synthesis, the resulting (+) amplicon strand contains the entire forward primer sequence, including the 5 ’-tag sequence at its 5’ end. Subsequently, these (+) amplicons serve as templates for the reverse allelediscriminating primer. The resulting (-) amplicons will contain the complement of the 5 ’-tag sequence at their 3’ ends. It is the 3’ complement of the 5’ tag sequence that is the target of the molecular beacon probes that are present in these reactions to light up the synthesized amplicons. Moreover, because the forward primer is present in limiting amount, single-stranded amplicons are made by extension of the excess allele-discriminatory reverse primer, ensuring that the molecular beacon probes can bind to their targets without any significant competition from collapsing amplicon double strands.

[0153] The target plasmid, containing the APC mutant was purchased from Integrated DNA Technologies, Coralville, Iowa (USA) and was prepared by inserting a 200-base-pair gene fragment into pIDTSmart Amp vectors. This plasmid DNA was linearized by incubation with restriction endonuclease Sea I (New England Biolabs, Ipswich, Massachusetts (USA)). The digestion mixture contained 10 units Sea I and 4 pg of mutant plasmid DNA in a 20-pL volume that contained 100 mM NaCl, 10 mM MgCh, 1 mM dithiothreitol, and 50 mM Tris-HCl (pH 7.9). The reactions were incubated for 120 min at 37 °C, followed by incubation for 20 min at 80 °C to inactivate the endonuclease.

[0154] The PCR assays were performed in 25-pL volumes containing either 500, 50, 5 or 0 copies of the mutant template in a mixture containing 30,000 copies of the wild-type template, as well as amplification buffer (50 mM KC1, 10 mM Tris-HCl (pH 8.0), 2.5 mM MgCh), 40 mM tetramethylammonium chloride (Sigma-Aldrich, St. Louis, Missouri (USA)), 0.5% Tween 20 (Si gm a- Aldrich), 1.5 Units Platinum Taq DNA polymerase (Thermo Fisher Scientific, Waltham, Massachusetts (USA)), 250 pM ATP, 250 pM CTP, 250 pM GTP, 250 pM TTP, 100 nM of APC mutant forward variant-signaling primer, 500 nM of APC mutant reverse variant-signaling primer and 400 nM of the fluorescein-labeled (FAM) molecular beacon probe. The amplification reactions were each run in five replicates. The amplifications were carried out using 0.2 ml white polypropylene tubes (USA Scientific, Ocala, Florida (USA)) in a Bio-Rad CFX-96™ Touch spectrofluorometric thermal cycler (Hercules, California (USA)). The thermal cycling program was 2 min at 95 °C, followed by 50 cycles of 95 °C for 20 sec, 60 °C for 20 sec, and 72 °C for 20 sec. Molecular beacon fluorescence intensity was measured in real time at the end of the 60 °C annealing stage of each thermal cycle.

[0155] The results are presented in FIG. 5B, which displays real-time fluorescence curves, showing fluorescence intensity plotted against the number of thermal cycles completed during PCR amplification. PCR amplification and detection assays were performed using a dilution series, consisting of 30,000 copies of wild-type genomic DNA combined with 500, 50, 5, or 0 copies of the mutant template, respectively.

[0156] Using a pair of variant-signaling primers, a long insertion can be detected even in the presence of a high abundance of wild-type targets. This strategy involves designing one primer to anneal specifically within the inserted region, which has been modified from the original gene. By targeting this unique sequence, the amplification is limited exclusively to the genetic rearrangement of interest, effectively eliminating even linear amplification of the wild-type gene or other unrelated sequences.

[0157] Example 2. Utilization of variant-signaling primers in real-time PCR assays for the detection of two small insertions (indels) in EGFR (Epidermal Growth Factor Receptor) Exon 20.

[0158] Table 2 shows the sequences of the primers and molecular beacons used in the PCR assays of this example.

[0159] Table 2. Variant-signaling primers, a conventional primer and molecular beacon sequences.

[0160] The 5 ’-tag sequence (where there is a tag) within the variant-signaling primer is underlined. The bridge nucleotides, demarcated from both the 5’ and 3’ domain nucleotides by dashes, are indicated by bold and italic letters. Within the 3' domain, nucleotides that do not match the corresponding nucleotides in the wild-type target, but align with those in the mutant target, are indicated with bold and underlined letters. A PCR assay for each EGFR mutant was carried out as described in Example 1 with the following modification for the primers and molecular beacon probes that were used. Variantsignaling primers comprising SEQ ID NO: 4 and SEQ ID NO: 5 were designed for the detection of two different specific EGFR (Epidermal Growth Factor Receptor) Exon 20 indels. These mutations, in combination with other EGFR Exon 20 mutations, which comprise 5% to 10% of all EGFR mutations, lead to protein overexpression, and they are implicated in driving the development of non-small cell lung cancer (NSCLC). These insertions impart resistance to first- and second-generation tyrosine kinase inhibitors (TKIs), which are utilized as targeted therapies that kill cancer cells that do not contain these insertions. Among NSCLC patients, these EGFR Exon 20 insertions correlate with inferior outcomes when they occur along with common mutations such as EGFR Exon 19 deletions or EGFR L858R mutations. The identification of these insertions in the EGFR gene, enables effective targeted therapies to be used (AACR Project GENIE Consortium. AACR Project GENIE: Powering Precision Medicine through an International Consortium. Cancer Discovery. 2017 Aug; 7(8): 818-831 ; and Oyamada Y et al. A Case of Advanced Lung Adenocarcinoma Harboring an Epidermal Growth Factor Receptor(EGFR) Exon 20 Insertion, D770 N771insSVD; and Gan To Kagaku Ryoho. 2021 Jun;48(6): 845-847).

[0161] The feet of variant-signaling primers of SEQ ID NO: 4 and SEQ ID NO: 5 overlap the junction region (containing the "breakpoint") where each insertion occurs within EGFR Exon 20. Due to the thermodynamic characteristics governing the circumference of the bubble formed between their bridges and the intervening sequences in the target, the hybrids that are formed between their feet and their corresponding mutant target sequences persist longer than the highly improbable hybrids formed between the feet and their mismatched wild-type targets.

[0162] The target plasmids, containing the EGFR mutants were purchased from Integrated DNA Technologies, Coralville, Iowa (USA) and were prepared by inserting 200-base-pair gene fragments into pIDTSmart Amp vectors. These plasmid DNAs were digested with restriction endonuclease Sea I (New England Biolabs, Ipswich, Massachusetts (USA)). The digestion mixture contained 10 units Sea I and 4 pg of mutant or wild-type plasmid DNA in a 20-pL volume that contained 100 mM NaCl, 10 mM MgCh, 1 mM dithiothreitol, and 50 mM Tris-HCl (pH 7.9). The reactions were incubated for 120 min at 37 °C, followed by incubation for 20 min at 80 °C to inactivate the endonuclease. The PCR assays were performed in 25-pL volumes containing either 500, 50, 5 or 0 copies of the mutant templates in a mixture containing 30,000 copies of the wild-type template, as well as amplification buffer (50 mM KC1, 10 mM Tris-HCl (pH 8.0), 2.5 mM MgCh), 40 mM tetramethylammonium chloride (Sigma-Aldrich, St. Louis, Missouri (USA)), 0.5% Tween 20 (Sigma-Aldrich), 1.5 Units Platinum Taq DNA polymerase (Thermo Fisher Scientific, Waltham, Massachusetts (USA)), 250 pM ATP, 250 pM CTP, 250 pM GTP, 250 pM TTP, 100 nM of one or the other EGFR mutant forward variant-signaling primer, 500 nM of EGFR common conventional reverse primer SEQ ID NO: 6, and 400 nM of the amplicon-specific molecular beacon probe SEQ ID NO: 7 or SEQ ID NO: 8. Each amplification reaction was run in five replicates. The amplifications were performed using 0.2 ml white polypropylene tubes (USA Scientific, Ocala, Florida (USA)) in a Bio-Rad CFX-96™ Touch spectrofluorometric thermal cycler (Hercules, California (USA)). The thermal cycling program was 2 min at 95 °C, followed by 50 cycles of 95 °C for 20 sec, 60 °C for 20 sec, and 72 °C for 20 sec. Molecular beacon fluorescein (FAM) fluorescence intensity was measured in real time at the end of the 60 °C annealing stage of each thermal cycle.

[0163] FIGS. 7A and 7B illustrate the real-time fluorescence curves, where fluorescence intensity is plotted against the number of thermal cycles completed during PCR amplification. FIG. 7A shows the results for the detection of a 3-nucleotides insertion in EGFR Exon 20 D770_N771 ins G mutation, while FIG. 7B displays the results for the detection of a 9-nucleotides insertion in EGFR Exon 20 D770_N771 ins SVD mutation. PCR amplification and detection assays were conducted using a dilution series, which included 30,000 copies of wild-type genomic DNA and varying amounts of mutant template (500, 50, 5, or 0 copies).

[0164] Using variant-signaling primers, small insertions of varying lengths, such as the 3-nucleotide and 9-nucleotide insertions used in this study, can be efficiently amplified with remarkable sensitivity. Importantly, this assay is capable of detecting as few as 5 copies of the mutant target DNA, even in the presence of a significantly higher abundance of wild-type sequences (in this case 30,000). The high sensitivity ensures the reliable detection of rare mutations under challenging sample conditions.

[0165] To assess the assay's specificity, control samples containing only wild-type targets without any mutant sequences were included. These controls consistently yielded no detectable signal, underscoring the assay’s ability to distinguish mutant sequences with high precision and to avoid false-positive results. This exceptional specificity highlights the assay's robustness and suitability for applications requiring the accurate identification of low-abundance mutations in complex genetic backgrounds.

[0166] Example 3. Variant-signaling primers for the detection of two distinct deletions in the BRCA2 (BReast CAncer Susceptibility 2) gene

[0167] BRCA1 and BRCA2 genes are crucial in maintaining genomic stability, and their role is especially significant given the high incidence and mortality rates of breast and ovarian cancers. In 2024, it is estimated that 310,720 women in the United States were diagnosed with breast cancer, leading to 42,250 deaths, with the highest incidence occurring in women aged 50 and older. For ovarian cancer, projections for 2025 indicate 20,890 new diagnoses and 12,730 deaths, with a lifetime risk of 1 .1% for women. BRCA genes play a critical role in maintaining genomic stability. Among younger women diagnosed with breast cancer, the prevalence of BRCA mutations is notably higher.

[0168] A study revealed that 5.9% of women diagnosed before the age of 36 carry BRCA mutations, with 3.5% attributed to BRCA1 and 2.4% to BRCA2. Similarly, in ovarian cancer patients, BRCA mutations occur at elevated rates, with 11.4% of unselected cases harboring BRCA1 or BRCA2 mutations, including 7.0% with BRCA1 and 4.4% with BRCA2. Women with a BRCA1 mutation have a 72% lifetime risk of developing breast cancer by age 80, while those with a BRCA2 mutation face a 69% risk (Peto et al. Prevalence of BRCA1 and BRCA2 gene mutations in patients with early-onset breast cancer. J Natl Cancer Inst. 1999 Jun 2;91(11):943-9). This underscores the importance of detecting genetic variations for early diagnosis and monitoring of patients.

[0169] Mutations, particularly in BRCA2, impair double-strand DNA break repair via homologous recombination, leading to genomic instability. The 6174delT mutation in BRCA2 is notably prevalent among Ashkenazi Jewish women and is associated with an increased cancer risk. Approximately 1% of individuals carry this mutation, with 8% of Ashkenazi Jewish women with early-onset breast cancer are affected, raising their breast cancer risk by about 9.3-fold by age 42. Around 29% of Jewish women diagnosed with ovarian cancer carry this mutation (Abeliovich et al. The founder mutations 185delAG and 5382insC in BRCA1 and 6174delT in BRCA2 appear in 60% of ovarian cancer and 30% of early-onset breast cancer patients among Ashkenazi women. Am J Hum Genet. 1997 Mar;60(3):505-14).

[0170] Poly ADP -Ribose Polymerase (PARP) inhibitors are a key treatment for / W -associated cancers by exploiting synthetic lethality. FDA-approved PARP inhibitors, such as olaparib and talazoparib, have proven effective in extending progression-free survival in BRCA -mutated breast cancer. These drugs are also used in other BRCA -associated cancers, including prostate cancer, with the recent approval of niraparib combined with abiraterone acetate in 2023.

[0171] PARP inhibitors target cancer cells deficient in DNA repair mechanisms due to BRCA1 or BRCA2 mutations, including the 6174delT mutation. However, not all patients with this mutation respond to PARP inhibitors equally. Some may develop resistance due to additional mutations that restore BRCA2 function.

[0172] A previous study observed that in patients UK1999 and UK2223, intragenetic deletions within BRCA2 restored the BRCA2 open reading frame (ORF), leading to resistance to carboplatin treatment (Resistance to therapy in BRCA2 mutant cells due to loss of the nucleosome remodeling factor CHD4. Genes Dev. 2015 Mar l;29(5):489-94). In UK1999, a 137-bp deletion within exon 11 (7 base pairs 3’ of the 6174delT mutation) restored the BRCA2 ORF, while in UK2223, an 8- base pair deletion (3 amino acids) in exon 11, 5’ from the 6174delT mutation, had a similar effect. These deletions were associated with resistance to carboplatin, suggesting that such mutations can suppress sensitivity to both platinum-based therapies and PARP inhibitors.

[0173] Accordingly, this study designed variant-signaling primers to detect these two deletions associated with therapeutic resistance. The aim of designing such primers was to detect novel mutations in BRCA mutation carriers that identify therapeutic resistance.

[0174] Table 3 shows the sequences of the primers and molecular beacons used in the PCR assays of Example 3.

[0175] Table 3. Variant-signaling primers, a SuperSelective primer, a conventional primer and molecular beacon sequences.

[0176] A PCR assay for each BRCA2 mutant was performed as described in Example 1, with the following modification for the primers and molecular beacon probes that were used. A forward variant-signaling primer comprising SEQ ID NO: 9 and a reverse SuperSelective primer comprising SEQ ID NO: 10 were designed to detect the BRCA2c. 6174delT & 8-bp deletion in exon 11, patient UK2223. Variant-signaling primers comprising SEQ ID NO: 12 and a conventional reverse primer SEQ ID NO: 13 were designed to detect the BRCA2c. 6174delT & 137-bp deletion in exon 11, patient UK1999.

[0177] In this study, the limiting forward primers contain a unique “5’-tag sequence.” When the forward allele-discriminating primer binds to a mutant (-) template and initiates synthesis, the resulting (+) amplicon strand contains the entire forward primer sequence, including the 5 ’-tag sequence at its 5’ end. Subsequently, these (+) amplicons serve as templates for the reverse primer (SuperSelective primer for the BRCA2c. 6174delT & 8-bp deletion in exon 11, patient UK2223 and a conventional primer for the BRCA2c. 6174delT & 137-bp deletion in exon 11, patient UK1999). The resulting (-) amplicons will contain the complement of the 5’-tag sequence at their 3’ ends. The 3’ complement of the 5’ tag sequence is the target of the molecular beacon probes that are present in these reactions to light up the synthesized amplicons.

[0178] For patient UK2223, the anchor sequence of the forward variant-signaling primer (SEQ ID NO: 9) binds upstream of the breakpoint, while its foot sequence overlaps the junction region containing the deletion breakpoint. The reverse SuperSelective primer (SEQ ID NO: 10) specifically detects targets harboring the BRCA2 c.6174delT mutation, while the forward variant-signaling primer selectively identifies the 8-bp deletion. This combination of allelespecific primers, including at least one variant-signaling primer, ensures highly specific detection of the intended mutations.

[0179] For patient UK19999, the anchor sequence of the variant-signaling primer SEQ ID NO: 12 is designed to bind upstream and close to the breakpoint region, while the foot sequence targets the junction region. In the wild-type target, the substantial undeleted sequence (137 bp) contributes to the formation of a thermodynamically unstable, rather large, bubble when interacting with the variant-signaling primer. This instability prevents the formation of a stable foot-target hybrid, thereby inhibiting amplification.

[0180] By contrast, the mutant strand lacks this sequence, resulting in a smaller and more stable bubble. This facilitates the hybridization of the foot sequence to the target, enabling efficient exponential amplification of the mutant target.

[0181] The target plasmids, containing the BRCA2 mutants were purchased from Integrated DNA Technologies, Coralville, Iowa (USA) and were prepared by inserting 200-base-pair gene fragments into pIDTSmart Amp vectors. These plasmid DNAs were digested with restriction endonuclease Sea I (New England Biolabs, Ipswich, Massachusetts (USA)). The digestion mixture contained 10 units Sea I and 4 .g of mutant plasmid DNA in a 20-pL volume that contained 100 mMNaCl, 10 mMMgCh, 1 mM dithiothreitol, and 50 mM Tris-HCl (pH 7.9). The reactions were incubated for 120 min at 37 °C, followed by incubation for 20 min at 80 °C to inactivate the endonuclease.

[0182] The PCR assays were performed in 25-pL volumes containing either 500, 50, 5 or 0 copies of the mutant templates in a mixture containing 30,000 copies of human genomic DNA, as well as amplification buffer (50 mM KC1, 10 mM Tris-HCl (pH 8.0), 2.5 mM MgCh), 40 mM tetramethylammonium chloride (Sigma-Aldrich, St. Louis, Missouri (USA)), 0.5% Tween 20 (Sigma-Aldrich), 1.5 Units Platinum Taq DNA polymerase (Thermo Fisher Scientific, Waltham, Massachusetts (USA)), 250 pM ATP, 250 pM CTP, 250 pM GTP, 250 pM TTP, 100 nM of BRCA2 of one or the other mutant forward variant-signaling primer, 500 nM of BRCA2 either the variant-signaling reverse primer or the conventional reverse primer, and 400 nM of a specific fluorescein-labeled molecular beacon probe designed to detect the corresponding amplicons. The amplification reactions were run in three replicates. The amplifications were performed using 0.2 ml white polypropylene tubes (USA Scientific, Ocala, Florida (USA)) in a Bio-Rad CFX-96™ Touch spectrofluorometric thermal cycler (Hercules, California (USA)). The thermal cycling program was 2 min at 95 °C, followed by 50 cycles of 95 °C for 20 sec, 60 °C for 20 sec, and 72 °C for 20 sec. Molecular beacon fluorescence intensity was measured in real time at the end of the 60 °C annealing stage of each thermal cycle.

[0183] FIGS. 8A and 8B present the real-time fluorescence curves, with fluorescence intensity plotted against the number of thermal cycles during PCR amplification. The top panel (FIG. 8A) depicts the detection of \h BRCA2 c.6174delT & 8-bp deletion in exon 11, patient UK2223, while the bottom panel (FIG. 8B) shows the detection of the BRCA2 c.6174delT & 137-bp deletion in exon 11, patient UK1999. PCR amplification and detection were performed using a dilution series containing 30,000 copies of wild-type genomic DNA, along with varying amounts of the mutant template (500, 50, 5, or 0 copies).

[0184] By employing variant-signaling primers, these assays enable the efficient amplification of small deletions of various lengths, such as the 8-nucleotide and 137-nucleotide deletions investigated in this study, demonstrating exceptional sensitivity.

[0185] This method is capable of detecting as few as 5 copies of the mutant target DNA in the presence of up to 30,000 copies of wild-type genomic DNA, which underscores the sensitivity of such method. Furthermore, the assay's specificity was assessed by including wild-type DNA samples without mutant targets as controls. The absence of any detectable signal in these controls consistently confirmed the assay’s exceptional precision in distinguishing mutant sequences, while effectively preventing false positives. This approach, leveraging combinations of allele-specific primers in which at least one is a variant-signaling primer, significantly enhances the specificity of deletion detection.

[0186] The foregoing examples and description of the preferred embodiments should be taken as illustrating, rather than as limiting the present invention as defined by the claims. As will be readily appreciated, numerous variations and combinations of the features set forth above can be utilized without departing from the present invention as set forth in the claims. Such variations are not regarded as a departure from the scope of the invention, and all such variations are intended to be included within the scope of the following claims. All references cited herein are incorporated by reference in their entireties.

Claims

CLAIMSWhat is claimed is:

1. An amplification and detection method that is capable of detecting as few as five copies of at least one rare mutant DNA target sequence in a mixture containing, for each mutant target sequence, 10,000 copies of a closely related wild-type DNA target sequence, comprising:(a) repeatedly cycling a reaction mixture in a primer-dependent amplification reaction having a primer-annealing temperature, said reaction mixture including said at least one mutant target sequence or its closely related wild-type target sequence or both, a DNA polymerase, other reagents needed for amplification, and for each mutant target sequence a primer pair that includes a variant-signaling primer comprising, in the 5' to 3' direction the following three contiguous DNA sequences: an anchor sequence that hybridizes with the mutant target sequence and with its closely related wild-type target sequence during primer annealing; a bridge sequence at least four nucleotides long that does not hybridize to either the mutant target sequence or its closely related wild-type target sequence during primer annealing; and a foot sequence that is at least five nucleotides long, perfectly complementary to the mutant sequence and mismatched to its wild-type sequence by at least three or more nucleotides, wherein:(i) if the anchor sequence and the foot sequence of the primer are hybridized to the mutant target sequence, there is in the target sequence an intervening sequence that does not hybridize to the primer's bridge sequence during primer-annealing, and the bridge sequence and the intervening sequence, together create a bubble in the hybrid having a circumference of at least twelve nucleotides, wherein the intervening sequence is at least four nucleotides long,(ii) the circumference of the bubble and the length of the foot sequence in combination result in a weak foot / mutant-target- sequence hybrid that makes copying the mutant target sequence unlikely,(iii) the bridge sequence and the foot sequence do not together prime non-target sequences in the mixture, and(iv) the probability that during said cycling a variant-signaling primer / wi Id-type target sequence hybrid will be extended is at least 10,000 times lower than the probability that during said cycling a variant-signaling primer / mutant target sequence hybrid will be extended, as evidenced by a ACrof at least 13.3 cycles; and(b) detecting amplified product or products with a dsDNA binding dye, or for each variantsignaling primer a fluorescent hybridization probe that signals upon hybridization to the amplification product of the primer, or for each variant-signaling primer a quenched, fluorescently labeled oligonucleotide hairpin at the primer's 5' end that fluoresces only when incorporated in or hybridized to the primer's amplified product, wherein copying is “unlikely” means that, if the reaction were begun with a reaction mixture containing 106copies of the at least one mutant DNA target sequence, no wild-type sequence and the variant-signaling primer, and separately with a reaction mixture containing 106copies of the at least one mutant DNA target sequence, no wild-type sequence and a corresponding conventional primer in place of the variant-signaling primer, the threshold cycle (CT) of the reaction utilizing the variant-signaling primer would be delayed by at least three cycles compared to the CT of the reaction utilizing the conventional primer.

2. The method of claim 1, wherein: i. the bridge sequence is at least 6 nucleotides long, the intervening sequence is at least 6 nucleotides long, and the bubble has a circumference of 28-52 nucleotides; or ii. for each of the at least one variant-signaling primers, the CT delay for a reaction begun with 106copies of the mutant DNA target sequence and the variant-signaling primer as compared to a reaction begun with 106copies of the mutant DNA target sequence and a conventional primer is at least 5 cycles.

3. The method of claims 1 or 2, wherein the bubble circumference is 28-44 nucleotides.

4. A primer-dependent amplification and detection method that is capable of amplifying and detecting in a sample as few as five copies of at least one rare DNA intended target sequence (“rare target sequence”) in a mixture containing, for each rare target sequence, 10,000 copies of a closely related unintended target sequence (“closely related sequence” or “unintendedtarget sequence”) that differs from the rare target sequence by at least three nucleotides, comprising:(a) preparing a primer-dependent amplification reaction mixture that includes the sample, a DNA polymerase, deoxyribonucleoside triphosphates, an amplification buffer, homogeneous fluorescence detection means for detecting amplification products, and for each rare target sequence a pair of a first primer and a second primer that are specific for the rare target sequence but mismatched to the closely related sequence,(b) repeatedly cycling the primer-dependent amplification reaction mixture by said primerdependent amplification method to amplify each rare target sequence present in the sample, and(c) detecting that rare target sequence by measuring the intensity of fluorescence from the homogeneous fluorescence detection means; wherein (i) the first primer is an allele-discriminating variant-signaling primer comprising from the 5 ' end to the 3 ' end a first anchor sequence, a first bridge sequence, and a first foot sequence that is mismatched to the closely related sequence by at least three nucleotides, and (ii) the second primer is an allele-discriminating primer.

5. The method according to claim 4, wherein each primer contains a 3 '-terminal interrogating nucleotide that is complementary to the rare target sequence, but mismatched to the unintended target sequence.

6. The method according to claims 4 or 5, wherein the at least one rare target sequence in the sample includes at least two different rare target sequences, and the homogeneous fluorescence detection means comprises at least two different homogeneous fluorescence detection probes for the at least two different rare target sequences respectively.

7. The method according to any one of claims 4-6, wherein each different unintended target sequence differs from its corresponding rare target sequence by at least three base pairs, and both the first and the second primers are mismatched to said base pairs.An amplification and detection method, contingent upon the identity of the mismatch, that is capable of detecting as few as five copies of at least one rare mutant DNA target sequence in a mixture containing, for each mutant target sequence, 10,000 copies of a closely related wildtype DNA target sequence, comprising:(a) repeatedly cycling a reaction mixture in a primer-dependent amplification reaction having a primer-annealing temperature, said reaction mixture including said at least one mutant target sequence or its closely related wild-type target sequence or both, a DNA polymerase, other reagents needed for amplification, and for each mutant target sequence a primer pair that includes a variant-signaling primer comprising, in the 5' to 3' direction the following three contiguous DNA sequences: an anchor sequence that hybridizes with the mutant target sequence and with its closely related wild-type target sequence during primer annealing; a bridge sequence at least four nucleotides long that does not hybridize to either the mutant target sequence or its closely related wild-type target sequence during primer annealing; and a foot sequence that is 5-14 nucleotides long, perfectly complementary to the mutant sequence and mismatched to its wild-type sequence by at least three or more nucleotides, wherein:(i) if the anchor sequence and the foot sequence of the primer are hybridized either to the mutant target sequence or to its closely related wild-type target sequence, there is in the target sequence an intervening sequence at least eight nucleotides long that does not hybridize to the primer's bridge sequence during primer-annealing, and the bridge sequence and the intervening sequence, together create a bubble in the hybrid having a circumference of 28-52 nucleotides,(ii) the circumference of the bubble and the length of the foot sequence in combination result in a weak foot / mutant-target-sequence hybrid that makes copying the intended target sequence unlikely as evidenced by a delay of at least five cycles in the threshold cycle (CT) of amplification of said at least one mutant target sequence using said variant-signaling primer as compared to using a conventional primer,(iii) the bridge sequence and the foot sequence do not together prime non-target sequences in the mixture, and(iv) the probability that during said cycling a variant-signaling primer / wild-type-target- sequence hybrid will be extended is at least 10,000 times lower than the probability that during said cycling a variant-signaling primer / mutant-target-sequence hybrid will be extended, as evidenced by a ACrof at least 13.3 cycles; and(b) detecting amplified product or products with a dsDNA binding dye, or for each variantsignaling primer a fluorescent hybridization probe that signals upon hybridization to the amplification product of the primer, or for each variant-signaling primer a quenched, fluorescently labeled oligonucleotide hairpin at the primer's 5' end that fluoresces only when incorporated in or hybridized to the primer's amplified product.

9. The method of claim 8, wherein the foot sequence of each variant-signaling primer is mismatched to its wild-type target at each or any one of the primer's 3' nucleotide, 3' penultimate nucleotide, and 3’ antepenultimate nucleotide.

10. The method of claims 8 or 9, wherein the at least one mutant target sequence is cDNA.

11. The method of any one of claims 8-10, wherein the variant-signaling primer for the mutant target sequence has a foot sequence that is 6-7 nucleotides long, wherein the bubble circumference is 28-44 nucleotides long, and wherein detection of amplified product is by SYBR Green or another dsDNA binding dye.

12. A multiplex assay method for amplifying and detecting in a sample copies of each of at least two different closely related, intended rare mutant DNA target sequences in the presence of copies of a wild-type allele of the mutant DNA target sequences (“a related wild-type DNA target sequence”), where the mutant DNA target sequences differ from each other and from the wild-type DNA target sequence, comprising:(a) preparing a non-symmetric primer-dependent amplification reaction mixture that includes the sample, a DNA polymerase, deoxyribonucleoside triphosphates, other reagents required for amplification, 10-70 mM tetramethylammonium chloride (TMAC), a distinguishably labeled homogeneous fluorescence detection probe that is specific for an amplification product of each rare mutant DNA target sequence, an excess concentration of a reverse primer for theclosely related mutant target sequences, and for each intended rare mutant target sequence, a limiting concentration of a unique variant-signaling primer: wherein the sequence of each variant-signaling primer comprises, in the 5' to 3' direction, the following three contiguous DNA sequences that are copied by extension of the reverse primer: an anchor DNA sequence that is sufficiently long so that it is able to hybridize with the closely related mutant DNA target sequences and with the related wild-type target sequence during primer annealing; a unique bridge DNA sequence at least four nucleotides long that does not hybridize during primer annealing to the unique variant-signaling primer's intended DNA target sequence, to any other closely related mutant target DNA sequences, or to the related wild-type DNA target sequence during primer annealing; and a unique foot DNA sequence that is at least 5 nucleotides long to 14 nucleotides long and that is perfectly complementary to the intended DNA target sequence but mismatches each other mutant target sequence and the related wild-type DNA sequence by three or more nucleotides, wherein all of the following requirements are met:(i) if the anchor DNA sequence and the foot DNA sequence of the variant-signaling primer are both hybridized to its intended target DNA sequences thereby creating a primer-target hybrid, the primer-target hybrid comprises in the 5' to 3' direction of the variant-signaling primer: an anchor-target hybrid, a bubble, and a foot-target hybrid, said bubble having a circumference of 24 to 40 nucleotides and being formed by an intervening DNA sequence in the target DNA sequence that is at least four nucleotides long and does not hybridize to the primer's bridge DNA sequence during primer annealing;(ii) the bubble isolates the foot-target hybrid from the function of the anchor-target hybrid;(iii) the variant-signaling primer that has generated an amplicon strand has bridge and foot DNA sequences that are perfectly complementary to the amplicon strand's complementary strand; and(b) repeatedly cycling the reaction mixture to amplify the closely related rare mutant target DNA sequences present in the sample and detecting the presence of those DNA sequences by measuring the intensity of fluorescence from each distinguishably labeled probe by real-time or end-point detection.

13. The method according to claim 12, wherein cycling is temperature cycling in a polymerase chain reaction (PCR) method.

14. The method according to claims 12 or 13, wherein a Ct value for one target DNA sequence represents the same number of starting templates as it does for any other target DNA sequence.

15. The method according to any one of claims 12-14, wherein: i. for at least one intended rare target sequence the variant-signaling primer includes a 5 '-tag sequence that is not complementary to any target sequence, that is unique for each target sequence or target-sequence group that is to be separately identified, and whose complement in the amplicon strand initiated by the reverse primer is the target of the probe; or ii. for at least one intended rare target sequence the target of the probe is the complement of the bridge sequence in the amplicon strand initiated by the reverse primer; or iii. for at least one intended rare target sequence the target of the probe is the interprimer sequence in the amplicon that lies between where the primers bind.

16. The method of any one of claims 12-15, wherein the foot DNA sequence mismatches the closely related wild-type DNA sequence by three or more nucleotides, and wherein at least one of which is the 3 '-terminal nucleotide.

17. The method according to any one of claims 12-16, wherein cycling the reaction mixture is performed by an instrument, wherein the number of different target DNA sequences exceeds the number of colors the instrument can separately detect, and wherein multiple different probes are thermospecific hybridization probes having the same fluorophore but the hybrids that they form have different melting temperatures.

18. A multiplexed assay method according to any one of claims 12-17, wherein amplification and detection are a digital PCR method.

19. A multiplexed assay method according to any one of claims 12-18, wherein the probes are color-coded molecular beacon probes.

20. A kit of reagents for detecting the amplification product of any one of the preceding claims by a fluorescent detection reagent.

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