US10385388B2 - Cleavable competitor polynucleotides - Google Patents

Cleavable competitor polynucleotides Download PDF

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US10385388B2
US10385388B2 US15/101,551 US201415101551A US10385388B2 US 10385388 B2 US10385388 B2 US 10385388B2 US 201415101551 A US201415101551 A US 201415101551A US 10385388 B2 US10385388 B2 US 10385388B2
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polynucleotide
target
sequence
competitor
dna
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US20160304949A1 (en
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Vladimir Makarov
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Integrated DNA Technologies Inc
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Swift Biosciences Inc
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6858—Allele-specific amplification
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2525/00—Reactions involving modified oligonucleotides, nucleic acids, or nucleotides
    • C12Q2525/10—Modifications characterised by
    • C12Q2525/137—Modifications characterised by incorporating/modifying moieties to eliminate restriction sites

Definitions

  • the invention relates to polynucleotide combinations and their use in allele-specific enrichment, amplification and detection.
  • Detection and amplification of nucleic acids play important roles in genetic analysis, molecular diagnostics, and drug discovery. Many such applications require specific, sensitive and cost effective quantitative detection of DNA mutations, copy number variants, gene expression or DNA methylation patterns that are present in a small fraction of total polynucleotides. In the field of cancer diagnosis for instance early detection of somatic mutations greatly increase the survival rate of cancer patients. Monitoring for occurrence of drug resistant mutations is also crucial in determining if a patient will have a relapse of the disease. An ideal example would be EGFR T790M mutation which occurs as a tyrosine kinase inhibitor resistant mutation in several non-small cell lung cancer (NSCLC) patients.
  • NSCLC non-small cell lung cancer
  • NSCLC patients harboring activating mutations in the epidermal growth factor receptor (EGFR) kinase domain tend to respond well to the tyrosine kinase inhibitors, gefitinib and erlotinib.
  • EGFR epidermal growth factor receptor
  • T790M tyrosine kinase inhibitors
  • Many of these early somatic mutations and drug resistant mutations are rare mutations which occur within a huge background of non-mutated DNA molecules.
  • Many current methods use polymerase chain reaction (PCR), quantitative PCR (qPCR) and Next Generation Sequencing (NGS) to detect and quantify DNA and RNA variants from clinical samples.
  • PCR polymerase chain reaction
  • qPCR quantitative PCR
  • NGS Next Generation Sequencing
  • the disclosure provides a polynucleotide competitor that comprises a sequence that is fully complementary to a first target DNA polynucleotide region (T 1 ) such that the competitor polynucleotide will hybridize to the first target DNA polynucleotide region under appropriate conditions.
  • the polynucleotide competitor comprises a mismatch to a non-target DNA polynucleotide that is a sequence variant of the first target DNA polynucleotide region (T 1 *).
  • the disclosure provides a polynucleotide competitor that comprises a sequence that is fully complementary to the non-target DNA polynucleotide region (T 1 *) such that the competitor polynucleotide will hybridize to the non-target DNA polynucleotide region under appropriate conditions.
  • the polynucleotide competitor comprises a mismatch to a target DNA polynucleotide region T 1 that is a sequence variant of the non-target DNA polynucleotide region.
  • the disclosure further contemplates an aspect wherein the polynucleotide competitor comprises a single RNA base or a plurality of consecutive RNA bases that are at the position of a mismatched base in the target DNA polynucleotide region (T 1 ) or the non-target DNA polynucleotide region (T 1 *) or alternatively the mismatched base is positioned 1, 2 or 3 DNA bases away either 5′ or 3′ to the RNA base(s).
  • RNA base(s) are located close to the central part of the polynucleotide competitor.
  • RNA base(s) are located within the 3′ portion of the polynucleotide competitor.
  • the polynucleotide competitor is comprised of RNA bases in its entirety.
  • a competitor comprising one or more RNA bases enables the polynucleotide competitor to be a substrate for cleavage by RNase H2 upon binding to its target DNA (T 1 ) and a less efficient substrate for cleavage by RNase H2 upon binding to its non-target DNA (T 1 *).
  • a competitor comprising 4 or more consecutive RNA bases enables the polynucleotide competitor to be a substrate for cleavage by RNase H1 upon binding to its target DNA (T 1 ) and a less efficient substrate for cleavage by RNase H1 upon binding to its non-target DNA (T 1 *).
  • the cleavage pattern is reversed wherein the polynucleotide competitor is a substrate for cleavage upon binding to the non-target T 1 * and is a less efficient substrate for cleavage upon binding to the target T 1 DNA region, utilizing either RNase H1 or RNase H2.
  • the polynucleotide competitor further comprises a modified nucleic acid, and includes a blocking group to prevent DNA polymerase extension from the 3′ end of the competitor polynucleotide, and optionally includes one or more cleavage-resistant linkages between RNA bases, DNA bases, and DNA-RNA junctions to eliminate cleavage by RNase H enzymes at other potential cleavage sites and to direct RNase H cleavage to the most mismatch sensitive position which is determined empirically for each sequence. Additional cleavage-resistant linkages, in various aspects, are incorporated at the 3′ terminal bases of the competitor polynucleotide to block 3′-5′ exonuclease activity by a proof-reading polymerase.
  • the polynucleotide competitor further comprises a 5′ sequence that is not complementary to either the target DNA T 1 or to the non-target DNA T 1 *.
  • the disclosure provides a polynucleotide combination comprising a polynucleotide competitor and flanking PCR amplification primers, wherein the polynucleotide competitor comprises a sequence that is fully complementary to a first target DNA polynucleotide region (T 1 ) such that the competitor polynucleotide will hybridize to its target under appropriate conditions.
  • the polynucleotide competitor comprises a mismatch to a non-target DNA polynucleotide that is a sequence variant of the first target DNA polynucleotide region (T 1 *).
  • the polynucleotide competitor sequence is fully complementary to the non-target T 1 * and harbors a mismatch to the target T 1 .
  • the polynucleotide competitor additionally comprises an RNA base, a modified base and a nuclease-resistant linkage as described above to enable the polynucleotide competitor to serve as a substrate for mismatch-sensitive RNase H1 or RNase H2 cleavage under appropriate hybridization and reaction conditions.
  • a PCR amplification primer pair comprises a sequence that is fully complementary to a second target DNA polynucleotide region (T 2 ) and a third target DNA polynucleotide region (T 3 ), thus comprising a forward primer where its target sequence T 2 is located 5′ to the competitor target sequence T 1 (and T 1 * mismatched non-target) and a reverse primer where its target sequence T 3 is located 3′ to the competitor target sequence T 1 (and T 1 * mismatched non-target), where the reverse primer is complementary to the strand that is the reverse complement of the strand which the forward primer and competitor hybridize.
  • the forward primer target region T 2 does not overlap with the competitor target region T 1 . In other aspects, the forward primer target region T 2 does overlap with the competitor target region T 1 , where the area of overlap between T 1 and T 2 is limited to sequences that do not vary between the target DNA T 1 and the non-target DNA T 1 * and where the area of overlap is not in sequences that do vary between the target DNA T 1 and the non-target DNA T 1 *, where such T 2 and T 3 regions correspond to PCR primers that amplify both T 1 and T 1 * equally.
  • the PCR primer pair further comprises a nucleic acid modification, and include, for example and without limitation, nuclease-resistant linkages at 3′ terminal bases to prevent 3′-5′ exonuclease activity by a proof-reading polymerase.
  • the forward primer target region T 2 overlaps with the competitor target region T 1 , where the area of overlap between T 1 and T 2 includes the sequence variant between the target DNA T 1 and the non-target DNA T 1 * and where such a T 2 region corresponds to an allele-specific forward primer that preferentially amplifies the fully complementary T 1 target sequence and either does not amplify T 1 * or does so at a reduced efficiency when the mismatch to the T 1 * non-target sequence is located at or near the 3′ terminus of the forward primer.
  • the allele-specific primer is comprised of two polynucleotides as previously disclosed in International Application No. PCT/US2010/054362, filed on Oct.
  • the PCR primer pair further comprise nucleic acid modifications, and include nuclease-resistant linkages at 3′ terminal bases to prevent 3′-5′ exonuclease activity by a proof-reading polymerase.
  • the primer pair target regions T 2 and T 3 correspond to endogenous genomic DNA sequences that are in proximity to an endogenous genomic DNA sequence that corresponds to the competitor target region T 1 and T 1 *, where such primers are designated forward and reverse primers.
  • the primer pair target regions T 2 and T 3 correspond to universal DNA library adaptor sequences that are in proximity to an endogenous genomic DNA sequence that corresponds to the competitor target regions T 1 and T 1 * when T 1 and T 1 * are included in a DNA library (for example, and without limitation, a Next Generation Sequencing or NGS library), where such primers are designated adaptor-specific Primer A and adaptor-specific Primer B.
  • a hairpin detection probe is additionally included, wherein the hairpin portion is comprised of a unique polynucleotide sequence that is not complementary to any target sequence of the disclosure and which forms a hairpin structure at its 5′ end through a self-complementary domain and loop sequence, and where the 3′ domain of the hairpin probe is single-stranded and complementary to the 5′ domain of a competitor polynucleotide of this disclosure, designated C1, and where the hairpin probe additionally includes a nucleic acid modification, including a 5′ terminal fluorophore (or quencher), an internal quencher (or fluorophore) at the junction of the single stranded domain and the self-complementary hairpin structure, and additionally the single stranded domain contains a modified base that increases binding affinity for its complementary sequence C1.
  • a 5′ domain that is not complementary to the T 1 target but is complementary to the 3′ end of the single stranded domain of the hairpin detection probe is additionally included on the competitor polynucleotide, such a C1 cleavage product would dissociate from the T 1 target at an appropriate reaction temperature and retain the ability to anneal to the single stranded portion of the hairpin detection probe due to the increased length of complementarity.
  • a hairpin detection probe is incorporated on an additional 5′ domain of the competitor polynucleotide, wherein the hairpin domain is comprised of a unique polynucleotide sequence that is not complementary to any target sequence of the disclosure and which forms a hairpin structure at its 5′ end through a self-complementary domain and loop sequence, and where the 3′ domain of the polynucleotide competitor/probe is single-stranded and at its 3′ portion corresponds to the competitor polynucleotide of this disclosure and the 5′ portion of the single-stranded domain corresponds to a C1′ reverse complement of the C1 competitor cleavage product, and where the hairpin probe domain additionally includes a nucleic acid modification, including a 5′ terminal quencher, an internal fluorophore at the junction of the single stranded domain and the self-complementary hairpin structure, and where additionally the single stranded C1′ domain
  • a hydrolysis detection probe or a beacon detection probe is additionally included, wherein the hydrolysis detection probe or the beacon detection probe is comprised of a unique polynucleotide sequence complementary to the genomic sequence located between the primer target sequences T 2 and T 3 .
  • the disclosure provides a plurality of polynucleotide combinations, each comprising a polynucleotide competitor and flanking PCR primers, where a first competitor comprises a sequence that is fully complementary to a first target DNA polynucleotide region (T 1 ) and comprises a mismatch to a non-target DNA polynucleotide that is a sequence variant of the first target DNA polynucleotide region (T 1 *), and the first PCR primer pair comprises sequences that are fully complementary to a second target DNA polynucleotide region (T 2 ) and a third target DNA polynucleotide region (T 3 ), thus comprising a forward primer where its target sequence T 2 is located 5′ to the competitor target sequence T 1 (where T 2 and T 1 are either distinct or partially overlapping), and a reverse primer where its target sequence T 3 is located 3′ to the competitor target sequence T 1 , where the 3′ reverse primer is complementary to the strand that is the reverse complement of the strand
  • a second competitor comprises a sequence that is fully complementary to a fourth target DNA polynucleotide region (T 4 ) and comprises a mismatch to a non-target DNA polynucleotide that is a sequence variant of the fourth target DNA polynucleotide region (T 4 *), and a second PCR amplification primer pair comprises sequences that are fully complementary to a fifth target DNA polynucleotide region (T 5 ) and a sixth target DNA polynucleotide region (T 6 ) that flank T 4 and T 4 *, and in an ‘n’ plurality of polynucleotide combinations where ‘n’ competitors comprise sequences that are fully complementary to ‘n’ target DNA polynucleotide regions (n 1 ) and comprise a mismatch to non-target DNA polynucleotides that are sequence variants (n 1 *), and ‘n’ PCR primer pairs comprise sequences that are fully complementary to (n 2 ) and (n 3 ) target DNA polynucleotide regions that flank
  • Also contemplated in this disclosure is a method of cleaving a polynucleotide competitor that comprises a sequence that is fully complementary to a first target DNA polynucleotide region (T 1 ) such that the competitor polynucleotide will hybridize to its target under appropriate conditions.
  • the method comprises (i) contacting a first target polynucleotide with a polynucleotide competitor under conditions wherein the polynucleotide competitor hybridizes to the first target polynucleotide sequence to form a first competitor/target complex; (ii) contacting the competitor/target complex with an enzyme that specifically identifies and cleaves the competitor/target complex when it is a fully complementary complex; and (iii) optionally detecting the enzyme cleavage.
  • the polynucleotide competitor comprises a mismatch to a non-target DNA polynucleotide that is a sequence variant of the first target DNA polynucleotide region (T 1 *).
  • the disclosure further contemplates an aspect wherein the polynucleotide competitor comprises an RNA base that is at the position of the mismatched base to the non-target DNA region (T 1 *), or alternatively the mutation lies 1, 2 or 3 bases away either 5′ or 3′ to the RNA base.
  • the RNA base enables the polynucleotide competitor to be a substrate for cleavage by RNase H2 upon binding to its target DNA (T 1 ) and a less efficient substrate for cleavage by RNase H2 upon binding to its non-target DNA (T 1 *), where under appropriate reaction conditions known in the art, the addition of RNase H2 enzyme will lead to cleavage of the competitor polynucleotide at the position of the RNA base when it is annealed to its fully complementary target sequence T 1 and not when it remains unbound in single-stranded form or if it is annealed to a mismatched non-target sequence T 1 * or other mismatched non-target sequence.
  • RNA base within the competitor polynucleotide that is a substrate for cleavage by RNase H2 upon binding to its target DNA (T 1 ) and a less efficient substrate for cleavage by RNase H2 upon binding to its non-target DNA (T 1 *).
  • the disclosure further provides a method of cleaving a polynucleotide competitor that comprises a sequence that is fully complementary to a first target DNA polynucleotide region (T 1 ) such that the competitor polynucleotide will hybridize to its target under appropriate conditions.
  • the method comprises (i) contacting a first target polynucleotide with a polynucleotide competitor under conditions wherein the polynucleotide competitor hybridizes to the first target polynucleotide sequence to form a first competitor/target complex; (ii) contacting the competitor/target complex with an enzyme that specifically identifies and cleaves the competitor/target complex when it is a fully complementary complex; and (iii) optionally detecting the enzyme cleavage.
  • the polynucleotide competitor comprises a mismatch to a non-target DNA polynucleotide that is a sequence variant of the first target DNA polynucleotide region (T 1 *).
  • the disclosure further contemplates an aspect wherein the polynucleotide competitor has a domain comprising 4 or more consecutive RNA bases, one of which is at the position of the mismatched base to the non-target DNA region (T 1 *) or alternatively the mismatch lies 1, 2 or 3 DNA bases away either 5′ or 3′ to the RNA domain.
  • the domain comprising 4 or more RNA bases enables the polynucleotide competitor to be a substrate for cleavage by RNase H1 or RNase H2 upon binding to its target DNA (T 1 ) and a less efficient substrate for cleavage by RNase H1 or RNase H2 upon binding to its non-target DNA (T 1 *), where under appropriate reaction conditions known in the art, the addition of RNase H1 or RNase H2 enzyme will lead to cleavage of the competitor polynucleotide at a position within the 4 or more consecutive RNA bases when it is annealed to its fully complementary target sequence T 1 and not when it remains unbound in single-stranded form or if it is annealed to a mismatched non-target sequence T 1 * or other mismatched non-target sequence.
  • the disclosure provides a method of allelic enrichment by amplification utilizing a polynucleotide combination comprising a cleavable competitor and flanking non-allele-specific PCR primers, where the cleavable competitor comprises a sequence that is fully complementary to a first target DNA polynucleotide region (T 1 ) such that the competitor polynucleotide will hybridize to its target under appropriate conditions.
  • the method comprises (i) contacting a first target polynucleotide with a polynucleotide competitor under conditions wherein the polynucleotide competitor hybridizes to the first target polynucleotide sequence to form a first competitor/target complex; (ii) contacting the competitor/target complex with an enzyme that specifically identifies and cleaves the competitor/target complex when it is a fully complementary complex, wherein competitor cleavage leads to cleavage product dissociation and enables a DNA polymerase to extend the forward primer and amplification of T 1 occurs via PCR; and (iii) optionally detecting the enzyme cleavage.
  • the polynucleotide competitor comprises a mismatch to a non-target DNA polynucleotide that is a sequence variant of the first target DNA polynucleotide region (T 1 *).
  • the disclosure further contemplates an aspect wherein the polynucleotide competitor comprises an RNA domain that is at the position of the mismatched base to the non-target DNA region (T 1 *), or alternatively the mutation lies 1, 2 or 3 bases away either 5′ or 3′ to the RNA domain.
  • the RNA domain enables the polynucleotide competitor to be a substrate for cleavage by RNase H1 or RNase H2 upon binding to its target DNA (T 1 ) and a less efficient substrate for cleavage upon binding to its non-target DNA (T 1 *).
  • a PCR primer pair comprising sequences that are fully complementary to second (T 2 ) and third (T 3 ) target DNA polynucleotide regions, thus comprising a forward primer where its target sequence T 2 is located 5′ to the competitor target sequence T 1 (where T 2 and T 1 are either distinct or partially overlapping but where T 2 does not include the variant base between T 1 and T 1 *), and a reverse primer where its target sequence T 3 is located 3′ to the competitor target sequence T 1 , where the 3′ reverse primer is complementary to the strand that is the reverse complement of the strand which the forward primer and competitor hybridize, and where T 2 and T 3 target DNA polynucleotide regions do not vary in sequence relative to the target DNA polynucleotide T 1 and the non-target DNA polynucleotide T 1 *, where under appropriate reaction conditions known in the art, the addition of RNase H1 or RNase H2 enzyme will lead to cleavage of the competitor polynucleotide at a
  • the RNase H1 or RNase H2 cleavage and simultaneous PCR amplification are performed at two reaction temperatures following denaturation, the first of which enables annealing of the intact, uncleaved competitor but which reaction temperature exceeds the annealing temperature of the forward and reverse primers and competitor cleavage products C1 and C2 which subsequently dissociate from their template following cleavage by RNase H1 or RNase H2, whereby the second, lower temperature that still exceeds the annealing temperature of the competitor cleavage products C1 and C2 but enables the forward and reverse primers to bind the target T 1 template and allele-enriched amplification can proceed, whereby on the non-target T 1 * strand the uncleaved competitor prevents the forward primer from annealing.
  • the RNase H1 or RNase H2 cleavage and simultaneous PCR amplification are also performed at two reaction temperatures following denaturation, the first of which enables annealing of the intact, uncleaved competitor but which reaction temperature exceeds the annealing temperature of the forward and reverse primers and competitor cleavage products C1 and C2 which subsequently dissociate from their template following cleavage by RNase H1 or RNase H2, whereby the second, lower temperature that still exceeds the annealing temperature of the competitor cleavage products C1 and C2 but enables the forward and reverse primers to bind the target T 1 and T 1 * templates and specifically enables forward primer extension on the T 1 template whereby on the T 1 * template forward primer extension is blocked by the uncleaved competitor.
  • a hairpin detection probe is additionally included, wherein the hairpin portion is comprised of a unique polynucleotide sequence that is not complementary to any target sequence of the disclosure and which forms a hairpin structure at its 5′ end through a self-complementary domain and loop sequence, and where the 3′ domain of the hairpin probe is single-stranded and complementary to the 5′ domain of a competitor polynucleotide of this disclosure, designated C1, and where the hairpin probe additionally includes nucleic acid modifications, including a 5′ terminal fluorophore (or quencher), an internal quencher (or fluorophore) at the junction of the single stranded domain and the self-complementary hairpin structure, and additionally the single stranded domain may contain modified bases or additional sequence complementary to the C1 cleavage
  • the hairpin detection probe can be incorporated into the 5′ domain of the competitor, where upon competitor cleavage, the C1 cleavage portion can anneal to its C1′ complement and polymerase extension of the C1 cleavage product on the hairpin detection probe unfolds the self-complementary portion of the probe, thus physically separating the fluorophore from the quencher which were previously juxtaposed and where the proximity results in quenching of the fluorophore signal, and where physical separation produces a fluorescence signal, and where fluorescence detection indicates the presence of the target sequence T 1 and which fluorescence signal is increased proportionally during PCR amplification of the target sequence T 1 .
  • the allele-enriched cleavable competitor PCR does not involve a simultaneous detection step, where a subsequent allele-specific qPCR is performed to detect the presence of target sequence T 1 , or, the allele-enriched competitor PCR is performed on an NGS library or an NGS library is simultaneously or subsequently made from the product of the allele-enriched competitor PCR and detection of target sequence T 1 is performed by NGS analysis.
  • the disclosure provides a multiplexed method of allele-enriched cleavable competitor PCR (or allele-enriched PCR with cleavable competitor) using a plurality of polynucleotide combinations, each comprising a cleavable competitor and flanking non-allele-specific PCR primers.
  • the method comprises (i) contacting each target polynucleotide of the ‘n’ plurality with ‘n’ polynucleotide competitors under conditions wherein each polynucleotide competitor hybridizes to its corresponding target polynucleotide sequence to form a competitor/target complex; (ii) contacting ‘n’ competitor/target complexes with an enzyme that specifically identifies and cleaves the competitor/target complexes when they are fully complementary complexes, wherein competitor cleavage leads to cleavage product dissociation and enables a DNA polymerase to extend ‘n’ forward primers and PCR amplification of ‘n’ target polynucleotides occurs; and (iii) optionally detecting the enzyme cleavage.
  • a first competitor comprises a sequence that is fully complementary to a first target DNA polynucleotide region (T 1 ) and comprises a mismatch to a non-target DNA polynucleotide that is a sequence variant of the first target DNA polynucleotide region (T 1 *), and the first PCR amplification primer pair comprises sequences that are fully complementary to second (T 2 ) and third (T 3 ) target DNA polynucleotide regions, thus comprising a forward primer where its target sequence T 2 is located 5′ to the competitor target sequence T 1 (where T 2 and T 1 are either distinct or partially overlapping but where T 2 does not include the variant base between T 1 and T 1 *), and a reverse primer where its target sequence T 3 is located 3′ to the competitor target sequence T 1 , where the 3′ reverse primer is complementary to the strand that is the reverse complement of the strand to which the forward primer and competitor hybridize.
  • a second comprises a sequence that is fully complementary to a fourth target DNA polynucleotide region (T 4 ) and comprises a mismatch to a non-target DNA polynucleotide that is a sequence variant of the fourth target DNA polynucleotide region (T 4 *), and a second PCR amplification primer pair comprises sequences that are fully complementary to fifth (T 5 ) and sixth (T 6 ) target DNA polynucleotide regions that flank T 4 and T 4 *, and in an ‘n’ plurality of polynucleotide combinations where ‘n’ competitors comprise sequences that are fully complementary to ‘n’ target DNA polynucleotide regions (n 1 ) and comprise a mismatch to non-target DNA polynucleotides that are sequence variants (n 1 *), and ‘n’ PCR primer pairs comprise sequences that are fully complementary to (n 2 ) and (n 3 ) target DNA polynucleotide regions that flank n 1 and n 1 *, where under appropriate reaction conditions known
  • the RNase H1 or RNase H2 cleavage and simultaneous PCR amplification are performed at two reaction temperatures following denaturation, the first of which enables annealing of the intact, uncleaved competitor but which reaction temperature exceeds the annealing temperature of the forward and reverse primers and competitor cleavage products C1 and C2 which subsequently dissociate from their template following cleavage by RNase H1 or RNase H2, whereby the second, lower temperature that still exceeds the annealing temperature of the competitor cleavage products C1 and C2 but enables the forward and reverse primers to bind the target T 1 template and allele-enriched amplification can proceed, whereby on the non-target T 1 * strand the uncleaved competitor prevents the forward primer from annealing.
  • the RNase H1 or RNase H2 cleavage and simultaneous PCR amplification are also performed at two reaction temperatures following denaturation, the first of which enables annealing of the intact, uncleaved competitor but which reaction temperature exceeds the annealing temperature of the forward and reverse primers and competitor cleavage products C1 and C2 which subsequently dissociate from their template following cleavage by RNase H1 or RNase H2, whereby the second, lower temperature that still exceeds the annealing temperature of the competitor cleavage products C1 and C2 but enables the forward and reverse primers to bind the target T 1 and T 1 * templates and specifically enables forward primer extension on the T 1 template whereby on the T 1 * template forward primer extension is blocked by the uncleaved competitor.
  • an aspect of the method wherein a plurality of ‘n’ hairpin detection probes are additionally included for multiplexed detection, wherein each hairpin detection probe of the plurality utilizes a unique fluorophore that can be distinguished during the detection step of the reaction, where under appropriate reaction conditions, upon allele-specific cleavage of ‘n’ competitor polynucleotides, annealing and extension of ‘n’ C1 competitor cleavage products on ‘n’ hairpin detection probes unfolds the self-complementary portion of ‘n’ hairpin detection probes, thus physically separating ‘n’ fluorophores from their quenchers, and where physical separation produces a fluorescence signal, and where multiplexed fluorescence detection indicates the presence of target sequences ‘n 1 ’ and which fluorescence signals are increased proportionally during PCR amplification of target sequences ‘n 1 ’.
  • ‘n’ hairpin detection probes are additionally included for multiplexed detection, wherein each hairpin detection probe of the plurality utilizes a unique fluorophor
  • the multiplexed allele-enriched cleavable competitor PCR does not involve a simultaneous detection step, where a subsequent single or multiplexed allele-specific qPCR is performed to detect the presence of target sequences ‘n 1 ’, or, the multiplexed allele-enriched competitor PCR is performed on an NGS library or an NGS library is simultaneously or subsequently made from the product of the multiplexed allele-enriched competitor PCR and detection of target sequences ‘n 1 ’ is performed by NGS analysis.
  • the disclosure provides a method of allele-specific amplification utilizing a polynucleotide combination comprising a cleavable competitor and flanking allele-specific PCR primers, where the cleavable competitor comprises a sequence that is fully complementary to a first target DNA polynucleotide region (T 1 ) such that the competitor polynucleotide will hybridize to its target under appropriate conditions.
  • T 1 first target DNA polynucleotide region
  • the method comprises (i) contacting a first target polynucleotide with a polynucleotide competitor under conditions wherein the polynucleotide competitor hybridizes to the first target polynucleotide sequence to form a first competitor/target complex; (ii) contacting the competitor/target complex with an enzyme that specifically identifies and cleaves the competitor/target complex when it is a fully complementary complex, wherein competitor cleavage on the T 1 strand will lead to cleavage product dissociation and enable a DNA polymerase to extend the forward primer and amplification of T 1 occurs via PCR; and (iii) optionally detecting the enzyme cleavage.
  • the polynucleotide competitor comprises a mismatch to a non-target DNA polynucleotide that is a sequence variant of the first target DNA polynucleotide region (T 1 *).
  • the disclosure further contemplates an aspect wherein the polynucleotide competitor comprises an RNA domain that is at the position of the mismatched base to the non-target DNA region (T 1 *), or alternatively the mutation lies 1, 2 or 3 bases away either 5′ or 3′ to the RNA domain.
  • the RNA domain enables the polynucleotide competitor to be a substrate for cleavage by RNase H1 or RNase H2 upon binding to its target DNA (T 1 ) and a less efficient substrate for cleavage upon binding to its non-target DNA (T 1 *).
  • an allele-specific PCR primer pair where the primer pair comprises sequences that are fully complementary to second (T 2 ) and third (T 3 ) target DNA polynucleotide regions, thus comprising a forward primer where its target sequence T 2 is located 5′ to the competitor target sequence T 1 and overlaps with T 1 to include the variant base at the 3′ terminus of the forward primer (which results in an allele-specific primer), and a reverse primer where its target sequence T 3 is located 3′ to the competitor target sequence T 1 , where the 3′ reverse primer is complementary to the strand that is the reverse complement of the strand which the forward primer and competitor hybridize, and where the T 2 target DNA polynucleotide region varies in sequence relative to the target DNA polynucleotide T 1 and the non-target DNA polynucleotide T 1 *, where under appropriate reaction conditions known in the art, the addition of RNase H1 or RNase H2 enzyme will lead to cleavage of the competitor polynucleotide at a
  • the RNase H1 or RNase H2 cleavage and simultaneous PCR amplification are performed at two reaction temperatures following denaturation, the first of which enables annealing of the intact, uncleaved competitor but which reaction temperature exceeds the annealing temperature of the forward and reverse primers and competitor cleavage products C1 and C2 which subsequently dissociate from their template following cleavage by RNase H1 or RNase H2, whereby the second, lower temperature that still exceeds the annealing temperature of the competitor cleavage products C1 and C2 but enables the forward and reverse primers to bind the target T 1 template and allele-enriched amplification can proceed, whereby on the non-target T 1 * strand the uncleaved competitor prevents the forward primer from annealing.
  • a hairpin detection probe is additionally included, wherein the hairpin portion is comprised of a unique polynucleotide sequence that is not complementary to any target sequence of the disclosure and which forms a hairpin structure at its 5′ end through a self-complementary domain and loop sequence, and where the 3′ domain of the hairpin probe is single-stranded and complementary to the 5′ domain of a competitor polynucleotide of this disclosure, designated C1, and where the hairpin probe additionally includes nucleic acid modifications, including a 3′ terminal fluorophore (or quencher), an internal quencher (or fluorophore) at the junction of the single stranded domain and the self-complementary hairpin structure, and additionally the single stranded domain contains modified bases or additional 5′ sequence that increases the binding affinity for its complementary sequence C1, where under appropriate reaction conditions, upon allele-specific cleavage of the competitor
  • the hairpin detection probe can be incorporated into the 5′ domain of the competitor, where upon competitor cleavage, the C1 cleavage portion can anneal to its C1′ complement and polymerase extension of the C1 cleavage product on the hairpin detection probe unfolds the self-complementary portion of the probe, thus physically separating the fluorophore from the quencher which were previously juxtaposed and where the proximity results in quenching of the fluorophore signal, and where physical separation produces a fluorescence signal, and where fluorescence detection indicates the presence of the target sequence T 1 and which fluorescence signal is increased proportionally during PCR amplification of the target sequence T 1 .
  • the disclosure provides a multiplexed method of allele-specific competitor PCR using a plurality of polynucleotide combinations, each comprising a cleavable competitor and flanking allele-specific PCR primers.
  • the method comprises (i) contacting each target polynucleotide of the ‘n’ plurality with ‘n’ polynucleotide competitors under conditions wherein each polynucleotide competitor hybridizes to its corresponding target polynucleotide sequence to form a competitor/target complex; (ii) contacting ‘n’ competitor/target complexes with an enzyme that specifically identifies and cleaves the competitor/target complexes when they are fully complementary complexes, wherein competitor cleavage leads to cleavage product dissociation and enables a DNA polymerase to extend ‘n’ forward primers and PCR amplification of ‘n’ target polynucleotides occurs; and (iii) optionally detecting the enzyme cleavage.
  • a first competitor comprises a sequence that is fully complementary to a first target DNA polynucleotide region (T 1 ) and comprises a mismatch to a non-target DNA polynucleotide that is a sequence variant of the first target DNA polynucleotide region (T 1 *), and the first allele-specific PCR amplification primer pair comprises sequences that are fully complementary to second (T 2 ) and third (T 3 ) target DNA polynucleotide regions, thus comprising a forward primer where its target sequence T 2 is located 5′ to the competitor target sequence T 1 and overlaps with the variant base such that the forward primer has the variant base at its 3′ terminus, and a reverse primer where its target sequence T 3 is located 3′ to the competitor target sequence T 1 , where the 3′ reverse primer is complementary to the strand that is the reverse complement of the strand which the forward primer and competitor hybridize.
  • a second polynucleotide competitor comprises a sequence that is fully complementary to a fourth target DNA polynucleotide region (T 4 ) and comprises a mismatch to a non-target DNA polynucleotide that is a sequence variant of the fourth target DNA polynucleotide region (T 4 *), and a second allele-specific PCR primer pair comprises sequences that are fully complementary to fifth (T 5 ) and sixth (T 6 ) target DNA polynucleotide regions that flank T 4 and T 4 *, where the forward primer contains the variant base at its 3′ terminus, and in an ‘n’ plurality of polynucleotide combinations where ‘n’ competitors comprise sequences that are fully complementary to ‘n’ target DNA polynucleotide regions (n 1 ) and comprise a mismatch to non-target DNA polynucleotides that are sequence variants (n 1 *), and ‘n’ allele-specific PCR primer pairs comprise sequences that are fully complementary to (n 2 ) and (n 3
  • the RNase H1 or RNase H2 cleavage and simultaneous PCR amplification are performed at two reaction temperatures following denaturation, the first of which enables annealing of the intact, uncleaved competitor but which reaction temperature exceeds the annealing temperature of the forward and reverse primers and competitor cleavage products C1 and C2 which subsequently dissociate from their template following cleavage by RNase H1 or RNase H2, whereby the second, lower temperature that still exceeds the annealing temperature of the competitor cleavage products C1 and C2 but enables the forward and reverse primers to bind the target T 1 template and allele-enriched amplification can proceed, whereby on the non-target T 1 * strand the uncleaved competitor prevents the forward primer from annealing.
  • a plurality of ‘n’ hairpin detection probes are additionally included for multiplexed detection, wherein each hairpin detection probe of the plurality utilizes a unique fluorophore that can be distinguished during the detection step of the reaction, where under appropriate reaction conditions, upon allele-specific cleavage of ‘n’ competitor polynucleotides, annealing and extension of ‘n’ C1 competitor cleavage products on ‘n’ hairpin detection probes unfolds the self-complementary portion of ‘n’ hairpin detection probes, thus physically separating ‘n’ fluorophores from their quenchers, and where physical separation produces a fluorescence signal, and where multiplexed fluorescence detection indicates the presence of target sequences ‘n 1 ’ and which fluorescence signals are increased proportionally during PCR amplification of target sequences ‘n 1 ’.
  • ‘n’ hairpin detection probes are additionally included for multiplexed detection, wherein each hairpin detection probe of the plurality utilizes a unique fluorophore that can be distinguished during the
  • the disclosure provides a method of allelic enrichment amplification utilizing a polynucleotide combination comprising an extendable cleavable competitor and flanking PCR primers, where the extendable cleavable competitor comprises a sequence that is fully complementary to a non-target DNA polynucleotide region (T 1 *) such that the competitor polynucleotide will hybridize to the non-target under appropriate conditions.
  • the method comprises (i) contacting a first non-target polynucleotide with a polynucleotide competitor under conditions wherein the polynucleotide competitor hybridizes to the first non-target polynucleotide sequence to form a first competitor/non-target complex; (ii) contacting the competitor/non-target complex with an enzyme that specifically identifies and cleaves the competitor/non-target complex when it is a fully complementary complex, wherein competitor cleavage on the non-target strand will lead to 3′ cleavage product dissociation and enable a DNA polymerase to extend the 5′ cleavage product; and wherein when the reaction temperature is raised above the non-cleaved and non-extended cleavable competitor but remains below that of the cleaved and extended competitor, the non-cleaved and non-extended competitor dissociates from the target DNA strand enabling a forward primer to be extended by a DNA polymerase and target-specific PCR amplification
  • the polynucleotide competitor comprises a mismatch to a target DNA polynucleotide that is a sequence variant of the first non-target DNA polynucleotide region (T 1 ).
  • the extendable cleavable competitor comprises an RNA domain that is at the position of the mismatched base to the target DNA region (T 1 ), or alternatively the mutation lies 1, 2 or 3 bases away either 5′ or 3′ to the RNA domain.
  • the RNA domain enables the polynucleotide competitor to be a substrate for cleavage by RNase H1 or RNase H2 upon binding to the fully complementary non-target DNA (T 1 *) and a less efficient substrate for cleavage upon binding to the mismatched target DNA (T 1 ), and where the RNA domain is placed such that the 5′ cleavage product C1 remains bound following cleavage and the 3′ cleavage product C2 dissociates.
  • a PCR primer pair where the primer pair comprises sequences that are fully complementary to second (T 2 ) and third (T 3 ) target DNA polynucleotide regions, thus comprising a forward primer where its target sequence T 2 is located 5′ to the extendable competitor target sequence T 1 * and a reverse primer where its target sequence T 3 is located 3′ to the competitor target sequence T 1 *, where the 3′ reverse primer is complementary to the strand that is the reverse complement of the strand which the forward primer and competitor hybridize, and where T 2 and T 3 target DNA polynucleotide regions do not vary in sequence relative to the target DNA polynucleotide T 1 and the non-target DNA polynucleotide T 1 *, where under appropriate reaction conditions known in the art, the addition of RNase H1 or RNase H2 enzyme will lead to cleavage of the competitor polynucleotide at a position within the RNA domain or at the DNA-RNA junction when it is annealed to the fully complementary non-target sequence T
  • the RNase H1 or RNase H2 cleavage and simultaneous PCR amplification are performed at multiple reaction temperatures following denaturation, the first of which enables annealing of the intact, uncleaved competitor at temperature t 1 and is followed by annealing and partial extension of the forward primer, annealing and extension of the reverse primer and at the same time competitor cleavage and extension on the T 1 * non-target strand at temperature t 2 , then when the temperature t 3 exceeds the annealing temperature of the intact competitor which subsequently dissociates from the T 1 target template, allowing the forward primer to complete extension on the T 1 target strand but not the T 1 * non-target strand due to the stabilized extended competitor.
  • the allelic enriched extendable competitor PCR does not involve a simultaneous detection step, where a subsequent allele-specific qPCR is performed to detect the presence of target sequence T 1 , or, the allelic enriched extendable competitor PCR is performed on an NGS library or an NGS library is simultaneously or subsequently made from the product of the allelic enriched extendable competitor PCR and detection of target sequence T 1 is performed by NGS analysis.
  • the disclosure provides a multiplexed method of allelic enriched extendable competitor PCR using a plurality of polynucleotide combinations, each comprising an extendable cleavable competitor and flanking PCR primers.
  • the method comprises (i) contacting ‘n’ non-target polynucleotides with polynucleotide competitors under conditions wherein the polynucleotide competitors hybridize to their corresponding non-target polynucleotide sequences to form ‘n’ competitor/non-target complexes; (ii) contacting the competitor/non-target complexes with an enzyme that specifically identifies and cleaves the competitor/non-target complexes when they are fully complementary complexes, wherein competitor cleavage of ‘n’ non-target strands will lead to 3′ cleavage product dissociation and enable a DNA polymerase to extend ‘n’ 5′ cleavage products, and wherein when the reaction temperature is raised above the non-cleaved and non-extended
  • a first competitor comprises a sequence that is fully complementary to a first non-target DNA polynucleotide region (T 1 *) and comprises a mismatch to a target DNA polynucleotide that is a sequence variant of the first target DNA polynucleotide region (T 1 ), and the first PCR amplification primer pair comprises sequences that are fully complementary to second (T 2 ) and third (T 3 ) target DNA polynucleotide regions, thus comprising a forward primer where its target sequence T 2 is located 5′ to the competitor target sequence T 1 and a reverse primer where its target sequence T 3 is located 3′ to the competitor target sequence T 1 , where the 3′ reverse primer is complementary to the strand that is the reverse complement of the strand which the forward primer and competitor hybridize.
  • a second polynucleotide combination comprises a sequence that is fully complementary to a second non-target DNA polynucleotide region (T 4 *) and comprises a mismatch to a target DNA polynucleotide that is a sequence variant of the fourth target DNA polynucleotide region (T 4 ), and a second PCR amplification primer pair comprises sequences that are fully complementary to fifth (T 5 ) and sixth (T 6 ) target DNA polynucleotide regions that flank T 4 and T 4 *, and in an ‘n’ plurality of polynucleotide combinations where ‘n’ competitors comprise sequences that are fully complementary to ‘n’ non-target DNA polynucleotide regions (n 1 *) and comprise a mismatch to target DNA polynucleotides that are sequence variants (n 1 ), and ‘n’ PCR primer pairs comprise sequences that are fully complementary to (n 2 ) and (n 3 ) target DNA polynucleotide regions that flank n 1 and n 1
  • the target sequences ‘n 1 ’ will be selectively amplified over the non-target sequences ‘n 1 *’, where competitor cleavage on each ‘n 1 *’ strand will lead to 3′ competitor cleavage product dissociation and enable a DNA polymerase to extend the 5′ competitor cleavage product and increase its melting temperature, and as the temperature is elevated, the uncleaved competitor on the ‘n 1 ’ strand will dissociate to enable the forward primer to extend on the ‘n 1 ’ target strand and ‘n 1 ’ PCR amplification can occur, where on each non-target ‘n 1 *’ strand, the stabilized extended competitor polynucleotide will remain annealed and prevent a DNA polymerase from extending the forward primer on the non-target ‘n 1 *’ template and ‘n 1 *’ PCR amplification will be suppressed.
  • the RNase H1 or RNase H2 cleavage and simultaneous PCR amplification are performed at multiple reaction temperatures following denaturation, the first of which enables annealing of the intact, uncleaved competitors at temperature t 1 and is followed by annealing and partial extension of the forward primers, annealing and extension of the reverse primers and at the same time competitor cleavage and extension on the T 1 * non-target strands at temperature t 2 , then when the temperature t 3 exceeds the annealing temperature of the intact competitors which subsequently dissociate from the T 1 target templates, allowing the forward primer to complete extension on the T 1 target strand but not the T 1 * non-target strand due to the stabilized extended competitor.
  • the allele enriched extendable competitor PCR involves a simultaneous detection step, or alternatively, a subsequent allele-specific qPCR is performed to detect the presence of target sequence T 1 , or, the allelic enriched extendable competitor PCR is performed on an NGS library or an NGS library is simultaneously or subsequently made from the product of the allele enriched extendable competitor PCR and detection of target sequence T 1 is performed by NGS analysis.
  • any of the polynucleotide combinations provided herein comprise a modified nucleic acid.
  • the competitor further comprises a modified nucleic acid, and in various embodiments of these aspects, the modified nucleic acid is in the PCR amplification primer pair and/or the modified nucleic acid is in the hairpin detection probe.
  • the competitor comprises a plurality of modified nucleic acids
  • the PCR amplification primer pairs and hairpin detection probe comprise a plurality of modified nucleic acids
  • the polynucleotide competitor further comprises a blocking group at its 3′ end which blocks extension from a DNA polymerase.
  • the blocking group is selected from the group consisting of a 3′ phosphate group, a 3′ amino group, a dideoxy nucleotide, a C3 spacer and an inverted deoxythymidine (dT).
  • the polynucleotide competitor further comprises a modified internucleotide linkage which blocks cleavage by RNase H1 or RNase H2.
  • the nuclease resistant linkages are selected from the group consisting of a 2′-propoxyamine, 2′-methoxy, 2′-propoxy, 2′-methoxy-ethoxy, 2′-fluoro, phosphorothioate, methylene methylimino substitution of the phosphodiester linkage.
  • the amplification primer pair further comprises nuclease resistant linkages between bases at their 3′ ends or nuclease resistant nucleotides which block exonuclease cleavage by a proofreading DNA polymerase.
  • the nuclease resistant linkages are selected from the group consisting of a 3′ phosphorothioate and a 2′-O methyl RNA.
  • the hairpin detection probe further comprises modified nucleic acids that increase the binding affinity of the probe to its complementary sequence, the C1 competitor cleavage product.
  • the modified bases are selected from the group consisting of a locked nucleic acid (LNA), a minor groove binder (MGB), or a peptide nucleic acid (PNA).
  • the hairpin detection probe comprises a label.
  • the label is located in the hairpin detection probe at its 3′ end and/or the label is quenchable.
  • the hairpin detection probe also comprises a quencher and/or the quencher is located at the junction of the single-stranded domain and the double-stranded hairpin domain.
  • the quencher is selected from the group consisting of Black Hole Quencher 1, Black Hole Quencher-2, Iowa Black FQ, Iowa Black RQ, and Dabcyl. G-base.
  • a composition comprising a first polynucleotide and a second polynucleotide, wherein: (A) the first polynucleotide comprises a sequence such that: (i) the first polynucleotide has a fully complementary domain to a target polynucleotide (T1) such that the first polynucleotide is able to hybridize to T1 under appropriate conditions, and the sequence comprises a RNA base that is susceptible to cleavage by a ribonuclease when the RNA base is hybridized to T1; and (ii) the first polynucleotide is mismatched to a non-target polynucleotide region (T1*) at the position of the RNA base or 1, 2 or 3 nucleotides adjacent to the RNA base; and (iii) T1* is a sequence variant of T1; and (B) the second polynucleotide comprises a sequence such that: (iv) the second polynucleo
  • Paragraph 2 The composition of paragraph 1, wherein the RNA base on the first polynucleotide is located at the midpoint of the first polynucleotide.
  • Paragraph 3 The composition of paragraph 1 or paragraph 2, further comprising at least one additional RNA base on the first polynucleotide located immediately adjacent to the first RNA base.
  • Paragraph 4 The composition of paragraph 3 wherein the first polynucleotide comprises at least 4 consecutive RNA bases.
  • Paragraph 5 The composition of paragraph 4 wherein one or more RNA bases are susceptible to cleavage by a ribonuclease when the polynucleotide is hybridized to the target sequence T1.
  • Paragraph 6 The composition of paragraph 5 wherein modified nucleotides at one or more RNA bases renders the one or more bases resistant to cleavage by a ribonuclease.
  • Paragraph 7 The composition of paragraphs 1-6 wherein the 3′ terminus of the first polynucleotide is blocked from initiation of extension by a DNA polymerase.
  • Paragraph 8 The composition of any one of paragraphs 1-7 wherein the first polynucleotide comprises a detectable marker and a moiety that quenches the detectable marker.
  • Paragraph 9 The composition of paragraph 8 wherein the detectable marker and the moiety are on opposite sides of the RNA base, and in a configuration that prevents detection of the detectable marker.
  • Paragraph 10 The composition of paragraph 9 wherein cleavage of the first polynucleotide results in detection of the detectable marker.
  • Paragraph 11 The composition of any one of paragraphs 1-10 wherein T2 overlaps T1 and T1* by at least about 1 to at least about 50 nucleotides.
  • Paragraph 12 The composition of any one of paragraphs 1-11 wherein the ribonuclease includes but is not limited to RNase H2 or RNase H1.
  • Paragraph 13 A method of initiating polymerase extension on a target polynucleotide in a sample using the composition of any one of paragraphs 1-12; wherein the sample comprises a target polynucleotide that comprises (i) a sequence T1 in a first region that is fully complementary to the sequence of a domain in the first polynucleotide; and (ii) a sequence T2 that is fully complementary to the sequence in the second polynucleotide; the method comprising the step of (a) contacting the sample with the composition and a polymerase under conditions that allow extension of a sequence from T2 following cleavage and dissociation of the first polynucleotide.
  • Paragraph 14 A method of amplifying a target polynucleotide in a sample using the composition of any one of paragraphs 1-12, wherein: the sample comprises a mixture of (i) a target polynucleotide having a sequence in a first region (T1) that is fully complementary to the sequence of a domain in the first polynucleotide, and a sequence in a second region (T2) that is fully complementary to the sequence in the second polynucleotide; and (ii) one or more non-target polynucleotides that are not fully complementary to T1; the method comprising the steps of: (a) contacting the sample with the composition and a polymerase under conditions that allow extension of a sequence (S) from T2, wherein the sequence is complementary to the target polynucleotide when the target polynucleotide is present in the sample; (b) denaturing the sequence (S) extended from T2 from the target polynucleotide, and (c) repeating step (
  • Paragraph 15 The method of paragraph 14, further comprising: (iii) a fourth polynucleotide having a sequence that is fully complementary to a region T4 in a second target polynucleotide in the sample, such that the fourth polynucleotide is able to hybridize to T4 under appropriate conditions, and the sequence comprises a RNA base that is susceptible to cleavage by a ribonuclease when the RNA base is hybridized to T4; and (iv) a fifth polynucleotide having a sequence that is fully complementary to a region T5 in a second target polynucleotide in the sample, wherein T5 overlaps T4 and T4* by at least one nucleotide, and wherein T5 is upstream of T4 and T4*; wherein (v) the fourth polynucleotide is mismatched to a non-target polynucleotide region (T4*) at the position of the RNA base or 1, 2 or 3 bases
  • Paragraph 16 The method of any one of paragraphs 13-15 further comprising the step of adding a ribonuclease at step (a).
  • FIG. 1 depicts various example compositions of a cleavable competitor polynucleotide.
  • FIG. 2 depicts annealing of a cleavable competitor to its matched target (T1) and to its mismatched non-target (T1*) templates, where RNase H2 cleavage occurs only on the fully complementary target template.
  • FIG. 3 depicts how RNase H1 enzyme cleaves the matched hybrid on the target sequence T1 whereas the mismatched non-target hybrid T1* is cleavage-resistant.
  • FIG. 4 depicts how RNase H2 enzyme cleaves the matched hybrid on the target sequence T1 whereas the mismatched non-target hybrid T1* is cleavage-resistant.
  • FIG. 5 depicts how RNase H1 enzyme cleaves the matched hybrid on the target sequence T1 whereas the mismatched non-target hybrid T1* is cleavage-resistant.
  • FIG. 6 depicts various cleavable competitor and primer combinations disclosed herein.
  • FIG. 7 depicts how target-enriched PCR amplification can be achieved using a cleavable polynucleotide competitor.
  • FIG. 8 a - d provide a detailed description of how target-enriched PCR amplification can be achieved using a cleavable polynucleotide competitor.
  • FIG. 9 depicts the thermocycling profile for the example outlined in FIGS. 7 and 8 .
  • FIG. 10 depicts how target-enriched PCR amplification can be achieved using a cleavable polynucleotide competitor and overlapping forward primer.
  • FIG. 11 depicts how target-specific PCR amplification can be achieved using a cleavable polynucleotide competitor and an overlapping target-specific forward primer.
  • FIG. 12 depicts how target-specific PCR amplification can be achieved using a cleavable polynucleotide competitor and an overlapping target-specific 2-polynucleotide primer.
  • FIG. 13 a - d provide a detailed description of how target-enriched PCR amplification can be achieved using a cleavable polynucleotide competitor and an overlapping forward primer.
  • FIG. 14 depicts the thermocycling profile for the example outlined in FIGS. 10 and 13 .
  • FIG. 15 depicts target-enriched PCR using a non-target specific extendable cleavable competitor.
  • FIG. 16 a - d provides a detailed description of how target-enriched PCR can be achieved using a non-target specific extendable cleavable competitor.
  • FIG. 17 depicts a thermocycling profile for the method outlined in FIGS. 15 and 16 .
  • FIG. 18 depicts multiplexed target-enriched PCR amplification for 3 target sequences in an NGS library.
  • FIG. 19 depicts multiplexed target-enriched PCR amplification for ‘n’ target sequences in an NGS library.
  • FIG. 20 depicts how competitor cleavage product C1 anneals and extends on the hairpin probe to generate a detectable signal.
  • FIG. 21 depicts target-specific PCR amplification combined with hairpin probe detection.
  • FIG. 22 depicts how a hairpin detection probe can be incorporated onto a portion of the competitor polynucleotide when it is designed to cleave on the target strand T 1 .
  • FIG. 23 depicts an NGS amplicon where adaptor sequence A overlaps with the Competitor sequence.
  • FIG. 24 depicts a Competitor with a 5′ non-genomic domain to increase stability of the C1/Hairpin Probe interaction.
  • FIG. 25 depicts a Cleavable Competitor with a 5′ non-genomic domain and Cleavable Probe.
  • FIG. 26 depicts a Cleavable Competitor as a Cleavable Probe.
  • FIG. 27 shows the sequence specificity of RNase H1 for a RNA/DNA hybrid for a sequence with efficient cleavage versus a sequence with inefficient cleavage. It also demonstrates the mismatch discrimination ability of RNase H1 as it cleaves the matched hybrids much more efficiently than mismatched hybrids.
  • FIG. 28 depicts the kinetics of cleavage of RNase H1 for an RNA/DNA hybrid sequence with efficient cleavage versus an RNA/DNA hybrid sequence with inefficient cleavage.
  • FIG. 29 depicts how an overlapping primer is better than a non-overlapping primer in terms of inhibition of the wild-type signal with a competitor.
  • FIG. 30 depicts how competitor and RNase H1 can be used in PCR to discriminate between wild type and mutant templates.
  • the disclosure is based on the discovery of a method by which RNase H1 and RNaseH2 can be used to enrich or amplify target DNA molecules and prevent amplification of non-target DNA molecules.
  • the disclosure also provides a method to multiplex various target DNA molecules in a single tube with high sensitivity and specificity.
  • the disclosure further provides the use of RNAseH1 and H2 for a novel detection method using hairpin shaped probes which can be used in qPCR to quantify amplified target DNA. These aspects are useful in qPCR and NGS diagnostic assays.
  • “fully complementary” means that two polynucleotides share 100% complementarity over the full length nucleotide sequence of any of the polynucleotides disclosed herein.
  • a cleavable competitor that is 20 nucleotides in length is fully complementary to a target polynucleotide if all 20 nucleotides can base pair with a region of the target polynucleotide.
  • polynucleotide either as a component of a polynucleotide combination, including cleavable competitor polynucleotides, primers and probes, or as a target molecule, is used interchangeably with the term oligonucleotide.
  • nucleotide or its plural as used herein is interchangeable with modified forms as discussed herein and otherwise known in the art.
  • base which embraces naturally-occurring nucleotides as well as modifications of nucleotides that can be polymerized.
  • methods provided include use of polynucleotides which are DNA oligonucleotides, RNA oligonucleotides, or combinations of the two types. Modified forms of oligonucleotides are also contemplated which include those having at least one modified internucleotide linkage. Modified polynucleotides or oligonucleotides are described in detail herein below.
  • oligonucleotides include those containing modified backbones or non-natural internucleoside linkages. Oligonucleotides having modified backbones include those that retain a phosphorus atom in the backbone and those that do not have a phosphorus atom in the backbone. Modified oligonucleotides that do not have a phosphorus atom in their internucleoside backbone are considered to be within the meaning of “oligonucleotide.”
  • the competitor polynucleotide comprises phosphorothioate linkages.
  • Modified oligonucleotide backbones containing a phosphorus atom include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates including 3′-alkylene phosphonates, 5′-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates including 3′-amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, selenophosphates and boranophosphates having normal 3′-5′ linkages, 2′-5′ linked analogs of these, and those having inverted polarity wherein one or more internucleotide linkages is a 3′ to 3′, 5′ to 5′ or 2′ to 2′ linkage.
  • oligonucleotides having inverted polarity comprising a single 3′ to 3′ linkage at the 3′-most internucleotide linkage, i.e. a single inverted nucleoside residue which may be abasic (the nucleotide is missing or has a hydroxyl group in place thereof). Salts, mixed salts and free acid forms are also contemplated. Representative United States patents that teach the preparation of the above phosphorus-containing linkages include, U.S. Pat. Nos.
  • Modified oligonucleotide backbones that do not include a phosphorus atom therein have backbones that are formed by short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkyl or cycloalkyl internucleoside linkages, or one or more short chain heteroatomic or heterocyclic internucleoside linkages.
  • oligonucleotide mimetics wherein both one or more sugar and/or one or more internucleotide linkage of the nucleotide units are replaced with “non-naturally occurring” groups.
  • this embodiment contemplates a peptide nucleic acid (PNA).
  • PNA compounds the sugar-backbone of an oligonucleotide is replaced with an amide containing backbone. See, for example U.S. Pat. Nos. 5,539,082; 5,714,331; and 5,719,262, and Nielsen et al., 1991 , Science, 254: 1497-1500, the disclosures of which are herein incorporated by reference.
  • oligonucleotides are provided with phosphorothioate backbones and oligonucleosides with heteroatom backbones, and including —CH 2 —NH—O—CH 2 —, —CH 2 —N(CH 3 )—O—CH 2 —, —CH 2 —O—N(CH 3 )—CH 2 —, —CH 2 —N(CH 3 )—N(CH 3 )—CH 2 — and —O—N(CH 3 )—CH 2 —CH 2 — described in U.S. Pat. Nos. 5,489,677, and 5,602,240. Also contemplated are oligonucleotides with morpholino backbone structures described in U.S. Pat. No. 5,034,506.
  • the linkage between two successive monomers in the oligo consists of 2 to 4, desirably 3, groups/atoms selected from —CH 2 —, —O—, —S—, —NR H —, >C ⁇ O, >C ⁇ NR H , >C ⁇ S, —Si(R′′) 2 —, —SO—, —S(O) 2 —, —P(O) 2 —, —PO(BH 3 )—, —P(O,S)—, —P(S) 2 —, —PO(R′′)—, —PO(OCH 3 )—, and —PO(NHR H )—, where RH is selected from hydrogen and C 1-4 -alkyl, and R′′ is selected from C 1-6 -alkyl and phenyl.
  • linkages are —CH 2 —CH 2 —CH 2 —, —CH 2 —CO—CH 2 —, —CH 2 —CHOH—CH 2 —, —O—CH 2 —O—, —O—CH 2 —CH 2 —, —O—CH 2 —CH ⁇ (including R 5 when used as a linkage to a succeeding monomer), —CH 2 —CH 2 —O—, —NR H —CH 2 —CH 2 —, —CH 2 —CH 2 —NR H —, —CH 2 —NR H —CH 2 —, —O—CH 2 —CH 2 —NR H —, —NR H —CO—O—, —NR H —CO—NR H —, —NR H —CS—NR H —, —NR H —C( ⁇ NR H )—NR H —, —NR H —CO—CH 2 —NR H —O—CO
  • Modified oligonucleotides may also contain one or more substituted sugar moieties.
  • oligonucleotides comprise one of the following at the 2′ position: OH; F; O—, S—, or N-alkyl; O—, S—, or N-alkenyl; O—, S- or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl, alkenyl and alkynyl may be substituted or unsubstituted C 1 to C 10 alkyl or C 2 to C 10 alkenyl and alkynyl.
  • oligonucleotides comprise one of the following at the 2′ position: C 1 to C 10 lower alkyl, substituted lower alkyl, alkenyl, alkynyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH 3 , OCN, Cl, Br, CN, CF 3 , OCF 3 , SOCH 3 , SO 2 CH 3 , ONO 2 , NO 2 , N 3 , NH2, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, an RNA cleaving group, a reporter group, an intercalator, a group for improving the pharmacokinetic properties of an oligonucleotide, or a group for improving the pharmacodynamic properties of an oligonucleotide, and other substituents having similar properties.
  • a modification includes 2′-methoxyethoxy (2′-O—CH 2 CH 2 OCH 3 , also known as 2′-O-(2-methoxyethyl) or 2′-MOE) (Martin et al., 1995 , Helv. Chim. Acta, 78: 486-504) i.e., an alkoxyalkoxy group.
  • 2′-dimethylaminooxyethoxy i.e., a O(CH 2 ) 2 ON(CH 3 ) 2 group, also known as 2′-DMAOE, as described in examples herein below
  • 2′-dimethylaminoethoxyethoxy also known in the art as 2′-O-dimethyl-amino-ethoxy-ethyl or 2′-DMAEOE
  • 2′-O—CH 2 —O—CH 2 —N(CH 3 ) 2 also described in examples herein below.
  • Still other modifications include 2′-methoxy (2′-O—CH 3 ), 2′-aminopropoxy (2′-OCH 2 CH 2 CH 2 NH 2 ), 2′-allyl (2′-CH 2 —CH ⁇ CH 2 ), 2′-O-allyl (2′-O—CH 2 —CH ⁇ CH 2 ) and 2′-fluoro (2′-F).
  • the 2′-modification may be in the arabino (up) position or ribo (down) position.
  • a 2′-arabino modification is 2′-F.
  • Oligonucleotides may also have sugar mimetics such as cyclobutyl moieties in place of the pentofuranosyl sugar. See, for example, U.S. Pat. Nos.
  • a modification of the sugar includes Locked Nucleic Acids (LNAs) in which the 2′-hydroxyl group is linked to the 3′ or 4′ carbon atom of the sugar ring, thereby forming a bicyclic sugar moiety.
  • LNAs Locked Nucleic Acids
  • the linkage in certain aspects is a methylene (—CH 2 —) n group bridging the 2′ oxygen atom and the 4′ carbon atom wherein n is 1 or 2.
  • LNAs and preparation thereof are described in WO 98/39352 and WO 99/14226, the disclosures of which are incorporated by reference in their entireties herein.
  • the hairpin probe polynucleotide comprises a locked nucleic acid.
  • the hairpin probe polynucleotide comprises a plurality of locked nucleic acids.
  • Polynucleotides may also include base modifications or substitutions.
  • “unmodified” or “natural” bases include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) and uracil (U).
  • Modified bases include other synthetic and natural bases such as 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl uracil and cytosine and other alkynyl derivatives of pyrimidine bases, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo particularly 5-bromo, 5-trifluoromethyl
  • Further modified bases include tricyclic pyrimidines such as phenoxazine cytidine(1H-pyrimido[5,4-b][1,4]benzoxazin-2(3H)-one), phenothiazine cytidine (1H-pyrimido[5,4-b][1,4]benzothiazin-2(3H)-one), G-competitors such as a substituted phenoxazine cytidine (e.g.
  • Modified bases may also include those in which the purine or pyrimidine base is replaced with other heterocycles, for example 7-deaza-adenine, 7-deazaguanosine, 2-aminopyridine and 2-pyridone. Further bases include those disclosed in U.S. Pat. No.
  • Certain of these bases are useful for increasing the binding affinity and include 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and O-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine.
  • 5-methylcytosine substitutions have been shown to increase nucleic acid duplex stability by 0.6-1.2° C. and are, in certain aspects combined with 2′-O-methoxyethyl sugar modifications. See, U.S. Pat. No. 3,687,808, U.S. Pat. Nos.
  • a “modified base” or other similar term refers to a composition which can pair with a natural base (e.g., adenine, guanine, cytosine, uracil, and/or thymine) and/or can pair with a non-naturally occurring base.
  • the modified base provides a T m differential of 15, 12, 10, 8, 6, 4, or 2° C. or less.
  • Exemplary modified bases are described in EP 1 072 679 and WO 97/12896.
  • nucleobase is meant the naturally occurring nucleobases adenine (A), guanine (G), cytosine (C), thymine (T) and uracil (U) as well as non-naturally occurring nucleobases such as xanthine, diaminopurine, 8-oxo-N 6 -methyladenine, 7-deazaxanthine, 7-deazaguanine, N 4 ,N 4 -ethanocytosin, N′,N′-ethano-2,6-diaminopurine, 5-methylcytosine (mC), 5-(C 3 -C 6 )-alkynyl-cytosine, 5-fluorouracil, 5-bromouracil, pseudoisocytosine, 2-hydroxy-5-methyl-4-triazolopyridin, isocytosine, isoguanine, inosine and the “non-naturally occurring” nucleobases described in Benner et al.
  • nucleobase thus includes not only the known purine and pyrimidine heterocycles, but also heterocyclic analogues and tautomers thereof. Further naturally and non-naturally occurring nucleobases include those disclosed in U.S. Pat. No. 3,687,808 (Merigan, et al.), in Chapter 15 by Sanghvi, in Antisense Research and Application, Ed. S. T. Crooke and B.
  • nucleosidic base or “base unit” is further intended to include compounds such as heterocyclic compounds that can serve like nucleobases including certain “universal bases” that are not nucleosidic bases in the most classical sense but serve as nucleosidic bases.
  • universal bases are 3-nitropyrrole, optionally substituted indoles (e.g., 5-nitroindole), and optionally substituted hypoxanthine.
  • Other desirable universal bases include, pyrrole, diazole or triazole derivatives, including those universal bases known in the art.
  • a cleavable competitor polynucleotide has 10 nucleotides that are complementary to a target polynucleotide region.
  • the cleavable competitor polynucleotide has at least 11 nucleotides, at least 12 nucleotides, at least 13 nucleotides, at least 14 nucleotides, at least 15 nucleotides, at least 16 nucleotides, at least 17 nucleotides, at least 18 nucleotides, at least 19 nucleotides, at least 20 nucleotides, at least 21 nucleotides, at least 22 nucleotides, at least 23 nucleotides, at least 24 nucleotides, at least 25 nucleotides, at least 26 nucleotides, at least 27 nucleotides, at least 28 nucleotides, at least 29 nucleotides, at least 30 nucleotides, at least 31 nucleotides, at least 32 nucle
  • the PCR amplification primers each comprise at least 10 nucleotides in unique DNA sequence that are sufficiently complementary to second and third target polynucleotide regions as to allow hybridization between these complementary sequences under appropriate conditions.
  • the PCR amplification primer polynucleotides comprise at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24 nucleotides, at least 25, at least about 30, at least about 35, at least about 40, at least about 45, at least about 50, at least about 60 or more nucleotides of a unique DNA sequence that is sufficiently complementary to the second and third target polynucleotide regions as to allow hybridization between the complementary sequences under appropriate conditions.
  • the reverse primer polynucleotide is sufficiently complementary to a region of a polymerase-extended first polynucleotide so as to allow hybridization under appropriate conditions.
  • the reverse primer when the target polynucleotide is a double-stranded polynucleotide, the reverse primer is complementary to a complementary strand of the target polynucleotide.
  • the reverse primer is a combination of first and second polynucleotides, as defined herein.
  • the hairpin probe polynucleotide comprises a first domain containing about 5 nucleotides, this first domain of the hairpin probe polynucleotide being complementary to a target DNA region C1 that is the cleavage product from a corresponding competitor.
  • the second polynucleotide comprises a first domain containing at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, at least 31, at least 32, at least 33, at least 34, at least 35, at least 36, at least 37, at least 38, at least 39, at least 40, at least 41, at least 42, at least 43, at least 44, at least 45, at least 46, at least 47, at least 48, at least 49, at least 50 or more nucleotides, the first domain of this hairpin probe polynucleotide being complementary, or sufficiently complementary, so as to recognize and bind to a C1 target DNA region that is derived from cleavage of its corresponding competitor polynucleotide.
  • the second domain of the hairpin probe polynucleotide comprises 10 nucleotides of a unique DNA sequence that is sufficiently self-complementary so as to allow hairpin formation under appropriate conditions.
  • the second domain of the second polynucleotide comprises at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least about 30, at least about 35, at least 40, at least about 45, at least about 50, at least about 60 or more nucleotides of a unique DNA sequence that is sufficiently self-complementary so as to allow hairpin formation between the two sufficiently complementary sequences under appropriate conditions.
  • compositions and methods described herein include a second set of polynucleotides with the characteristics described above for competitor, primer and probe polynucleotides.
  • a plurality of sets is contemplated. These additional sets of competitor, primer and probe polynucleotides can have any of the characteristics described for competitor, primer and probe polynucleotides.
  • the competitor polynucleotide is comprised of DNA, modified DNA, RNA, modified RNA, PNA, or combinations thereof.
  • the primer and probe polynucleotides are comprised of DNA, modified DNA, RNA, modified RNA, PNA, or combinations thereof.
  • Blocking groups are incorporated as needed when polymerase extension from a 3′ region of a polynucleotide is undesirable.
  • the competitor and probe polynucleotides in another aspect, further comprise a blocking group at the 3′ end to prevent extension by an enzyme that is capable of synthesizing a nucleic acid.
  • Blocking groups useful in the practice of the methods include but are not limited to a 3′ phosphate group, a 3′ amino group, a dideoxy nucleotide, a six carbon glycol spacer (and in one aspect the six carbon glycol spacer is hexanediol) and inverted deoxythymidine (dT).
  • Stringent conditions can be determined empirically by the worker of ordinary skill in the art and will vary based on, e.g., the length of the primer, complementarity of the primer, concentration of the primer, the salt concentration (i.e., ionic strength) in the hybridization buffer, the temperature at which the hybridization is carried out, length of time that hybridization is carried out, and presence of factors that affect surface charge of the polynucleotides.
  • stringent conditions are those in which the polynucleotide is able to bind to its complementary sequence preferentially and with higher affinity relative to any other region on the target.
  • Exemplary stringent conditions for hybridization to its complement of a polynucleotide sequence having 20 bases include without limitation about 50% G+C content, 50 mM salt (Na + ), and an annealing temperature of 60° C. For a longer sequence, specific hybridization is achieved at higher temperature. In general, stringent conditions are such that annealing is carried out about 5° C. below the melting temperature of the polynucleotide.
  • the “melting temperature” is the temperature at which 50% of polynucleotides are complementary to a target polynucleotide in equilibrium at definite ion strength, pH and polynucleotide concentration.
  • polynucleotide primer combinations of the present invention can be used to prime either one or both ends of a given PCR amplicon.
  • an “amplicon” is understood to mean a portion of a polynucleotide that has been synthesized using amplification techniques. It is contemplated that any of the methods of the present invention that comprise more than one polynucleotide combination may utilize any combination of standard primer and polynucleotide combination, provided at least one of the primers is a polynucleotide combination as described herein.
  • the target polynucleotide includes but is not limited to chromosomal DNA, genomic DNA, plasmid DNA, cDNA, RNA, a synthetic polynucleotide, a single stranded polynucleotide, or a double stranded polynucleotide.
  • multiplex PCR is performed using at least two polynucleotide primers to amplify more than one polynucleotide product.
  • each polynucleotide primer used for multiplex PCR is a polynucleotide combination as disclosed herein.
  • at least one polynucleotide primer used for multiplex PCR is a polynucleotide combination as disclosed herein.
  • Primer combinations with cleavable competitors and hairpin probes are useful for real-time PCR.
  • Analysis and quantification of rare transcripts, detection of limiting pathogens, diagnostics of rare cancer cells with mutations, or low levels of aberrant gene methylation in cancer patients are the problems that can be solved by improved real-time PCR assays that combine high sensitivity and specificity of target amplification, high specificity of target detection, the ability to selectively amplify and detect a small number of cancer-specific mutant alleles or abnormally methylated promoters in the presence of thousands of copies of normal DNA, analysis and quantification of low copy number RNA transcripts, detection of fluorescence traces the ability to multiplex 4-5 different targets in one assay to maximally utilize capabilities of current real-time thermal cyclers.
  • a fluorophore is positioned at the 3′ end of the hairpin probe polynucleotide, and a quencher is positioned at the junction of the single-stranded and hairpin portions of the probe polynucleotide.
  • a quencher is positioned at the junction of the single-stranded and hairpin portions of the probe polynucleotide.
  • no fluorescence is detected when the self-complementary hairpin sequences are hybridized (since the fluorophore is positioned adjacent to the quencher).
  • distance between the fluorophore and the quencher occurs, resulting in a detectable fluorescent signal.
  • the above embodiments further comprise a reverse primer polynucleotide.
  • the reverse primer is complementary to a region in the polynucleotide created by extension of the first polynucleotide.
  • the reverse primer is also complementary to the complementary strand of the target polynucleotide when the target polynucleotide is one strand of a double-stranded polynucleotide. Inclusion of a reverse primer allows for amplification of the target polynucleotide.
  • the reverse primer is a “simple” primer wherein the sequence of the reverse primer is designed to be sufficiently complementary over its entire length to hybridize to a target sequence over the entire length of the primer.
  • a simple primer of this type is in one aspect, 100% complementary to a target sequence, however, it will be appreciated that a simple primer with complementarity of less than 100% is useful under certain circumstances and conditions.
  • a reverse primer is a separate polynucleotide primer combination that specifically binds to regions in a sequence produced by extension of a polynucleotide from the first domain of the first polynucleotide in a primer pair combination used in a first reaction.
  • the methods described herein provide a change in sequence detection from a sample with a non-target polynucleotide compared to sequence detection from a sample with a target polynucleotide.
  • the change is an increase in detection of a target polynucleotide in a sample compared to sequence detection from a sample with a non-target polynucleotide.
  • the change is a decrease in detection of a target polynucleotide in a sample compared to sequence detection from a sample with a non-target polynucleotide.
  • the extension is performed by an enzyme that is capable of synthesizing a nucleic acid.
  • the enzymes useful in the practice of the invention include but are not limited to a DNA polymerase (which can include a thermostable DNA polymerase, e.g., a Taq DNA polymerase), RNA polymerase, and reverse transcriptase.
  • Non-limiting examples of enzymes that may be used to practice the present invention include but are not limited to Deep VentRTM DNA Polymerase, LongAmpTM Taq DNA Polymerase, PhusionTM High-Fidelity DNA Polymerase, PhusionTM Hot Start High-Fidelity DNA Polymerase, Kapa High-Fidelity DNA Polymerase, Q5 High-Fidelity DNA Polymerase, Platinum Pfx High-Fidelity Polymerase, Pfu High-Fidelity DNA Polymerase, Pfu Ultra High-Fidelity DNA Polymerase, KOD High-Fidelity DNA Polymerase, iProof High-Fidelity Polymerase, High-Fidelity 2 DNA Polymerase, Velocity High-Fidelity DNA Polymerase, ProofStart High-Fidelity DNA Polymerase, Tigo High-Fidelity DNA Polymerase, Accuzyme High-Fidelity DNA Polymerase, VentR® DNA Polymerase, DyNAzymeTM II Hot Start DNA Poly
  • coli DNA Polymerase I, Large (Klenow) Fragment, Klenow Fragment (3′ ⁇ 5′ exo-), phi29 DNA Polymerase, T4 DNA Polymerase, T7 DNA Polymerase (unmodified), Terminal Transferase, Reverse Transcriptases and RNA Polymerases, E. coli Poly(A) Polymerase, AMV Reverse Transcriptase, M-MuLV Reverse Transcriptase, phi6 RNA Polymerase (RdRP), Poly(U) Polymerase, SP6 RNA Polymerase, and T7 RNA Polymerase.
  • A Polymerase
  • AMV Reverse Transcriptase AMV Reverse Transcriptase
  • M-MuLV Reverse Transcriptase M-MuLV Reverse Transcriptase
  • phi6 RNA Polymerase RdRP
  • Poly(U) Polymerase Poly(U) Polymerase
  • SP6 RNA Polymerase and T7 RNA Polymerase.
  • the hairpin probe polynucleotide comprises a label.
  • the label is fluorescent. Methods of labeling oligonucleotides with fluorescent molecules and measuring fluorescence are well known in the art.
  • Fluorescent labels useful in the practice of the invention include but are not limited to 1,8-ANS (1-Anilinonaphthalene-8-sulfonic acid), 1-Anilinonaphthalene-8-sulfonic acid (1,8-ANS), 5-(and-6)-Carboxy-2′, 7′-dichlorofluorescein pH 9.0, 5-FAM pH 9.0, 5-ROX (5-Carboxy-X-rhodamine, triethylammonium salt), 5-ROX pH 7.0, 5-TAMRA, 5-TAMRA pH 7.0, 5-TAMRA-MeOH, 6 JOE, 6,8-Difluoro-7-hydroxy-4-methylcoumarin pH 9.0, 6-Carboxyrhodamine 6G pH 7.0, 6-Carboxyrhodamine 6G, hydrochloride, 6-HEX, SE pH 9.0, 6-TET, SE pH 9.0, 7-Amino-4-methylcoumarin pH 7.0, 7-Hydroxy-4-methylcoumarin,
  • fluorescent molecules Other labels besides fluorescent molecules can be used, such as chemiluminescent molecules, which will give a detectable signal or a change in detectable signal upon hybridization, and radioactive molecules.
  • the hairpin probe polynucleotide comprises a quencher that attenuates the fluorescence signal of a label.
  • Quenchers contemplated for use in practice of the methods of the invention include but are not limited to Black Hole Quencher 1, Black Hole Quencher-2, Iowa Black FQ, Iowa Black RQ, Zen quencher, and Dabcyl. G-base.
  • Modified polynucleotides that are more sensitive to changes in template polynucleotide sequence than the basic polynucleotides can be used for development of more specific PCR-based diagnostic assays and for more sensitive PCR detection of rare DNA mutations in, e.g., cancer tissues.
  • primers or primer combinations are referred to as being in “forward” or “reverse” orientations, these designations are arbitrary conventions used in describing PCR reactions and the structural relationship of the primers and the template.
  • forward primers becoming “reverse” primers
  • reverse reverse primers
  • designation of, for example, one primer combination as a forward primer or a reverse primer is not a limitation on the structure or use of that particular primer combination.
  • RNase H1 cleavage assay was performed in 25 ul reactions containing 10 pmol of RNA oligo, 15 pmol of DNA oligo, 1 ⁇ iTaq buffer, 3 mM Mg and DNA resuspension buffer. Cleavage assay was carried out in the presence of 5U of Hybridase at 95 C for 20 seconds followed by 65 C for 2 minutes. Samples were then immediately put on ice and re-suspended in formamide loading buffer.
  • Samples were boiled for 2 minutes and run under denaturing conditions on a pre-cast 15% TBE-Urea polyacrylamide gel (Invitrogen, Cat # EC68852Box), stained with SYBR Gold stain (Invitrogen, Cat # S11494), visualized on a Dark Reader light box (Clare Chemical Research) and photographed using a digital camera.
  • the match DNA oligo is depicted by M while the mismatch DNA oligo is depicted by MM.
  • the DNA oligos are 34 bp long while the RNA oligos are 38 bp long.
  • RNase H1 cleaves the 4 RNA bases at the center. This results in cleavage of the rC rA rUrG containing oligo in a match RNA/DNA hybrid (lane 1) in the middle (between second and third RNA bases) which gives 2 cleavage products which are both 19 bp in length.
  • RNA containing oligo in a mismatch RNA/DNA hybrid (lane 2) which has a rA:C mismatch does not cleave as efficiently.
  • rGrC rA rU containing oligo in a match hybrid gets cleaved to produce 2 products of 18 bp and 20 bp length (can't be easily separated on gel) while the rA:C mismatch (lane 4) is not cleaved as efficiently.
  • rUrGrC rA sequence is not cleaved at all by RNase H1 under these reaction conditions whether it is present in a match (lane 5) or a mismatch RNA/DNA hybrid (lane 6).
  • rA rUrGrA oligo is cleaved in a match (lane 7) and gives 2 products which are 18 bp and 20 bp in length.
  • the mismatch rA:C does not get cleaved at all by RNase H1 (lane 8). 17 bp, 19 bp and 21 bp oligos are shown as reference markers.
  • RNase H1 cleavage assay was performed in 25 ul reactions containing 10 pmol of RNA oligo, 15 pmol of DNA oligo, lx iTaq buffer, 3 mM Mg and DNA resuspension buffer. Cleavage assay was carried out in the presence of 5U of Hybridase at 95 C for 20 seconds followed by 65 C for either 0 secs, 30 secs, 1 minute, 5 minute and 10 minutes. Samples were then immediately put on ice and re-suspended in formamide loading buffer.
  • Samples were boiled for 2 minutes and run on a pre-cast 15% TBE-Urea polyacrylamide gel (Invitrogen, Cat # EC68852Box), stained with SYBR Gold stain (Invitrogen, Cat # S11494), visualized on a Dark Reader light box (Clare Chemical Research) and photographed using a digital camera.
  • the DNA oligos are 34 bp long while the RNA oligos are 38 bp long.
  • rC rA rUrG containing oligo in a match RNA/DNA hybrid (lane 2-6) is fully cleaved at 30 sec incubation (lane 2) while rArUrGrC containing oligo in a match RNA/DNA hybrid (lane 7-11) takes about 5 minutes to cleave partially (lane 10) and 10 minutes to cleave significantly (lane 11).
  • 19 bp oligo is used as a marker.
  • Competitor Mediated Inhibition of the Wild-Type Signal Using Forward Primers which are Either Overlapping or Non-Overlapping with the Competitor
  • PCR was set-up in 25 ul reactions using 200 nM of forward and reverse primers, 1600 nM of competitor, 1 ⁇ Phusion HF Buffer, 200 uM of dNTP, 0.5 U of Phusion high fidelity DNA polymerase, 4% Glycerol, 1.6 uM of SYTO® 9, DNA resuspension buffer and 1000 copies of wild-type EFGR template containing plasmid. Cycling conditions were as follows: 1. 95 C for 3 minutes, 2. 95 C for 10 seconds, 3. 75 C for 15 seconds, 4. 65 C for 1 minute, Go-to 2 repeat 6 cycles, 5. 90 C for 10 seconds, 6. 75 C for 15 seconds, 7. 65 C for 1 minute, Go to 5 repeat 54 cycles. PCR products were detected with SYTO® 9 dye under the SYBR/FAM filter in Bio-Rad CFX-96 thermocycler.
  • the PCR assay in FIG. 29 was carried out using Phusion which is a HiFi polymerase lacking strand displacement activity. This will prevent the forward primer from displacing the competitor during extension. Even in this scenario the inhibition of an overlapping forward primer is much more efficient than a non-overlapping primer which suggests that an overlapping primer and competitor combination would be much better in inhibiting the wild-type DNA signal as compare to a non-overlapping primer and competitor.
  • PCR was set-up in 25 ul reactions using 100 nM of forward and reverse primers, 240 nM of Taq-Man probe, 800 nM of competitor, 1 ⁇ iQ supermix, DNA resuspension buffer, 10U Hybridase or 4% Glycerol and 1000 copies of either wild-type EGFR template containing plasmid or mutant EGFR T790M template containing plasmid. Cycling conditions were as follows: 1. 95 C for 3 minutes, 2. 95 C for 10 seconds, 3. 75 C for 15 seconds, 4. 65 C for 1 minute, Go-to 2 repeat 6 cycles, 5. 90 C for 10 seconds, 6. 75 C for 15 seconds, 7. 65 C for 1 minute, Go to 5 repeat 54 cycles. PCR products were detected under the SYBR/FAM filter in Bio-Rad's CFX-96 thermocycler.
  • locus specific PCR was used to detect 1000 copies of EGFR wild-type plasmid and 1000 copies of EGFR T790M plasmid.
  • the Ct values for both were very close with the average wild-type Ct coming up at 21.11 and the average mutant Ct coming up at 21.16.
  • FIG. 30 B when competitor and RNase H1 were added to this assay the wild-type EGFR signal was inhibited much more significantly (average Ct of 34.76) than the mutant T790M EGFR signal (average Ct of 23.74).
  • the Ct difference between wild-type and mutant signal was 11.02 which translates into a difference of about 2000 fold.
  • the PCR assay results depicted in FIG. 30 indicate that RNase H1 can distinguish between a mutant and a wild-type signal even in a PCR assay.
  • the discrimination of the wild-type signal leads to an amplification fold difference of 2000 between mutant and wild-type.

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