WO2022060982A1

WO2022060982A1 - Occlusion primers and occlusion probes

Info

Publication number: WO2022060982A1
Application number: PCT/US2021/050668
Authority: WO
Inventors: Kerou ZHANG; Alessandro Pinto; David Zhang
Original assignee: Nuprobe Usa, Inc.; William Marsh Rice University
Priority date: 2020-09-17
Filing date: 2021-09-16
Publication date: 2022-03-24

Abstract

This invention describes new primers and probes for amplifying and detecting target nucleic acids. In quantitative PCR embodiments, the invention allows real-time fluorescence detection of amplicon products in PCR reaction utilizing DNA polymerases without 5' to 3' exonuclease activity, such as high-fidelity proofreading DNA polymerases. In next-generation sequencing embodiments, the invention facilitates highly multiplexed PCR amplification of selected target regions through the reduction of potential primer dimer formation associated with primer 5' adapter overhangs.

Description

OCCLUSION PRIMERS AND OCCLUSION PROBES

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 63/079,717, filed September 17, 2020, and U.S. Provisional Patent Application No.

63/084,322, filed September 28, 2020, both of which are incorporated herein in their entireties.

FIELD

[0002] 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.

INCORPORATION OF SEQUENCE LISTING

[0003] The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on September 13, 2021, is named P34851WOOO_SL.txt and is 53,618 bytes in size measured in Microsoft Windows®.

[0004] Table 1 provides nucleic acid sequences used in this application, including fluorophores and quenchers.

Table 1. Nucleic acid sequences

BACKGROUND

[0005] The 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). PCR can also be used upstream of a high- throughput sequencing reaction for target enrichment.

[0006] In 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. , TaqMan™ probes). TaqMan™ probes are generally favored over intercalating dyes because multiple different spectrally distinct fluorophores can be used on different TaqMan™ probes to allows multiplexed detection and quantitation of two to six distinct DNA target species in a single reaction. However, TaqMan™ probes function through the 5' to 3' exonuclease activity of DNA polymerases such as Taq. Other DNA polymerases used in PCR lack 5' to 3' exonuclease activity, and thus will not produce a detectable fluorescence signal with TaqMan™ probes. Importantly, many high-fidelity DNA polymerases such as Phusion® and Q5®, which have 100-fold or lower error rates as compared to Taq, lack 5' to 3' exonuclease activity.

[0007] In high-throughput sequencing, including next-generation sequencing (NGS) such as sequencing-by-synthesis (SBS), and third generation sequence such as nanopore sequencing, adapter sequences are needed on the 5' and 3' ends of the library to facilitate sequencing. Multiplex PCR target enrichment uses gene-specific primers with 5' overhang sequencing corresponding to sequencing adapters (e.g. Truseq or Nextera adapters). However, the addition of these 5' overhang sequences on the primer increase the likelihood of primer dimer formation and nonspecific amplification of other DNA sequences. [0008] Here, we present Occlusion Primers and Occlusion Probes that, among other benefits, overcome the above challenges for qPCR and multiplex PCR.

SUMMARY

[0009] In one aspect, this disclosure provides a system comprising: (a) a first Occlusion Primer, where the first Occlusion Primer comprises, in 5' to 3' order, a first probe attachment sequence and a first template binding sequence; and (b) a first Occlusion Probe, where the first Occlusion Probe comprises, in 5' to 3' order, a first primer attachment sequence and a first hairpin sequence.

[0010] In one aspect, this disclosure provides a kit comprising: (a) a first Occlusion Primer, wherein the first Occlusion Primer comprises, in 5' to 3' order, a first probe attachment sequence and a first template binding sequence; (b) a first Occlusion Probe, where the first Occlusion Probe comprises, in 5' to 3' order, a first primer attachment sequence and a first hairpin sequence; and (c) at least one DNA polymerase.

[0011] In one aspect, this disclosure provides a method for amplifying a template nucleic acid molecule, the method comprising: (a) mixing a sample comprising the template nucleic acid molecule with (i) at least one DNA polymerase; (ii) a first Occlusion Primer, where the first Occlusion Primer comprises, in 5' to 3' order, a first probe attachment sequence and a first template binding sequence; (iii) a first Occlusion Probe, where the first Occlusion Probe comprises, in 5' to 3' order, a first primer attachment sequence and a first hairpin sequencer; and (iv) a second primer; to generate a mixture; and (b)subjecting the mixture to at least one cycle of thermal cycling, where the template nucleic acid molecule is amplified to produce at least one amplicon.

[0012] In one aspect, this disclosure provides a method for multiplex amplification of a plurality of nucleic acid molecules, the method comprising: (a) obtaining a mixture comprising (i) a plurality of template nucleic acid molecules; (ii) at least one DNA polymerase; (iii) a first Occlusion Primer, where the first Occlusion Primer comprises, in 5' to 3' order, a first probe attachment sequence and a first template binding sequence; (iv) a first Occlusion Probe, where the first Occlusion Probe comprises, in 5' to 3' order, a first primer attachment sequence and a first hairpin sequence; (v) a second Occlusion Primer, where the second Occlusion Primer comprises, in 5' to 3' order, a second probe attachment sequence and a second template binding sequence; and (vi) a second Occlusion Probe, where the second Occlusion Probe comprises, in 5' to 3' order, a second primer attachment sequence and a second hairpin sequencer; to generate the mixture; and (b) subjecting the mixture to at least one cycle of thermal cycling, where the plurality of template nucleic acid molecules are amplified to produce a plurality of amplicons.

[0013] In one aspect, this disclosure provides a method for multiplex amplification of a plurality of nucleic acid molecules, the method comprising: (a) obtaining a mixture comprising (i) a plurality of template nucleic acid molecules; (ii) at least one DNA polymerase; (iii) a first Occlusion Primer, where the first Occlusion Primer comprises, in 5' to 3' order, a first probe attachment sequence and a first template binding sequence; (iv) a first Occlusion Probe, where the first Occlusion Probe comprises, in 5' to 3' order, a first primer attachment sequence and a first hairpin sequence; (v) a first primer, where the first primer produces an amplicon in conjunction with the first Occlusion Primer; (vi) a second Occlusion Primer, where the second Occlusion Primer comprises, in 5' to 3' order, a second probe attachment sequence and a second template binding sequence; (vii) a second Occlusion Probe, where the second Occlusion Probe comprises, in 5' to 3' order, a second primer attachment sequence and a second hairpin sequencer; to generate the mixture; and (viii) a second primer, where the second primer produces an amplicon in conjunction with the second Occlusion Primer; and (b) subjecting the mixture to at least one cycle of thermal cycling, where the plurality of template nucleic acid molecules are amplified to produce a plurality of amplicons.

[0014] In one aspect, this disclosure provides a method for selective amplification of at least one target DNA template, the method comprising: (a) obtaining a mixture comprising (i) at least one target DNA template; (ii) at least one background DNA template; (iii) at least one DNA polymerase; (iv) a forward primer, where the forward primer comprises a sequence that is at least 80% complementary to a sequence of the at least one target DNA template; (v) a forward blocker, where the forward blocker comprises a sequence that is at least 80% complementary to a sequence of the at least one background DNA template; (vi) a first Occlusion Primer, where the first Occlusion Primer comprises, in 5' to 3' order, a first probe attachment sequence and a first template binding sequence; and (vii) a first Occlusion Probe, where the first Occlusion Probe comprises, in 5' to 3' order, a first primer attachment sequence and a first hairpin sequencer; to produce the mixture; and (b) subjecting the mixture to at least five cycles of thermal cycling, where the at least one target DNA template is amplified to produce at least one target DNA amplicon. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 depicts a schematic of Occlusion Primers and Occlusion Probes. The light grey shaded region shows the Occlusion Primer. Occlusion Primers and Occlusion Probes are intended for use in conjunction with a DNA polymerase.

[0016] Figure 2 comprises Figure 2A and Figure 2B. Figure 2A depicts an Occlusion Primer comprising a fluorophore modification (grey star) at its 5' end in a complex with an Occlusion Probe comprising a quencher modification (grey circle) at its 3' end. Figure 2B depicts the Occlusion Primer comprising a quencher modification at its 5' end, and the Occlusion Probe comprising a fluorophore modification at its 3' end.

[0017] Figure 3 comprises Figure 3A and Figure 3B. Figures 3A and 3B depict Occlusion Probes with different hairpin sequences (SEQ ID NO: 183 in Figure 3A; SEQ ID NO: 184 in Figure 3B).

[0018] Figure 4 comprises Figure 4A, Figure 4B, Figure 4C, and Figure 4D. Figures 4A and 4B depict two Occlusion Primer and Occlusion Probe complexes, where the Occlusion Primer comprises a Cy5™ fluorophore (gray star) on the 5' end, and the Occlusion Probe comprises an Iowa Black® RQ quencher on the 3' end. Figures 4C and 4D depict normal fluorescence accumulation detected in quantitative PCR (qPCR) using the Occlusion Primers and Probes depicted in Figures 4A and 4B. See Examples 2 and 3, respectively, for additional details.

[0019] Figure 5 depicts an illustration of the Occlusion Primer and Occlusion Probe before and after PCR amplification. In the PCR amplicon products, the extended Occlusion Primer is paired with the extended amplicon from a second primer, and no longer able to efficiently bind the Occlusion Probe. If the Occlusion Primer is fluorophore labeled (gray star), the solution fluorescence increases relative to the beginning of the PCR reaction, because the quencher (gray circle) on the Occlusion Probe is delocalized from the fluorophore on the Occlusion Primer.

[0020] Figure 6 comprises Figure 6A, Figure 6B, Figure 6C, Figure 6D, Figure 6E, Figure 6F, Figure 6G, Figure 6H, Figure 61, and Figure 6J. Figure 6A compares the efficacy of Occlusion Primers and Occlusion Probes with TaqMan™ probes in qPCR when using a high- fidelity DNA polymerase having 3' to 5' exonuclease activity and lacking 5' to 3' exonuclease activity. Use of Occlusion Primer with an Occlusion Probe results in a normal accumulation of fluorescence during qPCR, while use of a TaqManTM probe results in abnormal fluorescence accumulation. Figure 6B depicts a non-functional design where the complex lacks a hairpin, but the three 3 '-most nucleotides of Probe D comprise a phosphorothioate backbone modification. Figure 6C depicts abnormal accumulation of fluorescence during qPCR when using the complex depicted in Figure 6B. Notably, no exponential phase of accumulation is detected. Figure 6D depicts anon-functional design non-functional design in which the primer (Probe B) comprises a fluorophore and hairpin sequence at its 5' end. Figure 6E depicts abnormal accumulation of fluorescence during the baseline stage of qPCR when using the complex depicted in Figure 6D. Figure 6F depicts anon-functional design in which the primer (Probe C) has a Probe Attachment Sequence but the probe (Probe A) does not comprise a Hairpin Sequence. Figure 6G depicts abnormal accumulation of fluorescence during qPCR when using the complex depicted in Figure 6F. Figure 6H depicts Sunrise primers with 5' quencher modifications and internal fluorophore modifications. Figure 61 depicts abnormal accumulation of fluorescence during qPCR when using the RP-SR-1 and RP-SR-2 primers, while Figure 6J depicts abnormal accumulation of fluorescence during qPCR when using the RP-SR-3. In all parts of Figure 6, a grey star represents a fluorophore, and a grey circle represents a quencher.

[0021] Figure 7 comprises Figures 7A and 7B. Figures 7A and 7B depict normal accumulation of fluorescence during qPCR using Occlusion Primers and Occlusion Probes with a Taq polymerase and SYBR™ Green intercalating dye. Because the First Primer is not functionalized with any Cy5™ fluorophores, no signal appears in the Cy5™ channel in Figure 7B

[0022] Figure 8 depicts an Occlusion Primer and Occlusion Probe complex where the Primer Attachment Sequence and the Probe Attachment Sequence have one or a small number of mismatches.

[0023] Figure 9 comprises Figure 9A, Figure 9B, Figure 9C, Figure 9D, and Figure 9E. Figure 9A depicts an Occlusion Primer and Occlusion Probe complex that comprises a single mismatch within the Primer Attachment Sequence. Figure 9B depicts an Occlusion Primer and Occlusion Probe complex that comprises two mismatches within the Primer Attachment Sequence. Figure 9C depicts normal accumulation of fluorescence during qPCR when using the Occlusion Primer and Occlusion Probe depicted in Figure 9A. Figure 9D depicts normal accumulation of fluorescence during qPCR when using the Occlusion Primer and Occlusion Probe depicted in Figure 9B. Figure 9E depicts normal accumulation of fluorescence during qPCR when using the Occlusion Primer and Occlusion Probe complex from Figure 9A (dashed line), from Figure 9B (solid black line), and an Occlusion Primer and Occlusion Probe complex that has zero mismatches within the Primer Attachment sequence (gray line) (see Figures 4A and 4C).

[0024] Figure 10 comprises Figure 10A and 10B. Figure 10A depicts an Occlusion Probe comprising a Cy5™ fluorophore at its 3' end, and an Occlusion Primer comprising an Iowa Black® RQ quencher at its 5' end. Figure 10B depicts normal accumulation of fluorescence during qPCR when using the Occlusion Primer and Occlusion Probe depicted in Figure 10A.

[0025] Figure 11 depicts normal accumulation of fluorescence during qPCR using an Occlusion Primer and Occlusion Probe system when varying the concentration stoichiometry of the Occlusion Probe.

[0026] Figure 12 comprises Figure 12A, Figure 12B, Figure 12C, and Figure 12D. Figure 12A depicts an Occlusion Primer and Occlusion Probe complex, where the Occlusion Primer comprises a 3' single-stranded overhang. Figure 12B depicts an Occlusion Primer and Occlusion Probe complex, where the Occlusion Primer comprises a 5' single-stranded overhang. Figure 12C depicts an Occlusion Primer (SEQ ID NO: 18) and Occlusion Probe (SEQ ID NO: 196) complex, where the Occlusion Primer comprises a 3' single-stranded overhang. The star represents a fluorophore, and the circle represents a quencher in each of Figures 12A-12C. Figure 12D depicts normal accumulation of fluorescence during qPCR when using the Occlusion Primer and Occlusion Probe depicted in Figure 12C.

[0027] Figure 13 depicts the use of Occlusion Probes and many Occlusion Primers for multiplex PCR target enrichment for next generation sequencing (NGS) library preparation. Here, all forward Occlusion Primers contain the same forward Probe Attachment Sequence, a forward adapter for NGS. Thus, the same forward Occlusion Probe is used for all forward Occlusion Primers. Likewise, all reverse Occlusion Primers contain the same reverse Probe Attachment Sequence, a reverse adapter for NGS. Thus, the same reverse Occlusion Probe is used for all reverse Occlusion Primers.

[0028] Figure 14 depicts the use of an Occlusion Primer as a reverse primer for selective variant amplification by Blocker Displacement Amplification (BDA).

[0029] Figure 15 depicts the use of an Occlusion Primer as a reverse primer for selective allelespecific blocker PCR amplification. DETAILED DESCRIPTION

[0030] Unless defined otherwise, all technical and scientific terms used have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Where a term is provided in the singular, the inventors also contemplate aspects of the disclosure described by the plural of that term. Where there are discrepancies in terms and definitions used in references that are incorporated by reference, the terms used in this application shall have the definitions given herein. Other technical terms used have their ordinary meaning in the art in which they are used, as exemplified by various art-specific dictionaries, for example, “The American Heritage® Science Dictionary” (Editors of the American Heritage Dictionaries, 2011, Houghton Mifflin Harcourt, Boston and New York), the “McGraw-Hill Dictionary of Scientific and Technical Terms” (6th edition, 2002, McGraw- Hill, New York), or the “Oxford Dictionary of Biology” (6th edition, 2008, Oxford University Press, Oxford and New York).

[0031] Any references cited herein, including, e.g., all patents, published patent applications, and non-patent publications, are incorporated herein by reference in their entirety.

[0032] Any composition provided herein is specifically envisioned for use with any applicable method provided herein.

[0033] When a grouping of alternatives is presented, 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.

[0034] 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. For example, 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.

[0035] When a range of numbers is provided herein, 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. [0036] As used herein, the singular form “a,” “an,” and “the” include plural references unless the context clearly dictates otherwise. For example, the term “a compound” or “at least one compound” may include a plurality of compounds, including mixtures thereof. As used herein, the term “plurality” refers to any number greater than one.

[0037] The terms “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. If 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%.

[0038] The terms “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 base-pairing 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. Thus, for purposes of the present application, when two sequences (query and subject) are optimally base-paired (with allowance for mismatches or non-base-paired nucleotides), 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%.

[0039] As used herein, a “mismatch” refers to an alignment of two sequences that pairs two uncomplimentary nucleotides. Non-limiting examples of 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. Conversely, “matched” alignments of nucleotides refer to complimentary pairs such as G-C, A-T, and A-U.

[0040] As a non-limiting example, the complement of the sequence 5'-ATGC-3' is 3'-TACG- 5', and the reverse complement of 5'-ATGC-3' is 5'-GCAT-3'. Notably, the complement and reverse complement sequences are identical to each other when viewed in the 5' to 3' direction.

[0041] For optimal alignment of sequences to calculate their percent complementarity, various pair-wise or multiple sequence alignment algorithms and programs are known in the art, such as ClustalW or Basic Local Alignment Search Tool® (BLAST™), etc., that can be used to compare the sequence complementarity or identity between two or more nucleotide sequences. Although other alignment and comparison methods are known in the art, the alignment and percent identity between two sequences (including the percent identity ranges described above) can be as determined by the ClustalW algorithm, see, e.g, Chenna et al., “Multiple sequence alignment with the Clustal series of programs, ” Nucleic Acids Research 31 : 3497-3500 (2003); Thompson et al., “Clustal W: Improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice,” Nucleic Acids Research 22: 4673-4680 (1994); Larkin MA et al., “Clustal W and Clustal X version 2.0,” Bioinformatics 23: 2947-48 (2007); and Altschul et al. "Basic local alignment search tool." J. Mol. Biol. 215:403-410 (1990), the entire contents and disclosures of which are incorporated herein by reference.

[0042] As used herein, 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. Without being limiting, a primer is used in PCR to initiate DNA synthesis. In an aspect, a primer is a DNA molecule. In an aspect, a primer is an RNA molecule. In an aspect, a primer comprises between 6 nucleotides and 70 nucleotides. In an aspect, a primer comprises between 10 nucleotides and 50 nucleotides. In an aspect, a primer comprises between 15 nucleotides and 30 nucleotides. In an aspect, 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. [0043] In an aspect, a primer comprises a sequence at least 80% identical or complementary to a template nucleic acid molecule. In an aspect, a primer comprises a sequence at least 85% identical or complementary to a template nucleic acid molecule. In an aspect, a primer comprises a sequence at least 90% identical or complementary to a template nucleic acid molecule. In an aspect, a primer comprises a sequence at least 95% identical or complementary to a template nucleic acid molecule. In an aspect, a primer comprises a sequence at least 99% identical or complementary to a template nucleic acid molecule. In an aspect, a primer comprises a sequence 100% identical or complementary to a template nucleic acid molecule.

[0044] The polymerase chain reaction (PCR) is a molecular biology technique that allows one to generate multiple copies of a targeted region of DNA. Copies of DNA made via PCR are termed “amplicons.” Amplicons can then be sequenced, analyzed (e.g, gel electrophoresis), or cloned into a plasmid or vector. In an aspect, an amplicon comprises a length of between 10 nucleotides and 7500 nucleotides. In an aspect, an amplicon comprises a length of between 20 nucleotides and 5000 nucleotides. In an aspect, an amplicon comprises a length of between 30 nucleotides and 5000 nucleotides. In an aspect, 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. In an aspect, 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. In an aspect, 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.

[0045] In an aspect, 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 re-amplification of at least one amplicon using fluorescent dideoxynucleotide triphosphates (ddNTPs). In an aspect, an amplicon is sequenced via Sanger sequencing. See Sanger and Coulson, J. Mol. Biol., 94:441-446 (1975). In an aspect, an amplicon is sequenced via next-generation sequencing. Non-limiting examples of 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.

[0046] In an aspect, a method comprises high-throughput sequencing. In an aspect, a method comprises subjecting a plurality of amplicons to high-throughput sequencing. As used herein, “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. In an aspect, Sanger sequencing is not high-throughput sequencing. In an aspect, high- throughput sequencing comprises the use of a sequencing-by-synthesis (SBS) flow cell. In an aspect, an SBS flow cell is selected from the group consisting of an Illumina SBS flow cell and a Pacific Biosciences (PacBio) SBS flow cell. In an aspect, high-throughput sequencing is performed via electrical current measurements in conjunction with an Oxford nanopore.

[0047] PCR requires a mixture comprising a targeted region of DNA to be amplified, a set of oligonucleotide primers that flank the targeted region of DNA, a thermostable DNA polymerase, and nucleotides. In general, the mixture is subjected to thermal cycling in order to amplify the targeted region of DNA. Without being limiting, thermal cycling often includes a denaturation stage to separate double-stranded DNA (dsDNA) into single strands; an annealing stage, which allows the primers to hybridize with the targeted region of DNA; and an extension stage, which allows the DNA polymerase to extend DNA from the primers, generating new dsDNA. In some protocols, the annealing and extension stages can be combined into a single stage.

[0048] As used herein, “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. In an aspect, a cycle further comprises a third stage comprising a third temperature maintained for a desired amount of time. In an aspect, a cycle further comprises a fourth stage comprising a fourth temperature maintained for a desired amount of time. Often, thermal cycling comprises repeating the same cycle several times.

[0049] In an aspect, 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. In an aspect, 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.

[0050] In an aspect, 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. In an aspect, 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. [0051] In an aspect, 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. In an aspect, 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.

[0052] In an aspect, 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.

[0053] In an aspect, 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 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 2 cycles and 40 cycles. In an aspect, 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. [0054] In an aspect, 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 atemperature of less than 75°C for between one second and two hours. In an aspect, 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. In an aspect, 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.

[0055] Quantitative PCR (qPCR) 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. However, 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.

[0056] Probes (e.g. TaqMan™ 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. During PCR, the probe, which binds between the two primers on the targeted region of DNA, is hydrolyzed during primer extension, allowing an amplificationdependent increase in fluorescence. Therefore, the measured fluorescence is proportional to the amount of the probe target sequence in a sample. However, this type of system will not work with DNA polymerases that lack 5' to 3' exonuclease activity.

[0057] This disclosure provides a system using Occlusion Primers and Occlusion Probes to overcome the shortcomings of qPCR described above. The provided system also reduces the likelihood of primer dimer formation and nonspecific amplification of non-targeted DNA sequences during multiplex PCR.

[0058] In an aspect, this disclosure provides an Occlusion Primer. Non-limiting examples of Occlusion Primers include SEQ ID NOs: 3, 8, and 21-180. An “Occlusion Primer” refers to a synthetic DNA molecule comprising two regions: a probe attachment sequence positioned 5' to a template binding sequence. See Figure 1. The template binding sequence is complementary to, and capable of hybridizing with, a target nucleic acid molecule’s initiation subsequence. The probe attachment sequence is an artificially designed sequence that is complementary to, and capable of hybridizing with, an Occlusion Probe’s primer attachment sequence. In an aspect, the probe attachment sequence is heterologous to the template binding sequence. In an aspect, an Occlusion Primer comprises DNA. In an aspect, an Occlusion Primer comprises RNA.

[0059] Each Occlusion Primer has a corresponding Occlusion Probe with which it can form a complex. See Figure 1. In an aspect, this disclosure provides an Occlusion Probe. Non-limiting examples of Occlusion Probes include SEQ ID NOs: 9-13, 181, and 182. An “Occlusion Probe” refers to a synthetic DNA molecule comprising two regions: a primer attachment sequence positioned 5' to a hairpin sequence. See Figure 1. The primer attachment sequence is complementary to, and capable of hybridizing with, an Occlusion Primer’s probe attachment sequence. The hairpin sequence comprises three regions in 5' to 3' order: a first subsequence, a loop subsequence, and a second subsequence. The first and second subsequences of the hairpin sequence are complementary to each other and form the stem of a stem-and-loop structure, with the loop subsequence forming the loop part of the structure. Non-limiting examples of hairpin sequences include SEQ ID NOs: 183 and 184. In an aspect, the primer attachment sequence is heterologous to the hairpin sequence. In an aspect, an Occlusion Probe comprises DNA. In an aspect, an Occlusion Probe comprises RNA.

[0060] In an aspect, this disclosure provides a system comprising: (a) a first Occlusion Primer, where the first Occlusion Primer comprises, in 5' to 3' order, a first probe attachment sequence and a first template binding sequence; and (b) a first Occlusion Probe, where the first Occlusion Probe comprises, in 5' to 3' order, a first primer attachment sequence and a first hairpin sequence. In an aspect, a system further comprises (c) a second Occlusion Primer, where the second Occlusion Primer comprises, in 5' to 3' order, a second probe attachment sequence and a second template binding sequence; and (d) a second Occlusion Probe, where the second Occlusion Probe comprises, in 5' to 3' order, a second primer attachment sequence and a second hairpin sequence. In an aspect, a system further comprises (e) a third Occlusion Primer, where the third Occlusion Primer comprises, in 5' to 3' order, a third probe attachment sequence and a third template binding sequence; and (I) a third Occlusion Probe, where the third Occlusion Probe comprises, in 5' to 3' order, a third primer attachment sequence and a third hairpin sequence. In an aspect, a system further comprises (g) a fourth Occlusion Primer, where the fourth Occlusion Primer comprises, in 5' to 3' order, a fourth probe attachment sequence and a fourth template binding sequence; and (h) a fourth Occlusion Probe, where the fourth Occlusion Probe comprises, in 5' to 3' order, a fourth primer attachment sequence and a fourth hairpin sequence. In an aspect, a system further comprises (i) a N^th Occlusion Primer, where the N¹¹¹ Occlusion Primer comprises, in 5' to 3' order, a N^th probe attachment sequence and a N^th template binding sequence; and (j) a N^th Occlusion Probe, where the N^th Occlusion Probe comprises, in 5' to 3' order, a N^th primer attachment sequence and a N^th hairpin sequence, where N refers to any whole number between 5 and 1000.

[0061] In an aspect, a primer attachment sequence is at least 50% complementary to a probe attachment sequence. In an aspect, a primer attachment sequence is at least 60% complementary to a probe attachment sequence. In an aspect, a primer attachment sequence is at least 70% complementary to a probe attachment sequence. In an aspect, a primer attachment sequence is at least 80% complementary to a probe attachment sequence. In an aspect, a primer attachment sequence is at least 90% complementary to a probe attachment sequence. In an aspect, a primer attachment sequence is at least 95% complementary to a probe attachment sequence. In an aspect, a primer attachment sequence is at least 99% complementary to a probe attachment sequence. In an aspect, a primer attachment sequence is 100% complementary to a probe attachment sequence.

[0062] In an aspect, a primer attachment sequence is between 50% and 100% complementary to a probe attachment sequence. In an aspect, a primer attachment sequence is between 60% and 100% complementary to a probe attachment sequence. In an aspect, a primer attachment sequence is between 70% and 100% complementary to a probe attachment sequence. In an aspect, a primer attachment sequence is between 80% and 100% complementary to a probe attachment sequence. In an aspect, a primer attachment sequence is between 90% and 100% complementary to a probe attachment sequence. In an aspect, a primer attachment sequence is between 95% and 100% complementary to a probe attachment sequence. In an aspect, a primer attachment sequence is between 50% and 95% complementary to a probe attachment sequence. In an aspect, a primer attachment sequence is between 60% and 95% complementary to a probe attachment sequence. In an aspect, a primer attachment sequence is between 70% and 95% complementary to a probe attachment sequence. In an aspect, a primer attachment sequence is between 75% and 95% complementary to a probe attachment sequence. In an aspect, a primer attachment sequence is between 80% and 95% complementary to a probe attachment sequence. In an aspect, a primer attachment sequence is between 90% and 95% complementary to a probe attachment sequence. In an aspect, a primer attachment sequence is between 70% and 90% complementary to a probe attachment sequence.

[0063] In an aspect, a probe attachment sequence comprises a length of between 6 nucleotides and 200 nucleotides. In an aspect, a probe attachment sequence comprises a length of between 6 nucleotides and 150 nucleotides. In an aspect, a probe attachment sequence comprises a length of between 6 nucleotides and 100 nucleotides. In an aspect, a probe attachment sequence comprises a length of between 6 nucleotides and 75 nucleotides. In an aspect, a probe attachment sequence comprises a length of between 6 nucleotides and 50 nucleotides. In an aspect, a probe attachment sequence comprises a length of between 6 nucleotides and 25 nucleotides. In an aspect, a probe attachment sequence comprises a length of between 6 nucleotides and 10 nucleotides. In an aspect, a probe attachment sequence comprises a length of between 10 nucleotides and 100 nucleotides. In an aspect, a probe attachment sequence comprises a length of between 10 nucleotides and 75 nucleotides. In an aspect, a probe attachment sequence comprises a length of between 10 nucleotides and 50 nucleotides. In an aspect, a probe attachment sequence comprises a length of between 10 nucleotides and 25 nucleotides.

[0064] In an aspect, a probe attachment sequence comprises a length of at least 5 nucleotides.

In an aspect, a probe attachment sequence comprises a length of at least 6 nucleotides. In an aspect, a probe attachment sequence comprises a length of at least 10 nucleotides. In an aspect, a probe attachment sequence comprises a length of at least 15 nucleotides. In an aspect, a probe attachment sequence comprises a length of at least 20 nucleotides. In an aspect, a probe attachment sequence comprises a length of at least 25 nucleotides. In an aspect, a probe attachment sequence comprises a length of at least 30 nucleotides. In an aspect, a probe attachment sequence comprises a length of at least 40 nucleotides. In an aspect, a probe attachment sequence comprises a length of at least 50 nucleotides. In an aspect, a probe attachment sequence comprises a length of at least 75 nucleotides. In an aspect, a probe attachment sequence comprises a length of at least 150 nucleotides. In an aspect, a probe attachment sequence comprises a length of at least 200 nucleotides.

[0065] In an aspect, a probe attachment sequence comprises a high-throughput sequencing adapter sequence. In an aspect, a first probe attachment sequence and a second probe attachment sequence both comprise high-throughput sequencing adapter sequences. In an aspect, a first probe attachment sequence and a second probe attachment sequence comprise different high-throughput sequencing adapter sequences. In an aspect, a first high-throughput sequencing adapter sequence and a second high-throughput adapter sequence are not identical. Non-limiting examples of high-throughput sequencing adapter sequences are provided as SEQ ID NOs: 185-195. “High-throughput sequencing adapters” refer to platform-specific (e.g, Illumina, Pacific Biosciences) sequences for fragment recognition by specific sequencing instruments. [0066] In an aspect, a method comprises ligating at least one sequence index to a plurality of amplicons. As used herein, a “sequence index” refers to a unique sequence added to a sample that enables the sequence to be sorted prior to sequence analysis. Sequence indexes are often added to DNA fragments during NGS library preparation.

[0067] In an aspect, a probe attachment sequence comprises a sequence at least 85% identical or complementary to a sequence selected from the group consisting of SEQ ID NOs: 185-195. In an aspect, a probe attachment sequence comprises a sequence at least 90% identical or complementary to a sequence selected from the group consisting of SEQ ID NOs: 185-195. In an aspect, a probe attachment sequence comprises a sequence at least 95% identical or complementary to a sequence selected from the group consisting of SEQ ID NOs: 185-195. In an aspect, a probe attachment sequence comprises a sequence at least 99% identical or complementary to a sequence selected from the group consisting of SEQ ID NOs: 185-195. In an aspect, a probe attachment sequence comprises a sequence 100% identical or complementary to a sequence selected from the group consisting of SEQ ID NOs: 185-195.

[0068] In an aspect, a primer attachment sequence comprises a length of between 6 nucleotides and 200 nucleotides. In an aspect, a primer attachment sequence comprises a length of between 6 nucleotides and 150 nucleotides. In an aspect, a primer attachment sequence comprises a length of between 6 nucleotides and 100 nucleotides. In an aspect, a primer attachment sequence comprises a length of between 6 nucleotides and 75 nucleotides. In an aspect, a primer attachment sequence comprises a length of between 6 nucleotides and 50 nucleotides. In an aspect, a primer attachment sequence comprises a length of between 6 nucleotides and 25 nucleotides. In an aspect, a primer attachment sequence comprises a length of between 6 nucleotides and 10 nucleotides. In an aspect, a primer attachment sequence comprises a length of between 10 nucleotides and 100 nucleotides. In an aspect, a primer attachment sequence comprises a length of between 10 nucleotides and 75 nucleotides. In an aspect, a primer attachment sequence comprises a length of between 10 nucleotides and 50 nucleotides. In an aspect, a primer attachment sequence comprises a length of between 10 nucleotides and 25 nucleotides.

[0069] In an aspect, a primer attachment sequence comprises a length of at least 5 nucleotides. In an aspect, a primer attachment sequence comprises a length of at least 6 nucleotides. In an aspect, a primer attachment sequence comprises a length of at least 10 nucleotides. In an aspect, a primer attachment sequence comprises a length of at least 15 nucleotides. In an aspect, a primer attachment sequence comprises a length of at least 20 nucleotides. In an aspect, a primer attachment sequence comprises a length of at least 25 nucleotides. In an aspect, a primer attachment sequence comprises a length of at least 30 nucleotides. In an aspect, a primer attachment sequence comprises a length of at least 40 nucleotides. In an aspect, a primer attachment sequence comprises a length of at least 50 nucleotides. In an aspect, a primer attachment sequence comprises a length of at least 75 nucleotides. In an aspect, a primer attachment sequence comprises a length of at least 150 nucleotides. In an aspect, a primer attachment sequence comprises a length of at least 200 nucleotides.

[0070] In an aspect, a probe attachment sequence is the same length as a primer attachment sequence. In an aspect, a probe attachment sequence comprises a different length as a primer attachment sequence. In an aspect, a sequence alignment of a primer attachment sequence and a probe attachment sequence comprises at least 1 mismatch. In an aspect, a sequence alignment of a primer attachment sequence and a probe attachment sequence comprises at least 2 mismatches. In an aspect, a sequence alignment of a primer attachment sequence and a probe attachment sequence comprises at least 3 mismatches. In an aspect, a sequence alignment of a primer attachment sequence and a probe attachment sequence comprises at least 4 mismatches. In an aspect, a sequence alignment of a primer attachment sequence and a probe attachment sequence comprises at least 5 mismatches. In an aspect, a sequence alignment of a primer attachment sequence and a probe attachment sequence comprises between 0 mismatches and 7 mismatches. In an aspect, a sequence alignment of a primer attachment sequence and a probe attachment sequence comprises between 0 mismatches and 5 mismatches. In an aspect, a sequence alignment of a primer attachment sequence and a probe attachment sequence comprises between 0 mismatches and 3 mismatches. In an aspect, a sequence alignment of a primer attachment sequence and a probe attachment sequence comprises between 1 mismatches and 7 mismatches. In an aspect, a sequence alignment of a primer attachment sequence and a probe attachment sequence comprises between 1 mismatches and 5 mismatches. In an aspect, a sequence alignment of a primer attachment sequence and a probe attachment sequence comprises between 1 mismatches and 3 mismatches. In an aspect, a sequence alignment of a primer attachment sequence and a probe attachment sequence comprises between 2 mismatches and 4 mismatches.

[0071] In an aspect, a template binding sequence comprises a length of between 5 nucleotides and 100 nucleotides. In an aspect, a template binding sequence comprises a length of between 6 nucleotides and 75 nucleotides. In an aspect, a template binding sequence comprises a length of between 6 nucleotides and 50 nucleotides. In an aspect, a template binding sequence comprises a length of between 6 nucleotides and 40 nucleotides. In an aspect, a template binding sequence comprises a length of between 6 nucleotides and 30 nucleotides. In an aspect, a template binding sequence comprises a length of between 6 nucleotides and 20 nucleotides.

[0072] In an aspect, a template binding sequence comprises a length of at least 5 nucleotides. In an aspect, a template binding sequence comprises a length of at least 6 nucleotides. In an aspect, a template binding sequence comprises a length of at least 10 nucleotides. In an aspect, a template binding sequence comprises a length of at least 15 nucleotides. In an aspect, a template binding sequence comprises alength of at least 20 nucleotides. In an aspect, atemplate binding sequence comprises a length of at least 25 nucleotides. In an aspect, a template binding sequence comprises a length of at least 30 nucleotides. In an aspect, a template binding sequence comprises a length of at least 40 nucleotides. In an aspect, a template binding sequence comprises a length of at least 50 nucleotides. In an aspect, a template binding sequence comprises a length of at least 75 nucleotides.

[0073] In an aspect, this disclosure provides a template nucleic acid molecule. As used herein, 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 Occlusion Primer and/or at least one Occlusion Probe. In an aspect, a system 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, 41:D36-42 (2013).

[0074] In an aspect, a template nucleic acid molecule is a DNA molecule. In an aspect, a template nucleic acid molecule is an RNA molecule. In an aspect, a template nucleic acid molecule is a genomic DNA molecule. In an aspect, a template nucleic acid molecule is an organellar DNA molecule. In an aspect, an organellar DNA molecule is selected from the group consisting of a mitochondrial DNA molecule and a plastid DNA molecule. In an aspect, a template nucleic acid molecule is a complementary DNA (cDNA) molecule.

[0075] In an aspect, a template nucleic acid molecule is a eukaryotic nucleic acid molecule. In an aspect, 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. In an aspect, an animal nucleic acid molecule is a human nucleic acid molecule. In an aspect, a template nucleic acid molecule is a prokaryotic nucleic acid molecule. In an aspect, a prokaryotic nucleic acid molecule is selected from the group consisting of a bacteria nucleic acid molecule and an archaea nucleic acid molecule. [0076] In an aspect, a template nucleic acid molecule is a virus nucleic acid molecule. In an aspect, 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 nucleic acid molecule, a Geminiviridae nucleic acid molecule, a Flaviviridae nucleic acid molecule, a Filoviridae nucleic acid molecule, a Paramyxoviridae nucleic acid molecule, and a Pneumoviridae nucleic acid molecule. In an aspect, 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.

[0077] In an aspect, a template nucleic acid molecule is a viroid nucleic acid molecule. In an aspect, a viroid nucleic acid molecule is selected from the group consisting of a Pospiviroidae nucleic acid molecule, and a Avsunviroidae nucleic acid molecule.

[0078] In an aspect, a template binding sequence is at least 70% complementary to a template nucleic acid molecule. In an aspect, a template binding sequence is at least 80% complementary to a template nucleic acid molecule. In an aspect, a template binding sequence is at least 85% complementary to a template nucleic acid molecule. In an aspect, a template binding sequence is at least 90% complementary to a template nucleic acid molecule. In an aspect, a template binding sequence is at least 95% complementary to a template nucleic acid molecule. In an aspect, a template binding sequence is at least 99% complementary to a template nucleic acid molecule. In an aspect, a template binding sequence is 100% complementary to a template nucleic acid molecule.

[0079] In an aspect, an Occlusion Primer and a template nucleic acid molecule exhibit a standard free energy of hybridization AG° 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. In an aspect, a primer and a template nucleic acid molecule exhibit a standard free energy of hybridization AG° 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. In an aspect, an Occlusion Primer and an Occlusion Probe exhibit a standard free energy of hybridization AG° between -7 and -100, between -7 and -90, between -7 and -80, between -7 and -70, between -7 and -60, between -7 and -50, between -7 and -40, between -7 and -30, between -7 and -20, between -7 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.

[0080] In an aspect, a template nucleic acid molecule comprises an initiation subsequence. As used herein, an “initiation subsequence” refers to a nucleic acid sequence that is complementary, and is capable of binding, to the template binding sequence of an Occlusion Primer.

[0081] In an aspect, 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 aspect, 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.

[0082] In an aspect, 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. In an aspect, 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.

[0083] In an aspect, a template binding sequence is at least 70% complementary to an initiation subsequence. In an aspect, a template binding sequence is at least 80% complementary to an initiation subsequence. In an aspect, a template binding sequence is at least 85% complementary to an initiation subsequence. In an aspect, a template binding sequence is at least 90% complementary to an initiation subsequence. In an aspect, a template binding sequence is at least 95% complementary to an initiation subsequence. In an aspect, a template binding sequence is at least 99% complementary to an initiation subsequence. In an aspect, a template binding sequence is 100% complementary to an initiation subsequence.

[0084] In an aspect, a template binding sequence is between 70% and 100% complementary to an initiation subsequence. In an aspect, a template binding sequence is between 70% and 99% complementary to an initiation subsequence. In an aspect, a template binding sequence is between 70% and 95% complementary to an initiation subsequence. In an aspect, a template binding sequence is between 70% and 90% complementary to an initiation subsequence. In an aspect, a template binding sequence is between 70% and 85% complementary to an initiation subsequence. In an aspect, a template binding sequence is between 70% and 80% complementary to an initiation subsequence. In an aspect, a template binding sequence is between 80% and 100% complementary to an initiation subsequence. In an aspect, a template binding sequence is between 85% and 100% complementary to an initiation subsequence. In an aspect, a template binding sequence is between 90% and 100% complementary to an initiation subsequence. In an aspect, a template binding sequence is between 95% and 100% complementary to an initiation subsequence.

[0085] Each Occlusion Probe comprises, in part, a hairpin sequence. In an aspect, the hairpin sequence comprises a first subsequence, a loop sequence, and a second subsequence in order from 5' to 3'. In an aspect, a second subsequence is at least 60% complementary to a first subsequence. In an aspect, a second subsequence is at least 70% complementary to a first subsequence. In an aspect, a second subsequence is at least 80% complementary to a first subsequence. In an aspect, a second subsequence is at least 85% complementary to a first subsequence. In an aspect, a second subsequence is at least 90% complementary to a first subsequence. In an aspect, a second subsequence is at least 95% complementary to a first subsequence. In an aspect, a second subsequence is at least 99% complementary to a first subsequence. In an aspect, a second subsequence is 100% complementary to a first subsequence.

[0086] In an aspect, a second subsequence is between 60% and 100% complementary to a first subsequence. In an aspect, a second subsequence is between 70% and 100% complementary to a first subsequence. In an aspect, a second subsequence is between 80% and 100% complementary to a first subsequence. In an aspect, a second subsequence is between 85% and 100% complementary to a first subsequence. In an aspect, a second subsequence is between 90% and 100% complementary to a first subsequence. In an aspect, a second subsequence is between 95% and 100% complementary to a first subsequence. In an aspect, a second subsequence is between 75% and 99% complementary to a first subsequence. In an aspect, a second subsequence is between 90% and 99% complementary to a first subsequence.

[0087] In an aspect, a first subsequence comprises the same number of nucleotides as compared to a second subsequence. In an aspect, a first subsequence comprises a different number of nucleotides as compared to a second subsequence. In an aspect, a first subsequence and a second subsequence differ in length by at least 1 nucleotide. In an aspect, a first subsequence and a second subsequence differ in length by at least 2 nucleotides.

[0088] In an aspect, a first subsequence comprises between 2 nucleotides and 25 nucleotides. In an aspect, a first subsequence comprises between 2 nucleotides and 20 nucleotides. In an aspect, a first subsequence comprises between 2 nucleotides and 15 nucleotides. In an aspect, a first subsequence comprises between 2 nucleotides and 12 nucleotides. In an aspect, a first subsequence comprises between 2 nucleotides and 10 nucleotides. In an aspect, a first subsequence comprises between 2 nucleotides and 7 nucleotides. In an aspect, a first subsequence comprises between 2 nucleotides and 5 nucleotides. In an aspect, a first subsequence comprises between 5 nucleotides and 15 nucleotides. In an aspect, a first subsequence comprises between 5 nucleotides and 10 nucleotides.

[0089] In an aspect, a first subsequence comprises at least 2 nucleotides. In an aspect, a first subsequence comprises at least 3 nucleotides. In an aspect, a first subsequence comprises at least 4 nucleotides. In an aspect, a first subsequence comprises at least 5 nucleotides. In an aspect, a first subsequence comprises at least 6 nucleotides. In an aspect, a first subsequence comprises at least 7 nucleotides. In an aspect, a first subsequence comprises at least 8 nucleotides. In an aspect, a first subsequence comprises at least 9 nucleotides. In an aspect, a first subsequence comprises at least 10 nucleotides. In an aspect, a first subsequence comprises at least 12 nucleotides. In an aspect, a first subsequence comprises at least 15 nucleotides. In an aspect, a first subsequence comprises at least 20 nucleotides. In an aspect, a first subsequence comprises at least 25 nucleotides.

[0090] In an aspect, a second subsequence comprises between 2 nucleotides and 25 nucleotides. In an aspect, a second subsequence comprises between 2 nucleotides and 20 nucleotides. In an aspect, a second subsequence comprises between 2 nucleotides and 15 nucleotides. In an aspect, a second subsequence comprises between 2 nucleotides and 12 nucleotides. In an aspect, a second subsequence comprises between 2 nucleotides and 10 nucleotides. In an aspect, a second subsequence comprises between 2 nucleotides and 7 nucleotides. In an aspect, a second subsequence comprises between 2 nucleotides and 5 nucleotides. In an aspect, a second subsequence comprises between 5 nucleotides and 15 nucleotides. In an aspect, a second subsequence comprises between 5 nucleotides and 10 nucleotides.

[0091] In an aspect, a second subsequence comprises at least 2 nucleotides. In an aspect, a second subsequence comprises at least 3 nucleotides. In an aspect, a second subsequence comprises at least 4 nucleotides. In an aspect, a second subsequence comprises at least 5 nucleotides. In an aspect, a second subsequence comprises at least 6 nucleotides. In an aspect, a second subsequence comprises at least 7 nucleotides. In an aspect, a second subsequence comprises at least 8 nucleotides. In an aspect, a second subsequence comprises at least 9 nucleotides. In an aspect, a second subsequence comprises at least 10 nucleotides. In an aspect, a second subsequence comprises at least 12 nucleotides. In an aspect, a second subsequence comprises at least 15 nucleotides. In an aspect, a second subsequence comprises at least 20 nucleotides. In an aspect, a second subsequence comprises at least 25 nucleotides.

[0092] In an aspect, a first subsequence and a second subsequence form the stem of a stemloop structure in conjunction with a loop sequence.

[0093] In an aspect, a loop sequence comprises between 3 nucleotides and 30 nucleotides. In an aspect, a loop sequence comprises between 3 nucleotides and 20 nucleotides. In an aspect, a loop sequence comprises between 3 nucleotides and 15 nucleotides. In an aspect, a loop sequence comprises between 3 nucleotides and 10 nucleotides. In an aspect, a loop sequence comprises between 3 nucleotides and 8 nucleotides. In an aspect, a loop sequence comprises between 5 nucleotides and 10 nucleotides. [0094] In an aspect, a loop sequence comprises at least 3 nucleotides. In an aspect, a loop sequence comprises at least 4 nucleotides. In an aspect, a loop sequence comprises at least 5 nucleotides. In an aspect, a loop sequence comprises at least 6 nucleotides. In an aspect, a loop sequence comprises at least 7 nucleotides. In an aspect, a loop sequence comprises at least 8 nucleotides. In an aspect, a loop sequence comprises at least 9 nucleotides. In an aspect, a loop sequence comprises at least 10 nucleotides. In an aspect, a loop sequence comprises at least 12 nucleotides. In an aspect, a loop sequence comprises at least 15 nucleotides. In an aspect, a loop sequence comprises at least 20 nucleotides.

[0095] In an aspect, a hairpin sequence comprises a sequence at least 85% identical or complementary to a sequence selected from the group consisting of SEQ ID NOs: 183 and 184. In an aspect, a hairpin sequence comprises a sequence at least 90% identical or complementary to a sequence selected from the group consisting of SEQ ID NOs: 183 and 184. In an aspect, a hairpin sequence comprises a sequence at least 95% identical or complementary to a sequence selected from the group consisting of SEQ ID NOs: 183 and 184. In an aspect, a hairpin sequence comprises a sequence 100% identical or complementary to a sequence selected from the group consisting of SEQ ID NOs: 183 and 184.

[0096] In an aspect, an Occlusion Probe comprises a 3' single-stranded overhang. In an aspect, an Occlusion Probe comprises a 5' single-stranded overhang. In an aspect, an Occlusion Primer comprises a 3' single-stranded overhang. In an aspect, an Occlusion Primer comprises a 5' single-stranded overhang.

[0097] As used herein, in reference to an Occlusion Primer, a “5' single-stranded overhang” refers to an extension of at least one nucleotide between a fluorophore or quencher and a probe attachment sequence of the Occlusion Primer, where the 5' single-stranded overhang does not hybridize with any part of an Occlusion Probe when the Occlusion Primer and Occlusion Probe form a complex.

[0098] As used herein, in reference to an Occlusion Primer, a “3' single-stranded overhang” refers to a portion of a probe attachment sequence that does not hybridize with a primer attachment sequence of an Occlusion Probe when the Occlusion Primer and Occlusion Probe form a complex.

[0099] As used herein, in reference to an Occlusion Probe, a “3' single-stranded overhang” refers to an extension of at least one nucleotide between a second subsequence and a fluorophore or quencher, where the 3' single-stranded overhang does not hybridize with a first subsequence or a probe attachment sequence of an Occlusion Primer when the Occlusion Primer and Occlusion Probe form a complex. [0100] As used herein, in reference to an Occlusion Probe, a “5' single-stranded overhang” refers to an extension on the 5' end of a primer attachment sequence, where the extension does not hybridize with the probe attachment sequence of an Occlusion Primer when the Occlusion Primer and Occlusion Probe form a complex.

[0101] In an aspect, a 3' single-stranded overhang comprises at least 1 nucleotide. In an aspect, a 3' single-stranded overhang comprises at least 2 nucleotides. In an aspect, a 3' single-stranded overhang comprises at least 3 nucleotides. In an aspect, a 3' single-stranded overhang comprises at least 4 nucleotides. In an aspect, a 3' single-stranded overhang comprises at least 5 nucleotides. In an aspect, a 3' single-stranded overhang comprises at least 6 nucleotides. In an aspect, a 3' single-stranded overhang comprises at least 7 nucleotides. In an aspect, a 3' single-stranded overhang comprises at least 8 nucleotides. In an aspect, a 3' single-stranded overhang comprises at least 9 nucleotides. In an aspect, a 3' single-stranded overhang comprises at least 10 nucleotides. In an aspect, a 3' single-stranded overhang comprises at least 15 nucleotides. In an aspect, a 3' single-stranded overhang comprises at least 20 nucleotides. In an aspect, a 3' single-stranded overhang comprises at least 25 nucleotides. In an aspect, a 3' single-stranded overhang comprises at least 50 nucleotides.

[0102] In an aspect, a 3' single-stranded overhang comprises between 1 nucleotide and 100 nucleotides. In an aspect, a 3' single-stranded overhang comprises between 1 nucleotide and 50 nucleotides. In an aspect, a 3' single-stranded overhang comprises between 1 nucleotide and 25 nucleotides. In an aspect, a 3' single-stranded overhang comprises between 1 nucleotide and 20 nucleotides. In an aspect, a 3' single-stranded overhang comprises between 1 nucleotide and 15 nucleotides. In an aspect, a 3' single-stranded overhang comprises between 1 nucleotide and 10 nucleotides. In an aspect, a 3' single-stranded overhang comprises between 1 nucleotide and 5 nucleotides. In an aspect, a 3' single-stranded overhang comprises between 2 nucleotides and 50 nucleotides. In an aspect, a 3' single-stranded overhang comprises between 2 nucleotides and 25 nucleotides. In an aspect, a 3' single-stranded overhang comprises between 2 nucleotides and 20 nucleotides. In an aspect, a 3' single-stranded overhang comprises between 2 nucleotides and 15 nucleotides. In an aspect, a 3' single-stranded overhang comprises between 2 nucleotides and 10 nucleotides.

[0103] In an aspect, a 5' single-stranded overhang comprises at least 1 nucleotide. In an aspect, a 5' single-stranded overhang comprises at least 2 nucleotides. In an aspect, a 5' single-stranded overhang comprises at least 3 nucleotides. In an aspect, a 5' single-stranded overhang comprises at least 4 nucleotides. In an aspect, a 5' single-stranded overhang comprises at least 5 nucleotides. In an aspect, a 5' single-stranded overhang comprises at least 6 nucleotides. In an aspect, a 5' single-stranded overhang comprises at least 7 nucleotides. In an aspect, a 5' single-stranded overhang comprises at least 8 nucleotides. In an aspect, a 5' single-stranded overhang comprises at least 9 nucleotides. In an aspect, a 5' single-stranded overhang comprises at least 10 nucleotides. In an aspect, a 5' single-stranded overhang comprises at least 15 nucleotides. In an aspect, a 5' single-stranded overhang comprises at least 20 nucleotides. In an aspect, a 5' single-stranded overhang comprises at least 25 nucleotides. In an aspect, a 5' single-stranded overhang comprises at least 50 nucleotides.

[0104] In an aspect, a 5' single-stranded overhang comprises between 1 nucleotide and 100 nucleotides. In an aspect, a 5' single-stranded overhang comprises between 1 nucleotide and 50 nucleotides. In an aspect, a 5' single-stranded overhang comprises between 1 nucleotide and 25 nucleotides. In an aspect, a 5' single-stranded overhang comprises between 1 nucleotide and 20 nucleotides. In an aspect, a 5' single-stranded overhang comprises between 1 nucleotide and 15 nucleotides. In an aspect, a 5' single-stranded overhang comprises between 1 nucleotide and 10 nucleotides. In an aspect, a 5' single-stranded overhang comprises between 1 nucleotide and 5 nucleotides. In an aspect, a 5' single-stranded overhang comprises between 2 nucleotides and 50 nucleotides. In an aspect, a 5' single-stranded overhang comprises between 2 nucleotides and 25 nucleotides. In an aspect, a 5' single-stranded overhang comprises between 2 nucleotides and 20 nucleotides. In an aspect, a 5' single-stranded overhang comprises between 2 nucleotides and 15 nucleotides. In an aspect, a 5' single-stranded overhang comprises between 2 nucleotides and 10 nucleotides.

[0105] In an aspect, this disclosure provides at least one DNA polymerase. In an aspect, a system comprises at least one DNA polymerase. As used herein, a “DNA polymerase” refers to an enzyme that is capable of catalyzing the synthesis of a DNA molecule from nucleoside triphosphates. 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.

[0106] DNA polymerases are not perfect at making copies of a DNA strand. In general, a DNA polymerase generates an error (e.g. , a non-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. [0107] 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.

[0108] In an aspect, 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.

[0109] In an aspect, 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, Accuris™ DNA polymerase, PowerUp™ DNA Polymerase, and Taq DNA polymerase. In an aspect, a DNA polymerase is a thermostable DNA polymerase.

[0110] The use of fluorophores during qPCR enables the quantification of a targeted DNA molecule. Either the absolute amount of a target DNA molecule, or the relative amount of a target molecule between samples, can be measured. A measurement called the threshold cycle (Ct) is often used to measure the relative concentration of a targeted DNA molecule in a qPCR sample. As used herein, “Ct” refers to the number of thermal cycles required for a fluorescent signal to cross the threshold (e.g, background fluorescence level) during qPCR. Ct levels are inversely proportional to the amount of target nucleic acid in a sample (e.g, the lower the Ct level, the greater the amount of target DNA molecule in the sample). In an aspect, a method provided herein comprises determining the concentration of at least one amplicon by calculating a cycle threshold (Ct) value.

[OHl] As used herein, a “fluorophore” refers to any fluorescent compound that can re-emit light upon light excitation.

[0112] In an aspect, a fluorophore is a peptide or protein. In an aspect, a fluorophore is a small organic compound. In an aspect, a fluorophore is a synthetic oligomer or polymer. In an aspect, a fluorophore is a multi-component system. In an aspect, 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. In an aspect, a fluorophore is an organic dye. [0113] Non-limiting examples of peptide or protein fluorophores included green fluorescence protein (GFP), yellow fluorescence protein (YFP), and red fluorescence protein (RFP).

[0114] 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.

[0115] In an aspect, a fluorophore is selected from the group consisting of Cy3™, FAM, VIC, HEX, Cy5™, ROX, and Quasar 705.

[0116] In an aspect, a fluorophore is selected from the group consisting of a Freedom™ dye, an ATTO™ dye, an Alexa Fluor® dye, a LI-COR IRDye®, and a Rhodamine dye. Freedom™ dyes, ATTO™ 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.

[0117] In an aspect, an Occlusion Primer comprises at least one fluorophore. See Figure 2A. In an aspect, at least one fluorophore is positioned at, or near, the 5' end of an Occlusion Primer. In an aspect, at least one fluorophore is positioned at, or near, the 3' end of an Occlusion Primer.

[0118] In an aspect, an Occlusion Primer comprises at least one fluorophore. See Figure 2B. In an aspect, at least one fluorophore is positioned at, or near, the 5' end of an Occlusion Primer. In an aspect, at least one fluorophore is positioned at, or near, the 3' end of an Occlusion Primer.

[0119] As used herein, “at” the 5' or 3' end of an oligonucleotide refers to being attached to the 5 '-most or 3 '-most nucleotide of the oligonucleotide. As used herein, “near” the 5' or 3' end of an oligonucleotide refers to being attached within the 10 5 '-most or 3 '-most nucleotides of the oligonucleotide.

[0120] Quenchers can be used to decrease the fluorescence intensity of a given substance, such as a fluorophore. As used herein, a “quencher” refers to any substance that absorbs the excitation energy from a fluorophore, thereby reducing or eliminating the fluorescence intensity of the fluorophore. In an aspect, a quencher dissipates the light energy from a fluorophore as heat. In an aspect, a quencher is a dye that lacks native fluorescence.

[0121] In an aspect, a quencher is selected from the group consisting of MGB, Black Hole Quencher, Iowa Black® RQ, and Iowa Black® FQ. [0122] In an aspect, an Occlusion Primer comprises at least one quencher. See Figure 2B. In an aspect, at least one quencher is positioned at, or near, the 5' end of an Occlusion Primer. In an aspect, at least one quencher is positioned at, or near, the 3' end of an Occlusion Primer.

[0123] In an aspect, an Occlusion Primer comprises at least one quencher. See Figure 2A. In an aspect, at least one quencher is positioned at, or near, the 5' end of an Occlusion Primer. In an aspect, at least one quencher is positioned at, or near, the 3' end of an Occlusion Primer.

[0124] In an aspect, an Occlusion Primer comprises at least one non-natural nucleoside. In an aspect, an Occlusion Primer comprises at least two non-natural nucleosides. In an aspect, an Occlusion Primer comprises at least three non-natural nucleosides. In an aspect, an Occlusion Primer comprises at least four non-natural nucleosides. In an aspect, an Occlusion Primer comprises at least five non-natural nucleosides. In an aspect, an Occlusion Primer comprises at least one non-natural nucleoside within a probe attachment sequence. In an aspect, an Occlusion Primer comprises at least one non-natural nucleoside within a template binding sequence. In an aspect, a primer comprises at least one non-natural nucleoside. In an aspect, a primer comprises at least two non-natural nucleosides. In an aspect, a primer comprises at least three non-natural nucleosides. In an aspect, a primer comprises at least four non-natural nucleosides. In an aspect, a primer comprises at least five non-natural nucleosides. In an aspect, a forward blocker comprises at least one non-natural nucleoside. In an aspect, a forward blocker comprises at least two non-natural nucleosides. In an aspect, a forward blocker comprises at least three non-natural nucleosides. In an aspect, a forward blocker comprises at least four non- natural nucleosides. In an aspect, a forward blocker comprises at least five non-natural nucleosides.

[0125] In an aspect, a non-natural nucleoside 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.

[0126] In an aspect, an Occlusion Primer comprises at least one backbone modification. In an aspect, an Occlusion Primer comprises at least two backbone modifications. In an aspect, an Occlusion Primer comprises at least three backbone modifications. In an aspect, an Occlusion Primer comprises at least four backbone modifications. In an aspect, an Occlusion Primer comprises at least five backbone modifications. In an aspect, an Occlusion Primer comprises at least one backbone modification within a probe attachment sequence. In an aspect, an Occlusion Primer comprises at least one backbone modification within a template binding sequence. In an aspect, a primer comprises at least one backbone modification. In an aspect, a primer comprises at least two backbone modifications. In an aspect, a primer comprises at least three backbone modifications. In an aspect, a primer comprises at least four backbone modifications. In an aspect, a primer comprises at least five backbone modifications. In an aspect, a forward blocker comprises at least one backbone modification. In an aspect, a forward blocker comprises at least two backbone modifications. In an aspect, a forward blocker comprises at least three backbone modifications. In an aspect, a forward blocker comprises at least four backbone modifications. In an aspect, a forward blocker comprises at least five backbone modifications.

[0127] As used herein, 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.

[0128] In an aspect, 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

[0129] In an aspect, this disclosure provides a kit comprising: (a) a first Occlusion Primer, wherein the first Occlusion Primer comprises, in 5' to 3' order, a first probe attachment sequence and a first template binding sequence; (b) a first Occlusion Probe, where the first Occlusion Probe comprises, in 5' to 3' order, a first primer attachment sequence and a first hairpin sequence; and (c) at least one DNA polymerase. In an aspect, a kit further comprises a second primer.

[0130] In an aspect, an Occlusion Primer is suspended in a liquid. In an aspect, an Occlusion Primer is lyophilized. In an aspect, an Occlusion Probe is suspended in a liquid. In an aspect, an Occlusion Probe is lyophilized. In an aspect, a DNA polymerase is suspended in a liquid. In an aspect, a DNA polymerase is lyophilized.

[0131] In an aspect, a system provided herein comprises at least one buffer. In an aspect, a kit provided herein comprises at least one buffer. In an aspect, a kit provided herein comprises at least one reagent. As used herein, 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. In an aspect, a reagent comprises a component selected from the group consisting of magnesium, at least one dNTP, phosphatase, betaine, dimethyl sulfoxide (DMSO), and tetramethyl ammonium chloride (TMAC). In an aspect, a reagent is a liquid. In an aspect, a reagent is lyophilized.

[0132] In an aspect, this disclosure provides a method for amplifying a template nucleic acid molecule, the method comprising: (a) mixing a sample comprising the template nucleic acid molecule with (i) at least one DNA polymerase; (ii) a first Occlusion Primer, where the first Occlusion Primer comprises, in 5' to 3' order, a first probe attachment sequence and a first template binding sequence; (iii) a first Occlusion Probe, where the first Occlusion Probe comprises, in 5' to 3' order, a first primer attachment sequence and a first hairpin sequencer; and (iv) a second primer; to generate a mixture; and (b)subjecting the mixture to at least one cycle of thermal cycling, where the template nucleic acid molecule is amplified to produce at least one amplicon. In an aspect, a method further comprises a second Occlusion Primer, where the second Occlusion Primer comprises, in 5' to 3' order, a second probe attachment sequence and a second template binding sequence. In an aspect, a method further comprises a third Occlusion Primer, where the third Occlusion Primer comprises, in 5' to 3' order, a third probe attachment sequence and a third template binding sequence. In an aspect, a method further comprises a fourth Occlusion Primer, where the fourth Occlusion Primer comprises, in 5' to 3' order, a fourth probe attachment sequence and a fourth template binding sequence. In an aspect, a method further comprises aN^th Occlusion Primer, where the N^th Occlusion Primer comprises, in 5' to 3' order, a N^th probe attachment sequence and a N^th template binding sequence, where N refers to any whole number between 5 and 1000. In an aspect, a method further comprises a second Occlusion Probe, where the second Occlusion Probe comprises, in 5' to 3' order, a second primer attachment sequence and a second hairpin sequence. In an aspect, a method further comprises a third Occlusion Probe, where the third Occlusion Probe comprises, in 5' to 3' order, a third primer attachment sequence and a third hairpin sequence. In an aspect, a method further comprises a fourth Occlusion Probe, where the fourth Occlusion Probe comprises, in 5' to 3' order, a fourth primer attachment sequence and a fourth hairpin sequence. In an aspect, a method further comprises aN¹¹¹ Occlusion Probe, where the N^th Occlusion Probe comprises, in 5' to 3' order, a N^th primer attachment sequence and a N^th hairpin sequence, where N refers to any whole number between 5 and 1000.

[0133] In an aspect, this disclosure provides a method for multiplex amplification of a plurality of nucleic acid molecules, the method comprising: (a) obtaining a mixture comprising (i) a plurality of template nucleic acid molecules; (ii) at least one DNA polymerase; (iii) a first Occlusion Primer, where the first Occlusion Primer comprises, in 5' to 3' order, a first probe attachment sequence and a first template binding sequence; (iv) a first Occlusion Probe, where the first Occlusion Probe comprises, in 5' to 3' order, a first primer attachment sequence and a first hairpin sequence; (v) a second Occlusion Primer, where the second Occlusion Primer comprises, in 5' to 3' order, a second probe attachment sequence and a second template binding sequence; and (vi) a second Occlusion Probe, where the second Occlusion Probe comprises, in 5' to 3' order, a second primer attachment sequence and a second hairpin sequencer; to generate the mixture; and (b) subjecting the mixture to at least one cycle of thermal cycling, where the plurality of template nucleic acid molecules are amplified to produce a plurality of amplicons. [0134] In an aspect, this disclosure provides a method for multiplex amplification of a plurality of nucleic acid molecules, the method comprising: (a) obtaining a mixture comprising (i) a plurality of template nucleic acid molecules; (ii) at least one DNA polymerase; (iii) a first Occlusion Primer, where the first Occlusion Primer comprises, in 5' to 3' order, a first probe attachment sequence and a first template binding sequence; (iv) a first Occlusion Probe, where the first Occlusion Probe comprises, in 5' to 3' order, a first primer attachment sequence and a first hairpin sequence; (v) a first primer, where the first primer produces an amplicon in conjunction with the first Occlusion Primer; (vi) a second Occlusion Primer, where the second Occlusion Primer comprises, in 5' to 3' order, a second probe attachment sequence and a second template binding sequence; (vii) a second Occlusion Probe, where the second Occlusion Probe comprises, in 5' to 3' order, a second primer attachment sequence and a second hairpin sequencer; to generate the mixture; and (viii) a second primer, where the second primer produces an amplicon in conjunction with the second Occlusion Primer; and (b) subjecting the mixture to at least one cycle of thermal cycling, where the plurality of template nucleic acid molecules are amplified to produce a plurality of amplicons.

[0135] In an aspect, a first probe attachment sequence and a second probe attachment sequence are identical sequences. In an aspect, a first probe attachment sequence, a second probe attachment sequence, and a third probe attachment sequence are identical sequences. In an aspect, a first probe attachment sequence, a second probe attachment sequence, a third probe attachment sequence, and a fourth probe attachment sequence are identical sequences. In an aspect, a first probe attachment sequence, a second probe attachment sequence, a third probe attachment sequence, a fourth probe attachment sequence, and aN^th probe attachment sequence are identical sequences. In an aspect, at least two of a first probe attachment sequence, a second probe attachment sequence, and a third probe attachment sequence are identical sequences. In an aspect, at least two of a first probe attachment sequence, a second probe attachment sequence, a third probe attachment sequence, and a fourth probe attachment sequence are identical sequences. In an aspect, at least three of a first probe attachment sequence, a second probe attachment sequence a third probe attachment sequence, and a fourth probe attachment sequence are identical sequences. In an aspect, a first probe attachment sequence and a second probe attachment sequence are not identical sequences. [0136] In an aspect, any Occlusion Primer used in a method provided herein further comprises at least one fluorophore. In an aspect, any Occlusion Primer used in a method provided herein further comprises at least one quencher. In an aspect, any Occlusion Probe used in a method provided herein further comprises at least one fluorophore. In an aspect, any Occlusion Probe used in a method provided herein further comprises at least one quencher.

[0137] In an aspect, a first Occlusion Primer and a second Occlusion Primer comprise the same fluorophore. In an aspect, a first Occlusion Primer, a second Occlusion Primer, and a third Occlusion Primer comprise the same fluorophore. In an aspect, a first Occlusion Primer, a second Occlusion Primer, a third Occlusion Primer, and a fourth Occlusion Primer comprise the same fluorophore. In an aspect, a first Occlusion Primer, a second Occlusion Primer, a third Occlusion Primer, a fourth Occlusion Primer, and a N¹¹¹ Occlusion Primer comprise the same fluorophore. In an aspect, at least two of a first Occlusion Primer, a second Occlusion Primer, and a third Occlusion Primer comprise the same fluorophore. In an aspect, at least two of a first Occlusion Primer, a second Occlusion Primer, a third Occlusion Primer, and a fourth Occlusion Primer comprise the same fluorophore. In an aspect, at least three of a first Occlusion Primer, a second Occlusion Primer a third Occlusion Primer, and a fourth Occlusion Primer comprise the same fluorophore. In an aspect, a first Occlusion Primer and a second Occlusion Primer comprise the same quencher. In an aspect, a first Occlusion Primer, a second Occlusion Primer, and a third Occlusion Primer comprise the same quencher. In an aspect, a first Occlusion Primer, a second Occlusion Primer, a third Occlusion Primer, and a fourth Occlusion Primer comprise the same quencher. In an aspect, a first Occlusion Primer, a second Occlusion Primer, a third Occlusion Primer, a fourth Occlusion Primer, and a N¹¹¹ Occlusion Primer comprise the same quencher. In an aspect, at least two of a first Occlusion Primer, a second Occlusion Primer, and a third Occlusion Primer comprise the same quencher. In an aspect, at least two of a first Occlusion Primer, a second Occlusion Primer, a third Occlusion Primer, and a fourth Occlusion Primer comprise the same quencher. In an aspect, at least three of a first Occlusion Primer, a second Occlusion Primer a third Occlusion Primer, and a fourth Occlusion Primer comprise the same quencher.

[0138] In an aspect, a first Occlusion Probe and a second Occlusion Probe comprise the same fluorophore. In an aspect, a first Occlusion Probe, a second Occlusion Probe, and a third Occlusion Probe comprise the same fluorophore. In an aspect, a first Occlusion Probe, a second Occlusion Probe, a third Occlusion Probe, and a fourth Occlusion Probe comprise the same fluorophore. In an aspect, a first Occlusion Probe, a second Occlusion Probe, a third Occlusion Probe, a fourth Occlusion Probe, and aN^th Occlusion Probe comprise the same fluorophore. In an aspect, at least two of a first Occlusion Probe, a second Occlusion Probe, and a third Occlusion Probe comprise the same fluorophore. In an aspect, at least two of a first Occlusion Probe, a second Occlusion Probe, a third Occlusion Probe, and a fourth Occlusion Probe comprise the same fluorophore. In an aspect, at least three of a first Occlusion Probe, a second Occlusion Probe a third Occlusion Probe, and a fourth Occlusion Probe comprise the same fluorophore. In an aspect, a first Occlusion Probe and a second Occlusion Probe comprise the same quencher. In an aspect, a first Occlusion Probe, a second Occlusion Probe, and a third Occlusion Probe comprise the same quencher. In an aspect, a first Occlusion Probe, a second Occlusion Probe, a third Occlusion Probe, and a fourth Occlusion Probe comprise the same quencher. In an aspect, a first Occlusion Probe, a second Occlusion Probe, a third Occlusion Probe, a fourth Occlusion Probe, and a N¹¹¹ Occlusion Probe comprise the same quencher. In an aspect, at least two of a first Occlusion Probe, a second Occlusion Probe, and a third Occlusion Probe comprise the same quencher. In an aspect, at least two of a first Occlusion Probe, a second Occlusion Probe, a third Occlusion Probe, and a fourth Occlusion Probe comprise the same quencher. In an aspect, at least three of a first Occlusion Probe, a second Occlusion Probe a third Occlusion Probe, and a fourth Occlusion Probe comprise the same quencher.

[0139] In an aspect, an Occlusion Primer is provided in a mixture. In an aspect, an Occlusion Probe is provided in a mixture. In an aspect, a primer is provided in a mixture.

[0140] In an aspect, the concentration of an Occlusion Primer in a mixture is between 10 pM and 10 pM. In an aspect, the concentration of an Occlusion Primer in a mixture is between 100 pM and 5 pM. In an aspect, the concentration of an Occlusion Primer in a mixture is between 100 pM and 1 pM. In an aspect, the concentration of an Occlusion Primer in a mixture is between 100 pM and 500 nM. In an aspect, the concentration of an Occlusion Primer in a mixture is at least 100 pM. In an aspect, the concentration of an Occlusion Primer in a mixture is at least 500 pM. In an aspect, the concentration of an Occlusion Primer in a mixture is at least 1 nM. In an aspect, the concentration of an Occlusion Primer in a mixture is at least 500 nM. In an aspect, the concentration of an Occlusion Primer in a mixture is at least 1 pM.

[0141] In an aspect, the concentration of an Occlusion Probe in a mixture is between 10 pM and 10 pM. In an aspect, the concentration of an Occlusion Probe in a mixture is between 100 pM and 5 pM. In an aspect, the concentration of an Occlusion Probe in a mixture is between 100 pM and 1 pM. In an aspect, the concentration of an Occlusion Probe in a mixture is between 100 pM and 500 nM. In an aspect, the concentration of an Occlusion Probe in a mixture is at least 100 pM. In an aspect, the concentration of an Occlusion Probe in a mixture is at least 500 pM. In an aspect, the concentration of an Occlusion Probe in a mixture is at least 1 nM. In an aspect, the concentration of an Occlusion Probe in a mixture is at least 500 nM. In an aspect, the concentration of an Occlusion Probe in a mixture is at least 1 pM.

[0142] In an aspect, the concentration of a primer in a mixture is between 10 pM and 10 pM. In an aspect, the concentration of a primer in a mixture is between 100 pM and 5 pM. In an aspect, the concentration of a primer in a mixture is between 100 pM and 1 pM. In an aspect, the concentration of a primer in a mixture is between 100 pM and 500 nM. In an aspect, the concentration of a primer in a mixture is at least 100 pM. In an aspect, the concentration of a primer in a mixture is at least 500 pM. In an aspect, the concentration of a primer in a mixture is at least 1 nM. In an aspect, the concentration of a primer in a mixture is at least 500 nM. In an aspect, the concentration of an Occlusion Probe in a mixture is at least 1 pM.

[0143] In an aspect, the stoichiometric ratio of an Occlusion Probe to an Occlusion Primer in a mixture is between 0.1 and 100. In an aspect, the stoichiometric ratio of an Occlusion Probe to an Occlusion Primer in a mixture is between 0.25 and 100. In an aspect, the stoichiometric ratio of an Occlusion Probe to an Occlusion Primer in a mixture is between 0.5 and 100. In an aspect, the stoichiometric ratio of an Occlusion Probe to an Occlusion Primer in a mixture is between 1 and 100. In an aspect, the stoichiometric ratio of an Occlusion Probe to an Occlusion Primer in a mixture is between 0.1 and 100. In an aspect, the stoichiometric ratio of an Occlusion Probe to an Occlusion Primer in a mixture is between 0.25 and 10. In an aspect, the stoichiometric ratio of an Occlusion Probe to an Occlusion Primer in a mixture is between 0.5 and 10. A stoichiometric ratio of 1 refers to when the Occlusion Probe and Occlusion Primer are present in equal concentrations.

[0144] In an aspect, a mixture further comprises at least one intercalating dye. As used herein, an “intercalating dye” refers to a fluorescent dye that is capable of inserting between nucleotides in a nucleic acid molecule. In an aspect, an intercalating dye is selected from the group consisting of SYBR® Green, EvaGreen®, and SYTO™ dyes.

[0145] In an aspect, this disclosure provides a method for selective amplification of at least one target DNA template, the method comprising: (a) obtaining a mixture comprising (i) at least one target DNA template; (ii) at least one background DNA template; (iii) at least one DNA polymerase; (iv) a forward primer, where the forward primer comprises a sequence that is at least 80% complementary to a sequence of the at least one target DNA template; (v) a forward blocker, where the forward blocker comprises a sequence that is at least 80% complementary to a sequence of the at least one background DNA template; (vi) a first Occlusion Primer, where the first Occlusion Primer comprises, in 5' to 3' order, a first probe attachment sequence and a first template binding sequence; and (vii) a first Occlusion Probe, where the first Occlusion Probe comprises, in 5' to 3' order, a first primer attachment sequence and a first hairpin sequencer; to produce the mixture; and (b) subjecting the mixture to at least five cycles of thermal cycling, where the at least one target DNA template is amplified to produce at least one target DNA amplicon.

[0146] As used herein, a “target DNA template” refers to a region of DNA that is desired to be amplified. As used herein, a “background DNA template” refers to DNA that is not desired to be amplified. In an aspect, the sequence of a target DNA template and a background DNA template differ by at least 1 nucleotide. In an aspect, the sequence of a target DNA template and a background DNA template differ by at least 2 nucleotides. In an aspect, the sequence of a target DNA template and a background DNA template differ by at least 3 nucleotides. In an aspect, the sequence of a target DNA template and a background DNA template differ by at least 4 nucleotides. In an aspect, the sequence of a target DNA template and a background DNA template differ by at least 5 nucleotides. In an aspect, the sequence of a target DNA template and a background DNA template differ by at least 10 nucleotides. In an aspect, the sequence of a target DNA template and a background DNA template differ by at least 15 nucleotides. In an aspect, the sequence of a target DNA template and a background DNA template differ by at least 20 nucleotides. In an aspect, the sequence of a target DNA template and a background DNA template differ by at least 25 nucleotides. In an aspect, the sequence of a target DNA template and a background DNA template differ by between 1 nucleotide and 25 nucleotides. In an aspect, the sequence of a target DNA template and a background DNA template differ by between 1 nucleotide and 21 nucleotides. In an aspect, the sequence of a target DNA template and a background DNA template differ by between 1 nucleotide and 20 nucleotides. In an aspect, the sequence of a target DNA template and a background DNA template differ by between 1 nucleotide and 15 nucleotides. In an aspect, the sequence of a target DNA template and a background DNA template differ by between 1 nucleotide and 10 nucleotides. In an aspect, the sequence of a target DNA template and a background DNA template differ by between 1 nucleotide and 5 nucleotides.

[0147] In an aspect, a forward primer comprises a sequence at least 80% identical or complementary to a target DNA template. In an aspect, a forward primer comprises a sequence at least 85% identical or complementary to a target DNA template. In an aspect, a forward primer comprises a sequence at least 90% identical or complementary to a target DNA template. In an aspect, a forward primer comprises a sequence at least 95% identical or complementary to a target DNA template. In an aspect, a forward primer comprises a sequence at least 99% identical or complementary to a target DNA template. In an aspect, a forward primer comprises a sequence 100% identical or complementary to a target DNA template.

[0148] As used herein, a “forward blocker” refers to an oligonucleotide that is designed to selectively bind to background DNA templates bearing a wildtype sequence. Forward primers cannot bind to the background DNA due to an overlap in binding region. See Figures 13 and 14. On a target DNA template 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.

[0149] In an aspect, a forward blocker comprises a sequence at least 80% identical or complementary to a background DNA template. In an aspect, a forward blocker comprises a sequence at least 85% identical or complementary to a background DNA template. In an aspect, a forward blocker comprises a sequence at least 90% identical or complementary to a background DNA template. In an aspect, a forward blocker comprises a sequence at least 95% identical or complementary to a background DNA template. In an aspect, a forward blocker comprises a sequence at least 99% identical or complementary to a background DNA template. In an aspect, a forward blocker comprises a sequence 100% identical or complementary to a background DNA template.

[0150] In an aspect, a forward primer comprises a sequence that overlaps with a sequence of a forward blocker. 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 50 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 40 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 30 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 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.

[0151] In an aspect, a sequence that overlaps between a forward primer and a forward blocker is positioned on the 3' half of the forward primer sequence.

[0152] In an aspect, a forward primer comprises a sequence that is not 100% identical or complementary to a background DNA template. In an aspect, a forward primer comprises a sequence that is 100% complementary to both a target DNA template and a background DNA template.

[0153] In an aspect, a target DNA amplicon comprises a sequence positioned on a target DNA template between a forward primer binding site and an Occlusion Primer binding site.

[0154] The following non-limiting embodiments are envisioned:

1. A system comprising:

(a) a first Occlusion Primer, wherein the first Occlusion Primer comprises, in 5' to 3' order, a first probe attachment sequence and a first template binding sequence; and

(b) a first Occlusion Probe, wherein the first Occlusion Probe comprises, in 5' to 3' order, a first primer attachment sequence and a first hairpin sequence.

2. The system of embodiment 1, wherein the first primer attachment sequence is at least 50% complementary to the first probe attachment sequence.

3. The system of embodiment 1, wherein the first primer attachment sequence is between 50% and 100% complementary to the first probe attachment sequence.

4. The system of embodiment 1, wherein the first primer attachment sequence is between 75% complementary and 95% complementary to the first probe attachment sequence.

5. The system of any one of embodiments 1-4, wherein the system further comprises at least one template nucleic acid molecule.

6. The system of embodiment 5, wherein the at least one template nucleic acid molecule is a DNA molecule.

7. The system of embodiment 5, wherein the at least one template nucleic acid molecule is a genomic DNA molecule.

8. The system of embodiment 5, wherein the at least one template nucleic acid molecule is an organellar DNA molecule.

9. The system of embodiment 5, wherein the at least one template nucleic acid molecule is a cDNA molecule.

10. The system of any one of embodiments 5-9, wherein the at least one template nucleic acid molecule is a eukaryotic nucleic acid molecule.

11. The system of any one of embodiments 5-7 or 9, wherein the at least one template nucleic acid molecule is a prokaryotic nucleic acid molecule.

12. The system of any one of embodiments 5, 6, or 9, wherein the at least one template nucleic acid molecule is a virus nucleic acid molecule.

13. The system of any one of embodiments 5, 6, or 9, wherein the at least one template nucleic acid molecule is a viroid nucleic acid molecule. 14. The system of embodiment 10, wherein the 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.

15. The system of embodiment 14, wherein the animal nucleic acid molecule is a human nucleic acid molecule.

16. The system of embodiment 11, wherein the prokaryotic nucleic acid molecule is selected from the group consisting of a bacteria nucleic acid molecule and an archaea nucleic acid molecule.

17. The system of embodiment 12, wherein the 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 nucleic acid molecule, a Geminiviridae nucleic acid molecule, a Flaviviridae nucleic acid molecule, a Filoviridae nucleic acid molecule, a Paramyxoviridae nucleic acid molecule, and a Pneumoviridae nucleic acid molecule.

18. The system of embodiment 12, wherein the 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.

19. The system of embodiment 13, wherein the viroid nucleic acid molecule is selected from the group consisting of a Pospiviroidae nucleic acid molecule, and a Avsunviroidae nucleic acid molecule.

20. The system of embodiment 5, wherein the at least one template nucleic acid molecule is an RNA molecule.

21. The system of any one of embodiments 5-20, wherein the at least one template nucleic acid molecule comprises an initiation subsequence.

22. The system of embodiment 21, wherein the initiation subsequence comprises a length of between 7 nucleotides and 100 nucleotides.

23. The system of embodiment 21, wherein the initiation subsequence comprises a length of at least 7 nucleotides. 24. The system of any one of embodiments 1-23, wherein the first probe attachment sequence comprises a length of between 6 nucleotides and 100 nucleotides.

25. The system of any one of embodiments 1-23, where in the first probe attachment sequence comprises a length of at least 6 nucleotides.

26. The system of any one of embodiments 1-25, wherein the first probe attachment sequence comprises a sequence selected from the group consisting of SEQ ID NOs: 185-195.

27. The system of any one of embodiments 1-26, wherein the first template binding sequence comprises a length of between 6 nucleotides and 50 nucleotides.

28. The system of any one of embodiments 1-26, wherein the first template binding sequence comprises a length of at least 6 nucleotides.

29. The system of embodiment 21, wherein the first template binding sequence is at least 70% complementary to the first initiation subsequence.

30. The system of any one of embodiments 1-29, wherein the first primer attachment sequence comprises a length of between 6 nucleotides and 100 nucleotides.

31. The system of any one of embodiments 1-29, wherein the first primer attachment sequence comprises a length of at least 6 nucleotides.

32. The system of any one of embodiments 1-31, wherein a sequence alignment of the first primer attachment sequence and the first probe attachment sequence comprises at least one nucleotide mismatch.

33. The system of any one of embodiments 1-32, wherein the first hairpin sequence comprises a first subsequence, a loop sequence, and a second subsequence, wherein the second subsequence is at least 80% complementary to the first subsequence.

34. The system of embodiment 33, wherein the first subsequence comprises between 2 nucleotides and 15 nucleotides.

35. The system of embodiment 33, wherein the first subsequence comprises at least 2 nucleotides.

36. The system of any one of embodiments 33-35, wherein the second subsequence comprises between 2 nucleotides and 15 nucleotides.

37. The system of any one of embodiments 33-35, wherein the second subsequence comprises at least 2 nucleotides.

38. The system of any one of embodiments 33-37, wherein the loop sequence comprises between 3 nucleotides and 10 nucleotides.

39. The system of any one of embodiments 33-37, wherein the loop sequence comprises at least 3 nucleotides. 40. The system of any one of embodiments 1-39, wherein the first hairpin sequence comprises a sequence at least 85% identical or complementary to a sequence selected from the group consisting of SEQ ID NOs: 183 and 184.

41. The system of any one of embodiments 1-40, wherein the system further comprises at least one DNA polymerase.

42. The system of any one of embodiments 1-41, wherein the system further comprises at least one buffer.

43. The system of any one of embodiments 1-42, wherein the system further comprises:

(c) a second Occlusion Primer, wherein the second Occlusion Primer comprises, in 5' to 3' order, a second probe attachment sequence and a second template binding sequence; and

(d) a second Occlusion Probe, wherein the second Occlusion Probe comprises, in 5' to 3' order, a second primer attachment sequence and a second hairpin sequence.

44. The system of embodiment 43, wherein the system further comprises:

(e) a third Occlusion Primer, wherein the third Occlusion Primer comprises, in 5' to 3' order, a third probe attachment sequence and a third template binding sequence; and

(1) a third Occlusion Probe, wherein the third Occlusion Probe comprises, in 5' to 3' order, a third primer attachment sequence and a third hairpin sequence.

45. The system of embodiment 44, wherein the system further comprises:

(g) a fourth Occlusion Primer, wherein the fourth Occlusion Primer comprises, in 5' to 3' order, a fourth probe attachment sequence and a third template binding sequence; and

(h) a fourth Occlusion Probe, wherein the fourth Occlusion Probe comprises, in 5' to 3' order, a fourth primer attachment sequence and a fourth hairpin sequence.

46. The system of any one of embodiments 1-45, wherein the first Occlusion Primer comprises at least one fluor ophore.

47. The system of embodiment 46, wherein the at least one fluorophore is positioned at, or near, the 5' end of the first Occlusion Primer.

48. The system of embodiment 46, wherein the at least one fluorophore is positioned at, or near, the 3' end of the first Occlusion Primer.

49. The system of any one of embodiments 1-48, wherein the first Occlusion Primer comprises at least one quencher.

50. The system of embodiment 49, wherein the at least one quencher is positioned at, or near, the 5' end of the first Occlusion Primer. 51. The system of embodiment 49, wherein the at least one quencher is positioned at, or near, the 3' end of the first Occlusion Primer.

52. The system of any one of embodiments 1-51, wherein the first Occlusion Probe comprises at least one fluor ophore.

53. The system of embodiment 52, wherein the at least one fluorophore is positioned at, or near, the 5' end of the first Occlusion Probe.

54. The system of embodiment 52, wherein the at least one fluorophore is positioned at, or near, the 3' end of the first Occlusion Probe.

55. The system of any one of embodiments 1-54, wherein the first Occlusion Probe comprises at least one quencher.

56. The system of embodiment 55, wherein the at least one quencher is positioned at, or near, the 5' end of the first Occlusion Probe.

57. The system of embodiment 55, wherein the at least one quencher is positioned at, or near, the 3' end of the first Occlusion Probe.

58. The system of any one of embodiments 46-48 or 52-54, wherein the at least one 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.

59. The system of any one of embodiments 46-48 or 52-54, wherein the at least one fluorophore is selected from the group consisting of Cy3™, FAM, VIC, HEX, Cy5™, ROX, and Quasar 705.

60. The system of any one of embodiments 46-48 or 52-54, wherein the at least one fluorophore is an organic dye.

61. The system of any one of embodiments 49-51 or 55-57, wherein the at least one quencher is selected from the group consisting of MGB, Black Hole Quencher, Iowa Black® RQ, and Iowa Black® FQ.

62. The system of embodiment 41, wherein the at least one DNA polymerase lacks 5' to 3' exonuclease activity.

63. The system of embodiment 41, wherein the at least one DNA polymerase comprises 3' to 5' exonuclease activity.

64. The system of embodiment 63, wherein the at least one DNA polymerase further comprises kinetic proofreading.

65. The system of embodiment 41, wherein the at least one 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, Accuris™ DNA polymerase, PowerUp™ DNA Polymerase, and Taq DNA polymerase.

66. The system of any one of embodiments 1-65, wherein the first Occlusion Probe comprises DNA.

67. The system of any one of embodiments 1-65, wherein the first Occlusion Probe comprises RNA.

68. The system of any one of embodiments 1-67, wherein the first Occlusion Primer comprises DNA.

69. The system of any one of embodiments 1-67, wherein the first Occlusion Primer comprises RNA.

70. The system of any one of embodiments 1-69, wherein the first Occlusion Primer comprises at least one non-natural nucleoside.

71. The system of embodiment 70, wherein the at least one non-natural nucleoside 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.

72. The system of any one of embodiments 1-71, wherein the first Occlusion Primer comprises at least one backbone modification.

73. The system of embodiment 72, wherein the at least one 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.

74. A kit comprising:

(a) a first Occlusion Primer, wherein the first Occlusion Primer comprises, in 5' to 3' order, a first probe attachment sequence and a first template binding sequence;

(b) a first Occlusion Probe, wherein the first Occlusion Probe comprises, in 5' to 3' order, a first primer attachment sequence and a first hairpin sequence; and

(c) at least one DNA polymerase.

75. The kit of embodiment 74, wherein the kit further comprises at least one reagent.

76. The kit of embodiment 74 or 75, wherein the kit further comprises at least one buffer.

77. The kit of any one of embodiments 74-76, wherein the first Occlusion Primer is suspended in a liquid.

78. The kit of any one of embodiments 74-73, wherein the first Occlusion Primer is lyophilized. The kit of any one of embodiments 74-78, wherein the first Occlusion Probe is suspended in a liquid. The kit of any one of embodiments 74-78, wherein the first Occlusion Probe is lyophilized. The kit of any one of embodiments 74-80, wherein the at least one DNA polymerase is suspended in a liquid. The kit of any one of embodiments 74-80, wherein the at least one DNA polymerase is lyophilized. The kit of embodiment 75, wherein the at least one reagent comprises a component selected from the group consisting of magnesium, at least one dNTP, phosphatase, betaine, dimethyl sulfoxide (DMSO), and tetramethylammonium chloride (TMAC). The kit of claim 74, wherein the kit further comprises a second primer. A method for amplifying a template nucleic acid molecule, the method comprising:

(a) mixing a sample comprising the template nucleic acid molecule with

(i) at least one DNA polymerase;

(ii) a first Occlusion Primer, wherein the first Occlusion Primer comprises, in 5' to 3' order, a first probe attachment sequence and a first template binding sequence;

(iii) a first Occlusion Probe, wherein the first Occlusion Probe comprises, in 5' to 3' order, a first primer attachment sequence and a first hairpin sequencer; and

(iv) a second primer; to generate a mixture; and

(b) subjecting the mixture to at least one cycle of thermal cycling, wherein the template nucleic acid molecule is amplified to produce at least one amplicon. The method of embodiment 85, wherein the at least one 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. The method of embodiment 85 or 86, wherein step (b) comprises at least five cycles of thermal cycling, and wherein each 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 method of any one of embodiments 85-87, wherein the at least one DNA polymerase is a thermostable DNA polymerase. 89. The method of any one of embodiments 85-88, wherein the first template binding sequence is at least 70% complementary to the template nucleic acid molecule.

90. The method of any one of embodiments 85-89, wherein the second primer comprises a length of between 6 nucleotides and 70 nucleotides.

91. The method of any one of embodiments 85-89, wherein the second primer comprises a length of at least 6 nucleotides.

92. The method of any one of embodiments 85-91, wherein the second primer comprises a sequence at least 80% identical or complementary to the template nucleic acid molecule.

93. The method of any one of embodiments 85-92, wherein the method further comprises a second Occlusion Primer, wherein the second Occlusion Primer comprises, in 5' to 3' order, a second probe attachment sequence and a second template binding sequence.

94. The method of embodiment 93, wherein the method further comprises a third Occlusion Primer, wherein the third Occlusion Primer comprises, in 5 ' to 3 ' order, a third probe attachment sequence and a third template binding sequence.

95. The method of embodiment 94, wherein the method further comprises a fourth Occlusion Primer, wherein the fourth Occlusion Primer comprises, in 5' to 3' order, a fourth probe attachment sequence and a fourth template binding sequence.

96. The method of any one of embodiments 85 or 93-95, wherein the first Occlusion Primer, the second Occlusion Primer, the third Occlusion Primer, or the fourth Occlusion Primer comprises at least one fluorophore.

97. The method of any one of embodiments 85 or 93-95, wherein the first Occlusion Primer, the second Occlusion Primer, the third Occlusion Primer, or the fourth Occlusion Primer comprises at least one quencher.

98. The method of embodiment 85, wherein the method further comprises a second Occlusion Probe, wherein the second Occlusion Probe comprises, in 5' to 3' order, a second primer attachment sequence and a second hairpin sequencer.

99. The method of embodiment 98, wherein the method further comprises a third Occlusion Probe, wherein the second Occlusion Probe comprises, in 5' to 3' order, a third primer attachment sequence and a third hairpin sequencer.

100. The method of embodiment 99, wherein the method further comprises a fourth Occlusion Probe, wherein the fourth Occlusion Probe comprises, in 5' to 3' order, a fourth primer attachment sequence and a fourth hairpin sequencer. 101. The method of any one of embodiments 85 or 98-100, wherein the first Occlusion Probe, the second Occlusion Probe, the third Occlusion Probe, or the fourth Occlusion Probe comprises at least one fluorophore.

102. The method of any one of embodiments 85 or 98-100, wherein the first Occlusion Probe, the second Occlusion Probe, the third Occlusion Probe, or the fourth Occlusion Probe comprises at least one quencher.

103. The method of any one of embodiments 93-95, wherein at least two of the first probe attachment sequence, the second probe attachment sequence, the third probe attachment sequence, and the fourth probe attachment sequence are identical sequences.

104. The method of embodiment 96, wherein at least two of the first Occlusion Primer, the second Occlusion Primer, the third Occlusion Primer, and the fourth Occlusion Primer comprise the same at least one fluorophore.

105. The method of embodiment 97, wherein at least two of the first Occlusion Primer, the second Occlusion Primer, the third Occlusion Primer, and the fourth Occlusion Primer comprise the same at least one quencher.

106. The method of embodiment 101, wherein at least two of the first Occlusion Probe, the second Occlusion Probe, the third Occlusion Probe, and the fourth Occlusion Probe comprise the same at least one fluorophore.

107. The method of embodiment 102, wherein at least two of the first Occlusion Probe, the second Occlusion Probe, the third Occlusion Probe, and the fourth Occlusion Probe comprise the same at least one quencher.

108. The method of any one of embodiments 85-92, wherein the first Occlusion Primer and the template nucleic acid molecule exhibit a standard free energy of hybridization AG° between -7 kcal/mol and -20 kcal/mol during step (b) at a temperature of 60°C and a salinity concentration of 0.2 M sodium.

109. The method of any one of embodiments 85-92 or 108, wherein the second primer and the template nucleic acid molecule exhibit a standard free energy of hybridization AG° between -7 kcal/mol and -20 kcal/mol during step (b) at a temperature of 60°C and a salinity concentration of 0.2 M sodium.

110. The method of any one of embodiments 85-92, 108, or 109, wherein the first Occlusion Primer and the first Occlusion Probe exhibit a standard free energy of hybridization AG° between -8 kcal/mol and -100 kcal/mol during step (b) at a temperature of 60°C and a salinity concentration of 0.2 M sodium. 111. The method of any one of embodiments 85-110, wherein the amplicon comprises a length of between 50 nucleotides and 5000 nucleotides.

112. The method of any one of embodiments 85-110, wherein the amplicon comprises a length of at least 30 nucleotides.

113. The method of any one of embodiments 85-112, wherein the concentration of the first Occlusion Primer in the mixture is between 100 pM and 5 pM.

114. The method of any one of embodiments 85-113, wherein the concentration of the first Occlusion Probe in the mixture is between 100 pM and 5 pM.

115. The method of any one of embodiments 85-114, wherein the concentration of the second primer in the mixture is between 100 pM and 5 pM.

116. The method of any one of embodiments 85-115, wherein the stoichiometric ratio of the first Occlusion Probe to the first Occlusion Primer is between 0.5 and 100.

117. The method of any one of embodiments 85-116, wherein the mixture further comprises at least one intercalating dye.

118. The method of embodiment 117, wherein the at least one intercalating dye is selected from the group consisting of SYBR® Green, EvaGreen®, and SYTO™.

119. The method of any one of embodiments 85-118, wherein a concentration of the at least one amplicon is determined by calculating a cycle threshold (Ct) value.

120. The method of any one of embodiments 85-119, wherein the method further comprises purifying the at least one amplicon.

121. The method of any one of embodiments 85-120, wherein the method further comprises sequencing the at least one amplicon.

122. The method of embodiment 120, wherein the method further comprises reamplification using fluorescent dideoxynucleotide triphosphates (ddNTPs).

123. The method of embodiment 121, wherein the sequencing is Sanger sequencing.

124. The method of any one of embodiments 85-123, wherein the template nucleic acid molecule is a DNA molecule.

125. A method for multiplex amplification of a plurality of nucleic acid molecules, the method comprising:

(a) obtaining a mixture comprising

(i) a plurality of template nucleic acid molecules;

(ii) at least one DNA polymerase; (iii) a first Occlusion Primer, wherein the first Occlusion Primer comprises, in 5' to 3' order, a first probe attachment sequence and a first template binding sequence;

(iv) a first Occlusion Probe, wherein the first Occlusion Probe comprises, in 5' to 3' order, a first primer attachment sequence and a first hairpin sequence;

(v) a second Occlusion Primer, wherein the second Occlusion Primer comprises, in 5' to 3' order, a second probe attachment sequence and a second template binding sequence; and

(vi) a second Occlusion Probe, wherein the second Occlusion Probe comprises, in 5' to 3' order, a second primer attachment sequence and a second hairpin sequencer; to generate the mixture; and

(b) subjecting the mixture to at least one cycle of thermal cycling, wherein the plurality of template nucleic acid molecules are amplified to produce a plurality of amplicons.

126. A method for multiplex amplification of a plurality of nucleic acid molecules, the method comprising:

(a) obtaining a mixture comprising

(i) a plurality of template nucleic acid molecules;

(ii) at least one DNA polymerase;

(iii) a first Occlusion Primer, where the first Occlusion Primer comprises, in 5' to 3' order, a first probe attachment sequence and a first template binding sequence;

(iv) a first Occlusion Probe, where the first Occlusion Probe comprises, in 5' to 3' order, a first primer attachment sequence and a first hairpin sequence;

(v) a first primer, wherein the first primer produces an amplicon in conjunction with the first Occlusion Primer;

(vi) a second Occlusion Primer, where the second Occlusion Primer comprises, in 5' to 3' order, a second probe attachment sequence and a second template binding sequence;

(vii) a second Occlusion Probe, where the second Occlusion Probe comprises, in 5' to 3' order, a second primer attachment sequence and a second hairpin sequencer; to generate the mixture; and

(viii) a second primer, wherein the second primer produces an amplicon in conjunction with the second Occlusion Primer; and (b) subjecting the mixture to at least one cycle of thermal cycling, where the plurality of template nucleic acid molecules are amplified to produce a plurality of amplicons.

127. The method of embodiment 125 or 126, wherein the at least one 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.

128. The method of embodiment 125 or 126, wherein the at least one cycle of thermal cycling comprises between one cycle and five cycles of thermal cycling, wherein each cycle 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.

129. The method of any one of embodiments 125-128, wherein the first probe attachment sequence and the second probe attachment sequence are identical sequences.

130. The method of any one of embodiments 125-128, wherein the first probe attachment sequence and the second probe attachment sequence are not identical sequences.

131. The method of any one of embodiments 125-130, wherein the first probe attachment sequence comprises a first high-throughput sequencing adapter sequence.

132. The method of embodiment 131, wherein the second probe attachment sequence comprises a second high-throughput sequencing adapter sequence.

133. The method of embodiment 132, wherein the first high-throughput sequencing adapter sequence and the second high-throughput sequencing adapter sequence are not identical.

134. The method of any one of embodiments 125-133, wherein the method further comprises ligating at least one sequence index to the plurality of amplicons.

135. The method of any one of embodiments 125-134, wherein the method further comprises subjecting the plurality of amplicons to high-throughput sequencing.

136. The method of embodiment 135, wherein high-throughput sequencing comprises the use of a sequencing-by-synthesis (SBS) flow cell

137. The method of embodiment 136, wherein the SBS flow cell is an Illumina SBS flow cell or a Pacific Biosciences SBS flow cell.

138. The method of embodiment 135, wherein the high-throughput sequencing is performed via electrical current measurements in conjunction with an Oxford nanopore.

139. A method for selective amplification of at least one target DNA template, the method comprising:

(a) obtaining a mixture comprising (i) at least one target DNA template;

(ii) at least one background DNA template;

(iii) at least one DNA polymerase;

(iv) a forward primer, wherein the forward primer comprises a sequence that is at least 80% complementary to a sequence of the at least one target DNA template;

(v) a forward blocker, wherein the forward blocker comprises a sequence that is at least 80% complementary to a sequence of the at least one background DNA template;

(vi) a first Occlusion Primer, wherein the first Occlusion Primer comprises, in 5' to 3' order, a first probe attachment sequence and a first template binding sequence; and

(vii) a first Occlusion Probe, wherein the first Occlusion Probe comprises, in 5' to 3' order, a first primer attachment sequence and a first hairpin sequencer; to produce the mixture; and

(b) subjecting the mixture to at least five cycles of thermal cycling, wherein the at least one target DNA template is amplified to produce at least one target DNA amplicon.

140. The method of embodiment 139, wherein each of the at least five cycles of the 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.

141. The method of embodiment 139 or 140, wherein the sequence of the at least one target DNA template and the at least one background DNA template differ by between 1 nucleotide and 21 nucleotides.

142. The method of any one of embodiments 139-141, wherein the forward primer comprises DNA.

143. The method of any one of embodiments 139-142, wherein the forward blocker comprises DNA.

144. The method of any one of embodiments 139-143, wherein the at forward primer, the forward blocker, or both, comprises one or more non-natural nucleosides.

145. The method of embodiment 144, wherein the one or more non-natural nucleosides are 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. 146. The method of any one of embodiments 139-145, wherein the at least one forward primer, the at least one forward blocker, or both, comprises at least one non-natural backbone modification.

147. The method of embodiment 146, wherein the at least one non-natural 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.

148. The method of any one of embodiments 139-147, wherein the forward primer comprises sequence that overlaps with a sequence of the forward blocker, wherein the overlap sequence is identical to between 3 nucleotides and 40 nucleotides of the forward blocker.

149. The method of embodiment 148, wherein the overlap sequence is positioned on the 3' half of the forward primer.

150. The method of any one of embodiments 139-149, wherein the forward primer comprises a sequence that is 100% complementary to the at least one target DNA template.

151. The method of any one of embodiments 139-149, wherein the forward primer comprises a sequence that is not 100% complementary to the at least one background DNA template.

152. The method of any one of embodiments 139-149, wherein the forward primer comprises a sequence that is 100% complementary to both the target DNA template and the background DNA template.

153. The method of any one of embodiments 139-152, wherein the at least one target DNA amplicon comprises a sequence positioned on the target DNA template between a binding site of the forward primer site and a binding site of the first Occlusion Primer.

[0155] Having now generally described the disclosure, the same will be more readily understood through reference to the following examples that are provided by way of illustration, and are not intended to be limiting of the present disclosure, unless specified.

EXAMPLES

Example 1. Design of Occlusion Primer and Occlusion Probes

[0156] An Occlusion Primer (SEQ ID NO: 3) is designed such that the 3' end of the Occlusion Primer is 100% complementary to an initiation subsequence within a specific human genomic DNA sequence (e.g., template binding sequence; SEQ ID NO: 2). The Occlusion Primer also comprises an artificially designed probe attachment sequence (SEQ ID NO: 196). The 5' end of the Occlusion Primer also comprises a Cy5™ molecule (e.g, fluorophore).

[0157] The Occlusion Primer has a corresponding first Occlusion Probe (SEQ ID NO: 9; see Figure 4A) that comprises a sequence (e.g, primer attachment sequence; SEQ ID NO: 197) on its 5' end that is the reverse complement of the Occlusion Primer’s probe attachment sequence. A hairpin sequence (SEQ ID NO: 183) is added to the 3' side of the primer attachment sequence, and an Iowa Black® RQ molecule (e.g., quencher) is added to the 3' end of the Occlusion Probe.

[0158] A second Occlusion Probe (SEQ ID NO: 10; see Figure 4B) is also generated. The second Occlusion Probe comprises the same primer attachment sequence and quencher as the first Occlusion Probe, but the hairpin sequence (SEQ ID NO: 184) has been altered as compared to the first Occlusion Probe.

[0159] As shown in Figures 4A and 4B, the primer attachment sequence of the Occlusion Probes and the probe attachment sequence of the Occlusion Primers creates a double-stranded nucleic acid molecule, which brings the Cy5™ molecules and Iowa Black ® RQ molecules into close proximity. When an Occlusion Primer is bound to an Occlusion Probe, the Iowa Black® RQ molecule will quench the Cy5™ fluorophore, and the pair has a low state of fluorescence. During PCR amplification, upon synthesis of a new strand that displace an Occlusion Probe from its complementary Occlusion Primer, the quenching effect is lost and fluorescence is emitted. See Figure 5.

Example 2. Testing of Occlusion Primer and First Occlusion Probe

[0160] In order to test the ability of the Occlusion Primer generated in Example 1 to function in a polymerase chain reaction, a second primer (SEQ ID NO: 1) is generated that anneals to the opposite strand of the human genomic DNA sequence. Use of the Occlusion Primer together with the second primer is intended to amplify a target region of the human genomic DNA sequence.

[0161] Two separate mixtures are prepared for use in PCR. The first mixture comprises 15 ng of human genomic DNA; 400 nM of the Occlusion Primer from Example 1; 400 nM of the first Occlusion Probe from Example 1; 400 nM of the second primer; and 0.3 units/reaction of Phusion® High-fidelity DNA Polymerase. [0162] The second mixture comprises 0 ng of human genomic DNA; 400 nM of the Occlusion Primer from Example 1; 400 nM of the first Occlusion Probe from Example 1; 400 nM of the second primer; and 0.3 units/reaction of Phusion® High-fidelity DNA Polymerase.

[0163] As shown in Figure 4C, the first mixture produces an increase in measured fluorescence, while the second mixture produces no measurable fluorescence. Three technical replicates are tested for each mixture.

Example 3. Testing of Occlusion Primer and Second Occlusion Probe

[0164] Two separate mixtures are prepared. The first mixture comprises 15 ng of human genomic DNA; 400 nM of the Occlusion Primer from Example 1; 400 nM of the second Occlusion Probe from Example 1; 400 nM of the second primer from Example 2; and 0.3 units/reaction of Phusion® High-fidelity DNA Polymerase.

[0165] The second mixture comprises 15 ng of human genomic DNA; 400 nM of a first primer (SEQ ID NO: 2); 400 nM of the second primer from Example 2; and 0.3 units/reaction of Phusion® High-fidelity DNA Polymerase.

[0166] As shown in Figure 4D, the first mixture produces an increase in measured fluorescence during PCR, while the second mixture produces no measurable fluorescence. Three technical replicates are tested for each mixture.

Example 4. Comparison of Occlusion Primer and Probe to TaqMan™ Probe

[0167] The efficacy of an Occlusion Primer and the first Occlusion Probe from Example 1 are compared to a TaqMan™ probe when using Phusion® High-fidelity DNA Polymerase.

[0168] TaqMan™ probes comprise a fluorophore and a quencher on opposite ends of an oligonucleotide that binds a template DNA molecule between a forward primer and a reverse primer. The fluorophore and quencher are in close enough proximity that the TaqMan™ probe does not emit any fluorescence until a DNA polymerase extends one of the primers, and the 5' to 3' exonuclease activity of the DNA polymerase degrades the TaqMan™ probe. This degradation releases the fluorophore from the quenching effects of the quencher, and allows fluorescence to be detected.

[0169] Two separate mixtures are prepared for use in PCR to compare Occlusion Primers and probes to TaqMan™ probes.

[0170] The first mixture comprises 400 nM of an Occlusion Primer (SEQ ID NO: 18); 400 nM of the first Occlusion Probe from Example 1; 400 nM of a forward primer (SEQ ID NO: 15); 15 ng of human genomic DNA; and 0.3 units/reaction of Phusion® High-fidelity DNA Polymerase.

[0171] The second mixture comprises 200 nM of a TaqMan™ probe (SEQ ID NO: 14) comprising a Cy5™ molecule on its 5' end and an Iowa Black® RQ molecule on its 3' end; 400 nM of the forward primer (SEQ ID NO: 15); 400 nM of a reverse primer (SEQ ID NO: 16); 15 ng of human genomic DNA; and 0.3 units/reaction of Phusion® High-fidelity DNA Polymerase. The forward and reverse primers are designed to amplify the same target region of the human genomic DNA sequence as the Occlusion Primer and the second primer.

[0172] As shown in Figure 6A, the use of TaqMan™ probe with Phusion® High-fidelity DNA

Polymerase produces a much slower increase in fluorescence and a much lower amount of fluorescence overall during PCR, as compared to the use of the Occlusion Probe and primer. Three technical replicates are tested for each mixture.

[0173] Without being limited by any theory, it is believed that the 3' to 5' exonuclease activity of Phusion® High-fidelity DNA Polymerase is incompatible with TaqMan™ probes.

Example 5. Testing additional primer/probe designs for use with Phusion® High-fidelity DNA Polymerase

[0174] Several additional fluorescent primer/probe designs are tested. Three technical replicates are tested for every mixture in this Example.

[0175] The first design utilizes a first forward primer (SEQ ID NO: 15), and two probes: probe D (SEQ ID NO: 17) and probe E (SEQ ID NO: 18), which are complementary to each other. Probe D comprises an Iowa Black® RQ quencher molecule on its 3' end, and probe E comprises a Cy5™ molecule on its 5’ end. Probe D also comprises a phosphorothioate backbone modification to its three 3'-most nucleotides. See Figure 6B. A mixture for PCR is prepared comprising 400 nM of the first forward primer; 400 nM of probe D; 400 nM of probe E; 15 ng of human genomic DNA; and 0.3 units/reaction of Phusion® High-fidelity DNA Polymerase. As shown in Figure 6C, this first primer/probe design produces almost no fluorescence even after over 50 thermal cycles.

[0176] The second design utilizes a second forward primer (SEQ ID NO: 1) and two probes: probe A (SEQ ID NO: 5) and probe B (SEQ ID NO: 4). Probe A comprises an Iowa Black® RQ quencher molecule on its 3' end. Probe B comprises a stem-loop structure (e.g., a hairpin) on its 5' end, with a Cy5™ molecule attached to the 5'-most end. Probe B also comprises a segment that is 100% complementary to probe A, followed by a template binding sequence on its 3' end. See Figure 6D. A mixture for PCR is prepared comprising 400 nM of the second forward primer; 400 nM of probe A; 400 nM of probe B; 15 ng of human genomic DNA; and 0.3 units/reaction of Phusion® High-fidelity DNA Polymerase. As shown in Figure 6E, this second primer/probe design produces a high baseline fluorescence.

[0177] The third design utilizes the second forward primer and two probes: probe A and probe C (SEQ ID NO: 3). Probe C is identical to probe B, except probe C lacks the hairpin of probe B, and thus the location of the Cy5™ molecule has moved to the 5' end of the segment that is 100% complementary to probe A. See Figure 6F. A mixture for PCR is prepared comprising 400 nM of the second forward primer; 400 nM of probe A; 400 nM of probe C; 15 ng of human genomic DNA; and 0.3 units/reaction of Phusion® High-fidelity DNA Polymerase. As shown in Figure 6G, this second primer/probe design produces a high baseline fluorescence.

[0178] The fourth design utilizes different scorpion primers. Sunrise primers combine a primer region with a hairpin probe region, where the sunrise probe region comprises both a fluorophore and a quencher. If a target nucleic acid molecule (e.g., template) is not present, the fluorophore is quenched by the quencher. However, when a target nucleic acid is present in PCR, the fluorophore and quencher are separated, allowing the fluorophore to fluoresce. Three sunrise primers are prepared: RP-SR-1 (SEQ ID NO: 6), RP-SR-2 (SEQ ID NO: 7), and RP- SR-3 (SEQ ID NO: 20). Each sunrise probe region comprises a Cy5™ molecule at the junction between the primer region and the hairpin probe region, and an Iowa Black® RQ quencher molecule at the primer’s 5' end. See Figure 6H. Three mixtures for PCR are prepared. The first mixture comprises 400 nM of the second forward primer; 400 nM of RP-SR1; 15 ng of human genomic DNA; and 0.3 units/reaction of Phusion® High-fidelity DNA Polymerase. The second mixture comprises 400 nM of the second forward primer; 400 nM of RP-SR2; 15 ng of human genomic DNA; and 0.3 units/reaction of Phusion® High-fidelity DNA Polymerase. The third mixture comprises 400 nM of a third forward primer (SEQ ID NO: 19); 400 nM of RP-SR3; 15 ng of human genomic DNA; and 0.3 units/reaction of Phusion® High-fidelity DNA Polymerase.

[0179] As shown in Figures 61 and 6J, all of the sunrise primers fail to produce ample or efficient amplification signals. Three technical replicates are tested for each mixture.

Example 6. Testing Occlusion Primers and Probes with Taq polymerase

[0180] The Occlusion Primer and probe from Example 1 are tested for efficacy using Taq polymerase in place of Phusion® High-fidelity DNA Polymerase. [0181] A first PCR mixture comprises 15 ng of human genomic DNA; 400 nM of the Occlusion Primer from Example 1; 400 nM of the first Occlusion Probe from Example 1; 400 nM of the second primer from Example 2; and Taq polymerase. A second PCR mixture comprises 15 ng of human genomic DNA; 400 nM of a first primer (SEQ ID NO: 1); 400 nM of the second primer from Example 2; and Taq polymerase.

[0182] As shown in Figure 7A, both the first and second mixtures generate increases in measured fluorescence when examining the subtracted FAM channel.

[0183] As shown in Figure 7B, the third mixture generates an increase in measured fluorescence during PCR, when examining the unsubtracted Cy5™ channel, while the fourth mixture does not produce any measurable fluorescence during PCR. Three technical replicates are tested for each mixture.

Example 7. Testing Occlusion Primers and Probes comprising mismatches

[0184] It was observed that long continuous stretches of double-stranded DNA can inhibit PCR. Without being limited by any scientific theory, this inhibition may be due to non-specific reversible binding on the DNA polymerase to double-stranded DNA. Therefore, the doublestranded DNA formed through the binding of the probe attachment sequence of an Occlusion Primer and the primer attachment sequence of an Occlusion Probe may result in a delay of observed threshold cycle (Ct) values.

[0185] In order to overcome this, Occlusion Primers and probes are designed to incorporate one or more mismatches between the probe attachment sequence and the primer attachment sequence. See Figure 8.

[0186] Two Occlusion Probes are designed to comprise one (Occlusion Probe Ml; SEQ ID NO: 11; see Figure 9A) or two (Occlusion Probe M2; SEQ ID NO: 12; see Figure 9B) mismatches within their primer attachment sequences as compared to the probe attachment sequence of an Occlusion Primer (SEQ ID NO: 196). The Occlusion Probes comprise an Iowa Black® RQ quencher on their 3' end, and the Occlusion Primer comprises a Cy5™ molecule on its 5' end.

[0187] Three separate PCR mixtures are prepared. The first mixture comprises 15 ng of human genomic DNA; 400 nM of the Occlusion Primer (SEQ ID NO: 18); 400 nM of a second primer (SEQ ID NO: 15); 400 nM of Occlusion Probe Ml; and 0.3 units/reaction of Phusion® High- fidelity DNA Polymerase. The second mixture comprises 15 ng of human genomic DNA; 400 nM of the Occlusion Primer; 400 nM of the second primer; 400 nM of Occlusion Probe M2; and 0.3 units/reaction of Phusion® High-fidelity DNA Polymerase. The third mixture comprises 15 ng of human genomic DNA; 400 nM of the Occlusion Primer; 400 nM of the second primer; 400 nM of an Occlusion Probe (SEQ ID NO: 9); and 0.3 units/reaction of Phusion® High-fidelity DNA Polymerase. In the third mixture, the Occlusion Probe’s primer attachment sequence has perfect complementarity to the Occlusion Primer’s probe attachment sequence.

[0188] Both the first mixture (see Figure 9C) and second mixture (see Figure 9D) generate increases in measured fluorescence during PCR. Also, as shown in Figure 9E, the use of one or two mismatches between the probe attachment sequence and the primer attachment sequence results in an earlier observed Ct value during PCR as compared to a probe attachment sequence and primer attachment sequence that have perfect complementarity. Therefore, mismatches are observed to reduce the Ct value of qPCR, implying increased PCR efficiency and decreased nonspecific inhibition of PCR by double-stranded DNA. Three technical replicates are tested for each mixture.

Example 8. Quencher and fluorophore molecule positions are interchangeable.

[0189] An experiment is performed to determine if the fluorophore and quencher molecules can be placed on either the Occlusion Probe or the Occlusion Primer. In Example 2, it is demonstrated that fluorescence levels accumulate during PCR when the Occlusion Primer comprises a Cy5™ molecule and the Occlusion Probe comprises an Iowa Black ® RQ molecule.

[0190] The Occlusion Primer from Examples 1 and 2 (SEQ ID NO: 8) is redesigned to comprise an Iowa Black® RQ molecule in place of the original Cy5™ molecule, and the first Occlusion Probe (SEQ ID NO: 13) is redesigned to comprise a Cy5™ molecule in place of the original Iowa Black® RQ molecule. See Figure 10A.

[0191] A PCR mixture is prepared comprising 15 ng of human genomic DNA; 400 nM of the Occlusion Primer with Iowa Black® RQ; 400 nM of the second primer from Example 2; 400 nM of the Occlusion Probe with Cy5™; and 0.3 units/reaction of Phusion® High-fidelity DNA Polymerase. Three technical replicates are tested for the mixture.

[0192] As shown in Figure 10B, fluorescence accumulates during PCR. Example 9. Occlusion Probe concentration.

[0193] The Occlusion Probe, Occlusion Primer, and second primer from Example 2 are used to test whether the concentration of the Occlusion Probe impacts PCR.

[0194] Two PCR mixtures are prepared. The first mixture comprises 15 ng of human genomic DNA, 400 nM of the Occlusion Primer; 800 nM of the Occlusion Probe; 400 nM of the second primer; and 0.3 units/reaction of Phusion® High-fidelity DNA Polymerase. The second mixture comprises 15 ng of human genomic DNA, 400 nM of the Occlusion Primer; 400 nM of the Occlusion Probe; 400 nM of the second primer; and 0.3 units/reaction of Phusion® High- fidelity DNA Polymerase. Three technical replicates are tested for each mixture.

[0195] As shown in Figure 11, normal accumulation of fluorescence during qPCR is detected when the Occlusion Probe concentration is doubled to 800 nM while the Occlusion Primer concentration remains at 400 nM.

Example 10. Single-stranded overhangs.

[0196] Occlusion Primers can be designed to comprise a single-stranded overhang on either its 3' end (see Figure 12A) or its 5' end (see Figure 12B) when in a complex with an Occlusion Probe.

[0197] One PCR mixture is prepared to test the functionality of an Occlusion Primer having a 5' single-stranded overhang when in a complex with an Occlusion Probe. See Figure 12C. The mixture comprises 50 ng of human genomic DNA, 400 nM of an Occlusion Primer (SEQ ID NO: 18; 400 nM of an Occlusion Probe (SEQ ID NO: 196); 400 nM of a second primer (SEQ ID NO: X); and 0.4 units/reaction of Phusion® High-fidelity DNA Polymerase. Three technical replicates are tested for the mixture.

[0198] As shown in Figure 12D, normal accumulation of fluorescence during qPCR is detected when using an Occlusion Primer having a single-stranded overhang when in a complex with an Occlusion Probe.

Example 11. Multiplex Occlusion Primers and Occlusion Probes for targeted high- throughput sequencing.

[0199] High-throughput sequencing generally require that DNA molecules to be sequenced within a sample have adapter sequences at their 5' and/or 3' ends. DNA molecules in a sample that do not have the required adapter sequences are not sequenced. One way to selectively append adapter sequences to DNA regions of interest is through multiplexed PCR using primers with 5' overhangs corresponding to the adapter sequence. Through the course of PCR, amplicons are generated with the adapter sequences at the 5' and 3' ends. Unfortunately, amplicons unintentionally formed from primer dimers will also bear the adapter sequences, and can comprise a significant fraction of the DNA molecules in a library with the adapter sequences. Similarly, amplicons unintentionally formed from nonspecific genomic amplification by the adapter primers will also have the adapters at the 5' and 3' ends. Both primer dimers and nonspecific amplification amplicons will reduce the “on-target” rate of high- throughput sequencing, reducing sensitivity to sequence variants and increasing effective sequencing cost.

[0200] The likelihood of primer dimer formation and nonspecific genomic amplification increase with the length and binding strength of the primer. For example, a 20 nucleotide long primer can result in nonspecific amplification if a non-cognate DNA target matches 18 nucleotides of the 20 nucleotides in the primer. When the primer is extended at the 5' end to include an adapter, there will be many more non-cognate DNA targets that match 18 nucleotides of the extended primer's 40 nucleotides. Thus, in commercial multiplex PCR methods for target enrichment in next generation sequencing (NGS), the protocols often include many amplicon size selection steps to remove primer dimers and nonspecific amplicons in order to improve NGS on-target rate. Even with these methods, the on-target rate for larger multiplex PCR panels can be low.

[0201] Eighty forward (e.g., first) Occlusion Primers (SEQ ID NOs: 21-100) are designed to comprise an Illumina adapter sequence (SEQ ID NO: 198) within the primer’s probe attachment sequence. Each Occlusion Primer comprises the same probe attachment sequence. A forward (e.g, first) Occlusion Probe (SEQ ID NO: 181) is designed to hybridize with the forward Occlusion Primers and create a double-stranded DNA region. Eighty reverse (e.g., second) Occlusion Primers (SEQ ID NOs: 101-180) also comprise the same probe attachment sequences (SEQ ID NO: 99), although the reverse Occlusion Primers do not have the same probe attachment sequence as the forward Occlusion Primers. A reverse (e.g, second) Occlusion Probe (SEQ ID NO: 182) is designed to hybridize with the reverse Occlusion Primers. Neither the Occlusion Probes nor the Occlusion Primers comprises either a fluorophore or a quencher. See Figure 13.

[0202] Because double-stranded DNA regions have much lower nonspecific binding to non- cognate DNA sequences than single-stranded DNA regions, the Occlusion Probe/Occlusion Primer system is expected to exhibit primer dimer and non-specific amplification rates more similar to standard PCR primers without 5' adapter sequences, improving the NGS on-target rates of multiplex PCR libraries.

[0203] An 80-amplicon NGS panel is generated with and without the use of Occlusion Primers (SEQ ID NOs: 21-180) and probes (SEQ ID NOs: 181 and 182), and both libraries use the same Occlusion Primers comprising 5’ Illumina adapter sequences. See Table X.

Table 2. Results of NGS with and without Occlusion Probes (13 thermal cycles per library).

[0204] The use of Occlusion Probes and Occlusion Primers improves the on-target rate and decreases the off-target rate as compared to using only Occlusion Primers.

Example 12. Use of Occlusion Primers and probes in qPCR assays for DNA sequence variants.

[0205] Occlusion Primers and Occlusion Probes can be used in conjunction with other PCR- based methods for selective amplification and/or detection of DNA sequence variants. Figure 14 depicts a schematic in which the Occlusion Primer serves as the reverse primer of a Blocker Displacement Amplification (BDA) reaction. In BDA, a wildtype-specific blocker oligonucleotide selectively binds to background DNA molecules bearing a wildtype sequence. The forward primer cannot bind to the background DNA due to the overlap in binding region. On a target DNA molecule bearing a variant sequence, the blocker is mismatched in its binding, and can be effectively displaced by the forward primer, resulting in effective PCR amplification.

[0206] Occlusion Primers and Occlusion Probes can also be used for allele-specific blocker PCR (asbPCR), in which the Occlusion Primer serves as the reverse primer. See Figure 15. Like BDA, asbPCR uses a blocker that is perfectly matched to a background DNA molecule with a wildtype sequence. Unlike BDA, the forward primer for asbPCR contains at its 3' end the nucleotide corresponding to suspected DNA sequence variant specific to the target DNA. Thus, asbPCR allows specific detection of a known DNA sequence variant, while BDA allows broad amplification of all DNA sequence variants within a region.

[0207] Without being limited by any scientific theory, in both BDA and asbPCR, the variant allele frequency limit of detection for a single nucleotide variant is suspected to be bottlenecked by the DNA polymerase's misincorporation error rate. To be compatible with TaqMan™ probes for fluorescence multiplexing, both BDA and asbPCR typically use Ta -based DNA polymerases with error rates of about 1 in 8,000 nucleotides. The use of occlusion Primers and occlusion Probes allows fluorescence multiplexing for BDA and asbPCR in conjunction with high-fidelity DNA polymerases such as Phusion®, which have error rates of less than 1 in 100,000 nucleotides. Thus, occlusion Primers and occlusion Probes can improve the sensitivity and limit of detection of BDA and asbPCR.

Example 13. PCR conditions.

[0208] In Examples 2 to 10, all PCR mixture volumes are approximately 15 pL. The Phusion® High-fidelity DNA Polymerase, and required reagents, is from New England BioLabs Inc. Human genomic DNA is NA18537 from Coriell Cell Repositories. Thermal cycling and fluorescence measurements are performed using a Bio-Rad CFX96 qPCR instrument.

[0209] The thermal cycle used for Phusion® DNA Polymerase experiments is: 98°C for 30 seconds; then 55 cycles of (98°C for 10 seconds; 60°C for 30 seconds; 72°C for 30 seconds). Fluorescence measurements are taken at 60°C.

[0210] In Example 6, PowerUp™ SYBR™ Green Master Mix (ThermoFisher Scientific) is used for Tag-based experiments. Human genomic DNA is NA18537 from Coriell Cell Repositories. Thermal cycling and fluorescence measurements are performed using a Bio-Rad CFX96 qPCR instrument.

[0211] The thermal cycle used for Tag-based experiments is: 95°C for 3 minutes; then 55 cycles of (95°C for 15 seconds; 60°C for 30 seconds). Fluorescence measurements are taken at 60°C.

Example 14. NGS library preparation and analysis.

[0212] Data provided in Examples 11 and 12 were collected using an Illumina MiSeq instrument and a MiSeq v3 single-end (150 cycle) kit. Each library utilized 25 ng input human genomic DNA in a 50 pL reaction mixture. [0213] The library preparation protocol, briefly, comprises:

(a) Perform 13 cycles (98 °C for 10 seconds; 60 °C for 5 minutes; 72 °C for 2 minutes) of PCR using Phusion® Hot Start Flex 2X Master Mix (New England Biolabs, Inc.), using the forward Occlusion Primer, reverse Occlusion Primer, forward Occlusion Probe and reverse Occlusion Probe oligonucleotide using the concentrations listed above for each in Table 2.

(b) PCR output is purified using a DNA Clean & Concentrator kit (Zymo Research).

(c) Option 1 : For libraries without Occlusion Probes, 15 cycles of index PCR is performed using a concentration of 500 nM index primers and Phusion® Hot Start Flex 2X Master Mix. Each PCR cycle comprises (98 °C for 10 seconds, 60 °C for 60 seconds, 72 °C for 30 seconds).

Option 2: For libraries with Occlusion Probes, 29 cycles of index PCR is performed using a concentration of 500 nM index primers and Phusion® Hot Start Flex 2X Master Mix. Each PCR cycle comprises (98 °C for 10 seconds, 60 °C for 60 seconds, 72 °C for 30 seconds).

(d) Index PCR output is purified using l. lx SPRI beads.

[0214] After obtaining Illumina sequences, the resulting FASTQ file is processed. The processing is briefly described here:

(a) Trim adapter sequences from each read.

(b) Count the number of insert reads that perfectly match the amplicon (e.g., on-target reads). Any degenerate nucleotides (e.g., an “N”) are considered mismatches and are not reported as on-target reads. The fraction of all reads in the libraries that can be counted as an on-target read for any locus is defined as the “on-target rate.” Any sequences that are not considered “on-target reads” are considered to be off-target reads.

Claims

CLAIMS A system comprising:

(b) a first Occlusion Probe, wherein the first Occlusion Probe comprises, in 5' to 3' order, a first primer attachment sequence and a first hairpin sequence. The system of claim 1, wherein the first primer attachment sequence is at least 50% complementary to the first probe attachment sequence. The system of claim 1, wherein the system further comprises at least one template nucleic acid molecule. The system of claim 3, wherein the at least one template nucleic acid molecule comprises an initiation subsequence. The system of claim 1, wherein the first hairpin sequence comprises a sequence at least 85% identical or complementary to a sequence selected from the group consisting of SEQ ID NOs: 183 and 184. The system of claim 1, wherein the system further comprises:

(d) a second Occlusion Probe, wherein the second Occlusion Probe comprises, in 5' to 3' order, a second primer attachment sequence and a second hairpin sequence. The system of claim 1, wherein the first Occlusion Primer comprises at least one fluorophore. The system of claim 1, wherein the first Occlusion Primer comprises at least one quencher. The system of claim 1, wherein the first Occlusion Probe comprises at least one fluorophore. The system of claim 1, wherein the first Occlusion Probe comprises at least one quencher. A kit comprising:

(c) at least one DNA polymerase. The kit of claim 10, wherein the kit further comprises (i) at least one reagent, (ii) at least one buffer, or (iii) at least one reagent and at least one buffer.

72 The kit of claim 10, wherein the at least one reagent comprises a component selected from the group consisting of magnesium, at least one dNTP, phosphatase, betaine, dimethyl sulfoxide (DMSO), and tetramethylammonium chloride (TMAC). A method for amplifying a template nucleic acid molecule, the method comprising:

(a) mixing a sample comprising the template nucleic acid molecule with

(i) at least one DNA polymerase;

(iv) a second primer; to generate a mixture; and

(b) subjecting the mixture to at least one cycle of thermal cycling, wherein the template nucleic acid molecule is amplified to produce at least one amplicon. The method of claim 14, wherein the method further comprises a second Occlusion Primer, wherein the second Occlusion Primer comprises, in 5' to 3' order, a second probe attachment sequence and a second template binding sequence. The method of claim 15, wherein the first Occlusion Primer or the second Occlusion Primer comprises at least one fluorophore. The method of claim 15, wherein the first Occlusion Primer or the second Occlusion Primer comprises at least one quencher. The method of claim 14, wherein the method further comprises a second Occlusion Probe, wherein the second Occlusion Probe comprises, in 5' to 3' order, a second primer attachment sequence and a second hairpin sequencer. The method of claim 18, wherein the first Occlusion Probe or the second Occlusion Probe comprises at least one fluorophore. The method of claim 18, wherein the first Occlusion Probe or the second Occlusion Prob comprises at least one quencher.

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