Off-target block sequences improve target discrimination by polymerase chain reaction

Abstract

Provided herein are methods and kits for distinguishing between a target sequence and a reference sequence in a polymerase chain reaction (PCR), comprising a labeled probe comprising a fluorophore and a quencher that is complementary to and specifically binds to the target sequence and capable of non-specifically binding to the reference sequence, and a promiscuity block nucleotide doubler oligonucleotide (PBNJ) that specifically binds to the reference sequence and capable of non-specifically binding to the target sequence, wherein the PBNJ comprises a reference binding region and an extension blocker that prevents extension by a polymerase.

Description

[Technical field]

[0001] [CROSS REFERENCE TO RELATED APPLICATIONS]

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 271,522, filed October 25, 2021, which is incorporated by reference herein to the extent not inconsistent herewith.

[0002] [Incorporation by reference of electronically submitted material]

[0002] The computer-readable nucleotide / amino acid sequence listing submitted concurrently with this specification is incorporated by reference in its entirety and is identified as follows: One 71 KB XML file entitled "97-21WO_Seq_Listing_10-24-2022.XML", created at WIPO Sequence on October 24, 2022.

[0003] [Field of the Invention]

[0003] This application relates generally to assays for the detection and quantification of PCR products using oligonucleotides to reduce or avoid detection of off-target amplification products. [Background technology]

[0004]

[0004] The following discussion provides a brief summary of information relevant to the present application and is not an admission that any of the information provided or publications referenced herein is prior art to the present application.

[0005] PCR is a method for making copies of a DNA sample, using a primer for the template strand and a DNA polymerase for primer extension to create a new DNA strand complementary to the template DNA or RNA strand. Quantitative PCR (qPCR, also called real-time PCR) is a PCR-based technique that couples the amplification of a target DNA sequence in a reaction with quantification of the DNA concentration. Digital PCR (dPCR) is an improved version of the traditional PCR method, applying the division of the sample into subsamples or droplet formation, and can be used to directly quantify or clonally amplify nucleic acid strands. Reverse transcription PCR (RT-PCR) combines reverse transcription of RNA into complementary DNA with PCR, and PCR can be either dPCR or qPCR. In dPCR or qPCR, a labeled probe designed to specifically hybridize to the DNA target region is used to detect and quantify the amplified DNA. However, the probe may hybridize nonspecifically to non-target regions, which can significantly reduce the accuracy of DNA / RNA detection and quantification. Non-specific hybridization to non-target regions is called promiscuity. In addition, some off-target amplification is expected, which may also affect accuracy. Provided herein are methods, kits, and related compositions that address the need in the art for methods and kits to accurately detect and quantify DNA in dPCR or qPCR, or RNA by RT-PCR (e.g., RT-dPCR and RT-qPCR), containing very small amounts of a target nucleic acid of interest. Summary of the Invention

[0006]

[0006] The accuracy of dPCR and qPCR assays (in the case of complementary DNA reverse transcribed from DNA or RNA) is limited by non-specific binding of labeled probes and / or off-target amplification, leading to inaccuracies in detection and quantification of target sequences. The present disclosure provides methods and kits for distinguishing target and reference sequences in biological samples by PCR, including dPCR or qPCR, by reducing non-specific binding of labeled probes. The methods, kits and compositions provided herein are also useful for distinguishing biological materials from any of a variety of organisms in which the target sequence originates from natural variations or artificial modifications such as mutagenesis or genome editing. The difference between the target and reference may be as small as a single base pair (bp) substitution or may represent an indel (insertion or deletion). Provided herein is a platform that improves the accuracy of dPCR and / or qPCR to reliably detect any of a variety of differences in sequence between the target and reference sequences that cannot be reliably detected without the platform provided herein due to off-target binding and its associated amplification and detection, regardless of the type of nucleic acid difference. By reducing the detection of non-target sequences, on-target amplification is more reliably detected, even at very low concentrations compared to the reference sequence. These methods, kits and compositions are particularly useful because they can be incorporated into conventional "probe-based" PCR assays, thereby providing highly efficient and accurate detection without the need for additional complex, time-consuming or expensive components.

[0007]

[0007] These methods, kits and compositions are compatible with a variety of targets used in PCR-based detection and diagnosis, where detection of at least one nucleotide difference is desirable and therefore suppression of off-target amplification is important to better distinguish between target and reference sequences.

[0008]

[0008] Provided herein are various methods described in this section as "Aspects", including, for example, Aspect 1: A method for identifying a target sequence and a reference sequence in a biological sample by polymerase chain reaction (PCR), comprising: a. providing a labeled probe comprising a fluorophore and a quencher, said labeled probe being complementary to and capable of specifically binding to said target sequence and non-specifically binding to said reference sequence; b. providing a Promiscuity-Blocking Nucleotide Juror oligonucleotide (PBNJ), the PBNJ capable of specifically binding to the reference sequence and non-specifically binding to the target sequence, the PBNJ comprising a reference binding region and an extension blocker that prevents extension by a polymerase; c. Below: i. a sample comprising said reference sequence and / or said target sequence; ii. the labeled probe; iii. a competitive concentration of said PBNJ relative to the concentration of said labeled probe; iv. PCR reagents and performing PCR on a solution comprising: Specific binding of the PBNJ to the reference sequence competes with the labeled probe binding to the reference sequence and inhibits the labeled probe binding to the reference sequence for amplification; thereby identifying said target sequence and said reference sequence. The competitive concentration of PBNJ:probe depends on the intended application, and therefore various concentrations are suitable. Examples include, but are not limited to, ratios between 0.2 and 20.

[0009] 2. The biological sample is a. a virus, wherein the reference sequence is derived from a wild-type or parent virus and the target sequence comprises at least one mutation in the reference sequence; b. a mammalian cell having one or more nucleotide changes in the reference sequence, wherein the reference sequence reflects a low disease state and the target sequence reflects an increased risk of a disease or the presence of a disease; c. circulating cell-free tumor DNA, wherein the reference sequence is a wild-type sequence from a somatic cell and the target sequence is derived from a tumor or cancerous cell and has one or more nucleotide changes in the reference sequence that reflect an increased risk of a pathology or the presence of a disease; d. circulating cell-free fetal DNA, wherein the reference sequence reflects a maternal DNA sequence and the target sequence reflects a fetal DNA sequence, with one or more nucleotide changes in the reference sequence; e. a bacterium, wherein the reference sequence is derived from a wild-type bacterium or a species of bacterium, and the target sequence comprises at least one variation in the reference sequence; f. a fungus, wherein the reference sequence is derived from a wild-type fungus or a species of fungus, and the target sequence comprises at least one variation in the reference sequence; and g. A plant, wherein the reference sequence is derived from a wild-type plant or a species of plant, and the target sequence comprises at least one variation in the reference sequence: 2. The method of aspect 1, wherein the compound is selected from the group consisting of:

[0010]

[0010] Of course, the present invention is also compatible with other applications such as "genetic testing" (for various diseases such as Alzheimer's disease, cancer, cystic fibrosis, sickle cell anemia, Duchenne muscular dystrophy, thalassemia, Huntington's disease, rare diseases, and other diseases) and sequences related to various applications (cancer diagnosis, genetic disease diagnosis, cardiovascular disease diagnosis, and other applications). Another important application is the detection of antimicrobial resistance (AMR) genes, where small changes in microbial genes can lead to antimicrobial resistance. Such rapid and reliable early detection of AMR is important for improving health outcomes and safety in the food, beverage, and agriculture (including cannabis) industries, especially for E. coli, Salmonella, and any of the various bacterial targets identified as concerns by the CDC.

[0011] 3. The method of aspect 1 or 2, wherein the PBNJ eliminates detection of non-specific amplification of the reference sequence by 90% or more. Of course, the methods and kits provided herein are compatible with less than 90% elimination, based on obtaining a meaningful threshold.

[0012] 4. The reference sequence and the target sequence are a.1 nucleotide substitution; b. a nucleotide insertion of one or more nucleotides; and / or C. a nucleotide deletion of one or more nucleotides 4. The method according to any one of aspects 1 to 3, depending on the

[0013]

[0013] 5. The method of any one of aspects 1 to 4, wherein the reference sequence and the target sequence are DNA sequences or RNA sequences.

[0014]

[0014] 6. The method according to any one of aspects 1 to 5, wherein the PCR is selected from the group consisting of dPCR, qPCR, RT-dPCR and RT-qPCR.

[0015]

[0015] 7. The method according to any one of aspects 1 to 6, wherein the labeled probe is a dual-labeled probe comprising a fluorescent molecule and a quencher molecule.

[0016]

[0016] 8. The method according to any one of aspects 1 to 7, wherein the labeled probe is a single-nucleotide variant (SNV)-specific TaqMan (registered trademark) probe (a fluorophore covalently attached to the 5' end of the probe and a quencher at the 3' end of the probe, or an internal quencher).

[0017]

[0017] 9. A method according to any one of aspects 1 to 8 for detecting DNA or RNA sequences containing SNVs, insertions or deletions.

[0018]

[0018] 10. A method according to any one of aspects 1 to 9, wherein the PCR is dPCR, the dPCR comprising compartment- or droplet-based PCR, and the PBNJ reduces or eliminates signals associated with low efficiency non-specific off-target amplification, thereby increasing the signal-to-noise ratio for specific amplification of the target sequence.

[0019]

[0019] 11. a. adjusting the probe output amplitude by providing the PBNJ at a lower concentration; b. detecting multiple probe output amplitudes for multiplexed detection of multiple target sequences in a single or multi-channel fluorescence detector; 11. The method of any one of aspects 1 to 10, further comprising:

[0020] 12. The method of any one of aspects 1 to 11, wherein the target sequence and the reference sequence differ by a single nucleotide mismatch that is a single nucleotide polymorphism (SNP) or is part of a short nucleotide polymorphism.

[0021] 13. The method of any one of aspects 1 to 11, wherein the target sequence and the reference sequence differ by an insertion.

[0022] 14. The method of any one of aspects 1 to 11, wherein the target sequence and the reference sequence differ by a deletion.

[0023] 15. The method of any one of aspects 1 to 14, wherein the extension blocker is a 3' carbon-based spacer, such as C3, C6, or C12, or a 3' quencher, such as a black hole quencher.

[0024]

[0024] 16. The method according to any one of aspects 1 to 15, wherein the PBNJ comprises a locked nucleic acid (LNA) at the SNP position.

[0025]

[0025] 17. The above PBNJ is having a length between 10 and 50 nucleotides; having between 90% and 95% target sequence complementarity to at least a portion of the target sequence; By having a reference sequence complementarity that is greater than the target sequence complementarity, the binding affinity of the PBNJ for the reference sequence is greater than the binding affinity of the PBNJ for the target sequence; the binding affinity of the PBNJ for the reference sequence is greater than the binding affinity of the labeled probe for the reference sequence; and / or 17. The method of any one of aspects 1 to 16, wherein the binding affinity of the PBNJ to the target sequence is less than the binding affinity of the labeled probe to the target sequence.

[0026]

[0026] 18. The method of any one of aspects 1 to 17, comprising a plurality of PBNJs that specifically bind to any possible SNP at a particular position in the target sequence.

[0027]

[0027] 19. The method according to any one of aspects 1 to 18, wherein the length of the target sequence is between 10 and 50 nucleotides, and the length of the probe and the PBNJ are each between 10 and 50 nucleotides.

[0028]

[0028] 20. The method of any one of aspects 1 to 19, used in a biological sample to test for mutations associated with an increased risk of cancer or the presence of cancer.

[0029]

[0029] 21. The method of any one of aspects 1 to 19, used in a biological sample to test for variants of a pathogen, including a pathogen that is a virus, a bacteria, or a fungus.

[0030]

[0030] 22. The method according to any one of aspects 1 to 21, wherein the biological sample is derived from wastewater, an environmental sample, a body fluid, a tissue, a cell culture or a tumor.

[0031]

[0031] 23. The method of any one of aspects 1 to 22, wherein the labeled probe has a polynucleotide sequence that differs from the PBNJ sequence by one or more nucleotides.

[0032]

[0032] 24. The method of any one of aspects 1 to 23, wherein the PBNJ is a PCR blocker during PCR amplification cycles to effect enrichment of a target sequence that is part of a mutant sequence.

[0033]

[0033] 25. The method according to any one of claims 1 to 24, wherein the ratio of the PBNJ concentration to the labeled probe concentration is equimolar or greater (e.g., 1:1 to 16:1); or less than equimolar (e.g., 0.1:1 to 0.99:1).

[0034] 26: A kit for identifying a target sequence and a reference sequence in a biological sample by polymerase chain reaction (PCR), comprising: a forward primer and a reverse primer that serve to amplify both the reference strand and the target strand; a label probe comprising a fluorophore and a quencher capable of specifically binding to the target sequence and non-specifically binding to the reference sequence; a promiscuity blocked nucleotide doubler oligonucleotide (PBNJ) capable of specifically binding to said reference sequence and non-specifically binding to said target sequence; Optionally, a positive control of said reference sequence; Optionally, a positive control for the target sequence. Including the kit.

[0035]

[0035] 27: The forward primer was provided at a concentration between 200 nM and 1100 nM; The reverse primer was provided at a concentration between 200 nM and 1100 nM; The labeled probe is provided at a concentration of between 50 and 800 nM; The PBNJ is provided at a concentration between 0.25 and 16 times the concentration of the labeled probe. 27. The method or kit according to any one of aspects 1 to 26.

[0036]

[0036] 28. A kit according to aspect 26 or 27, further comprising reagents for dPCR, qPCR, RT-dPCR or RT-qPCR.

[0037]

[0037] 29. The kit of any one of aspects 26 to 28, wherein assay reagents are provided for a first reaction that does not include PBNJ and a second reaction that includes at least one PBNJ.

[0038]

[0038] 30. A kit described in any one of aspects 26 to 29, wherein PBNJ is provided for a wild-type reference sequence of a SARS-CoV-2 mutation selected from the group consisting of spike residues HV69-70, R408, K417, L452, T478, N501, N679, L704, Q954 and L981, and optionally also K417, 478 and L452.

[0039] 31. The reference sequence is: a. the parent SARS-CoV-2, wherein the target sequence comprises a variant of SARS-CoV-2 selected from the group consisting of an alpha variant, a beta variant, a gamma variant, a delta variant, a delta plus variant, a mu variant, a lambda variant and an omicron variant; or b. a proto-oncogene, wherein the target sequence has a mutation that converts the proto-oncogene into an oncogene that indicates a higher risk of developing cancer or the presence of cancer; 31. The method or kit according to any one of aspects 1 to 30.

[0040]

[0040] 32. The method or kit according to aspect 31, wherein the reference sequence is a proto-oncogene.

[0041]

[0041] 33. The method or kit according to aspect 32, wherein the protooncogene is KRAS.

[0042]

[0042] 34. The method or kit according to aspect 32, wherein the target is a KRAS mutation selected from G12C, G12A, G12D, G12R or G13D.

[0043]

[0043] 35. The method or kit of any one of aspects 1 to 34, wherein the PBNJ is provided at a concentration such that one or more non-specifically amplified populations are optically indistinguishable from negative compartment populations.

[0044]

[0044] 36. The method or kit according to any one of aspects 1 to 35, wherein the PBNJ is provided at a concentration such that the threshold cut-off value for detection of the target sequence is reduced compared to the threshold cut-off value of a method or kit that does not include the PBNJ.

[0045]

[0045] 37. The method or kit according to any one of aspects 1 to 36, wherein the PBNJ has a length between 80% and 100% of the length of the labeled probe.

[0046] The assays and components described herein may be used with conventional digital PCR platforms including the QIAgen QIAcuity® One Digital PCR System or Bio-Rad QX200 Droplet Digital PCR System, as well as conventional quantitative PCR systems including the Bio-Rad CFX96 and Thermo Scientific QuantStudio5. The assays and components described herein may be used with conventional RT-PCR platforms.

[0047]

[0047] Also provided herein are compositions of matter useful for carrying out any of the methods described herein, including any one or more PBNJs, with or without LNA, with or without quenchers and with or without other components such as one or more primers, probes and PCR solutions, alone or in combination (see, e.g., Tables 1-6 for representative sequences, with and without LNA (indicated by +), extension (e.g., C3, etc.), quencher and / or optically detectable tag).

[0048]

[0048] While not wishing to be bound by any particular theory, one may discuss herein a belief or understanding of the underlying principles relating to the devices and methods disclosed herein. It is recognized that regardless of the ultimate correctness of any mechanistic explanation or hypothesis, embodiments of the invention may still be functional and useful. [Brief description of the drawings]

[0049] [Figure 1] Reagents are shown, including a reference sequence ("WT template"), a target sequence ("mutant template"), a primer pair, a dual-labeled probe, and a promiscuity-blocked nucleotide multiplexer oligonucleotide ("PBNJ"). Figure 1 further illustrates a PCR reaction using these same reagents, and the reduced affinity of the labeled probe for the reference sequence in the presence of PBNJ, and the reduced affinity of PBNJ for the target sequence in the presence of the labeled probe.

[0050] [Figure 2A] The amplification results detected by the fluorescent signal are shown in Figure 2. Figure 2A shows the results of specific and non-specific amplification, where the probe binds to both on-target and off-target molecules in the reaction. [Figure 2B] The amplification results detected by fluorescent signals are shown in Figure 2. Figure 2B illustrates the improved detection of specific amplification, with the disappearance of non-specific signals by PBNJ.

[0051] [Figure 3A] 3A shows the amplification results detected by fluorescent signals in the presence of PBNJ, including multiplexed results. FIG. 3A shows the specific amplification of a specific signal A, including the absence of non-specific amplification. [Figure 3B] Figure 3B shows the amplification results detected by fluorescent signals in the presence of PBNJ, including multiplexed results. Figure 3B shows the specific amplification of specific signal B, including the absence of non-specific amplification. [Figure 3C] Figure 3C shows the amplification results detected by the fluorescent signal in the presence of PBNJ, including multiplexed results. Figure 3C shows specific amplification for specific signal A, specific amplification for specific signal B, and the absence of non-specific amplification, demonstrating amplitude adjustment for the multiplex strategy using a single channel.

[0052] [Figure 4A] Figure 4 shows amplification results detected by fluorescent signal in the presence of PBNJ, including multiplexed results: Figure 4A shows amplification with two probes in the same channel. [Figure 4B] Figure 4B shows amplification results detected by fluorescent signal in the presence of PBNJ, including multiplexed results. Figure 4B shows amplification with two probes in the same alternate channel. [Figure 4C] Amplification results detected by fluorescent signals in the presence of PBNJ, including multiplexed results, are shown in Figure 4C, which shows amplitude adjustment for a multiplex strategy using multiple channels and two-dimensional based analysis.

[0053] [Figure 5A] The concentration of PBNJ relative to the dual-labeled probe for the 484K mutation of SARS CoV-2 shows that nonspecific signals are reduced when the PBNJ:probe concentration ratio is between 2:1 and 8:1. In Figure 5A, [PBNJ] = 0, and the nonspecific signals are indicated by arrows. [Figure 5B]The concentration of PBNJ for the dual-labeled probe for the 484K mutation of SARS CoV-2 shows that nonspecific signals are reduced at PBNJ:probe concentration ratios between 2:1 and 8:1. In Figure 5B, [PBNJ] = 400 nM. [Figure 5C] The concentration of PBNJ for the dual-labeled probe for the 484K mutation of SARS CoV-2 shows that nonspecific signals are reduced at PBNJ:probe concentration ratios between 2:1 and 8:1. In Figure 5C, [PBNJ] = 800 nM. [Figure 5D] The concentration of PBNJ for the dual-labeled probe for the 484K mutation of SARS CoV-2 shows that nonspecific signals are reduced at PBNJ:probe concentration ratios between 2:1 and 8:1. In Figure 5D, [PBNJ] = 1600 nM.

[0054] [Figure 6] We demonstrate titration of PBNJ to reduce the fluorescence levels of off-target amplification using a SARS-CoV-2 variant mutation assay, enabling improved discrimination of the 417N mutation.

[0055] [Figure 7] We show that PBNJ can be used to reduce the fluorescence levels of off-target amplification of oncogenic KRAS, allowing improved discrimination of the G12C allele.

[0056] [Figure 8] FIG. 1 shows channel assignments for detection of different SARS-CoV-2 variants in either the first or second reaction.

[0057] [Figure 9]An example of the use of RT-qPCR with PBNJ in SARS-CoV-2 variant discrimination is shown. As seen in the top panel, the HEX (upper curve) trace from the amplification curve on the left shows non-specific hybridization of the 484Q probe to the E484 template. As seen in the bottom panel, the addition of PBNJ completely inhibits non-specific hybridization of the probe.

[0058] [Figure 10] We summarize the genes and cancer-associated mutations relevant to the analysis of the minimal requirements for PBNJ.

[0059] [Figure 11A] The effects of temperature, sequence length and the presence of LNA on specificity are illustrated. FIG. 11A: Effect of temperature (Tm) on target specificity in the absence of PBNJ for mixed WT and mutant synthetic DNA KRAS-G12C (FAM, blue; top panel) and EGFR-T790M (HEX, green; bottom panel). Specificity is only achieved at high Tm, but at the expense of PCR efficiency and signal-to-noise ratio. FIG. 11B: Effect of PBNJ length at 14-fold concentration and 59° C. annealing / extension with and without LNA at the SNP position. Specificity is improved and non-specific signals are reduced when the PBNJ length is at least 80% of the probe length. Specificity is improved in all cases with the presence of LNA. At 100% length, complete specificity is achieved with the presence of LNA, eliminating non-specific signals for both targets. Non-specific signals are represented by dashed boxes. [Figure 11B]The effects of temperature, sequence length and the presence of LNA on specificity are illustrated. FIG. 11A: Effect of temperature (Tm) on target specificity in the absence of PBNJ for mixed WT and mutant synthetic DNA KRAS-G12C (FAM, blue; top panel) and EGFR-T790M (HEX, green; bottom panel). Specificity is only achieved at high Tm, but at the expense of PCR efficiency and signal-to-noise ratio. FIG. 11B: Effect of PBNJ length at 14-fold concentration and 59° C. annealing / extension with and without LNA at the SNP position. Specificity is improved and non-specific signals are reduced when the PBNJ length is at least 80% of the probe length. Specificity is improved in all cases with the presence of LNA. At 100% length, complete specificity is achieved with the presence of LNA, eliminating non-specific signals for both targets. Non-specific signals are represented by dashed boxes.

[0060] [Figure 12A] Illustrated are the effects of PBNJ:probe ratio, length and the presence of LNA on specificity. The effect of PBNJ ratio and the presence of LNA on KRAS-G12C (FAM, blue; top panel) and EGFR-T790M (HEX, green; bottom panel) target specificity of mixed WT and mutant synthetic DNA at 80% PBNJ length (FIG. 12A) and 100% PBNJ length (FIG. 12B). Non-specific signals are represented by red dashed boxes. [Figure 12B] Illustrated are the effects of PBNJ:probe ratio, length and the presence of LNA on specificity. The effect of PBNJ ratio and the presence of LNA on KRAS-G12C (FAM, blue; top panel) and EGFR-T790M (HEX, green; bottom panel) target specificity of mixed WT and mutant synthetic DNA at 80% PBNJ length (FIG. 12A) and 100% PBNJ length (FIG. 12B). Non-specific signals are represented by red dashed boxes.

[0061] [Figure 13A]13A-B illustrate that PBNJ does not affect target amplification of pure KRAS-G12C (FIG. 13A) or EGFR-T790M (FIG. 13B) synthetic DNA. Target DNA concentrations are quantified. The difference in concentration of PBNJ is not statistically different compared to the absence of PBNJ, as measured by z-test. ns, not significant. [Figure 13B] 13A-B illustrate that PBNJ does not affect target amplification of pure KRAS-G12C (FIG. 13A) or EGFR-T790M (FIG. 13B) synthetic DNA. Target DNA concentrations are quantified. The difference in concentration of PBNJ is not statistically different compared to the absence of PBNJ, as measured by z-test. ns, not significant.

[0062] [Figure 14A] Figure 14 illustrates that PBNJ-G inhibits off-target amplification in RT-PCR amplification of SARS-CoV2 variant templates. Plots show relative fluorescence units (RFU) as a function of amplification cycle number for no PBNJ (Figure 14A), PBNJ-G C3 (Figure 14B), and PBNJ-G BHQ-1 (Figure 14C) against off-target (top panel) and on-target (bottom panel) templates. Both types of PBNJ inhibit off-target amplification without adversely affecting on-target amplification. [Figure 14B] Figure 14 illustrates that PBNJ-G inhibits off-target amplification in RT-PCR amplification of SARS-CoV2 variant templates. Plots show relative fluorescence units (RFU) as a function of amplification cycle number for no PBNJ (Figure 14A), PBNJ-G C3 (Figure 14B), and PBNJ-G BHQ-1 (Figure 14C) against off-target (top panel) and on-target (bottom panel) templates. Both types of PBNJ inhibit off-target amplification without adversely affecting on-target amplification. [Figure 14C]Figure 14 illustrates that PBNJ-G inhibits off-target amplification in RT-PCR amplification of SARS-CoV2 variant templates. Plots show relative fluorescence units (RFU) as a function of amplification cycle number for no PBNJ (Figure 14A), PBNJ-G C3 (Figure 14B), and PBNJ-G BHQ-1 (Figure 14C) against off-target (top panel) and on-target (bottom panel) templates. Both types of PBNJ inhibit off-target amplification without adversely affecting on-target amplification. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0063]

[0063] Reference will now be made in detail to exemplary embodiments of the invention. While the invention will be described in conjunction with the enumerated embodiments, it will be understood that it is not intended to limit the invention to those embodiments. On the contrary, the invention is intended to cover all alternatives, modifications, and equivalents which may be included within the scope of the present invention as defined by the claims.

[0064]

[0064] One skilled in the art will recognize many methods and materials similar or equivalent to those described herein that can be used in and are within the scope of the present invention, and the present invention is in no way limited to the methods and materials described.

[0065]

[0065] Generally, the terms and phrases used herein have their art-recognized meanings, which can be found by reference to standard texts, references, and contexts known to those of skill in the art. The following definitions are provided to clarify the specific use of terms and phrases in the context of the present invention.

[0066]

[0066] All publications, published patent documents, and patent applications cited in this application are indicative of the level of skill in the art to which this application pertains. All publications, published patent documents, and patent applications cited in this specification are herein incorporated by reference to the same extent as if each individual publication, published patent document, or patent application was specifically and individually indicated to be incorporated by reference.

[0067]

[0067] As used in this specification, including the accompanying claims, the singular forms "a," "an," and "the" include plural references and are used interchangeably with "at least one" and "one or more," unless the content clearly dictates otherwise.

[0068]

[0068] As used herein, the term "about" refers to a small modification or variation of a numerical value that does not change the basic function of the item to which the numerical value is associated. In certain aspects, about refers to 90% of the stated value.

[0069] As used herein, the terms "biological sample", "sample" and "test sample" are used interchangeably herein and refer to any material, body fluid, tissue or cell obtained from or derived from an individual. This includes blood (including whole blood, white blood cells, peripheral blood mononuclear cells, buffy coat, plasma and serum), dried blood spots (such as those obtained from infants), sputum, tears, mucus, nasal washings, nasal aspirates, breath, urine, semen, saliva, peritoneal washings, ascites, cyst fluid, cerebrospinal fluid, glandular fluid, pancreatic juice, lymphatic fluid, pleural fluid, nipple aspirate, bronchial aspirate, bronchial scraping, synovial fluid, joint aspirate, organ secretions, cells, cell extracts and cerebrospinal fluid. This also includes experimentally isolated portions of all of the above. For example, a blood sample can be fractionated into serum, plasma, or fractions containing specific types of blood cells, such as red blood cells or white blood cells (leucocytes). Optionally, the sample can be a combination of samples from an individual, such as a combination of a tissue sample and a bodily fluid sample. The term "biological sample" also includes material containing homogenized solid material, such as from a stool sample, tissue sample, or tissue biopsy. The term "biological sample" also includes material derived from tissue or cell culture. Any suitable method for obtaining a biological sample can be used; exemplary methods include, for example, bloodletting, swabs (e.g., swabs of the inside of the cheek), and fine needle aspiration biopsy procedures. Examples of tissues from which fine needle aspiration is often performed include lymph nodes, lungs, lung lavage fluid, bronchoalveolar lavage (BAL), thyroid, breast, pancreas, and liver. Samples may also be collected, for example, by microdissection (e.g., laser capture micro dissection (LCM) or laser micro dissection (LMD)), bladder washing, smear (e.g., PAP smear) or ductal washing. A "biological sample" obtained or derived from an individual includes any sample that has been processed in any suitable manner after being obtained from that individual.In certain aspects, the biological sample is used to test for mutations associated with increased disease risk. In certain further aspects, when testing for disease risk, the reference sequence reflects a low disease state and the target sequence has one or more nucleotide changes in the reference sequence that reflect increased disease risk or the presence of the disease. In certain aspects, the biological sample is used to test for mutations associated with increased risk of cancer, dementia, and / or cardiovascular conditions. In certain aspects, the biological sample is used to test for pathogen variants, including pathogens that are viruses. In certain further aspects, when testing for viral variants, the reference sequence is derived from a wild type or parent virus and the target sequence contains at least one mutation in the reference sequence. In certain aspects, the biological sample is derived from wastewater, an environmental sample, a bodily fluid, a tissue, a cell culture, or a tumor.

[0070]

[0070] As used in this specification, the terms "comprise," "comprising," "include," "including," "contain," and "containing" and any variations thereof are intended to include a non-exclusive inclusion, and a process, method, product-by-process, or composition of matter that comprises, includes, or includes an element or list of elements does not include only those elements, but may include other elements that are not expressly listed or that are not inherent to such process, method, product-by-process, or composition of matter.

[0071]

[0071] As used herein, "distinguish" refers to distinguishing or discriminating between two or more things. In certain aspects, "distinguish" refers to distinguishing between a reference oligonucleotide sequence and a target oligonucleotide sequence.

[0072]

[0072] As used herein, "dPCR" refers to the division of a sample, where each divided sample is analyzed for the presence or absence of amplicons, and a statistical analysis of amplicon detection is performed for all compartments to correct for multiple targets delivered to each compartment. In this way, high sensitivity and quantitation are achieved. dPCR can be applied to samples that originally contained DNA or samples that contain complementary DNA obtained from reverse transcription of RNA.

[0073] As used herein, "non-specifically binds" refers to binding or hybridization of a binding agent that is not correlated with the specificity of the binding agent. In certain aspects, a binding agent is an oligonucleotide that non-specifically hybridizes to an oligonucleotide sequence that is not completely complementary to the sequence of the oligonucleotide binding agent.

[0074]

[0074] As used herein, "promiscuity" refers to the non-specific hybridization of nucleic acids.

[0075]

[0075] As used herein, "quantitative PCR," "qPCR," or "real-time PCR" is a PCR-based technique that couples the amplification of a target DNA sequence with quantification of the DNA concentration in the reaction. qPCR can be applied to samples that originally contained DNA or to samples that contain complementary DNA obtained by reverse transcription of RNA.

[0076]

[0076] As used herein, "reference sequence" refers to an oligonucleotide sequence selected as a basis for comparison with a target sequence. In certain aspects, a target sequence is a sequence that differs from a reference sequence by one or more single nucleotide polymorphisms, insertions, or deletions. However, the term reference sequence is intended to be used broadly herein. For example, the use of PBNJ has application to parent or wild-type sequences, but PBNJ can also be used for any mutation that occurs within the probe binding region. As an illustrative example, PBNJ designed as a G12C probe, which may also include wild-type PBNJ, may also include other different reference sequences, such as PBNJ of G12A, G12R, G13D, etc. In the broadest sense, "reference sequence" simply refers to a sequence that has one or more nucleotide differences compared to a target sequence.

[0077] As used herein, "target" or "target sequence" are used interchangeably and refer to a nucleic acid that hybridizes to a primer and can be detected and quantified by dPCR and qPCR analysis. Target or target sequence is used broadly to refer to any oligonucleotide sequence of interest, including sequences associated with a pathogen (e.g., a virus or bacteria) and sequences associated with a patient's genome (e.g., a mammal, such as a human). A labeled probe specifically binds to, and therefore has complementarity to, a target or target sequence. In certain aspects, a target or target sequence is an oligonucleotide sequence that differs from a selected reference sequence by one or more single nucleotide polymorphisms, insertions, or deletions. In certain aspects, the length of the target or target sequence is between 10 and 50 nucleotides. In certain further aspects, the target or target sequence is a region of 10 to 50 nucleotides within a substantially longer polynucleotide.

[0078]

[0078] A target or target sequence specifically hybridizes to a single-stranded target sequence in the presence of corresponding primers and probes, a polymerase, optionally reverse transcriptase, and nucleotides at appropriate pH, temperature, metal and ion concentrations to prime the synthesis of a second strand complementary to the target. This amplification can be repeated by cycling temperature for repeated hybridization and separation, thereby amplifying any target sequence.

[0079]

[0079] The primer need not reflect the exact sequence of the template, but must be sufficiently complementary to hybridize to the template. The primer may further comprise a "tail" at the 5' end of the primer that contains additional nucleotides that are non-complementary to the template. Typically, the length of the primer is in the range of between 7 and 100 nucleotides, such as 10-30, 15-60, 20-40, and more typically in the range of between 15-35 nucleotides, and any subranges thereof. In general, the shorter the primer molecule, the lower the temperature required to form a sufficiently stable hybrid complex with the template. The term "primer site" or "primer binding site" refers to the segment of the target DNA to which the primer hybridizes. Typically, a set of primers is used for amplification of a target polynucleotide, including a 5' "upstream primer" or "forward primer" that hybridizes to a complementary sequence at the 5' end of the DNA sequence to be amplified, and a 3' "downstream primer" or "reverse primer" that hybridizes to the 3' end of the sequence to be amplified. Useful primers include different 5' tail lengths to facilitate differentiation of amplicons of a first target and different targets based on amplicon length, and can be designed according to any of the teachings provided in U.S. Pat. No. 10,465,238, specifically incorporated herein by reference.

[0080] As used herein, "sequence complementarity" refers to the standard arrangement of nucleotide bases relative to opposite base pairs, such as thymine pairs with adenine, cytosine pairs with guanine, etc. In certain aspects, sequence complementarity is complete or exact complementarity at all base positions within an oligonucleotide. RNA has uracil in place of thymine.

[0081]

[0081] As used herein, "specifically binds" refers to hybridization between complementary oligonucleotide or nucleotide sequences. In certain aspects, a probe is designed to be specific for a target region, and the probe has a sequence complementary to a nucleotide sequence of the target region. In one aspect, a probe can be completely (e.g., 100%) complementary to the target.

[0082]

[0082] As used herein, "suppressing a labeled probe" refers to competition with a competitor (e.g., PBNJ) for probe binding at a region of hybridization that is not perfectly complementary to the probe, thereby reducing such non-specific probe binding and detection of the signal from such non-specific probe binding.

[0083]

[0083] As used herein, "substantially eliminate" refers to a reduction of at least 90%, at least 95%, or at least 99%. In the context of using a threshold, different populations are defined by the threshold setting (see, for example, dashed boxes in Figures 11-13 and line 200 in Figure 2B and similar lines in the figures depicting populations), and even a 20% knockdown of the PBNJ signal may be sufficient to reliably distinguish between target and off-target.

[0084]

[0084] The present disclosure provides a method for distinguishing between a target sequence and a reference sequence in a biological sample by PCR. In certain aspects, the PCR is dPCR. In certain further aspects, the discrimination between the target sequence and the reference sequence is achieved by the introduction into the PCR reaction of a promiscuity block nucleotide doubler oligonucleotide (PBNJ) that specifically binds to the reference sequence and may non-specifically bind to the target sequence and includes a reference binding region and an extension blocker that prevents extension by a polymerase.

[0085] As shown in Figure 1, PBNJ can compete for binding to a reference sequence with a labeled probe that is specific for a target sequence but can bind non-specifically to a reference sequence. The reduced affinity of the labeled probe for the reference sequence, brought about by competition with PBNJ, reduces the error signal generated by non-specific binding of the probe to the reference sequence.

[0086]

[0086] In certain aspects, the PBNJ sequence is similar to the sequence of a probe oligonucleotide labeled with both a fluorophore and a quencher, i.e., a dual-labeled probe. As shown in Figure 1, in certain aspects, only the single nucleotide polymorphism (SNP) base is changed (the SNP can be located either internally or at the 3' end). Thus, the target sequence and the reference sequence may have a single nucleotide mismatch that is part of a single nucleotide polymorphism (SNP) or a short nucleotide polymorphism such as a deletion or insertion.

[0087] In certain aspects, the probe labeled with both a fluorophore and a quencher is a conventional probe used in qPCR that contains a fluorophore (F) at the 5' end and a quencher (Q) usually at the 3' end, see Figure 1. In certain aspects, the length of the probe is 10-50 nucleotides.

[0088] In certain aspects, the PBNJ has a C3 spacer modification at the 3' end to prevent 5' to 3' extension by DNA polymerase. In certain further aspects, the PBNJ and the probe compete during hybridization, and as shown in FIG. 1, the change in affinity for each target increases the specificity of the probe for the target, especially when PBNJ is in excess. In certain aspects, the modification at the 3' end of the PBNJ is a 3' quencher, such as a C3 spacer or a black hole quencher. The PBNJ can also be modified with an extended carbon spacer (C6, C9, C12, etc.). The PBNJ can have any length useful in the methods of the invention, including, but not limited to, a length of 10 to 50 nucleotides. In certain aspects, the PBNJ has target sequence complementarity (e.g., one or more mismatches over the entire length of the PBNJ) with at least a portion of the target sequence that is equal to or less than the range between 9 / 10 nucleotides and 49 / 50 nucleotides. In certain aspects, PBNJ has a reference sequence complementarity that is greater than the target sequence complementarity, such that the binding affinity of PBNJ to the reference sequence is greater than the binding affinity of PBNJ to the target sequence; the binding affinity of PBNJ to the reference sequence is greater than the binding affinity of the labeled probe to the reference sequence, and / or the binding affinity of PBNJ to the target sequence is less than the binding affinity of the labeled probe to the target sequence. In certain aspects, PBNJ is a PCR blocker during PCR amplification cycles to provide enrichment of target sequences that are part of mutant alleles. In certain aspects, PBNJ can have modifications at the 5' end to increase its stability by preventing digestion by DNA polymerase.

[0089] In certain aspects, the ratio of probe to PBNJ is such that the concentration of PBNJ exceeds the concentration of the probe. In certain aspects, the ratio of PBNJ concentration to dual-labeled probe concentration is at least 1.5:1. In certain further aspects, PBNJ acts as a PCR blocker during the amplification cycle, leading to enrichment of mutant alleles. In certain aspects, the reaction can be multiplexed to utilize more than one PBNJ.

[0090] One practical impact of the methods, kits and compositions provided herein is illustrated in Figures 2A-4C. In particular, the use of PBNJ effectively reduces the detection of undesired amplicons, as shown by the amplification results detected by fluorescent signals (compare Figure 2A without PBNJ and Figure 2B with PBNJ). Interfering signals that may be associated with non-specific amplification are reduced by the use of PBNJ provided herein. This improves the ability to properly set the threshold at 200. Figure 2A shows the results of specific and non-specific amplification, where the probe binds to both on-target and off-target molecules in the reaction. Figure 2B illustrates the improved detection of specific amplification, where the disappearance of non-specific signals by PBNJ is seen.

[0091]

[0091] In certain aspects, the reference sequence and the target sequence differ by a single nucleotide; a nucleotide insertion of one or more nucleotides; and / or a nucleotide deletion of one or more nucleotides.

[0092]

[0092] The methods and kits disclosed herein can be used with any oligonucleotide, including, but not limited to, DNA and / or RNA. In certain aspects, the methods and kits disclosed herein are applied to dPCR, qPCR, RT-dPCR and RT-qPCR.

[0093]

[0093] In certain aspects, the labeled probe is a single base variation (SNV)-specific TaqMan® probe (a fluorophore covalently attached to the 5' end of the probe and a quencher at the 3' end of the probe).

[0094]

[0094] In certain aspects, dPCR includes compartment- and droplet-based PCR, and PBNJ eliminates signals associated with low efficiency non-specific off-target amplification, thereby increasing the signal-to-noise ratio for specific amplification of target sequences.

[0095] In certain aspects, the probe output amplitude is adjusted by providing a lower concentration of PBNJ. In certain further aspects, multiple probe output amplitudes are adjusted for multiplex detection of multiple target sequences in a single or multi-channel fluorescent detector.

[0096] In certain aspects, the methods or kits disclosed herein are designed for a reference sequence that is the parent SARS-CoV-2 and the target sequence includes variants of SARS-CoV-2 such as alpha, beta, gamma, delta and omicron. In certain aspects, the methods or kits disclosed herein are designed for a reference sequence that is a proto-oncogene and a target sequence that has a mutation that converts the proto-oncogene into an oncogene that indicates a higher risk of developing cancer or the presence of cancer.

[0097]

[0097] In certain aspects, the methods or kits disclosed herein provide PBNJ at a concentration such that one or more non-specifically amplified populations are optically indistinguishable from negative compartment populations.

[0098]

[0098] Disclosed are kits for carrying out the methods disclosed herein. Kits are provided for identifying target and reference sequences in biological samples by dPCR or RT-PCR. In certain aspects, the kits include a buffer, a primer, a polymerase, one or more labeled probes, and one or more PBNJs. In certain aspects, the forward primer is provided at a concentration between 50 nM and 1100 nM. In certain aspects, the reverse primer is provided at a concentration between 50 nM and 1100 nM. In certain aspects, the labeled probe is provided at a concentration between 20 and 800 nM. In certain aspects, the one or more PBNJs are provided at a concentration between 0.25 and 16 times the concentration of the labeled probe.

[0099] In one aspect, a kit is provided that contains 200 x 26k reactions (400 x 8.5k compartment reactions) per assay solution. In certain further aspects, the kit contains all primers, probes and controls for detection and differentiation of alpha, beta, gamma, delta, delta plus, mu and lambda variants of SARS-CoV-2 in two multiplexed dPCR wells.

[0100]

[0100] In certain further aspects, the kit comprises reagents for a reaction as depicted in Figure 8, wherein the oligonucleotides comprise sequences specified in Table 1. In certain further aspects, the kit comprises: assay solution for reaction 1 - 20x (400 μL); assay solution for reaction 2 - 20x (400 uL); parental (non-variant / Wuhan) positive control - sufficient for at least 20 tests, and a mixed variant positive control containing all seven variant templates - sufficient for at least 20 tests.

[0101] In certain aspects, channel assignments can be made as depicted in the first reaction (reaction 1) and / or the second reaction (reaction 2), as shown in FIG. 8. Table 1 provides sequences useful for reaction 1 or reaction 2 according to the methods disclosed herein, as shown in FIG. 8. Reaction 1 controls can include a parent SARS-CoV-2 positive control. Reaction 2 controls can include an alpha variant positive control, a delta variant positive control, a delta plus variant positive control, a mu variant positive control, a lambda variant positive control, a gamma variant positive control, and a beta variant positive control. Unless otherwise noted, the symbol + refers to the position of the LNA (+A refers to the LNA on an adenine).

[0102]

[0102] [Table 1] EXAMPLES

[0103]

[0103] The following examples are provided for illustrative purposes only and are not intended to limit the scope of this application, which is defined by the appended claims. All examples described herein were carried out using standard techniques that are well known and routine to those skilled in the art. The routine molecular biology techniques described in the following examples can be carried out as described in standard laboratory manuals such as Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, (2001).

[0104]

[0104] Example 1: Reduction of off-target E484 signal by PBNJ

[0105]

[0105] This example describes the use of PBNJ to reduce off-target amplification when using E484K probes. Dual-labeled probes were designed to the mutant 484K sequence. The concentrations of reagents used were as follows: common forward primer - 500 nM; common reverse primer - 500 nM; dual-labeled probe to 484K - 200 nM; and PBNJ concentrations were variable. The assay was performed on a Qiagen Qiacuity® instrument.

[0106]

[0106] Figure 5A shows that the probe detects 484K in SARS-CoV-2 variant "beta" but causes off-target amplification in the absence of PBNJ (i.e., [PBNJ] = 0). The arrows in Figure 5A indicate a secondary, less efficient, lower amplitude population in the "parental / wild type" and "alpha" controls that are wild type at locus 484.

[0107]

[0107] Figures 5B-5D show the results of increasing the concentration of PBNJ. Figure 5B shows that the addition of PBNJ at a competitive concentration of 400 nM, corresponding to a ratio of [2:1] (PBNJ:probe), eliminates the off-target E484 signal. Figure 5C shows a similar result at [PBNJ] = 800 nM. Figure 5D shows a similar result at [PBNJ] = 1600 nM. Thus, excess PBNJ (up to a ratio of 8:1) does not affect on-target detection while eliminating non-specific signals.

[0108]

[0108] Example 2: Reduction of off-target 417N signal by PBNJ

[0109] This example describes the use of PBNJ to reduce off-target amplification when using 417N probes. Dual-labeled probes were designed for the mutant 417N sequence. Dual-labeled probes were used on a mixture of templates consisting of parental, beta, gamma and delta variants of SARS-CoV-2. In well G02, the probes generated a high amplitude droplet population resulting from on-target amplification of templates from the beta variant of SARS-CoV-2 containing the 417N mutation (highlighted by the arrow in FIG. 6) and a low amplitude compartment population of off-targets due to less efficient amplification of off-targets. The assay was performed on a Qiagen-Qiacuity® instrument.

[0110]

[0110] Upon titration of PBNJ, designed to contain a sequence complementary to the wild-type K417 allele, the amplitude of the off-target amplified population (highlighted by an arrow in Figure 6) was reduced to the point where the non-specific amplified population was indistinguishable from negative droplets (also highlighted by an arrow in Figure 6).

[0111]

[0111] Example 3: Reduction of off-target human KRAS G12C signal by PBNJ

[0112] This example describes the use of PBNJ to reduce off-target amplification when using a KRAS G12C probe. A dual-labeled probe was designed to the mutant KRAS G12C sequence. The concentrations of reagents used were as follows: common forward primer-900 nM; common reverse primer-900 nM; dual-labeled probe to G12C-250 nM; and PBNJ-500 nM. The assay was performed on a Bio-Rad Droplet Digital QX200 instrument. See Tables 2-5 for sequence information and concentrations tested.

[0113]

[0113] [Table 2]

[0114]

[0114] [Table 3]

[0115]

[0115] [Table 4]

[0116]

[0116] [Table 5]

[0117]

[0117] The probe generated a high amplitude droplet population for templates containing the G12C mutation. As shown in Figure 7, the probe also generated a lower amplitude off-target droplet population for WT and G12R templates, as demonstrated by the presence of a lower amplitude off-target population in the wild-type template (B01, E01) and the template containing the alternative G12R mutation (E01).

[0118]

[0118] Using a PBNJ designed to contain a sequence complementary to the G12R template, the lower amplitude droplet population of the G12R off-target is eliminated (D01). A second PBNJ complementary to the wild-type sequence of this locus could also be used to eliminate the lower amplitude droplet population of that off-target. This usage shows that PBNJ can be designed to selectively remove the off-target population of the user's choice.

[0119]

[0119] Example 4: Characterization of PCR and PBNJ parameters for target specificity (cancer-associated mutations).

[0120]

[0120] Figure 10 summarizes target genes and their mutations associated with cancer (KRAS and EGFR are shown as examples herein) with corresponding mutations and SNPs associated with Figures 11A-13B. Exemplary sequences for use in the methods described herein are provided in Table 6 (EGFR) and Table 7 (KRAS).

[0121]

[0121] [Table 6] TIFF2024538973000008.tif202149 TIFF2024538973000009.tif205149 TIFF2024538973000010.tif190149 TIFF2024538973000011.tif41149

[0122]

[0122] Figure 11A shows the effect of Tm on target amplification of KRAS-G12C (top panel) and EGFR-T790M (bottom panel) without PBNJ in mixed samples of wild-type (WT) and mutant synthetic DNA. Specificity (e.g., on-target amplification) improves with increasing Tm, but at the expense of a concomitant decrease in PCR efficiency (e.g., lower signal output) and lower signal-to-noise (STN) ratio. Figure 11B shows an equivalent PCR experiment, but with PBNJ at 59°C (annealing / extension). We also varied the length of PBNJ (relative to probe length), the range of PBNJ from 0% (e.g., no PBNJ) to 100%, and the presence (+) / absence (-) of LNA (at the SNP position). The PBNJ:probe ratio is 14-fold. Thus, a preferred embodiment for reducing non-specific signals is at least 80% PBNJ length with LNA, including 100% PBNJ length (relative to probe length) with LNA, which removes at least 95% of the non-specific signal, including at least 99%.

[0123] 12A-12B show the effect of various PBNJ ratios from 0.25x to 14x (relative to probe), with or without LNA, and two different PBNJ lengths (80% - FIG. 12A; 100% - FIG. 12B) for KRAS-G12C target (top panel of each figure) and EGFR-T790M target (bottom panel of each figure) in samples with mixed WT and mutant synthetic DNA. Reduction of off-target amplification is demonstrated for increasing PBNJ:probe amount, which may be reflected in terms of concentration in PCR assay, as well as for 100% length PBNJ (relative to probe) for both KRAS and EGFR targets. Thus, preferred embodiments are 100% length and at least 0.5x to 1x PBNJ relative to probe. Especially for short length PBNJ, the presence of LNA is preferred.

[0124]

[0124] Figures 13A-13B confirm that PBNJ does not adversely affect target amplification of pure KRAS-G12C (Figure 13A) or EGFR-T790M (Figure 13B) synthetic DNA for either 80% or 100% PBNJ length (relative to the probe). The bottom panel illustrates that no significant difference in quantified amplicon concentration occurs at any PBNJ concentration (ranging from 0-fold to 6-fold).

[0125]

[0125] This example demonstrates that the present invention is compatible across a range of PCR parameters (melting temperature (Tm), target sequence) and PBNJ-related parameters (LNA+ / -; PBNJ vs. probe length; and PBNJ:probe concentration), and that the choice of one parameter influences the choice of another. Although these experiments are based on DNA targets (KRAS and EGFR genes), the methods are certainly compatible with RT-PCR on RNA targets, as described in Example 5.

[0126]

[0126] Example 5: Use of PBNJ for RT-PCR

[0127] 14A-14C demonstrate that PBNJ is well suited for RT-PCR, as reflected by targets from SARS-CoV-2 variants, and that the amplification and concomitant detection of non-target sequences is reduced when PBNJ is used, resulting in more reliably detectable on-target amplification, even at very low concentrations compared to the reference sequence.

[0128] As discussed, the main obstacle in short nucleotide variant assay development is off-target amplification and detection. In many cases, nucleic acids differ by only one nucleotide. The PBNJ blocking technology described herein is particularly suited for this type of application, as demonstrated by PBNJ applied using SARS-CoV-2 variant templates that differ by one nucleotide in the spike protein gene. Specifically, a probe is designed against the E484Q mutation and a PBNJ is designed against the E484 wild-type sequence (denoted as PBNJ-G). The PBNJ is synthesized with either 3'C3 or BHQ-1 polymerase extension blocker and tested against on-target and off-target templates by RT-PCR using TaqPath® (Thermo) on a Bio-Rad CFX-96. As shown in Figures 14A-14C, E484 PBNJ can efficiently block non-specific amplification without any loss of on-target detection.

[0129]

[0129] This example demonstrates that PBNJ can be modified with either C3 (and longer chains, such as C6) and BHQ-1 and exhibits efficient non-specific amplification blocking. Moreover, PBNJ is compatible with RT-PCR.

[0130] [INCORPORATION BY REFERENCE AND VARIATION STATEMENT]

[0130] All references throughout this application, e.g., patent documents, including issued or granted patents or equivalents; patent application publications; and non-patent literature or other source materials; are incorporated by reference in their entirety herein, as if each such reference were individually incorporated by reference, to the extent that it is not at least partially inconsistent with the disclosure of this application (e.g., a partially inconsistent reference is incorporated by reference except for the partially inconsistent portion of the reference).

[0131]

[0131] The terms and expressions used in this specification are used as terms of description, not as terms of limitation, and in the use of such terms and expressions, there is no intention to exclude any equivalents of the features shown and described or portions of such equivalents, and it is recognized that various modifications are possible within the scope of the invention as claimed. Thus, although the present invention has been specifically disclosed by preferred embodiments, exemplary embodiments, and optional features, it should be understood that modifications and variations of the concepts disclosed herein may be made by those skilled in the art, and such modifications and variations are considered to be within the scope of the invention as defined by the appended claims. The specific embodiments provided herein are examples of useful embodiments of the invention, and it will be apparent to one skilled in the art that the invention can be implemented using numerous variations of the apparatus, apparatus components, and method steps set forth in this description. As will be apparent to one skilled in the art, the methods and apparatus useful in the methods of the present invention may include numerous optional compositions, processing elements, and steps.

[0132]

[0132] When a group of substituents is disclosed herein, it is to be understood that all individual members and all subgroups of that group are separately disclosed. When Markush-type groups or other groupings are used herein, all individual members of the group, all possible combinations and subcombinations of the groups are intended to be individually included in the disclosure.

[0133]

[0133] Unless otherwise stated, any formulation or combination of components described or exemplified herein can be used to practice the present invention.

[0134]

[0134] Whenever a range is given herein, for example, a temperature range, a ratio range, a time range, or a concentration range, all intermediate and subranges, as well as all individual values ​​within the given range, are intended to be included in the disclosure. It is understood that any subrange or individual value within a range or subrange included in the description herein may be excluded from the claims herein.

[0135]

[0135] All patents and publications referred to in this specification are indicative of the degree of skill of those skilled in the art to which the present invention pertains. References cited in this specification are incorporated herein by reference in their entirety to indicate the state of the art as of their publication or filing dates, and it is intended that this information may be used herein, if necessary, to exclude certain embodiments that exist in the prior art.

[0136]

[0136] As used herein, "comprising" is synonymous with "including," "containing," or "characterized by," and is inclusive or open-ended, and does not exclude additional, unrecited elements or method steps. As used herein, "consisting of" excludes any element, step, or ingredient not specified in the claim element. As used herein, "consisting essentially of" does not exclude materials or steps that do not materially affect the basic novel characteristics of the claim. In each instance herein, any of "comprising," "consisting essentially of," and "consisting of" may be substituted with either of the other two terms. The invention as illustratively described herein may be practiced in the absence of any element or elements, or limitation or limitations not specifically disclosed herein.

[0137]

[0137] All functional equivalents known in the art of all such materials and methods are intended to be included in the present invention. The terms and expressions used are used as terms of description, not as terms of limitation, and in using such terms and expressions, there is no intention to exclude any equivalents or portions of such equivalents of the features shown and described, but it is recognized that various modifications are possible within the scope of the invention as claimed. Thus, although the present invention has been specifically disclosed by preferred embodiments and optional features, it should be understood that modifications and variations of the concepts disclosed herein may be made by those skilled in the art, and that such modifications and variations are considered to be within the scope of the invention as defined by the appended claims.

Claims

1. 1. A method for distinguishing between a target sequence and a reference sequence in a biological sample by polymerase chain reaction (PCR), comprising: providing a labeled probe comprising a fluorophore and a quencher, wherein the labeled probe is complementary to and capable of specifically binding to the target sequence and non-specifically binding to the reference sequence; providing a promiscuity-blocked nucleotide chain oligonucleotide (PBNJ), wherein the PBNJ is capable of specifically binding to the reference sequence and non-specifically binding to the target sequence, the PBNJ comprising a reference binding region and an extension blocker that prevents extension by a polymerase; below: a sample comprising the reference sequence and / or the target sequence; the labeled probe; a competitive concentration of said PBNJ relative to the concentration of said labeled probe; PCR reagents; performing PCR on a solution comprising: The specific binding of the PBNJ to the reference sequence competes with the labeled probe binding to the reference sequence, thereby inhibiting the labeled probe bound to the reference sequence from amplifying; thereby distinguishing between said target sequence and said reference sequence. A method comprising:

2. the biological sample a virus, wherein the reference sequence is derived from a wild-type or parental virus and the target sequence comprises at least one mutation in the reference sequence; a mammalian cell having one or more nucleotide changes in said reference sequence, wherein said reference sequence reflects a low disease state and said target sequence reflects an increased risk of disease or the presence of a disease; circulating cell-free tumor DNA, wherein the reference sequence is a wild-type sequence from a somatic cell and the target sequence is derived from a tumor or cancerous cell and has one or more nucleotide changes in the reference sequence that reflect an increased risk of a pathological condition or the presence of a disease; circulating cell-free fetal DNA with one or more nucleotide changes in the reference sequence, wherein the reference sequence reflects a maternal DNA sequence and the target sequence reflects a fetal DNA sequence; a bacterium, wherein the reference sequence is derived from a wild-type bacterium or a species of bacterium, and the target sequence comprises at least one variation in the reference sequence; a fungus, wherein the reference sequence is derived from a wild-type fungus or a species of fungus, and the target sequence comprises at least one variation in the reference sequence; and A plant, wherein the reference sequence is derived from a wild-type plant or a species of plant, and the target sequence comprises at least one variation in the reference sequence:

2. The method of claim 1, wherein the compound is selected from the group consisting of:

3. 2. The method of claim 1, wherein said PBNJ eliminates detection of non-specific amplification of said reference sequence by 90% or more.

4. The reference sequence and the target sequence are 1 nucleotide substitution; a nucleotide insertion of one or more nucleotides; and / or Nucleotide deletion of one or more nucleotides The method of claim 1 , wherein the difference is

5. The method of claim 1 , wherein the reference sequence and the target sequence are DNA sequences or RNA sequences.

6. 2. The method of claim 1, wherein the PCR is selected from the group consisting of dPCR, qPCR, RT-dPCR, and RT-qPCR.

7. The method of claim 1 , wherein the labeled probe is a dual-labeled probe comprising a fluorescent molecule and at least one quencher molecule.

8. 2. The method of claim 1, wherein the labeled probe is a single base variation (SNV)-specific probe having a fluorophore covalently attached to the 5' end of the probe and a quencher at the 3' end of the probe or an internal quencher.

9. 10. The method of claim 1 for detecting DNA or RNA sequences containing SNVs, insertions or deletions.

10. 2. The method of claim 1, wherein the PCR is dPCR, the dPCR comprising compartment- or droplet-based PCR, and the PBNJ reduces or eliminates signals associated with low-efficiency non-specific off-target amplification, thereby increasing the signal-to-noise ratio for specific amplification of the target sequence.

11. adjusting the probe output amplitude by providing the PBNJ at a lower concentration; detecting multiple probe output amplitudes for multiplexed detection of multiple target sequences in a single or multi-channel fluorescence detector; The method of claim 1 further comprising:

12. 10. The method of claim 1, wherein the target sequence and the reference sequence differ by a single nucleotide mismatch that is a single nucleotide polymorphism (SNP) or part of a short nucleotide polymorphism.

13. The method of claim 1 , wherein the target sequence and the reference sequence differ by an insertion.

14. The method of claim 1 , wherein the target sequence and the reference sequence differ by a deletion.

15. 2. The method of claim 1, wherein the extension blocker is a 3' carbon-based spacer such as C3, C6, or C12 or a 3' quencher such as a black hole quencher.

16. 2. The method of claim 1, wherein the PBNJ comprises a locked nucleic acid (LNA) at the SNP position.

17. The PBNJ is having a length of between 10 and 50 nucleotides; having between 90% and 95% target sequence complementarity to at least a portion of said target sequence; having a reference sequence complementarity greater than the target sequence complementarity, the binding affinity of the PBNJ for the reference sequence is greater than the binding affinity of the PBNJ for the target sequence; the binding affinity of the PBNJ for the reference sequence is greater than the binding affinity of the labeled probe for the reference sequence; and / or The method of claim 1, wherein the binding affinity of the PBNJ to the target sequence is less than the binding affinity of the labeled probe to the target sequence.

18. 2. The method of claim 1, comprising a plurality of PBNJs that specifically bind to any possible SNP at a particular position in the target sequence.

19. 2. The method of claim 1, wherein the length of the target sequence is between 10 and 50 nucleotides, and the length of the probe and the PBNJ are each between 10 and 50 nucleotides.

20. 10. The method of claim 1, wherein the method is used in a biological sample to test for mutations associated with an increased risk of or the presence of cancer.

21. 10. The method of claim 1, wherein the method is used in a biological sample to test for variants of a pathogen, including pathogens that are viral, bacterial, or fungal.

22. 10. The method of claim 1, wherein the biological sample is derived from wastewater, an environmental sample, a body fluid, a tissue, a cell culture, or a tumor.

23. 2. The method of claim 1, wherein the labeled probe has a polynucleotide sequence that differs from the PBNJ sequence by one or more nucleotides.

24. 2. The method of claim 1, wherein the PBNJ is a PCR blocker during PCR amplification cycles to result in enrichment of a target sequence that is part of a variant sequence.

25. 2. The method of claim 1, wherein the ratio of PBNJ concentration to labeled probe concentration is equal to or greater than equimolar; or equal to or less than equimolar.

26. 1. A kit for distinguishing between a target sequence and a reference sequence in a biological sample by polymerase chain reaction (PCR), comprising: a forward primer and a reverse primer that serve to amplify both the reference strand and the target strand; a labeled probe comprising a fluorophore and a quencher, capable of specifically binding to the target sequence and non-specifically binding to the reference sequence; a promiscuity-blocked nucleotide-based oligonucleotide (PBNJ) capable of specifically binding to the reference sequence and non-specifically binding to the target sequence; optionally, a positive control of said reference sequence; Optionally, a positive control for said target sequence; Includes a kit.

27. The forward primer is provided at a concentration between 50 nM and 1100 nM; The reverse primer is provided at a concentration between 50 nM and 1100 nM; the labeled probe is provided at a concentration of between 20 and 800 nM; and / or the PBNJ is provided at a concentration between 0.25 and 16 times the concentration of the labeled probe; 27. The kit of claim 26.

28. 27. The kit of claim 26, further comprising reagents for dPCR, qPCR, RT-dPCR, or RT-qPCR.

29. 27. The kit of claim 26, wherein assay reagents are provided for a first reaction that does not include PBNJ and a second reaction that includes at least one PBNJ.

30. 27. The kit of claim 26, wherein PBNJ is provided relative to a wild-type reference sequence of a SARS-CoV-2 mutation selected from the group consisting of spike residues HV69-70, R408, K417, L452, T478, N501, N679, L704, Q954, and L981.

31. The reference sequence: the parent SARS-CoV-2, wherein the target sequence comprises a variant of SARS-CoV-2 selected from the group consisting of an alpha variant, a beta variant, a gamma variant, a delta variant, a delta plus variant, a mu variant, a lambda variant, an omicron variant, and an omicron subvariant; or 27. The kit of claim 26, wherein the target sequence is a proto-oncogene and the target sequence has a mutation that converts the proto-oncogene into an oncogene that indicates a higher risk of developing cancer or the presence of cancer.

32. 32. The kit of claim 31 , wherein the reference sequence is a proto-oncogene.

33. 33. The kit of claim 32, wherein the proto-oncogene is KRAS.

34. 34. The kit of claim 33, wherein the target is a KRAS mutation selected from G12C, G12A, G12D, G12R, or G13D.

35. 27. The kit of claim 26, wherein the PBNJ is provided at a concentration such that one or more non-specifically amplified populations are optically indistinguishable from negative compartment populations.

36. 27. The kit of claim 26, wherein the PBNJ is provided at a concentration such that the threshold cutoff value for detection of the target sequence is reduced compared to the threshold cutoff value of a method or kit that does not include the PBNJ.

37. 37. The method or kit of any one of claims 1 to 36, wherein the PBNJ has a length between 80% and 100% of the length of the labeled probe.

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