WO2023108145A1 - Sondes universelles pour l'amplification et la détection d'acides nucléiques - Google Patents

Sondes universelles pour l'amplification et la détection d'acides nucléiques Download PDF

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WO2023108145A1
WO2023108145A1 PCT/US2022/081306 US2022081306W WO2023108145A1 WO 2023108145 A1 WO2023108145 A1 WO 2023108145A1 US 2022081306 W US2022081306 W US 2022081306W WO 2023108145 A1 WO2023108145 A1 WO 2023108145A1
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nucleotides
sequence
hairpin
mixture
stranded
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PCT/US2022/081306
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English (en)
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David Y. Zhang
Alessandro Pinto
Cailin B. WELLER
Deepak THIRUNAVUKARASU
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Nuprobe Usa, Inc.
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Publication of WO2023108145A1 publication Critical patent/WO2023108145A1/fr

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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
    • C12Q1/686—Polymerase chain reaction [PCR]

Definitions

  • the present disclosure relates to the field of molecular biology. More particularly, it relates to methods and compositions useful for the detection, amplification, and quantification of nucleic acid molecules.
  • PCR polymerase chain reaction
  • PCR uses a thermostable DNA polymerase and pairs of design oligonucleotide primers to amplify specific DNA target sequences of interest that are potentially present in a nucleic acid sample.
  • PCR can be used in conjunction with fluorophore-labeled probes to allow quantitative and real-time detection of DNA targets using a fluorescence detection device (e.g, qPCR).
  • fluorescence detection device e.g, qPCR
  • PCR can also be used upstream of a high-throughput sequencing reaction for target enrichment.
  • qPCR the most commonly used methods for detection and quantitation of PCR amplification products are through the use of an intercalating dye such as SYBR® Green, or through the use of target-specific oligonucleotide probes dual-labeled with fluorophores and quenchers (e.g, TaqManTM probes).
  • TaqManTM probes are generally favored over intercalating dyes because multiple different spectrally distinct fluorophores can be used on different TaqManTM probes to allows multiplexed detection and quantitation of two to six distinct DNA target species in a single reaction. TaqManTM probes must be individually designed and tested for different target sequences.
  • Universal Probes that, among other benefits, enables a faster turnaround time when designing and developing assays as compared to TaqManTM probes. Universal probes can be added onto primers for amplifying and detecting nucleic acids, where they serve as alternatives to TaqManTM probes.
  • this disclosure provides a mixture comprising a primer, where the primer comprises, from 3' to 5': (a) a single-stranded extensible sequence comprising between 7 nucleotides and 100 nucleotides, where the single-stranded extensible sequence is at least 90% complementary to an initiation subsequence of a template nucleic acid molecule; and (b) a double-stranded universal signal probe comprising: (i) a first strand comprising a universal probe attachment sequence conjugatable to the single-stranded extensible sequence, where the universal probe attachment sequence comprises between 6 nucleotides and 100 nucleotides; and (ii) a second strand comprising a hairpin sequence comprising between 7 nucleotides and 40 nucleotides, where the hairpin sequence further comprises: (1) a first hairpin subsequence comprising between 2 nucleotides and 30 nucleotides, where the first hairpin sequence is at least 70% identical or complementary to the reverse complement of the primer
  • this disclosure provides a kit comprising: (a) single-stranded extensible sequence comprising between 7 nucleotides and 100 nucleotides, where the single-stranded extensible sequence is at least 90% complementary to an initiation subsequence of a template nucleic acid molecule; and (b) a double-stranded universal signal probe comprising: (i) a first strand comprising a universal probe attachment sequence conjugated to the single-stranded extensible sequence, where the universal probe attachment sequence comprises between 6 nucleotides and 100 nucleotides; and (ii) a second strand comprising a hairpin sequence comprising between 7 nucleotides and 40 nucleotides, where the hairpin sequence further comprises: (1) a first hairpin subsequence comprising between 2 nucleotides and 30 nucleotides, where the first hairpin subsequence is at least 70% identical or complementary to the reverse complement of the universal probe attachment sequence; (2) a loop subsequence
  • this disclosure provides a method for amplifying a template nucleic acid molecule, the method comprising: (a) generating a mixture by mixing a sample comprising the template nucleic acid molecule with: (i) a first primer, where the first primer comprises, from 3' to 5': (1) a single-stranded extensible sequence comprising between 7 nucleotides and 100 nucleotides, where the single-stranded extensible sequence is at least 90% complementary to an initiation subsequence of a template nucleic acid molecule; and (2) a double-stranded universal signal probe comprising: (A) a first strand comprising a universal probe attachment sequence conjugated to the single-stranded extensible sequence, where the universal probe attachment sequence comprises between 6 nucleotides and 100 nucleotides; and (B) a second strand comprising a hairpin sequence comprising between 7 nucleotides and 40 nucleotides, where the hairpin sequence further comprises: (I) a first primer, where the first primer comprises
  • this disclosure provides a method for multiplexed amplification of nucleic acids, the method comprising: (a) generating a mixture by mixing a sample comprising a template nucleic acid molecule with: (i) a plurality of first primers, where each primer of the plurality of first primers comprises, from 3' to 5': (1) a single-stranded extensible sequence comprising between 7 nucleotides and 100 nucleotides, where the single-stranded extensible sequence is at least 90% complementary to an initiation subsequence of a template nucleic acid molecule; and (2) a double-stranded universal signal probe comprising: (A) a first strand comprising a universal probe attachment sequence conjugated to the single-stranded extensible sequence, where the universal probe attachment sequence comprises between 6 nucleotides and 100 nucleotides; and (B) a second strand comprising a hairpin sequence comprising between 7 nucleotides and 40 nucleotides, where the
  • this disclosure provides a method for selective amplification of a template nucleic acid molecule via polymerase chain reaction, the method comprising: (a) generating a mixture by mixing a sample comprising one or both of a target nucleic acid template molecule and a background nucleic acid template molecule with: (i) a thermostable DNA polymerase enzyme: (ii) at least one reagent, at least one buffer, or at least one reagent and at least one buffer needed for the DNA polymerase enzyme to function; (iii) a forward primer comprising between 6 nucleotides and 70 nucleotides, where the forward primer comprises a sequence that is the reverse complement of a subsequence of the target nucleic acid template molecule; (iv) a forward blocker, where the forward blocker comprises a sequence that is the reverse complement of a subsequence of the background nucleic acid template molecule, and where the forward blocker comprises a 3' sequence or a chemical modification that
  • Figure 1 comprises panels 1A and IB. Both panels depict universal signal probes for amplification.
  • the universal signal probe is a double-stranded nucleic acid with two components: a Universal Attachment Sequence and a Hairpin Sequence.
  • the Hairpin Sequence hybridizes to the Universal Attachment Sequence, bringing the fluorophore (gray star) and quencher (gray circle) in close proximity resulting in little to no fluorescence.
  • the Universal Attachment Sequence comprises a chemical modification at the 3' end for conjugation to any target-specific extensible primer with a suitable 5' end. Two formats for the probes are depicted.
  • the fluorophore can be on the Universal Attachment Sequence while the quencher is on the Hairpin Sequence (as shown in 1A), or the fluorophore can be on the Hairpin Sequence, while the quencher is on the Universal Attachment Sequence (as shown in IB).
  • Figure 2 comprises panels 2A and 2B. Both panels depict the conjugation of a Universal Attachment Sequence to a target-specific extensible primer.
  • Panel 2A The Universal Attachment Sequence can be conjugated to any Primer via a chemical reaction between reactive chemical groups on the 3' end of the Universal Attachment Probe and the 5' end of the Primer.
  • Panel 2B Enzymatic ligation can also be used to conjugate a Universal Attachment Sequence to a Primer.
  • the Primer comprises a phosphorylated 5' phosphate.
  • a splint oligo is used to bring together the two sequences together to maximize the ligation yield.
  • Figure 3 depicts a non-limiting mechanism for Universal Probe function.
  • the Universal Attachment Sequence conjugated with an Extensible Primer (e.g., sequence) hybridizes to the Hairpin Sequence through a first subsequence, while the target-specific Extensible Sequence hybridizes to the target region on the template.
  • a DNA polymerase extends the 3' end of the Extensible Sequence in the first cycle of PCR.
  • the Second Primer hybridizes to the product from the first cycle. As the polymerase extends the Second Primer, it releases the Hairpin Sequence from the Universal Attachment Sequence, generating a fluorescent signal.
  • composition provided herein is specifically envisioned for use with any applicable method provided herein. Any composition provided herein is specifically envisioned for use with any kit provided herein.
  • any and all combinations of the members that make up that grouping of alternatives is specifically envisioned. For example, if an item is selected from a group consisting of A, B, C, and D, the inventors specifically envision each alternative individually (e.g., A alone, B alone, etc.), as well as combinations such as A, B, and D; A and C; B and C; etc.
  • the term “and/or” when used in a list of two or more items means any one of the listed items by itself or in combination with any one or more of the other listed items.
  • the expression “A and/or B” is intended to mean either or both of A and B - i.e., A alone, B alone, or A and B in combination.
  • the expression “A, B and/or C” is intended to mean A alone, B alone, C alone, A and B in combination, A and C in combination, B and C in combination, or A, B, and C in combination.
  • the range is understood to inclusive of the edges of the range as well as any number between the defined edges of the range. For example, “between 1 and 10” includes any number between 1 and 10, as well as the number 1 and the number 10.
  • a compound or “at least one compound” may include a plurality of compounds, including mixtures thereof.
  • plural refers to any number greater than one.
  • this disclosure provides a mixture comprising a primer, where the primer comprises, from 3' to 5': (a) a single-stranded extensible sequence comprising between 7 nucleotides and 100 nucleotides, where the single-stranded extensible sequence is at least 90% complementary to an initiation subsequence of a template nucleic acid molecule; and (b) a double-stranded universal signal probe comprising: (i) a first strand comprising a universal probe attachment sequence conjugatable to the single-stranded extensible sequence, where the universal probe attachment sequence comprises between 6 nucleotides and 100 nucleotides; and (ii) a second strand comprising a hairpin sequence comprising between 7 nucleotides and 40 nucleotides, where the hairpin sequence further comprises: (1) a first hairpin subsequence comprising between 2 nucleotides and 30 nucleotides, where the first hairpin sequence is at least 70% identical or complementary to the reverse complement of the primer
  • this disclosure provides a kit comprising: (a) single-stranded extensible sequence comprising between 7 nucleotides and 100 nucleotides, where the single-stranded extensible sequence is at least 90% complementary to an initiation subsequence of a template nucleic acid molecule; and (b) a double-stranded universal signal probe comprising: (i) a first strand comprising a universal probe attachment sequence conjugated to the single-stranded extensible sequence, where the universal probe attachment sequence comprises between 6 nucleotides and 100 nucleotides; and (ii) a second strand comprising a hairpin sequence comprising between 7 nucleotides and 40 nucleotides, where the hairpin sequence further comprises: (1) a first hairpin subsequence comprising between 2 nucleotides and 30 nucleotides, where the first hairpin subsequence is at least 70% identical or complementary to the reverse complement of the universal probe attachment sequence; (2) a loop subsequence
  • a “mixture” refers to a composition comprising a plurality of components.
  • a mixture is partitioned into a plurality of lipid droplets.
  • a double-stranded universal signal probe comprises deoxyribonucleic acid (DNA). In an aspect, a double-stranded universal signal probe comprises ribonucleic acid (RNA). In an aspect, a double-stranded universal signal probe comprises DNA and RNA. In an aspect, a single-stranded extensible sequence comprises DNA. In an aspect, a single-stranded extensible sequence comprises RNA. In an aspect, a single-stranded extensible sequence comprises DNA and RNA. In an aspect, a primer comprises DNA. In an aspect, a primer comprises RNA. In an aspect, a universal probe attachment sequence comprises DNA. In an aspect, a universal probe attachment sequence comprises RNA. In an aspect, a universal probe attachment sequence comprises DNA and RNA. In an aspect, a hairpin sequence comprises DNA. In an aspect, a hairpin sequence comprises RNA. In an aspect, a hairpin sequence comprises DNA and RNA.
  • percent identity or “percent identical” as used herein in reference to two or more nucleotide or amino acid sequences is calculated by (i) comparing two optimally aligned sequences (nucleotide or amino acid) over a window of comparison (the “alignable” region or regions), (ii) determining the number of positions at which the identical nucleic acid base (for nucleotide sequences) or amino acid residue (for proteins and polypeptides) occurs in both sequences to yield the number of matched positions, (iii) dividing the number of matched positions by the total number of positions in the window of comparison, and then (iv) multiplying this quotient by 100% to yield the percent identity.
  • the percent identity is being calculated in relation to a reference sequence without a particular comparison window being specified, then the percent identity is determined by dividing the number of matched positions over the region of alignment by the total length of the reference sequence. Accordingly, for purposes of the present application, when two sequences (query and subject) are optimally aligned (with allowance for gaps in their alignment), the “percent identity” for the query sequence is equal to the number of identical positions between the two sequences divided by the total number of positions in the query sequence over its length (or a comparison window), which is then multiplied by 100%.
  • percent complementarity or “percent complementary” as used herein in reference to two nucleotide sequences refers to the percentage of nucleotides of a query sequence that optimally base-pair or hybridize to nucleotides a subject sequence when the query and subject sequences are linearly arranged and optimally base paired without secondary folding structures, such as loops, stems or hairpins. Such a percent complementarity can be between two DNA strands, two RNA strands, or a DNA strand and a RNA strand.
  • the “percent complementarity” can be calculated by (i) optimally basepairing or hybridizing the two nucleotide sequences in a linear and fully extended arrangement (i.e., without folding or secondary structures) over a window of comparison, (ii) determining the number of positions that base-pair between the two sequences over the window of comparison to yield the number of complementary positions, (iii) dividing the number of complementary positions by the total number of positions in the window of comparison, and (iv) multiplying this quotient by 100% to yield the percent complementarity of the two sequences.
  • Optimal base pairing of two sequences can be determined based on the known pairings of complementary nucleotide bases, such as guanine (G)-cytosine (C), adenine (A)-thymine (T), and A-uracil (U), through hydrogen binding. If the “percent complementarity” is being calculated in relation to a reference sequence without specifying a particular comparison window, then the percent identity is determined by dividing the number of complementary positions between the two linear sequences by the total length of the reference sequence.
  • complementary nucleotide bases such as guanine (G)-cytosine (C), adenine (A)-thymine (T), and A-uracil (U)
  • the “percent complementarity” for the query sequence is equal to the number of base-paired positions between the two sequences divided by the total number of positions in the query sequence over its length, which is then multiplied by 100%.
  • mismatch refers to an alignment of two sequences that pairs two uncomplimentary nucleotides.
  • mismatches include G-A, G- T, G-U, G-G, C-A, C-T, C-U, C-C, A-A, T-T, and T-U.
  • matched alignments of nucleotides refer to complimentary pairs such as G-C, A-T, and A-U.
  • the complement of the sequence 5'-ATGC-3' is 3'- TACG-5'
  • the reverse complement of 5'-ATGC-3' is 5'-GCAT-3'
  • the complement and reverse complement sequences are identical to each other when viewed in the 5' to 3' direction.
  • various pair-wise or multiple sequence alignment algorithms and programs are known in the art, such as ClustalW or Basic Local Alignment Search Tool® (BLASTTM), etc., that can be used to compare the sequence complementarity or identity between two or more nucleotide sequences.
  • a “primer” refers to a chemically synthesized single-stranded oligonucleotide which is designed to anneal to a specific site on a template nucleic acid molecule.
  • a primer is used in PCR to initiate DNA synthesis.
  • a primer is a DNA molecule.
  • a primer is an RNA molecule.
  • a primer comprises between 6 nucleotides and 70 nucleotides.
  • a primer comprises between 10 nucleotides and 50 nucleotides.
  • a primer comprises between 15 nucleotides and 30 nucleotides.
  • a primer comprises between 18 nucleotides and 25 nucleotides. In an aspect, a primer comprises at least 6 nucleotides. In an aspect, a primer comprises at least 10 nucleotides. In an aspect, a primer comprises at least 15 nucleotides. In an aspect, a primer comprises at least 20 nucleotides. In an aspect, a primer is a forward primer. In an aspect, a primer is a reverse primer. As used herein, a “forward primer” hybridizes to the anti-sense strand of dsDNA, and a “reverse primer” hybridizes to the sense strand of dsDNA. In an aspect, a forward primer comprises DNA. In an aspect, a reverse primer comprises DNA. In an aspect, a forward primer comprises RNA. In an aspect, a reverse primer comprises RNA. In an aspect, a second primer is a forward primer. In an aspect, a second primer is a reverse primer.
  • a “second primer,” a “forward primer,” or a “reverse primer” is a primer that does not comprise a double-stranded universal signal probe.
  • a primer comprises a single-stranded extensible sequence.
  • a primer comprises a target-specific single-stranded extensible sequence.
  • an “extensible sequence” refers to a nucleic acid molecule that is cable of being extended by a DNA polymerase (e.g, the extensible sequence can initiate DNA synthesis).
  • a single-stranded extensible sequence comprises at least 5 nucleotides.
  • a single-stranded extensible sequence comprises at least 6 nucleotides. In an aspect, a single-stranded extensible sequence comprises at least 7 nucleotides. In an aspect, a single-stranded extensible sequence comprises at least 8 nucleotides. In an aspect, a single-stranded extensible sequence comprises at least 9 nucleotides. In an aspect, a singlestranded extensible sequence comprises at least 10 nucleotides. In an aspect, a singlestranded extensible sequence comprises at least 12 nucleotides. In an aspect, a singlestranded extensible sequence comprises at least 15 nucleotides. In an aspect, a singlestranded extensible sequence comprises at least 18 nucleotides.
  • a singlestranded extensible sequence comprises at least 20 nucleotides. In an aspect, a singlestranded extensible sequence comprises at least 25 nucleotides. In an aspect, a singlestranded extensible sequence comprises at least 50 nucleotides. In an aspect, a singlestranded extensible sequence comprises at least 75 nucleotides. In an aspect, a singlestranded extensible sequence comprises at least 100 nucleotides. In an aspect, a singlestranded extensible sequence comprises at least 150 nucleotides. In an aspect, a singlestranded extensible sequence comprises at least 200 nucleotides.
  • a single-stranded extensible sequence comprises between 5 nucleotides and 200 nucleotides. In an aspect, a single-stranded extensible sequence comprises between 5 nucleotides and 150 nucleotides. In an aspect, a single-stranded extensible sequence comprises between 5 nucleotides and 100 nucleotides. In an aspect, a single-stranded extensible sequence comprises between 7 nucleotides and 200 nucleotides. In an aspect, a single-stranded extensible sequence comprises between 7 nucleotides and 150 nucleotides. In an aspect, a single-stranded extensible sequence comprises between 7 nucleotides and 100 nucleotides.
  • a single-stranded extensible sequence comprises between 7 nucleotides and 75 nucleotides. In an aspect, a single-stranded extensible sequence comprises between 7 nucleotides and 50 nucleotides. In an aspect, a single-stranded extensible sequence comprises between 7 nucleotides and 40 nucleotides. In an aspect, a single-stranded extensible sequence comprises between 7 nucleotides and 30 nucleotides. In an aspect, a single-stranded extensible sequence comprises between 15 nucleotides and 200 nucleotides. In an aspect, a single-stranded extensible sequence comprises between 15 nucleotides and 100 nucleotides.
  • a single-stranded extensible sequence comprises between 15 nucleotides and 50 nucleotides. In an aspect, a single-stranded extensible sequence comprises between 20 nucleotides and 50 nucleotides. [0037] In an aspect, a single-stranded extensible sequence is at least 70% complementary to an initiation subsequence of a template nucleic acid molecule. In an aspect, a singlestranded extensible sequence is at least 75% complementary to an initiation subsequence of a template nucleic acid molecule. In an aspect, a single-stranded extensible sequence is at least 80% complementary to an initiation subsequence of a template nucleic acid molecule.
  • a single-stranded extensible sequence is at least 85% complementary to an initiation subsequence of a template nucleic acid molecule. In an aspect, a single-stranded extensible sequence is at least 90% complementary to an initiation subsequence of a template nucleic acid molecule. In an aspect, a single-stranded extensible sequence is at least 91% complementary to an initiation subsequence of a template nucleic acid molecule. In an aspect, a single-stranded extensible sequence is at least 92% complementary to an initiation subsequence of a template nucleic acid molecule.
  • a single-stranded extensible sequence is at least 93% complementary to an initiation subsequence of a template nucleic acid molecule. In an aspect, a single-stranded extensible sequence is at least 94% complementary to an initiation subsequence of a template nucleic acid molecule. In an aspect, a single-stranded extensible sequence is at least 95% complementary to an initiation subsequence of a template nucleic acid molecule. In an aspect, a single-stranded extensible sequence is at least 96% complementary to an initiation subsequence of a template nucleic acid molecule.
  • a single-stranded extensible sequence is at least 97% complementary to an initiation subsequence of a template nucleic acid molecule. In an aspect, a single-stranded extensible sequence is at least 98% complementary to an initiation subsequence of a template nucleic acid molecule. In an aspect, a single-stranded extensible sequence is at least 99% complementary to an initiation subsequence of a template nucleic acid molecule. In an aspect, a single-stranded extensible sequence is at least 99.5% complementary to an initiation subsequence of a template nucleic acid molecule. In an aspect, a single-stranded extensible sequence is 100% complementary to an initiation subsequence of a template nucleic acid molecule.
  • a sample provided herein is mixed with a plurality of single-stranded extensible sequences. In an aspect, a sample provided herein is mixed with a plurality of unique single-stranded extensible sequences. In an aspect, a sample provided herein is mixed with at least 3 unique single-stranded extensible sequences. In an aspect, a sample provided herein is mixed with at least 4 unique single-stranded extensible sequences. In an aspect, a sample provided herein is mixed with at least 5 unique single-stranded extensible sequences. In an aspect, a sample provided herein is mixed with at least 6 unique singlestranded extensible sequences.
  • a sample provided herein is mixed with at least 7 unique single-stranded extensible sequences. In an aspect, a sample provided herein is mixed with at least 8 unique single-stranded extensible sequences. In an aspect, a sample provided herein is mixed with at least 9 unique single-stranded extensible sequences. In an aspect, a sample provided herein is mixed with at least 10 unique single-stranded extensible sequences. In an aspect, a sample provided herein is mixed with at least 15 unique singlestranded extensible sequences. In an aspect, a sample provided herein is mixed with at least 20 unique single-stranded extensible sequences. In an aspect, a sample provided herein is mixed with at least 25 unique single-stranded extensible sequences.
  • a sample provided herein is mixed with at least 30 unique single-stranded extensible sequences. In an aspect, a sample provided herein is mixed with at least 40 unique single-stranded extensible sequences. In an aspect, a sample provided herein is mixed with at least 50 unique single-stranded extensible sequences. In an aspect, a sample provided herein is mixed with at least 75 unique single-stranded extensible sequences. In an aspect, a sample provided herein is mixed with at least 100 unique single-stranded extensible sequences.
  • unique single-stranded extensible sequences are single-stranded extensible sequences that are not identical to each other.
  • two unique single-stranded extensible sequences could bind to the same locus (e.g, different locations within a single gene) or to two different loci (e.g, two different genes).
  • a plurality of single-stranded extensible sequences are each at least 85% complementary to a plurality of initiation subsequences. In an aspect, a plurality of single-stranded extensible sequences are each at least 90% complementary to a plurality of initiation subsequences. In an aspect, a plurality of single-stranded extensible sequences are each at least 92.5% complementary to a plurality of initiation subsequences. In an aspect, a plurality of single-stranded extensible sequences are each at least 95% complementary to a plurality of initiation subsequences.
  • a plurality of singlestranded extensible sequences are each at least 97.5% complementary to a plurality of initiation subsequences. In an aspect, a plurality of single-stranded extensible sequences are each at least 99% complementary to a plurality of initiation subsequences. In an aspect, a plurality of single-stranded extensible sequences are each 99.5% complementary to a plurality of initiation subsequences. In an aspect, a plurality of single-stranded extensible sequences are each 100% complementary to a plurality of initiation subsequences.
  • an “initiation subsequence” refers to a nucleic acid sequence that is complementary, and is capable of binding, to a single-stranded extensible sequence or a primer.
  • an initiation subsequence and a complementary single-stranded extensible sequence or a primer will have lengths that are the same or about the same (e.g. , their lengths differ by no more than 10%, which allows for short indels).
  • an initiation subsequence comprises a length of at least 5 nucleotides. In an aspect, an initiation subsequence comprises a length of at least 7 nucleotides. In an aspect, an initiation subsequence comprises a length of at least 15 nucleotides. In an aspect, an initiation subsequence comprises a length of at least 20 nucleotides. In an aspect, an initiation subsequence comprises a length of at least 25 nucleotides. In an initiation subsequence comprises a length of at least 30 nucleotides. In an aspect, an initiation subsequence comprises a length of at least 50 nucleotides. In an aspect, an initiation subsequence comprises a length of at least 75 nucleotides.
  • an initiation subsequence comprises a length of between 5 nucleotides and 100 nucleotides. In an aspect, an initiation subsequence comprises a length of between 7 nucleotides and 100 nucleotides. In an aspect, an initiation subsequence comprises a length of between 15 nucleotides and 100 nucleotides. In an aspect, an initiation subsequence comprises a length of between 25 nucleotides and 100 nucleotides. In an aspect, an initiation subsequence comprises a length of between 50 nucleotides and 100 nucleotides.
  • an initiation subsequence comprises a length of between 75 nucleotides and 100 nucleotides. In an aspect, an initiation subsequence comprises a length of between 7 nucleotides and 75 nucleotides. In an aspect, an initiation subsequence comprises a length of between 7 nucleotides and 50 nucleotides. In an aspect, an initiation subsequence comprises a length of between 7 nucleotides and 30 nucleotides. In an aspect, an initiation subsequence comprises a length of between 7 nucleotides and 25 nucleotides. In an aspect, an initiation subsequence comprises a length of between 7 nucleotides and 20 nucleotides. In an aspect, an initiation subsequence comprises a length of between 15 nucleotides and 30 nucleotides.
  • a primer comprises a double-stranded universal signal probe.
  • a double-stranded universal signal probe comprises (i) a first strand comprising a universal probe attachment sequence conjugatable to a single-stranded extensible sequence and (ii) a second strand comprising a hairpin sequence.
  • a double-stranded universal signal probe comprises (i) a first strand comprising a universal probe attachment sequence conjugated to a single-stranded extensible sequence and (ii) a second strand comprising a hairpin sequence.
  • a sample is mixed with a plurality of double-stranded universal signal probes. In an aspect, a sample is mixed with a plurality of unique double-stranded universal signal probes. In an aspect, a sample is mixed with at least 3 unique double-stranded universal signal probes. In an aspect, a sample is mixed with at least 4 unique doublestranded universal signal probes. In an aspect, a sample is mixed with at least 5 unique double-stranded universal signal probes. In an aspect, a sample is mixed with at least 6 unique double-stranded universal signal probes. In an aspect, a sample is mixed with at least 7 unique double-stranded universal signal probes.
  • a sample is mixed with at least 8 unique double-stranded universal signal probes. In an aspect, a sample is mixed with at least 9 unique double-stranded universal signal probes. In an aspect, a sample is mixed with at least 10 unique double-stranded universal signal probes. In an aspect, a sample is mixed with at least 15 unique double-stranded universal signal probes. In an aspect, a sample is mixed with at least 20 unique double-stranded universal signal probes. In an aspect, a sample is mixed with at least 25 unique double-stranded universal signal probes. In an aspect, a sample is mixed with at least 30 unique double-stranded universal signal probes. In an aspect, a sample is mixed with at least 40 unique double-stranded universal signal probes.
  • a sample is mixed with at least 50 unique doublestranded universal signal probes. In an aspect, a sample is mixed with at least 60 unique double-stranded universal signal probes. In an aspect, a sample is mixed with at least 70 unique double-stranded universal signal probes. In an aspect, a sample is mixed with at least 80 unique double-stranded universal signal probes. In an aspect, a sample is mixed with at least 90 unique double-stranded universal signal probes. In an aspect, a sample is mixed with at least 100 unique double-stranded universal signal probes. In an aspect, a sample is mixed with at least 200 unique double-stranded universal signal probes.
  • a “plurality of unique double-stranded universal signal probes” refers to two or more non-identical double-stranded universal signal probes.
  • a plurality of unique double-stranded universal signal probes comprise a single type of fluorophore. In an aspect, a plurality of unique double-stranded universal signal probes comprise a plurality of fluorophore types.
  • a plurality of unique double-stranded universal signal probes comprise a single type of quencher. In an aspect, a plurality of unique double-stranded universal signal probes comprise a plurality of quencher types.
  • a universal probe attachment sequence comprises at least 5 nucleotides. In an aspect, a universal probe attachment sequence comprises at least nucleotides. In an aspect, a universal probe attachment sequence comprises at least 7 nucleotides. In an aspect, a universal probe attachment sequence comprises at least 8 nucleotides. In an aspect, a universal probe attachment sequence comprises at least 9 nucleotides. In an aspect, a universal probe attachment sequence comprises at least 10 nucleotides. In an aspect, a universal probe atachment sequence comprises at least 15 nucleotides. In an aspect, a universal probe atachment sequence comprises at least 20 nucleotides. In an aspect, a universal probe atachment sequence comprises at least 25 nucleotides.
  • a universal probe atachment sequence comprises at least 50 nucleotides. In an aspect, a universal probe atachment sequence comprises at least 75 nucleotides. In an aspect, a universal probe atachment sequence comprises at least 100 nucleotides. In an aspect, a universal probe atachment sequence comprises at least 150 nucleotides. In an aspect, a universal probe atachment sequence comprises at least 200 nucleotides.
  • a universal probe atachment sequence comprises between 5 nucleotides and 200 nucleotides. In an aspect, a universal probe atachment sequence comprises between 5 nucleotides and 150 nucleotides. In an aspect, a universal probe atachment sequence comprises between 5 nucleotides and 100 nucleotides. In an aspect, a universal probe atachment sequence comprises between 5 nucleotides and 50 nucleotides. In an aspect, a universal probe atachment sequence comprises between 6 nucleotides and 200 nucleotides. In an aspect, a universal probe atachment sequence comprises between 6 nucleotides and 150 nucleotides.
  • a universal probe atachment sequence comprises between 6 nucleotides and 100 nucleotides. In an aspect, a universal probe atachment sequence comprises between 6 nucleotides and 75 nucleotides. In an aspect, a universal probe atachment sequence comprises between 6 nucleotides and 50 nucleotides. In an aspect, a universal probe atachment sequence comprises between 6 nucleotides and 25 nucleotides. In an aspect, a universal probe atachment sequence comprises between 10 nucleotides and 200 nucleotides. In an aspect, a universal probe atachment sequence comprises between 10 nucleotides and 100 nucleotides.
  • a universal probe atachment sequence comprises between 10 nucleotides and 75 nucleotides. In an aspect, a universal probe atachment sequence comprises between 10 nucleotides and 50 nucleotides. In an aspect, a universal probe atachment sequence comprises between 25 nucleotides and 100 nucleotides. In an aspect, a universal probe atachment sequence comprises between 50 nucleotides and 100 nucleotides.
  • a sample provided herein is mixed with a plurality of universal probe atachment sequences. In an aspect, a sample provided herein is mixed with a plurality of unique universal probe atachment sequences. In an aspect, a sample provided herein is mixed with at least 3 unique universal probe atachment sequences. In an aspect, a sample provided herein is mixed with at least 4 unique universal probe atachment sequences. In an aspect, a sample provided herein is mixed with at least 5 unique universal probe attachment sequences. In an aspect, a sample provided herein is mixed with at least 6 unique universal probe attachment sequences. In an aspect, a sample provided herein is mixed with at least 7 unique universal probe attachment sequences.
  • a sample provided herein is mixed with at least 8 unique universal probe attachment sequences. In an aspect, a sample provided herein is mixed with at least 9 unique universal probe attachment sequences. In an aspect, a sample provided herein is mixed with at least 10 unique universal probe attachment sequences.
  • a plurality of unique universal probe attachment sequences comprise a single type of fluorophore. In an aspect, a plurality of unique universal probe attachment sequences comprise a plurality of fluorophore types.
  • a “type” of fluorophore refers to a unique fluorophore. Therefore, a “plurality of fluorophore types” refers to two or more non-identical fluorophores.
  • a plurality of unique universal probe attachment sequences comprise a single type of quencher. In an aspect, a plurality of unique universal probe attachment sequences comprise a plurality of quencher types.
  • a “type” of quencher refers to a unique quencher. Therefore, a “plurality of quencher types” refers to two or more non-identical quenchers.
  • unique universal probe attachment sequences refers to two or more universal probe attachment sequences that are not identical.
  • a hairpin sequence comprises at least 6 nucleotides. In an aspect, a hairpin sequence comprises at least 7 nucleotides. In an aspect, a hairpin sequence comprises at least 8 nucleotides. In an aspect, a hairpin sequence comprises at least 9 nucleotides. In an aspect, a hairpin sequence comprises at least 10 nucleotides. In an aspect, a hairpin sequence comprises at least 11 nucleotides. In an aspect, a hairpin sequence comprises at least 12 nucleotides. In an aspect, a hairpin sequence comprises at least 13 nucleotides. In an aspect, a hairpin sequence comprises at least 14 nucleotides.
  • a hairpin sequence comprises at least 15 nucleotides. In an aspect, a hairpin sequence comprises at least 16 nucleotides. In an aspect, a hairpin sequence comprises at least 17 nucleotides. In an aspect, a hairpin sequence comprises at least 18 nucleotides. In an aspect, a hairpin sequence comprises at least 19 nucleotides. In an aspect, a hairpin sequence comprises at least 20 nucleotides. In an aspect, a hairpin sequence comprises at least 25 nucleotides. In an aspect, a hairpin sequence comprises at least 30 nucleotides. In an aspect, a hairpin sequence comprises at least 35 nucleotides. In an aspect, a hairpin sequence comprises at least 40 nucleotides.
  • a hairpin sequence comprises at least 45 nucleotides. In an aspect, a hairpin sequence comprises at least 50 nucleotides. In an aspect, a hairpin sequence comprises at least 60 nucleotides. In an aspect, a hairpin sequence comprises at least 70 nucleotides. In an aspect, a hairpin sequence comprises at least 80 nucleotides. In an aspect, a hairpin sequence comprises at least 90 nucleotides. In an aspect, a hairpin sequence comprises at least 100 nucleotides.
  • a hairpin sequence comprises between 5 nucleotides and 100 nucleotides. In an aspect, a hairpin sequence comprises between 5 nucleotides and 50 nucleotides. In an aspect, a hairpin sequence comprises between 5 nucleotides and 45 nucleotides. In an aspect, a hairpin sequence comprises between 5 nucleotides and 40 nucleotides. In an aspect, a hairpin sequence comprises between 5 nucleotides and 30 nucleotides. In an aspect, a hairpin sequence comprises between 7 nucleotides and 100 nucleotides. In an aspect, a hairpin sequence comprises between 7 nucleotides and 50 nucleotides.
  • a hairpin sequence comprises between 7 nucleotides and 45 nucleotides. In an aspect, a hairpin sequence comprises between 7 nucleotides and 40 nucleotides. In an aspect, a hairpin sequence comprises between 7 nucleotides and 30 nucleotides. In an aspect, a hairpin sequence comprises between 7 nucleotides and 25 nucleotides. In an aspect, a hairpin sequence comprises between 15 nucleotides and 35 nucleotides.
  • a hairpin sequence comprises: (1) a first hairpin subsequence; (2) a loop subsequence; and (3) a second hairpin subsequence, where the second hairpin subsequence is the reverse complement of the first hairpin subsequence.
  • a “hairpin sequence” refers to a sequence that is capable of forming a stem-loop structure.
  • a “loop subsequence” refers to a sequence that forms an unpaired loop as part of the stem-loop structure.
  • a “first hairpin subsequence” and/or a “second hairpin subsequence” refers to a sequence that forms part of a stem as part of the stem-loop structure.
  • a stem-loop structure comprises a hairpin sequence and a universal attachment sequence.
  • a stem-loop structure comprises a hairpin sequence.
  • a first hairpin subsequence comprises at least 2 nucleotides. In an aspect, a first hairpin subsequence comprises at least 3 nucleotides. In an aspect, a first hairpin subsequence comprises at least 4 nucleotides. In an aspect, a first hairpin subsequence comprises at least 5 nucleotides. In an aspect, a first hairpin subsequence comprises at least 6 nucleotides. In an aspect, a first hairpin subsequence comprises at least 7 nucleotides. In an aspect, a first hairpin subsequence comprises at least 8 nucleotides.
  • a first hairpin subsequence comprises at least 9 nucleotides. In an aspect, a first hairpin subsequence comprises at least 10 nucleotides. In an aspect, a first hairpin subsequence comprises at least 11 nucleotides. In an aspect, a first hairpin subsequence comprises at least 12 nucleotides. In an aspect, a first hairpin subsequence comprises at least 13 nucleotides. In an aspect, a first hairpin subsequence comprises at least 14 nucleotides. In an aspect, a first hairpin subsequence comprises at least 15 nucleotides. In an aspect, a first hairpin subsequence comprises at least 20 nucleotides.
  • a first hairpin subsequence comprises at least 25 nucleotides. In an aspect, a first hairpin subsequence comprises at least 30 nucleotides. In an aspect, a first hairpin subsequence comprises at least 35 nucleotides. In an aspect, a first hairpin subsequence comprises at least 40 nucleotides. In an aspect, a first hairpin subsequence comprises at least 45 nucleotides. In an aspect, a first hairpin subsequence comprises at least 50 nucleotides.
  • a first hairpin subsequence comprises between 2 nucleotides and 50 nucleotides. In an aspect, a first hairpin subsequence comprises between 2 nucleotides and 40 nucleotides. In an aspect, a first hairpin subsequence comprises between 2 nucleotides and 35 nucleotides. In an aspect, a first hairpin subsequence comprises between 2 nucleotides and 30 nucleotides. In an aspect, a first hairpin subsequence comprises between 2 nucleotides and 25 nucleotides. In an aspect, a first hairpin subsequence comprises between 2 nucleotides and 20 nucleotides.
  • a first hairpin subsequence comprises between 2 nucleotides and 15 nucleotides. In an aspect, a first hairpin subsequence comprises between 5 nucleotides and 50 nucleotides. In an aspect, a first hairpin subsequence comprises between 5 nucleotides and 40 nucleotides. In an aspect, a first hairpin subsequence comprises between 5 nucleotides and 35 nucleotides. In an aspect, a first hairpin subsequence comprises between 5 nucleotides and 30 nucleotides. In an aspect, a first hairpin subsequence comprises between 5 nucleotides and 25 nucleotides.
  • a first hairpin subsequence comprises between 5 nucleotides and 20 nucleotides. In an aspect, a first hairpin subsequence comprises between 5 nucleotides and 15 nucleotides. In an aspect, a first hairpin subsequence comprises between 10 nucleotides and 50 nucleotides. In an aspect, a first hairpin subsequence comprises between 10 nucleotides and 40 nucleotides. In an aspect, a first hairpin subsequence comprises between 10 nucleotides and 35 nucleotides. In an aspect, a first hairpin subsequence comprises between 10 nucleotides and 30 nucleotides.
  • a first hairpin subsequence comprises between 10 nucleotides and 25 nucleotides. In an aspect, a first hairpin subsequence comprises between 10 nucleotides and 20 nucleotides. In an aspect, a first hairpin subsequence comprises between 10 nucleotides and 15 nucleotides. In an aspect, a first hairpin subsequence comprises between 15 nucleotides and 30 nucleotides. [0064] In an aspect, a first hairpin subsequence is at least 60% identical or complementary to the reverse complement of a universal probe attachment sequence. In an aspect, a first hairpin subsequence is at least 65% identical or complementary to the reverse complement of a universal probe attachment sequence.
  • a first hairpin subsequence is at least 70% identical or complementary to the reverse complement of a universal probe attachment sequence. In an aspect, a first hairpin subsequence is at least 75% identical or complementary to the reverse complement of a universal probe attachment sequence. In an aspect, a first hairpin subsequence is at least 80% identical or complementary to the reverse complement of a universal probe attachment sequence. In an aspect, a first hairpin subsequence is at least 85% identical or complementary to the reverse complement of a universal probe attachment sequence. In an aspect, a first hairpin subsequence is at least 90% identical or complementary to the reverse complement of a universal probe attachment sequence.
  • a first hairpin subsequence is at least 91% identical or complementary to the reverse complement of a universal probe attachment sequence. In an aspect, a first hairpin subsequence is at least 92% identical or complementary to the reverse complement of a universal probe attachment sequence. In an aspect, a first hairpin subsequence is at least 93% identical or complementary to the reverse complement of a universal probe attachment sequence. In an aspect, a first hairpin subsequence is at least 94% identical or complementary to the reverse complement of a universal probe attachment sequence. In an aspect, a first hairpin subsequence is at least 95% identical or complementary to the reverse complement of a universal probe attachment sequence.
  • a first hairpin subsequence is at least 96% identical or complementary to the reverse complement of a universal probe attachment sequence. In an aspect, a first hairpin subsequence is at least 97% identical or complementary to the reverse complement of a universal probe attachment sequence. In an aspect, a first hairpin subsequence is at least 98% identical or complementary to the reverse complement of a universal probe attachment sequence. In an aspect, a first hairpin subsequence is at least 99% identical or complementary to the reverse complement of a universal probe attachment sequence. In an aspect, a first hairpin subsequence is at least 99.5% identical or complementary to the reverse complement of a universal probe attachment sequence. In an aspect, a first hairpin subsequence is 100% identical or complementary to the reverse complement of a universal probe attachment sequence.
  • a universal probe attachment sequence and a first hairpin subsequence comprise 0 mismatches when optimally aligned. In an aspect, a universal probe attachment sequence and a first hairpin subsequence comprise at least 1 mismatch when optimally aligned. In an aspect, a universal probe attachment sequence and a first hairpin subsequence comprise at least 2 mismatches when optimally aligned. In an aspect, a universal probe attachment sequence and a first hairpin subsequence comprise at least 3 mismatches when optimally aligned. In an aspect, a universal probe attachment sequence and a first hairpin subsequence comprise at least 4 mismatches when optimally aligned. In an aspect, a universal probe attachment sequence and a first hairpin subsequence comprise at least 5 mismatches when optimally aligned.
  • a universal probe attachment sequence and a first hairpin subsequence comprise between 0 mismatches and 7 mismatches when optimally aligned. In an aspect, a universal probe attachment sequence and a first hairpin subsequence comprise between 0 mismatches and 5 mismatches when optimally aligned. In an aspect, a universal probe attachment sequence and a first hairpin subsequence comprise between 0 mismatches and 3 mismatches when optimally aligned. In an aspect, a universal probe attachment sequence and a first hairpin subsequence comprise between 0 mismatches and 2 mismatches when optimally aligned.
  • a universal probe attachment sequence and a first hairpin subsequence comprise between 1 mismatch and 7 mismatches when optimally aligned. In an aspect, a universal probe attachment sequence and a first hairpin subsequence comprise between 1 mismatch and 5 mismatches when optimally aligned. In an aspect, a universal probe attachment sequence and a first hairpin subsequence comprise between 1 mismatch and 3 mismatches when optimally aligned. In an aspect, a universal probe attachment sequence and a first hairpin subsequence comprise between 2 mismatches and 4 mismatches when optimally aligned.
  • a loop subsequence comprises at least 3 nucleotides. In an aspect, a loop subsequence comprises at least 4 nucleotides. In an aspect, a loop subsequence comprises at least 5 nucleotides. In an aspect, a loop subsequence comprises at least 6 nucleotides. In an aspect, a loop subsequence comprises at least 7 nucleotides. In an aspect, a loop subsequence comprises at least 8 nucleotides. In an aspect, a loop subsequence comprises at least 9 nucleotides. In an aspect, a loop subsequence comprises at least 10 nucleotides.
  • a loop subsequence comprises at least 11 nucleotides. In an aspect, a loop subsequence comprises at least 12 nucleotides. In an aspect, a loop subsequence comprises at least 15 nucleotides.
  • a loop subsequence comprises between 3 nucleotides and 15 nucleotides. In an aspect, a loop subsequence comprises between 3 nucleotides and 12 nucleotides. In an aspect, a loop subsequence comprises between 3 nucleotides and 10 nucleotides. In an aspect, a loop subsequence comprises between 3 nucleotides and 8 nucleotides. In an aspect, a loop subsequence comprises between 3 nucleotides and 6 nucleotides. In an aspect, a loop subsequence comprises between 5 nucleotides and 15 nucleotides.
  • a loop subsequence comprises between 5 nucleotides and 10 nucleotides. In an aspect, a loop subsequence comprises between 8 nucleotides and 15 nucleotides. In an aspect, a loop subsequence comprises between 8 nucleotides and 10 nucleotides.
  • a second hairpin subsequence comprises at least 2 nucleotides. In an aspect, a second hairpin subsequence comprises at least 3 nucleotides. In an aspect, a second hairpin subsequence comprises at least 4 nucleotides. In an aspect, a second hairpin subsequence comprises at least 5 nucleotides. In an aspect, a second hairpin subsequence comprises at least 6 nucleotides. In an aspect, a second hairpin subsequence comprises at least 7 nucleotides. In an aspect, a second hairpin subsequence comprises at least 8 nucleotides.
  • a second hairpin subsequence comprises at least 9 nucleotides. In an aspect, a second hairpin subsequence comprises at least 10 nucleotides. In an aspect, a second hairpin subsequence comprises at least 11 nucleotides. In an aspect, a second hairpin subsequence comprises at least 12 nucleotides. In an aspect, a second hairpin subsequence comprises at least 13 nucleotides. In an aspect, a second hairpin subsequence comprises at least 14 nucleotides. In an aspect, a second hairpin subsequence comprises at least 15 nucleotides. In an aspect, a second hairpin subsequence comprises at least 20 nucleotides. In an aspect, a second hairpin subsequence comprises at least 25 nucleotides.
  • a second hairpin subsequence comprises between 2 nucleotides and 25 nucleotides. In an aspect, a second hairpin subsequence comprises between 2 nucleotides and 20 nucleotides. In an aspect, a second hairpin subsequence comprises between 2 nucleotides and 18 nucleotides. In an aspect, a second hairpin subsequence comprises between 2 nucleotides and 15 nucleotides. In an aspect, a second hairpin subsequence comprises between 2 nucleotides and 12 nucleotides. In an aspect, a second hairpin subsequence comprises between 2 nucleotides and 10 nucleotides.
  • a second hairpin subsequence comprises between 2 nucleotides and 7 nucleotides. In an aspect, a second hairpin subsequence comprises between 5 nucleotides and 25 nucleotides. In an aspect, a second hairpin subsequence comprises between 5 nucleotides and 20 nucleotides. In an aspect, a second hairpin subsequence comprises between 5 nucleotides and 15 nucleotides. In an aspect, a second hairpin subsequence comprises between 5 nucleotides and 10 nucleotides. In an aspect, a second hairpin subsequence comprises between 10 nucleotides and 20 nucleotides.
  • a second hairpin subsequence comprises between 10 nucleotides and 15 nucleotides.
  • a hairpin sequence comprises SEQ ID NO: 1 (GTTCGCAAGAAC).
  • a hairpin sequence comprises the sequence 5' - GTCGAAGAC - 3'.
  • a hairpin sequence comprises SEQ ID NO: 1 or 5' - GTCGAAGAC - 3'.
  • a universal probe attachment sequence comprises a linking moiety at or near its 3' end.
  • a single-stranded extensible sequence comprises a linking moiety at or near its 5' end.
  • linking moiety refers to any composition that can directly or indirectly bind/attach a universal probe attachment sequence to a single-stranded extensible sequence.
  • the linking moiety is attached to a universal probe attachment sequence via an affinity interaction selected from the group consisting of ligand-receptor, ligand-substrate, hydrogen bonds, van der Waals bonds, ionic bonds, and hydrophobic interactions.
  • the linking moiety is covalently attached to a universal probe attachment sequence.
  • the linking moiety is attached to a single-stranded extensible sequence via an affinity interaction selected from the group consisting of ligand-receptor, ligandsubstrate, hydrogen bonds, van der Waals bonds, ionic bonds, and hydrophobic interactions.
  • the linking moiety is covalently attached to a single-stranded extensible sequence.
  • Non-limiting examples of non-covalent affinity interactions include: biotin-avidin; biotin-streptavidin; a protein-protein interaction; a nucleic acid-protein interaction; a substrate-ligand interaction; an antigen-antibody interaction; an antigen-single chain antibody interaction; a hapten-antibody interaction; a hapten-single chain antibody interaction; a hormone-hormone binding protein interaction; an agonist-receptor interaction; a protein A-IgG interaction; an enzyme cofactor-enzyme interaction; a singled- stranded DNA-VirE2 interaction; a nucleic acid- viral coat protein interaction; and an Agrobacterium VirD2-VirD2 binding protein interaction.
  • a linking moiety is a chemical moiety.
  • a “chemical moiety” refers to a molecule that can directly or indirectly bind/attach a universal probe attachment sequence to a single-stranded extensible sequence.
  • a singlestranded extensible sequence comprises a chemical moiety at or near its 5' end.
  • a universal probe attachment sequence comprises a chemical moiety at or near its 3' end.
  • a chemical moiety is selected from the group consisting of an alkyne, a strained alkyne, an azide, an amide, a carboxyl, a phosphate, a hydroxyl, and a thiol.
  • a chemical moiety is an alkyne.
  • a chemical moiety is a strained alkyne.
  • a chemical moiety is an azide.
  • a chemical moiety is an amide.
  • a chemical moiety is a carboxyl.
  • a chemical moiety is a phosphate.
  • a chemical moiety is a hydroxyl.
  • a chemical moiety is a thiol.
  • a linking moiety comprises an alkyne group.
  • an alkyne group is selected from the group consisting of a strained alkyne and an azide group.
  • a linking moiety comprises a phosphodiester group.
  • a linking moiety comprises a thioether group.
  • a linking moiety comprises a biotin-streptavidin interaction.
  • a linking moiety comprises an antibody-antigen interaction.
  • nucleic acid molecule refers to being attached to the 5'-most or 3'-most nucleotide of the nucleic acid molecule.
  • near the 5' or 3' end of an nucleic acid molecule refers to being attached within the 10 5 '-most or 3 '-most nucleotides of the nucleic acid molecule.
  • a universal probe attachment sequence comprises a fluorophore at or near its 5’ end.
  • a hairpin sequence comprises a fluorophore at or near its 3’ end.
  • Quantitative PCR is used to quantify nucleic acid molecules in a given sample.
  • qPCR can be performed using a fluorescent dye that binds to dsDNA. During each cycle of thermal cycling the dye binds to the newly formed dsDNA, which produces more fluorescence that can be measured. The fluorescence signal increases proportionally to the amount of replicated DNA, enabling quantification.
  • this approach only allows for one target to be examined at a time and the dye will indiscriminately bind to any dsDNA present in the sample.
  • Probes can also be used in qPCR in place of a fluorescent dye. Probes often comprise both a fluorophore and a quencher. Fluorescence resonance energy transfer prevents the emission of the fluorophore when the fluorophore and quencher are both present on the probe.
  • the probe which binds between the two primers on the targeted region of DNA, is hydrolyzed during primer extension, allowing an amplification-dependent increase in fluorescence. Therefore, the measured fluorescence is proportional to the amount of the probe target sequence in a sample.
  • this type of system will not work with DNA polymerases that lack 5' to 3' exonuclease activity.
  • a method provided herein comprises determining the quantity or concentration of at least one amplicon by calculating a cycle threshold (Ct) value.
  • a “fluorophore” refers to any fluorescent compound that can reemit light upon light excitation.
  • a fluorophore is a peptide or protein.
  • a fluorophore is a small organic compound.
  • a fluorophore is a synthetic oligomer or polymer.
  • a fluorophore is a multi-component system.
  • a fluorophore is selected from the group consisting of a peptide or protein, a small organic compound, a synthetic oligomer or polymer, and a multi-component system.
  • a fluorophore is an organic dye.
  • Non-limiting examples of peptide or protein fluorophores included green fluorescence protein (GFP), yellow fluorescence protein (YFP), and red fluorescence protein (RFP).
  • Non-limiting examples of small organic compound fluorophores include xanthene derivatives (e.g, fluorescein, rhodamine, Oregon green, eosin, Texas red), cyanine derivatives (e.g. cyanine, indocarbocyanine, merocyanine), squaraine derivatives, squararine rotaxane derivatives, naphthalene derivatives, coumarin derivatives, oxadiazole derivatives, anthracene derivatives, pyrene derivatives (e.g, Cascade blue), oxazine derivatives (e.g, Nile red, Nile blue, cresyl violet), acridine derivatives (e.g, proflavine, acridine orange, acridine yellow), arylmethine derivatives (e.g. auramine, crystal violet, malachite green), tetrapyrrole derivatives, and dipyrromethene derivatives.
  • a fluorophore is selected from the group consisting of Cy3TM, FAM, VIC, HEX, Cy5TM, ROX, and Quasar 705.
  • a fluorophore is selected from the group consisting of a FreedomTM dye, an ATTOTM dye, an Alexa Fluor® dye, a LI-COR IRDye®, and a Rhodamine dye. FreedomTM dyes, ATTOTM dyes, Alexa Fluor® dyes, LI-COR IRDyes®, and Rhodamine dyes are obtainable from Integrated DNA Technologies (Coralville, Iowa). See, for example idtdna[dot]com/site/Catalog/Modifications/Category/3.
  • a universal probe attachment sequence comprises a quencher at or near its 5’ end.
  • a hairpin sequence comprises a quencher at or near its 3’ end.
  • Quenchers can be used to decrease the fluorescence intensity of a given substance, such as a fluorophore.
  • a quencher refers to any substance that absorbs the excitation energy from a fluorophore, thereby reducing or eliminating the fluorescence intensity of the fluorophore.
  • a quencher dissipates the light energy from a fluorophore as heat.
  • a quencher is a dye that lacks native fluorescence.
  • a quencher is selected from the group consisting of MGB, Black Hole Quencher, Iowa Black® RQ, and Iowa Black® FQ.
  • a double-stranded universal signal probe comprises a non-natural oligonucleotide comprising a non-natural nucleoside base.
  • a universal probe attachment sequence comprises a non-natural oligonucleotide comprising at least one non- natural nucleoside base.
  • a hairpin sequence comprises a non-natural oligonucleotide comprising at least one non-natural nucleoside base.
  • a primer comprises a non-natural oligonucleotide comprising at least one non-natural nucleoside base.
  • a forward primer comprises a non-natural oligonucleotide comprising at least one non-natural nucleoside base.
  • a forward blocker comprises a non- natural oligonucleotide comprising at least one non-natural nucleoside base.
  • a non-natural nucleoside base is selected from the group consisting of a 2'-O-methoxy-ethyl base, a 2'-O-methyl RNA base, a 2' fluoro base, 2-Aminopurine, 5- Bromo-deoxyuridine, deoxyUridine, 2,6-Diaminopurine, dideoxycytidine, deoxyinosine, hydroxymethyl dC, inverted dT, Iso-dG, Iso-dC, inverted dideoxy-T, 5-methyl dC, Super T®, Super G®, and 5-nitroindole.
  • a double-stranded universal signal probe comprises at least 1 non- natural nucleoside base. In an aspect, a double-stranded universal signal probe comprises at least 2 non-natural nucleoside bases. In an aspect, a double-stranded universal signal probe comprises at least 3 non-natural nucleoside bases. In an aspect, a double-stranded universal signal probe comprises at least 4 non-natural nucleoside bases. In an aspect, a double-stranded universal signal probe comprises at least 5 non-natural nucleoside bases. In an aspect, a double-stranded universal signal probe comprises at least 6 non-natural nucleoside bases. In an aspect, a double-stranded universal signal probe comprises at least 7 non-natural nucleoside bases.
  • a universal probe attachment sequences comprises a backbone modification.
  • a primer comprises a backbone modification.
  • a forward primer comprises a backbone modification.
  • a forward blocker comprises a backbone modification.
  • a single-stranded non-extensible sequence comprises a backbone modification.
  • a “backbone modification” refers to a chemical modification to the intemucleotide linkage and/or to a sugar moiety of an oligonucleotide. Without being limited by any scientific theory, backbone modifications increase the stability of oligonucleotides.
  • a backbone modification comprises a modification selected from the group consisting of a locked nucleic acid, a phosphorothioate modification, a phosphorodithioate modification, a methylphosphonate modification, and a phosphoramidate modification.
  • a mixture further comprises an intercalating dye.
  • an “intercalating dye” refers to a fluorescent dye that is capable of inserting between nucleotides in a nucleic acid molecule.
  • an intercalating dye is selected from the group consisting of SYBR® Green, EvaGreen®, and SYTOTM dyes.
  • a kit comprises an intercalating dye.
  • a mixture comprises a template nucleic acid molecule.
  • a kit comprises a template nucleic acid molecule.
  • a “template nucleic acid molecule” refers to a nucleic acid molecule that comprises a sequence that is desired to be detected and/or amplified using PCR-based techniques in conjunction with at least one universal signal probe.
  • a mixture comprises at least one template nucleic acid molecule.
  • a kit comprises at least one template nucleic acid molecule. Any nucleic acid molecule that is desired to be detected or amplified can serve as a suitable template nucleic acid molecule. Numerous potential template nucleic acid molecules can be found in publicly available databases such as GenBank®. See Nucleic Acids Research, 4ED36-42 (2013).
  • a template nucleic acid molecule is a DNA molecule.
  • a template nucleic acid molecule is an RNA molecule.
  • a template nucleic acid molecule is a genomic DNA molecule.
  • a template nucleic acid molecule is an organellar DNA molecule.
  • an organellar DNA molecule is selected from the group consisting of a mitochondrial DNA molecule and a plastid DNA molecule.
  • a template nucleic acid molecule is a complementary DNA (cDNA) molecule.
  • a template nucleic acid molecule is a eukaryotic nucleic acid molecule.
  • a eukaryotic nucleic acid molecule is selected from the group consisting of an animal nucleic acid molecule, a plant nucleic acid molecule, and a fungi nucleic acid molecule.
  • an animal nucleic acid molecule is a human nucleic acid molecule.
  • a template nucleic acid molecule is a prokaryotic nucleic acid molecule.
  • a prokaryotic nucleic acid molecule is selected from the group consisting of a bacteria nucleic acid molecule and an archaea nucleic acid molecule.
  • a template nucleic acid molecule is a virus nucleic acid molecule.
  • a virus nucleic acid molecule is selected from the group consisting of an Adenoviridae nucleic acid molecule, a Herpesviridae nucleic acid molecule, a Poxviridae nucleic acid molecule, a Papillomaviridae nucleic acid molecule, a Parvoviridae nucleic acid molecule, a Reoviridae nucleic acid molecule, a Coronaviridae nucleic acid molecule, a Picomaviridae nucleic acid molecule, a Togaviridae nucleic acid molecule, an Orthomyxoviridae nucleic acid molecule, a Rhabdoviridae nucleic acid molecule, a Retroviridae nucleic acid molecule, a Hepadnaviridae nucleic acid molecule, a Baculoviridae nu
  • a virus nucleic acid molecule is selected from the group consisting of a human orthopnemovirus (HRSV) nucleic acid molecule, an influenza virus nucleic acid molecule, a human immunodeficiency virus (HIV) nucleic acid molecule, a hepatitis B virus (HBV) nucleic acid molecule, and a human papillomavirus (HPV) nucleic acid molecule.
  • HRSV human orthopnemovirus
  • HAV human immunodeficiency virus
  • HBV hepatitis B virus
  • HPV human papillomavirus
  • a template nucleic acid molecule is a viroid nucleic acid molecule.
  • a viroid nucleic acid molecule is selected from the group consisting of a Pospiviroidae nucleic acid molecule, and a Avsunviroidae nucleic acid molecule.
  • a mixture comprises a DNA polymerase enzyme.
  • a kit comprises a DNA polymerase enzyme.
  • a DNA polymerase enzyme is suspended in a liquid.
  • a DNA polymerase enzyme is lyophilized.
  • DNA polymerase refers to an enzyme that is capable of catalyzing the synthesis of a DNA molecule from nucleoside triphosphates (the terms “DNA polymerase” and “DNA polymerase enzyme” are used interchangeably herein).
  • DNA polymerases add a nucleotide to the 3' end of a DNA strand one nucleotide at a time, creating an antiparallel DNA strand as compared to a template DNA strand. DNA polymerases are unable to begin a new DNA molecule de novo; they require a primer to which it can add a first new nucleotide.
  • DNA polymerases are not perfect at making copies of a DNA strand. In general, a DNA polymerase generates an error (e.g., anon-complementary nucleotide is added to the new DNA strand) once in every 100 million to 1 billion nucleotides. However, some DNA polymerases contain proofreading capabilities in the form of 3' to 5' exonuclease activity. DNA polymerases with 3' to 5' exonuclease activity are able to excise an incorrectly placed nucleotide, reinsert the correct nucleotide, and continue with replication.
  • an error e.g., anon-complementary nucleotide is added to the new DNA strand
  • some DNA polymerases contain proofreading capabilities in the form of 3' to 5' exonuclease activity. DNA polymerases with 3' to 5' exonuclease activity are able to excise an incorrectly placed nucleotide, reinsert the
  • Some DNA polymerases such as Taq, possess 5' to 3' exonuclease activity. This 5' to 3' exonuclease activity allows the polymerase to remove primers at the 5' ends of newly synthesized DNA so that polymerase activity can fill in gaps.
  • a DNA polymerase provided herein lacks 5' to 3' exonuclease activity. In an aspect, a DNA polymerase provided herein comprises 5' to 3' exonuclease activity. In an aspect, a DNA polymerase provided herein lacks 3' to 5' exonuclease activity. In an aspect, a DNA polymerase provided herein comprises 3' to 5' exonuclease activity. In an aspect, a DNA polymerase provided herein comprises kinetic proofreading. As used herein, “kinetic proofreading” refers to a mechanism for error correction where a DNA polymerase can discriminate between two possible reaction pathways leading to correct or incorrect products with an accuracy higher than one would predict based on the difference in the activation energy between these two pathways.
  • a DNA polymerase is selected from the group consisting of Phusion® DNA polymerase, Phusion® U DNA polymerase, Q5® DNA polymerase, Q5U® DNA polymerase, PfuUltra II DNA polymerase, KAPA HiFi DNA polymerase, AccurisTM DNA polymerase, PowerUpTM DNA Polymerase, and Taq DNA polymerase.
  • a DNA polymerase is a thermostable DNA polymerase.
  • a mixture comprises at least one reagent needed for a DNA polymerase enzyme to function.
  • a mixture comprises at least one buffer needed for a DNA polymerase enzyme to function.
  • a mixture comprises at least one reagent and at least one buffer needed for a DNA polymerase enzyme to function.
  • a mixture comprises at least one reagent, at least one buffer, or at least one reagent and at least one buffer needed for a DNA polymerase enzyme to function.
  • a kit comprises at least one reagent needed for a DNA polymerase enzyme to function. In an aspect, a kit comprises at least one buffer needed for a DNA polymerase enzyme to function. In an aspect, a kit comprises at least one reagent and at least one buffer needed for a DNA polymerase enzyme to function. In an aspect, a kit comprises at least one reagent, at least one buffer, or at least one reagent and at least one buffer needed for a DNA polymerase enzyme to function.
  • a “reagent” refers to any substance or compound added to a mixture to cause a chemical reaction or to test if a chemical reaction occurs.
  • a reagent comprises a component selected from the group consisting of magnesium, at least one deoxyribose nucleotide triphosphate (dNTP), phosphatase, betaine, dimethyl sulfoxide (DMSO), and tetramethylammonium chloride (TMAC).
  • dNTP deoxyribose nucleotide triphosphate
  • DMSO dimethyl sulfoxide
  • TMAC tetramethylammonium chloride
  • a reagent is a liquid.
  • a reagent is lyophilized.
  • a “buffer” refers to any solution useful for maintaining a relatively constant pH over a given range by neutralizing the effects of hydrogen ions.
  • a buffer comprises a component selected from the group consisting of Tris-HCl, potassium chloride (KC1), a detergent (e.g., without being limiting, Tween®-20, Tween®-80, TritonTM X-100, TritonTM X-114, NP-40, Brij-35, Brij-58, n-dodecyl-beta-maltoside, octyl-beta-glucoside, octylthio glucoside, sodium dodecyl sulfate (SDS), 3-((3- cholamidopropyl) dimethylammonio)-!
  • SDS sodium dodecyl sulfate
  • CHAPS -propanesulfonate magnesium chloride
  • MgCh magnesium chloride
  • EDTA ethylenediaminetetraacetic acid
  • BSA bovine serum albumin
  • DTT dithiothreitol
  • this disclosure provides a method for amplifying a template nucleic acid molecule, the method comprising: (a) generating a mixture by mixing a sample comprising the template nucleic acid molecule with: (i) a first primer, where the first primer comprises, from 3' to 5': (1) a single-stranded extensible sequence comprising between 7 nucleotides and 100 nucleotides, where the single-stranded extensible sequence is at least 90% complementary to an initiation subsequence of a template nucleic acid molecule; and (2) a double-stranded universal signal probe comprising: (A) a first strand comprising a universal probe attachment sequence conjugated to the single-stranded extensible sequence, where the universal probe attachment sequence comprises between 6 nucleotides and 100 nucleotides; and (B) a second strand comprising a hairpin sequence comprising between 7 nucleotides and 40 nucleotides, where the hairpin sequence further comprises: (I) a first primer, where the first primer comprises
  • this disclosure provides a method for multiplexed amplification of nucleic acids, the method comprising: (a) generating a mixture by mixing a sample comprising a template nucleic acid molecule with: (i) a plurality of first primers, where each primer of the plurality of first primers comprises, from 3' to 5': (1) a single-stranded extensible sequence comprising between 7 nucleotides and 100 nucleotides, where the single-stranded extensible sequence is at least 90% complementary to an initiation subsequence of a template nucleic acid molecule; and (2) a double-stranded universal signal probe comprising: (A) a first strand comprising a universal probe attachment sequence conjugated to the single-stranded extensible sequence, where the universal probe attachment sequence comprises between 6 nucleotides and 100 nucleotides; and (B) a second strand comprising a hairpin sequence comprising between 7 nucleotides and 40 nucleotides, where the
  • the method further comprises (c) sequencing the plurality of amplicons using high-throughput sequencing.
  • the plurality of first primers comprises at least 2 first primers, where each of the at least 2 first primers comprises a different single-stranded extensible sequence.
  • the plurality of first primers comprises at least 3 first primers, where each of the at least 3 first primers comprises a different single-stranded extensible sequence.
  • the plurality of first primers comprises at least 2 different universal probe attachment sequences.
  • the plurality of first primers comprises at least 3 different universal probe attachment sequences.
  • the plurality of first primers comprises at least 2 different hairpin sequences.
  • the plurality of first primers comprises at least 3 different hairpin sequences.
  • the plurality of first primers comprises a first universal probe attachment sequence and a second universal probe attachment sequence, where the first universal probe attachment sequence comprises a first high-throughput sequencing adapter sequence and the second universal probe attachment sequence comprises a second high-throughput sequencing adapter sequence, and where the first high-throughput sequencing adapter sequence and the second high-throughput adapter sequence are not identical.
  • the plurality of first primers comprises a first universal probe attachment sequence and a second universal probe attachment sequence, where the first universal probe attachment sequence comprises a first high-throughput sequencing adapter sequence and the second universal probe attachment sequence comprises a second high-throughput sequencing adapter sequence, and where the first high-throughput sequencing adapter sequence and the second high-throughput adapter sequence are identical.
  • the plurality of first primers comprises a first group comprising at least one single-stranded extensible sequence conjugated to a first universal probe attachment sequence and a second group comprising at least one single-stranded extensible sequence conjugated to a second universal probe attachment sequence, and where the first universal probe attachment second and the second universal probe attachment sequence are not identical.
  • the plurality of first primers comprises a first group comprising at least one single-stranded extensible sequence conjugated to a first universal probe attachment sequence and a second group comprising at least one single-stranded extensible sequence conjugated to a second universal probe attachment sequence, and where the first universal probe attachment second and the second universal probe attachment sequence are identical.
  • the concentration of each first primer from the plurality of first primers in the mixture is between 1 pM and 10 pM. In an aspect, the concentration of each first primer from the plurality of first primers in the mixture is between 1 pM and 5 pM. In an aspect, the concentration of each first primer from the plurality of first primers in the mixture is between 1 pM and 1 pM.
  • the concentration of each first primer from the plurality of first primers in the mixture is between 1 pM and 0.5 pM. In an aspect, the concentration of each first primer from the plurality of first primers in the mixture is between 1 pM and 0. 1 pM. In an aspect, the concentration of each first primer from the plurality of first primers in the mixture is between 1 pM and 0.05 pM.
  • thermal cycling refers to a controlled set of timed temperature changes.
  • One “cycle” of thermal cycling comprises at least two stages. The first stage of a cycle comprises a first temperature maintained for a desired amount of time, and the second stage of a cycle comprises a second temperature maintained for a desired amount of time.
  • a cycle further comprises a third stage comprising a third temperature maintained for a desired amount of time.
  • a cycle further comprises a fourth stage comprising a fourth temperature maintained for a desired amount of time.
  • thermal cycling comprises repeating the same cycle several times.
  • a first, second, third, or fourth stage of a cycle comprises a temperature of less than 60°C. In an aspect, a first, second, third, or fourth stage of a cycle comprises a temperature of less than 70°C. In an aspect, a first, second, third, or fourth stage of a cycle comprises a temperature of less than 75°C. In an aspect, a first, second, third, or fourth stage of a cycle comprises a temperature of less than 80°C. In an aspect, a first, second, third, or fourth stage of a cycle comprises a temperature of less than 90°C. In an aspect, a first, second, third, or fourth stage of a cycle comprises a temperature of greater than 60°C.
  • a first, second, third, or fourth stage of a cycle comprises a temperature of greater than 70°C. In an aspect, a first, second, third, or fourth stage of a cycle comprises a temperature of greater than 75°C. In an aspect, a first, second, third, or fourth stage of a cycle comprises a temperature of greater than 80°C. In an aspect, a first, second, third, or fourth stage of a cycle comprises a temperature of greater than 90°C.
  • a first, second, third, or fourth stage of a cycle lasts for at least 1 second. In an aspect, a first, second, third, or fourth stage of a cycle lasts for at least 10 seconds. In an aspect, a first, second, third, or fourth stage of a cycle lasts for at least 30 seconds. In an aspect, a first, second, third, or fourth stage of a cycle lasts for at least 1 minute. In an aspect, a first, second, third, or fourth stage of a cycle lasts for at least 2 minutes. In an aspect, a first, second, third, or fourth stage of a cycle lasts for at least 10 minutes.
  • a first, second, third, or fourth stage of a cycle lasts for at least 15 minutes. In an aspect, a first, second, third, or fourth stage of a cycle lasts for at least 30 minutes. In an aspect, a first, second, third, or fourth stage of a cycle lasts for at least 1 hour. In an aspect, a first, second, third, or fourth stage of a cycle lasts for at least 2 hours.
  • a first, second, third, or fourth stage of a cycle lasts for between 1 second and 3 hours. In an aspect, a first, second, third, or fourth stage of a cycle lasts for between 1 second and 2 hours. In an aspect, a first, second, third, or fourth stage of a cycle lasts for between 1 second and 1 hour. In an aspect, a first, second, third, or fourth stage of a cycle lasts for between 1 second and 30 minutes. In an aspect, a first, second, third, or fourth stage of a cycle lasts for between 1 second and 20 minutes. In an aspect, a first, second, third, or fourth stage of a cycle lasts for between 1 second and 15 minutes.
  • a first, second, third, or fourth stage of a cycle lasts for between 1 second and 10 minutes. In an aspect, a first, second, third, or fourth stage of a cycle lasts for between 1 second and 5 minutes. In an aspect, a first, second, third, or fourth stage of a cycle lasts for between 1 second and 2 minutes. In an aspect, a first, second, third, or fourth stage of a cycle lasts for between 1 second and 1 minute. In an aspect, a first, second, third, or fourth stage of a cycle lasts for between 1 second and 30 seconds.
  • thermal cycling comprises at least 1 cycle. In an aspect, thermal cycling comprises at least 2 cycles. In an aspect, thermal cycling comprises at least 3 cycles. In an aspect, thermal cycling comprises at least 4 cycles. In an aspect, thermal cycling comprises at least 5 cycles. In an aspect, thermal cycling comprises at least 6 cycles. In an aspect, thermal cycling comprises at least 7 cycles. In an aspect, thermal cycling comprises at least 8 cycles. In an aspect, thermal cycling comprises at least 9 cycles. In an aspect, thermal cycling comprises at least 10 cycles. In an aspect, thermal cycling comprises at least 15 cycles. In an aspect, thermal cycling comprises at least 20 cycles. In an aspect, thermal cycling comprises at least 25 cycles. In an aspect, thermal cycling comprises at least 30 cycles. In an aspect, thermal cycling comprises at least 40 cycles.
  • thermal cycling comprises between 1 cycle and 60 cycles. In an aspect, thermal cycling comprises between 1 cycle and 50 cycles. In an aspect, thermal cycling comprises between 1 cycle and 40 cycles. In an aspect, thermal cycling comprises between
  • thermal cycling comprises between 1 cycle and 30 cycles. In an aspect, thermal cycling comprises between 1 cycle and 20 cycles. In an aspect, thermal cycling comprises between 1 cycle and 10 cycles. In an aspect, thermal cycling comprises between 1 cycle and 5 cycles. In an aspect, thermal cycling comprises between 2 cycles and 60 cycles. In an aspect, thermal cycling comprises between
  • thermal cycling comprises between 2 cycles and 20 cycles. In an aspect, thermal cycling comprises between 2 cycles and 10 cycles. In an aspect, thermal cycling comprises between 2 cycles and 8 cycles. In an aspect, thermal cycling comprises between 20 cycles and 60 cycles. In an aspect, thermal cycling comprises between 20 cycles and 40 cycles.
  • each cycle of thermal cycling comprises (a) a first stage comprising a temperature of at least 70°C for between one second and one hour; and (b) a second stage comprising a temperature of less than 70°C for between one second and two hours.
  • each cycle of thermal cycling comprises (a) a first stage comprising a temperature of at least 75°C for between one second and one hour; and (b) a second stage comprising a temperature of less than 75°C for between one second and two hours.
  • each cycle of thermal cycling comprises (a) a first stage comprising a temperature of at least 80°C for between one second and one hour; and (b) a second stage comprising a temperature of less than 80°C for between one second and two hours.
  • each cycle of thermal cycling comprises (a) a first stage comprising a temperature of at least 90°C for between one second and one hour; and (b) a second stage comprising a temperature of less than 90°C for between one second and two hours.
  • a first primer and a template nucleic acid molecule comprise a standard of free energy of hybridization (AG°1) between -7 kcal/mol and -20 kcal/mol; and/or (b) a second primer and the template nucleic acid molecule comprise a standard of free energy of hybridization (AG°2) between -7 kcal/mol and -20 kcal/mol; and/or (c) a universal probe attachment sequence and a hairpin sequence comprise a standard free energy of hybridization (AG°3) between -8 kcal/mol and -100 kcal/mol where an amplicon comprises a length of between 50 nucleotides and 5000 nucleotides, and where AG°1, AG°2, and AG°3 are calculated at a temperature of 60°C and a salinity of 0.2M sodium.
  • AG°1 is between -7 and -20, between -8 and -20, between -9 and -20, between -10 and -20, between -11 and -20, between -12 and -20, between -13 and -20, between -14 and -20, between -15 and -20, between -16 and -20, between -17 and -20, between -18 and -20, between -19 and -20, between -8 and -19, between -8 and -18, between -8 and -17, between -8 and -16, between -8 and -15, between -8 and -14, between -8 and -13, between -8 and -12, between -8 and -11, between -8 and -10, between -8 and - 9, between -7 and -8, between -9 and -18, between -10 and -17, between -11 and -16, or between -12 and -15 kcal/mol at a temperature of 60°C and a salinity concentration of 0.2M sodium.
  • AG°2 is between -7 and -20, between -8 and -20, between -9 and -20, between -10 and -20, between -11 and -20, between -12 and -20, between -13 and -20, between -14 and -20, between -15 and -20, between -16 and -20, between -17 and -20, between -18 and -20, between -19 and -20, between -8 and -19, between -8 and -18, between -8 and -17, between -8 and -16, between -8 and -15, between -8 and -14, between -8 and -13, between -8 and -12, between -8 and -11, between -8 and -10, between -8 and - 9, between -7 and -8, between -9 and -18, between -10 and -17, between -11 and -16, or between -12 and -15 kcal/mol at a temperature of 60°C and a salinity concentration of 0.2M sodium.
  • AG°3 is between -8 and -100, between -8 and -90, between -8 and - 80, between -8 and -70, between -8 and -60, between -8 and -50, between -8 and -40, between -8 and -30, between -8 and -20, between -8 and -10, between -10 and -100, between -20 and -100, between -30 and -100, between -40 and -100, between -50 and -100, between -60 and -100, between -70 and -100, between -80 and -100, between -90 and -100, between -10 and -90, between -20 and -80, between -30 and -70, or between -40 and -60 kcal/mol at a temperature of 60°C and a salinity concentration of 0.2M sodium.
  • a primer (e.g., without being limiting, a second primer) comprises a sequence at least 75% identical or complementary to a template nucleic acid molecule.
  • a primer comprises a sequence at least 80% identical or complementary to a template nucleic acid molecule.
  • a primer comprises a sequence at least 85% identical or complementary to a template nucleic acid molecule.
  • a primer comprises a sequence at least 90% identical or complementary to a template nucleic acid molecule.
  • a primer comprises a sequence at least 95% identical or complementary to a template nucleic acid molecule.
  • a primer comprises a sequence at least 99% identical or complementary to a template nucleic acid molecule.
  • a primer comprises a sequence 100% identical or complementary to a template nucleic acid molecule.
  • a sample is mixed with a plurality of primers. In an aspect, a sample is mixed with a plurality of second primers. In an aspect, a sample is mixed with a plurality of forward primers. In an aspect, a sample is mixed with a plurality of reverse primers.
  • a plurality of primers refers to two or more primers that are unique. In an aspect, a plurality of primers refers to two or more second primers that are unique. In an aspect, a plurality of primers refers to two or more forward primers that are unique. In an aspect, a plurality of primers refers to two or more reverse primers that are unique. Unique primers are capable of binding different loci. As a non-limiting examples, two unique primers can either bind different locations within a single gene or they can bind to two different genes.
  • a sample is mixed with at least 3 unique second primers. In an aspect, a sample is mixed with at least 4 unique second primers. In an aspect, a sample is mixed with at least 5 unique second primers. In an aspect, a sample is mixed with at least 6 unique second primers. In an aspect, a sample is mixed with at least 7 unique second primers. In an aspect, a sample is mixed with at least 8 unique second primers. In an aspect, a sample is mixed with at least 9 unique second primers. In an aspect, a sample is mixed with at least 10 unique second primers. In an aspect, a sample is mixed with at least 15 unique second primers. In an aspect, a sample is mixed with at least 20 unique second primers.
  • a sample is mixed with at least 25 unique second primers. In an aspect, a sample is mixed with at least 30 unique second primers. In an aspect, a sample is mixed with at least 40 unique second primers. In an aspect, a sample is mixed with at least 50 unique second primers. In an aspect, a sample is mixed with at least 60 unique second primers. In an aspect, a sample is mixed with at least 70 unique second primers. In an aspect, a sample is mixed with at least 80 unique second primers. In an aspect, a sample is mixed with at least 90 unique second primers. In an aspect, a sample is mixed with at least 100 unique second primers. In an aspect, a sample is mixed with at least 200 unique second primers.
  • a sample is mixed with at least 3 unique forward primers. In an aspect, a sample is mixed with at least 4 unique forward primers. In an aspect, a sample is mixed with at least 5 unique forward primers. In an aspect, a sample is mixed with at least 6 unique forward primers. In an aspect, a sample is mixed with at least 7 unique forward primers. In an aspect, a sample is mixed with at least 8 unique forward primers. In an aspect, a sample is mixed with at least 9 unique forward primers. In an aspect, a sample is mixed with at least 10 unique forward primers. In an aspect, a sample is mixed with at least 15 unique forward primers. In an aspect, a sample is mixed with at least 20 unique forward primers.
  • a sample is mixed with at least 25 unique forward primers. In an aspect, a sample is mixed with at least 30 unique forward primers. In an aspect, a sample is mixed with at least 40 unique forward primers. In an aspect, a sample is mixed with at least 50 unique forward primers. In an aspect, a sample is mixed with at least 60 unique forward primers. In an aspect, a sample is mixed with at least 70 unique forward primers. In an aspect, a sample is mixed with at least 80 unique forward primers. In an aspect, a sample is mixed with at least 90 unique forward primers. In an aspect, a sample is mixed with at least 100 unique forward primers. In an aspect, a sample is mixed with at least 200 unique forward primers.
  • a sample is mixed with at least 3 unique reverse primers. In an aspect, a sample is mixed with at least 4 unique reverse primers. In an aspect, a sample is mixed with at least 5 unique reverse primers. In an aspect, a sample is mixed with at least 6 unique reverse primers. In an aspect, a sample is mixed with at least 7 unique reverse primers. In an aspect, a sample is mixed with at least 8 unique reverse primers. In an aspect, a sample is mixed with at least 9 unique reverse primers. In an aspect, a sample is mixed with at least 10 unique reverse primers. In an aspect, a sample is mixed with at least 15 unique reverse primers. In an aspect, a sample is mixed with at least 20 unique reverse primers.
  • a sample is mixed with at least 25 unique reverse primers. In an aspect, a sample is mixed with at least 30 unique reverse primers. In an aspect, a sample is mixed with at least 40 unique reverse primers. In an aspect, a sample is mixed with at least 50 unique reverse primers. In an aspect, a sample is mixed with at least 60 unique reverse primers. In an aspect, a sample is mixed with at least 70 unique reverse primers. In an aspect, a sample is mixed with at least 80 unique reverse primers. In an aspect, a sample is mixed with at least 90 unique reverse primers. In an aspect, a sample is mixed with at least 100 unique reverse primers. In an aspect, a sample is mixed with at least 200 unique reverse primers.
  • PCR polymerase chain reaction
  • amplicons can then be sequenced, analyzed (e.g., gel electrophoresis), or cloned into a plasmid or vector.
  • an amplicon comprises a length of between 10 nucleotides and 7500 nucleotides.
  • an amplicon comprises a length of between 20 nucleotides and 5000 nucleotides.
  • an amplicon comprises a length of between 30 nucleotides and 5000 nucleotides.
  • an amplicon comprises a length of between 50 nucleotides and 5000 nucleotides. In an aspect, an amplicon comprises a length of between 75 nucleotides and 5000 nucleotides. In an aspect, an amplicon comprises a length of between 100 nucleotides and 5000 nucleotides. In an aspect, an amplicon comprises a length of between 250 nucleotides and 5000 nucleotides. In an aspect, an amplicon comprises a length of between 500 nucleotides and 5000 nucleotides. In an aspect, an amplicon comprises a length of between 1000 nucleotides and 5000 nucleotides.
  • an amplicon comprises a length of between 2500 nucleotides and 5000 nucleotides. In an aspect, an amplicon comprises a length of between 30 nucleotides and 1000 nucleotides. In an aspect, an amplicon comprises a length of between 30 nucleotides and 750 nucleotides. In an aspect, an amplicon comprises a length of between 30 nucleotides and 500 nucleotides. In an aspect, an amplicon comprises a length of between 30 nucleotides and 250 nucleotides. In an aspect, an amplicon comprises a length of between 30 nucleotides and 100 nucleotides.
  • an amplicon comprises a length of at least 10 nucleotides. In an aspect, an amplicon comprises a length of at least 20 nucleotides. In an aspect, an amplicon comprises a length of at least 30 nucleotides. In an aspect, an amplicon comprises a length of at least 50 nucleotides. In an aspect, an amplicon comprises a length of at least 75 nucleotides. In an aspect, an amplicon comprises a length of at least 100 nucleotides. In an aspect, an amplicon comprises a length of at least 250 nucleotides. In an aspect, an amplicon comprises a length of at least 500 nucleotides. In an aspect, an amplicon comprises a length of at least 1000 nucleotides.
  • a method comprises purifying at least one amplicon. In an aspect, a method comprises sequencing at least one amplicon. In an aspect, a method comprises purification of at least one amplicon and obtaining the nucleotide sequence of the at least one amplicon.
  • a method comprises re-amplification of at least one amplicon using fluorescent dideoxyribose nucleotide triphosphates (ddNTPs).
  • ddNTPs dideoxyribose nucleotide triphosphates
  • an amplicon is sequenced via Sanger sequencing. See Sanger and Coulson, J. Mol. Biol., 94:441-446 (1975).
  • an amplicon is sequenced via next-generation sequencing.
  • next-generation sequencing include single-molecule real-time sequencing (e.g., Pacific Biosciences), Ion Torrent sequencing, sequencing-by-synthesis (e.g., Illumina), sequencing by ligation (SOLiD sequencing), nanopore sequencing, and GenapSys sequencing.
  • a method comprises high-throughput sequencing.
  • a method comprises subjecting a plurality of amplicons to high-throughput sequencing.
  • “high-throughput sequencing” refers to any sequences method that is capable of sequencing multiple (e.g, tens, hundreds, thousands, millions, hundreds of millions) DNA molecules in parallel.
  • Sanger sequencing is not high-throughput sequencing.
  • high-throughput sequencing comprises sequencing-by-synthesis.
  • high-throughput sequencing comprises the use of a sequencing-by-synthesis (SBS) flow cell.
  • an SBS flow cell is selected from the group consisting of an Illumina SBS flow cell and a Pacific Biosciences (PacBio) SBS flow cell.
  • high-throughput sequencing is performed via electrical current measurements in conjunction with a nanopore.
  • the concentration of each single-stranded extensible sequence in a mixture is between 10 pM and 15 pM. In an aspect, the concentration of each singlestranded extensible sequence in a mixture is between 10 pM and 10 pM. In an aspect, the concentration of an each single-stranded extensible sequence in a mixture is between 100 pM and 5 pM. In an aspect, the concentration of each single-stranded extensible sequence in a mixture is between 100 pM and 1 pM. In an aspect, the concentration of each singlestranded extensible sequence in a mixture is between 100 pM and 500 nM.
  • the concentration of each double-stranded universal signal probe in a mixture is between 1 pM and 5 pM. In an aspect, the concentration of each single-stranded extensible sequence in a mixture is at least 10 pM. In an aspect, the concentration of each single-stranded extensible sequence in a mixture is at least 50 pM. In an aspect, the concentration of each single-stranded extensible sequence in a mixture is at least 100 pM. In an aspect, the concentration of each single-stranded extensible sequence in a mixture is at least 500 pM. In an aspect, the concentration of each single-stranded extensible sequence in a mixture is at least 1 nM.
  • the concentration of each single-stranded extensible sequence in a mixture is at least 500 nM. In an aspect, the concentration of each single-stranded extensible sequence in a mixture is at least 1 pM. In an aspect, the concentration of each single-stranded extensible sequence in a mixture is at least 5 pM. In an aspect, the concentration of each single-stranded extensible sequence in a mixture is at least 10 pM. [0136] In an aspect, the concentration of a single-stranded extensible sequence conjugated to a universal probe attachment sequence in a mixture is between 10 pM and 15 pM.
  • the concentration of a single-stranded extensible sequence conjugated to a universal probe attachment sequence in a mixture is between 10 pM and 10 pM. In an aspect, the concentration of an a single-stranded extensible sequence conjugated to a universal probe attachment sequence in a mixture is between 100 pM and 5 pM. In an aspect, the concentration of a single-stranded extensible sequence conjugated to a universal probe attachment sequence in a mixture is between 100 pM and 1 pM. In an aspect, the concentration of a single-stranded extensible sequence conjugated to a universal probe attachment sequence in a mixture is between 100 pM and 500 nM.
  • the concentration of each double-stranded universal signal probe in a mixture is between 1 pM and 5 pM. In an aspect, the concentration of a single-stranded extensible sequence conjugated to a universal probe attachment sequence in a mixture is at least 10 pM. In an aspect, the concentration of a single-stranded extensible sequence conjugated to a universal probe attachment sequence in a mixture is at least 50 pM. In an aspect, the concentration of a single-stranded extensible sequence conjugated to a universal probe attachment sequence in a mixture is at least 100 pM. In an aspect, the concentration of a single-stranded extensible sequence conjugated to a universal probe attachment sequence in a mixture is at least 500 pM.
  • the concentration of a single-stranded extensible sequence conjugated to a universal probe attachment sequence in a mixture is at least 1 nM. In an aspect, the concentration of a single-stranded extensible sequence conjugated to a universal probe attachment sequence in a mixture is at least 500 nM. In an aspect, the concentration of a single-stranded extensible sequence conjugated to a universal probe attachment sequence in a mixture is at least 1 pM. In an aspect, the concentration of a single-stranded extensible sequence conjugated to a universal probe attachment sequence in a mixture is at least 5 pM. In an aspect, the concentration of a single-stranded extensible sequence conjugated to a universal probe attachment sequence in a mixture is at least 10 pM.
  • the concentration of each double-stranded universal signal probe in a mixture is between 10 pM and 15 pM. In an aspect, the concentration of each doublestranded universal signal probe in a mixture is between 10 pM and 10 pM. In an aspect, the concentration of an each double-stranded universal signal probe in a mixture is between 100 pM and 5 pM. In an aspect, the concentration of each double-stranded universal signal probe in a mixture is between 100 pM and 1 pM. In an aspect, the concentration of each double-stranded universal signal probe in a mixture is between 100 pM and 500 nM.
  • the concentration of each double-stranded universal signal probe in a mixture is between 1 pM and 5 pM. In an aspect, the concentration of each double-stranded universal signal probe in a mixture is at least 10 pM. In an aspect, the concentration of each doublestranded universal signal probe in a mixture is at least 50 pM. In an aspect, the concentration of each double-stranded universal signal probe in a mixture is at least 100 pM. In an aspect, the concentration of each double-stranded universal signal probe in a mixture is at least 500 pM. In an aspect, the concentration of each double-stranded universal signal probe in a mixture is at least 1 nM.
  • the concentration of each double-stranded universal signal probe in a mixture is at least 500 nM. In an aspect, the concentration of each double-stranded universal signal probe in a mixture is at least 1 pM. In an aspect, the concentration of each double-stranded universal signal probe in a mixture is at least 5 pM. In an aspect, the concentration of each double-stranded universal signal probe in a mixture is at least 10 pM.
  • the concentration of each hairpin sequence in a mixture is between 10 pM and 15 pM. In an aspect, the concentration of each hairpin sequence in a mixture is between 10 pM and 10 pM. In an aspect, the concentration of an each hairpin sequence in a mixture is between 100 pM and 5 pM. In an aspect, the concentration of each hairpin sequence in a mixture is between 100 pM and 1 pM. In an aspect, the concentration of each hairpin sequence in a mixture is between 100 pM and 500 nM. In an aspect, the concentration of each double-stranded universal signal probe in a mixture is between 1 pM and 5 pM.
  • the concentration of each hairpin sequence in a mixture is at least 10 pM. In an aspect, the concentration of each hairpin sequence in a mixture is at least 50 pM. In an aspect, the concentration of each hairpin sequence in a mixture is at least 100 pM. In an aspect, the concentration of each hairpin sequence in a mixture is at least 500 pM. In an aspect, the concentration of each hairpin sequence in a mixture is at least 1 nM. In an aspect, the concentration of each hairpin sequence in a mixture is at least 500 nM. In an aspect, the concentration of each hairpin sequence in a mixture is at least 1 pM. In an aspect, the concentration of each hairpin sequence in a mixture is at least 5 pM. In an aspect, the concentration of each hairpin sequence in a mixture is at least 10 pM.
  • the concentration of each second primer in a mixture is between 10 pM and 15 pM. In an aspect, the concentration of each second primer in a mixture is between 10 pM and 10 pM. In an aspect, the concentration of an each second primer in a mixture is between 100 pM and 5 pM. In an aspect, the concentration of each second primer in a mixture is between 100 pM and 1 pM. In an aspect, the concentration of each second primer in a mixture is between 100 pM and 500 nM. In an aspect, the concentration of each second primer in a mixture is at least 10 pM. In an aspect, the concentration of each second primer in a mixture is at least 50 pM.
  • the concentration of each second primer in a mixture is at least 100 pM. In an aspect, the concentration of each second primer in a mixture is at least 500 pM. In an aspect, the concentration of each second primer in a mixture is at least 1 nM. In an aspect, the concentration of each second primer in a mixture is at least 500 nM. In an aspect, the concentration of each second primer in a mixture is at least 1 pM. In an aspect, the concentration of each second primer in a mixture is at least 5 pM. In an aspect, the concentration of each second primer in a mixture is at least 10 pM.
  • the concentration of a forward primer in a mixture is between 10 pM and 15 pM. In an aspect, the concentration of a forward primer in a mixture is between 10 pM and 10 pM. In an aspect, the concentration of an a forward primer in a mixture is between 100 pM and 5 pM. In an aspect, the concentration of a forward primer in a mixture is between 100 pM and 1 pM. In an aspect, the concentration of a forward primer in a mixture is between 100 pM and 500 nM. In an aspect, the concentration of a forward primer in a mixture is between 1 pM and 5 pM. In an aspect, the concentration of a forward primer in a mixture is at least 10 pM.
  • the concentration of a forward primer in a mixture is at least 50 pM. In an aspect, the concentration of a forward primer in a mixture is at least 100 pM. In an aspect, the concentration of a forward primer in a mixture is at least 500 pM. In an aspect, the concentration of a forward primer in a mixture is at least 1 nM. In an aspect, the concentration of a forward primer in a mixture is at least 500 nM. In an aspect, the concentration of a forward primer in a mixture is at least 1 pM. In an aspect, the concentration of a forward primer in a mixture is at least 5 pM. In an aspect, the concentration of a forward primer in a mixture is at least 10 pM.
  • the concentration of a forward blocker in a mixture is between 10 pM and 15 pM. In an aspect, the concentration of a forward blocker in a mixture is between 10 pM and 10 pM. In an aspect, the concentration of an a forward blocker in a mixture is between 100 pM and 5 pM. In an aspect, the concentration of a forward blocker in a mixture is between 100 pM and 1 pM. In an aspect, the concentration of a forward blocker in a mixture is between 100 pM and 500 nM. In an aspect, the concentration of a forward blocker in a mixture is between 1 pM and 5 pM.
  • the concentration of a forward blocker in a mixture is at least 10 pM. In an aspect, the concentration of a forward blocker in a mixture is at least 50 pM. In an aspect, the concentration of a forward blocker in a mixture is at least 100 pM. In an aspect, the concentration of a forward blocker in a mixture is at least 500 pM. In an aspect, the concentration of a forward blocker in a mixture is at least 1 nM. In an aspect, the concentration of a forward blocker in a mixture is at least 500 nM. In an aspect, the concentration of a forward blocker in a mixture is at least 1 pM. In an aspect, the concentration of a forward blocker in a mixture is at least 5 pM. In an aspect, the concentration of a forward blocker in a mixture is at least 10 pM.
  • the stoichiometric ratio of a single-stranded extensible sequence conjugated to a universal probe attachment sequence to a hairpin sequence in a mixture is between 0.01 and 100. In an aspect, the stoichiometric ratio of a single-stranded extensible sequence conjugated to a universal probe attachment sequence to a hairpin sequence in a mixture is between 0.1 and 100. In an aspect, the stoichiometric ratio of a single-stranded extensible sequence conjugated to a universal probe attachment sequence to a hairpin sequence in a mixture is between 0.25 and 100.
  • the stoichiometric ratio of a single-stranded extensible sequence conjugated to a universal probe attachment sequence to a hairpin sequence in a mixture is between 0.5 and 100. In an aspect, the stoichiometric ratio of a single-stranded extensible sequence conjugated to a universal probe attachment sequence to a hairpin sequence in a mixture is between 0.5 and 75. In an aspect, the stoichiometric ratio of a single-stranded extensible sequence conjugated to a universal probe attachment sequence to a hairpin sequence in a mixture is between 0.5 and 50.
  • the stoichiometric ratio of a single-stranded extensible sequence conjugated to a universal probe attachment sequence to a hairpin sequence in a mixture is between 0.5 and 25. In an aspect, the stoichiometric ratio of a single-stranded extensible sequence conjugated to a universal probe attachment sequence to a hairpin sequence in a mixture is between 0.5 and 10. In an aspect, the stoichiometric ratio of a single-stranded extensible sequence conjugated to a universal probe attachment sequence to a hairpin sequence in a mixture is between 0.5 and 5.
  • the stoichiometric ratio of a single-stranded extensible sequence conjugated to a universal probe attachment sequence to a hairpin sequence in a mixture is between 0.5 and 2.5. In an aspect, the stoichiometric ratio of a single-stranded extensible sequence conjugated to a universal probe attachment sequence to a hairpin sequence in a mixture is between 0.5 and 1. In an aspect, the stoichiometric ratio of a single-stranded extensible sequence conjugated to a universal probe attachment sequence to a hairpin sequence in a mixture is between 1 and 100.
  • the stoichiometric ratio of a single-stranded extensible sequence conjugated to a universal probe attachment sequence to a hairpin sequence in a mixture is between 1 and 50. In an aspect, the stoichiometric ratio of a single-stranded extensible sequence conjugated to a universal probe attachment sequence to a hairpin sequence in a mixture is between 1 and 25. In an aspect, the stoichiometric ratio of a single-stranded extensible sequence conjugated to a universal probe attachment sequence to a hairpin sequence in a mixture is between 1 and 10.
  • the stoichiometric ratio of a single-stranded extensible sequence conjugated to a universal probe attachment sequence to a hairpin sequence in a mixture is between 1 and 5. In an aspect, the stoichiometric ratio of a single-stranded extensible sequence conjugated to a universal probe attachment sequence to a hairpin sequence in a mixture is between 1 and 2.5.
  • the stoichiometric ratio of all of the a single-stranded extensible sequences conjugated to a universal probe attachment sequence to a hairpin sequence in a mixture is between 0.01 and 100. In an aspect, the stoichiometric ratio of all of the a singlestranded extensible sequences conjugated to a universal probe attachment sequence to a hairpin sequence in a mixture is between 0.1 and 100. In an aspect, the stoichiometric ratio of all of the a single-stranded extensible sequences conjugated to a universal probe attachment sequence to a hairpin sequence in a mixture is between 0.25 and 100.
  • the stoichiometric ratio of all of the a single-stranded extensible sequences conjugated to a universal probe attachment sequence to a hairpin sequence in a mixture is between 0.5 and 100. In an aspect, the stoichiometric ratio of all of the a single-stranded extensible sequences conjugated to a universal probe attachment sequence to a hairpin sequence in a mixture is between 0.5 and 75. In an aspect, the stoichiometric ratio of all of the a single-stranded extensible sequences conjugated to a universal probe attachment sequence to a hairpin sequence in a mixture is between 0.5 and 50.
  • the stoichiometric ratio of all of the a single-stranded extensible sequences conjugated to a universal probe attachment sequence to a hairpin sequence in a mixture is between 0.5 and 25. In an aspect, the stoichiometric ratio of all of the a single-stranded extensible sequences conjugated to a universal probe attachment sequence to a hairpin sequence in a mixture is between 0.5 and 10. In an aspect, the stoichiometric ratio of all of the a single-stranded extensible sequences conjugated to a universal probe attachment sequence to a hairpin sequence in a mixture is between 0.5 and 5.
  • the stoichiometric ratio of all of the a single-stranded extensible sequences conjugated to a universal probe attachment sequence to a hairpin sequence in a mixture is between 0.5 and 2.5. In an aspect, the stoichiometric ratio of all of the a single-stranded extensible sequences conjugated to a universal probe attachment sequence to a hairpin sequence in a mixture is between 0.5 and 1. In an aspect, the stoichiometric ratio of all of the a single-stranded extensible sequences conjugated to a universal probe attachment sequence to a hairpin sequence in a mixture is between 1 and 100.
  • the stoichiometric ratio of all of the a single-stranded extensible sequences conjugated to a universal probe attachment sequence to a hairpin sequence in a mixture is between 1 and 50. In an aspect, the stoichiometric ratio of all of the a single-stranded extensible sequences conjugated to a universal probe attachment sequence to a hairpin sequence in a mixture is between 1 and 25. In an aspect, the stoichiometric ratio of all of the a single-stranded extensible sequences conjugated to a universal probe attachment sequence to a hairpin sequence in a mixture is between 1 and 10.
  • the stoichiometric ratio of all of the a single-stranded extensible sequences conjugated to a universal probe attachment sequence to a hairpin sequence in a mixture is between 1 and 5. In an aspect, the stoichiometric ratio of all of the a single-stranded extensible sequences conjugated to a universal probe attachment sequence to a hairpin sequence in a mixture is between 1 and 2.5.
  • the stoichiometric ratio of a first primer to a second primer in a mixture is between 0.01 and 100. In an aspect, the stoichiometric ratio of a first primer to a second primer in a mixture is between 0.05 and 100. In an aspect, the stoichiometric ratio of a first primer to a second primer in a mixture is between 0.1 and 100. In an aspect, the stoichiometric ratio of a first primer to a second primer in a mixture is between 0.5 and 100. In an aspect, the stoichiometric ratio of a first primer to a second primer in a mixture is between 1 and 100.
  • the stoichiometric ratio of a first primer to a second primer in a mixture is between 0.01 and 50. In an aspect, the stoichiometric ratio of a first primer to a second primer in a mixture is between 0.01 and 25. In an aspect, the stoichiometric ratio of a first primer to a second primer in a mixture is between 0.01 and 10. In an aspect, the stoichiometric ratio of a first primer to a second primer in a mixture is between 0.01 and 1.
  • the stoichiometric ratio of a forward primer to a forward blocker in a mixture is between 0.01 and 100. In an aspect, the stoichiometric ratio of a forward primer to a forward blocker in a mixture is between 0.05 and 100. In an aspect, the stoichiometric ratio of a forward primer to a forward blocker in a mixture is between 0.1 and 100. In an aspect, the stoichiometric ratio of a forward primer to a forward blocker in a mixture is between 0.5 and 100. In an aspect, the stoichiometric ratio of a forward primer to a forward blocker in a mixture is between 1 and 100.
  • the stoichiometric ratio of a forward primer to a forward blocker in a mixture is between 0.01 and 50. In an aspect, the stoichiometric ratio of a forward primer to a forward blocker in a mixture is between 0.01 and 25. In an aspect, the stoichiometric ratio of a forward primer to a forward blocker in a mixture is between 0.01 and 10. In an aspect, the stoichiometric ratio of a forward primer to a forward blocker in a mixture is between 0.01 and 1.
  • this disclosure provides a method for selective amplification of a template nucleic acid molecule via polymerase chain reaction, the method comprising: (a) generating a mixture by mixing a sample comprising one or both of a target nucleic acid template molecule and a background nucleic acid template molecule with: (i) a thermostable DNA polymerase enzyme: (ii) at least one reagent, at least one buffer, or at least one reagent and at least one buffer needed for the DNA polymerase enzyme to function; (iii) a forward primer comprising between 6 nucleotides and 70 nucleotides, where the forward primer comprises a sequence that is the reverse complement of a subsequence of the target nucleic acid template molecule; (iv) a forward blocker, where the forward blocker comprises a sequence that is the reverse complement of a subsequence of the background nucleic acid template molecule, and where the forward blocker comprises a 3' sequence or a chemical modification that
  • a “forward blocker” refers to an oligonucleotide that is designed to selectively bind to background nucleic acid template molecules bearing a wildtype sequence. Forward primers cannot bind to the background DNA due to an overlap in binding region. See for example, US Patent Application Publication No. 2017/0067090. On a target nucleic acid template molecule bearing a variant sequence, the blocker is mismatched in its binding, and can be effectively displaced by a forward primer, resulting in effective PCR amplification.
  • a forward blocker comprises DNA.
  • a forward blocker comprises a sequence at least 80% identical or complementary to a background nucleic acid template molecule.
  • a forward blocker comprises a sequence at least 85% identical or complementary to a background nucleic acid template molecule. In an aspect, a forward blocker comprises a sequence at least 90% identical or complementary to a background nucleic acid template molecule. In an aspect, a forward blocker comprises a sequence at least 95% identical or complementary to a background nucleic acid template molecule. In an aspect, a forward blocker comprises a sequence at least 99% identical or complementary to a background nucleic acid template molecule. In an aspect, a forward blocker comprises a sequence 100% identical or complementary to a background nucleic acid template molecule.
  • a forward primer comprises a sequence that overlaps with a sequence of a forward blocker.
  • a forward primer comprises a sequence that overlaps with a sequence of the forward blocker, where the overlap sequence comprises between 3 nucleotides and 50 nucleotides.
  • a forward primer comprises a sequence that overlaps with a sequence of the forward blocker, where the overlap sequence comprises between 3 nucleotides and 40 nucleotides.
  • a forward primer comprises a sequence that overlaps with a sequence of the forward blocker, where the overlap sequence comprises between 3 nucleotides and 30 nucleotides.
  • a forward primer comprises a sequence that overlaps with a sequence of the forward blocker, where the overlap sequence comprises between 3 nucleotides and 20 nucleotides. In an aspect, a forward primer comprises a sequence that overlaps with a sequence of the forward blocker, where the overlap sequence comprises between 3 nucleotides and 10 nucleotides.
  • a sequence that overlaps between a forward primer and a forward blocker is positioned on the 3' half of the forward primer sequence.
  • a forward primer comprises a sequence that is not 100% identical or complementary to a background nucleic acid template molecule. In an aspect, a forward primer comprises a sequence that is 100% complementary to both a target nucleic acid template molecule and a background nucleic acid template molecule.
  • a forward primer comprises a sequence that is complementary to a portion of a target nucleic acid template molecule, but not a background nucleic acid template molecule.
  • a forward primer comprises a sequence that is present in both a target nucleic acid template molecule and a background nucleic acid template molecule, and where the forward primer does not comprise a sequence that is present in the target nucleic acid template molecule but is absent in the background nucleic acid template molecule.
  • a forward primer is designed to detect a specific mutation.
  • a specific mutation is a single point mutation.
  • a specific mutation comprises a deletion of one or more nucleotides as compared to a wildtype sequence.
  • a specific mutation comprises an insertion of one or more nucleotides as compared to a wildtype sequence. In an aspect, a specific mutation comprises a substitution of one or more nucleotides as compared to a wildtype sequence. In an aspect, a specific mutation comprises an inversion of two or more sequences as compared to a wildtype sequence.
  • a mutation is in a gene that is linked to an increased risk of cancer when mutated as compared to a wildtype copy of the gene.
  • genes that have been linked to an increased risk of cancer when mutated include: APC, ATM, BAP1, BARD1, BMPR1A, BRCA2, BRCA2, BRIP1, CHEK2, CDH1, CDK4, CDKN2A, EP300, EPCAM, ETV6, FHIT, FLCN, FLT3, HOXB13, KIT, MET, MLH1, MLL, MSH2, MSH6, MUTYH, NBN, NF1, NT3, NTRK1, PALB2, PDGFRA, PMS1, PMS2, PPARy, PRCC, PTEN, RAD51C, RAD51D, RBI, RET, RUNXBP2, SMAD4, STK11, TFE3, TGF-P, TGF-piII, TP53, TSC1, TSC2, and WWOX.
  • a mutation is in a gene selected from the group consisting of APC, ATM, BAP1, BARD1, BMPR1A, BRCA2, BRCA2, BRIP1, CHEK2, CDH1, CDK4, CDKN2A, EP300, EPCAM, ETV6, FHIT, FLCN, FLT3, HOXB13, KIT, MET, MLH1, MLL, MSH2, MSH6, MUTYH, NBN, NF1, NT3, NTRK1, PALB2, PDGFRA, PMS1, PMS2, PPARy, PRCC, PTEN, RAD51C, RAD51D, RBI, RET, RUNXBP2, SMAD4, STK11, TFE3, TGF-P, TGF-piII, TP53, TSC1, TSC2, and WWOX.
  • a mutation is in a gene linked to a disease selected from the group consisting of breast cancer, colorectal cancer, endometrial cancer, fallopian tube cancer, ovarian cancer, primary peritoneal cancer, gastric cancer, melanoma, pancreatic cancer, prostate cancer, sarcoma, carcinoma, leukemia, brain cancer, central nervous system cancer, adrenal cortex cancer, gallbladder cancer, urinary tract cancer, thyroid cancer, liver cancer, kidney cancer, eye cancer, and lymphoma.
  • a disease selected from the group consisting of breast cancer, colorectal cancer, endometrial cancer, fallopian tube cancer, ovarian cancer, primary peritoneal cancer, gastric cancer, melanoma, pancreatic cancer, prostate cancer, sarcoma, carcinoma, leukemia, brain cancer, central nervous system cancer, adrenal cortex cancer, gallbladder cancer, urinary tract cancer, thyroid cancer, liver cancer, kidney cancer, eye cancer, and lymphoma.
  • a forward primer is designed for a clinical patient sample for personalized medicine.
  • a “clinical patient sample” refers to a sample obtained from a cell, blood, plasma, tissue, organ, or combination thereof from a patient, where the same comprises at least one nucleic acid template molecule.
  • a patient is a human.
  • a patient is a non-human animal.
  • a patient is a mammal.
  • a patient is selected from the group consisting of a mouse, a rat, a cat, a dog, a monkey, a chimpanzee, a cow, and a horse.
  • a clinical patient sample is obtained from a tissue selected from the group consisting of epithelial tissue, connective tissue, muscle tissue, and nervous tissue.
  • a clinical patient sample is obtained from a cell of an organ system selected from the group consisting of the respiratory system, the digestive or excretory system, the circulatory system, the urinary system, the integumentary system, the skeletal system, the muscular system, the endocrine system, the lymphatic system, the nervous system, and the reproductive system.
  • Nucleic acid molecules can be isolated from a clinical patient sample.
  • a sample is a clinical patient sample.
  • a sample is a biological sample.
  • a sample is a clinical patient sample and a biological sample.
  • a “biological sample” refers to a material obtained or isolated from a fresh or preserved biological sample or synthetically created source that contains nucleic acids.
  • Samples can include at least one cell, fetal cell, cell culture, tissue specimen, blood, serum, plasma, saliva, urine, tear, vaginal secretion, sweat, lymph fluid, cerebrospinal fluid, mucosa secretion, peritoneal fluid, ascites fluid, fecal matter, body exudates, umbilical cord blood, chorionic villi, amniotic fluid, embryonic tissue, multicellular embryo, lysate, extract, solution, or reaction mixture suspected of containing nucleic acids.
  • a biological sample can comprise DNA, RNA, or both.
  • a biological sample is from a healthy organism.
  • a biological sample is from a diseased organism.
  • a biological sample is from a mutagenized sample.
  • the nucleic acids obtained from a biological sample can be converted to cDNA.
  • a “target nucleic acid template molecule” refers to a region of a nucleic acid molecule that is desired to be amplified.
  • a “background nucleic acid template molecule” refers to a nucleic acid molecule that is not desired to be amplified.
  • a target nucleic acid template molecule is a DNA molecule.
  • a background nucleic acid template molecule is a DNA molecule.
  • a target nucleic acid template molecule is a cDNA molecule.
  • a background nucleic acid template molecule is a cDNA molecule.
  • a target nucleic acid template molecule is an RNA molecule.
  • a background nucleic acid template molecule is an RNA molecule.
  • sequence of a target nucleic acid template molecule and a background nucleic acid template molecule differ by at least 1 nucleotide. In an aspect, the sequence of a target nucleic acid template molecule and a background nucleic acid template molecule differ by at least 2 nucleotides. In an aspect, the sequence of a target nucleic acid template molecule and a background nucleic acid template molecule differ by at least 3 nucleotides. In an aspect, the sequence of a target nucleic acid template molecule and a background nucleic acid template molecule differ by at least 4 nucleotides.
  • sequence of a target nucleic acid template molecule and a background nucleic acid template molecule differ by at least 5 nucleotides. In an aspect, the sequence of a target nucleic acid template molecule and a background nucleic acid template molecule differ by at least 10 nucleotides. In an aspect, the sequence of a target nucleic acid template molecule and a background nucleic acid template molecule differ by at least 15 nucleotides. In an aspect, the sequence of a target nucleic acid template molecule and a background nucleic acid template molecule differ by at least 20 nucleotides.
  • sequence of a target nucleic acid template molecule and a background nucleic acid template molecule differ by at least 25 nucleotides. In an aspect, the sequence of a target nucleic acid template molecule and a background nucleic acid template molecule differ by between 1 nucleotide and 25 nucleotides. In an aspect, the sequence of a target nucleic acid template molecule and a background nucleic acid template molecule differ by between 1 nucleotide and 21 nucleotides. In an aspect, the sequence of a target nucleic acid template molecule and a background nucleic acid template molecule differ by between 1 nucleotide and 20 nucleotides.
  • sequence of a target nucleic acid template molecule and a background nucleic acid template molecule differ by between 1 nucleotide and 15 nucleotides. In an aspect, the sequence of a target nucleic acid template molecule and a background nucleic acid template molecule differ by between 1 nucleotide and 10 nucleotides. In an aspect, the sequence of a target nucleic acid template molecule and a background nucleic acid template molecule differ by between 1 nucleotide and 5 nucleotides.
  • a mixture comprising: a primer, wherein the primer comprises, from 3' to 5':
  • a first strand comprising a universal probe attachment sequence conjugatable to the single-stranded extensible sequence, wherein the probe attachment sequence comprises between 6 nucleotides and 100 nucleotides; and (ii) a second strand comprising a hairpin sequence comprising between 7 nucleotides and 40 nucleotides, wherein the hairpin sequence further comprises:
  • a first hairpin subsequence comprising between 2 nucleotides and 30 nucleotides, wherein the first hairpin subsequence is at least 70% identical or complementary to the reverse complement of the universal probe attachment sequence;
  • the at least one reagent is selected from the group consisting of magnesium, at least one dNTP, phosphatase, betaine, dimethyl sulfoxide, and tetramethylammonium chloride.
  • the single-stranded extensible sequence comprises a chemical moiety at or near its 5' end.
  • the chemical moiety is selected from the group consisting of an alkyne, a strained alkyne, an azide, an amide, a carboxyl, a phosphate, a hydroxyl, and a thiol.
  • DNA polymerase enzyme is selected from the group consisting of Phusion® DNA polymerase, Phusion® U DNA polymerase, Q5® DNA polymerase, Q5U® DNA polymerase, PfuUltra II DNA polymerase, KAPA HiFi DNA polymerase, AccurisTM DNA polymerase, PowerUpTM DNA Polymerase, and Taq DNA polymerase.
  • the at least one non-natural nucleoside base is selected from the group consisting of a 2'-O-methoxy-ethyl base, a 2'-O-methyl RNA base, a 2' fluoro base, 2-Aminopurine, 5-Bromo-deoxyuridine, deoxyUridine, 2,6- Diaminopurine, dideoxy cytidine, deoxyinosine, hydroxymethyl dC, inverted dT, Iso-dG, Iso-dC, inverted dideoxy -T, 5-methyl dC, Super T®, Super G®, and 5-nitroindole.
  • a kit comprising:
  • a first strand comprising a universal probe attachment sequence conjugated to the single-stranded extensible sequence, wherein the probe attachment sequence comprises between 6 nucleotides and 100 nucleotides;
  • a second strand comprising a hairpin sequence comprising between 7 nucleotides and 40 nucleotides, wherein the hairpin sequence further comprises:
  • a first hairpin subsequence comprising between 2 nucleotides and 30 nucleotides, wherein the first hairpin subsequence is at least 70% identical or complementary to the reverse complement of the universal probe attachment sequence;
  • a single-stranded extensible sequence comprising between 7 nucleotides and 100 nucleotides, wherein the single-stranded extensible sequence is at least 90% complementary to an initiation subsequence of a template nucleic acid molecule;
  • a second primer wherein the second primer comprises between 6 nucleotides and 70 nucleotides, and wherein the second primer is at least 80% identical or complementary to a subsequence of the template nucleic acid molecule;
  • each cycle of the at least seven cycles of thermal cycling comprises: (a) a first stage comprising a temperature of at least 75°C for between one second and one hour; and (b) a second stage comprising a temperature of less than 75°C for between one second and two hours.
  • DNA polymerase enzyme is selected from the group consisting of Phusion® DNA polymerase, Phusion® U DNA polymerase, Q5® DNA polymerase, Q5U® DNA polymerase, PfuUltra II DNA polymerase, KAPA HiFi DNA polymerase, AccurisTM DNA polymerase, PowerUpTM DNA Polymerase, and Taq DNA polymerase.
  • quencher is selected from the group consisting of MGB, Black Hole Quencher, Iowa Black® RQ, and Iowa Black® FQ.
  • the first primer and the template nucleic acid molecule comprise a standard of free energy of hybridization (AG°1) between -7 kcal/mol and -20 kcal/mol; and/or
  • the second primer and template nucleic acid molecule comprise a standard of free energy of hybridization (AG°2) between -7 kcal/mol and -20 kcal/mol; and/or
  • the universal probe attachment sequence and the hairpin sequence comprise a standard free energy of hybridization (AG°3) between -8 kcal/mol and -100 kcal/mol wherein the at least one amplicon comprises a length of between 50 nucleotides and 5000 nucleotides, and wherein AG°1, AG°2, and AG°3 are calculated at a temperature of 60°C and a salinity of 0.2M sodium.
  • AG°3 standard free energy of hybridization
  • intercalating dye is selected from the group consisting of SYBR® Green, EvaGreen®, and SYTOTM dyes.
  • nucleotide sequence of the at least one amplicon is obtained via Sanger sequencing.
  • a method for multiplexed amplification of nucleic acids comprising: (a) generating a mixture by mixing a sample comprising a template nucleic acid molecule with:
  • each primer of the plurality of first primers comprises, from 3' to 5':
  • a single-stranded extensible sequence comprising between 7 nucleotides and 100 nucleotides, wherein the single-stranded extensible sequence is at least 90% complementary to an initiation subsequence of a template nucleic acid molecule;
  • thermostable DNA polymerase enzyme (iii) a thermostable DNA polymerase enzyme; and (iv) at least one reagent, at least one buffer, or at least one reagent and at least one buffer needed for the DNA polymerase enzyme to function;
  • each cycle of the at least five cycles of thermal cycling comprises: (a) a first stage comprising a temperature of at least 75°C for between one second and one hour; and (b) a second stage comprising a temperature of less than 75°C for between one second and two hours.
  • the plurality of first primers comprises at least 2 first primers, wherein each of the at least 2 first primers comprises a different single-stranded extensible sequence.
  • the plurality of first primers comprises a first universal probe attachment sequence and a second universal probe attachment sequence
  • the first universal probe attachment sequence comprises a first high-throughput sequencing adapter sequence
  • the second universal probe attachment sequence comprises a second high-throughput sequencing adapter sequence
  • the first high-throughput sequencing adapter sequence and the second high- throughput sequencing adapter sequence are not identical.
  • the plurality of first primers comprises a first group comprising at least one single-stranded extensible sequence conjugated to a first universal probe attachment sequence and a second group comprising at least one single-stranded extensible sequence conjugated to a second universal probe attachment sequence, and wherein the first universal probe attachment sequence and the second universal probe attachment sequence are not identical.
  • a method for selective amplification of template nucleic acid molecules via polymerase chain reactions comprising:
  • thermostable DNA polymerase enzyme (i) a thermostable DNA polymerase enzyme
  • a forward primer comprising between 6 nucleotides and 70 nucleotides, wherein the forward primer comprises a sequence that is the reverse complement of a subsequence of the target nucleic acid template molecule;
  • a forward blocker wherein the forward blocker comprises a sequence that is the reverse complement of a subsequence of the background nucleic acid template molecule, and wherein the forward blocker comprises a 3’ sequence or a chemical modification that reduces DNA polymerase enzyme extension efficiency;
  • a single-stranded extensible sequence comprising between 7 nucleotides and 100 nucleotides, wherein the single-stranded extensible sequence is at least 90% complementary to an initiation subsequence of the template nucleic acid molecule;
  • each cycle of the at least five cycles of thermal cycling comprises: (a) a first stage comprising a temperature of at least 75°C for between one second and one hour; and (b) a second stage comprising a temperature of less than 75°C for between one second and two hours.
  • the forward primer comprises a non-natural oligonucleotide comprising at least one non-natural nucleoside base.
  • the forward blocker comprises a non-natural oligonucleotide comprising at least one non-natural nucleoside base.
  • the at least one non-natural nucleoside base is selected from the group consisting of a 2'-O-methoxy-ethyl base, a 2'- O-methyl RNA base, a 2' fluoro base, 2- Aminopurine, 5-Bromo-deoxyuridine, deoxyUridine, 2,6-Diaminopurine, dideoxycytidine, deoxyinosine, hydroxymethyl dC, inverted dT, Iso-dG, Iso-dC, inverted dideoxy-T, 5-methyl dC, Super T®, Super G®, and 5 -nitroindole.
  • the forward primer comprises a backbone modification.
  • the backbone modification comprises a modification selected from the group consisting of a locked nucleic acid, a phosphorothioate modification, a phosphorodithioate modification, a methylphosphonate modification, and a phosphoramidate modification.
  • the forward primer comprises a subsequence at its 3' end that comprises between 3 nucleotides and 40 nucleotides that are identical to a subsequence of the forward blocker.
  • quencher is selected from the group consisting of MGB, Black Hole Quencher, Iowa Black® RQ, and Iowa Black® FQ.
  • thermostable DNA polymerase enzyme lacks 5' to 3' exonuclease activity.
  • thermostable DNA polymerase enzyme comprises 5' to 3' exonuclease activity.
  • thermostable DNA polymerase enzyme is capable of performing kinetic proofreading.
  • thermostable DNA polymerase enzyme is selected from the group consisting of Phusion® DNA polymerase, Phusion® U DNA polymerase, Q5® DNA polymerase, Q5U® DNA polymerase, PfuUltra II DNA polymerase, KAPA HiFi DNA polymerase, AccurisTM DNA polymerase, PowerUpTM DNA Polymerase, and Taq DNA polymerase.
  • the forward primer comprises a sequence that is present in both the target nucleic acid template molecule and the background nucleic acid template molecule, and wherein the forward primer does not comprise a sequence that is present in the target nucleic acid template molecule but is absent in the background nucleic acid template molecule.

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Abstract

La présente divulgation concerne des sondes universelles qui peuvent être ajoutées sur des amorces pour amplifier et détecter des acides nucléiques cibles. L'invention permet la détection par fluorescence en temps réel de produits d'amplicon à l'aide d'une sonde universelle dans des réactions de PCR. Les sondes universelles sont des alternatives de sonde TaqManTM où une séquence spécifique d'un locus individuel est conjuguée à un duplex d'ADN universel qui émet une fluorescence lors de l'amplification. Cette technologie permet un temps de rotation plus rapide lors de la conception et du développement de dosages.
PCT/US2022/081306 2021-12-10 2022-12-09 Sondes universelles pour l'amplification et la détection d'acides nucléiques WO2023108145A1 (fr)

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US20160298197A1 (en) * 2013-10-01 2016-10-13 Queensland University Of Technology Kits and methods for diagnosis, screening, treatment and disease monitoring
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