Synthetic polynucleotides and methods for selectively amplifying alleles - Patents.com

Abstract

In alternative embodiments, provided are primer-based nucleic acid amplification methods that can select for the amplification or recovery of specific alleles while not interfering with the amplification or recovery of any alternative alleles or mutations at specific nucleotide positions within the target sequence, or in other words, provided are methods that selectively suppress one allele while simultaneously amplifying any alternative alleles, or provided are methods that suppress wild-type sequences while simultaneously amplifying point mutations, including amplifying single base variants (SNVs), insertions and deletions. In alternative embodiments, a portion of the nucleic acid composition does not selectively suppress the amplification of specific nucleic acid target sequences, thereby providing an internal standard useful for determining the success of amplification and for determining the relative ratios of specific nucleic acid target sequences to those encoding alternative alleles or mutations.

Description

[Technical field]

[0001] This Patent Cooperation Treaty (PCT) international application claims the benefit of priority under 35 U.S.C. § 119(e) of U.S. Provisional Patent Application No. (USSN) 63 / 144,723, filed February 2, 2021. The foregoing application is expressly incorporated herein by reference in its entirety and for all purposes. All publications, patents, and patent applications cited herein are expressly incorporated herein by reference for all purposes.

[0002] The present invention generally relates to molecular biology and high-throughput sequencing, such as Next Generation Sequencing (NGS) of nucleic acids from genomes. In alternative embodiments, what is provided is a primer-based nucleic acid amplification method that can select for the amplification or recovery of a specific allele while not interfering with the amplification or recovery of any alternative alleles or mutations at a specific nucleotide position within the target sequence, or in other words, what is provided is a method that selectively suppresses one allele while simultaneously amplifying any alternative alleles, or what is provided is a method that suppresses wild-type sequences while simultaneously amplifying point mutations, including single base variants (SNVs), insertions and deletions. In alternative embodiments, cDNA fusions can also be detected. In alternative embodiments, a portion of the nucleic acid composition does not selectively suppress the amplification of a specific nucleic acid target sequence, thereby providing an internal standard useful for determining the success of the amplification and for determining the relative ratio of a specific nucleic acid target sequence to one encoding an alternative allele or mutation. In alternative embodiments, provided are methods of diagnosing a disease or condition comprising the use of synthetic DNA polynucleotides (referred to as selector polynucleotides) and / or the methods provided herein. In alternative embodiments, provided are methods of treating, ameliorating, or preventing a disease or condition comprising the use of synthetic DNA polynucleotides (referred to as selector polynucleotides) and / or the methods provided herein, including the diagnostic methods provided herein. [Background technology]

[0003] With the advent of liquid biopsy, detection of rare alleles has become increasingly important. Circulating cell-free DNA (ccfDNA) is present in every person's blood and is mostly derived from healthy normal cells. However, in pregnant women, small amounts of fetal DNA are also present and can be used to look for genetic diseases in a procedure known as non-invasive prenatal testing (NIPT); similarly, in solid organ transplantation (SOT), minute amounts of donor-derived extracellular DNA (dd-cfDNA) can be used to detect acute rejection of various organs, and in cancer, minute levels of circulating tumor DNA (ctDNA) are used to detect early recurrence of disease. By using error correction methods (e.g., molecular beacons) that improve sensitivity, specificity, and quantification, high-throughput next-generation sequencing (NGS) is now the detection device of choice for all liquid biopsy applications. However, the vast majority of sequence reads are uninformative: material reads from NIPT, recipient reads from dd-cfDNA, and wild-type reads from ccfDNA. A large number of uninformative reads reduces throughput, increases costs, and prevents detection.

[0004] Allele-specific PCR (AS-PCR) can be used to selectively amplify the desired target (specific allele or somatic mutation), but proofreading polymerases cannot be used, mispriming can lead to false positives, and different primers are required for each of the desired alleles. Blockers can also be used for undesired alleles, but blockers increase the "footprint" of the PCR assay, which involves short pieces of DNA (median size approximately 168 bp, even shorter if derived from fetal or tumor DNA) of ccfDNA, lowering the detection rate. It is also desirable to dramatically reduce the number of uninformative reads, but a set number of such reads are useful as internal standards for comparison and to ensure that the entire reaction worked well. Neither AS-PCR nor blockers can do this reliably. Summary of the Invention [Means for solving the problem]

[0005] Summary of the Invention In an alternative embodiment, provided are primer-based nucleic acid compositions useful in methods for selectively suppressing amplification of specific nucleic acid target sequences while selectively amplifying alternative alleles or mutations present in nucleic acid (or polynucleotide, or deoxyribonucleic acid (DNA), including cDNA) sequences that differ from the suppression target by as little as a single nucleotide, allowing for analysis and measurement of the alternative alleles or mutations. In an alternative embodiment, a portion of the nucleic acid composition does not selectively suppress amplification of specific nucleic acid target sequences, thereby providing an internal standard useful for determining the success of amplification and for determining the relative ratios of specific nucleic acid target sequences to those encoding alternative alleles or mutations.

[0006] In an alternative embodiment, provided is a method for selectively inhibiting isolation of a nucleic acid target by selectively dissociating a binding moiety from the nucleic acid target, such that the recovery or retention of alternative alleles or mutations of the nucleic acid target that selectively retain the binding moiety is not affected. In an alternative embodiment, a portion of the nucleic acid composition used is not selectively dissociated from the binding moiety, thereby providing an internal standard useful for determining successful recovery and for determining the relative ratio of a particular nucleic acid target sequence to those encoding alternative alleles or mutations.

[0007] In alternative embodiments, allele-specific or mutation-specific or methylation-specific amplification or capture is used to determine the molecular haplotype.

[0008] In alternative embodiments, the difference is detected by sequencing, hybridization, mass spectrometry, or any other technique capable of detecting sequence variations, including nucleotide resolution.

[0009] In an alternative embodiment, what is provided is a synthetic DNA polynucleotide (termed a selector polynucleotide) that includes at least a single residue (termed a selector nucleotide) that is located at the first (last), second (penultimate), third (third last), fourth (fourth last), fifth (fifth last) or sixth (one but one to the fifth last) position relative to the 3' end, or at a position even more distal from the 3' end, at least a single residue selector nucleotide is structurally or chemically different from any other residue within a region or section of the selector polynucleotide that is required to bind the selector polynucleotide to a target nucleic acid; The synthetic DNA polynucleotide (called the selector polynucleotide) has a 3' end that can be extended by a DNA polymerase, or is processed to have a 3' end that can be extended by a DNA polymerase, or A synthetic DNA polynucleotide is not blocked at the 3' end for its ability to be enzymatically extended, optionally by a DNA polymerase enzyme.

[0010] In an alternative embodiment of the synthetic DNA polynucleotides (i.e., selector polynucleotides) provided herein, a single selector nucleotide residue is positioned at the second position from the 3' end or at the penultimate position of the synthetic DNA polynucleotide (i.e., the selector polynucleotide); a single selector nucleotide residue is positioned at the third position from the 3' end or at the penultimate position of the synthetic DNA polynucleotide (i.e., the selector polynucleotide); the selector nucleotide comprises or consists of at least one ribonucleotide or at least one synthetic or non-natural nucleotide; and / or The selector nucleotide is a ribonucleotide.

[0011] In an alternative embodiment, provided is a nucleic acid amplification method for distinguishing a first nucleic acid sequence from a second nucleic acid sequence, wherein the first and second nucleic acids are in the same amplification reaction mixture; (a) providing or having provided a synthetic DNA polynucleotide (i.e., a selector polynucleotide) as provided or described herein, wherein the synthetic DNA polynucleotide (i.e., the selector polynucleotide) contains a selector nucleotide residue (optionally, a single ribonucleotide residue); (b) providing or having provided a DNA polynucleotide or a plurality of DNA polynucleotides, optionally wherein the DNA polynucleotide or a plurality of DNA polynucleotides comprises or is derived from a genome (optionally a cellular, microbial or viral genome), a cDNA library or a genomic library, optionally wherein the genome, cDNA library or genomic library is derived from a eukaryote or prokaryote, a plant or an animal (optionally a human), a microorganism (optionally a bacterium, algae, protist, Archea or fungus), or a virus or bacteriophage; (c) contacting, annealing or hybridizing a synthetic DNA polynucleotide (i.e., a selector polynucleotide) with a DNA polynucleotide or plurality of DNA polynucleotides, wherein the DNA polynucleotide or plurality of DNA polynucleotides acts as a template (template DNA polynucleotide) for the synthetic DNA polynucleotide (i.e., the selector polynucleotide) under conditions in which the synthetic DNA polynucleotide (i.e., the selector polynucleotide) anneals or specifically hybridizes to a complementary or substantially complementary sequence in the DNA polynucleotide (e.g., a sequence is substantially complementary if it has about 95%-99% sequence identity), thereby generating a nucleic acid duplex; wherein the synthetic DNA polynucleotide (i.e., the selector polynucleotide) either pairs with the template DNA polynucleotide at a selector nucleotide residue (optionally a single ribonucleotide residue) (see, e.g., FIG. 2A, where "R" refers to the position of a single ribonucleotide residue that pairs with the template DNA polynucleotide) or does not pair with the template DNA polynucleotide at a selector nucleotide residue (optionally a single ribonucleotide residue) (see, e.g., FIG. 2B, where "R" refers to the position of a single ribonucleotide residue that does not pair with the template DNA polynucleotide (denoted by "X"); (d) contacting the duplex with a DNA polymerase enzyme having 5' to 3' extension activity and with 3' to 5' exonuclease activity, and / or a DNA polymerase enzyme having 5' to 3' extension activity and an enzyme having 3' to 5' exonuclease activity, under conditions in which the polymerase enzyme having 5' to 3' extension activity and the enzyme having 3' to 5' exonuclease activity are enzymatically active, wherein (i) a selector nucleotide residue (optionally, a single ribonucleotide residue) is mismatched between the synthetic DNA polynucleotide (i.e., the selector polynucleotide) and the DNA polynucleotide or DNA polynucleotides, and a 3' to 5' exonuclease activity results in enzymatically removing from the 3' end a portion of the synthetic DNA polynucleotide (i.e., the selector polynucleotide) that includes the selector nucleotide residue (optionally, a single ribonucleotide residue) and all nucleotides 3' of the selector nucleotide, before the DNA polymerase extends from the remainder of the synthetic DNA polynucleotide (i.e., the selector polynucleotide) to a newly extended DNA polynucleotide that does not retain the selector nucleotide residue (optionally, a single ribonucleotide residue); or (ii) the DNA polymerase extends a synthetic DNA polynucleotide (i.e., a selector polynucleotide) that is base-paired to the DNA polynucleotide or DNA polynucleotides at a selector nucleotide residue (optionally, a single ribonucleotide residue) without removing the selector nucleotide residue (optionally, a single ribonucleotide residue), thereby retaining or incorporating the selector nucleotide residue (optionally, a single ribonucleotide residue) of the synthetic DNA polynucleotide (i.e., the selector polynucleotide) into the newly extended DNA polynucleotide; (e) contacting the newly created nucleic acid duplex with a ribonuclease enzyme, where optionally the ribonuclease enzyme is thermostable, and the thermostable ribonuclease is a thermostable ribonuclease H2 enzyme, under conditions in which the thermostable ribonuclease enzyme is active, where (i) when a selector nucleotide residue (optionally a single ribonucleotide residue) is removed by 3' to 5' exonuclease activity present in the reaction, the portion of the synthetic DNA polynucleotide (i.e. the selector polynucleotide) that was 5' to the single selector nucleotide residue (optionally a single ribonucleotide residue) is retained in the extended synthetic polynucleotide; or (ii) if a selector nucleotide residue (optionally a single ribonucleotide residue) is retained within the extended synthetic DNA polynucleotide (i.e., the selector polynucleotide) and matches a deoxyribonucleotide residue, the thermostable ribonuclease enzyme cleaves at the selector nucleotide residue (optionally a single ribonucleotide residue), thereby releasing the portion of the extended synthetic DNA polynucleotide (i.e., the selector polynucleotide) that was 5' to the selector nucleotide residue (optionally a single ribonucleotide residue); wherein an extended synthetic DNA polynucleotide (or extended selector polynucleotide) that retains a portion of the synthetic DNA polynucleotide (i.e., selector polynucleotide) that was 5' to a selector nucleotide residue (optionally, a single ribonucleotide residue) can be exponentially amplified, and an extended DNA polynucleotide (extended selector polynucleotide) that had a portion of the synthetic DNA polynucleotide (i.e., selector polynucleotide) that was 5' to a selector nucleotide residue (optionally, a single ribonucleotide residue) that was displaced from the extended synthetic DNA polynucleotide (i.e., selector polynucleotide) cannot be exponentially amplified, thereby selectively suppressing their amplification and distinguishing a first nucleic acid sequence from a second nucleic acid sequence. This is the method.

[0012] In an alternative embodiment, provided is a nucleic acid amplification method for distinguishing a first nucleic acid sequence from a second nucleic acid sequence, the first nucleic acid and the second nucleic acid being in the same amplification reaction mixture; (a) providing or having provided a synthetic DNA polynucleotide (i.e., a selector polynucleotide) as provided or described herein, wherein the synthetic DNA polynucleotide (i.e., the selector polynucleotide) contains a selector nucleotide residue (optionally a single selector ribonucleotide residue); (b) providing or having provided a DNA polynucleotide or a plurality of DNA polynucleotides, optionally wherein the DNA polynucleotide or a plurality of DNA polynucleotides comprises or is derived from a genome (optionally a cellular, microbial or viral genome), a cDNA library or a genomic library, optionally wherein the genome, cDNA library or genomic library is derived from a eukaryote or prokaryote, a plant or an animal (optionally a human), a microorganism (optionally a bacterium, algae, protist, archaea or fungus), or a virus or bacteriophage; (c) contacting, annealing or hybridizing a synthetic DNA polynucleotide (i.e., a selector polynucleotide) with a DNA polynucleotide or plurality of DNA polynucleotides, wherein the DNA polynucleotide or plurality of DNA polynucleotides acts as a template (template DNA polynucleotide) for the synthetic DNA polynucleotide (i.e., the selector polynucleotide) under conditions in which the synthetic DNA polynucleotide (i.e., the selector polynucleotide) anneals or specifically hybridizes to a complementary or substantially complementary sequence in the DNA polynucleotide, thereby generating a nucleic acid duplex; wherein the synthetic DNA polynucleotide (i.e., the selector polynucleotide) either pairs with the template DNA polynucleotide at a selector nucleotide residue (optionally a single ribonucleotide residue) (see, e.g., FIG. 10A, where "R" refers to the position of a single ribonucleotide residue that pairs with the template DNA polynucleotide) or does not pair with the template DNA polynucleotide at a selector nucleotide residue (optionally a single ribonucleotide residue) (see, e.g., FIG. 10B, where "R" refers to the position of a single ribonucleotide residue that does not pair with the template DNA polynucleotide (indicated by an "X"); (d) contacting the nucleic acid duplex with a DNA polymerase enzyme having 5' to 3' extension activity and with 3' to 5' exonuclease activity and / or a DNA polymerase enzyme having 5' to 3' extension activity and an enzyme having 3' to 5' exonuclease activity under conditions in which the DNA polymerase enzyme having 5' to 3' extension activity and the enzyme having 3' to 5' exonuclease activity are enzymatically active, wherein (i) a selector nucleotide residue (optionally a single ribonucleotide residue) is mismatched between the synthetic DNA polynucleotide (i.e., the selector polynucleotide) and the DNA polynucleotide or DNA polynucleotides, and 3' to 5' exonuclease activity results in enzymatically removing from the 3' end a portion of the synthetic DNA polynucleotide (i.e., the selector polynucleotide) that includes the selector nucleotide residue (optionally a single ribonucleotide residue) and all nucleotides 3' of the selector nucleotide before the DNA polymerase extends the remainder of the synthetic DNA polynucleotide (i.e., the selector polynucleotide) to a newly extended DNA polynucleotide that does not retain the selector nucleotide residue (optionally a single ribonucleotide residue); or (ii) the DNA polymerase extends a synthetic DNA polynucleotide (i.e., a selector polynucleotide) that is base-paired to the DNA polynucleotide or multiple DNA polynucleotides at a selector nucleotide residue (optionally, a single ribonucleotide residue) without removing the selector nucleotide residue (optionally, a single ribonucleotide residue), thereby retaining or incorporating the selector nucleotide residue (optionally, a single ribonucleotide residue) of the synthetic DNA polynucleotide (i.e., the selector polynucleotide) into the newly extended DNA polynucleotide; and (e) after amplification, treating the amplicon (or the newly extended synthetic DNA polynucleotide, i.e., the selector polynucleotide) with a reagent or enzyme that cleaves 5' or 3' to the selector nucleotide residue (optionally a single ribonucleotide residue), or, if present, within 3 nucleotides of the selector nucleotide residue (optionally a single ribonucleotide residue); wherein, optionally, a reagent or enzyme displaces the binding moiety or a substantial amount of incorporated synthetic DNA polynucleotide (i.e., selector nucleotide) or primer from the amplicon (or newly extended DNA polynucleotide) that retains the selector nucleotide residue (optionally, a single ribonucleotide residue), thereby allowing the amplicon or newly extended DNA polynucleotide that does not have the selector nucleotide residue (optionally, a single ribonucleotide residue) and thus retains the binding moiety or a substantial amount of incorporated synthetic DNA polynucleotide (i.e., selector nucleotide) or primer to be preferentially captured (or physically isolated) or subsequently preferentially amplified. This is the method.

[0013] In an alternative embodiment, the methods provided herein further comprise: the reagent used to cleave the 5' end of the single selector ribonucleotide is RNase H2 or the reagent used to cleave the 3' end of the single selector ribonucleotide is sodium hydroxide in the presence of heat; the method further comprises denaturing the nucleic acid duplex to produce single stranded DNA, the single stranded DNA being treated with a ribonuclease that cleaves 3' of the single selector ribonucleotide; The polymerase and 3' to 5' exonuclease activities are provided by different enzymes; the synthetic DNA polynucleotide (i.e., the selector polynucleotide) is or comprises a primer used in a nucleic acid amplification method, optionally the amplification method comprises polymerase chain reaction (PCR); during amplification, the extended synthetic DNA polynucleotide (i.e., selector polynucleotide) or primer is treated with an enzyme that cleaves 5' or 3' to the selector nucleotide residue (optionally, a single ribonucleotide residue) or, if present, within 3 nucleotides of the selector nucleotide residue (optionally, a single ribonucleotide residue), thereby releasing the portion of the synthetic DNA polynucleotide (i.e., selector polynucleotide) or primer from the amplicon (or newly extended DNA polynucleotide) that retains the selector nucleotide residue (optionally, a single ribonucleotide residue), preventing the synthetic DNA polynucleotide (i.e., selector polynucleotide) or primer from being completely copied by extension of the return primer in the amplification reaction, and allowing amplicons that do not have the selector nucleotide residue (optionally, a single ribonucleotide residue) to be preferentially amplified since they retain sufficient synthetic DNA polynucleotide (i.e., selector polynucleotide) or primer sequence to support exponential amplification; the synthetic DNA polynucleotide (i.e., the selector polynucleotide) or primer comprises a ribonucleotide, and the enzyme that cleaves 5' to the selector nucleotide is ribonuclease H2, optionally, the ribonuclease H2 is thermostable, and optionally, the thermostable ribonuclease H2 is Pyrococcus abysii RNase H2; the method further comprises a second synthetic DNA polynucleotide or primer identical to the first synthetic DNA polynucleotide (i.e., the selector polynucleotide) except that the selector nucleotide residue (optionally, a single ribonucleotide residue) has been replaced by a corresponding normal deoxyribonucleotide to create a DNA amplification primer, and a certain amount of this DNA amplification primer is mixed with the first synthetic DNA polynucleotide (i.e., the selector polynucleotide) or primer containing the selector nucleotide residue (optionally, a single ribonucleotide residue) to allow production of a certain amount of an amplicon that would otherwise contain the selector nucleotide residue (optionally, a single ribonucleotide residue) but now lacks the selector nucleotide residue (optionally, a single ribonucleotide residue) and is thereby now resistant to cleavage by a reagent or enzyme specific for the selector nucleotide residue (optionally, a single ribonucleotide residue), the amplicon produced by the second synthetic DNA polynucleotide is used as an internal reaction standard to demonstrate that the amplification worked well and as an internal reference against which the amount of amplicon or newly extended DNA polynucleotide produced by the first synthetic DNA polynucleotide (i.e. the selector polynucleotide) can be compared; The sequence of the amplicon or newly extended DNA polynucleotide is optionally determined by DNA sequencing, using methods including the use of Sanger sequencing, next generation sequencing (NGS), single molecule real-time (SMRT) sequencing, nanopore DNA sequencing, reversible terminated chemistry (e.g., SOLEXA technology (Illumina)), combinatorial probe anchor synthesis (cPAS), mass spectrometry sequencing, or massively parallel signature sequencing (MPSS); the identity of the nucleotide corresponding to the position of the original selector nucleotide residue (optionally a single ribonucleotide residue) is determined by primer extension across the target site; the identity and relative amount of the nucleotides at the site of interest are determined by or through the use of labels or masses, optionally the identity and relative amount of the nucleotides at the site of interest are determined by a method comprising single base extension of a primer across the site of interest in an amplicon created using a synthetic DNA polynucleotide (i.e., a selector polynucleotide); and / or The production of the amplicon or newly extended DNA polynucleotide of interest is determined by quantitative PCR (qPCR), digital PCR, genotyping methods or their equivalents, or a combination thereof.

[0014] In alternative embodiments, what is provided is a kit or article of manufacture that includes materials for practicing the methods provided or described herein, optionally enzymes and / or synthetic DNA polynucleotides, optionally selector polynucleotides (optionally one or more synthetic DNA polynucleotides (i.e., selector polynucleotides) provided herein), and optionally further includes instructions for practicing the methods provided or described herein.

[0015] In an alternative embodiment, what is provided is a kit that contains materials for practicing the methods provided herein, and optionally also contains instructions for practicing the methods provided herein.

[0016] In an alternative embodiment, provided is a method of diagnosing a disease or condition comprising determining whether an individual in need thereof has the disease or condition by determining the presence or absence of an allele or genomic sequence associated with or having a diagnosis of the disease or condition, The presence or absence of an allele or genomic sequence associated with a disease or condition, or having a diagnosis of a disease or condition, is determined using the methods provided herein. The disease can be cancer.

[0017] In an alternative embodiment, provided is a method of treating, ameliorating, or preventing a disease or condition comprising treating an individual in need thereof with a drug, drug combination, or treatment regimen indicated for the disease or condition, wherein the individual in need thereof is diagnosed as having or predisposed to having the disease or condition using the diagnostic methods provided herein. The disease can be cancer, or the condition is a genetic disease or condition.

[0018] In an alternative embodiment, provided is the use of a synthetic DNA polynucleotide provided herein to diagnose a disease or condition, wherein the disease or condition is diagnosed by the presence of an allele or genomic sequence associated with or having a diagnosis of the disease or condition, and the presence or absence of an allele or genomic sequence associated with or having a diagnosis of the disease or condition is determined using the methods provided herein.

[0019] In an alternative embodiment, the synthetic DNA polynucleotides provided herein are for use in the diagnosis of a disease or condition, wherein the disease or condition is diagnosed by the presence of an allele or genomic sequence associated with or having a diagnosis of the disease or condition, and the presence or absence of the allele or genomic sequence associated with or having a diagnosis of the disease or condition is determined using the methods provided herein.

[0020] In alternative embodiments, provided are methods and synthetic DNA polynucleotides for detecting the presence or absence of rare alleles in a biological specimen, comprising use of the methods provided herein, optionally the biological specimen comprising or derived from a biopsy, or a tissue or blood sample, or a liquid sample, of an individual in need thereof.

[0021] In alternative embodiments, the detection of the presence or absence of rare alleles in a biological specimen is for non-invasive prenatal testing (NIPT), or to assess histocompatibility or detect donor-derived nucleic acids after organ transplantation (optionally solid organ or bone marrow transplantation), or to assess early detection of antimicrobial resistance (AMR) or bacterial resistance in an individual in need thereof, or to assess the presence of minimal residual disease (MRE), optionally after bone marrow ablation.

[0022] The details of one or more exemplary embodiments of the invention are set forth in the accompanying drawings and set forth below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims.

[0023] All publications, patents, and patent applications cited herein are hereby expressly incorporated by reference in their entirety for all purposes.

[0024] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0025] The drawings described herein are for the purpose of illustrating exemplary embodiments provided herein and are not intended to limit the scope of the invention as encompassed by the claims. [Brief description of the drawings]

[0026] [Figure 1] Illustrated are possible placements of single residue ribonucleotide selector nucleotides within a selector primer, where the ribonucleotide selector nucleotide is designated with an "r" that is placed in front of the nucleotide letter (in other words, the "r" is not itself a nucleotide residue, but rather is included only to clearly specify that the "G" nucleotide underlined with the "r" is a ribonucleotide). The selector nucleotide may be placed at the first (last) (SEQ ID NO:1), second (penultimate) (SEQ ID NO:2), third (third-last) (SEQ ID NO:3), fourth (fourth-last) (SEQ ID NO:4), fifth (fifth-last) (SEQ ID NO:5) or sixth (next to penultimate) (SEQ ID NO:6) position relative to the 3' end. The ribonucleotide selector nucleotide is designated with an "r" that is placed in front of the nucleotide letter and is underlined. Not shown are positions even more distal from the 3' end at which the selector nucleotide may be placed. [ka] [Figure 2A]Schematically illustrates an exemplary method provided herein that suppresses one allele at the same nucleotide position while simultaneously allowing amplification of alternative alleles or mutations, e.g., only the mutant allele is amplified. For the allele to be suppressed (in this example, the wild-type (WT) nucleotide), a primer, in which "R" points to the position of a single ribonucleotide residue that pairs with the template DNA polynucleotide, is extended by a proofreading polymerase that has an exact sequence match with the WT nucleotide of interest and has 3' to 5' exonuclease activity. As the primer is extended, a thermostable ribonuclease H2 (e.g., Pyrococcus abyssi RNase H2) cleaves at the 5' side of the ribonucleotide at the position marked with "R" (underlined), and most of the primer sequence (5' end) is removed or released. The return primer cannot copy most of the first primer because the sequence has been removed or released by the thermostable ribonuclease H2. This stops the allele to be suppressed (in this example the WT allele) from being exponentially amplified, greatly reducing the number of WT allele sequences produced in the amplification reaction. [Figure 2B]Schematically illustrates an exemplary method provided herein that suppresses one allele at the same nucleotide position while simultaneously allowing amplification of alternative alleles or mutations, e.g., only mutant alleles are amplified. In contrast, if a single ribonucleotide in the selector primer, where "R" refers to the position of the single ribonucleotide residue that pairs with the template DNA polynucleotide, is mismatched with the template (in this example, the WT allele), the 3' to 5' exonuclease activity of the proofreading polymerase consumes the annealed primer through the mismatch, the single residue ribonucleotide is eliminated, and the correct deoxyribonucleotide (i.e., the one that matches the mutant) is used when the shortened primer is extended across the mutant site. Note that any alternative allele or any mutant at that site can be mismatched with the wild-type ribonucleotide, and therefore, for all alternative alleles or mutants, when the shortened primer is extended across the mutant site, the single residue mismatch ribonucleotide is removed from the primer bound to the WT allele and replaced with the exact match of the specific mutation. Loss of a single residue ribonucleotide in the selector primer renders the incorporated (annealed) primer sequence resistant to removal by ribonucleases. The return primer can then copy over the incorporated (i.e., partially consumed and then extended) first primer, and any alternative alleles or mutant sequences will be exponentially amplified, greatly increasing the number of alternative alleles or mutant sequences produced in the reaction that should be present. There are no more mutant sequences introduced by the selector primer. Any mutant sequences detected by subsequent analysis pass two quality checks.In the first, the variant is detected by a hybridization mismatch resulting in the removal of a single residue ribonucleotide; loss of the ribonucleotide indicates that an alternative allele or variant was present; in the second, a high fidelity polymerase copies the alternative allele or variant site with high fidelity (where, optionally, high fidelity means that the polymerase copies across the alternative allele or variant site with higher fidelity than TAQ polymerase), and sequencing (or other methods, such as primer extension or hybridization) reveals the identity of the alternative allele or variant. [Diagram 3] Selector primers with identical 3' ends and 5' ends of different lengths are illustrated. The selector single residue ribonucleotide is underlined and preceded by an "r" and is positioned to match the wild type nucleotide at nucleotide 38 of the coding sequence in the human gene Kirsten rat sarcoma (KRAS). The predicted TM difference between the wild type and mutant sequences (38G>A is used here) under standard PCR conditions is noted. As the length of the selector primer increases, the TM difference decreases. Reduction in TM difference can also be achieved using locked nucleic acid (LNA) (also known as bridged nucleic acid (BNA) or sequestered RNA (not consumed by RNase H2) or modified RNA nucleotides in which the ribose moiety has been modified with an extra bridge connecting the 2' oxygen and the 4' carbon) and / or other modified nucleotides, such as modified nucleotides that increase TM in the sequence 5' of the selector nucleotide, for example using 2,6-diaminopurine which can base pair with dT (the increase can be as little as 1-2°C per residue) or 5-methyldeoxycytidine which base pairs with dG and increases TM by as much as 0.5°C per residue. [ka] [Figure 4]Illustrated are Sanger sequencing traces of amplicons produced by PCR runs with each of the primers depicted in FIG. 3 and the common return primer. In both the top and bottom rows, arrows indicate the position of various nucleotides in the target sequence (templates used are DNA extracted from cell lines heterozygous for wild type and KRAS 38G>A mutant). The top Sanger sequencing trace shows the results of each reaction in which RNase H2 was not present during PCR. The longer and therefore more stable the primer, the better the mutant signal (green trace, also highlighted by the arrows) is seen, but even with a 60-mer primer, the green mutant "A" signal is significantly smaller than the black wild type "G" signal. Further stabilization, for example by LNA or other nucleotide modifications increasing the TM, improves the results with the mutant allele. In the absence of RNase H2, no primer portion (no ribonucleotide or nucleotide residue) anneals to the wild type sequence and is removed from the extended primer, resulting in wild type exponential amplification. When thermostable RNase H2 is included in otherwise identical reactions (bottom), the mutant signal becomes progressively more pronounced compared to the wild-type signal as a function of primer stability to the mutant template. In the wild-type case, the presence of RNase H2 results in the detachment or removal of most of the primer sequence (5' end) from the extended primer, impairing exponential amplification. In the mutant case, a single residue ribonucleotide is removed from the primer annealed to the mutant sequence by the 3' to 5' exonuclease activity of the proofreading polymerase prior to extension, thus rendering the primer extended across the mutant site resistant to RNase H2 and amplified exponentially. [Diagram 5]Illustrates the results of Sanger sequencing using the 51-mer selector primer and common return primer depicted in FIG. 3 in titration of mutant (allele A) DNA template with wild-type (allele G) DNA template. The calculated number of haploid genome equivalents for both wild-type (WT) and mutant (MUT) KRAS alleles is shown above each Sanger sequencing trace. Even with a calculated 2 copies of mutant DNA against a calculated background of 2,998 wild-type DNA, a clear mutant (A) signal is seen (bottom, far right). At very low amounts of mutant DNA (bottom), a very faint "G" signal can be seen, and what is seen is not the encoded template, but a "T" signal added by the sequencing polymerase (e.g., SEQUENASE™ (ThermoFisher Scientific)) to the end of the template. The added "T" signal being comparable in magnitude to the "G" signal indicates that the primer site has been cleaved from all or virtually (or substantially) all of the WT "G" containing amplicon. Subsequent PCR that adds, for example, a barcode (e.g., an ILLUMINA sample barcode) will not amplify these WT sequences. Samples that go to ILLUMINA® NGS are not cross-amplified and are not sequenced. Thus, any amount of mutant DNA can be detected without or virtually (or substantially) having any wild-type background. [Figure 6A-B]Figures 6A-B display the results when wild type KRAS is amplified using a selector primer in the absence of RNase H2. After amplification, in Figure 6A, RNase H2 is added to cleave 5' of the ribonucleotide G (rG) and remove the primer sequence upstream of the WT "G", or in Figure 6B, NaOH and heat are used to cleave 3' of the ribonucleotide G (rG) and remove the primer sequence upstream of the WT "C" that is 3' of the ribonucleotide G (rG). In both cases, a polymerase (SEQUENASE™) added "T" is seen, indicating the end of the processed DNA strand. Any binding moiety, e.g., biotin or ILLUMINA® capture sequence, is removed from the WT-containing amplicon. Recovery using streptavidin (for biotin) or an ILLUMINA® flow cell (for ILLUMINA® capture sequence) captures only the mutant amplicon (if present). [Figure 7]Depicts a 51-mer selector primer containing a G ribonucleotide (rG) in the third-to-last position (SEQ ID NO:11) followed by a counterpart with a normal deoxyribonucleotide in the third-to-last position (SEQ ID NO:13). A unique feature of the selector primer is that it can be fine-tuned to allow for a limited number of wild-type sequences. Primers containing G ribonucleotides (rG) can be selected for PCR in the presence of, for example, RNase H2, while primers lacking ribonucleotides (replaced with deoxyribonucleotides) are resistant to RNase H2 and can be amplified or recovered. The value of this is that it provides an internal standard to demonstrate that a particular assay is working well, and also allows for quantitative comparison of WT and MUT sequences between samples. An exemplified sequence is GCCTGCTGAAAATGACTGAATATAAACTTGTGGTAGTTGGAGCTGGTGrGCG (SEQ ID NO: 3), where the ribonucleotide selector nucleotide is designated an "r" which is placed before the nucleotide letter (bold and underlined residues are ribonucleotides). In Figure 7, below is the same sequence except that the nucleotide in the third to last position is a deoxyribonucleotide instead of a ribonucleotide (SEQ ID NO: 13). Shorter sequences can be used, as shown in Figures 3 and 4. [ka] [Figure 8]The utility of the methods provided herein in performing molecular haplotyping is illustrated. In this example, the genotype of an individual is heterozygous at each of the four sites of the polymorphism. By using appropriate selector primers, the amplification of the top DNA is suppressed while the amplification of the bottom DNA is not hindered. Genotyping by various means reveals one haplotype while the other haplotype can be derived from the overall genotype if known. If desired, the reverse can be done in a separate reaction, where the amplification of the bottom DNA is suppressed by appropriate selector primers while the amplification of the top DNA is not hindered. Genotyping of this amplicon confirms the haplotype composition. [Figure 9] Illustrating the results of Sanger sequencing using amplicons obtained with the selector polynucleotides listed in FIG. 1, results obtained in the absence of RNase H2 (top) often show two traces at position 38, a wild type "G" and a mutant "A", where the wild type "G" signal is roughly comparable to the mutant "A" signal when the selector nucleotide is in the last position in this example, and in all other cases the "A" signal is smaller in magnitude than the "G" signal. In the presence of thermostable RNase H2 (bottom), all selector polynucleotides are effective in suppressing or partially suppressing the amplification of the wild type "G" containing sequence at position 38 (marked with an arrow) while not interfering with the mutant "A" signal (in this case). [Figure 10A]10A illustrates an exemplary method provided herein, in which all possible alleles at the same nucleotide position are labeled with a binding moiety during amplification, but only one of the alleles, e.g., the wild-type allele, is dislodged from the binding moiety to prevent its purification. In the case of an allele that is not desired to be purified (in this example, the wild-type (WT) nucleotide), a primer in which "R" pairs with the template DNA polynucleotide, e.g., (see also FIG. 1 ) points to the position of a single ribonucleotide residue that exactly matches the WT nucleotide of interest, is extended by a proofreading polymerase with 3' to 5' exonuclease activity. The primer is labeled with a binding moiety, e.g., biotin, represented by "B" in a circle in FIG. 10A, and the binding moiety can also be a specific nucleotide sequence or any other moiety that allows capture and purification (as well as subsequent detection). After amplification, the amplicon is treated with an agent or enzyme that cleaves at the 5' or 3' side of the selector nucleotide (represented in this example by "R" and underlined), which displaces the capture moiety from the extended primer containing the wild-type allele and prevents subsequent capture and purification. If the selector nucleotide is a ribonucleotide, the enzyme that cleaves at the 5' side may be RNase H2 if the amplicon is still double-stranded, or ribonuclease I, which cleaves at the 3' side of the ribonucleotide if it is denatured to a single-stranded state. Sodium hydroxide in the presence of heat can also be used to cleave at the 3' side of the ribonucleotide. [Figure 10B]The exemplary method provided herein is illustrated in a schematic manner, where all possible alleles at the same nucleotide position are labeled with a binding moiety during amplification, but only one of the alleles, e.g., the wild-type allele, is dislodged from the binding moiety, preventing its purification. In contrast, if a single ribonucleotide in the selector primer mismatches with the template (in this example, the WT allele), the 3' to 5' exonuclease activity of the proofreading polymerase consumes the annealed primer via the mismatch, the single residue ribonucleotide is eliminated, and the correct deoxyribonucleotide (i.e., the one that matches the mutant) is used when the shortened primer is extended across the mutant site. Note that any alternative allele or any mutant at that site can be mismatched with the wild-type ribonucleotide, and therefore for all alternative alleles or mutants, the single residue mismatch ribonucleotide is removed from the primer bound to the WT allele and replaced with the correct (deoxyribonucleotide) match of the specific mutation when the shortened primer is extended across the mutant site. The loss of a single residue ribonucleotide in the selector primer renders the incorporated (annealed) primer sequence resistant to removal by ribonucleases or any other process that depends on the presence of the selector nucleotide. Because the capture moiety remains bound, the extended primer can be preferentially captured (or physically isolated) or subsequently preferentially amplified. There is no longer any mutant sequence introduced by the selector primer. Any mutant sequence detected by subsequent analysis passes two quality checks. First, the variant is detected by a hybridization mismatch resulting in the removal of a single residue ribonucleotide; the loss of the ribonucleotide indicates that an alternative allele or variant was present; second, a high fidelity polymerase copies the alternative allele or variant site with high fidelity, and its sequencing (or other methods, e.g., primer extension or hybridization) reveals the identity of the alternative allele or variant. [Figure 11]The results from the use of the exemplary methods provided herein are graphically depicted, demonstrating that an exemplary single selector primer (in this case the selector nucleotide is specific for KRAS c.35) can detect any of the three mutations occurring at that site, in exactly the same way that the master mix was applied to each of the three different templates, and in each case the KRAS c.35 selector primer detected the exact mutation in the presence or absence of a KRAS c.35G>A mutant (corresponding to a G12D amino acid change), a c.35G>T mutant (corresponding to a G12V amino acid change) or a c.35G>C mutant (corresponding to a G12A amino acid change), all three of which are frequently mutated in colon cancer and found in pancreatic and lung cancers. [Figure 12] A graphical representation of results from use of the exemplary methods provided herein demonstrates that the exemplary selector primer (in this case the selector nucleotide is specific for EGFR c.2573T, a T>G mutation resulting in the EGFR p.L858R mutation) can detect the mutant G allele at a frequency as low as 0.1%, with the unshaded columns outlined in red equaling 5-fold the wild-type signal, demonstrating that even at a mutant allele frequency of 0.1%, the mutant signal is well over 5-fold the wild-type value. [Figure 13] Results from use of the exemplary methods provided herein are displayed, demonstrating that the exemplary selector primer (in this case the selector nucleotide is specific for EGFR c.2236G, a c.2236_2250 deletion mutation resulting in the EGFR p.E746_A750del mutation) can detect mutant deletions at frequencies as low as 0.1%, with unshaded columns outlined in red equaling 5-fold the wild-type signal, demonstrating that even at a mutant allele frequency of 0.1%, the mutant signal is well over 5-fold the wild-type value. [Figure 14]Results from use of the exemplary methods provided herein are presented, demonstrating that an exemplary selector primer (in this case the selector nucleotide is specific for KRAS c.37, a G>T mutation resulting in a KRAS p.G13C mutation) can detect mutant T alleles at frequencies as low as 0.02%, equating to finding 2 mutant molecules out of 10,000 wild type molecules. [Figure 15] Results from the use of exemplary methods provided herein are presented, demonstrating the ability of an exemplary selector primer (in this case the selector nucleotide is specific for KRAS c.38, a G>A mutation resulting in the KRAS p.G13D mutation) to suppress unwanted signal to a "tunable" level; a primer identical to the selector primer except containing a normal deoxyribonucleotide instead of the selector nucleotide (in this case a ribonucleotide) is used in various mixtures with selector primer ranging from 0% selector primer (referred to herein as a PointSuppressor Primer or PSP), i.e., 100% selector primer; when no selector primer is used, amplification of the wild type G sequence predominates, and the mutant A allele is amplified to a greater extent as the percentage of selector primer increases. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0027] Like reference symbols in the various drawings indicate like elements.

[0028] In an alternative embodiment, provided is a nucleic acid amplification method that can select for a specific allele while not interfering with the amplification or recovery of any alternative alleles or mutations at a particular nucleotide position within a target sequence, e.g., a genomic target sequence. In an alternative embodiment, provided is a nucleic acid amplification method that suppresses wild-type sequences while simultaneously amplifying point mutations, including single nucleotide variants, insertions and deletions, and in the case of cDNA, also fusions.

[0029] In alternative embodiments, what is provided is a method that is "tunable," meaning that unwanted alleles can be suppressed (in the amplification or recovery process) by 10 or 100 fold, or any amount desired. This reduces the number of uninformative reads, which increases throughput, lowers the cost per sample, and enhances detection of desired targets since sequencing rate competition is reduced.

[0030] In an alternative embodiment, the methods provided herein have the ability to allow for some uninformative readouts, which is unique and allows for an internal standard to demonstrate that the assay is working well (versus a "no result" when an alternative allele or variant is not present in the sample) and can also serve as an internal standard for quantification.

[0031] In an alternative embodiment, the method provided herein can be performed with any primer-based approach, and by using the method provided herein, variant sequences are not introduced into the amplification reaction. As an example, when looking for somatic mutations, the wild type allele may be G, and the possible mutant alleles are A, C, and T, and by using the method provided herein, G-containing reads are reduced (e.g., 100-fold) without reducing mutant reads, and any of the possible mutants are amplified with the same single primer. The number of wild type reads is significantly reduced if present, increasing the chance that mutant reads are sequenced and detected, and if they are absent, a set number of wild type reads ensures that the reaction has worked well.

[0032] In an alternative embodiment, the methods provided herein may have unique advantages over current methods, including never introducing a primer containing the sequence of interest to detect, and the mutants may be: (1) mismatches between the selector primer and the mutant template are recognized and removed by the 3' to 5' exonuclease activity of the proofreading polymerase; and (2) It is detected only when the high-fidelity polymerase copies the mutant template. In contrast, allele-specific amplification introduces variant sequences that are interesting to detect, which can result in false positives.

[0033] In an alternative embodiment, the methods provided herein can have the advantage that a single primer detects any variant at a particular nucleotide position. In contrast, allele-specific PCR requires primers specific for each allelic variant or mutation, see, for example, van Mansfeld AD, Bos JL. PCR-based approaches for detection of mutated ras genes. PCR Methods Appl. 1992 May;1(4):211-6; Darawi,MN, Ai-Vyrn,C., Ramasamy,K,et al., Allele-specific polymerase chain reaction for the detection of Alzheimer's disease-related single nucleotide polymorphisms. BMC Med Genet 14, 27(2013); Lang AH, Drexel H, Geller-Rhomberg S,et al., Optimized allele-specific real-time PCR assays for the detection of common mutations in KRAS and BRAF. J Mol Diagn. 2011;13(1):23-28; Dobosy JR, Rose SD,et al., (2011) RNase H-dependent PCR (rhPCR): improved specificity and single nucleotide polymorphism detection using blocked cleavable primers. BMC Biotechnol, 11:80.

[0034] Nucleic acid amplification In an alternative embodiment, provided is a method that involves the use of DNA amplification techniques, such as the polymerase chain reaction (PCR) or other primer-based amplification methods.

[0035] Any known protocol or material (including medical kit or enzymes) used in the practice of DNA amplification techniques can be used to practice the methods provided herein, including, for example, the use of a proofreading enzyme with 5' to 3' extension activity and 3' to 5' exonuclease activity, and / or a DNA polymerase enzyme with 5' to 3' extension activity and an enzyme with 3' to 5' exonuclease activity. Standard materials and methods for polymerase chain reaction used in the practice of the methods provided herein can be found, for example, in Dieffenbach and Dveksler (1995) PCR Primer: A Laboratory Manual, Cold Spring Harbor Laboratory Press and McPherson et al., (2000) PCR-Basics: From Background to Bench, 1st Edition, Springer Verlag, Germany.

[0036] An exemplary PCR protocol is shown in the two tables below, the first showing the reaction set-up and the second showing the cycling conditions.

[0037] [Table 1]

[0038] [Table 2]

[0039] Various DNA concentrations can be used and are known to those skilled in the art. Similarly, primer concentrations can vary. Any source of Pyrococcus abyssi RNase H2 and / or other thermostable RNase H2 enzymes can be used. Thermostable proofreading DNA polymerases from other sources can be used, including but not limited to Q5® Hot Start High-Fidelity® DNA Polymerase from New England Biolabs and Platinum SuperFi II® DNA Polymerase-High-Fidelity PCR Enzyme from ThermoFisher.

[0040] The following are general exemplary cycling conditions, not intended to limit the implementation of the amplification: denaturation temperature in the range of 94°C-98°C for times ranging from 10 seconds to 30 seconds, annealing temperature in the range of 55°C-72°C for 5 seconds to 30 seconds, extension temperature in the range of 55°C-72°C or higher for 15 seconds to 30 seconds, per kilobase of DNA to be amplified. A general discussion of PCR methods that can be used in the practice of the methods provided herein can be found, for example, in Wikipedia (https: / / en.wikipedia.org / wiki / Polymerase_chain_reaction, or Valones MA, Guimaraes RL, Brandao LA, de Souza PR, de Albuquerque Tavares Carvalho A, Crovela S. Principles and applications of polymerase chain reaction in medical diagnostic fields: a review. Braz J Microbiol. 2009; 40(1): 1-11; Garibyan L, Avashia N. Polymerase chain reaction. J Invest Dermatol. 2013; 133(3): 1-4; Dieffenbach CW, Lowe TM, Dveksler GS. General concepts for PCR primer design. PCR Methods Appl. 1993 December; 3(3): S30-7). Exemplary conditions for use of RNase H2 enzyme are included on the IDT website (https: / / www.idtdna.com / pages / products / qpcr-and-pcr / master-mixes-reagents / rnase-h-enzyme) and / or in Dobosy JR, Rose SD, et al. (2011) RNase H-dependent PCR (rhPCR): improved specificity and single nucleotide polymorphism detection using blocked cleavable primers, BMC Biotechnol, 11:80.Exemplary enzymes that can be used in the practice of the methods provided herein include, for example, Phusion Hot Start II®, a commercially available thermostable DNA polymerase, which is one of many that are available and can be used in the practice of the methods provided herein; see, for example, Ishino S, Ishino Y, DNA polymerases as useful reagents for biotechnology - the history of developmental research in the field, Front Microbiol. 2014 Aug. 29;5:465. Exemplary conditions for Phusion Hot Start II® are available from the ThermoFisher website (https: / / www.thermofisher.com / order / catalog / product / F549L# / F549L).

[0041] In alternative embodiments, provided are methods that include the use of digital PCR (see, e.g., Morley AA. Digital PCR: A brief history. Biomol Detect Quantif. 2014 Aug. 15;1(1):1-2), or digital PCR or dPCR, including, for example, Droplet Digital PCR (ddPCR), for use in the methods provided herein, e.g., to generate an amplicon of interest or to generate new extended DNA polynucleotides, and the methods provided herein can be practiced using any method known in the art, e.g., the methods described in U.S. Pat. No. 9,797,007.

[0042] synthetic nucleic acid In an alternative embodiment, provided is a synthetic DNA polynucleotide that includes at least a single ribonucleotide residue (referred to as a selector nucleotide) that can be located at the first (last), second (second to last), third (third to last), fourth (fourth to last), fifth (fifth to last) or sixth (next to the fifth to last) position relative to the 3' end of the synthetic DNA polynucleotide (i.e., the selector nucleotide), and in an alternative embodiment, a second and a third additional ribonucleotide residue are included in the provided synthetic DNA polynucleotide.

[0043] In an alternative embodiment, the selector polynucleotide is used as a primer in an amplification method that can select for the amplification of a specific allele while not interfering with the amplification or recovery of any alternative alleles or mutations at a particular nucleotide position within the target sequence. The design, composition and manufacture of the selector polynucleotide is based on the allele to be suppressed. As an example, if one is interested in a single nucleotide mutation occurring at c.38 of the human oncogene KRAS, a chimeric DNA / RNA polynucleotide is created that perfectly matches the wild-type sequence and contains a single ribonucleotide at the position corresponding to the site of interest. With reference to FIG. 1, all sequences shown are identical to the wild-type sequence, and each sequence includes the nucleotide that is red and underlined (this is the selector nucleotide, the single ribonucleotide in the sequence). The selector nucleotide can be at any of the positions depicted in FIG. 1, although the last position is not preferred, and positions even more distal from the 3' end can sometimes act as selector nucleotides. With reference to FIG. 3, the selector polynucleotide can be of various lengths, including but not limited to those depicted. Figure 9 displays the results obtained using the selector polynucleotides listed in Figure 1. In the presence of thermostable RNase H2, all selector polynucleotides are effective at suppressing or partially suppressing amplification of the wild type "G" containing sequence at position 38 (marked with an arrow), while not interfering with the mutant "A" signal (in this case).

[0044] As described in the Examples, the design, composition and production of selector polynucleotides to suppress wild-type sequences can also be achieved without interfering with the detection of other alternatives, including but not limited to deletions, insertions and, in the case of cDNA, fusions. Because suppression is based on the sequence of the allele being suppressed, the design of alternatives can be achieved by one of skill in the art.

[0045] Nucleic acid sequencing In alternative embodiments, provided are methods in which the sequence of the amplicon or newly extended DNA polynucleotide is determined by DNA sequencing, optionally using methods including the use of Sanger sequencing, or next generation sequencing (NGS), single molecule real-time (SMRT) sequencing, nanopore DNA sequencing, reversible end chemistry (e.g., SOLEXA technology (Illumina)), combinatorial probe anchor synthesis (cPAS), mass spectrometry sequencing, or massively parallel signature sequencing (MPSS), or any equivalent thereof or any combination thereof.

[0046] Any sequencing method known in the art can be used to sequence the amplicons or newly extended DNA polynucleotides produced by the methods provided herein.

[0047] Products and Kits Provided are articles of manufacture and kits for practicing the methods provided herein, and optionally the articles of manufacture and kits can further include instructions for practicing the methods provided herein.

[0048] Any of the above aspects and embodiments may be combined with any other aspect or embodiment disclosed herein in the Summary, Drawings, and / or Detailed Description.

[0049] As used in this application and the claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0050] Unless specifically stated or clear from the context, as used herein, the term "or" is understood to be inclusive and to cover both "or" and "and."

[0051] Unless specifically stated or clear from the context, the term "about" as used herein is understood to be within the normal tolerance of the art, for example, within 2 standard deviations of the mean. About can be understood to be within 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the stated value. Unless clear from the context, all numerical values ​​provided herein are modified by the term "about".

[0052] Unless specifically stated or clear from the context, as used herein, the terms "substantially all," "substantially most of," "substantially all of," or "majority of" include at least about 90%, 95%, 97%, 98%, 99%, or 99.5% or more of a reference amount of a composition.

[0053] The entirety of each patent, patent application, publication, or document referenced herein is incorporated herein by reference. Citation of any of the above patents, patent applications, publications, or documents does not constitute an admission that any of the foregoing pertains to prior art, nor an admission of the contents or dates of such publications or documents. The incorporation by reference of such documents should not, in and of itself, be construed as an assertion or admission that any portion of the contents of any document is considered essential material to satisfy any national or local statutory disclosure requirements for patent applications. The right nevertheless reserves the right to rely on any such document, where appropriate, by an examining body or court to provide material deemed essential to the claimed subject matter.

[0054] Modifications may be made in the foregoing without departing from the basic aspects of the invention. Although the invention has been described in considerable detail with reference to one or more specific embodiments, those skilled in the art will recognize that variations may be made to the embodiments specifically disclosed in this application, and that these modifications and improvements are still within the scope and spirit of the invention. The invention illustratively described herein may suitably be practiced in the absence of any element not specifically disclosed herein. Thus, for example, in each instance herein, any of the terms "comprising," "consisting essentially of," and "consisting of" may be replaced with any of the other two terms. Thus, the terms and expressions used are used as descriptive terms and not as limitations, and do not exclude equivalents of the features shown and described or portions thereof, recognizing that various modifications are possible within the scope of the invention. Embodiments of the invention are described in the following claims.

[0055] The present invention will be further described with reference to the examples described herein, but it should be understood that the invention is not limited to such examples. EXAMPLES

[0056] Unless described in the examples, all recombinant DNA techniques are carried out according to standard protocols, e.g. as described in Sambrook et al. (2012) Molecular Cloning: A Laboratory Manual, 4th edition, Cold Spring Harbor Laboratory Press, NY, and Ausubel et al. (1994) Current Protocols in Molecular Biology, Current Protocols, USA, volumes 1 and 2. Other references for standard molecular biology techniques include Sambrook and Russell (2001) Molecular Cloning: A Laboratory Manual, 3rd edition, Cold Spring Harbor Laboratory Press, NY, Brown (1998) Molecular Biology LabFax, 2nd edition, Academic Press (UK), volumes I and II. Standard materials and methods for the polymerase chain reaction can be found in Dieffenbach and Dveksler (1995) PCR Primer: A Laboratory Manual, Cold Spring Harbor Laboratory Press and McPherson et al. (2000) PCR-Basics: From Background to Bench, 1st ed., Springer Verlag, Germany.

[0057] In alternative embodiments, the differences noted in the examples are detected by sequencing, hybridization, mass spectrometry, or any other technique capable of detecting sequence variations, including nucleotide resolution.

[0058] Example 1: Suppressing one allele while simultaneously amplifying any alternative allele This example describes and demonstrates the efficacy of an exemplary method provided herein to selectively suppress one allele while simultaneously amplifying any alternative alleles. This example also describes and demonstrates the efficacy of an exemplary method to suppress a wild-type sequence while simultaneously amplifying a point mutation.

[0059] In an alternative embodiment, an exemplary method provided herein involves suppressing exponential amplification of KRAS DNA encoding a wild-type "G" nucleotide at coding position 35 (in this example, KRAS c.35), while simultaneously allowing exponential amplification of any of the three possible mutations at this position. Mutations at this position are the most frequent oncogenic KRAS mutations, representing approximately 65% ​​of point mutations at KRAS codons 12 and 13. The selector primer used contains a "G" ribonucleotide (riboG or rG) at the third-to-last position, which matches perfectly to the wild-type template, and is extended by a proofreading DNA polymerase enzyme. Because the newly formed double-stranded DNA retains riboG, the thermostable ribonuclease H2 (TS RNase H2) present in the reaction cleaves 5' to the ribonucleotide, releasing most of the primer sequence and suppressing exponential amplification. In contrast, templates encoding the c.35G>A (p.G12D) or c.35G>T (p.G12V) or c.35G>C (p.G12A) mutations do not match the selector primer at the penultimate position, and as a result, the 3' to 5' exonuclease activity of the proofreading polymerase consumes the primer through the mismatch, eliminating a ribonucleotide before the primer can be extended across the mutation site. The loss of a ribonucleotide renders the incorporated primer sequence resistant to removal by the ribonuclease. The return primer copies the incorporated (i.e., partially consumed and then extended) first primer, and any mutant sequence is exponentially amplified.

[0060] Non-limiting examples of proofreading DNA polymerases that can be used in the methods provided herein include Phusion Hot Start II DNA Polymerase (ThermoFisher Scientific, Catalog #F549L), Q5® Hot Start High-Fidelity DNA Polymerase (New England Biolabs, Catalog #M0493L), and Platinum SuperFi II DNA Polymerase (ThermoFisher Scientific, Catalog #12361050). A non-limiting example of a thermostable ribonuclease H2 that can be used in the methods provided herein is RNase H2 enzyme (IDT, Catalog #11-03-02-03).

[0061] This exemplary method includes unique advantages over current methods, including never introducing a primer containing the sequence of interest to detect; a variant is only detected if: 1. a mismatch between the selector primer and the mutant template is recognized and removed by the 3' to 5' exonuclease activity of the proofreading polymerase; and 2. a high fidelity polymerase copies the mutant template. In contrast, allele-specific amplification introduces variant sequences of interest to detect, which can result in false positives. Another advantage is that a single primer detects any variant at a particular nucleotide position, in contrast to allele-specific PCR, which requires a primer specific for each allelic variant or mutation.

[0062] Example 2: Suppressing wild-type sequences while simultaneously amplifying DNA deletions This example demonstrates an exemplary method to suppress amplification of wild-type DNA while simultaneously amplifying a template containing a deletion.

[0063] In an alternative embodiment, an exemplary method provided herein involves suppressing exponential amplification of wild-type epidermal growth factor receptor (EGFR) DNA while simultaneously allowing exponential amplification of DNA with the in-frame deletion c.2235_2249del (p.E746_A750del). In-frame deletions represent approximately 41% of all EGFR mutations in cancer, with this particular mutation being one of the more frequent.

[0064] The 3' end of the selector primer terminus with GAA contains a "G" ribonucleotide (riboG or rG) in the third to last position, which is a perfect match with the wild-type EGFR codon encoding a glutamic acid residue at amino acid position 746. Because the newly formed duplex DNA retains riboG, the thermostable ribonuclease H2 (TS RNase H2) present in the reaction cleaves 5' to the ribonucleotide, liberating most of the primer sequence and thus suppressing exponential amplification.

[0065] In contrast, the c.2235_2249del (p.E746_A750del) deletion template does not match the selector primer at the penultimate position, and as a result, the 3' to 5' exonuclease activity of the proofreading polymerase consumes the primer through the mismatch, eliminating a ribonucleotide before the primer can be extended across the mutation site. The loss of a ribonucleotide renders the incorporated primer sequence resistant to removal by ribonucleases. The return primer copies the incorporated (i.e., partially consumed and then extended) first primer, and any mutant sequence is exponentially amplified.

[0066] Non-limiting examples of proofreading polymerases that can be used in the practice of the methods provided herein include Phusion Hot Start II DNA Polymerase (ThermoFisher Scientific, Catalog #F549L), Q5® Hot Start High-Fidelity DNA Polymerase (New England Biolabs, Catalog #M0493L), and Platinum SuperFi II DNA Polymerase (ThermoFisher Scientific, Catalog #12361050). A non-limiting example of a thermostable ribonuclease H2 that can be used in the practice of the methods provided herein is RNase H2 enzyme (IDT, Catalog #11-03-02-03).

[0067] This exemplary method includes unique advantages over current methods, including never introducing primers containing sequences of interest to detect, in contrast to allele-specific amplification, which introduces variant sequences of interest to detect, which can result in false positives. Another advantage is that a single primer detects any variant at a particular nucleotide position, in contrast to allele-specific PCR, which requires primers specific for each allelic variant or mutation.

[0068] Example 3: Suppressing wild-type sequences while simultaneously amplifying DNA insertions This example demonstrates an exemplary method to suppress amplification of wild-type DNA while simultaneously amplifying a template containing an insertion.

[0069] A non-limiting example is suppressing exponential amplification of wild-type EGFR DNA while simultaneously allowing exponential amplification of DNA with the c.2300_2308dup (p.A767_V769dup) insertion. This insertion is a duplication and patients with it often have reduced sensitivity to first and second generation EGFR tyrosine kinase inhibitors (TKIs). The 3' end of the selector primer terminus with ACA contains an "A" ribonucleotide (riboA or rA) at the third-to-last position, which perfectly matches the wild-type EGFR codon encoding an aspartic acid residue at amino acid position 770. Because the newly formed duplex DNA retains riboA, the thermostable ribonuclease H2 (TS RNase H2) present in the reaction cleaves 5' to the ribonucleotide, releasing most of the primer sequence and suppressing exponential amplification. In contrast, the template containing the c.2300_2308dup (p.A767_V769dup) insertion does not match the selector primer at the penultimate position, and as a result, the 3' to 5' exonuclease activity of the proofreading polymerase consumes the primer through the mismatch, eliminating a ribonucleotide before the primer can be extended across the mutation site. The loss of a ribonucleotide makes the incorporated primer sequence resistant to removal by ribonucleases. The return primer copies the incorporated (i.e., partially consumed and then extended) first primer, and any mutant sequence is exponentially amplified.

[0070] Non-limiting examples of proofreading polymerases that can be used in the practice of the methods provided herein include Phusion Hot Start II DNA Polymerase (ThermoFisher Scientific, Catalog #F549L), Q5® Hot Start High-Fidelity DNA Polymerase (New England Biolabs, Catalog #M0493L), and Platinum SuperFi II DNA Polymerase (ThermoFisher Scientific, Catalog #12361050). A non-limiting example of a thermostable ribonuclease H2 that can be used in the practice of the methods provided herein is RNase H2 enzyme (IDT, Catalog #11-03-02-03).

[0071] This exemplary method includes unique advantages over current methods, including never introducing primers containing sequences of interest to detect, in contrast to allele-specific amplification, which introduces variant sequences of interest to detect, which can result in false positives. Another advantage is that a single primer detects any variant at a particular nucleotide position, in contrast to allele-specific PCR, which requires primers specific for each allelic variant or mutation.

[0072] Example 4: Suppressing wild-type cDNA sequences while simultaneously amplifying cDNAs encoding fusion proteins This example demonstrates an exemplary method for suppressing amplification of wild-type cDNA while simultaneously amplifying a template encoding a fusion protein.

[0073] A non-limiting example is the suppression of exponential amplification of wild-type echinoderm microtubule-associated protein-like 4 (EML4), while simultaneously allowing exponential amplification of cDNA encoding the EML4-ALK fusion protein (ALK stands for anaplastic lymphoma kinase, also known as ALK tyrosine kinase receptor or CD246). The EML4-ALK fusion protein is found in non-small cell lung cancer (NSCLC) and is the target of crizotinib, among other targeted therapies. cDNA is first prepared, followed by amplification using a selector primer with the sequence GTG at its 3' end, where the penultimate "G" is a ribonucleotide (riboG or rG), which perfectly matches the wild-type EML4 sequence. Because the newly formed double-stranded DNA retains riboG, the thermostable ribonuclease H2 (TS RNase H2) present in the reaction cleaves at the 5' side of the ribonucleotide, releasing most of the primer sequence and suppressing exponential amplification. In contrast, the EML4-ALK fusion template does not match the selector primer at the penultimate position, and as a result, the 3' to 5' exonuclease activity of the proofreading polymerase consumes the primer through the mismatch, eliminating the ribonucleotide before the primer extends across the mutation site. The loss of the ribonucleotide makes the incorporated primer sequence resistant to removal by the ribonuclease. The return primer copies the incorporated (i.e., partially consumed and then extended) first primer, and any mutant sequence is exponentially amplified. Non-limiting examples of proofreading polymerases include Phusion Hot Start II DNA Polymerase (ThermoFisher Scientific, Catalog #F549L), Q5® Hot Start High-Fidelity DNA Polymerase (New England Biolabs, Catalog #M0493L), and Platinum SuperFi II DNA Polymerase (ThermoFisher Scientific, Catalog #12361050).

[0074] A non-limiting example of a thermostable ribonuclease H2 that can be used in the practice of the methods provided herein is RNase H2 enzyme (IDT, catalog #11-03-02-03). A non-limiting example of a reverse transcriptase used to generate cDNA from an mRNA template that can be used in the practice of the methods provided herein is SuperScript™ III Reverse Transcriptase (ThermoFisher Scientific, catalog #18080085).

[0075] This exemplary method includes unique advantages over current methods, including never introducing primers containing sequences of interest to detect; in contrast, allele-specific amplification introduces variant sequences of interest to detect, which can result in false positives.

[0076] Example 5: Suppressing one allele while simultaneously amplifying any alternative allele: Application to modified or unmodified nucleotide status determination This example demonstrates an exemplary method to suppress amplification of one allele while simultaneously amplifying any alternative alleles present at a nucleotide position, particularly when one or the other allele is created based on the presence or absence of a nucleotide modification. A non-limiting example is when an unmodified cytosine nucleotide is changed to a deoxyuridine (dU) while a methylated or hydroxymethylated cytosine remains unchanged.

[0077] A non-limiting example is the use of a selector primer that contains a "G" ribonucleotide (riboG or rG) in the penultimate position, which matches perfectly with the processed template (where the complementary position remains "C" since it has been modified and therefore unchanged) and is extended by a proofreading enzyme. Because the newly formed duplex DNA retains riboG, the thermostable ribonuclease H2 (TS RNase H2) present in the reaction cleaves 5' to the ribonucleotide, liberating most of the primer sequence and suppressing exponential amplification. In contrast, the processed template, in which the "C" has been changed to a "dU", does not match the selector primer in the penultimate position, and as a result, the 3' to 5' exonuclease activity of the proofreading polymerase consumes the primer through the mismatch, eliminating the ribonucleotide before the primer can be extended across the "dU" site (pairing with an "A"). The loss of ribonucleotides renders the incorporated primer sequence resistant to removal by ribonucleases. The return primer copies the incorporated (i.e., partially consumed and then extended) first primer, and the "dU" sequence is exponentially amplified (changing to a "T" during amplification).

[0078] A non-limiting example is the conversion described above where the selector primer now contains an "A" ribonucleotide (riboA or rA) in the penultimate position, which matches perfectly with the processed template (where the complementary position is unmodified and therefore altered to "dU") and is extended by the proofreading enzyme. Because the newly formed duplex DNA retains riboA, the thermostable ribonuclease H2 (TS RNase H2) present in the reaction cleaves 5' to the ribonucleotide, liberating most of the primer sequence and suppressing exponential amplification. In contrast, the processed template, where the "C" is unchanged (protected by the modification), does not match the selector primer in the penultimate position, and as a result, the 3' to 5' exonuclease activity of the proofreading polymerase consumes the primer through the mismatch, eliminating the ribonucleotide before the primer can be extended across the mutation site. The loss of the ribonucleotide renders the incorporated primer sequence resistant to removal by the ribonuclease. The return primer copies the first primer that was incorporated (ie, partially consumed and then extended), resulting in exponential amplification of the sequence.

[0079] Non-limiting examples of proofreading polymerases that can be used in the practice of the methods provided herein include Phusion Hot Start II DNA Polymerase (ThermoFisher Scientific, catalog #F549L), Q5® Hot Start High-Fidelity DNA Polymerase (New England Biolabs, catalog #M0493L), and Platinum SuperFi II DNA Polymerase (ThermoFisher Scientific, catalog #12361050).

[0080] This exemplary method includes unique advantages over current methods, including never introducing primers containing sequences of interest to detect; in contrast, allele-specific amplification introduces variant sequences of interest to detect, which can result in false positives.

[0081] Example 6: Suppressing one allele while simultaneously amplifying any alternative allele: Application to molecular haplotyping This example demonstrates an exemplary method to suppress the amplification of one allele, while simultaneously amplifying any alternative alleles present at a nucleotide position, along with alleles that are located on the same DNA homolog as the alternative alleles, thus enabling molecular haplotyping.

[0082] A non-limiting example is determining the presence or absence of the AGT haplotype in the ITGA4 gene. Single nucleotide polymorphisms within the ITGA4 gene include c.1845G>A, c.2633A>G and c.2883C>T, with the haplotype AGT being associated with the occurrence of antibody-mediated rejection in heart transplant patients. If genotyping reveals that an individual is heterozygous at two or three sites, the haplotype can be determined using a selector primer at the appropriate heterozygous site and a return primer positioned beyond the most distal heterozygous site. As an example, if the DNA is heterozygous at c.1845, the selector primer contains a "G" ribonucleotide (riboG or rG) at the penultimate position corresponding to the c.1845 position, and the return primer is downstream of the c.2883 position. This results in suppression of the c.1845G allele while allowing exponential amplification of the c.1845A allele. Due to the high fidelity of the proofreading enzyme used and the very low level of strand-switching, the alleles present in the same exponentially amplified DNA homolog can be determined and the haplotype of the 1845A DNA homolog can be found. The haplotype of other homologs can be inferred from the genotype or confirmed in a separate reaction using a selector primer containing an "A" ribonucleotide (riboA or rA) in the penultimate third position.

[0083] Non-limiting examples of proofreading polymerases that can be used in the practice of the methods provided herein include Phusion Hot Start II DNA Polymerase (ThermoFisher Scientific, Catalog #F549L), Q5® Hot Start High-Fidelity DNA Polymerase (New England Biolabs, Catalog #M0493L), and Platinum SuperFi II DNA Polymerase (ThermoFisher Scientific, Catalog #12361050). A non-limiting example of a thermostable ribonuclease H2 that can be used in the practice of the methods provided herein is RNase H2 enzyme (IDT, Catalog #11-03-02-03).

[0084] This exemplary method includes unique advantages over current methods, including never introducing primers containing sequences of interest to detect, in contrast to allele-specific amplification, which introduces variant sequences of interest to detect, which can result in false positives. Another advantage is that a single primer detects any variant at a particular nucleotide position, in contrast to allele-specific PCR, which requires primers specific for each allelic variant or mutation.

[0085] Example 7: Partially suppressing one allele while simultaneously amplifying any alternative allele This example demonstrates an exemplary method to partially suppress the amplification of one allele while simultaneously amplifying any alternative alleles present at a nucleotide position. This can also be applied to partially suppress the wild type sequence while simultaneously amplifying a point mutation. Referring to the latter case, which only partially suppresses the amplification of the wild type while not interfering with the amplification of any mutant, two conclusions can be reached: 1. In the absence of a mutant, the partial amplification of the wild type provides an internal standard that confirms that the overall reaction worked well, and 2. Controlling the level of suppression of the wild type provides an internal standard against which the amplification level of the mutant (if present) can be compared.

[0086] A non-limiting example would be to partially suppress exponential amplification of KRAS DNA encoding a wild-type "G" nucleotide at coding position 35, while simultaneously allowing exponential amplification of any of the three possible mutations at this position. The selector primer contains a "G" ribonucleotide (riboG or rG) at the third-to-last position, which matches perfectly to the wild-type template and is extended by a proofreading enzyme. Mixed with the selector primer in a predetermined amount is its counterpart that is identical to the selector primer except that the "G" at the third-to-last position is a normal deoxyribonucleotide rather than a ribonucleotide. The newly formed duplex DNA that retains riboG is cleaved by the thermostable ribonuclease H2 (TS RNase H2) present in the reaction, thereby releasing most of the primer sequence and suppressing exponential amplification. However, the newly formed duplex DNA that contains the counterpart of the selector primer (i.e., a primer that contains a normal deoxynucleotide counterpart at the selector nucleotide position) is not cleaved and the entirety of the primer is retained. Wild-type exponential amplification is only partially suppressed. Templates encoding the c.35G>A (p.G12D) or c.35G>T (p.G12V) or c.35G>C (p.G12A) mutations do not match the selector primer (or its counterpart) at the penultimate position, and as a result, the 3' to 5' exonuclease activity of the proofreading polymerase consumes the primer through the mismatch, eliminating the ribonucleotide or its deoxyribonucleotide counterpart before the primer can be extended across the mutation site. The absence of any ribonucleotides renders the incorporated primer sequence resistant to removal by ribonucleases. The return primer copies the incorporated (i.e., partially consumed and then extended) first primer, and any mutant sequence is exponentially amplified.

[0087] Non-limiting examples of proofreading polymerases that can be used in the practice of the methods provided herein include Phusion Hot Start II DNA Polymerase (ThermoFisher Scientific, Catalog #F549L), Q5® Hot Start High-Fidelity DNA Polymerase (New England Biolabs, Catalog #M0493L), and Platinum SuperFi II DNA Polymerase (ThermoFisher Scientific, Catalog #12361050). A non-limiting example of a thermostable ribonuclease H2 that can be used in the practice of the methods provided herein is RNase H2 enzyme (IDT, Catalog #11-03-02-03).

[0088] This exemplary method includes unique advantages over current methods, including never introducing a primer containing the sequence of interest to detect; a variant is only detected if: 1. a mismatch between the selector primer and the mutant template is recognized and removed by the 3' to 5' exonuclease activity of the proofreading polymerase; and 2. a high fidelity polymerase copies the mutant template. In contrast, allele-specific amplification introduces the variant sequence of interest to detect, which can result in false positives. Another advantage is that a single primer detects any variant at a specific nucleotide position; in contrast, allele-specific PCR requires a primer specific for each allelic variant or mutation. Another advantage described in this example, as well as applicable to other examples and generally to all methods provided herein, is the ability to allow amplification of a set amount of a particular allele or wild type, which serves as an internal standard to demonstrate that the reaction worked well, and to which the amplification levels of alternative alleles or mutants (if any) can be compared.

[0089] A number of embodiments of the invention have been described. Nevertheless, it will be understood that various modifications may be made without departing from the spirit and scope of the invention. Accordingly, other embodiments are within the scope of the following claims.

Claims

1. A synthetic DNA polynucleotide (termed a selector polynucleotide) comprising at least a single residue (termed a selector nucleotide) located at the first (last), second (second to last), third (third to last), fourth (fourth to last), fifth (fifth to last) or sixth (one residue before the fifth to last) position relative to the 3' end, or at a position even more distal from the 3' end, the at least single residue selector nucleotide is structurally or chemically distinct from any other residue within a region or section of the selector polynucleotide that is required for binding of the selector polynucleotide to a target nucleic acid; the synthetic DNA polynucleotide (called the selector polynucleotide) has a 3' end that can be extended by a DNA polymerase or is processed to have a 3' end that can be extended by a DNA polymerase; Synthetic DNA polynucleotides.

2. 2. The synthetic DNA polynucleotide (i.e., selector polynucleotide) of claim 1, wherein a single selector nucleotide residue is located at the second position from the 3' end or at the penultimate position of the synthetic DNA polynucleotide (i.e., selector polynucleotide).

3. 2. The synthetic DNA polynucleotide (i.e., selector polynucleotide) of claim 1, wherein a single selector nucleotide residue is located at the third position from the 3' end or at the penultimate position of the synthetic DNA polynucleotide (i.e., selector polynucleotide).

4. 2. The synthetic DNA polynucleotide (i.e., the selector polynucleotide) of claim 1, wherein a single selector nucleotide residue is located at the first position from the 3' end or at the last position of the synthetic DNA polynucleotide (i.e., the selector polynucleotide).

5. 2. The synthetic DNA polynucleotide (i.e., selector polynucleotide) of claim 1, wherein a single selector nucleotide residue is located at the fourth position from the 3' end or at the penultimate position of the synthetic DNA polynucleotide (i.e., selector polynucleotide).

6. 2. The synthetic DNA polynucleotide (i.e., selector polynucleotide) of claim 1, wherein a single selector nucleotide residue is located at the fifth position from the 3' end or at the fifth position penultimate to the end of the synthetic DNA polynucleotide (i.e., selector polynucleotide).

7. 2. The synthetic DNA polynucleotide (i.e., selector polynucleotide) of claim 1, wherein a single selector nucleotide residue is located at the sixth position from the 3' end or at the penultimate to fifth residue position of the synthetic DNA polynucleotide (i.e., selector polynucleotide).

8. The synthetic DNA polynucleotide (ie, the selector polynucleotide) of any one of claims 1 to 7, wherein the selector nucleotide is a ribonucleotide.

9. A synthetic DNA polynucleotide (ie, a selector polynucleotide) according to any one of claims 1 to 8, wherein the selector nucleotide comprises or is composed of at least one ribonucleotide.

10. A synthetic DNA polynucleotide (ie, a selector polynucleotide) according to any one of claims 1 to 9, wherein the selector nucleotide comprises or consists of at least one synthetic or non-naturally occurring nucleotide.

11. 1. A nucleic acid amplification method for distinguishing a first nucleic acid sequence from a second nucleic acid sequence, the first nucleic acid and the second nucleic acid being in the same amplification reaction mixture; (a) providing or having provided a synthetic DNA polynucleotide (i.e., a selector polynucleotide) according to any one of claims 1 to 10, wherein said synthetic DNA polynucleotide (i.e., a selector polynucleotide) contains a selector nucleotide residue (optionally a single selector ribonucleotide residue); (b) providing or having provided a DNA polynucleotide or a plurality of DNA polynucleotides, optionally wherein said DNA polynucleotide or a plurality of DNA polynucleotides comprises or is derived from a genome (optionally a cellular, microbial or viral genome), a cDNA library or a genomic library, optionally wherein said genome, cDNA library or genomic library is derived from a eukaryote or prokaryote, a plant or an animal (optionally human), a microorganism (optionally a bacterium, algae, protist, archaea or fungus), or a virus or bacteriophage; (c) contacting, annealing or hybridizing said synthetic DNA polynucleotide (i.e., selector polynucleotide) with said DNA polynucleotide or DNA polynucleotides, wherein said DNA polynucleotide or DNA polynucleotides act as a template (template DNA polynucleotide) for said synthetic DNA polynucleotide (i.e., selector polynucleotide) under conditions where said synthetic DNA polynucleotide (i.e., selector polynucleotide) anneals or specifically hybridizes to a complementary or substantially complementary sequence in said DNA polynucleotide, thereby generating a nucleic acid duplex; wherein the synthetic DNA polynucleotide (i.e., selector polynucleotide) either pairs with a template DNA polynucleotide at a selector nucleotide residue (optionally a single selector ribonucleotide residue) (see, e.g., FIG. 2A ) or does not pair with a template DNA polynucleotide at a selector nucleotide residue (optionally a single ribonucleotide residue) (see, e.g., FIG. 2B ). (d) contacting the duplex with a DNA polymerase enzyme having 5' to 3' extension activity and with 3' to 5' exonuclease activity, and / or the DNA polymerase enzyme having 5' to 3' extension activity and the enzyme having 3' to 5' exonuclease activity under conditions in which the DNA polymerase enzyme having 5' to 3' extension activity and the enzyme having 3' to 5' exonuclease activity are enzymatically active, wherein (i) a selector nucleotide residue (optionally a single ribonucleotide residue) is mismatched between the synthetic DNA polynucleotide (i.e., selector polynucleotide) and the DNA polynucleotide or DNA polynucleotides, and the 3' to 5' exonuclease activity results in enzymatically removing from the 3' end a portion of the synthetic DNA polynucleotide (i.e., selector polynucleotide) that includes the selector nucleotide residue (optionally a single ribonucleotide residue) and all nucleotides 3' to the selector nucleotide, before the DNA polymerase extends from the remainder of the synthetic DNA polynucleotide (i.e., selector polynucleotide) to a newly extended DNA polynucleotide that does not retain the selector nucleotide residue (optionally a single ribonucleotide residue); or (ii) the DNA polymerase extends a synthetic DNA polynucleotide (i.e., a selector polynucleotide) that is base-paired to the DNA polynucleotide or DNA polynucleotides at a selector nucleotide residue (optionally, a single ribonucleotide residue) without removing the selector nucleotide (optionally, a single ribonucleotide residue), thereby retaining or incorporating the selector nucleotide residue (optionally, a single ribonucleotide residue) of the synthetic DNA polynucleotide (i.e., a selector polynucleotide) into the newly extended DNA polynucleotide; (e) contacting the newly created nucleic acid duplex with a ribonuclease enzyme, where, optionally, the ribonuclease enzyme is thermostable, and where the thermostable ribonuclease is a thermostable ribonuclease H2 enzyme, under conditions in which the thermostable ribonuclease enzyme is active, where (i) when the selector nucleotide residue (optionally a single ribonucleotide residue) is removed by 3' to 5' exonuclease activity present in the reaction, the portion of the synthetic DNA polynucleotide (i.e., selector polynucleotide) that was 5' to the single selector nucleotide residue (optionally a single ribonucleotide residue) is retained in the extended synthetic polynucleotide; or (ii) if the selector nucleotide residue (optionally a single ribonucleotide residue) is retained within the extended synthetic DNA polynucleotide (i.e., selector polynucleotide) and matches a deoxyribonucleotide residue, the thermostable ribonuclease enzyme cleaves at the selector nucleotide residue (optionally a single ribonucleotide residue), thereby releasing the portion of the extended synthetic DNA polynucleotide (i.e., selector polynucleotide) that was 5' to the selector nucleotide residue (optionally a single ribonucleotide residue); wherein the extended synthetic DNA polynucleotide (or extended selector polynucleotide) that retains a portion of the synthetic DNA polynucleotide (i.e., selector polynucleotide) that was 5' to the selector nucleotide residue (optionally, a single ribonucleotide residue) can be exponentially amplified, and the extended DNA polynucleotide (extended selector polynucleotide) that had a portion of the synthetic DNA polynucleotide (i.e., selector polynucleotide) that was 5' to the selector nucleotide residue (optionally, a single ribonucleotide residue) that was detached from the extended synthetic DNA polynucleotide (i.e., selector polynucleotide) cannot be exponentially amplified, thereby selectively suppressing their amplification and distinguishing a first nucleic acid sequence from a second nucleic acid sequence; A method comprising:

12. 1. A nucleic acid amplification method for distinguishing a first nucleic acid sequence from a second nucleic acid sequence, the first nucleic acid and the second nucleic acid being in the same amplification reaction mixture; (a) providing or having provided a synthetic DNA polynucleotide (i.e., a selector polynucleotide) according to any one of claims 1 to 10, wherein said synthetic DNA polynucleotide (i.e., a selector polynucleotide) contains a selector nucleotide residue (optionally a single ribonucleotide residue); (b) providing or having provided a DNA polynucleotide or a plurality of DNA polynucleotides, optionally wherein said DNA polynucleotide or a plurality of DNA polynucleotides comprises or is derived from a genome (optionally a cellular, microbial or viral genome), a cDNA library or a genomic library, optionally wherein said genome, cDNA library or genomic library is derived from a eukaryote or prokaryote, a plant or an animal (optionally a human), a microorganism (optionally a bacterium, algae, protist, archaea or fungus), or a virus or bacteriophage; (c) contacting, annealing or hybridizing said synthetic DNA polynucleotide (i.e., selector polynucleotide) with said DNA polynucleotide or DNA polynucleotides, wherein said DNA polynucleotide or DNA polynucleotides act as a template (template DNA polynucleotide) for said synthetic DNA polynucleotide (i.e., selector polynucleotide) under conditions where said synthetic DNA polynucleotide (i.e., selector polynucleotide) anneals or specifically hybridizes to a complementary or substantially complementary sequence in said DNA polynucleotide, thereby generating a nucleic acid duplex; wherein the synthetic DNA polynucleotide (i.e., selector polynucleotide) either pairs with the template DNA polynucleotide at the selector nucleotide residue (optionally a single ribonucleotide residue) (see, e.g., FIG. 10A ) or does not pair with the template DNA polynucleotide at the selector nucleotide residue (optionally a single ribonucleotide residue) (see, e.g., FIG. 10B ). (d) contacting the nucleic acid duplex with a DNA polymerase enzyme having 5' to 3' extension activity and with 3' to 5' exonuclease activity and / or the DNA polymerase enzyme having 5' to 3' extension activity and the enzyme having 3' to 5' exonuclease activity under conditions in which the DNA polymerase enzyme having 5' to 3' extension activity and the enzyme having 3' to 5' exonuclease activity are enzymatically active, wherein (i) the selector nucleotide residue (optionally a single ribonucleotide residue) is mismatched between the synthetic DNA polynucleotide (i.e., selector polynucleotide) and the DNA polynucleotide or DNA polynucleotides, and a 3' to 5' exonuclease activity results in enzymatically removing from the 3' end a portion of the synthetic DNA polynucleotide (i.e., selector polynucleotide) that includes the selector nucleotide residue (optionally a single ribonucleotide residue) and all nucleotides 3' of the selector nucleotide before the DNA polymerase extends the remainder of the synthetic DNA polynucleotide (i.e., selector polynucleotide) to a newly extended DNA polynucleotide that does not retain the selector nucleotide residue (optionally a single ribonucleotide residue); or (ii) the DNA polymerase extends a synthetic DNA polynucleotide (i.e., a selector polynucleotide) that is base-paired to the DNA polynucleotide or DNA polynucleotides at the selector nucleotide residue (optionally, a single ribonucleotide residue) without removing the selector nucleotide (optionally, a single ribonucleotide residue), thereby retaining or incorporating the selector nucleotide residue (optionally, a single ribonucleotide residue) of the synthetic DNA polynucleotide (i.e., a selector polynucleotide) into the newly extended DNA polynucleotide; and (e) after amplification, treating the amplicon (or the newly extended synthetic DNA polynucleotide, i.e., the selector polynucleotide) with a reagent or enzyme that cleaves 5' or 3' to said selector nucleotide residue (optionally a single ribonucleotide residue), or, if present, within 3 nucleotides of said selector nucleotide residue (optionally a single ribonucleotide residue); wherein, optionally, the reagent or enzyme displaces a binding moiety or a substantial amount of incorporated synthetic DNA polynucleotide (i.e., selector nucleotide) or primer from an amplicon (or newly extended DNA polynucleotide) that retains the selector nucleotide residue (optionally, a single ribonucleotide residue), thereby allowing an amplicon or newly extended DNA polynucleotide that does not have the selector nucleotide residue (optionally, a single ribonucleotide residue) and thus retains a binding moiety or a substantial amount of incorporated synthetic DNA polynucleotide (i.e., selector nucleotide) or primer to be preferentially captured (or physically isolated) or subsequently preferentially amplified; A method comprising:

13. 13. The method of claim 11 or 12, wherein the reagent used to cleave the 5' end of the single selector ribonucleotide is ribonuclease H2.

14. 13. The method of claim 12, wherein the reagent used to cleave the 3' end of the single selector ribonucleotide is sodium hydroxide in the presence of heat.

15. 13. The method of claim 11 or 12, further comprising denaturing the nucleic acid duplex to produce single stranded DNA, wherein the single stranded DNA is treated with a ribonuclease that cleaves 3' to the single selector ribonucleotide.

16. The method of any of claims 11 to 15, wherein the polymerase and 3' to 5' exonuclease activities are provided by different enzymes.

17. The method of any of claims 11 to 16, wherein the synthetic DNA polynucleotide (i.e. the selector polynucleotide) is or comprises a primer used in a nucleic acid amplification method.

18. 20. The method of claim 17, wherein the amplification method comprises polymerase chain reaction (PCR).

19. 19. The method of any of claims 11-18, wherein during amplification, the extended synthetic DNA polynucleotide (i.e. the selector polynucleotide) or primer is treated with an enzyme that cleaves 5' or 3' to the selector nucleotide residue (optionally a single ribonucleotide residue) or, if present, within 3 nucleotides of the selector nucleotide residue (optionally a single ribonucleotide residue), thereby releasing the portion of the synthetic DNA polynucleotide (i.e. the selector polynucleotide) or primer from the amplicon (or newly extended DNA polynucleotide) that retains said selector nucleotide residue (optionally a single ribonucleotide residue), preventing said synthetic DNA polynucleotide (i.e. the selector polynucleotide) or primer from being completely copied by extension of a return primer in the amplification reaction, and allowing amplicons that do not have said selector nucleotide residue (optionally a single ribonucleotide residue) to be preferentially amplified by retaining sufficient synthetic DNA polynucleotide (i.e. the selector polynucleotide) or primer sequence to support exponential amplification.

20. The method of any one of claims 11 to 19, wherein the synthetic DNA polynucleotide (i.e. the selector polynucleotide) or the primer comprises a ribonucleotide, and the enzyme that cleaves at the 5' side of the selector nucleotide is ribonuclease H2.

21. 21. The method of claim 20, wherein the ribonuclease H2 is thermostable.

22. 22. The method of claim 21 , wherein the thermostable ribonuclease H2 is Pyrococcus abyssi RNase H2.

23. 23. The method of any of claims 11 to 22, further comprising a second synthetic DNA polynucleotide or primer identical to the first synthetic DNA polynucleotide (i.e. selector polynucleotide) except that the selector nucleotide residue (optionally a single ribonucleotide residue) has been replaced by a corresponding normal deoxyribonucleotide to create a DNA amplification primer, and a certain amount of this DNA amplification primer is mixed with the first synthetic DNA polynucleotide (i.e. selector polynucleotide) or primer containing a selector nucleotide residue (optionally a single ribonucleotide residue) to allow production of a certain amount of an amplicon that would otherwise contain the selector nucleotide residue (optionally a single ribonucleotide residue) but now lacks the selector nucleotide residue (optionally a single ribonucleotide residue) and is thereby now resistant to cleavage by a reagent or enzyme specific for the selector nucleotide residue (optionally a single ribonucleotide residue).

24. 24. The method of claim 23, wherein the amplicon produced by the second synthetic DNA polynucleotide is used as an internal reaction standard to demonstrate that the amplification worked well and as an internal reference against which the amount of amplicon or newly extended DNA polynucleotide produced by the first synthetic DNA polynucleotide (i.e. the selector polynucleotide) can be compared.

25. 25. The method of any of claims 11 to 24, wherein the sequence of the amplicon or newly extended DNA polynucleotide is determined by DNA sequencing, optionally using methods including the use of Sanger sequencing, next generation sequencing (NGS), single molecule real-time (SMRT) sequencing, nanopore DNA sequencing, reversible end chemistry (e.g. SOLEXA technology (Illumina)), combinatorial probe anchor synthesis (cPAS), mass spectrometry sequencing or massively parallel signature sequencing (MPSS).

26. 26. The method of any of claims 11 to 25, wherein the identity of the nucleotide corresponding to the position of the original selector nucleotide residue (optionally a single ribonucleotide residue) is determined by primer extension across the site of interest.

27. 27. The method of claim 26, wherein the identity and relative amount of the nucleotides at the site of interest are determined by or by means of the use of labels or masses, optionally the identity and relative amount of the nucleotides at the site of interest are determined by a method comprising single base extension of a primer across the site of interest in an amplicon created using a synthetic DNA polynucleotide (i.e. a selector polynucleotide).

28. The method of any of claims 11 to 27, wherein the production of the amplicon or newly extended DNA polynucleotide of interest is determined by quantitative PCR (qPCR), digital PCR, or equivalent.

29. 29. A kit or article of manufacture comprising materials for practicing the method of any one of claims 11 to 28, optionally enzymes and / or synthetic DNA polynucleotides, optionally a selector polynucleotide (optionally one or more synthetic DNA polynucleotides (i.e. selector polynucleotides) according to any one of claims 1 to 10), and optionally further comprising instructions for practicing the method of any one of claims 11 to 28.

30. 1. A method of providing data for diagnosing a disease or condition, comprising determining the presence or absence of an allele or genomic sequence associated with or having a diagnosis of the disease or condition, thereby providing data for determining whether an individual in need of diagnosis has the disease or condition, The presence or absence of an allele or genomic sequence associated with a disease or condition or having a diagnosis of a disease or condition is determined using a method according to any of claims 11 to 28. method.

31. 31. The method of claim 30, wherein the disease is cancer.

32. 29. A method for detecting the presence or absence of a rare allele in a biological specimen, comprising the use of a method according to any of claims 11 to 28, optionally wherein the biological specimen comprises or is derived from a biopsy, or a tissue or blood sample, or a liquid sample, of an individual in need thereof.

33. 33. The method of claim 32, wherein detecting the presence or absence of rare alleles in a biological specimen is for non-invasive prenatal testing (NIPT), or for assessing histocompatibility or detecting donor-derived nucleic acids after organ transplantation (optionally of solid organ or bone marrow), or for early detection of antimicrobial resistance (AMR) or bacterial resistance in an individual in need thereof, or for assessing the presence of minimal residual disease (MRE), optionally after bone marrow ablation MRE.

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