Suppression of off-target signals in nucleic acid amplification reactions

Abstract

Disclosed are compositions and methods for suppressing off-target signal generation in nucleic acid amplification processes. The compositions and methods use a detector probe having a detectable label and a dark probe that omits the detectable label. The detector probe is configured to specifically interact with a target nucleic acid. The dark probe is configured to specifically interact with an off-target sequence similar to the target nucleic acid, thereby limiting off-target interactions between the detector probe and the off-target sequence.

Description

[Technical Field]

[0001] (Related Applications) This application claims priority to U.S. Provisional Patent Application No. 63 / 430,256, filed December 5, 2022, the disclosure of which is incorporated herein by reference in its entirety for all purposes.

[0002] FIELD OF THE INVENTION The present disclosure is directed to compositions and methods for suppressing interactions between target nucleic acid probes and off-target nucleic acids in a reaction mixture. [Background technology]

[0003] For many medical, diagnostic, and forensic applications, amplification of a specific nucleic acid sequence is essential to enable its detection in, or isolation from, a sample where it is present in very small amounts.

[0004] The polymerase chain reaction (PCR) is an in vitro method for the enzymatic synthesis of specific DNA sequences using two oligonucleotide primers that hybridize to opposite strands and flank a region of interest in the target DNA. A repetitive series of reaction steps, including template denaturation, primer annealing, and extension of the annealed primers by DNA polymerase, results in the exponential accumulation of a specific fragment whose termini are defined by the primers. PCR allows for the selective enrichment of specific DNA sequences by several orders of magnitude.

[0005] In some assays using PCR or other methods of nucleic acid amplification, the target nucleic acid is mixed with similar nucleic acid sequences in the test sample. In some cases, PCR components intended to interact with the target nucleic acid instead interact with similar off-target sequences. This can detrimentally introduce errors into the associated detection and quantification analysis.

[0006] Therefore, there is a continuing need for nucleic acid amplification assays that substantially reduce or eliminate the problem of cross-reactivity in nucleic acid amplification reactions. Summary of the Invention

[0007] Disclosed are compositions and methods for suppressing off-target signal generation in nucleic acid amplification processes. The compositions and methods utilize a detector probe having a detectable label and a dark probe that omits the detectable label. The detector probe is configured to specifically interact with a target nucleic acid. The dark probe is configured to specifically interact with off-target sequences similar to the target nucleic acid, thereby limiting off-target interactions between the detector probe and the off-target sequence.

[0008] In one embodiment, a composition for suppressing off-target signal generation in a nucleic acid amplification process includes a detector probe containing a detectable label. The detector probe is configured to specifically interact with a nucleic acid target to enable detection and / or quantification of the nucleic acid target. The composition also includes a dark probe that omits the detectable label. The dark probe is configured to specifically interact with an off-target sequence, thereby blocking or limiting interaction between the detectable label and the off-target sequence.

[0009] In some embodiments, the detector probe is configured to target a first allele of a particular single nucleotide polymorphism (SNP) position and the dark probe is configured to target a second, different allele of the SNP position, in some embodiments, the SNP position is associated with an oncogenic mutation.

[0010] In some embodiments, the concentration of the dark probe provided in the composition is at least the same as the amount of the detector probe.

[0011] In one embodiment, a method for suppressing off-target signal generation in a nucleic acid amplification process includes providing a composition comprising: a detector probe having a detectable label configured to specifically interact with a nucleic acid target to enable detection and / or quantification of the nucleic acid target; and a dark probe omitting the detectable label and configured to specifically interact with an off-target sequence, thereby blocking or limiting interaction between the detectable label and the off-target sequence. The method further includes forming a reaction mixture comprising the sample and the composition, subjecting the reaction mixture to an amplification process, and detecting or quantifying the nucleic acid target. In some embodiments, the amplification process is digital PCR (dPCR).

[0012] This Summary is provided to introduce a selection of concepts in a simplified form that are further described below in the Detailed Description. This Summary is not intended to identify key features or essential features of the claimed subject matter, nor is it intended to be used as an index of the scope of the claimed subject matter.

[0013] Various objects, features, characteristics, and advantages of the present invention will become apparent and more readily understood from the following description of embodiments taken in conjunction with the accompanying drawings and appended claims, all of which form a part hereof. In the drawings, like reference numerals may be utilized to designate corresponding or similar parts in the various views, and the various elements depicted are not necessarily drawn to scale. [Brief explanation of the drawings]

[0014] [Figure 1A] 1 is an overview of a conventional nucleic acid amplification process in which a detector probe is intended to specifically interact with a nucleic acid target, resulting in the generation of a corresponding signal; the schematic illustrates that the detector probe, or some portion of the detector probe, may instead interact with an off-target sequence and erroneously add to the resulting signal.

[0015] [Figure 1B] This is an overview of an improved nucleic acid amplification process in which a "dark probe" is also provided in the reaction mixture, the dark probe being configured to specifically interact with off-target sequences, thereby blocking the detector probe from interacting with the off-target sequences and thus suppressing cross-reactivity of the detector probe.

[0016] [Figure 2A] 1 illustrates examples of detector probe and dark probe pairs with different types of overlap. [Figure 2B] 1 illustrates examples of detector probe and dark probe pairs with different types of overlap. [Figure 2C] 1 illustrates examples of detector probe and dark probe pairs with different types of overlap.

[0017] [Figure 3A] Comparing the results of separate conventional KRAS516 assays, Figure 3A shows an assay in which KRAS516 served as the template, and Figure 3B shows an assay in which KRAS518 served as the template, the results show that the KRAS516 assay probe has substantial cross-reactivity with off-target KRAS518 templates, which can result in false positive and / or erroneously increased measurements for KRAS516. [Figure 3B] Comparing the results of separate conventional KRAS516 assays, Figure 3A shows an assay in which KRAS516 served as the template, and Figure 3B shows an assay in which KRAS518 served as the template, the results show that the KRAS516 assay probe has substantial cross-reactivity with off-target KRAS518 templates, which can result in false positive and / or erroneously increased measurements for KRAS516.

[0018] [Figure 4A]The results of separate KRAS518 assays enriched to include a dark probe to block off-target KRAS516 templates were compared; Figure 4A shows the assay in which KRAS516 served as the template, and Figure 4B shows the assay in which KRAS518 served as the template. The results show that the inclusion of a dark probe to block off-target KRAS518 templates effectively suppressed the cross-reactivity of the KRAS518 target probe with the off-target KRAS516 template. [Figure 4B] The results of separate KRAS518 assays enriched to include a dark probe to block off-target KRAS516 templates were compared; Figure 4A shows the assay in which KRAS516 served as the template, and Figure 4B shows the assay in which KRAS518 served as the template. The results show that the inclusion of a dark probe to block off-target KRAS518 templates effectively suppressed the cross-reactivity of the KRAS518 target probe with the off-target KRAS516 template.

[0019] [Figure 5A] 1 shows the results of a dark probe titration test for two exemplary dark probes. [Figure 5B] 1 shows the results of a dark probe titration test for two exemplary dark probes. [Figure 5C] 1 shows the results of a dark probe titration test for two exemplary dark probes. [Figure 5D] 1 shows the results of a dark probe titration test for two exemplary dark probes. DETAILED DESCRIPTION OF THE INVENTION

[0020] Overview of off-target suppression using dark probes

[0021] 1A illustrates a conventional nucleic acid amplification process in which a labeled detector probe is intended to specifically interact with a nucleic acid target and generate a corresponding signal. As shown, the detector probe (or some portion of multiple detector probes) may instead interact with off-target sequences, increasing the resulting signal. This cross-reactivity can result in, for example, false-positive detection of the target and / or an increase in the calculated amount of the target.

[0022] FIG. 1B illustrates an improved nucleic acid amplification process in which a "dark probe" is also provided in the reaction mixture. The dark probe is configured to specifically interact with the off-target sequence, thereby blocking the detector probe from interacting with the off-target sequence. This beneficially reduces the amount of cross-reactivity between the detector probe and the off-target sequence, thereby allowing the signal to better correspond to the amount of target. In other words, by limiting the interaction between the detector probe and the off-target sequence, the amount of signal associated with the detectable label better corresponds to the amount of nucleic acid target in the sample, thus allowing for more accurate detection and / or quantification of the target nucleic acid.

[0023] The use of an off-target suppression embodiment such as that shown in Figure 1B is beneficial when the test sample has (or is suspected to have) one or more sequences similar enough to the target nucleic acid to cause some degree of cross-reactivity with the detector probe. As an example, one or more dark probes can be used in an assay designed to amplify and detect a specific allele of a nucleic acid target that is often present along with other alleles in a sample. In such an assay, the detector probe is designed to interact specifically with the specific targeted allele, while each dark probe is designed to interact specifically with other alleles, thereby reducing interactions between the detector probe and the off-target nucleic acid of the other alleles.

[0024] The problem of cross-reactivity with off-target sequences is particularly serious in rare mutation assays designed to detect and / or quantify rare alleles that may exist together with other more abundant alleles. For example, the target allele may exist in a background of wild-type and other alleles. In these applications, even limited cross-reactivity with off-target sequences can have a substantial effect on the results, simply because the amount of off-target nucleic acid is significantly greater than the amount of target nucleic acid containing the intended allele. That is, even if the detector probe only interacts with a small proportion of off-target sequences, the abundance of off-target nucleic acid templates relative to the target nucleic acid templates in the reaction mixture can result in excessive disruption of the results.

[0025] Rare mutation assays can include, for example, assays designed to detect and / or quantitate oncogenic mutations. In these and other rare mutation assays, samples often contain relatively high levels of other alleles (e.g., wild-type or other alleles), which can affect the results even when the detector probe is designed to specifically interact with the target allele.

[0026] In some embodiments, the sample comprises genomic DNA having a target locus with a mutant allele frequency (MAF) of 5% or less, 4% or less, 3% or less, 2% or less, 1% or less, 0.75% or less, 0.5% or less, 0.25% or less, 0.15% or less, or 0.1% or less, and the method can effectively detect rare mutant alleles with greater accuracy than other similar assays that omit dark probes and / or can effectively quantitate rare mutant alleles with greater accuracy than other similar assays that omit dark probes.

[0027] In some embodiments, the off-target nucleic acid sequence is similar to the target sequence except for a few nucleotides (e.g., 2-3 nucleotides). Often, the target sequence and the off-target sequence differ by only a single nucleotide. For example, the target may be a first allele at a particular single nucleotide polymorphism (SNP) position, and the off-target sequence is the other allele at the same SNP position. In these embodiments, the detector probe is configured to target a first allele at the SNP position, and the dark probe is configured to target a second, different allele at the SNP position. Some embodiments may include an additional dark probe, such as a dark probe for targeting a third allele at the SNP position. While many of the examples described herein relate to SNP mutations, it is understood that the same principles can be applied to other types of mutations, including deletion mutations, insertion mutations, and multi-nucleotide base substitutions.

[0028] In some embodiments, the off-target sequence is not necessarily a different allele of the target sequence, but is still sufficiently similar to the target to cause cross-reactivity with the detector probe. For example, the detector probe can be configured to target a specific allele at a specific nucleotide position. The off-target nucleic acid can match the target nucleic acid at the mutation locus, but can have another nucleotide that differs from the target, located a certain number of nucleotides (e.g., 1-20) away from the mutation locus.

[0029] The detector probe and the corresponding dark probe have similar sequences because the off-target sequence is close enough to the target sequence to cause potential cross-reactivity with the detector probe. The dark probe is adjusted relative to the detector probe so that it specifically targets the off-target sequence rather than the target sequence itself. In many cases, the detector probe and the corresponding dark probe are the same size, but this need not be the case in all embodiments.

[0030] 2A-2C illustrate an exemplary detector probe and dark probe pair. The detector probe and corresponding dark probe each have an "overlapping sequence portion" that represents the portion of their respective sequences that overlap when the detector probe and dark probe are aligned. In many cases, the detector probe and dark probe are the same size and will completely overlap each other, as shown in FIG. 2A. In FIG. 2B, the dark probe is shorter than the detector probe, so the overlapping portion is the size of the dark probe (i.e., the detector probe has a 5' portion that is not within the overlapping portion). In FIG. 2C, the probes are substantially the same size, but the dark probe is shifted relative to the detector probe such that the detector probe includes a 5' section that is not within the overlapping portion and the dark probe includes a 3' section that is not within the overlapping portion.

[0031] As shown, even when the probes differ in length and / or are shifted relative to one another, the distinguishing feature of the probes (here, the difference in A or G nucleotides) is within the overlapping sequence portion, which is preferably about 10-30 nucleotides in length to ensure that the dark probe can adequately block interactions between the detector probe and off-target sequences.

[0032] In the example shown in Figures 2A-2C, the overlapping portions of the detector probe and dark probe differ by only a single nucleotide; at the corresponding position, the detector probe contains an A (for example) and the dark probe contains a G (for example). This is the case when the target sequence and the off-target sequence are different alleles of a particular SNP. In other embodiments, the overlapping sequence portions of the detector probe and dark probe may differ by two or more nucleotides, may contain one or more deletions, and / or may contain one or more insertions relative to the opposing probe. Typically, however, the detector probe and dark probe have about 80%, about 85%, about 90%, or about 95% identity, or an identity within a range defined by any two of the above values.

[0033] Some embodiments may include a single detector probe and a single dark probe. Such embodiments may be considered singleplex with respect to the detector probe and singleplex with respect to the dark probe. Other embodiments may include one or more additional detector probes (each typically uniquely labeled and directed to a specific target) and thus be multiplex with respect to the detector probe, and / or may include one or more additional dark probes directed to other off-target sequences and thus be multiplex with respect to the dark probe.

[0034] For embodiments in which the detector probes are multiplexed, each detector probe may have a single corresponding dark probe or multiple corresponding dark probes. For example, in a duplex assay using a first detector probe and a second detector probe, the assay may include two dark probes (i.e., dark probe "1a" and dark probe "1b") associated with the first detector probe, each designed to block different off-target sequences that may cross-react with the first detector probe, and a single dark probe (i.e., dark probe 2) associated with the second detector probe, designed to block off-target sequences that may cross-react with the second detector probe. In other words, the number of dark probes associated with each different detector probe need not be consistent; instead, the number of dark probes utilized with each detector probe can be selected based on the number of off-target sequences to be blocked from that particular detector probe.

[0035] Some embodiments may include one or more detector probes that do not have a corresponding dark probe. For example, in a multiplex assay using first, second, and third detector probes, the assay may include two dark probes associated with the first detector probe, each designed to block a different off-target sequence that may cross-react with the first detector probe (i.e., dark probe "1a" and dark probe "1b"), a single dark probe associated with the second detector probe, designed to block an off-target sequence that may cross-react with the second detector probe (i.e., dark probe 2), and any dark probe associated with the third detector probe may be omitted. This may be the case if significant off-target interactions of the third detector probe are not expected.

[0036] In some embodiments, the dark probe is provided in a concentration at least equal to that of the detector probe. In some embodiments, the dark probe is provided in a concentration greater than that of the detector probe. In embodiments including multiple dark probes, each associated with a single detector probe, each of these dark probes may be provided in a concentration equal to or greater than that of the detector probe. Characteristics of detector probe and dark probe

[0037] As discussed above, the detector probe includes a detectable label, while the dark probe omits a detectable label. The detectable label of the detector probe can be a fluorescent label. Examples of fluorescent labels are known in the art, and include, for example, VIC, FAM, JUN, ABY, Alexa Fluor dye labels (e.g., AF647 and AF676), and combinations thereof.

[0038] Exemplary detectable labels that can be utilized with the embodiments described herein include, for example, the following:

[0039] fluorescein (e.g., 5-carboxy-2,7-dichlorofluorescein, 5-carboxyfluorescein (5-FAM), 6-JOE, 6-carboxyfluorescein (6-FAM), VIC, FITC, 6-carboxy-4',5'-dichloro-2',7'-dimethoxy-fluorescein (JOE)), 5-carboxy-1,4-dichloro-2',7'-dichloro-fluorescein and 6-carboxy-1,4-dichloro-2',7'-dichloro-fluorescein (TET), 5-carboxy-1,4-dichloro-2',4',5',7'-tetra-chlorofluorescein and 6-carboxy-1,4-dichloro-2',4',5',7'-tetra-chlorofluorescein, HEX, PET, NED, Oregon Green (e.g., 488, 500, 514)),

[0040] pyrene (e.g., Cascade Blue, Alexa Fluor 405),

[0041] Coumarins (e.g., Pacific Blue, Atto 425, Alexa Fluor 350, Alexa Fluor 430),

[0042] cyanine dyes (e.g., Cy dyes such as Cy3, Cy3.18, Cy3.5, Cy5, Cy5.18, Cy5.5, and Cy7);

[0043] Rhodamines (e.g., 110, 123, B, B200, BB, BG, B extra, 5-carboxytetramethylrhodamine and 6-carboxytetramethylrhodamine (5-TAMRA, 6-TAMRA), 5-carboxyrhodamine 6G and 6-carboxyrhodamine 6G, Lissamine, Lissamine rhodamine B, Rhod-2, ROX (6-carboxy-X-rhodamine), 5-ROX (carboxy-X-rhodamine) and 6-ROX (carboxy-X-rhodamine), sulforhodamine B can C, sulforhodamine G Extra, 5TAMRA and 6TAMRA (carboxytetramethyl-rhodamine), (TRITC), ABY, JUN, LIZ, RAD, RXJ, Texas Red; and Texas Red-X),

[0044] Alexa Fluor fluorophores, a broad class that includes numerous dye types such as cyanines (e.g., Alexa350, Alexa405, Alexa430, Alexa488, Alexa500, Alexa514, Alexa532, Alexa546, Alexa555, Alexa568, Alexa594, Alexa610, Alexa633, Alexa635, Alexa647, Alexa660, Alexa676, Alexa680, Alexa700, and Alexa750);

[0045] FRET donor / acceptor pairs (e.g., fluorescein / fluorescein, fluorescein / rhodamine, fluorescein / cyanine, rhodamine / cyanine, fluorescein / Alexa Fluor, Alexa Fluor / rhodamine), as well as other types of dyes known to those skilled in the art.

[0046] The fluorophore label may be conjugated to a quencher such as a dark fluorescent quencher (DFQ), a black hole quencher (BHQ), Iowa Black, QSY7, QSY21 quencher, Dabsyl sulfonic acid / carboxylic acid quencher and Dabcel sulfonic acid / carboxylic acid quencher, and MGB-NFQ quencher. The fluorophore label may also include, for example, sulfonate derivatives of fluorescein dyes with SO3 in place of the carboxylate group, phosphoramidite forms of fluorescein, and / or phosphoramidite forms of Cy5.

[0047] The detector probe can be configured as a TaqMan probe, which is known in the art and described in more detail below. Such a probe can hybridize to the target downstream from the primer so that the exonuclease activity of the polymerase during subsequent primer extension separates the dye label from the quencher, increasing the dye signal.

[0048] The detector probe and dark probe can be about 10 to about 40 nucleotides in length, more preferably about 15 to about 35 nucleotides in length, more preferably about 18 to about 30 nucleotides in length.

[0049] Dark probes as disclosed herein preferably include a 3' block to prevent or limit the dark probe from acting as a primer. The 3' block may include, for example, an alkyl spacer, a terminal 3' phosphate, a dideoxynucleotide, an inverted 3' end, or other extension block known in the art. [Example]

[0050] The following example illustrates the effective use of off-target suppression assays to detect KRAS516 mutations. KRAS is a proto-oncogene, and specific KRAS mutations are involved in various cancers. The KRAS516 mutation is a 34G>T nucleotide mutation that causes a G12C amino acid change. As with many rare mutation assays, assays for detecting and / or quantifying KRAS516 mutations can be obscured by similar off-target sequences, including the KRAS517 (34G>A) and / or KRAS518 (34G>C) alleles.

[0051] In the following exemplary assays using dark probes, the dark probes include one or both of those shown in Table 1. Exemplary detector probes are also shown. Other detector probes for KRAS516 are known in the art (e.g., detector probe with Assay ID Hs000000047_rm available from Thermo Fisher Scientific). Alternatively, reverse complements of these sequences may be utilized. [Table 1] Example 1: Cross-reactivity of KRAS516 assay with KRAS518 template

[0052] Figures 3A and 3B compare the results of separate conventional KRAS516 dPCR assays. Figure 3A shows an assay in which KRAS516 served as the template (at 0.5% MAF), and Figure 3B shows an assay in which KRAS518 served as the template (at 1.75% MAF). In Figures 3A and 3B, VIC fluorescence corresponds to the wild-type allele, and FAM fluorescence corresponds to the mutant allele. Quantification results are shown in Table 2. [Table 2]

[0053] The results indicate that the KRAS516 assay probe has substantial cross-reactivity with off-target KRAS518 templates, which can result in false positive and / or erroneously inflated measurements for KRAS516. Example 2: Effective suppression of off-target KRAS518 templates

[0054] Figures 4A and 4B compare the results of separate KRAS518 dPCR assays enriched with a dark probe (at 1 μM) to block off-target KRAS516 templates, with other conditions maintained the same as in Example 1. Figure 4A shows the assay in which KRAS516 served as the template, and Figure 4B shows the assay in which KRAS518 served as the template. The results demonstrate that the inclusion of a dark probe to block off-target KRAS518 templates effectively suppressed cross-reactivity of the KRAS518-targeted probe with off-target KRAS516 templates. Quantification results are shown in Table 3. [Table 3] Example 3: Dark Probe Titration Test

[0055] Figures 5A-5D show the results of dark probe dPCR titration studies. Figure 5A shows the results using the dark probe of SEQ ID NO: 1 with the KRAS517 template (5% MAF). Figure 5B shows the results using the dark probe of SEQ ID NO: 2 with the KRAS517 template (5% MAF). Figure 5C shows the results using the dark probe of SEQ ID NO: 1 with the KRAS518 template (18% MAF). Figure 5D shows the results using the dark probe of SEQ ID NO: 2 with the KRAS518 template (18% MAF).

[0056] The results show that blocking of off-target templates begins even at concentrations as low as about 0.5 μM, with more effective blocking occurring at a concentration of 1 μM and very effective blocking occurring at a concentration of 2 μM. Example 4: Accurate quantification in the presence of off-target templates

[0057] The dark probe of SEQ ID NO:2 was provided at 2 μM in a reaction mixture containing a 10-fold higher concentration of off-target template relative to the target template. Two pools of templates were formed. Pool 1 contained target templates KRAS516, 520, and 521, each at 0.5% MAF. Pool 2 contained the same target template at the same MAF and off-target templates KRAS517 and 518, each at 5% MAF. These pools were subjected to a multiplex dPCR assay targeting the KRAS516, 520, and 521 mutations. The results are shown in Table 4. [Table 4]

[0058] The results were substantially similar using Pool 1 or Pool 2, indicating that the dark probes advantageously limit cross-reactivity while not interfering with the quantification of the target template. Additional reaction mixture details

[0059] Assays utilizing the off-target suppression embodiments described herein may further include one or more primer sets to enable amplification of a target nucleic acid. For example, the assay may include at least one pair of primers configured to amplify a nucleic acid target. Multiplexed embodiments may include additional sets of primers, each set designed to enable amplification of a different target.

[0060] Other amplification reaction mixture components known in the art may also be included in the assay compositions and / or assay kits, such as, for example, a polymerase, nucleotides, one or more buffers, and / or one or more salts to facilitate amplification of the target when the mixture and associated sample are exposed to amplification conditions.

[0061] The dark probe may be provided at a concentration of at least about 0.5 μM, or at least about 0.75 μM, or at least about 1 μM, or at least about 1.25 μM, or at least about 1.5 μM, or at least about 1.75 μM, or at least about 2 μM.

[0062] The source of the sample is often a clinical sample, such as a blood sample. Other sources of samples include, but are not limited to, forensic or environmental samples (e.g., clothing, soil, paper, surfaces, water), plants, human and / or animal skin, hair, blood, serum, feces, milk, saliva, urine, and / or other secretions. More details on amplification

[0063] Amplification products obtained by using one or more embodiments described herein can be generated, detected, and / or analyzed on any suitable platform. In some embodiments, the nucleic acid target can be single-stranded, double-stranded, or any other nucleic acid molecule of any size or conformation. The amplification process described herein can include PCR (see, e.g., U.S. Pat. No. 4,683,202). In some embodiments, the PCR is real-time or quantitative PCR (qPCR). In some embodiments, the PCR is end-point PCR. In some embodiments, the PCR is digital PCR (dPCR). Other amplification methods, such as loop-mediated isothermal amplification ("LAMP") and other isothermal methods, are also contemplated for use with the assay embodiments described herein.

[0064] Rare mutation assays commonly utilize dPCR. In dPCR, the reaction mixture is divided into many small reaction volumes (i.e., partitions), so that the target nucleic acid is present in some, but not all, of the reaction volumes / partitions. The reaction volumes are subjected to thermal cycling, and the proportion of "positive" partitions that generate a signal (usually a fluorescent signal) indicating the presence of the target is determined. Quantification is based on the application of Poisson statistics, using the number of negative / non-reactive reaction volumes to determine the initial number of copies distributed throughout the reaction volume, assuming a Poisson distribution.

[0065] Embodiments involving dPCR may utilize a variety of distribution mechanisms or devices known in the art or that may be developed in the future. For example, some conventional dPCR systems utilize multiple droplets encapsulated by an oil phase to form multiple partitions / reaction volumes. Other embodiments may utilize an array of microchambers. An example of such a system is the QuantStudio Absolute Q system available from Thermo Fisher Scientific, which uses a microfluidic array plate to perform sample compartmentalization / distribution.

[0066] In some qPCR embodiments, the nucleic acid amplification assays described herein are performed using a qPCR instrument, including, for example, a QuantStudio Real-Time PCR system, such as the QuantStudio 5 RealTime PCR System (QS5), the QuantStudio 7 RealTime PCR System (QS7), and / or the QuantStudio 12K Flex System (QS12K), or a 7500 Real-Time PCR system, such as the 7500 Fast Dx system from Thermo Fisher Scientific. Selected List of Terms and Definitions

[0067] As used herein, "nucleic acid" includes compounds having multiple natural nucleotide and / or non-natural (or "derivative") nucleotide units. "Nucleic acid" may further include non-nucleotide units, such as peptides. Thus, "nucleic acid" encompasses compounds such as DNA, RNA, peptide nucleic acids, phosphothioate-containing nucleic acids, phosphonate-containing nucleic acids, and the like. There is no particular limitation regarding the number of units in a nucleic acid, provided that the nucleic acid contains two or more nucleotides, nucleotide derivatives, or combinations thereof, specifically 5, 10, 15, 25, 50, 100, or more. Nucleic acids can include both single-stranded and double-stranded forms, as well as fully or partially duplex hybrids (e.g., RNA-DNA, RNA-PNA, or DNA-PNA).

[0068] The term "primer" may refer to more than one primer, whether naturally occurring, such as in a purified restriction digest, or synthetically produced, capable of acting as a point of initiation of synthesis along a complementary strand when placed under conditions that catalyze the synthesis of a primer extension strand complementary to the nucleic acid strand. Such conditions include the presence of four different deoxyribonucleoside triphosphates and a polymerization-inducing agent (e.g., DNA polymerase or reverse transcriptase) in a suitable buffer and at a suitable temperature. Primers are typically 11 bases or longer, more specifically, primers are 17 bases or longer, although shorter or longer primers can be used depending on the needs of a particular application.

[0069] As used herein, the terms "target," "target sequence," "nucleic acid target," "target nucleic acid," and similar terms refer to a region of nucleic acid that is to be amplified, detected, or both. The target sequence is located between two primer sequences used for amplification.

[0070] For any given element of a component of a described embodiment, unless stated otherwise, either implicitly or explicitly, any of the possible alternatives listed for that element or component may generally be used individually or in combination with each other.

[0071] Additionally, unless otherwise indicated, numbers expressing quantities, components, distances, or other measurements used in the specification and claims are to be understood as being optionally modified by the term "about" or its equivalents. When terms such as "about," "approximately," or "substantially" are used in connection with a stated quantity, value, or condition, they may be interpreted to mean an amount, value, or condition that deviates by less than 20%, less than 10%, less than 5%, less than 1%, less than 0.1%, or less than 0.01% from the stated quantity, value, or condition. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, each numerical parameter should be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.

[0072] The headings and sub-headings used herein are for organizational purposes only and are not meant to be used to limit the scope of the specification or the claims.

[0073] It should also be noted that as used in this specification and the appended claims, the singular forms "a," "an," and "the" do not exclude plural referents unless the context clearly dictates otherwise. Thus, for example, embodiments referring to a singular referent (e.g., a "widget") may also include two or more such referents.

[0074] It will also be understood that the embodiments described herein may include properties and / or characteristics (e.g., components, components, members, elements, parts, and / or portions) described in one or more separate embodiments, and are not necessarily limited to the precise features explicitly described for that particular embodiment. Accordingly, various features of a given embodiment can be combined with and / or incorporated into other embodiments of the present disclosure. Thus, the disclosure of a particular feature in connection with a particular embodiment of the present disclosure should not be construed as limiting the application or inclusion of that feature to the particular embodiment. Rather, it will be understood that other embodiments may also include such features.

Claims

1. 1. A composition for suppressing off-target signal generation in a nucleic acid amplification process, the composition comprising: a detector probe comprising a detectable label, said detector probe configured to specifically interact with a nucleic acid target to enable detection and / or quantification of said nucleic acid target; a dark probe omitting a detectable label, said dark probe configured to specifically interact with an off-target sequence; The composition, wherein the detector probe is capable of interacting with the off-target sequence and the dark probe therefore functions to limit interactions between the detectable label and the off-target sequence.

2. 10. The composition of claim 1, wherein the detector probe and the dark probe comprise overlapping sequence portions, and the overlapping sequence portions of the detector probe and the overlapping sequence portions of the dark probe differ by a single nucleotide.

3. 10. The composition of claim 1, wherein the detector probe and the dark probe are the same length and share the same sequence except for a single nucleotide.

4. 4. The composition of claim 1, further comprising a plurality of dark probes, each omitting a label and each comprising an overlapping sequence portion that differs from a corresponding overlapping portion of the detector probe by a single nucleotide.

5. 5. The composition of any one of claims 1 to 4, wherein the detector probe is configured to target a first allele of a particular single nucleotide polymorphism (SNP) position and the dark probe is configured to target a second, different allele of the SNP position.

6. The composition of claim 5 , wherein the SNP position is associated with an oncogenic mutation.

7. The composition of claim 6 , wherein the SNP position is in the KRAS gene.

8. The composition according to any one of claims 1 to 7, wherein the overlapping sequence portion is 10 to 30 nucleotides in length.

9. The composition of any one of claims 1 to 8, wherein the dark probe comprises a 3' block.

10. The composition of any one of claims 1 to 9, wherein the detector probe further comprises a quencher.

11. The composition of claim 10 , wherein the detector probe is a TaqMan probe.

12. The composition of any one of claims 1 to 11, further comprising a primer pair configured to amplify the nucleic acid target.

13. 13. The composition of claim 12, wherein the nucleic acid target is at least a portion of a proto-oncogene.

14. The composition of any one of claims 1 to 13, wherein the amount of dark probe provided in the composition is at least the same as the amount of detector probe.

15. 15. The composition of claim 14, wherein the amount of dark probe is greater than the amount of detector probe.

16. The composition of any one of claims 1 to 15, wherein the nucleic acid target comprises a KRAS516 allele.

17. 17. The composition of claim 16, wherein the dark probe comprises SEQ ID NO: 1 or SEQ ID NO:

2.

18. 1. A method for suppressing off-target signal generation in a nucleic acid amplification process, the method comprising: Providing a composition according to any one of claims 1 to 17; forming a reaction mixture comprising the sample and the composition; and subjecting said reaction mixture to an amplification process.

19. 20. The method of claim 18, wherein the amplification process is polymerase chain reaction (PCR).

20. 20. The method of claim 19, wherein the PCR is digital PCR (dPCR).

21. The method of any one of claims 18 to 20, further comprising detecting and / or quantifying the nucleic acid target.

22. 22. The method of claim 21, wherein quantifying the nucleic acid target comprises determining a variant allele frequency.

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