WO2024123672A1 - Off-target signal suppression in nucleic acid amplification reactions - Google Patents

Abstract

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

Description

OFF-TARGET SIGNAL SUPPRESSION IN NUCLEIC ACID AMPLIFICATION REACTIONS

RELATED APPLICATIONS

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

TECHNICAL FIELD

[0002] This disclosure is directed to compositions and methods for suppressing interaction of a target nucleic acid probe with off-target nucleic acids in the reaction mixture.

RELATED TECHNOLOGY

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

[0004] 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 the region of interest in target DNA. A repetitive series of reaction steps involving template denaturation, primer annealing, and the 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 can selectively enrich a specific DNA sequence by several orders of magnitude.

[0005] In some assays that utilize 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 instances, the PCR components intended for interacting with the target nucleic acid instead interact with the similar, off-target sequences. This can detrimentally introduce error into the associated detection and quantification analyses.

[0006] Accordingly, there is an ongoing need for nucleic acid amplification assays that substantially reduce or eliminate the problem of cross-reactivity in nucleic acid amplification reactions. SUMMARY

[0007] Disclosed are compositions and methods for suppressing off-target signal generation in a nucleic acid amplification process. The compositions and methods utilize a detector probe with a detectable label and a dark probe that omits a detectable label. The detector probe is configured to specifically interact with a target nucleic acid target. The dark probe is configured to specifically interact with an off-target sequence that is similar to the target nucleic acid, the dark probe thereby limiting off- target interaction 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 comprises a detector probe comprising 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 a detectable label. The dark probe is configured to specifically interact with an off-target sequence to thereby block or limit 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) location and the dark probe is configured to target a second, different allele at the SNP location. In some embodiments, the SNP location is associated with an oncogenic mutation.

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

[0011] In one embodiment, a method for suppressing off-target signal generation in a nucleic acid amplification process comprises providing a composition that includes a detector probe with a detectable label, the detector probe configured to specifically interact with a nucleic acid target to enable detection and/or quantification of the nucleic acid target, and a dark probe that omits a detectable label and is configured to specifically interact with an off-target sequence to thereby block or limit interaction between the detectable label and the off-target sequence. The method further comprises forming a reaction mixture comprising a 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 indication of the scope of the claimed subject matter.

BRIEF DESCRIPTION OF THE DRAWINGS

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

[0014] Figure 1 A 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 and generate a corresponding signal as a result, the schematic showing that the detector probe or some portion of detector probes may instead interact with off- target sequences, falsely adding to the resulting signal;

[0015] Figure IB 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 the off-target sequence and thereby block the detector probe from interacting with the off-target sequence, thus suppressing cross-reactivity of the detector probe;

[0016] Figures 2A-2C illustrate examples of detector probe and dark probe pairs having different types of overlapping portions;

[0017] Figures 3 A and 3B compare results of separate conventional KRAS 516 assays, Figure 3A showing an assay in which KRAS 516 was provided as template, and Figure 3B showing an assay in which KRAS 518 was provided as template, the results showing that the KRAS 516 assay probe has substantial cross-reactivity with the off-target KRAS 518 template and can thereby lead to false positives and/or falsely inflated measurement values for KRAS 516;

[0018] Figures 4A and 4B compare results of separate KRAS 516 assays enhanced to include dark probes for blocking the off-target KRAS 518 template, Figure 4 A showing an assay in which KRAS 516 was provided as template and Figure 4B showing an assay in which KRAS 518 was provided as template, the results showing that the inclusion of dark probes for blocking the off-target KRAS 518 template effectively suppressed cross-reactivity of the KRAS 516 target probe with the off-target KRAS 518 template; and

[0019] Figures 5A-5D show results of a dark probe titration test for two example dark probes.

DETAILED DESCRIPTION

Overview of Off-Target Suppression Using Dark Probes

[0020] Figure 1A illustrates a conventional nucleic acid amplification process in which a labelled 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 a plurality of detector probes) may instead interact with an off-target sequence, adding to the resulting signal. This cross-reactivity can lead to, for example, false positive detection of the target and/or an inflated calculated amount of the target.

[0021] Figure IB 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 and thereby block 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, which in turn enables the signal to better correspond to the amount of target. In other words, by limiting interaction between the detector probe and the off-target sequence, the amount of signal associated with the detectable label will better correspond to the amount of nucleic acid target in the sample and will therefore enable more accurate detection and/or quantification of the target nucleic acid.

[0022] The use of an off-target suppression embodiment such as shown in Figure IB is beneficial where a test sample has (or is suspected of having) one or more sequences that are similar enough to the target nucleic acid to cause some amount of cross-reactivity with the detector probe. As an example, one or more dark probes may be used in assays that are designed to amplify and detect a specific allele of a nucleic acid target that is often present with other alleles in the sample. In such an assay, the detector probe is designed to specifically interact with the targeted specific allele, while each dark probe is designed to specifically interact with other alleles to thereby reduce interaction between the detector probe and the off-target nucleic acids of the other alleles.

[0023] The problem of cross-reactivity with off-target sequences is particularly acute in rare mutation assays designed to detect and/or quantify rare alleles that may be present with other, more abundant alleles. For example, the targeted allele may be present within 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 interacts with only a small percentage of the off-target sequences, the abundance of off-target nucleic acid templates relative to target nucleic acid templates in the reaction mixture can lead to excessive disruption of the results.

[0024] Rare mutation assays may include assays designed to detect and/or quantify oncogenic mutations, for example. In these and other rare mutation assays, the sample will often include relatively high levels of other alleles (e.g., wild type or other alleles) that can affect the results even where the detector probe is designed to specifically interact with the target allele.

[0025] In some embodiments, the sample comprises genomic DNA with a target locus having 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 is capable of effectively detecting the rare mutant allele with greater accuracy than otherwise similar assays omitting dark probes, and/or is capable of effectively quantifying the rare mutant allele with greater accuracy than otherwise similar assays omitting dark probes.

[0026] In some embodiments, the off-target nucleic acid sequences are similar to the target sequences except for a few nucleotides (e.g., 2-3 nucleotides). Often, the target sequence and the off-target sequence(s) differ by only a single nucleotide. For example, the target may be a first allele of a particular single nucleotide polymorphism (SNP) location, and the off-target sequence(s) are other alleles of the same SNP location. In these embodiments, the detector probe is configured to target the first allele of the SNP location, and a dark probe is configured to target a second, different allele at the SNP location. Some embodiments may include additional dark probes, such as a dark probe for targeting a third allele at the SNP location. Although many of the examples described herein relate to SNP mutations, it will be understood that the same principles may be applied to other types of mutations, including deletion mutations, insertion mutations, and multi-nucleotide base substitutions.

[0027] In some embodiments, an off-target sequence is not necessarily a different allele of the target sequence, but still resembles the target enough to cause crossreactivity with the detector probe. For example, the detector probe may be configured to target a particular allele at a specified nucleotide location. An off-target nucleic acid may match the target nucleic acid at the mutation locus but have another nucleotide that differs from the target located some number of nucleotides (e.g., 1 to 20) away from the mutation locus.

[0028] Because the off-target sequence is close enough to the target sequence to cause potential cross-reactivity with the detector probe, a detector probe and a corresponding dark probe will have similar sequences. The dark probe is adjusted relative to the detector probe to specifically target an off-target sequence rather than the target sequence itself. Often, a detector probe and a corresponding dark probe are the same size, though this need not be the case in all embodiments.

[0029] Figures 2A-2C illustrate example detector probe and dark probe pairs. A detector probe and a corresponding dark probe will each have an “overlapping sequence portion” representing the respective sequence portions that overlap when the detector probe and dark probe are aligned. Often, the detector probe and the dark probe are the same size and will fully overlap one another, as shown in Figure 2A. In Figure 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 not within the overlapping portion). In Figure 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 not within the overlapping portion and the dark probe includes a 3’ section not within the overlapping portion. [0030] As shown, even if the probes are different in length and/or are shifted relative to each other, the differentiating feature(s) of the probes (here, the A or G nucleotide difference) lie within the overlapping sequence portion. The overlapping sequence portions are preferably about 10-30 nucleotides in length to ensure the dark probes can properly block interaction between the detector probes and the off-target sequences.

[0031] In the examples shown in Figures 2A-2C, the overlapping portions of the detector probe and the dark probe differ by only a single nucleotide, with the detector probe including an A (as an example) and the dark probe including a G (as an example) at the corresponding location. This will be the case where the target and off- target sequences are different alleles of a particular SNP. In other embodiments, the overlapping sequence portions of the detector probe and the dark probe may differ at more than one nucleotide, may include one or more deletions, and/or may include one or more insertions relative to the opposing probe. Typically, however, the detector probe and dark probe will have about 80%, about 85%, about 90%, or about 95% identity, or will have identity within a range defined by any two of the foregoing values.

[0032] Some embodiments may include a single detector probe and a single dark probe. Such embodiments may be considered single-plex with respect to the detector probe and single-plex with respect to the dark probe. Other embodiments may include one or more additional detector probes (each typically uniquely labelled and directed to a specific target), and thus be multi-plex with respect to the detector probes, and/or may include one or more additional dark probes directed to other off-target sequences, and thus be multi-plex with respect to the dark probes.

[0033] For embodiments that are multiplex with respect to detector probes, each detector probe may have a single corresponding dark probe or multiple corresponding dark probes. For example, in a duplex assay with a first detector probe and second detector probe, the assay may include two dark probes (i.e., dark probe “la” and dark probe “lb”) associated with the first detector probe and each designed to block a different off-target sequence that could cross-react with the first detector probe, and include a single dark probe (i.e., dark probe 2) associated with the second detector probe and designed to block an off-target sequence that could 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, and the number of dark probes utilized with each detector probe can instead be chosen based on the number of off-target sequences to be blocked from that particular detector probe.

[0034] Some embodiments may include one or more detector probes without a corresponding dark probe. For example, in a multiplex assay with first, second, and third detector probes, the assay may include two dark probes (i.e., dark probe “la” and dark probe “lb”) associated with the first detector probe and each designed to block a different off-target sequence that could cross-react with the first detector probe, a single dark probe (i.e., dark probe 2) associated with the second detector probe and designed to block an off-target sequence that could cross-react with the second detector probe, and may omit any dark probes associated with the third detector probe. This may be the case where significant off-target interaction of the third detector probe is not expected.

[0035] In some embodiments , the dark probe is provided at a concentration at least as great as the concentration of detector probe. In some embodiments, the dark probe is provided at a concentration greater than the concentration of the detector probe. In embodiments that include multiple dark probes each associated with a single detector probe, each of those dark probes may be provided at a concentration that is equal to or greater than the concentration of the detector probe.

Detector Probe & Dark Probe Features

[0036] As discussed above, the detector probe includes a detectable label, whereas the dark probe omits a detectable label. The detectable label of the detector probe may 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.

[0037] Exemplary detectable labels that may be utilized with the embodiments described herein include, for example:

[0038] Fluoresceins (e.g., 5-carboxy-2,7-dichlorofluorescein, 5 Carboxyfluorescein (5-FAM), 6-JOE, 6-carboxyfhiorescein (6-FAM), VIC, FITC, 6- carboxy-4’,5’-dichloro-2’,7’-dimethoxy-fluorescein (JOE)), 5 and 6-carboxy-l,4- dichloro-2’,7’-dichloro-fluorescein (TET), 5 and 6-carboxy-l,4-dichloro-2’,4’,5’,7’- tetra-chlorofhiorescein, HEX, PET, NED, Oregon Green (e.g. 488, 500, 514));

[0039] Pyrenes; (e.g. Cascade Blue; Alexa Fluor 405);

[0040] Coumarins; (e.g. Pacific Blue, Atto 425, Alexa Fluor 350, Alexa Fluor 430);

[0041] Cyanine Dyes; (e.g. Cy dyes such as Cy3, Cy3.18, Cy3.5, Cy5, Cy5.18, Cy5.5, Cy7);

[0042] Rhodamines; (e.g., 110, 123, B, B 200, BB, BG, B extra, 5 and 6- carboxytetramethylrhodamine (5-TAMRA, 6-TAMRA), 5 and 6-Carboxyrhodamine 6G, Lissamine, Lissamine Rhodamine B, Rhod-2, ROX (6-carboxy-X-rhodamine), 5 and 6-ROX (carboxy-X-rhodamine), Sulphorhodamine B can C, Sulphorhodamine G Extra, 5 and 6 TAMRA (carboxytetramethyl-rhodamine), (TRITC), ABY, JUN, LIZ, RAD, RXJ, Texas Red; and Texas Red-X);

[0043] Alexa Fluor fhiorophores (which is a broad class including many dye types such as cyanines) (e.g., Alexa 350, 405, 430, 488, 500, 514, 532, 546, 555, 568, 594, 610, 633, 635, 647, 660, 676, 680, 700, 750);

[0044] FRET donor/acceptor pairs (e.g., fluorescein/fluorescein, fluorescein/rhodamine, fluorescein/cyanine, rhodamine/cyanine, fluorescein/ Alexa Fluor, Alexa Fluor/rhodamine); and other types of dyes known to those of skill in the art.

[0045] Fluorophore labels may be associated with quenchers such as dark fluorescent quencher (DFQ), black hole quenchers (BHQ), Iowa Black, QSY7, QSY21 quencher, Dabsyl and Dabcel sulfonate/carboxylate quenchers, and MGB- NFQ quenchers. Fluorophore labels may also include sulfonate derivatives of fluorescein dyes with SO3 instead of the carboxylate group, phosphoramidite forms of fluorescein, and/or phosphoramidite forms of Cy5, for example.

[0046] Detector probes may be configured as TaqMan probes, which are known in the art and described in greater detail below. Such probes are able to hybridize to a target downstream from a primer such that exonuclease activity of the polymerase during subsequent primer extension separates a dye label from a quencher to increase the dye signal. [0047] Detector probes and dark probes may 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.

[0048] Dark probes as disclosed herein preferably include a 3 ’ block to prevent or limit the dark probes from acting as primers. The 3’ block may include, for example, an alkyl spacer, a terminal 3 ’ phosphate, a dideoxy nucleotide, an inverted 3 ’ end, or other extension blocks as known in the art.

Examples

[0049] The following examples illustrate effective use of off-target suppression assays for detecting the KRAS 516 mutation. KRAS is a proto-oncogene, and certain KRAS mutations are implicated in a variety of cancers. The KRAS 516 mutation is a 34 G>T nucleotide mutation which causes a G12C amino acid change. As with many rare mutation assays, assays for detecting and/or quantifying the KRAS 5156 mutation can be obscured by similar off-target sequences, including the KRAS 517 (34 G>A) and/or KRAS 518 (34 G>C) alleles.

[0050] In the following example assays that use dark probes, the dark probes include one or both of those shown in Table 1. An example detector probe is also shown. Other detector probes for KRAS 516 are known in the art (e.g., the detector probe of Assay ID Hs000000047_rm, available from Thermo Fisher Scientific). The reverse complements of these sequences may alternatively be utilized.

Table 1: Example Probes

*M = Adenine (to block KRAS 517) or Cytosine (to block KRAS 518) Example 1: Cross-Reactivity of KRAS 516 Assay with KRAS 518 Template

[0051] Figures 3 A and 3B compare results of separate conventional KRAS 516 dPCR assays. Figure 3A shows an assay in which KRAS 516 was provided as template (at 0.5% MAF), and Figure 3B shows an assay in which KRAS 518 was provided as 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. The quantification results are tabulated in Table 2.

Table 2: Cross-Reactivity of KRAS 516 Assay with KRAS 518 Template

[0052] The results show that the KRAS 516 assay probe has substantial crossreactivity with the off-target KRAS 518 template and can thereby lead to false positives and/or falsely inflated measurement values for KRAS 516.

Example 2: Effective Suppression of Off-Target KRAS 518 Template

[0053] Figures 4A and 4B compare results of separate KRAS 516 dPCR assays enhanced to include dark probes (at 1 pM) for blocking the off-target KRAS 518 template, with conditions otherwise held the same as in Example 1. Figure 4A shows the assay in which KRAS 516 was provided as template and Figure 4B shows the assay in which KRAS 518 was provided as the template. The results show that the inclusion of dark probes for blocking the off-target KRAS 518 template effectively suppressed cross-reactivity of the KRAS 516 target probe with the off-target KRAS 518 template. The quantification results are tabulated in Table 3.

Table 3: Effective Suppression of Off-Target KRAS 518 Template

Example 3: Dark Probe Titration Test

[0054] Figures 5 A-5D show results of a dark probe dPCR titration test. Figure 5A shows results using the dark probe of SEQ ID NO: 1 against a KRAS 517 template (5% MAF). Figure 5B shows results using the dark probe of SEQ ID NO:2 against the KRAS 517 template (5% MAF). Figure 5C shows results using the dark probe of SEQ ID NO: 1 against a KRAS 518 template (18% MAF). Figure 5D shows results using the dark probe of SEQ ID NO:2 against a KRAS 518 template (18% MAF).

[0055] The results show that blocking of the off-target templates begins even at concentrations as low as about 0.5 pM, with more effective blocking resulting at concentrations of 1 pM, and very effective blocking resulting at concentrations of 2 pM.

Example 4: Accurate Quantification in the Presence of Off-Target Template

[0056] The dark probe of SEQ ID NO:2 was provided at 2 pM in a reaction mixture including 10X higher concentration of off- target template relative to target template. Two pools of template were formed. Pool 1 included target templates KRAS 516, 520, and 521 at 0.5% MAF each. Pool 2 included the same target templates at the same MAF and included off-target templates KRAS 517 and 518 at 5% MAF each. These pools were subjected to a multiplex dPCR assay targeting the KRAS 516, 520, and 521 mutations. The results are tabulated in Table 4.

[0057] The results were substantially similar whether Pool 1 or Pool 2 was used, indicating that the dark probe beneficially limited cross-reactivity while also not disrupting the quantification of the target templates.

Additional Reaction Mixture Details

[0058] Assays utilizing the off-target suppression embodiments described herein may additionally include one or more sets of primers to enable amplification of target nucleic acids. For example, an assay may include at least one pair of primers configured to amplify the nucleic acid target. Multiplex embodiments may include additional sets of primers, each set designed to enable amplification of a different target.

[0059] Other amplification reaction mixture components known in the art may also be included in an assay composition and/or in an assay kit. Such components may include, for example, polymerase, nucleotides, one or more buffers, and/or one or more salts to promote amplification of the target when the mixture and a sample combined therewith are exposed to amplification conditions.

[0060] Dark probes may be provided at concentrations of at least about 0.5 M, or at least about 0.75 pM, or at least about 1 pM, or at least about 1.25 pM, or at least about 1.5 pM, or at least about 1.75 pM, or at least about 2 pM.

[0061] The source for the sample will often be a clinical sample, such as a blood sample. Other sources for the sample 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 secretory fluids.

Additional Amplification Details

[0062] Amplified products resulting from use of one or more embodiments described herein can be generated, detected, and/or analyzed on any suitable platform. In some embodiments, the nucleic acid targets may be single-stranded, doublestranded, or any other nucleic acid molecule of any size or conformation. The amplification processes 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 an end point PCR. In some embodiments, the PCR is digital PCR (dPCR). Other amplification methods, such as, e.g., loop-mediated isothermal amplification (“LAMP”), and other isothermal methods are also contemplated for use with the assay embodiments described herein.

[0063] In rare mutation assays, dPCR is commonly utilized. In dPCR, the reaction mixture is partitioned into many small reaction volumes (i.e., partitions) so that the target nucleic acid is 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 fluorescence signal) indicative of the presence of the target is determined. Quantitation is based on application of Poisson statistics, using the number of negative/non-reactive reaction volumes and assuming a Poisson distribution to establish the number of initial copies that were distributed across all the reaction volumes.

[0064] Embodiments that include dPCR may utilize a variety of partitioning mechanisms or devices as known in the art or as may be developed in the future. For example, some conventional dPCR systems utilize a plurality of droplets encapsulated by an oil phase to form the plurality of parti tions/reaction volumes. Other embodiments may utilize an array of microchambers. As example of such a system is the QuantStudio Absolute Q system available from Thermo Fisher Scientific, which uses a microfluidic array plate to perform the compartmentalizing/partitioning of sample.

[0065] In some qPCR embodiments, the nucleic acid amplification assays as 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), QuantStudio 7 RealTime PCR System (QS7), and/or 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 & Definitions

[0066] As used herein, “nucleic acid” includes compounds having a plurality of natural nucleotides and/or non-natural (or “derivative”) nucleotide units. A “nucleic acid” can further comprise non-nucleotide units, for example peptides. “Nucleic acid” therefore encompasses compounds such as DNA, RNA, peptide nucleic acids, phosphothioate-containing nucleic acids, phosphonate-containing nucleic acids and the like. There is no particular limit as to the number of units in a nucleic acid, provided that the nucleic acid contains 2 more nucleotides, nucleotide derivatives, or combinations thereof, specifically 5, 10, 15, 25, 50, 100, or more. Nucleic acids can encompass both single and double-stranded forms, and fully or partially duplex hybrids (e.g., RNA-DNA, RNA-PNA, or DNA-PNA).

[0067] The term “primer” may refer to more than one primer and refers to an oligonucleotide, whether occurring naturally, as in a purified restriction digest, or produced synthetically, which is capable of acting as a point of initiation of synthesis along a complementary strand when placed under conditions in which synthesis of a primer extension product which is complementary to a nucleic acid strand is catalyzed. Such conditions include the presence of four different deoxyribonucleoside triphosphates and a polymerization-inducing agent such as DNA polymerase or reverse transcriptase, in a suitable buffer and at a suitable temperature. A primer is typically 11 bases or longer; more specifically, a primer is 17 bases or longer, although shorter or longer primers may be used depending on particular application needs.

[0068] As used herein, the term “target,” “target sequence,” “nucleic acid target,” “target nucleic acid,” and similar terms refer to a region of a nucleic acid which is to be either amplified, detected, or both. The target sequence resides between the two primer sequences used for amplification.

[0069] For any given element of component of a described embodiment, any of the possible alternatives listed for that element or component may generally be used individually or in combination with one another, unless implicitly or explicitly stated otherwise.

[0070] In addition, unless otherwise indicated, numbers expressing quantities, constituents, distances, or other measurements used in the specification and claims are to be understood as optionally being modified by the term “about” or its synonyms. When the terms “about,” “approximately,” “substantially,” or the like are used in conjunction with a stated amount, value, or condition, it may be taken 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 % of the stated amount, 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.

[0071] Any headings and subheadings used herein are for organizational purposes only and are not meant to be used to limit the scope of the description or the claims.

[0072] It will 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, an embodiment referencing a singular referent (e.g., “widget”) may also include two or more such referents.

[0073] It will also be appreciated that embodiments described herein may also include properties and/or features (e.g., ingredients, components, members, elements, parts, and/or portions) described in one or more separate embodiments and are not necessarily limited strictly to the features expressly described for that particular embodiment. Accordingly, the various features of a given embodiment can be combined with and/or incorporated into other embodiments of the present disclosure. Thus, disclosure of certain features relative to a specific embodiment of the present disclosure should not be construed as limiting application or inclusion of said features to the specific embodiment. Rather, it will be appreciated that other embodiments can also include such features.

Claims

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, the detector probe being configured to specifically interact with a nucleic acid target to enable detection and/or quantification of the nucleic acid target; and a dark probe that omits a detectable label, the dark probe being configured to specifically interact with an off-target sequence, wherein the detector probe is capable of interaction with the off-target sequence, the dark probe thus functioning to limit interaction between the detectable label and the off-target sequence.

2. The composition of claim 1 , wherein the detector probe and the dark probe include an overlapping sequence portion, the overlapping sequence portion of the detector probe and the overlapping sequence portion of the dark probe differing by a single nucleotide.

3. 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. The composition of any one of claims 1-3, further comprising a plurality of dark probes each omitting a label and each including an overlapping sequence portion that differs from a corresponding overlapping portion of the detector probe by a single nucleotide.

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

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

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

8. The composition of any one of claims 1-7, wherein the overlapping sequence portions are 10-30 nucleotides in length.

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

10. The composition of any one of claims 1-9, 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 1-11, further comprising a pair of primers configured to amplify the nucleic acid target.

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

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

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 1-15, wherein the nucleic acid target includes the KRAS 516 allele.

17. The composition of claim 16, wherein the dark probe comprises SEQ ID NO: 1 or SEQ ID NO:2.

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

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

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

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

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