Assay methods and kits for detecting rare sequence variants - Patents.com

Abstract

The present invention relates to a method for analyzing nucleic acids. The present disclosure provides a primer-dependent amplification and detection method that can amplify and detect as few as 10 copies of at least one rare intended target sequence in a sample in the presence of abundant closely related unintended target sequences. Reaction compositions and kits for carrying out the method are also provided.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 62 / 909,483, filed October 2, 2019, the disclosure of which is incorporated herein by reference. [Background technology]

[0002] Being able to detect and quantify the presence of extremely rare nucleic acid sequence variants in samples enriched for closely related sequences has been a long-sought goal; for example, detecting rare (<10 in 10,000) somatic mutations occurring in cancer cells in clinical samples (typically 10,000 or 100,000 cells) enriched in copies of wild-type sequences from normal cells. To achieve this goal, next-generation sequencing has been employed. However, the sensitivity of sequencing approaches is limited because they require extensive amplification of the nucleic acids in the sample and because nucleic acid polymerases can incorporate incorrect nucleotides (creating mutant sequences that were not present in the original sample). Furthermore, sequencing analysis requires expensive equipment, is time-consuming (days), and expensive, costing approximately $4,000 per assay.

[0003] Various designs of allele-discriminating primers have been adopted in exponential amplification assays, hoping to selectively initiate amplification of one or more rare nucleic acid variants while ignoring the abundant closely related wild-type nucleic acids. These designs have in common that they contain at least one nucleotide, called a detection nucleotide, which is complementary to the intended target sequence but mismatches closely related unintended target sequences. These include hairpin primers (published International Patent Application WO 2000 / 71562 (November 30, 2000) and corresponding U.S. Patent No. 6,365,729); Amplification Refractory Mutation System ("ARMS") primers (Newton et al. (1989) Nucleic Acids Research 17:2503-2516; Kwok et al. (1990) Nucleic Acids Research 18:999-1005); and multi-part primers that contain an internal sequence that is not complementary to the target sequence and is sandwiched between two target-complementary sequences. Multipart primers include our laboratory's highly selective ultraselective primers for closely related alleles, as disclosed in published international patent applications WO 2014 / 124290 (August 14, 2014) and WO 2017 / 176852 (October 12, 2017), and U.S. Patent No. 9,909,159. Allele-discriminating primers commonly contain a detection nucleotide that is complementary to the intended rare target sequence but mismatches the abundant unintended closely related sequence.

[0004] Superselective primers are highly selective (see, e.g., Vargas et al. (2016) PLoS ONE 11:e0156546 and Vargas et al. (2018) Journal of Molecular Diagnostics 20:415-427) and rarely copy abundant, closely related strands. When analyzing clinical samples containing only a very small number of mutant targets (e.g., a sample containing 10 or fewer mutant molecules in the presence of 10,000 closely related wild-type molecules), it is particularly desirable to ensure accurate determination of whether an amplified signal is due to the presence of those few mutant molecules or whether the signal is the result of unintended amplification initiated with abundant wild-type molecules, without averaging multiple parallel amplifications. Therefore, if only a single amplification is performed, samples containing only a very small number of mutant targets may be confused with samples containing no mutant targets (resulting in a false-negative conclusion), and samples containing no mutant targets may be confused with samples containing only a very small number of mutant targets (resulting in a false-positive conclusion).

[0005] There is a critical need for a highly sensitive, easy-to-use, low-cost, rapid (hours instead of days), and non-invasive assay for detecting very rare mutations. The required assay must be able to distinguish between samples containing only abundant nucleic acid sequences (e.g., wild-type sequences) and samples containing only 10 copies of a closely related rare nucleic acid sequence (e.g., mutant sequences) per 10,000 copies of the abundant nucleic acid sequence.

[0006] For assays utilizing allele-discriminating primers, a robust assay is needed to detect very few mutant target sequences in a background of abundant wild-type sequences, e.g., 10 mutant sequences in a mixture containing 10,000 wild-type sequences. Selectivity and sensitivity can be condensed into a single feature: the ability to detect as few as 10 copies of a rare target sequence in a mixture containing 10,000 copies of a closely related sequence that differs by one or two nucleotides. Summary of the Invention

[0007] The present invention addresses the above-mentioned needs in many aspects.

[0008] In one aspect, the present invention provides a primer-dependent amplification and detection method capable of amplifying and detecting in a sample as few as 10 copies of at least one intended target sequence of rare DNA ("rare target sequence") within a mixture containing, for each rare target sequence, 10,000 copies of closely related, unintended target sequences ("closely related sequences" or "unintended target sequences") that differ from the intended target sequence by only one or two base pairs. The method includes: (a) preparing a primer-dependent amplification reaction mixture containing the sample, DNA polymerase, deoxyribonucleoside triphosphates, an amplification buffer, a homogeneous fluorescent detection means for detecting the amplification products, and, for each rare target sequence, a pair of primers consisting 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 to amplify each rare target sequence present in the sample; and (c) detecting the rare target sequence by measuring the intensity of fluorescence from the homogeneous fluorescent detection means. The first primer may be an allele-discriminating multi-part primer, and may comprise, from the 5' to 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 its 3' or penultimate nucleotide. The second primer may be an allele-discriminating primer.

[0009] In the method, the first primer, the second primer, or both can be superselective primers that are mismatched to the closely related sequence by at least its 3' or penultimate nucleotide, and each primer can include a 3' terminal detection nucleotide that is complementary to the rare target sequence but mismatched to the non-intended target sequence.

[0010] In the above-described method, cycling can include temperature cycling in an asymmetric polymerase chain reaction (PCR). In one embodiment, the detecting step can include real-time detection. In another embodiment, the PCR can be digital PCR, and detecting can include end-point detection.

[0011] In the above-described method, the at least one rare target sequence in the sample may contain at least two different rare target sequences. In this case, the homogeneous fluorescent detection means may include at least two different homogeneous fluorescent detection probes, each for the at least two different rare target sequences. The at least two rare target sequences may include a group of rare target sequences, and the probes for the rare target sequences in the group may be labeled with the same color. The probes may be color-coded.

[0012] In some embodiments, each different unintended target sequence may differ from the corresponding rare target sequence by a single base pair, and both the first primer and the second primer are mismatched by the single base pair. Each of the second primers may be a multi-part primer comprising, from the 5' end to the 3' end, a second anchor sequence, a second bridging sequence, and a second foot sequence. The homogeneous fluorescent detection means may include a probe for each rare target sequence. The first primer or the second primer, or both the first primer and the second primer, for each rare target sequence may include a 5' tag sequence, and the complement of each 5' tag sequence is the target of the probe or probe pair.

[0013] Each of the above-mentioned probes may comprise a sequence complementary to the complement of the first bridge sequence or the complement of the second bridge sequence. Examples of such probes include shared stem molecular beacons.

[0014] In the above-described method, at least one of the rare target sequences may differ from a corresponding unintended target sequence by a first base pair and a second base pair occurring in cis. In this case, the first primer may be complementary to the first base pair, and the second primer may be complementary to the second base pair. In some embodiments, the at least one rare target sequence in the sample may include two or more rare target sequences, and the homogeneous fluorescent detection means may include at least one homogeneous fluorescent detection probe for each rare target sequence. An example of the homogeneous fluorescent detection means for each rare target sequence includes an inter-primer-specific molecular beacon probe.

[0015] The primer-dependent amplification reaction mixture described above may further comprise an effective amount of a selectivity enhancing reagent, such as a Hofmeister salt. Examples include tetramethylammonium chloride (TMAC) and bis-tetramethylammonium oxalate.

[0016] The present invention further provides a kit or composition of reagents (e.g., a reaction mixture) for carrying out the above-described method. The kit or composition may include one, two, or more reagents selected from the group consisting of the above-described primers, nucleic acid polymerase, deoxyribonucleoside triphosphates, and detection agents. Examples of detection agents include homogeneous fluorescent detection means, such as molecular beacon probes, for each rare target sequence.

[0017] The method of the present invention utilizes a pair of first and second allele-discriminating primers for each of at least one rare target sequence, at least one of which is a multipart first primer, preferably a superselective primer. Both primers in each pair of amplification primers are specific for the target sequence but are mismatched with closely related sequences. Each first multipart primer is mismatched with closely related sequences by at least its 3'-terminal or penultimate nucleotide. In certain preferred embodiments, each allele-discriminating second primer can be similar. In both the forward and reverse primers of each primer pair, the detection nucleotide is preferably the 3'-terminal nucleotide. The method of the present invention may also include the amplification and detection of unrelated wild-type gene sequences for quantification purposes.

[0018] The methods can be classified into two general types: In the first type, each rare target sequence contains a single difference, such as a mutation (resulting from a deletion, insertion, or nucleotide change such as a single nucleotide polymorphism (SNP)). In the case of an SNP, the intended rare target sequence contains a single base pair that differs from a closely related sequence, e.g., a mutant sequence differs from a wild-type sequence by a single base pair. In this case, both primers have a detection nucleotide specific to the single base pair that differs in the rare target sequence; i.e., the forward primer has a detection nucleotide at its 3' or penultimate position that is complementary to a base pair in one of the two strands of the mutant nucleic acid, and the reverse primer has a detection nucleotide at its 3' or penultimate position that is complementary to the other mutant nucleotide of that base pair in the other of the two strands of the mutant nucleic acid, such that one primer binds to the target strand and the other primer binds to the complementary target strand. The amplification reaction mixture containing the sample is subjected to multiple cycles of primer-dependent amplification, and fluorescence is detected in real time or after amplification (endpoint detection used in digital PCR). During amplification, the nucleic acid polymerase cannot incorporate incorrect nucleotides into the amplicon generated from the unintended target sequence (creating a mutant sequence that was not present in the original sample) because the primer will only initiate synthesis beyond the site where the target mutation is located.

[0019] The second type often addresses the need to determine whether two different mutations occur on different sister chromosomes (i.e., in trans), in which case two different versions of the encoded protein can be created, but each version is encoded by only one of these two mutations; or whether these two mutations exist and occur on the same chromosome (in cis), in which case the protein encoded by the gene on the mutant chromosome contains both mutations. In the second type, the rare target sequence contains two base pairs that differ from the wild-type sequence, and the base pairs can occur in the same or different exons, and can occur in either a cis or trans relationship. In this type of target, the forward primer has a detection nucleotide specific for one mutation, and the reverse primer has a detection nucleotide specific for the other mutation. For example, when a patient's non-small cell lung cancer harbors a T790M or C797S mutation in exon 20 of the EGFR gene, an amino acid substitution is introduced into the encoded EGFR protein, rendering first-line treatment (gefitinib or erlotinib) ineffective and suggesting that the use of osimertinib would be effective (Lamb and Scott (2017) Targeted Oncology 12:555-562). However, recent evidence has shown that when both the T790M and C797S mutations occur in the EGFR gene on the same chromosome (i.e., in cis), resulting in two amino acid substitutions in the same EGFR protein, osimertinib does not kill these cancer cells, and only brigitinib is effective (Uchibori et al. (2017) Nature Communications 18:14768). In the method of the present invention for making this determination, one primer of the pair detects the T790M mutation in one fragment strand, the (+) strand, and the other primer detects the C797S mutation in the complementary strand, the (-) strand, and only fragments containing both mutations are amplified.While the resulting amplicon can be probed to target the complement of the bridge sequence of the limiting primer or the complement of the 5' tag sequence of the limiting primer, our preferred embodiment utilizes a homogeneous fluorescent detection probe, preferably a molecular beacon probe that targets the portion of the amplicon between the sequences bound by the two probes (i.e., an interprimer-specific probe).

[0020] The method of the present invention has high selectivity, high sensitivity, and improved robustness. High selectivity refers to the ability to detect a rare sequence in a mixture with closely related sequences when the ratio is as low as 1 / 1,000. High sensitivity refers to the ability to detect only 10 copies of a rare sequence in such a mixture. Selectivity and sensitivity can be condensed into a single requirement: the ability to detect only 10 copies of a rare target sequence in a mixture containing 10,000 copies of closely related sequences that differ by one or two nucleotides. Published international patent applications WO 2014 / 124290 (August 14, 2014) and WO 2017 / 176852 (October 12, 2017) and U.S. Patent No. 9,909,159 describe real-time PCR methods utilizing a primer pair consisting of a highly selective forward primer and a conventional reverse primer, including methods capable of detecting as few as 10 copies of a rare target sequence in a mixture containing 10,000 copies of such a closely related sequence. The threshold cycle difference from a sample containing only 10,000 copies of the closely related sequence typically differs by two to several cycles from the threshold cycle (ΔCt) of a sample containing an additional 10 copies of the rare target sequence, often with some variability between replicates. The method according to the present invention offers improved robustness without sacrificing the aforementioned selectivity and sensitivity. By "improved robustness" it is meant that the method meets one of the following two criteria: a sample containing only 10,000 copies of a closely related sequence does not achieve the fluorescence intensity threshold for at least 55 amplification cycles, or the ΔCt between a sample containing only the closely related sequence and a sample containing an additional 10 copies of the rare target sequence is at least 5 cycles greater than when the primer pair contains the same superselective primer and a conventional primer.

[0021] As described above, the methods and kits of the present invention utilize a pair of allele-discriminating primers, each of which is complementary to a rare target sequence but mismatches one or two nucleotides with a closely related sequence that differs from the rare target sequence. In all cases, the first primer is a multipart primer, preferably a superselective primer. The second primer is an allele-discriminating primer. It can be, for example, a superselective primer or other multipart primer, an allele-discriminating hairpin primer, or an ARMS primer. In the first type of embodiment, the second primer, like the first primer, contains a detection nucleotide at or near its 3' end. Multipart primers and ARMS primers can meet this requirement, but allele-discriminating hairpin primers cannot. However, the second type of embodiment does not have this requirement for the second primer, so a multipart primer (preferably a superselective primer), an ARMS primer, or an allele-discriminating hairpin primer can be used.

[0022] The assay method of the present invention is a primer-dependent amplification and detection method. The primer-dependent amplification reaction useful in the method of the present invention can be any suitable exponential amplification method, such as polymerase chain reaction (PCR), ligase chain reaction (LCR), nicking enzyme amplification reaction (NEAR), strand displacement amplification (SDA), nucleic acid sequence-based amplification (NASBA), transcription-mediated amplification (TMA), and rolling circle amplification (RCA). A preferred method utilizes PCR.

[0023] The primer-dependent amplification and detection method of the present invention can utilize asymmetric DNA amplification, for example, asymmetric PCR.Although symmetric DNA amplification can also be used, asymmetric amplification is recommended.In asymmetric PCR amplification, one primer, the limiting primer, is present in a limiting amount so that it is used up before the completion of amplification, preferably at or just after the threshold cycle, and then linear amplification occurs using the remaining primer, the excess primer.The asymmetric PCR method useful in the present invention is LATE-PCR (see, for example, European Patent EP 1,468,114; and Pierce et al. (2005) Proceedings of the National Academy of Sciences of the United States of America 102:8609-8614).

[0024] Preferred methods also include digital PCR (see, e.g., Vogelstein and Kinzler (1999) Proceedings of the National Academy of Sciences of the United States of America 96:9236-9241), in which a single PCR assay mixture is divided into a large number of individual wells or droplets, with only one target molecule (or no target molecules) present in each well or droplet, so that it is desirable to detect amplicons from a single mutant template molecule present in each well or droplet that may contain the related wild-type molecule.

[0025] If the amplification reaction utilizes an RNA-dependent DNA polymerase (an example is NASBA), the amplification reaction can be isothermal. Repeated rounds of synthesis of amplification products are called "cycles," although NASBA is not a thermal cycle. For such amplifications, the "intended target sequence" and "unintended target sequence" primed by the multi-part primers of the present invention are RNA sequences present in the original sample and amplification reaction mixture, along with the DNA polymerase and multi-part primer.

[0026] When the amplification reaction utilizes a DNA-dependent DNA polymerase (e.g., PCR), the original sample may contain a DNA target or an RNA target. For such amplification, the "intended target sequence" and "unintended target sequence" primed by the multipart primers useful in the methods of the present invention are DNA sequences that occur in the original sample or are created by reverse transcribing RNA sequences that occur in the original sample. When multipart primers are used for reverse transcription, the "intended target sequence" and "unintended target sequence" are cDNA and RNA. When other external primers are used for reverse transcription, the "intended target sequence" and "unintended target sequence" are cDNA. In either case, the "intended target sequence" and "unintended target sequence" are nucleic acid sequences present in the amplification reaction mixture with the DNA polymerase and the multipart primer.

[0027] Primer-dependent amplification reactions involve repeated thermal cycles of primer annealing, primer extension, and strand denaturation (strand melting). Primer annealing can be performed at a temperature lower than the primer extension temperature (e.g., three-temperature PCR), or primer annealing and primer extension can be performed at the same temperature (e.g., two-temperature PCR). The overall thermal profile of the reaction can include repetition of specific cycles, or the temperature / time can be varied during one or more cycles. For example, once amplification is initiated and the priming sequence of a multi-part primer becomes longer, a higher annealing temperature appropriate for the longer primer can be used to complete the amplification reaction.

[0028] A preferred method according to the present invention is a primer-dependent amplification and detection method, most preferably an asymmetric method, capable of asymmetrically amplifying and detecting in a sample as few as 10 copies of at least one rare target sequence in a mixture containing, for each rare target sequence, 10,000 copies of a closely related unintended target sequence that differs from the intended target sequence of rare DNA by only one or two nucleotides; (a) preparing a primer-dependent amplification reaction mixture comprising the sample, a DNA polymerase, deoxyribonucleoside triphosphates, an amplification buffer, a homogeneous fluorescent probe for detecting amplification products, and a pair of primers for a rare target sequence, such as a pair of allele-specific amplification primers specific for the rare target sequence but mismatched to the closely related sequence, a first allele-discriminating multipart primer mismatched to the closely related sequence by at least its 3'-terminal or penultimate nucleotide, and a second allele-discriminating primer; (b) amplifying each rare DNA target sequence present in the sample by repeatedly cycling the reaction mixture through the primer-dependent amplification method, and detecting that sequence by measuring the intensity of fluorescence from distinguishably labeled probes targeted to that amplification product; wherein when the method is tested with a first sample containing 10,000 copies of an unintended target sequence and a second sample containing 10 copies of the intended target sequence in a mixture with 10,000 copies of the unintended target sequence, either (a) the fluorescent signal from the first sample is suppressed for 55 amplification cycles, or (b) the threshold cycle difference (ΔCt) between the two amplifications is at least 5 cycles greater than that obtained when the same test is performed replacing the second primer with a conventional primer.

[0029] If the second primer is a multipart primer, it is mismatched with the closely related sequence by at least its 3'-terminal or penultimate nucleotide. If the second primer is an ARMS primer, it is mismatched with the closely related sequence by its 3'-terminal nucleotide. If the second primer is a hairpin primer, it is mismatched with the closely related sequence by the nucleotides of its single-stranded loop.

[0030] In the examples provided below, a common, non-proprietary buffer containing KCl, Tris-HCl (pH 8.0), and MgCl2 was used. The contents of some amplification buffers are considered proprietary to the supplier. Such buffers may also be used, as they are functionally equivalent. In some preferred embodiments, the amplification reaction mixture contains an effective concentration of a selectivity enhancement reagent, preferably tetramethylammonium chloride (TMAC).

[0031] In certain preferred methods, each primer comprises a 3' terminal detector nucleotide that is complementary to the intended target sequence but mismatches the non-intended target sequence. In some embodiments, one primer of a pair of primers comprises a 5' tag sequence, the complement of which is the target of the probe.

[0032] Detection can be performed by a homogeneous detection means for detecting the amplified products. When there is no need to distinguish between the amplification products of multiple rare intended target sequences, such as when there is only one target or group of targets being detected, or when it is only desired to detect whether any of multiple targets is present, the detection means can include a homogeneous detection probe, multiple homogeneous detection probes all labeled with the same color; labeled primers, such as Scorpion primers or LUX primers, or intercalating DNA dyes, such as SYBR® Green. Otherwise, there is a homogeneous fluorescent detection probe of a different color for each target (or group of targets) being detected. The homogeneous fluorescent detection probe can be, for example, a TaqMan® probe, a minor groove binder (MGB) probe, a molecular beacon probe, or an MNAzyme / cleavable probe combination (WO 2013 / 123552). Our preferred detection is by molecular beacon probes that target amplicon sequences complementary to the 5' tag sequence contained in the sequence of the limiting multi-part primer, or that target amplicon sequences complementary to the bridge sequence of the limiting multi-part primer.

[0033] As shown above, the functional characteristics of such an assay are (a) the ability to detect as few as 10 copies of each rare target sequence in the presence of 10,000 copies of its abundant closely related sequence, and (b) improved robustness in distinguishing between (i) a sample containing only 10,000 copies of the closely related sequence and (ii) a sample containing 10,000 copies of the closely related sequence and 10 copies of the rare intended target sequence, by the following test: When a reaction mixture containing the two samples is subjected to real-time PCR utilizing first and second primers according to the present invention, and a similar method except that a conventional primer is used instead of the second, allele-discriminating primer, one of the following results is obtained: (1) for a sample containing only the closely related sequence, fluorescence does not rise above background for 55 cycles using a primer pair according to the present invention, or (2) the difference in appearance of a fluorescent signal whose intensity exceeds background (ΔCt) between the two samples is at least 5 amplification cycles greater using a primer pair according to the present invention than when conventional primers are substituted.

[0034] In preferred embodiments, sensitivity can be improved by including a selectivity-enhancing reagent, preferably tetramethylammonium chloride (TMAC) or other Hofmeister salts, as disclosed in International Patent Application WO 2017 / 176852 (October 12, 2017), in the amplification reaction mixture. Including this reagent in the amplification reaction mixture can improve selectivity for wild-type or other closely related unintended target sequences. Such reagents are added at effective concentrations determined by trial and error. Certain preferred superselective primers used in the methods of the present invention have relatively long footings, e.g., 9-11 nucleotides, and often form large gaps of 32-48 nucleotides, composed of long bridge sequences ranging from 15-24 nucleotides. In reaction mixtures containing such superselective primers, TMAC can be included at relatively high concentrations, e.g., 50 mM or 60 mM. For reaction mixtures containing shorter, ultraselective primers (e.g., 5-7 nucleotides), TMAC may be added at lower concentrations, e.g., 10 mM, 20 mM, or 30 mM, to avoid adverse effects on the amplification reaction. The test described in the previous paragraph can be used to determine whether and how much selectivity enhancing reagent is included in a particular reaction mixture, for example, by comparing the results achieved with various concentrations of TMAC (e.g., 0, 10 mM, 50 mM, and 60 mM). An "effective concentration" refers to a concentration that passes the test, optimally a concentration that most improves the results of the test and does not significantly interfere with the amplification reaction.

[0035] The method according to the present invention is particularly suitable for multiplexing, enabling the amplification of multiple rare target sequences that may be present in a sample. Different embodiments have different objectives and features. For example, certain embodiments may be designed to detect the presence of at least two mutations in a sample containing genomic DNA fragments in the presence of abundant wild-type sequences. A different primer pair may be used for each target sequence, using a uniquely colored fluorescent probe, preferably a molecular beacon probe, that targets an amplicon sequence that is not present in the probe itself or in the wild-type correctly amplified product. In the first type of method, the target of each probe is preferably the bridge complement or the 5' tag of each different limiting primer. In the second type of method, the target of each probe may be the bridge complement or the 5' tag, although it is recommended to use a unique probe between the primers. Alternatively, different primer pairs may be used for different groups of target sequences when the presence of one or more mutations within a group is technically important, for example, for the treatment of cancer patients. The monoplex and multiplex methods described above can typically be performed in a spectrofluorometric thermal cycler, which limits the number of distinguishable fluorescent labels (typically five-color thermal cyclers) to a maximum of seven, and sometimes ten. Reaction mixtures for assays to detect different groups of rare target sequences contain a different multipart limiting primer for each mutant target sequence and, optionally, an unrelated wild-type gene sequence for quantification purposes, with each multipart limiting primer within a group of primers having a similar 5' tag sequence and each group having a different 5' tag sequence.

[0036] The second type of method, i.e., detecting two mutations in a cis-relationship, can have multiple possibilities, even in cases where either or both of the two rare base pairs cause the same amino acid change. For example, one base pair change may be constant, while the other base pair change may be variable, e.g., an X change, a Y change, and a Z change, at the same or slightly different locations. When this occurs, a multiplex assay method can have allele-discriminating primers specific to each possibility; for example, one primer specific to the constant change, but three primers specific to the variable changes (one for X, one for Y, and one for Z). Alternatively, a unique inter-primer probe indicates the presence of the change but does not identify the change. For this purpose, each primer (for X, Y, and Z) can have a unique 5' tag or a unique bridge, the complement of which is targeted by a different probe of a unique color.

[0037] Yet another embodiment of a multiplex assay, particularly the first type of method, is a screening assay, whose purpose is to determine which mutant target sequences from a list of many different mutant target sequences are present in a sample, or to determine whether any of these mutant target sequences are present in the sample. In these assays, the mutant target sequences present are exponentially amplified, preferably by polymerase chain reaction, and the resulting amplicons (produced only if the mutant target sequence is present in the sample) are detected with a fluorescently labeled hybridization probe. In such embodiments, each possible mutant target sequence has a multipart limiting primer, preferably a superselective primer, each with a different 5' tag sequence, the complement of which is the target of the hybridization probe. Typically, the number of mutant target sequences on the list exceeds the number of different fluorescent colors that the detection instrument can distinguish, which can also occur in other multiplex assays. This problem is solved in one of two ways. One method is to use color-coded homogeneous detection probes, preferably color-coded molecular beacon probes, as disclosed in International Patent Publication WO 2004 / 099434 A3, U.S. Patent No. 7,385,043, and Marras et al. (2019) PloS ONE 14:e0213906. Briefly, a batch of probes is divided into a number of aliquots (usually two or three) of a coding scheme, each labeled with a different colored fluorophore, and the aliquots are recombined to create batches containing two or three color codes. A second method to overcome the color limitations of spectrofluorometric thermal cyclers is to use what are called "thermally specific" molecular beacon probes, in which the probe-target hybrids have different melting temperatures (Tm). For example, if a liquid biopsy sample is to be tested for the presence of one or more of 35 different target sequences in a five-color spectrofluorimetric thermal cycler, the 35 different multipart limiting primers, each specific for a different target sequence, may be separated into five sets of seven.Each of the five sets contains seven distinct 5' tags whose complementary sequences are targets for seven different thermally specific molecular beacon probes, all labeled with the same fluorophore but all generating probe-target hybrids with distinguishable melting temperatures (Tm). Thus, each of the 35 distinct target sequences, if present, can be distinguished by the combination of fluorescence color and Tm determined by post-amplification (endpoint) thermal analysis.

[0038] As an alternative to the first type of assay, which is performed using a real-time amplification and detection assay, they can also be performed using digital PCR assays, including assays performed in many different reaction wells of a thermal cycler, and droplet digital PCR (ddPCR) assays performed in many different droplets of a thermal cycler. In both cases, detection of the resulting amplicons after amplification (endpoint detection) is often performed using other detection instruments, such as the Bio-Rad QX200™ Droplet Digital PCR System or the Stilla Technologies Naica™ System. The basic principle underlying digital PCR assays is that the reaction mixture containing the sample may be diluted to such an extent that for each rare target molecule to be detected, only one target DNA molecule (either the rare intended target molecule or the abundant non-intended target molecule) is typically present in a well or droplet (or no target molecule is present in a well or droplet), and many wells or droplets may be present, although some wells or droplets may contain no target molecule, two or three non-intended target molecules, or the intended target molecule and one or two non-intended target molecules. Simultaneous PCR amplification is then performed in each well or droplet. Fluorescently labeled probes present in all wells or droplets bind to the amplicons generated in each well or droplet (if they contain the intended target molecule) and fluoresce brightly with a specific color or color code, indicating that the well or droplet contains the intended target molecule and identifying which intended target sequence it contained. Wells or droplets with a fluorescence intensity above a selected threshold intensity (background) upon completion of amplification are considered positive for a specific color or color code. The number of droplets or wells that light up with the same color or color code provides an accurate measure of the number of corresponding target molecules in the original sample. This approach is so sensitive that even a single DNA fragment containing the target sequence in a well or droplet can be detected.

[0039] Classical droplet digital PCR has been used to detect and quantify rare somatic mutations associated with cancer diagnosis, prognosis, and treatment. See Sanmamed et al. (2015) Clinical Chemistry 61:297-304. More than one million droplets are required to separate the rare mutant target molecules from the much more abundant related wild-type molecules. See, e.g., Hindson et al. (2011) Analytical Chemistry 83:8604-8610. This large number of droplets is necessary because there are far more wild-type targets in a sample (often differing from the wild-type target only by single nucleotide polymorphisms) than there are rare-associated mutant targets, and because the probes (designed to bind to subsequences within amplicons containing mutations) may bind to corresponding sequences in amplicons generated from the related wild-type targets. Therefore, it is desirable to have so many droplets that it is highly unlikely that a droplet containing a mutant target also contains one or more related wild-type targets. This ensures that no droplets contain enough wild-type target such that the signal intensity generated in that droplet is similar to the signal intensity that would have been generated if that droplet had been erroneously thought to contain the relevant mutant target. In other words, if the original sample was divided into too few droplets, droplets containing some wild-type target sequences but no relevant mutant target sequences could be mistaken for droplets containing mutant targets.

[0040] However, when digital PCR embodiments employing the methods of the present invention are used to detect rare mutant target molecules in a sample, far fewer droplets or wells (e.g., only 10,000 to 30,000 droplets) are required because the primer pairs used do not generate detectable amplicons from the relatively small number of closely related wild-type DNA molecules that may also be present in the well or droplet. In digital PCR assays according to the present invention, detection can occur in a thermal cycler, flow cytometer, or microscope, and one of the detection instruments described above.

[0041] The present invention also includes reagent kits for carrying out the aforementioned methods. Such kits may include one or more pairs of allele-specific primers for one or more intended rare target sequences, dNTPs, a primer-dependent polymerase, a detection probe for each intended rare target sequence (or for a group of rare target sequences in some embodiments), and other reagents necessary for amplification, particularly an amplification buffer. One primer in each primer pair may be a multipart primer, and the other primer in each pair may be a superselective or other multipart primer, or an ARMS primer.

[0042] The selectivity of superselective primers can be maximized for a particular target by adjusting the foot length and bubble circumference, with larger bubbles and shorter feet typically resulting in higher selectivity. Also, by adjusting the length and nucleotide sequence of the bridge sequence, superselective primers can be fine-tuned for maximum discrimination, and the specific Ct values ​​of any pair of primers in the same assay can be fine-tuned to reflect the same number of nucleic acid targets in a sample. [Brief explanation of the drawings]

[0043] [Figure 1] FIG. 1 shows the design of the method of Example 1 for detecting rare copies of a mutation utilizing a pair of superselective primers complementary to the mutation, with detection by a molecular beacon probe that targets the complement of the 5′ tag on the restrictive superselective primer. [Figure 2]Figures 2A and 2B show the results of the real-time PCR assay of Example 1, where the top panel A shows the results of amplification and detection using a superselective forward primer whose 3' interrogating nucleotide is complementary to the mutant sequence, and a conventional reverse primer that is complementary to both the mutant sequence and its wild-type closely related sequence downstream of the target mutation; the bottom panel B shows the results of an otherwise identical real-time PCR assay using a pair of superselective primers, both of which have their 3' detecting nucleotide complementary to the target mutation. [Figure 3] Figure 3 shows the design of the method of Example 2 for detecting rare copies of a mutation utilizing a pair of superselective primers complementary to the mutation, with detection by a shared stem molecular beacon probe that targets the complement of the bridge sequence of the restrictive superselective forward primer. [Figure 4] Figures 4A and 4B show the results of the real-time PCR assay of Example 2, where the top panel A shows amplification and detection using a superselective forward primer complementary to the EGF T790M mutation and a conventional reverse primer complementary to both the mutant sequence and its wild-type closely related sequence downstream of the target mutation; the bottom panel B shows the results of an otherwise identical real-time PCR assay using a pair of superselective primers, both of which have their 3' detection nucleotide complementary to the T790M target mutation. [Figure 5] Figure 5 shows the design of the method of Example 3 for determining whether two mutations occur on the same chromosome (in cis) using a superselective forward primer complementary to one mutation and a superselective reverse primer complementary to the other mutation. [Figure 6]Figures 6A, 6B, 6C, 6D, and 6E show the results of the real-time PCR assay of Example 3, in which the reaction mixture contained a superselective forward primer for the EGFR T790M mutation, a superselective forward primer for the EGFR C797S mutation, and a conventional reverse primer: (A) 10 copies of T790M, (B) 10 copies of C797S, (C) 10 copies of T790M and C797S (cis), (D) 10 copies of T790M and 10 copies of C797S (trans), and (E) wild-type alone. [Figure 7] Figures 7A, 7B, and 7C show the results of real-time PCR assays in Example 3, where the reaction mixture contained a superselective forward primer complementary to one mutation and a superselective reverse primer complementary to the other mutation: (A) 10 copies of T790M and C797S (cis), (B) 10 copies of T790M and 10 copies of C797S (trans), and (C) wild-type alone. [Figure 8] Figure 8 shows the design of the method of Example 4 for determining whether two mutations occur (in cis) on the same chromosome using a superselective forward primer complementary to one mutation and an ARMS reverse primer complementary to the other mutation. [Figure 9] Figures 9A, 9B, and 9C show the results of the real-time PCR assay of Example 4, where the reaction mixture contained a superselective forward primer for the EGFR T790M mutation and an ARMS reverse primer for the EGF C797S mutation: (A) 10 copies of T790M and C797S (cis), (B) 10 copies of T790M and 10 copies of C797S (trans), and (C) wild-type only. [Figure 10] Figure 10 shows the design of the method of Example 5 for detecting rare copies of a mutation using an ARMS forward primer complementary to that mutation and a superselective reverse primer complementary to that mutation, with detection by a molecular beacon probe targeting the complement of the 5' tag of the ARMS forward primer. [Figure 11] Figures 11A, 11B, and 11C show the results of the real-time PCR assay of Example 5, where the top panel A shows amplification and detection using a pair of superselective primers; the bottom left panel B shows amplification and detection using a restrictive superselective forward primer and an excess of ARMS reverse primer; and the bottom right panel C shows amplification and detection using a restrictive ARMS forward primer and an excess of superselective reverse primer; in all three cases, both primers are complementary to a single mutant base pair in the intended target sequence. [Figure 12] Figure 12 shows the design of the method of Example 6 for detecting rare copies of a mutation in the presence of abundant copies of normal human genomic DNA, utilizing a pair of superselective primers complementary to the mutation, where detection is performed with a conventional molecular beacon probe labeled with one fluorescent color that targets the complement of the 5' tag of the limiting superselective primer; these assays also include a superselective primer and a conventional reverse primer for the β-actin reference gene of normal human genomic DNA, and detection of the reference gene is achieved simultaneously with an inter-primer specific molecular beacon labeled with a different fluorescent color, thereby allowing the relative abundance of the rare mutation to be assessed by comparing the difference between the mutant and reference gene thresholds. [Figure 13] Figures 13A, 13B, 13C, and 13D show the results of the real-time PCR assay of Example 6, in which all samples contained the same number of abundant copies of the entire human genome, and each of the four panels shows results obtained with samples containing various amounts of mutant target DNA, including a sample that did not contain any mutant target DNA: (A) 500 copies of G719C, (B) 50 copies of G719C, (C) 5 copies of G719C, and (D) 0 copies of G719C.

[0044] Definitions and Nomenclature As used in this description and in the claims of this patent application, the following definitions apply:A "multipart primer" that distinguishes alleles refers to a nucleic acid (e.g., DNA) amplification primer that has an internal sequence, called a "bridge sequence," sandwiched between two target-complementary sequences, called an "anchor sequence" and a "foot sequence," that are not sufficiently complementary to the target sequence to hybridize under primer annealing conditions. The anchor sequence, like a conventional primer, is sufficiently complementary to the target sequence (both intended and unintended target sequences) to hybridize during the primer annealing step of a primer-dependent amplification reaction, for which primers are typically designed with 15-40 (e.g., 17-35, or 20-30) complementary nucleotides. The foot sequence is sufficiently complementary to the rare intended target sequence (e.g., a mutant sequence) to hybridize to it during primer annealing, when the anchor sequence hybridizes and initiates copying, but is mismatched to the abundant, closely related unintended target sequence (e.g., a wild-type sequence) at at least one of its 3' end and penultimate nucleotides. We refer to nucleotides complementary to the intended target sequence but mismatched to closely related non-intended target sequences as "detection nucleotides." The foot sequence may contain intentionally introduced nucleotides near its 3' end that mismatch both the intended and non-intended target sequences to destabilize the foot and enhance allelic discrimination. The foot sequence typically has 5-12 (e.g., 5-10, 6-12, 6-9, or most preferably 8-9) nucleotides complementary to the intended target sequence. The bridge sequence may be 1-50 (e.g., 5-40, 10-30, 15-30, 20-30, or most often 18-22 or 10-14) nucleotides in length. When a multipart primer hybridizes to its target sequence, the target sequence has a region opposite the bridge sequence that is not hybridized to the bridge, referred to as the "intervening sequence," which is 1-100 nucleotides in length.Together, the bridge sequence and the intervening sequence create a "bubble" in the primer-target hybrid whose circumference is the sum of the nucleotides of the length of the bridge sequence and the length of the intervening sequence plus 4 nucleotides, ignoring secondary structure.

[0045] A "superselective" primer is an allele-discriminating multipart primer structured to allow detection of as few as 10 copies of a rare target sequence in the presence of 10,000 copies of a closely related sequence that differs by as little as one base pair when the primer is used as a limiting primer in a PCR amplification. A superselective primer has a sequence that includes, in the 5' to 3' direction, the following three consecutive nucleic acid sequences (e.g., DNA sequences) that are copied by extension of other primers: an anchor sequence, typically in the range of 15-40 nucleotides in length, often 20-30 nucleotides, long enough to hybridize to the mutant or other closely related DNA target sequence and the wild-type or other abundant related DNA target sequence during primer annealing; a unique bridge sequence of at least 6 nucleotides in length that does not hybridize to the primer's intended target sequence or other closely related sequences during primer annealing; and A unique foot sequence that is 6 to 12 nucleotides in length and is perfectly complementary to the intended DNA target sequence, but that is mismatched to a closely related sequence by one or more nucleotides (one or more detection nucleotides), at least one of which is the 3'-terminal nucleotide or the penultimate nucleotide of the 3'-terminus.

[0046] Superselective primers may have one or more of the following structural and functional properties in polymerase chain reaction (PCR) amplification and detection assays: (i) when both the anchor sequence and the foot sequence hybridize to the intended target sequence of the primer, the primer-target hybrid comprises, in the 5' to 3' direction of the primer, an anchor-target hybrid, a single-stranded bubble, and a foot-target hybrid, the bubble having a circumference of 18 to 50 nucleotides, being at least 8 nucleotides in length, and being formed by intervening sequences in the target DNA sequence that do not hybridize to the bridge sequence during primer annealing; (ii) the bubble separates the foot-target hybrid from the anchor-target hybrid, such that the isolated foot-target hybrid is a weak hybrid that is less likely to copy the intended target DNA sequence, as evidenced by a delay in threshold (Ct) of at least 2, preferably at least 5, cycles compared to the Ct generated using conventional primers that do not contain any bridging DNA sequence; (iii) during PCR amplification, the probability that the multipart primer will initiate copying of a closely related mutant target DNA sequence or a related wild-type target DNA sequence is at least 1,000-fold lower than the probability that the multipart primer will initiate copying of its intended target sequence, as evidenced by a difference in threshold (ΔCt) values ​​over at least 10 thermal cycles; (iv) the multi-part primer that generated the amplicon strand has a bridge sequence and a foot sequence that are perfectly complementary to the complementary strand of the amplicon strand; and (v) The length and sequence of the bridge sequence of each multi-part primer, and the length of the intervening sequence of its intended target sequence, result in a threshold (Ct) at which a sample containing only 10 copies of its intended target DNA sequence is observed, which occurs within 40-65 cycles, preferably 55 cycles, of exponential amplification and is at least 2 cycles less than the Ct observed from a sample containing no copies.

[0047] Allele-discriminating "hairpin" primers, like molecular beacon probes, are stem-loop oligonucleotides that contain a single-stranded region (the "loop") flanked by complementary sequences (the "arms") that hybridize to each other to form a double-stranded region (the "stem"). The loop and 3' arm of a hairpin primer are sufficiently complementary to the intended target sequence to hybridize and initiate copying under primer annealing conditions. Allele-discriminating hairpin primers contain a detection nucleotide at or near the center of the loop sequence.

[0048] ARMS primers are conventional primers that discriminate between alleles because their 3' terminal nucleotide is the detection nucleotide. ARMS primers may contain intentionally introduced nucleotides near their 3' end that are mismatched with both the intended and non-intended target sequences to destabilize the primer and enhance its allele discrimination.

[0049] "Conventional" primers are single-stranded oligonucleotides 15-40 nucleotides in length, more commonly 20-30 nucleotides in length, that are perfectly complementary to the intended target. To design conventional PCR primers, any of several computer programs are commonly used.

[0050] Our convention in describing primer pairs is to refer to the limiting primer as the "forward" primer, complementary to the (-) template strand of the target, and the excess primer as the "reverse" primer, complementary to the (+) template strand of the target. This is done for convenience only. It will be understood that the limiting primer can be complementary to the (+) strand and the excess primer can be complementary to the (-) strand.

[0051] The primer nomenclature is illustrated by the limited superselective primers in Example 1, whose sequences are as follows:

[0052] [ka]

[0053] In the 5' to 3' direction, this primer contains four elements separated by dashes (-). In our nomenclature, this primer is 32 -28-20 / 13-8:1:0. 32 indicates a 32-nucleotide long 5' tag sequence; the next element, 28, indicates a 28-nucleotide long anchor sequence; and the final element, 8:1:0, indicates a 9-nucleotide long (8+1+0) foot sequence, with 8 nucleotides from the 5' end that are complementary to both the intended target sequence and a closely related non-intended target sequence, one detection nucleotide that is complementary to the intended target sequence but mismatches the non-intended target sequence, and zero nucleotides 3' from the detection nucleotide that is complementary to both the intended target sequence and the non-intended target sequence (i.e., the detection nucleotide of this primer is the 3'-terminal nucleotide). If the foot contains a destabilizing nucleotide, as is often the case with ARMS primers, it is italicized in the sequence and represented by "m" in the characterization. For example, the foot sequence

[0054] [ka]

[0055] is written as 6:m1:1:1:0, indicating six target-complementary nucleotides from the 5' end; followed by one nucleotide, designated "m," that is a mismatch with both the intended target sequence and the non-intended target sequence; followed by one target-complementary nucleotide; followed by the detection nucleotide; and finally, followed by the number of target-complementary 3' nucleotides (here, 0). Because the bridge sequence is characterized not only by its length but also by the length of the intervening sequence on the other side, the size of the circumference of the bubble can be determined as the length of the bridge sequence plus the length of the intervening sequence plus 4, so that each side of the bubble contains a hybridized base pair. In the example sequence above, the circumference of the bubble is 37 nucleotides (20 + 13 + 4 = 37). DETAILED DESCRIPTION OF THE INVENTION

[0056] [Detailed Description of the Invention] Figure 1 illustrates an embodiment of the first type of method of the present invention, in which the first primer is an allele-discriminating multipart primer and the second primer is an allele-discriminating primer, both complementary to a single base pair variation (SNP) in a rare mutant target sequence. In the top panel, the primers are shown hybridizing (indicated by a short vertical line) to a rare target sequence of a double-stranded template containing a plus (+) strand and a minus (-) strand. While only one primer must be a multipart primer, preferably a superselective primer, the illustrated embodiment has a pair of superselective primers. As shown in the top sketch, each primer has an anchor sequence, an unhybridized bridge sequence opposite an intervening sequence in the template, and a foot sequence. The intended target sequence (shown for illustrative purposes to be the EGFR gene, as in Example 1) contains a single base pair that differs from a closely related sequence. For illustrative purposes, the base pair is shown as an A nucleotide in the (-) template strand of the intended target sequence and a T nucleotide in the (+) template strand of the intended target sequence. That is, the mutation to be detected is a single nucleotide polymorphism occurring in exon 18 of the EGFR gene (EGFR G719C), the subject of Example 1. Each primer has a detection nucleotide (here, the 3'-terminal detection nucleotide) complementary to one nucleotide of its base pair. Figure 1 illustrates an embodiment in which the amplification reaction is asymmetric. Here, one primer, called the forward primer, is also the limiting primer, as shown in the center panel, and contains a 5' tag sequence that is not complementary to the target strand but is copied in the amplification reaction. Here, the other primer, called the reverse primer, is also the excess primer, as shown in the center panel. The sketch above also shows a homogeneous fluorescent detection probe, here a hairpin-shaped molecular beacon probe with a single-stranded loop and a double-stranded stem, one arm of the stem labeled with a fluorophore (○) and the other arm of the stem labeled with a quencher (●).In the illustrated embodiment, the molecular beacon is a "traditional" molecular beacon probe, i.e., a molecular beacon probe in which only its single-stranded loop is complementary to the probe's target (in this case, the complement of the 5' tag sequence). The sketch below illustrates detection, which can be real-time or end-point detection as used in digital PCR. The probe is shown hybridized to the (-) amplicon, i.e., the amplification product generated by extension of the excess primer, and the probe's target is the complement of the 5' tag sequence of the limiting primer. The probe's fluorophore is separated from the probe's quencher by hybridization of the probe to its target, resulting in fluorescence (Tyagi et al. (1998) Nature Biotechnology 16:49-53).

[0057] Figure 3 shows an embodiment similar to that shown in Figure 1, except that the target of the probe is the complement of the bridge sequence of the limiting primer, rather than the complement of the 5' tag sequence of the limiting primer; therefore, the limiting primer does not contain a 5' tag sequence. In the illustrated embodiment, a molecular beacon probe, sometimes called a "shared stem" molecular beacon (Tsourkas et al. (2002) Nucleic Acids Research 30:4208-4215), has one arm that is complementary to the target of the probe, in this case the arm labeled with a quencher. Thus, as shown in the sketch below, both the loop and that arm hybridize to the complement of the bridge of the limiting primer. For illustrative purposes, the base pair present in the rare target but absent in the closely related sequence is shown as an A nucleotide on the (-) strand of the intended target sequence and a T nucleotide on the (+) strand of the intended target sequence. That is, the mutation detected is a single nucleotide polymorphism occurring in exon 20 of the EGFR gene (EGFR T790M), which is the subject of Example 2.

[0058] In Figures 1 and 3, one detection probe is shown to hybridize to one sequence that is the target of the probe.This does not exclude the inclusion of two sequences that are the probe target.For example, if the complement of the bridge sequence of one primer is the target of the first probe, the complement of the 5' tag sequence of the other primer can be the target of a different probe of the same color, in which case twice the number of probe copies can bind and emit fluorescence.

[0059] Example 1 illustrates an embodiment of the method according to the present invention, as shown in Figure 1. The rare intended target sequence is the G719C mutation in the EGFR gene. Detecting this mutation allows for the use of particularly effective targeted therapies (such as erlotinib or gefitinib) to kill cancer cells containing this mutation present in patients with non-small cell lung cancer (Pao et al. (2004) Proceedings of the National Academy of Sciences of the United States of America 101:13306-13311; Kobayashi and Hagiwara (2013) Targeted Oncology 8:27-33).

[0060] The multipart first primer (here, the superselective limiting primer) and the allele-discriminating second primer (here, also the superselective primer) both detected a single base pair. The rare intended target sequence, in this case the G719C mutation in the EGFR gene, differed from the abundant, closely related unintended target sequence, in this case the wild-type sequence, by a single base pair change. In this case, the A:T of the mutant (see Figure 1) differs from the C:G of the wild-type. The amplification and detection method was a real-time PCR assay. For comparison, in Example 1, the excess superselective primer (for convenience, referred to as the reverse primer) was replaced with a conventional PCR reverse primer (in this case, the anchor sequence of the superselective reverse primer) complementary to both the intended and unintended target sequences.

[0061] In Example 1, the foot sequence of the superselective forward primer was 9 nucleotides long, and the circumference of the bubble formed when the primer hybridized to the intended target sequence was 37 nucleotides (20 + 13 + 4). The foot sequence of the reverse primer was also 9 nucleotides long, and the circumference of the bubble formed when the primer hybridized to the intended target sequence was 32 nucleotides (18 + 10 + 4). The inventors have found that methods of the present invention using superselective primers with relatively long (8-12 nucleotide) feet without destabilizing nucleotides, creating relatively large bubble circumferences (28-50 nucleotides), can benefit from the inclusion of selectivity-enhancing reagents. In Example 1, 50 mM tetramethylammonium chloride was included in each amplification reaction mixture as an effective amount of a selectivity-enhancing reagent.

[0062] The samples were subjected to PCR amplification with real-time fluorescence detection. One sample contained only 10,000 copies of the EGFR wild-type sequence. The second sample contained 10 copies of the G719C mutant sequence in a mixture containing 10,000 copies of the EGFR wild-type sequence. The third sample contained 100 copies of the G719C mutant sequence in a mixture containing 10,000 copies of the EGFR wild-type sequence. Each sample was tested in duplicate. Fluorescence intensity curves of the amplification reactions are shown in Figure 2. For each primer pair, the average Ct values ​​(of two replicates) for samples containing only the wild-type sequence and samples containing 10 copies of the mutant sequence, as well as the ΔCt values, are shown in Table 1. Comparing a method using two superselective primers, both of which select for a single SNP (bottom panel B of Figure 2), with a method using the same restrictive superselective forward primer and conventional reverse primer (top panel A of Figure 2), we see that while the latter can indeed distinguish between 10,000 wild-type and 10 mutants of the 10,000 wild-type, the method of the present invention is far more powerful. Table 1 shows that the ΔCt of the primer pair containing the conventional primer was 2.95, while the ΔCt of the primer pair containing two superselective primers was 12.87. This is an increase of nearly 10 cycles, demonstrating that the method of Example 1, utilizing a pair of superselective primers, is a method of the present invention.

[0063] Example 2 illustrates an embodiment of the method according to the present invention, as shown in Figure 3. The mutation in the target sequence was T790M in exon 20 of the EGFR gene. The mutation is a single base pair substitution (SNP) of an A:T base pair instead of a G:C base pair, and occurs in an otherwise identical, closely related wild-type sequence. Detection of this mutation indicates that commonly used targeted therapies (erlotinib or gefitinib) that kill cancer cells containing any of several different EGFR mutations (including G719C, G719S, L858R, L861Q, and E746-A750 deletions) do not work, but a different targeted therapy (osimertinib) can kill cancer cells in patients with non-small cell lung cancer (Lamb and Scott (2017) Targeted Oncology 12:555-562).

[0064] The amplification and detection method was a real-time PCR method similar to that described in Example 1, except that the target mutation was the EGFR mutation T790M. The uniform detection probe was a shared stem molecular beacon targeting the complement of the bridge sequence of a superselective forward primer without a 5' tag sequence. In this case, the A:T of the mutant (see Figure 3) differed from the G:C of the wild-type. As in Example 1, one set of reactions utilized a pair of superselective primers, each detecting the 3'-terminal nucleotide, while the second set of reactions utilized a restrictive superselective forward primer and a conventional reverse primer complementary to both the intended target sequence and the non-intended target sequence, in this case, the anchor sequence of the superselective reverse primer. Both pairs of primers were tested on triplicate samples. The samples contained 10,000 copies of a closely related wild-type non-intended target sequence, plus 0, 10, or 100 copies of the intended target sequence of the mutant. 50 mM TMAC was included in each amplification reaction mixture as an effective selectivity enhancement reagent.

[0065] Five replicates of each sample were subjected to PCR amplification with real-time fluorescence detection. The fluorescence intensity curves of the amplification reactions are shown in Figure 4. For each primer pair, the average Ct values ​​(of the five replicates) for the sample containing only the wild-type and for the sample containing 10 copies of the mutant in the presence of 10,000 wild-type are shown in Table 2, along with the delta Ct between these average Ct values. Table 2 shows that the method using the superselective forward limiting primer and a conventional reverse primer achieved a substantial average delta Ct of 5.35. However, the top panel A of Figure 4 shows that due to replicate-to-replicate variability, multiple replicates were required to achieve this result. In contrast, the method using two superselective primers was much more robust. The bottom panel B of Figure 4 shows that fluorescence from the wild-type-only sample was significantly delayed, with three of the five replicates failing to give a Ct through 55 cycles. To calculate and average the Ct, these replicates were assigned a Ct greater than 55. The average delta Ct was >14.00. This is an increase of almost 9 cycles, demonstrating that the method of Example 2 utilizing a pair of superselective reactions is a method according to the present invention.

[0066] Figure 5 shows an embodiment of the present invention having a pair of multipart primers (indicated by short vertical lines) hybridized to a rare target sequence in a double-stranded template containing a plus (+) strand and a minus (-) strand. While only one primer must be a multipart primer, the illustrated embodiment has a pair of highly selective primers. As shown in the sketch above, each primer has an anchor sequence, an unhybridized bridge sequence opposite an intervening sequence in the template, and a foot sequence. The intended target sequence contains two mutant base pairs that differ from closely related sequences. For illustrative purposes, the first mutant base pair is shown as an A nucleotide in the minus (-) template strand of the intended target and a T nucleotide in the plus (+) template strand of the intended target. That is, the first detected mutation is a single nucleotide polymorphism occurring in exon 20 of the EGFR gene (EGFR T790M). The second mutant base pair is shown as a G in the minus (-) template strand of the target and a C in the plus (+) template strand of the target. That is, the second mutation detected is a single nucleotide polymorphism that also occurs in exon 20 of the EGFR gene (EGFR C797S).

[0067] In the embodiment of the invention shown in Figure 5, the objective is to identify the presence in a sample of a chromosome containing both target mutations (in this case, the presence of both EGFR T790M and EGFR C797S). As described in Example 3, the occurrence of two target mutations on the same chromosome within a sample (i.e., in cis) is distinguished not only from samples containing only closely related wild-type sequences, but also from samples containing the same two mutations but on different sister chromosomes (i.e., in trans). Each primer has a detection nucleotide (here, the 3'-terminal detection nucleotide) complementary to one of the two mutations. One primer, referred to here as the forward primer, is also the limiting primer, as shown in the center panel, and has a detection nucleotide complementary to the T790M mutation on the (-) template strand. The other primer (referred to here as the reverse primer), as shown in the center panel, is the excess primer, and has a detection nucleotide complementary to the C797S mutation on the (+) template strand. The top sketch also shows a homogeneous fluorescent detection probe, here a molecular beacon probe with a single-stranded loop and a double-stranded stem, one arm of the stem labeled with a fluorophore (○) and the other arm labeled with a quencher (●). In the illustrated embodiment, the molecular beacon is a "traditional" molecular beacon probe, i.e., one in which only the single-stranded loop is complementary to the probe's target, in this case the complement of the region between the primer sequences of the amplification product. The bottom sketch shows detection, either real-time or end-point detection as used in digital PCR. This "inter-primer-specific" probe hybridizes to the (-) amplicon, and hybridization of the probe separates the probe's fluorophore from the probe's quencher.

[0068] Example 3 illustrates an embodiment of a method according to the present invention, as shown in Figure 5. Two different target mutations, T790M (having an A:T base pair instead of a G:C base pair in exon 20 of the EGFR gene) and C797S (having a G:C base pair instead of a C:G base pair in the same exon), are located 20 nucleotides apart from each other when they are on the same chromosome, i.e., when they are in cis. The purpose of the assay illustrated in Example 3 is to determine whether these two mutations (if both are present in the sample) actually occur on the same chromosome, or whether (if both are present) they occur on sister chromosomes, i.e., when they occur in trans. If only one of these two somatic mutations occurs in a patient sample with non-small cell lung cancer, or if both occur but in trans on sister chromosomes (Vokes and Janne (2017) Journal of Thoracic Oncology 12:1608-1610), osimertinib would be an effective targeted therapy (Lamb and Scott (2017) Targeted Oncology 12:555-562). However, if both of these mutations occur in cis on the same chromosome, the resulting EGFR protein contains two amino acid substitutions, and osimertinib is not an effective targeted therapy (Wang et al. (2016) Journal of Hematology and Oncology 9:59). Instead, brigutinib is an effective alternative (Uchibori et al. (2017) Nature Communications 8:14768).

[0069] To obtain a reference for assessing ΔCt and to illustrate why this method is necessary, a first series of amplifications was performed using a conventional primer as the reverse primer, and the real-time fluorescence curves are shown in Figure 6. Bottom panel E shows the fluorescence curves of four replicates of a sample containing only 10,000 copies of the closely related (wild-type) EGFR sequence; top left panel A shows the curves of four replicates of a sample containing 10,000 copies of the EGFR wild-type sequence and 10 copies of the EGFR T790M sequence; top right panel B shows the curves of four replicates of a sample containing 10,000 copies of the EGFR wild-type sequence and 10 copies of the EGFR C797S sequence; center right panel D shows the curves of four replicates of a sample containing 10,000 copies of the EGFR wild-type sequence plus 10 copies of the EGFR T790M sequence and 10 copies of the EGFR C797S sequence (i.e., two mutations in trans); center left panel C shows the curves of four replicates of a sample containing 10,000 copies of the EGFR wild-type sequence plus the EGFR T790M mutation and the EGFR The curves for four replicates of a sample containing 10 copies of a sequence containing both C797S mutations (i.e., both mutations are present in cis) are shown. As shown in Table 3, the average Ct value for the sample containing only 10,000 wild-type templates was 45.82, while the average Ct values ​​for four different types of samples containing 10 copies of 10,000 wild-type templates plus one or both mutant templates were 38.34, 39.95, 38.44, and 39.49 (for a total average Ct value of 39.05). Importantly, the presence of one or both mutations within a sample was indicated by a lower average Ct value than the Ct value for samples containing only the wild-type template (average ΔCt of 6.77). However, the average Ct value for the trans construct, whose replicate curve is shown in panel D (middle right), was 38.44, which is nearly identical to the average Ct value for the cis construct, whose replicate curve is shown in panel C (middle left). Therefore, it was not possible to distinguish between cis and trans conformations.

[0070] To distinguish between the two mutations, a second round of amplification was performed using the method shown in Figure 5. Real-time PCR amplification and detection were performed using a sample containing both mutations in cis configuration or both mutations in trans configuration, using the superselective primer pair shown in Figure 5 and the real-time fluorescence curves reported in Figure 7. Bottom panel C shows the fluorescence curves of four replicates of a sample containing only 10,000 copies of the wild-type sequence; top left panel B shows the curves of four replicates of a sample containing 10,000 copies of the wild-type sequence and 10 copies of the two mutations in cis configuration; top right panel B shows the curves of four replicates of a sample containing 10,000 copies of the wild-type sequence and 10 copies of the two mutations in trans configuration, respectively. The average Ct value of the cis sample was 41.31. Neither the trans sample nor the wild-type-only sample exhibited fluorescence above background throughout 55 amplification cycles. As defined by the inventors, when each was assigned a Ct value of >55, the ΔCt value was 13.69. This method qualifies as a method according to the present invention. This meets the criterion that the sample without the cis template (the intended target sequence) does not produce a Ct value above background within 55 cycles of amplification. Furthermore, the ΔCt value compared to the wild-type-only sample increased by 13.69 cycles, thus meeting the alternative criterion of an increase of at least 5 cycles.

[0071] In Example 4, the second round of amplification was repeated as in Example 3, using an ARMS primer as the reverse primer instead of the superselective reverse primer. The method for detecting the two cis mutations is shown in Figure 8, and the real-time fluorescence curves are shown in Figure 9. Panel C at the bottom shows the fluorescence curves of four replicates of a sample containing only 10,000 copies of the wild-type sequence; Panel A at the top left shows the curves of four replicates of a sample containing 10,000 copies of the wild-type sequence and 10 copies of the two cis mutations; and Panel B at the top right shows the curves of four replicates of a sample containing 10,000 copies of the wild-type sequence and 10 copies of the two trans mutations, respectively. The average Ct value of the cis sample was 43.31. Neither the trans sample nor the wild-type-only sample exhibited fluorescence above background throughout 55 amplification cycles. Assigning a Ct value of >55, as defined by the inventors, the delta Ct value was 11.69. This method qualifies as a method according to the present invention because it meets the criterion that the sample without cis template (the intended target sequence) does not produce a Ct value above background within 55 cycles of amplification. Furthermore, the delta Ct value compared to the wild-type only sample increased by 11.69 cycles, thus meeting the alternative criterion of an increase of at least 5 cycles.

[0072] Example 5 demonstrates the use of an ARMS primer as a second primer in a method for detecting a single base pair change. Assays were performed to demonstrate the use of an ARMS primer as either the limiting primer or the excess primer. As described in Example 5, a real-time PCR assay with real-time detection was performed using several different primer pairs: a limiting ARMS forward primer with an excess of a superselective reverse primer, a limiting superselective forward primer with an excess of an ARMS reverse primer, and a pair of superselective primers (as a control) to detect 10 copies of a rare KRAS G12D intended mutation target sequence in a mixture containing 10,000 copies of its closely related unintended wild-type target sequence. Each multipart primer and each ARMS primer had a detection nucleotide at its 3' end that was complementary to the nucleotide of the mutant base pair.

[0073] Methods in which the ARMS primer was the forward primer are shown in Figure 10. The results of these assays are shown in Figure 11. For all primer pairs containing a superselective primer as the multipart first primer, the fluorescence intensity of samples containing only the unintended (wild-type) template sequence did not rise above background through 55 amplification cycles. Thus, methods utilizing each of these primer pairs are methods in accordance with the present invention.

[0074] Example 6 demonstrates the selectivity and sensitivity of a real-time PCR assay, which is designed to detect the presence of rare mutant target DNA fragments and determine their relative abundance in samples containing abundant DNA fragments from the entire normal human genome.In particular, this example demonstrates the use of a pair of multi-part primers that distinguish alleles, each complementary to a single base pair mutation present in a rare DNA fragment, and by analyzing the sample containing abundant DNA fragments from the entire normal human genome in a real-time PCR assay, very few target fragments can be reliably detected.In particular, in the presence of DNA fragments from 10,000 copies of the entire normal human genome, all 10 samples each containing nominally five mutant DNA fragments showed positive signals for the presence of the mutant DNA fragments.As a control, all 10 samples that did not contain mutant DNA fragments but contained DNA fragments from 10,000 copies of the entire normal human genome did not give positive signals for the presence of the mutant DNA fragments.

[0075] These results mean that a positive result in an assay using a pair of allele-discriminating primers, such as superselective primers, both specific for the same mutant base pair, can be trusted to indicate a true positive result; a negative result in these same assays can be trusted to indicate a true negative result. This is an important criterion for highly sensitive PCR assays, such as those designed to detect the presence of rare mutant fragments in cell-free DNA isolated from the plasma of 10 mL blood samples obtained from cancer patients, where the presence of a particular mutation indicates that a particular targeted therapy will be effective (Sabari et al. (2019) Journal of the National Cancer Institute 111:575-583).

[0076] All assays performed in this example included DNA fragments from 10,000 copies of the entire normal human genome. This is greater than the amount of cell-free DNA fragments typically isolated from 1 mL of plasma obtained from a 10 mL patient blood sample (Meddeb et al. (2019) Scientific Reports 9:5220). However, because the actual amount of cell-free DNA fragments in a patient's blood sample can vary over time, it is important to include primers and probes to detect DNA fragments from a normal reference gene in the real-time PCR assay, thereby measuring the amount of DNA in the sample. The results indicate whether the sample contains sufficient DNA to detect rare mutant DNA fragments. Furthermore, the threshold cycle (Ct) obtained in the PCR assay for the mutant target fragment compared to the threshold cycle (ΔCt) obtained for the reference gene allows the results to be expressed as the relative abundance of that mutation in the patient's DNA, a clinically relevant result.

[0077] Figure 12 shows how a pair of superselective primers for the G719C mutation of the EGFR gene was utilized in the assay of Example 6. The limiting primer for the G719C gene contained a 5' tag sequence, and the FAM-labeled conventional molecular beacon present in the assay indicated the presence of amplicons generated as a result of the presence of mutant DNA fragments in the sample by binding to the complement of the 5' tag sequence incorporated into the 3' end of the excess (-) amplicon. All assays performed in Example 6 also included a limiting concentration of a superselective forward primer for the human β-actin gene, an excess concentration of a conventional reverse primer for the β4-actin gene, and a Quasar 705-labeled interprimer-specific molecular beacon to signal the presence of amplicons generated from the β-actin reference gene.

[0078] Four sets of 10 assays were performed, and all assays contained 10,000 copies of a DNA restriction fragment from normal human genomic DNA. In addition, in all assays, the first set also contained 500 copies of a linearized plasmid containing the mutant target sequence, the second set also contained 50 copies of the mutant DNA plasmid, the third set also contained 5 copies of the mutant DNA plasmid, and the fourth set served as a negative control without any mutant DNA plasmid.

[0079] The results of these 40 PCR assays are shown in Figure 13. Panel A, top left, of Figure 13, shows the results of assays containing 500 mutant plasmids each; Panel B, top right, shows the results of assays containing 50 mutant plasmids; Panel C, bottom left, shows the results of assays containing only the five mutant plasmids; and Panel D, bottom right, shows the results of assays without any mutant plasmids.

[0080] All of these assays showed positive FAM signals, with an average Ct value of 43.27. In comparison, all reactions without either mutant plasmid produced no FAM signals above background throughout 55 cycles of amplification. These results demonstrate the exceptional selectivity and sensitivity of exponential amplification assays using allele-specific primer pairs for single-nucleotide polymorphism detection and suggest that assays utilizing these primer pairs may enable highly sensitive clinical assays that can be performed relatively quickly and at low cost on widely available equipment.

[0081] Compositions and Kits The present invention encompasses compositions or reaction mixtures containing the aforementioned primers and reagents for carrying out the above-described methods. For example, the compositions may contain one or more reagents selected from the group consisting of a nucleic acid polymerase, a deoxyribonucleoside triphosphate, and a detection agent.

[0082] The detection agent can be an oligonucleotide probe, such as a molecular beacon probe or a Yin-Yang probe labeled with a fluorophore and a quencher. See, e.g., U.S. Patent Nos. 5,925,517, 6,103,476, 6,150,097, 6,270,967, 6,326,145, and 7,799,522. The composition may also include, in addition to the above-mentioned reagents, one or more of the following: salts, e.g., NaCl, MgCl2, KCl, MgSO4; buffers, e.g., Tris buffer, N-(2-hydroxyethyl)-piperazine-N'-(2-ethanesulfonic acid) (HEPES), 2-(N-morpholino)ethanesulfonic acid (MES), MES sodium salt, 3-(N-morpholino)propanesulfonic acid (MOPS), N-tris-[hydroxymethyl]-methyl-3-aminopropanesulfonic acid (TAPS); solubilizing agents; detergents, e.g., non-ionic detergents such as Tween-20; nuclease inhibitors; and the like.

[0083] The reaction components used in the amplification and / or detection process can be provided in a variety of forms. For example, the components (e.g., enzymes, deoxyribonucleoside triphosphates, adapters, blockers, and / or primers) can be suspended in aqueous solution or as lyophilized or freeze-dried powders, pellets, or beads. In the latter case, the components, upon reconstitution, form a complete mixture of components for use in the assay. [Example]

[0084] Example 1: Use of a pair of superselective primers in a real-time PCR assay for the detection of rare EGFR G719C mutant template in the presence of abundant wild-type template The design of this first example is shown in Figure 1. The target mutation (G719C), located in exon 18 of the human epidermal growth factor (EGFR) gene, is an A:T base pair instead of a C:G base pair and occurs in an otherwise identical wild-type gene sequence. A PCR assay was performed utilizing mutant and wild-type plasmids containing the target gene sequence, with the "anchor" sequence of one superselective primer (referred to as the "forward" primer) binding to all (-) template strands in the sample (both the rare intended target and the abundant unintended target).

[0085] In this first example, the limiting forward primer contains a unique "5' tag sequence." When the forward allele-discriminating primer binds to and initiates synthesis in the mutant (-) template, the resulting (+) amplicon strand contains the entire forward primer sequence, including the 5' tag sequence. These (+) amplicons then serve as templates for the reverse allele-discriminating primer or, in control experiments, the reverse conventional (non-discriminating) primer. The resulting (-) amplicons 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 probe present in these reactions to illuminate the synthesized amplicons. Furthermore, because the forward primer is present in limited amounts, a single-stranded amplicon is created by extension of the excess reverse primer (either the allele-discriminating primer or the conventional primer), ensuring that the molecular beacon probe can bind to its target without competing with disruption of the amplicon duplex. The sequences of the oligonucleotides used in this example are as follows:

[0086] [ka]

[0087] Target plasmids containing either the EGFR G719C mutation or the corresponding EGFR wild-type sequence were purchased from Integrated DNA Technologies (Coralville, IA, USA) and prepared by inserting a 211-base pair gene fragment into the pIDTSmart Amp vector. Mutant and wild-type plasmid DNA were digested with the restriction endonuclease ScaI (New England Biolabs, Ipswich, Massachusetts, USA). The digestion mixture contained 10 units of ScaI and 4 μg of mutant or wild-type plasmid DNA in a 20 μL volume containing 100 mM NaCl, 10 mM MgCl2, 1 mM dithiothreitol, and 50 mM Tris-HCl (pH 7.9). The reaction was incubated at 37°C for 120 minutes, followed by incubation at 80°C for 20 minutes to inactivate the endonuclease.

[0088] The PCR assays were performed in a volume of 30 μL containing either 10,000 copies of the wild-type template or 10 copies of the mutant template in a mixture containing 10,000 copies of the wild-type template, as well as amplification buffer (50 mM KCl, 10 mM Tris-HCl (pH 8.0), 2.5 mM MgCl), 50 mM tetramethylammonium chloride (Sigma-Aldrich, St. Louis, MO, USA), 0.5% Tween 20 (Sigma-Aldrich), 1.5 units Platinum Taq DNA polymerase (Thermo Fisher Scientific, Waltham, MA, USA), 250 μM ATP, 250 μM CTP, 250 μM GTP, 250 μM TTP, and 300 nM conventional molecular beacons. One set of reactions contained 60 nM of the EGFR G719C superselective forward primer and 300 nM of the EGFR Exon18 conventional reverse primer. The other set of reactions contained 60 nM of the EGFR G719C superselective forward primer and 300 nM of the EGFR G719C superselective reverse primer. Both sets of reactions consisted of overlapping amplifications of 10,000 wild-type copies and 10 mutant copies in a mixture containing 10,000 wild-type copies. The amplifications were performed in duplicate in a Bio-Rad CFX-96 Touch spectrofluorimetric thermal cycler (Hercules, California, USA) using 0.2 ml white polypropylene tubes (USA Scientific, Ocala, Florida, USA). The thermal cycling program was 95°C for 2 minutes, followed by 55 cycles of 95°C for 20 seconds, 60°C for 20 seconds, and 72°C for 20 seconds. The fluorescence intensity of the molecular beacons was measured in real time at the end of the 60°C annealing step of each thermal cycle. The threshold cycle (Ct value) was automatically calculated by the thermal cycler.

[0089] Figure 2 shows the relationship between fluorescence intensity readouts and thermal cycles completed in these real-time PCR amplification and detection assays. The curves for the reaction using a superselective (SSP) forward primer and a conventional reverse primer are plotted in panel A, while the curves for the reaction using the superselective (SSP) forward primer and the superselective (SSP) reverse primer are plotted in panel B. Comparing an assay containing 10 mutants in the presence of 10,000 wild-type nucleotides with an assay containing only 10,000 wild-type nucleotides, the mean threshold cycles (Ct values) for overlap amplification and the difference in mean Ct values ​​(ΔCt values) between the assays are listed in Table 1.

[0090] [Table 1]

[0091] Example 2: Use of a pair of superselective primers in a real-time PCR assay to detect rare EGFRT790M mutant templates in the presence of abundant wild-type templates This example of the method of the present invention utilizes the design shown in Figure 3, which differs from the design of Figure 1 with respect to detection. In this example, a molecular beacon variant, sometimes referred to as a "shared stem" molecular beacon probe (Tsourkas et al. (2002) Nucleic Acids Research 30:4208-4215), was utilized to target the complement of the bridge sequence of the restrictive superselective primer. Such a molecular beacon probe differs from a conventional molecular beacon probe (Example 1) in that the probe's target sequence is complementary to one arm of the stem and the single-stranded loop. The target sequence mutation (T790M), located in exon 20 of the EGFR gene, is a single base pair substitution (SNP) of an A:T base pair instead of a G:C base pair, occurring in an otherwise identical, closely related wild-type sequence. The sequences of the oligonucleotides used in this example are as follows:

[0092] [ka]

[0093] Target plasmids containing either the EGFR T790M mutation or the corresponding EGFR wild-type sequence were purchased from Integrated DNA Technologies (IDT) and prepared by inserting a 200-base pair gene fragment into the pIDTSmartAmp vector. Mutant and wild-type plasmid DNA were digested with the restriction endonuclease ScaI. The digestion mixture contained 10 units of ScaI and 4 μg of mutant or wild-type plasmid DNA in a 20 μL volume containing 100 mM NaCl, 10 mM MgCl2, 1 mM dithiothreitol, and 50 mM Tris-HCl (pH 7.9). The reaction was incubated at 37°C for 120 minutes, followed by incubation at 80°C for 20 minutes to inactivate the endonuclease.

[0094] PCR assays were performed in a 30 μL volume containing 50 mM KCl, 10 mM Tris-HCl (pH 8.0), 2.5 mM MgCl, 50 mM tetramethylammonium chloride, 0.5% Tween 20, 1.5 units Platinum Taq DNA polymerase, 250 μM ATP, 250 μM CTP, 250 μM GTP, 250 μM TTP, and 300 nM shared stem molecular beacons. One set of reactions contained 60 nM of superselective forward primer and 300 nM of conventional reverse primer. The other set of reactions contained 60 nM of superselective forward primer and 300 nM of superselective reverse primer. Each set or reaction contained five replicate amplifications of 10,000 wild-type copies and five replicate amplifications of 10 mutant copies in a mixture containing 10,000 wild-type copies. The amplification was carried out in a Bio-Rad CFX-96Touch spectrofluorimetric thermal cycler using 0.2 ml white polypropylene tubes. The thermal cycling program was 95°C for 2 minutes, followed by 55 cycles of 95°C for 20 seconds, 60°C for 20 seconds, and 72°C for 20 seconds. Molecular beacon fluorescence intensity was measured at the end of each annealing stage at 60°C. The threshold cycle was automatically calculated by the thermal cycler.

[0095] Figure 4 shows fluorescence intensity readouts for completed thermal cycles in PCR amplification using real-time fluorescence detection. The top panel A contains the curves for the replication reaction utilizing the limiting superselective (SSP) forward primer and an excess of conventional reverse primers. The bottom panel B depicts the curves for the replication reaction using the limiting superselective (SSP) forward primer and an excess of superselective (SSP) reverse primers. Comparing an assay containing 10 mutants in the presence of 10,000 wild-type DNA to an assay containing only 10,000 wild-type DNA, the mean threshold cycles (Ct values) for replicate amplifications and the difference in mean Ct values ​​(ΔCt values) between those assays are listed in Table 2. The fluorescence intensity in three of the five amplifications of wild-type-only templates using the superselective primer pair did not exceed background for 55 cycles, and each was assigned a Ct value of >55.

[0096] [Table 2]

[0097] Example 3: Use of a pair of superselective primers in a real-time PCR assay to determine whether two different somatic mutations in the same gene occur in cis on the same chromosome or whether they occur in trans on sister chromosomes. This example of a method of the present invention utilizes the method shown in Figure 5. Two different target mutations, T790M (which has an A:T base pair instead of a G:C base pair in exon 20 of the EGFR gene) and C797S (which has a G:C base pair instead of a C:G base pair in the same exon), are located 20 nucleotides apart from each other when they are on the same chromosome, i.e., in cis. The purpose of the assay shown in this example is to determine whether these two mutations (if both are present in the sample) actually occur on the same chromosome, or whether (if both are present) they occur on sister chromosomes, i.e., they occur in trans.

[0098] First, we performed a series of preliminary assays to demonstrate the type of results that can be obtained from multiplex assays of these individual mutations (or individual assays each searching for one or the other target mutation), with the results accompanying the cis or trans determination for identifying effective targeted therapies. In these assays, there were three primers: a superselective forward primer for EGFR T790M, a superselective forward primer for EGFR C797S, and a conventional reverse primer responsible for the synthesis of an amplicon, regardless of whether only one of these two mutations or both of these mutations are present in the sample. The sequences of the oligonucleotides used in these experiments are as follows:

[0099] [ka]

[0100] In this first series of experiments (which served as a control), the design of which is not shown in Figure 5, both superselective forward primers bound to the (-) template strand; the conventional reverse primer bound to the complementary (+) template strand downstream from where both forward primers bound on their complementary strands; and the molecular beacon probe bound to the (-) amplicon strand in the region between the binding sites of the superselective forward primer and the conventional reverse primer.

[0101] As in the previous example, three target plasmids were purchased from Integrated DNA Technologies and prepared by inserting 200-base-pair gene fragments into the pIDTSmartAmp vector. Each of these plasmid DNAs was digested with the restriction endonuclease ScaI. The digestion mixture contained 10 units of ScaI and 4 μg of mutant or wild-type plasmid DNA in a 20 μL volume containing 100 mM NaCl, 10 mM MgCl2, 1 mM dithiothreitol, and 50 mM Tris-HCl (pH 7.9). The reaction was incubated at 37°C for 120 minutes, followed by incubation at 80°C for 20 minutes to inactivate the endonuclease.

[0102] Five different amplification reaction mixtures were prepared in 30 µL volumes. Each contained 10,000 copies of the wild-type DNA template. One set of reactions contained only the wild-type template. The other four sets of reactions contained an additional 10 copies of one of the following target plasmids or plasmid combinations: C797S plasmid; T790M plasmid; C797S plasmid; T790M plasmid plus C797S plasmid (simulating a situation in which these two mutations occur in trans); or T790M-C797S plasmid, in which the two mutations are present in the same template (cis). All reaction mixtures contained 60 nM superselective T790M forward primer, 60 nM C797S superselective forward primer, 300 nM conventional reverse primer, 300 nM "amplicon-specific" molecular beacon, 50 mM KCl, 10 mM Tris-HCl (pH 8.0), 2.5 mM MgCl, 50 mM tetramethylammonium chloride (TMAC), 0.5% Tween 20, 1.5 units of Platinum Taq DNA polymerase, 250 μM ATP, 250 μM CTP, 250 μM GTP, and 250 μM TTP.

[0103] Four replicate amplifications of each of the five different reaction mixtures were performed in 0.2 ml white polypropylene tubes in a Bio-Rad CFX-96Touch spectrofluorimetric thermal cycler. The thermal cycling program was 95°C for 2 min, followed by 55 cycles of 95°C for 20 s, 60°C for 20 s, and 72°C for 20 s. Molecular beacon fluorescence intensity was measured at the end of each annealing stage at 60°C.

[0104] Figure 6 shows the relationship between fluorescence intensity readouts and thermal cycles for PCR amplification using real-time fluorescence detection. As mentioned above, all amplification reaction mixtures contained 10,000 wild-type copies (unintended target template). Panel A on the top left shows the results of a replicate reaction in which the amplification reaction mixture also contained 10 copies of the T790M mutant target template; Panel B on the top right shows the results of a replicate reaction in which the amplification reaction mixture also contained 10 copies of the C797S mutant target template; Panel C on the center left shows the results of a replicate reaction in which the amplification reaction mixture also contained 10 copies of the T790M mutant target template and 10 copies of the T790M-C797S mutant target template; Panel D on the center right shows the results of a replicate reaction in which the amplification reaction mixture also contained 10 copies of the T790M mutant target template and 10 copies of the C797S mutant target template; and Panel E on the bottom shows the results of a replicate reaction in which the amplification reaction mixture did not contain any copies of the mutant target template. The average threshold cycles (Ct values) for these replicate amplifications are shown in Table 3.

[0105] [Table 3]

[0106] Table 3 shows the results of control reactions containing the EGFRT790M and EGFRC797S superselective forward primers and the EGFRExon20 conventional reverse primer. The mean Ct value of the sample containing only the wild-type (45.82) was distinguishable from the sample containing two mutations in cis (39.49). However, this mean Ct value was virtually identical to the mean Ct value of the reaction containing two mutations in trans, which was virtually identical to the mean Ct value of the reaction containing only one of the two different mutations.

[0107] To illustrate how to determine whether these two mutations occur on the same template (i.e., in cis), three additional assay sets were performed according to embodiments of the present invention. These assays comprised a limited EGFR T790M superselective forward primer, an excess of EGFR C797S reverse primers, and an interprimer-specific molecular beacon probe that targets the region of the (-) amplicon between the sequences where the superselective primers bind, thus binding to both primers and emitting a signal only when extended with a template molecule containing both the EGFR T790M and EGFR C797S mutations (i.e., only when the two mutations are present in cis). The oligonucleotides utilized were as follows:

[0108] [ka]

[0109] The amplification reaction mixture contained 60 nM superselective forward primer, 300 nM superselective reverse primer, and 300 nM molecular beacon. All reaction mixtures contained 10,000 copies of the EGFR wild-type target template. One set of reactions also contained 10 copies of the T790M-C797S target template, a second set of reactions contained 10 copies of the T790M target template and 10 copies of the C797S target template, and a third set of reactions contained no copies of either mutant target template, i.e., only the wild-type template was present. Otherwise, the amplification reaction mixtures were as described above.

[0110] Four replicate amplifications of each of the three reaction mixtures with real-time detection were performed as described above. Real-time fluorescence measurements obtained during the first 55 amplification cycles are shown in the three graphs contained in Figure 7. Bottom panel C shows the results for a reaction mixture containing only 10,000 wild-type templates; the fluorescence of all four replicates remained above background throughout the 55 cycles; the average Ct of the four replicates, while undeterminable, was at least above 55. Top right panel B shows the results for a reaction mixture containing 10,000 wild-type templates and 10 copies each of the T790M and C797S templates (i.e., both mutations were present in trans); the fluorescence of all four replicates remained above background throughout the 55 cycles; the average Ct of the four replicates, while undeterminable, was at least above 55. Panel A, top left, shows the results for a reaction mixture containing 10,000 wild-type templates and 10 copies of the T790M-C797S template (i.e., two mutations were present in cis); all four of these reactions showed a positive signal, with an average Ct of 41.31. Thus, although the ΔCt between the 10 cis templates in the presence of 10,000 wild-type templates cannot be precisely determined, it was at least 13.69 when compared to reactions containing 10 copies each of the two different mutant templates present in trans in reactions that also contained 10,000 wild-type templates.

[0111] Example 4: Use of superselective primers as limiting primers in real-time PCR assays to determine whether two different somatic mutations on the same gene occur in cis on the same chromosome or whether they occur in trans on two different sister chromosomes. The method described in Example 3 was repeated utilizing an ARMS primer as the reverse primer instead of the reverse superselective primer. This alternative configuration is shown in Figure 8. The oligonucleotides in the amplification reaction mixture were as follows:

[0112] [ka]

[0113] In the sequence of the ARMS primer, the intentionally introduced nucleotide that is a mismatch with both the intended (mutated) target sequence and the closely related unintended (wild-type) target sequence is in bold and italic. This nucleotide is the third nucleotide from the 3' end and creates an A:C mismatch with both the intended target sequence and the closely related unintended target sequence.

[0114] Except for the use of the ARMS reverse primer instead of the superselective reverse primer, the reaction mixture was the same as that described in Example 3, as were the thermal cycling conditions and method of fluorescence detection. The design of this experiment is shown in Figure 8. Very similar results were obtained with the same series of amplifications as shown in Figure 7.

[0115] The results of these experiments involving ARMS primers are shown in Figure 9. For reactions containing only 10,000 wild-type templates, the fluorescent signal from all four replicates did not exceed background throughout 55 cycles, and the average Ct value for the four replicates, although not determinable, was at least above 55. For reactions containing 10,000 wild-type templates and 10 copies each of the T790M and C797S templates (present in trans), the fluorescent signal from all four replicates did not exceed background throughout 55 cycles, and the average Ct value for all four replicates, although not determinable, was at least above 55. For reactions containing 10,000 wild-type templates and 10 T790M-C797S templates (present in cis), the average Ct value for the four replicates was 43.31. Thus, the ΔCt value between the reaction containing 10 cis templates and 10,000 wild-type templates and the reaction containing 10 trans templates and 10,000 wild-type templates could not be determined precisely but was at least greater than 11.69.

[0116] Example 5: Comparison of the use of superselective primers as limiting or excess primers in real-time PCR assays containing ARMS primers to detect rare KRASG12D mutant templates in the presence of abundant wild-type templates In this example experiment, a superselective primer (referred to herein as a forward primer) was used as the restrictive first primer, and an ARMS primer (referred to herein as a reverse primer) was used as the excess second primer; a superselective primer (referred to herein as a reverse primer) was used as the excess first primer, and an ARMS primer (referred to herein as a forward primer) was used as the restrictive second primer. For comparison, pairs of superselective primers were also used in the experiment. In all primer pairs, the restrictive forward primer has a 5' tag sequence, the complementary sequence of which is the target of the molecular beacon probe. Figure 10 shows how the superselective primer is the reverse primer and the ARMS primer is the forward primer, where the ARMS forward primer contains the 5' tag sequence. The intended target in this example is a KRAS G12D mutant template, which differs from the closely related wild-type template by a single T:A mutant base pair. Both primers in all pairs have a 3'-terminal detection nucleotide complementary to the nucleotide in that base pair. The sequences of the oligonucleotides used in this example are as follows:

[0117] [ka]

[0118] The third nucleotide from the 3' end of each ARMS primer is shown in bold and italic because it did not match both the mutant and wild-type sequences. This mismatch is indicated by an "m" in the sequence name.

[0119] Target plasmids containing either the KRAS G12D mutation or the corresponding KRAS wild-type sequence were purchased from Integrated DNA Technologies and prepared by inserting a 390-base pair gene fragment into the pUCIDT vector. Mutant and wild-type plasmid DNA were digested with the restriction endonuclease DraI (New England Biolabs). The digestion mixture contained 10 units of DraI and 4 μg of mutant or wild-type plasmid DNA in a 20 μL volume containing 50 mM potassium acetate, 20 mM Tris acetate (pH 7.9), 10 mM magnesium acetate, and 100 μg / ml bovine serum albumin. The reaction was incubated at 37°C for 120 minutes, followed by incubation at 65°C for 20 minutes to inactivate the endonuclease.

[0120] PCR assays were performed in a 30 μL volume containing 50 mM KCl, 10 mM Tris-HCl (pH 8.0), 2.5 mM MgCl, 50 mM tetramethylammonium chloride (Sigma-Aldrich), 0.5% Tween 20 (Sigma-Aldrich), 1.5 units of Platinum Taq DNA polymerase (Thermo Fisher Scientific), 250 μM ATP, 250 μM CTP, 250 μM GTP, 250 μM TTP, 60 nM forward primer, 300 nM reverse primer for use with the superselective forward primer, and 300 nM conventional molecular beacon or 300 nM shared stem molecular beacon for use with the ARMS forward primer. Amplifications were performed in a Bio-Rad CFX-96Touch spectrofluorimetric thermal cycler using 0.2 ml white polypropylene tubes (USA Scientific). The thermal cycling program was 95°C for 2 min, followed by 55 cycles of 95°C for 20 s, 60°C for 20 s, and 72°C for 20 s. The fluorescence intensity of the molecular beacons was measured at the end of each annealing stage at 60°C.

[0121] The reaction mixture contained 10,000 copies of the wild-type target template and 10 or 0 copies of the mutant target template. Amplification reactions were performed in triplicate. The real-time fluorescence results are shown in Figure 11, where the top panel A shows the results obtained with the superselective primer pair, the bottom left panel B shows the results obtained with the primer pair containing the superselective forward restricting primer and an excess of ARMS reverse primer, and the bottom right panel C shows the results obtained with the primer pair containing the restricting ARMS forward primer and an excess of the superselective reverse primer. In all panels, the fluorescence of the wild-type sample did not exceed background throughout 55 cycles, while the Ct values ​​for all samples containing 10 copies of the mutant template were below 45.

[0122] Example 6: Duplex assay: Use of a pair of superselective primers in a real-time PCR assay for the detection of a rare EGFR G719C mutant template in the presence of abundant normal human genomic DNA template, and simultaneous use of a different superselective primer and a conventional primer to detect the β-actin reference gene. The design of these PCR assays for detecting G719C is shown in Figure 12. The G719C target mutation, located in exon 21 of the human epidermal growth factor receptor (EGFR) gene, is a T:A base pair instead of a G:C base pair and occurs in an otherwise identical wild-type gene sequence. The assay utilizes a linearized plasmid containing the mutant target sequence, and fragmented normal human genomic DNA containing the wild-type EGFR gene sequence to simulate cell-free DNA fragments isolated from the plasma of a liquid biopsy sample.

[0123] In this example, the limiting forward primer for detecting G719C contains a unique "5' tag sequence." When the forward allele-discriminating primer binds to and initiates synthesis of the mutant (-) template, the resulting (+) amplicon strand contains the entire forward primer sequence, including the 5' tag sequence. These (+) amplicons then serve as templates for the reverse allele-discriminating primer. The resulting (-) amplicons contain the complement of the 5' tag sequence at their 3' ends. It is the 3' complement of the 5' tag sequence that is targeted for binding by a conventional FAM-labeled molecular beacon probe. The forward superselective primer is present at a limiting concentration, and the reverse superselective primer is present in excess, ensuring that the molecular beacon probe can bind to the excess (-) amplicon target without significant competition from the limiting concentration of (+) amplicons.

[0124] Additionally, detection of a β-actin reference gene sequence occurring in normal human DNA (including the wild-type EGFR gene) was included in the assay to provide a reference threshold (Ct) reflecting the amount of DNA in the sample. The amplicon was detected using Quasar 705-labeled interprimer-specific molecular beacons that bind to excess β-actin (-) amplicons between the complement of the superselective primer sequence and the conventional primer sequence.

[0125] The sequences of the oligonucleotides used in this example are as follows:

[0126] [ka]

[0127] A target plasmid containing the EGFR G719C mutation was purchased from Integrated DNA Technologies and prepared by inserting a 211-base pair gene fragment into the pUCIDT vector. The mutant plasmid DNA was digested with the restriction endonuclease DraI (New England Biolabs). The digestion mixture contained 10 units of DraI and 4 μg of mutant plasmid DNA in a 20 μL volume containing 50 mM potassium acetate, 20 mM Tris acetate (pH 7.9), 10 mM magnesium acetate, and 100 μg / ml bovine serum albumin. The reaction was incubated at 37°C for 120 minutes, followed by incubation at 65°C for 20 minutes to inactivate the endonuclease.

[0128] Wild-type human DNA (from multiple anonymous donors), catalog number G1521, was purchased from Promega Corporation (Madison, WI). Approximately 9 μg of this DNA was digested with 10 units of restriction endonuclease MseI (New England Biolabs, Ipswich, MA) in 50 μL of buffer provided by New England Biolabs containing 100 μg / mL bovine serum albumin, 10 mM magnesium acetate, 50 mM potassium acetate, and 20 mM Tris acetate (pH 7.9) at 37°C for 120 minutes; the enzyme was then inactivated by incubation at 65°C for 20 minutes.

[0129] The PCR assay was performed in a volume of 30 μL containing 50 mM KCl, 10 mM Tris-HCl (pH 8.0), 2.5 mM MgCl, 60 mM tetramethylammonium chloride (Sigma-Aldrich), 0.5% Tween 20 (Sigma-Aldrich), 1.5 units of Platinum Taq DNA polymerase (Thermo Fisher Scientific), 250 μM ATP, 250 μM CTP, 250 μM GTP, 250 μM TTP, 60 nM each of two different superselective forward primers, 500 nM EGFR G719C superselective reverse primer, 500 nM β-actin conventional reverse primer, 300 nM conventional molecular beacon for detection of the EGFR G719C mutation amplicon, and 500 nM interprimer-specific molecular beacon for detection of the β-actin amplicon.

[0130] The amplification was carried out in a Bio-Rad CFX-96Touch spectrofluorimetric thermal cycler using 0.2 ml white polypropylene tubes (USA Scientific). The thermal cycling program was 95°C for 2 minutes, followed by 55 cycles of 95°C for 20 seconds, 60°C for 20 seconds, and 72°C for 20 seconds. Fluorescence intensity of the molecular beacons was measured in both the FAM and Quasar705 channels at the end of each 60°C annealing stage.

[0131] FIG. 13 shows the relationship between fluorescence intensity readings and thermal cycling.

[0132] The first set of 10 reactions (the results of which are shown in panel A, top left) contained 10,000 copies of wild-type genomic DNA and 500 copies of mutant plasmid DNA; the second set of 10 reactions (the results of which are shown in panel B, top right) contained 10,000 copies of wild-type genomic DNA and 50 copies of mutant plasmid DNA; the third set of 10 reactions (the results of which are shown in panel C, bottom left) contained 10,000 copies of wild-type genomic DNA and 5 copies of mutant plasmid DNA; and the fourth set of 10 reactions (the results of which are shown in panel D, bottom right) contained only 10,000 copies of wild-type genomic DNA and no copies of mutant plasmid DNA.

[0133] The foregoing examples and description of the preferred embodiments should be construed as illustrative, and not limiting, of the invention as defined by the claims. As will be readily appreciated, numerous variations and combinations of the features described above can be utilized without departing from the invention as set forth in the claims. Such variations are not to be considered a departure from the scope of the invention, and all such variations are intended to be included within the scope of the following claims.

Claims

1. 1. A primer-dependent method for amplifying and detecting only a mutant sequence in a sample containing 10,000 copies of DNA having a wild-type sequence and 10 or fewer copies of DNA having a mutant sequence that differs from the wild-type sequence by one or two base pairs, comprising: (a) comprising the sample, a DNA polymerase, deoxyribonucleoside triphosphates, an amplification buffer, and a homogeneous fluorescent detection means for detecting the amplification products, and a pair of forward and reverse primers; preparing a primer-dependent amplification reaction mixture, wherein the forward and reverse primers are specific for the mutant sequence but mismatched to the wild-type sequence; (b) amplifying each mutant sequence present in the sample by repeatedly cycling the primer-dependent amplification reaction mixture by a primer-dependent amplification method; and (c) detecting the mutant sequence by measuring the intensity of fluorescence from the homogeneous fluorescence detection means; (i) the forward primer is an allele-discriminating multi-part primer, and comprises, from the 5' end to the 3' end, a first anchor sequence, a first bridge sequence, and a first foot sequence, in this order; the first foot sequence is mismatched to the wild-type sequence by the 3' or penultimate nucleotide, and (ii) the reverse primer is an allele-discriminating primer, and the reverse primer is a multipart primer that is mismatched to the wild-type sequence by at least its 3'-end or penultimate nucleotide, and comprises, from its 5'-end to its 3'-end, a second anchor sequence, a second bridge sequence, and a second foot sequence, in this order; The method, wherein the first bridge sequence and the second bridge sequence each have a region that does not hybridize to the mutant sequence.

2. 2. The method of claim 1, wherein each primer comprises a 3' terminal detection nucleotide that is complementary to the mutant sequence but mismatched to the wild-type sequence.

3. 3. The method of claim 1 or 2, wherein the cycling comprises temperature cycling in an asymmetric polymerase chain reaction (PCR) method.

4. The method of claim 3 , wherein the detecting comprises real-time detecting.

5. The method of claim 3, wherein the PCR method is a digital PCR method and the detection comprises end-point detection.

6. The method of any one of claims 1 to 5, wherein at least one of the mutant sequences in the sample comprises at least two different mutant sequences, and the homogeneous fluorescent detection means comprises at least two different homogeneous fluorescent detection probes, each for one of the at least two different mutant sequences.

7. 7. The method of claim 6, wherein the at least two mutant sequences comprise a group of mutant sequences, and the probes of the mutant sequences in the group are labeled with the same color.

8. The method of claim 6 , wherein the probes are color-coded.

9. 9. The method of any one of claims 1 to 8, wherein each different wild-type sequence differs from the corresponding mutant sequence by a single base pair, and both the forward primer and the reverse primer are mismatched by the single base pair.

10. The method of claim 9, wherein each of the reverse primers is a multi-part primer comprising, from the 5' end to the 3' end, a second anchor sequence, a second bridge sequence, and a second foot sequence, in that order.

11. 11. The method of claim 9 or 10, wherein the homogeneous fluorescent detection means comprises a probe for each mutant sequence.

12. 12. The method of claim 11, wherein the forward primer or the reverse primer for each mutant sequence comprises a 5' tag sequence, and the complement of each 5' tag sequence is the target of the probe.

13. 12. The method of claim 6 or 11, wherein each of the probes comprises a sequence complementary to the complement of the first bridge sequence or the complement of the second bridge sequence.

14. The method of claim 13 , wherein the probe is a shared stem molecular beacon.

15. The method of claim 6 , wherein the primer-dependent amplification reaction mixture comprises an effective amount of a selectivity enhancement reagent.

16. 16. The method of claim 15, wherein the selectivity enhancing reagent is Hofmeister's salt.

17. 9. The method of any one of claims 1 to 8, wherein at least one of the mutant sequences differs from the corresponding wild-type sequence by a first base pair and a second base pair that occur in cis, and wherein the forward primer is complementary to the first base pair and the reverse primer is complementary to the second base pair.

18. 18. The method of claim 17, wherein at least one of the mutant sequences in the sample comprises two or more mutant sequences, and the homogeneous fluorescent detection means comprises at least one homogeneous fluorescent detection probe for each mutant sequence.

19. 19. The method of claim 17 or 18, wherein the homogeneous fluorescent detection means for each mutant sequence comprises an inter-primer specific molecular beacon probe.

20. The method of any one of claims 17 to 19, wherein the primer-dependent amplification reaction mixture comprises an effective amount of a selectivity enhancement reagent.

21. 21. The method of claim 20, wherein the selectivity enhancing reagent is Hofmeister's salt.

22. 22. The method of claim 16 or claim 21, wherein the Hofmeister salt comprises tetramethylammonium chloride (TMAC).

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