Compositions and methods for signal-amplified flap cleavage assays

Abstract

Methods and compositions for detection and measuring target nucleic acid molecules using a FEN-1 endonuclease to cleave a FEN-1 substrate padlock probe in the presence of the target nucleic acid, with ligation to form a circularized probe. The technology further pertains to detection of circularized probes using, e.g., rolling circle amplification.

Description

COMPOSITIONS AND METHODS FOR SIGNAL-AMPLIFIED FLAP CLEAVAGE ASSAYSThe present application claims priority to U.S. Provisional Application Serial No. 63 / 481,979, filed January 27, 2023, which is incorporated herein by reference.SEQUENCE LISTINGThe text of the computer readable sequence listing filed herewith, titled “41508- 601_SEQUENCE_LISTING”, created January 26, 2024, having a file size of 10,750 bytes, is hereby incorporated by reference in its entirety.FIELD OF THE INVENTIONThe invention relates to detection of nucleic acid molecules using FEN-1 substrate padlock probes that, after cleavage of the probe using a FEN-1 endonuclease, are ligatable to form a circle. The invention further pertains to detection of circularized probes using, e.g., rolling circle amplification.BACKGROUND OF THE INVENTIONDetection of target nucleic acids, e.g., for diagnostic purposes, typically requires a high degree of specificity, e.g., to distinguish between nucleic acids that may differ by a single nucleotide, and a high degree of sensitivity, e.g., to detect target nucleic acids that may be present in very low copy numbers. There is a need in the field for assays that combine specific recognition of target nucleic acids with signal amplification to produce sensitive reporting of the presence of the target nucleic acids.SUMMARY OF THE INVENTIONThe technology is in the field of nucleic acid detection and measuring using the specificity of FEN- 1 cleavage in the presence of a target nucleic acid, and signal amplification from a ligated probe, preferably a circularized probe, e.g., using rolling circle amplification. Embodiments of the technology include but are not limited to:1. A method, comprising: a) providing a padlock probe hybridized to a strand of target nucleic acid to form a FEN-1 cleavage substrate complex comprising a downstream duplex and an upstream duplex that define an invasive cleavage structure cleavable at a cleavage site in the padlockprobe on the 3' side of the first paired nucleotide of the downstream duplex by a strict FEN-1 endonuclease in a Mg++flap assay buffer, wherein the padlock probe comprises a 5' blocking group; b) cleaving the cleavage substrate complex with a strict FEN-1 endonuclease in conditions wherein cleavage of the invasive cleavage structure at the cleavage site produces: i) a cleaved substrate complex comprising a nick that is ligatable by a DNA ligase, and ii) a 5' cleavage product consisting of the 5' blocking group and the first paired nucleotide from the downstream duplex of the cleavage substrate complex; and c) treating the cleaved substrate complex with a ligase to form a circularized padlock probe.2. The method of embodiment 1, wherein in the cleavage substrate complex, the 5' blocking group is attached to the 5' position of the first paired nucleotide in the downstream duplex.3. The method of embodiment 1 or embodiment 2, wherein the 5' blocking group comprises a moiety selected from a hexane, a non-standard nucleotide, an abasic nucleotide, a fhiorophore, a flap nucleic acid sequence, a hairpin flap nucleic acid sequence, and an hydroxyl group.4. The method of embodiment 3, wherein the 5' blocking group is attached to the first paired nucleotide by a phosphodiester bond.5. The method of any one of embodiments 1-4, wherein the 5' blocking group comprises a non-standard nucleotide.6. The method of any one of embodiments 1-5, wherein the 5' blocking group does not comprise a standard nucleotide.7. The method of any one of embodiments 1-6, wherein the strict FEN-1 endonuclease is selected from the group consisting of Archaeoglobus fulgidus (Afu) FEN-1, Pyrococcus furiosus (Pfu) FEN-1, and Archaeoglobus veneficus (Ave) FEN-1, and CLEAVASE 2.0 nuclease.8. The method of any one of embodiments 1-7, further comprising detecting the circularized padlock probe in a nucleic acid detection assay.9. The method of embodiment 8, wherein the nucleic acid detection assay comprises rolling circle amplification.10. The method of any one of embodiments 1-9, wherein the target nucleic acid is extracted from a sample.11. The method of embodiment 10, wherein the target nucleic acid is treated with a methylation-specific reagent.12. The method of embodiment 11, wherein the methylation-specific reagent modifies a methylated nucleotide to produce a different nucleotide.13. The method of embodiment 12, wherein the methylation-specific reagent modifies a methylated cytosine to produce a dihydrouracil.14. The method of embodiment 11, wherein the methylation-specific reagent modifies an unmethylated nucleotide to produce a different nucleotide.15. The method of embodiment 14, wherein the methylation-specific reagent modifies an unmethylated cytosine to produce a uracil.16. The method of any one of embodiments 1-9, wherein the target nucleic acid is amplified nucleic acid.DEFINITIONSTo facilitate an understanding of the present technology, a number of terms and phrases are defined below. Additional definitions are set forth throughout the detailed description.Throughout the specification and claims, the following terms take the meanings explicitly associated herein, unless the context clearly dictates otherwise. The phrase “in one embodiment” as used herein does not necessarily refer to the same embodiment, though it may. Furthermore, the phrase “in another embodiment” as used herein does not necessarily refer to a different embodiment, although it may. Thus, as described below, various embodiments of the technology may be readily combined, without departing from the scope or spirit of the technology.In addition, as used herein, the term “or” is an inclusive “or” operator and is equivalent to the term “and / or” unless the context clearly dictates otherwise. The term “basedon” is not exclusive and allows for being based on additional factors not described, unless the context clearly dictates otherwise. In addition, throughout the specification, the meaning of “a”, “an”, and “the” include plural references. The meaning of “in” includes “in” and “on.”The transitional phrase “consisting essentially of’ as used in claims in the present application limits the scope of a claim to the specified materials or steps “and those that do not materially affect the basic and novel characteristic(s)” of the claimed invention, as discussed in In re Herz, 537 F.2d 549, 551-52, 190 USPQ 461, 463 (CCPA 1976). For example, a composition “consisting essentially of’ recited elements may contain an unrecited contaminant at a level such that, though present, the contaminant does not alter the function of the recited composition as compared to a pure composition, i.e., a composition “consisting of’ the recited components.The term “primer” refers to an oligonucleotide, whether occurring naturally as, e.g., a nucleic acid fragment from a restriction digest, or produced synthetically, that is capable of acting as a point of initiation of synthesis when placed under conditions in which synthesis of a primer extension product that is complementary to a nucleic acid template strand is induced, (e.g., in the presence of nucleotides and an inducing agent such as a DNA polymerase, and at a suitable temperature and pH). The primer is preferably single stranded for maximum efficiency in amplification, but may alternatively be double stranded. If double stranded, the primer is first treated to separate its strands before being used to prepare extension products. Preferably, the primer is an oligodeoxyribonucleotide. Generally, the primer is sufficiently long to prime the synthesis of extension products in the presence of the inducing agent. The exact lengths of the primers will depend on many factors, including temperature, source of primer, and the use of the method.As used herein, the term “nucleic acid detection assay” refers to any method of determining the nucleotide composition of a nucleic acid of interest. Nucleic acid detection assay include but are not limited to, DNA sequencing methods, probe hybridization methods, structure specific cleavage assays (e.g., the “INVADER” flap assay, or invasive cleavage assay, (Hologic, Inc.) described, e.g., in U.S. Patent Nos. 5,846,717, 5,985,557, 5,994,069, 6,001,567, 6,090,543, and 6,872,816; Lyamichev et al., Nat. Biotech., 17:292 (1999), Hall et al., PNAS, USA, 97:8272 (2000), and in combined PCR / invasive cleavage assays (Hologic, Inc., e.g., in U.S. Patent Publications 2006 / 0147955 and 2009 / 0253142), each of which is herein incorporated by reference in its entirety for all purposes); enzyme mismatch cleavage methods (e.g., Variagenics, U.S. Pat. Nos. 6,110,684, 5,958,692, 5,851,770, herein incorporated by reference in their entireties); polymerase chain reaction (PCR), describedabove; branched hybridization methods (e.g., Chiron, U.S. Pat. Nos. 5,849,481, 5,710,264, 5,124,246, and 5,624,802, herein incorporated by reference in their entireties); rolling circle replication (e.g., U.S. Pat. Nos. 6,210,884, 6,183,960 and 6,235,502, herein incorporated by reference in their entireties); the variation of rolling circle amplification called “RAM amplification” (see, e.g., US 5,942,391, incorporated herein by reference in its entirety; Fan Li, et al., J Clin Microbiol. 2005 Dec; 43(12): 6086-6090); NASBA (e.g., U.S. Pat. No. 5,409,818, herein incorporated by reference in its entirety); molecular beacon technology (e.g., U.S. Pat. No. 6,150,097, herein incorporated by reference in its entirety); E-sensor technology (U.S. Pat. Nos. 6,248,229, 6,221,583, 6,013,170, and 6,063,573, herein incorporated by reference in their entireties); cycling probe technology (e.g., U.S. Pat. Nos. 5,403,711, 5,011,769, and 5,660,988, herein incorporated by reference in their entireties); Dade Behring signal amplification methods (e.g., U.S. Pat. Nos. 6,121,001, 6,110,677, 5,914,230, 5,882,867, and 5,792,614, herein incorporated by reference in their entireties); ligase chain reaction (e.g., Barany Proc. Natl. Acad. Sci USA 88, 189-93 (1991)); and sandwich hybridization methods (e.g., U.S. Pat. No. 5,288,609, herein incorporated by reference in its entirety).As used herein, “rolling circle amplification” refers to in vitro rolling circle replication of a circular nucleic acid using a strand-displacing DNA polymerase to form a DNA molecule comprising tandem repeats of a sequence complementary to the circular nucleic acid, as described, e.g., in U.S. Pat. Nos. 6,210,884; 6,183,960; 6,235,502; 5,942,391; 6,316,229; 7,862,999; 11,186,863; U.S. Pat. Publication US 2015 / 0284786; and in M. Ali, et al. “Rolling circle amplification: a versatile tool for chemical biology, materials science and medicine”. Chemical Society Reviews. 43 (10): 3324-3341.In some embodiments, target nucleic acid is amplified (e.g., by PCR) and amplified nucleic acid is detected simultaneously using an invasive cleavage assay. Assays configured for performing a detection assay (e.g., invasive cleavage assay) in combination with an amplification assay are described in US Patent No. 9,096,893 B2 (App. Ser. No. 13 / 941,122), incorporated herein by reference in its entirety for all purposes, and as diagrammed in Figure 1. Because many copies of the FRET cassette are cleaved for each copy of the target amplicon produced, the assay is said to produce “signal amplification” in addition to target amplification. See also Allawi HT, et al. J of Clin Microbio 2006; vol 44, no. 9: 3443-3447. Additional amplification plus invasive cleavage detection configurations, termed theQUARTS method, are described in U.S. Pat. No. 8,361,720, 8,715,937, and 8,916,344, incorporated herein by reference in their entireties for all purposes.A “flap oligonucleotide” refers to an oligonucleotide cleavable in a detection assay, such as an invasive cleavage assay, by a flap endonuclease. In preferred embodiments, a flap oligonucleotide forms an invasive cleavage structure with other nucleic acids, e.g., a target or template nucleic acid and an invasive oligonucleotide. In some embodiments, a flap oligonucleotide comprises a 5' flap or arm that is not substantially complementary to the target strand. Flap assay reagents may optionally contain a target or template nucleic acid to which an invasive oligonucleotide and flap oligonucleotide bind. In particularly preferred embodiments, flap assay reagents comprise a Mg++flap assay buffer, as discussed herein.As used herein, the term “flap endonuclease” refers to a structure-specific nucleolytic enzyme that cleaves a nucleic acid flap structure, e.g., an invasive cleavage structure. Flap endonuclease include, e.g., 5' -exonuclease domains of the DNA polymerase I proteins of Eubacteria and the FEN-1 proteins of Eukarya and Archaea. (Kaiser M.W., et al. (1999) J. Biol. Chem., 274:21387). Flap endonuclease may cleave additional structures, e.g., pseudo- Y, 5' overhang, and gap structures. See, e.g., Shen, B., BioEssays 27:717-729 (2005), and Finger, LD., Subcell Biochem. 62:301-326 (2012), each of which is incorporated herein in its entirety. Flap endonucleases are reviewed by Ceska and Savers (Trends Biochem. Sci. 1998 23:331-336) and Liu, et al. (Annu. Rev. Biochem. 2004 73: 589-615), and Tsutakawa S, et al. (Cell vl45: 198-211 (2011)) each herein incorporated by reference in its entirety.The terms “FEN-1” as used herein in reference to an enzyme refers to a nonpolymerase flap endonuclease from a eukaryote or archaeal organism, as encoded by a FEN-1 (Flap Structure-Specific Endonuclease 1) gene. See, e.g., Kaiser, et al., supra, WO 02 / 070755, and US Patent No. US 7,122,364, which are incorporated by reference herein in their entireties for all purposes. The term “FEN-1 activity” refers to any enzymatic activity of a FEN-1 enzyme. FEN-1 endonucleases also comprise modified FEN-1 proteins, e.g., chimerical proteins comprising portions of FEN- 1 enzymes from different organisms, and enzymes comprising one or more mutations (e.g., substitutions, deletions, insertions, etc.), as described in WO 02 / 070755, and US Patent No. US 7,122,364. The term “FEN1” is used interchangeably with “FEN-1” herein.As used herein, the term “a strict FEN-1” refers to a FEN-1 enzyme, generally from an archaeal organism, that, under suitable conditions, will cleave an invasive cleavage structure but will show substantially no activity in cleaving non-invasive structures, e.g., agapped structure (having a single-stranded region of target or template strand between the upstream and downstream duplexes), Y-structure (having a 5' flap but no overlap between the upstream and downstream duplexes), pseudo- Y structure (having a 5' flap and an unduplexed template strand upstream of the downstream duplex). For example, Afu FEN-1 and Pfu FEN-1 are strict FEN-1 s when Mg++is the predominant or sole divalent cation in a cleavage buffer, e.g., in a flap assay buffer.As used herein, the term “PCR-flap assay” is used interchangeably with the term “PCR-invasive cleavage assay” and refers to an assay configuration combining PCR target amplification and detection of the amplified DNA by formation of a first overlap cleavage structure comprising amplified target DNA, and a second overlap cleavage structure comprising a cleaved 5' flap from the first overlap cleavage structure and a labeled reporter oligonucleotide, e.g., a “FRET cassette” or 5' hairpin FRET reporter oligonucleotide. In the PCR-flap assay as used herein, the assay reagents comprise a mixture containing DNA polymerase, FEN-1 endonuclease, a primary probe comprising a portion complementary to a target nucleic acid, and a FRET cassette or 5' hairpin FRET reporter, and the target nucleic acid is amplified by PCR and the amplified nucleic acid is detected simultaneously (i.e., detection occurs during the course of target amplification). PCR-flap assays include the QuARTS assays described in U.S. Pat. Nos. 8,361,720; 8,715,937; and 8,916,344, and the amplification assays of US Pat. No. 9,096,893 (for example, as diagrammed in Figure 1 of that patent.) LQAS assays are flap assays using probe oligonucleotides having a longer target-specific region (Long probe Quantitative Amplified Signal, “LQAS”) and TELQAS assays combine the LQAS probe oligos with a pre-amplification step (Target Enrichment Long probe Quantitative Amplified Signal), as is described in U.S. Pat. No. 10,648,025, and in WO 2020 / 112869, each of which is incorporated herein by reference in its entirety.As used herein, the term “PCR-flap assay reagents” refers to one or more reagents for detecting target sequences in a PCR-flap assay, the reagents comprising nucleic acid molecules capable of participating in amplification of a target nucleic acid and in formation of a flap cleavage structure in the presence of the target sequence, in a mixture containing DNA polymerase, FEN-1 endonuclease and a FRET cassette or 5' hairpin FRET reporter.As used herein, the term “invasive cleavage structure” refers to a cleavage structure comprising a template nucleic acid, an upstream nucleic acid (e.g., an invasive oligonucleotide, or a 3' portion of a template strand folded back and hybridized to form a hairpin, or a 3’ portion of a padlock probe ), and iii) a downstream nucleic acid (e.g., a probe,or a 5' portion of a template strand folded back and hybridized to form a hairpin, or a 5' portion of a padlock probe), where the upstream and downstream nucleic acids anneal to adjacent regions of the template strand (z.e., regions of a template strand that are next to each other on the strand, not separated by intervening nucleotides or base pairs) and where an overlap forms between the 3' end of the upstream nucleic acid and the duplex formed between the downstream nucleic acid and the template nucleic acid. An overlap occurs where one or more bases from the upstream and downstream nucleic acids occupy the same position with respect to a particular nucleotide in a template strand, whether or not the overlapping base(s) at the 3' end of the upstream nucleic acid are complementary with corresponding nucleotide in the template strand, and whether or not those bases are natural bases or nonnatural bases. In some embodiments, the 3' portion of the upstream nucleic acid that overlaps with the downstream duplex is a non-base chemical moiety such as an aromatic ring structure, e.g., as disclosed, for example, in U.S. Pat. No. 6,090,543, incorporated herein by reference in its entirety. As used herein in reference to an invasive cleavage structure, the term “upstream duplex” refers to a duplex formed between a template strand and a hybridized upstream nucleic acid strand having a 3' end, and the term “downstream duplex” refers to a duplex formed between a template strand and a hybridized downstream nucleic acid having a 5' end, such that an overlapping flap endonuclease substrate is formed by the adjacent hybridized regions of the upstream and downstream nucleic acid strands. In some embodiments, the upstream nucleic acid, the downstream nucleic acid, or both, are part of the same strand of nucleic acid as the template strand. See, e.g., Fig. 1 A and IB, 2A and 2B, which illustrate the upstream and downstream duplex regions of invasive cleavage structures formed from 3 strands or from 2 strands of nucleic acid, respectively. In some embodiments, one or more of the nucleic acid strands may be attached to each other through a non-nucleic acid chemical linkage (e.g., a multi-carbon chain). See, e.g., U.S. Pat. No. 8,445,238, which is incorporated herein by reference in its entirety. In some embodiments, two nucleic acid strands are attached to each other through a nucleic acid backbone region, forming a padlock probe. Preferably, a nucleic acid backbone in a padlock probe is substantially non- complementary to the target nucleic acid.As used herein, the term “padlock probe” refers to any oligonucleotide probe that forms a nicked circle by hybridization to a strand of nucleic acid, and which by treatment that includes a ligase forms a covalently closed circular molecule. See, e.g., M. Nilsson, et al. “Padlock probes: circularizing oligonucleotides for localized DNA detection”. Science. 265(5181): 2085-2088 (1994). In some embodiments, a padlock probe requires modification prior to being ligatable, e.g., by endo- or exonuclease digestion (e.g., to remove a flap sequence), polymerase extension (e.g., to fill gaps) or other treatment to produce a circular probe comprising a ligatable nick. Preferably, the modification to produce the ligatable nick is dependent on the padlock probe being hybridized to an intended target nucleic acid. Padlock probes are described in U.S. Pat. No. 5,854,033 (Lizardi), WO99 / 49079 (Landegren) and U.S. Pat. No. 5,871,921 (Landegren & Kwiatkowski). A version of a padlock probe known as an inversion probe is described in U.S. Pat. No. 6,858,412 (Willis et al.). Inversion probes are padlock probes containing a cleavage site in the probe backbone, allowing the circularized probe to be cleaved to form a linear product, which may then be amplified and detected.As used herein, “methylation” refers to cytosine methylation at positions C5 or N4 of cytosine, the N6 position of adenine, or other types of nucleic acid methylation. In vitro amplified DNA is usually unmethylated because typical in vitro DNA amplification methods do not retain the methylation pattern of the amplification template. However, “unmethylated DNA” or “methylated DNA” can also refer to amplified DNA whose original template was unmethylated or methylated, respectively.Accordingly, as used herein a “methylated nucleotide” or a “methylated nucleotide base” refers to the presence of a methyl moiety on a nucleotide base, where the methyl moiety is not present in a recognized typical nucleotide base. For example, cytosine does not contain a methyl moiety on its pyrimidine ring, but 5-methylcytosine contains a methyl moiety at position 5 of its pyrimidine ring. Therefore, cytosine is not a methylated nucleotide and 5-methylcytosine is a methylated nucleotide. In another example, thymine contains a methyl moiety at position 5 of its pyrimidine ring; however, for purposes herein, thymine is not considered a methylated nucleotide when present in DNA since thymine is a typical nucleotide base of DNA.As used herein, a “methylated nucleic acid molecule” refers to a nucleic acid molecule that contains one or more methylated nucleotides.As used herein, a “methylation state”, “methylation profile”, and “methylation status” of a nucleic acid molecule refers to the presence or absence of one or more methylated nucleotide bases in the nucleic acid molecule. For example, a nucleic acid molecule containing a methylated cytosine is considered methylated e.g., the methylation state of thenucleic acid molecule is methylated). A nucleic acid molecule that does not contain any methylated nucleotides is considered unmethylated. In some embodiments, a nucleic acid may be characterized as “unmethylated” if it is not methylated at a specific locus (e.g., the locus of a specific single CpG dinucleotide) or specific combination of loci, even if it is methylated at other loci in the same gene or molecule.The methylation state of a particular nucleic acid sequence (e.g., a gene marker or DNA region as described herein) can indicate the methylation state of every base in the sequence or can indicate the methylation state of a subset of the bases (e.g., of one or more cytosines) within the sequence, or can indicate information regarding regional methylation density within the sequence with or without providing precise information of the locations within the sequence the methylation occurs. As used herein, the terms “marker gene” and “marker” are used interchangeably to refer to DNA, RNA, or protein (or other sample components) that is associated with a condition, e.g., cancer or other disease, regardless of whether the marker region is in a coding region of DNA. Markers may include, e.g., regulatory regions, flanking regions, intergenic regions, etc. Similarly, the term “marker” used in reference to any component of a sample, e.g., protein, RNA, carbohydrate, small molecule, etc., refers to a component that can be assayed in a sample (e.g., measured or otherwise characterized) and that is associated with a condition of a subject, or of the sample from a subject. The term “methylation marker” refers to a gene or DNA in which the methylation state of the gene or DNA is associated with a condition, e.g., cancer or other disease.The methylation state of a nucleotide locus in a nucleic acid molecule refers to the presence or absence of a methylated nucleotide at a particular locus in the nucleic acid molecule. For example, the methylation state of a cytosine at the 7th nucleotide in a nucleic acid molecule is methylated when the nucleotide present at the 7th nucleotide in the nucleic acid molecule is 5-methylcytosine. Similarly, the methylation state of a cytosine at the 7th nucleotide in a nucleic acid molecule is unmethylated when the nucleotide present at the 7th nucleotide in the nucleic acid molecule is cytosine (and not 5-methylcytosine).The methylation status can optionally be represented or indicated by a “methylation value” (e.g., representing a methylation frequency, fraction, ratio, percent, etc.) A methylation value can be generated, for example, by quantifying the amount of intact nucleic acid present following restriction digestion with a methylation dependent restriction enzyme or by comparing amplification profiles after bisulfite reaction or by comparing sequences ofbisulfite-treated and untreated nucleic acids. Accordingly, a value, e.g., a methylation value, represents the methylation status and can thus be used as a quantitative indicator of methylation status across multiple copies of a locus. This is of particular use when it is desirable to compare the methylation status of a sequence in a sample to a threshold or reference value.As used herein, “methylation frequency” or “methylation percent (%)” refer to the number of instances in which a molecule or locus is methylated relative to the number of instances the molecule or locus is unmethylated.As such, the methylation state describes the state of methylation of a nucleic acid (e.g., a genomic sequence). In addition, the methylation state refers to the characteristics of a nucleic acid segment at a particular genomic locus relevant to methylation. Such characteristics include, but are not limited to, whether any of the cytosine (C) residues within this DNA sequence are methylated, the location of methylated C residue(s), the frequency or percentage of methylated C throughout any particular region of a nucleic acid, and allelic differences in methylation due to, e.g., difference in the origin of the alleles. The terms “methylation state”, “methylation profile”, and “methylation status” also refer to the relative concentration, absolute concentration, or pattern of methylated C or unmethylated C throughout any particular region of a nucleic acid in a biological sample. For example, if the cytosine (C) residue(s) within a nucleic acid sequence are methylated it may be referred to as “hypermethylated” or having “increased methylation”, whereas if the cytosine (C) residue(s) within a DNA sequence are not methylated it may be referred to as “unmethylated”, “hypomethylated” or having “decreased methylation”. Likewise, if the cytosine (C) residue(s) within a nucleic acid sequence are methylated as compared to another nucleic acid sequence (e.g., from a different region or from a different individual, etc.) that sequence is considered hypermethylated or having increased methylation compared to the other nucleic acid sequence. Alternatively, if the cytosine (C) residue(s) within a DNA sequence are not methylated as compared to another nucleic acid sequence (e.g., from a different region or from a different individual, etc.) that sequence is considered hypomethylated or having decreased methylation compared to the other nucleic acid sequence. Additionally, the term “methylation pattern” as used herein refers to the collective sites of methylated and unmethylated nucleotides over a region of a nucleic acid. Two nucleic acids may have the same or similar methylation frequency or methylation percent but have different methylation patterns when the number of methylated and unmethylated nucleotides is the same or similarthroughout the region but the locations of methylated and unmethylated nucleotides are different. Sequences are said to be “differentially methylated” or as having a “difference in methylation” or having a “different methylation state” when they differ in the extent (e.g., one has increased or decreased methylation relative to the other), frequency, or pattern of methylation. The term “differential methylation” refers to a difference in the level or pattern of nucleic acid methylation in a sample from a subject having a condition, e.g., cancer, as compared with the level or pattern of nucleic acid methylation in a sample from a subject who does not have the condition. It may also refer to the difference in levels or patterns between patients that have recurrence of the condition, e.g., after treatment or surgery, versus patients who do not have recurrence. Differential methylation and specific levels or patterns of DNA methylation are prognostic and predictive biomarkers, e.g., once the correct cut-off or predictive characteristics have been defined.Methylation state frequency can be used to describe a population of individuals or a sample from a single individual. For example, a nucleotide locus having a methylation state frequency of 50% is methylated in 50% of instances and unmethylated in 50% of instances. Such a frequency can be used, for example, to describe the degree to which a nucleotide locus or nucleic acid region is methylated in a population of individuals or a collection of nucleic acids. Thus, when methylation in a first population or pool of nucleic acid molecules is different from methylation in a second population or pool of nucleic acid molecules, the methylation state frequency of the first population or pool will be different from the methylation state frequency of the second population or pool. Such a frequency also can be used, for example, to describe the degree to which a nucleotide locus or nucleic acid region is methylated in a single individual. For example, such a frequency can be used to describe the degree to which a group of cells from a tissue sample are methylated or unmethylated at a nucleotide locus or nucleic acid region.As used herein a “nucleotide locus” refers to the location of a nucleotide in a nucleic acid molecule. A nucleotide locus of a methylated nucleotide refers to the location of a methylated nucleotide in a nucleic acid molecule.Typically, methylation of human DNA occurs on a dinucleotide sequence including an adjacent guanine and cytosine where the cytosine is located 5' of the guanine (also termed CpG dinucleotide sequences). Most cytosines within the CpG dinucleotides are methylated in the human genome, however some remain unmethylated in specific CpG dinucleotide richgenomic regions, known as CpG islands (see, e.g., Antequera, et al. (1990) Cell 62: 503- 514).As used herein, a “CpG island” refers to a G:C-rich region of genomic DNA containing an increased number of CpG dinucleotides relative to total genomic DNA. A CpG island can be at least 100, 200, or more base pairs in length, where the G:C content of the region is at least 50% and the ratio of observed CpG frequency over expected frequency is 0.6; in some instances, a CpG island can be at least 500 base pairs in length, where the G:C content of the region is at least 55% and the ratio of observed CpG frequency over expected frequency is 0.65. The observed CpG frequency over expected frequency can be calculated according to the method provided in Gardiner-Garden et al (1987) J. Mol. Biol. 196: 261- 281. For example, the observed CpG frequency over expected frequency can be calculated according to the formula R = (A x B) / (C x D), where R is the ratio of observed CpG frequency over expected frequency, A is the number of CpG dinucleotides in an analyzed sequence, B is the total number of nucleotides in the analyzed sequence, C is the total number of C nucleotides in the analyzed sequence, and D is the total number of G nucleotides in the analyzed sequence. Methylation state is typically determined in CpG islands, e.g., at promoter regions. It will be appreciated though that other sequences in the human genome are prone to DNA methylation such as CpA and CpT (see Ramsahoye (2000) Proc. Natl. Acad. Sci. USA 97: 5237-5242; Salmon and Kaye (1970) Biochim. Biophys. Acta. 204: 340-351; Grafstrom (1985) Nucleic Acids Res. 13: 2827-2842; Nyce (1986) Nucleic Acids Res. 14: 4353-4367; Woodcock (1987) Biochem. Biophys. Res. Commun. 145: 888-894).As used herein, the terms “methyl cytosine,” “methyl C,” “methylated cytosine,” “methylated C,” and “meC” are used interchangeably and encompass both 5-methylcytosine (5mC) and 5 -hydroxymethyl cytosine (5hmC).As used herein, the term “modified cytosine” or “modified C” refers to a cytosine nucleobase in which the base portion has a side group or other modification as compared to a standard cytosine nucleotide. Modified cytosines include but are not limited to 5- methylcytosine (5mC), 5-hydroxymethylcytosine (5hmC), 5-carboxylcytosine (5caC), and 5- formylcytosine (5fC).As used herein, a “methylation-specific reagent” refers to a reagent that modifies a nucleotide of the nucleic acid molecule as a function of the methylation state of the nucleic acid molecule. In particular embodiments the term refers to a compound or composition orother agent or collection or sequence thereof that can change the nucleotide sequence of a nucleic acid molecule in a manner that reflects the methylation state of the nucleic acid molecule. Methods of treating a nucleic acid molecule with such reagents can include contacting the nucleic acid molecule with the reagent, coupled with additional steps, if desired, to accomplish the desired change of nucleotide sequence. Such methods can be applied in a manner in which unmethylated nucleotides (e.g., each unmethylated cytosine) is modified to a different nucleotide. For example, in some embodiments, such a reagent can deaminate unmethylated cytosine nucleotides to produce deoxy uracil residues. An exemplary reagent is a bisulfite reagent.In addition, treatment with a “methylation-specific reagent” can be applied in a manner in which methylated nucleotides are modified to a different nucleotide. For example, methylated cytosines (including 5mC and 5hmC) in DNA can be converted by combining oxidation by ten-eleven translocation (TET) family dioxygenases with reduction by borane derivatives (e.g., pyridine borane and 2-picoline borane (pic-BEE)), in a process referred to herein as TAPS (TET Assisted Pyridine borane Sequencing). See, e.g., the TAPS method combining oxidation by TET enzymes with reduction by borane derivatives, described, e.g., in US 2020 / 0370114 Al, Application Ser. No. 16 / 960,510, filed July 7, 2020, which is incorporated herein by reference for all purposes. In embodiments of the TAPS method, methylated cytosines are converted to dihydro uracil. Other methods of converting methylated Cs include, for example:(Loise Williams, et al., Enzymatic Methyl-seq: The next generation of methylome analysis, New England Biolabs Expressions 2019. Feature Article.)In preferred embodiments, methylation-specific reagents modify one nucleotide of the four typically-occurring nucleotides in a nucleic acid molecule (C, G, T, and A for DNA and C, G, U, and A for RNA), such that the reagent modifies the one nucleotide without modifying the other three nucleotides. The nucleotides resulting from conversion are not limited to the four typically occurring nucleotides listed above, and may include, for example, modified or variant forms of purine or pyrimidine structures, including, e.g., nucleobase analogs discussed herein. In preferred embodiments, the nucleotides produced by conversion are recognized by DNA modifying enzymes, e.g., DNA polymerases, as one of the typically occurring nucleotides listed above, and can serve as templates for strand replication. Conversion of nucleotides by any of the methods described herein may be detected by determining the sequence of a resulting strand, e.g., using standard sequencing methods, or by interrogating single or a few specific nucleotide locations to determine the identity of the nucleobase at the select locations.As used herein, the term “converted” as used in reference to a nucleotide or DNA strand refers to a nucleotide or DNA strand that has been treated with a reagent or reagents under conditions in which some nucleotides are converted into other nucleotides. For example, in bisulfite conversion, cytosine bases in the DNA are typically deaminated, resulting in uracil bases at converted loci. While inefficient, bisulfite can also cause deamination of 5-methyl cytosine bases, resulting in thymine bases at converted loci. “Bisulfite-treated” and bisulfite-converted” are used interchangeably herein in reference to DNA or nucleotide loci that have been exposed to a bisulfite reagent under conditions in which cytosine is typically converted to uracil. In the bi sulfite-free TAPS process, methylated cytosines are selectively converted to dihydrouracil (DHU), while unmethylated Cs are not converted. The DHU nucleotides base pair with A nucleotides rather than G nucleotides, making them readily distinguishable from the unmethylated C bases in the converted DNA strands.As used herein, the term “poorly converted,” as used in reference to conversion of a nucleotide upon treatment with reagent(s) and / or conditions under which some nucleotides are converted into other nucleotides, refers to a nucleotide having a reduced rate of conversion (preferably less than 10%, more preferably less than 1% of the rate of conversion) under the given treatment, as compared to the rate of conversion of a nucleotide expected to convert under the same treatment. For example, bisulfite-mediated deamination of cytosine is greatly slowed down by the presence of a 5-methyl group, such that the rate for thedeamination of 5-methylcytosine to form thymine is about two orders of magnitude smaller than the rate for deamination of cytosine to form uracil (see, e.g., Hayatsu et al., Biochemistry 18:4:632-37 (1979); Hayatsu, Proc. Jpn. Acad 84(8):321-330 (2008), each of which is incorporated herein by reference). Thus, 5-methylcytosine is said to be poorly converted compared to cytosine under bisulfite treatment conditions typically used to convert cytosine to uracil.The term “bisulfite reagent” refers to a reagent comprising bisulfite, di sulfite, hydrogen sulfite, or combinations thereof, useful as disclosed herein to distinguish between methylated and unmethylated CpG dinucleotide sequences. Methods of said treatment are known in the art (e.g., PCT / EP2004 / 011715 and WO 2013 / 116375, each of which is incorporated by reference in its entirety). In some embodiments, bisulfite treatment is conducted in the presence of denaturing solvents such as but not limited to n-alkyleneglycol or diethylene glycol dimethyl ether (DME), or in the presence of dioxane or dioxane derivatives. In some embodiments the denaturing solvents are used in concentrations between 1% and 35% (v / v). In some embodiments, the bisulfite reaction is carried out in the presence of scavengers such as but not limited to chromane derivatives, e.g., 6-hydroxy-2, 5,7,8, - tetramethylchromane 2-carboxylic acid or trihydroxybenzone acid and derivatives thereof, e.g., Gallic acid (see: PCT / EP2004 / 011715, which is incorporated by reference in its entirety). In certain preferred embodiments, the bisulfite reaction comprises treatment with ammonium hydrogen sulfite, also referred to as ammonium bisulfite, e.g., as described in WO 2013 / 116375.The term “methylation assay” refers to any assay for determining the methylation state of one or more CpG dinucleotide sequences within a sequence of a nucleic acid.As used herein in reference to cleavage of an invasive cleavage structure, the term “target cleavage site” refers to a preferred site (or sites) of cleavage on a nucleic acid structure (e.g., an invasive cleavage structure) by a structure-specific nuclease (e.g., a FEN-1 endonuclease) that recognizes the structure as a cleavage substrate. For example, as discussed by Kaiser, etal., 5' flap endonucleases, including FEN-1 endonucleases, typically cleave an invasive cleavage structure in the downstream nucleic acid, generally after the first basepaired nucleotide, z.e., one nucleotide into the downstream duplex. In preferred embodiments described herein, a target cleavage site may be used as a reporting cleavage site in a flap assay. For example, in certain preferred embodiments, donor and acceptor moieties of a FRET labeling system (e.g., fluorophore and a quencher moieties) are positioned such thatthey are separated upon cleavage of the invasive cleavage structure at a target cleavage site, such that an increase in fluorescence in the reaction mixture reports the cleavage at the target cleavage site. The technology is not limited by any particular placement of moieties of the FRET system on the cleavage structure. For example, in some embodiments, moieties of a FRET system are attached to a probe or reporter at positions that closely flank the reporting cleavage site, e.g., that are within 1, 2, or a few nucleotides on either side of a reporting cleavage site, while in other embodiments, moieties of a FRET system are more widely spaced, e.g., 5, 6, 7, 8, 9, 10, etc., nucleotides apart on a nucleic acid strand. In some embodiments, one or more moieties of a FRET system are at or near the termini of a nucleic acid strand that is cleaved in an invasive cleavage structure. In preferred embodiments, an invasive cleavage structure is cleavable at a target cleavage site by a FEN-1 endonuclease from Archaeoglobus fulgidus ( Afu FEN-1”) in a Mg++flap assay buffer, as discussed below. See also, e.g., U.S. Pat. No. 6,562,611 to Kaiser, et al., and Kaiser M.W., et al. (1999) J. Biol. Chem., 274:21387, each of which is incorporated herein by reference in its entirety for all purposes.As used herein, “first paired nucleotide” and “first paired base,” as used in reference to a FEN-1 cleavage site of an invasive cleavage structure, e.g, in a padlock probe or flap oligonucleotide, refers to 5'-most base-paired nucleotide of the probe or oligonucleotide as hybridized in the downstream duplex, as illustrated, e.g, in Figs. 1A, IB, 2A, 3 A, 4A, 5 A, 6 A, and 7.The term “template” as used in reference to a nucleic acid strand of a flap structure, e.g., an invasive cleavage structure, may be used interchangeably with “target” and refers to the strand to which upstream and downstream nucleic acids or nucleic acid regions hybridize to form an invasive cleavage structure. While a template strand may serve as template for extension of a primer by a polymerase, e.g., in a PCR flap endonuclease assay, use of the term is not limited to polymerizing assays or reactions.As used herein, the term “3' end blocker” refers to a 3' end modification, e.g., on an oligonucleotide, that suppresses cleavage by a flap endonuclease of an invasive cleavage structure when the 3' end blocker is present on the 3' end of an upstream nucleic acid in the cleavage structure. For example, 3' amines, 3' phosphate (3'-PO4), 3' biotin, 3' Ce (a 6-carbon glycol spacer, also referred to as “3' hexanediol” ), and 3' dideoxynucleotides suppress flap cleavage if present on an upstream nucleic acid. See, e.g., Kaiser, et al., supra.As used herein, the term “5' blocking group” refers to a 5' end modification or moiety that prevents a phosphate at a 5' end of a nucleic acid strand or at the 5' end of a region of a nucleic acid strand (e.g., the 5' end of a duplexed portion of a padlock probe in a downstream duplex, when the 5' end of the padlock probe includes an unpaired 5' flap sequence) , from being available for enzymatic ligation, e.g., to an hydroxyl at the 3' end of a nucleic acid strand, e.g., the 3' terminal nucleotide of an adjacent duplexed portion of a padlock probe. Exemplary 5' blocking groups include but are not limited to a carbon chain (e.g., hexane), a non-standard nucleotide, an abasic nucleotide (e.g., a ribose or deoxyribose lacking a nucleobase), a dye tag, a flap nucleic acid sequence, a hairpin flap, etc. In preferred embodiments, a 5' blocking group is attached to the 5' position of a nucleotide (e.g., the 5' end of a duplexed portion of a padlock probe in a downstream duplex) via a phosphate, e.g., a phosphodiester bond. In particularly preferred embodiments the 5' blocking group is attached to the 5' position of a nucleotide that is 5' of a FEN-1 cleavage site in an invasive cleavage structure.As used herein, the term “Mg++flap assay buffer” refers to a buffer solution for a flap endonuclease that includes Mg++as the predominant or essentially only divalent cation in the buffer, such that assay reactions performed in the buffer, e.g., flap assays, exhibit characteristics of the presence of Mg++(e.g., high specificity of some flap endonucleases for invasive cleavage structures, to the exclusion of, e.g., Y-structures, pseudo Y structures), and do not exhibit characteristics associated with the presence of other divalent cations (e.g., alternative cleavage activities of flap endonucleases associated with use of Mn++with or in place of Mg++), as described, e.g., by Kaiser, et al., supra. In some embodiments the flap assay buffer has very low or no KC1 and comprises elevated Mg++as compared to standard PCR buffer, (e.g., a flap assay buffer of the technology comprises >6 mM, preferably > 7mM, more preferably 7.5mM Mg++, while PCR buffers typically comprise about 1.5 to 2.5 mM Mg++.)As used herein, the term “flap assay reagents” or “invasive cleavage assay reagents” refers to a collection of all reagents required for performing a flap assay or invasive cleavage assay on a substrate. As is known in the art, flap assays generally include oligonucleotides for forming an invasive cleavage structure, a flap endonuclease and, optionally, a FRET cassette or 5' hairpin FRET reporter.As used herein, the term “FRET” refers to fluorescence resonance energy transfer, a process in which moi eties e.g., fluorophores) transfer energy e.g., among themselves, or,from a fluorophore to a non-fluorophore (e.g., a quencher molecule). In some circumstances, FRET involves an excited donor fluorophore transferring energy to a lower-energy acceptor fluorophore via a short-range (e.g., about 10 nm or less) dipole-dipole interaction. In other circumstances, FRET involves a loss of fluorescence energy from a donor and an increase in fluorescence in an acceptor fluorophore. In still other forms of FRET, energy can be exchanged from an excited donor fluorophore to a non-fluorescing molecule e.g., a quenching molecule). FRET is known to those of skill in the art and has been described (See, e.g., Stryer et al., 1978, Ann. Rev. Biochem., 47:819; Selvin, 1995, Methods Enzymol., 246:300; Orpana, 2004 Biomol Eng 21, 45-50; Olivier, 2005 Mutant Res 573, 103-110, each of which is incorporated herein by reference in its entirety).As used herein, the term “FRET system” refers to a pair or group of moieties that together act as donor-acceptor or donor-quencher partners for FRET -based analysis of molecules, e.g., flap oligonucleotides or assay reporter molecules. While embodiments of the technology are illustrated with a fluorophore in one particular position and a quencher or other FRET acceptor moiety in a particular second position, e.g., in a FRET assay reporter as illustrated in Figs. 1-5, the illustrated embodiments provided as non-limiting examples of the technology. The moieties of a FRET system finding application in the technology are not limited to these placements and may, for example, be switched in position, may be positioned on different parts of the illustrated molecules, or may comprise additional donor or acceptor moieties that are the same or different.As used herein, the term “reporting cleavage site” refers to a cleavage site that generates signal in a flap endonuclease assay. For example, flap oligonucleotides configured to use a FRET effect to indicate release of a 5' flap by a flap endonuclease may comprise fluorophore moiety and quencher moieties that are positioned to be separated by flap endonuclease cleavage when the flap oligonucleotide hybridizes to a target molecule. Cleavage between the fluorophore and quencher moieties separates the FRET system moieties and removes the quenching effect from the fluorophore such that successful cleavage is reported, i.e., is made detectable, by an increase in fluorescence from the fluorophore. Thus, a site between fluorophore and quencher moieties of a FRET system is a reporting cleavage site, while any site on that oligonucleotide that may be cleaved by an activity of a flap endonuclease but that is not between the FRET moieties would not be a reporting cleavage site. In preferred embodiments, a “reporting cleavage site” in a substrate structure used in an assay is a target cleavage site for the flap endonuclease used in the assay.As used herein, the term “FRET cassette” refers to a hairpin oligonucleotide that contains a fluorophore moiety and a nearby quencher moiety that quenches the fluorophore. Hybridization of a cleaved flap (e.g., from cleavage of a target-specific probe in a PCR-flap assay assay) with a FRET cassette produces a secondary substrate for the flap endonuclease, e.g., a FEN-1 enzyme. Once this substrate is formed, the 5' fluorophore-containing base can be cleaved from the cassette by the flap endonuclease, thereby generating a fluorescence signal. In preferred embodiments, a FRET cassette comprises an unpaired 3' portion to which a cleavage product, e.g., a portion of a cleaved flap oligonucleotide, can hybridize to form an invasive cleavage structure cleavable by a FEN-1 endonuclease.As used herein, the phrase “not substantially complementary” as used in reference to a probe flap or arm means that the flap portion is sufficiently non-complementary not to hybridize selectively to a nucleic acid sequence, e.g, a target nucleic acid or amplified DNA, under the designated annealing conditions or stringent conditions, encompassing the terms “substantially non-complementary” and “perfectly non-complementary.”The term “signal” as used herein refers to any detectable effect, such as would be caused or provided by a label or by action or accumulation of a component or product in an assay reaction.The term “neoplasm” as used herein refers to any new and abnormal growth of tissue, including but not limited to a cancer. Thus, a neoplasm can be a premalignant neoplasm or a malignant neoplasm.The term “neoplasm-specific marker,” as used herein, refers to any biological material or element that can be used to indicate the presence of a neoplasm. Examples of biological materials include, without limitation, nucleic acids, polypeptides, carbohydrates, fatty acids, cellular components (e.g, cell membranes and mitochondria), and whole cells. In some instances, markers are particular nucleic acid regions (e.g., genes, intragenic regions, specific loci, etc.). Regions of nucleic acid that are markers may be referred to, e.g., as “marker genes,” “marker regions,” “marker sequences,” “marker loci,” etc.The term “sample” is used in its broadest sense. In one sense it can refer to an animal cell or tissue or fluid. In another sense, it refers to a specimen or culture obtained from any source, as well as biological and environmental samples. Biological samples may be obtained from plants or animals (including humans) and encompass, e.g., fluids, solids, tissues, and gases. Environmental samples include environmental material such as surface matter, soil, water, and industrial samples. These examples are not to be construed as limiting the sampletypes applicable to the present invention. As used herein in reference to samples, the term “a sample” collected from a source or subject, e.g., from a patient, is not limited to a single physical specimen but also encompasses a sample that is collected in multiple portions, e.g., “a sample” of blood may be collected in two, three, four or more different blood collection tubes or other blood collection devices (e.g., bags), or combinations of different blood collection devices. A sample “suspected of containing” a marker or other material may contain or not contain the marker.As used herein, the terms “patient” or “subject” refer to organisms to be subject to various tests provided by the technology. The term “subject” includes animals, preferably mammals, including humans. In a preferred embodiment, the subject is a primate. In an even more preferred embodiment, the subject is a human. Further with respect to diagnostic methods, a preferred subject is a vertebrate subject. A preferred vertebrate is warm-blooded; a preferred warm-blooded vertebrate is a mammal. A preferred mammal is most preferably a human. As used herein, the term “subject1includes both human and animal subjects. Thus, veterinary therapeutic uses are provided herein. As such, the present technology provides for the diagnosis of mammals such as humans, as well as those mammals of importance due to being endangered, such as Siberian tigers; of economic importance, such as animals raised on farms for consumption by humans; and / or animals of social importance to humans, such as animals kept as pets or in zoos. Examples of such animals include but are not limited to: carnivores such as cats and dogs; swine, including pigs, hogs, and wild boars; ruminants and / or ungulates such as cattle, oxen, sheep, giraffes, deer, goats, bison, and camels; pinnipeds; and horses. Thus, also provided is the diagnosis and treatment of livestock, including, but not limited to, domesticated swine, ruminants, ungulates, horses (including racehorses), and the like. The presently-disclosed subject matter further includes a system for analyzing nucleic acid from a sample or subject. The system can be provided, for example, as a commercial kit that can be used to screen nucleic acid from a subject for risk for a condition, e.g., for a risk of cancer, or to detect the presence of a condition, e.g., to diagnose a cancer, in a subject from whom a biological sample has been collected. An exemplary system provided in accordance with the present technology includes assessing the methylation state of a marker described herein.The term “real time” as used herein in reference to detection of nucleic acid amplification or signal amplification refers to the detection or measurement of the accumulation of products or signal in the reaction while the reaction is in progress, e.g.,during incubation or thermal cycling. Such detection or measurement may occur continuously, or it may occur at a plurality of discrete points during the progress of the amplification reaction, or it may be a combination. For example, in a polymerase chain reaction, detection (e.g., of fluorescence) may occur continuously during all or part of thermal cycling, or it may occur transiently, at one or more points during one or more cycles. In some embodiments, real time detection of PCR is accomplished by determining a level of fluorescence at the same point (e.g., a time point in the cycle, or temperature step in the cycle) in each of a plurality of cycles, or in every cycle. Real time detection of amplification may also be referred to as detection “during” the amplification reaction.As used herein, the term “abundance of nucleic acid” refers to the amount of a particular target nucleic acid sequence present in a sample or aliquot. The amount is generally referred to in terms of mass (e.g., pg), mass per unit of volume (e.g., pg / pL); copy number (e.g., 1000 copies, 1 attomole), or copy number per unit of volume (e.g, 1000 copies per mL, 1 attomole per pL). Abundance of a nucleic acid can also be expressed as an amount relative to the amount of a standard of known concentration or copy number. Measurement of abundance of a nucleic acid may be on any basis understood by those of skill in the art as being a suitable quantitative representation of nucleic acid abundance, including physical density or the sample, optical density, refractive property, staining properties, or on the basis of the intensity of a detectable label, e.g. a fluorescent label.The term “amplifying” or “amplification” in the context of nucleic acids refers to the production of multiple copies of a polynucleotide, or a portion of the polynucleotide, typically starting from a small amount of the polynucleotide (e.g., a single polynucleotide molecule), where the amplification products or amplicons are generally detectable. Amplification of polynucleotides encompasses a variety of chemical and enzymatic processes. The generation of multiple DNA copies from one or a few copies of a target or template DNA molecule during a polymerase chain reaction (PCR) or a ligase chain reaction (LCR; see, e.g., U.S. Patent No. 5,494,810; herein incorporated by reference in its entirety) are forms of amplification. Additional types of amplification include, but are not limited to, allele-specific PCR (see, e.g., U.S. Patent No. 5,639,611; herein incorporated by reference in its entirety), assembly PCR (see, e.g., U.S. Patent No. 5,965,408; herein incorporated by reference in its entirety), helicase-dependent amplification (see, e.g., U.S. Patent No. 7,662,594; herein incorporated by reference in its entirety), hot-start PCR (see, e.g., U.S. Patent Nos. 5,773,258 and 5,338,671; each herein incorporated by reference in their entireties), intersequence-specific PCR, inverse PCR (see, e.g., Triglia, et a / .(1988) NucleicAcids Res., 16:8186; herein incorporated by reference in its entirety), ligation-mediated PCR (see, e.g., Guilfoyle, R. et al., Nucleic Acids Research, 25: 1854-1858 (1997); U.S. Patent No. 5,508,169; each of which are herein incorporated by reference in their entireties), methylation-specific PCR (see, e.g., Herman, et al., (1996) PNAS 93(13) 9821-9826; herein incorporated by reference in its entirety), miniprimer PCR, multiplex ligation-dependent probe amplification (see, e.g., Schouten, et al., (2002) Nucleic Acids Research 30(12): e57; herein incorporated by reference in its entirety), multiplex PCR (see, e.g., Chamberlain, et al., (1988) Nucleic Acids Research 16(23) 11141-11156; Ballabio, et al., (1990) Human Genetics 84(6) 571-573; Hayden, et al., (2008) BMC Genetics 9:80; each of which are herein incorporated by reference in their entireties), nested PCR, overlap-extension PCR (see, e.g., Higuchi, et al., (1988) Nucleic Acids Research 16(15) 7351-7367; herein incorporated by reference in its entirety), real time PCR (see, e.g., Higuchi, et al., (1992) Biotechnology 10:413-417; Higuchi, et al., (1993) Biotechnology 11 : 1026-1030; each of which are herein incorporated by reference in their entireties), reverse transcription PCR (see, e.g., Bustin, S.A. (2000) J. Molecular Endocrinology 25: 169-193; herein incorporated by reference in its entirety), solid phase PCR, thermal asymmetric interlaced PCR, and Touchdown PCR (see, e.g., Don, et al., Nucleic Acids Research (1991) 19(14) 4008; Roux, K. (1994) Biotechniques 16(5) 812-814; Hecker, et al., (1996) Biotechniques 20(3) 478-485; each of which are herein incorporated by reference in their entireties). Polynucleotide amplification also can be accomplished using digital PCR (see, e.g., Kalinina, et al., Nucleic Acids Research. 25; 1999- 2004, (1997); Vogelstein and Kinzler, Proc Natl Acad Sci USA. 96; 9236-41, (1999);International Patent Publication No. W005023091 A2; US Patent Application Publication No. 20070202525; each of which are incorporated herein by reference in their entireties). In some embodiments, a portion of a target nucleic acid is copied in the amplification, and in some embodiments, a non-target polynucleotide is amplified in response to the presence of a target nucleic acid, (e.g., a cleaved flap, ligation product, a rolling circle replication product, etc.)The term “polymerase chain reaction” (“PCR”) refers to the method of K.B. Mullis U.S. Patent Nos. 4,683,195, 4,683,202, and 4,965,188, that describe a method for increasing the concentration of a segment of a target sequence in a mixture of genomic or other DNA or RNA, without cloning or purification. This process for amplifying the target sequence consists of introducing a large excess of two oligonucleotide primers to the DNA mixture containing the desired target sequence, followed by a precise sequence of thermal cycling in the presence of a DNA polymerase. The two primers are complementary to their respectivestrands of the double stranded target sequence. To effect amplification, the mixture is denatured and the primers then annealed to their complementary sequences within the target molecule. Following annealing, the primers are extended with a polymerase so as to form a new pair of complementary strands. The steps of denaturation, primer annealing, and polymerase extension can be repeated many times (e.g., denaturation, annealing and extension constitute one “cycle”; there can be numerous “cycles”) to obtain a high concentration of an amplified segment of the desired target sequence. The length of the amplified segment of the desired target sequence is determined by the relative positions of the primers with respect to each other, and therefore, this length is a controllable parameter. By virtue of the repeating aspect of the process, the method is referred to as the “polymerase chain reaction” (“PCR”). Because the desired amplified segments of the target sequence become the predominant sequences (in terms of concentration) in the mixture, they are said to be “PCR amplified” and are “PCR products” or “amplicons.” Those of skill in the art will understand the term “PCR” encompasses many variants of the originally described method using, e.g., real time PCR, nested PCR, reverse transcription PCR (RT-PCR), single primer and arbitrarily primed PCR, etc.As used herein, the term “primer annealing” refers to conditions that permit oligonucleotide primers to hybridize to template nucleic acid strands, preferably sufficiently to be extended by a DNA polymerase. Conditions for primer annealing vary with the length and sequence of the primer and are generally based upon the Tmthat is determined or calculated for the primer. For example, an annealing step in an amplification method that involves thermocycling involves reducing the temperature after a heat denaturation step to a temperature based on the Tmof the primer sequence, for a time sufficient to permit such annealing.As used herein, the term “amplifiable nucleic acid” is used in reference to nucleic acids that may be amplified by any amplification method. It is contemplated that “amplifiable nucleic acid” will usually comprise “sample template.”The term “amplicon” or “amplified product” refers to a segment of nucleic acid, generally DNA, generated by an amplification process such as the PCR process. The terms are also used in reference to RNA segments produced by amplification methods that employ RNA polymerases, such as NASB A, TMA, etc.The term “amplification plot” as used in reference to a thermal cycling amplification reaction refers to the plot of signal that is indicative of amplification, e.g., fluorescencesignal, versus cycle number. When used in reference to a non-thermal cycling amplification method, an amplification plot generally refers to a plot of the accumulation of signal as a function of time.The term “baseline” as used in reference to an amplification plot refers to the detected signal coming from assembled amplification reactions prior to incubation or, in the case of PCR, in the initial cycles, in which there is little change in signal.The term “Ct” or “threshold cycle” as used herein in reference to real time detection during an amplification reaction that is thermal cycled refers to the fractional cycle number at which the detected signal (e.g., fluorescence) passes the fixed threshold.The term “no template control” and “no target control” (or “NTC”) as used herein in reference to a control reaction refers to a reaction or sample that does not contain template or target nucleic acid. It is used to verify amplification quality.As used herein, the term “sample template” refers to nucleic acid originating from a sample that is analyzed for the presence of “target.” In contrast, “background template” is used in reference to nucleic acid other than sample template that may or may not be present in a sample. The presence of background template is most often inadvertent. It may be the result of carryover, or it may be due to the presence of nucleic acid contaminants sought to be purified away from the sample. For example, nucleic acids from organisms other than those to be detected may be present as background in a test sample.The present technology is not limited by the type of biological sample used or analyzed. The present technology is useful with a variety of biological samples including, but not limited to, tissue (e.g., organ (e.g., heart, liver, brain, lung, stomach, intestine, spleen, kidney, pancreas, and reproductive organs), glandular, skin, and muscle), cell (e.g., blood cell (e.g., lymphocyte or erythrocyte), muscle cell, tumor cell, and skin cell), gas, bodily fluid (e.g., blood or portion thereof, serum, plasma, urine, semen, saliva, etc.), or solid (e.g., stool) samples obtained from a human (e.g., adult, infant, or embryo) or animal (e.g., cattle, poultry, mouse, rat, dog, pig, cat, horse, and the like). In some embodiments, biological samples may be solid food and / or feed products and / or ingredients such as dairy items, vegetables, meat and meat by-products, and waste. Biological samples may be obtained from all of the various families of domestic animals, as well as feral or wild animals, including, but not limited to, such animals as ungulates, bear, fish, lagomorphs, rodents, pinnipeds, etc.Biological samples also include biopsies and tissue sections (e.g., biopsy or section of tumor, growth, rash, infection, or paraffin-embedded sections), medical or hospital samples (e.g, including, but not limited to, blood samples, saliva, buccal swab, cerebrospinal fluid,pleural fluid, milk, colostrum, lymph, sputum, vomitus, bile, semen, oocytes, cervical cells, amniotic fluid, urine, stool, hair, and sweat), laboratory samples (e.g., subcellular fractions), and forensic samples (e.g., blood or tissue (e.g., spatter or residue), hair and skin cells containing nucleic acids), and archeological samples (e.g, fossilized organisms, tissue, or cells).Environmental samples include, but are not limited to, environmental material such as surface matter, soil, water (e.g., freshwater or seawater), algae, lichens, geological samples, air containing materials containing nucleic acids, crystals, and industrial samples, as well as samples obtained from food and dairy processing instruments, apparatus, equipment, utensils, disposable and non-disposable items.Samples may be prepared by any desired or suitable method. In some embodiments, nucleic acids are analyzed directly from bodily fluids, stool, or other samples using the methods described in U.S. Pat. No. 9,000,146, which is herein incorporated by reference in its entirety for all purposes.The above-described examples are not, however, to be construed as limiting the sample (e.g., suspected of comprising a target sequence, gene, or template (e.g., the presence or absence of which can be determined using the compositions and methods of the present technology)) types applicable to the present technology.The term “nucleic acid” as used herein refers to any polynucleotide in which nucleosides are connected together via a sugar-phosphate backbone.The terms “nucleic acid sequence” and “nucleic acid molecule” as used herein refer to an oligonucleotide, nucleotide or polynucleotide, and fragments or portions thereof. The terms encompass sequences that include analogs of DNA and RNA nucleotides, including those listed above, and also including, but not limited to, 4-acetylcytosine, 8-hydroxy-N6- methyladenosine, aziridinylcytosine, pseudoisocytosine, 5-(carboxyhydroxyl-methyl)uracil, 5-fluorouracil, 5-bromouracil, 5-carboxymethylaminomethyl-2-thiouracil, 5-carboxymethyl- aminomethyluracil, dihydrouracil, inosine, N6-isopentenyladenine, 1 -methyladenine, 1- m ethylpseudo-uracil, 1-methylguanine, 1 -methylinosine, 2,2-dimethyl-guanine, 2- methyladenine, 2-methylguanine, 3-methyl-cytosine, 5-methylcytosine, N6-methyladenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxy-amino-methyl-2-thiouracil, beta- D-mannosylqueosine, 5 '-methoxy carbonylmethyluracil, 5 -methoxyuracil, 2-methylthio-N6- isopentenyladenine, uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid, oxybutoxosine, pseudouracil, queosine, 2-thiocytosine, 5-methyl-2 -thiouracil, 2-thiouracil, 4- thiouracil, 5 -methyluracil, N-uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid,pseudouracil, queosine, 2-thiocytosine, 2,6-diaminopurine, and pyrazolo[3,4-d]pyrimidines such as guanine analogue 6 amino lH-pyrazolo[3,4d]pyrimidin 4(5H) one (ppG or PPG, also Super G) and the adenine analogue 4 amino lH-pyrazolo[3,4d]pyrimidine (ppA or PPA). The xanthine analogue lH-pyrazolo[5,4d]pyrimidin 4(5H)-6(7H)-dione (ppX) can also be used. These base analogues, when present in an oligonucleotide, strengthen hybridization, and improve mismatch discrimination. All tautomeric forms of naturally occurring bases, modified bases and base analogues may be included in the oligonucleotide conjugates of the technology. Other modified bases useful in the present technology include 6-amino-3-prop-l- ynyl-5-hydropyrazolo[3,4-d]pyrimidine-4-one, PPPG; 6-amino-3 -(3 -hydroxyprop- l-yny)l-5- hydropyrazolo[3,4-d]pyrimidine-4-one, HOPPPG; 6-amino-3-(3-aminoprop-l-ynyl)-5- hydropyrazolo[3,4-d]pyrimidine-4- -one, NH2PPPG; 4-amino-3-(prop-l-ynyl)pyrazolo[3,4- d]pyrimidine, PPP A; 4-amino-3-(3-hydroxyprop-l-ynyl)pyrazolo[3,4-d]pyrimidine, HOPPPA; 4-amino-3 -(3 -aminoprop- l-ynyl)pyrazolo[3,4-d]pyrimidine, NH2 PPP A; 3 -prop- 1- ynylpyrazolo[3,4-d]pyrimidine-4,6-diamino, (NTfc)? PPP A; 2-(4,6-diaminopyrazolo[3,4- d]pyrimidin-3-yl)ethyn-l-ol, (NTfc)? PPPAOH; 3-(2-aminoethynyl)pyrazolo[3,4- d]pyrimidine-4,6-diamine, (NTfc)? PPPANH2; 5-prop-l-ynyl-l,3-dihydropyrimidine-2,4- dione, PU; 5-(3-hydroxyprop-l-ynyl)-l,3-dihydropyrimidine-2, 4-dione, HOPU; 6-amino-5- prop-l-ynyl-3-dihydropyrimidine-2-one, PC; 6-amino-5-(3-hydroxyprop-l-yny)-l,3- dihydropyrimidine-2-one, HOPC; and 6-amino-5-(3-aminoprop-l-yny)-l,3- dihy dropyrimidine-2-one, NH2PC ; 5 - [4-amino-3 -(3 -methoxyprop- 1 -ynyljpyrazol [3,4- d]pyrimidinyl]-2-(hydroxymethyl)oxolan-3-ol, CH3 OPPPA; 6-amino-l-[4-hydroxy-5- (hydroxymethyl)oxolan-2-yl]-3-(3-methoxyprop-l-ynyl)-5-hydropyrazolo[3,4-d]pyrimidin-4- one, CH3 OPPPG; 4, (4,6-Diamino-lH-pyrazolo[3,4-d]pyrimidin-3-yl)-but-3-yn-l-ol, Super A; 6-Amino-3-(4-hydroxy-but-l-ynyl)-l,5-dihydro-pyrazolo[3,4-d]pyrimidin-4-one; 5-(4- hydroxy-but-l-ynyl)-lH-pyrimidine-2, 4-dione, Super T; 3-iodo-lH-pyrazolo[3,4- d]pyrimidine-4,6-diamine ((NEh^PPAI); 3-bromo-lH-pyrazolo[3,4-d]pyrimidine-4,6- diamine ((NH2)2 PPABr); 3-chloro-lH-pyrazolo[3,4-d]pyrimidine-4,6-diamine ((NH2)2PPAC1); 3-Iodo-lH-pyrazolo[3,4-d]pyrimidin-4-ylamine (PPAI); 3-Bromo-lH- pyrazolo[3,4-d]pyrimidin-4-ylamine (PPABr); and 3-chloro-lH-pyrazolo[3,4-d]pyrimidin-4- ylamine (PPAC1). The term encompasses base analogs that provide alternative hydrogen bonding configurations (e.g., Iso-C and Iso-G and other non-standard base pairs described in U.S. Patent No. 6,001,983 to S. Benner); non-hydrogen bonding analogs (e.g., non-polar, aromatic nucleoside analogs such as 2,4-difluorotoluene, described by B.A. Schweitzer and E.T. Kool, J. Org. Chem., 1994, 59, 7238-7242, B.A. Schweitzer and E.T. Kool, J. Am.Chem. Soc., 1995, 117, 1863-1872); "universal" bases such as 5-nitroindole and 3- nitropyrrole; and universal purines and pyrimidines (such as "K" and "P" nucleotides, respectively; P. Kong, et al., Nucleic Acids Res., 1989, 17, 10373-10383, P. Kong et al., Nucleic Acids Res., 1992, 20, 5149-5152). Nucleotide analogs include modified forms of deoxyribonucleotides as well as ribonucleotides.A nucleic acid sequence or molecule may be DNA or RNA, of either genomic or synthetic origin, that may be single or double stranded, and represent the sense or antisense strand. Thus, nucleic acid sequence may be dsDNA, ssDNA, mixed ssDNA, mixed dsDNA, dsDNA made into ssDNA (e.g., through melting, denaturing, helicases, etc.), A-, B-, or Z- DNA, triple-stranded DNA, RNA, ssRNA, dsRNA, mixed ss and dsRNA, dsRNA made into ssRNA (e.g., via melting, denaturing, helicases, etc.), messenger RNA (mRNA), ribosomal RNA (rRNA), transfer RNA (tRNA), catalytic RNA, snRNA, microRNA, or protein nucleic acid (PNA).The present technology is not limited by the type or source of nucleic acid (e.g., sequence or molecule (e.g. target sequence and / or oligonucleotide)) utilized. For example, the nucleic acid sequence may be amplified or created sequence (e.g., amplification or creation of nucleic acid sequence via synthesis (e.g., polymerization (e.g., primer extension (e.g., RNA- DNA hybrid primer technology)) and reverse transcription (e.g., of RNA into DNA)) and / or amplification (e.g., polymerase chain reaction (PCR), rolling circle amplification (RCA), nucleic acid sequence based amplification (NASBA), transcription mediated amplification (TMA), ligase chain reaction (LCR), cycling probe technology, Q-beta replicase, strand displacement amplification (SDA), branched-DNA signal amplification (bDNA), hybrid capture, and helicase dependent amplification).The terms “nucleotide” and “base” are used interchangeably when used in reference to a nucleic acid sequence, unless indicated otherwise herein, and refer to a nucleoside comprising a sugar molecule and a nucleobase, and optionally a 5' phosphate. A “nucleobase” is a heterocyclic nitrogenous base such as adenine, guanine, cytosine, thymine, uracil, inosine, xanthine, hypoxanthine, or a heterocyclic derivative, analog, or tautomer thereof. A nucleobase can be naturally occurring or synthetic. Non-limiting examples of nucleobases are adenine, guanine, thymine, cytosine, uracil, xanthine, hypoxanthine, 8- azapurine, purines substituted at the 8 position with methyl or bromine, 9-oxo-N6- methyladenine, 2-aminoadenine, 7-deazaxanthine, 7-deazaguanine, 7-deaza-adenine, N4- ethanocytosine, 2,6-diaminopurine, N6-ethano-2,6-diaminopurine, 5-methylcytosine, 5-(C3- C6)-alkynylcytosine, 5 -fluorouracil, 5-bromouracil, thiouracil, pseudoisocytosine, 2-hydroxy-5-methyl-4-triazolopyridine, isocytosine, isoguanine, inosine, 7,8-dimethylalloxazine, 6- dihydrothymine, 5,6-dihydrouracil, 4-methyl-indole, ethenoadenine and the non-naturally occurring nucleobases described in U.S. Pat. Nos. 5,432,272 and 6,150,510 and PCT applications WO 92 / 002258, WO 93 / 10820, WO 94 / 22892, and WO 94 / 24144, and Fasman (“Practical Handbook of Biochemistry and Molecular Biology”, pp. 385-394, 1989, CRC Press, Boca Raton, LO), all herein incorporated by reference in their entireties.As used herein, the term “non-standard” as used in reference to nucleotides and nucleobases, e.g., in oligonucleotides, refers to nucleotides other than adenine, guanine, cytosine, thymine, and uracil deoxy- and ribonucleotides. Non-standard nucleotides include analog and derivative nucleobases, including but not limited to those described hereinabove in reference to “nucleic acid sequence” and “nucleotide” definitions.As used herein, the term “standard” as used in reference to nucleotides and nucleobases, e.g., in oligonucleotides, refers to adenine, guanine, cytosine, thymine, and uracil deoxy- and ribonucleotides.The term “oligonucleotide” as used herein is defined as a molecule comprising two or more nucleotides (e.g., deoxyribonucleotides or ribonucleotides), preferably at least 5 nucleotides, more preferably at least about 10-15 nucleotides and more preferably at least about 15 to 30 nucleotides, or longer (e.g., oligonucleotides are typically less than 200 residues long (e.g., between 15 and 100 nucleotides), however, as used herein, the term is also intended to encompass longer polynucleotide chains). The exact size will depend on many factors, which in turn depend on the ultimate function or use of the oligonucleotide. Oligonucleotides are often referred to by their length. For example, a 24-residue oligonucleotide is referred to as a “24-mer”. Oligonucleotides can form secondary and tertiary structures by self-hybridizing or by hybridizing to other polynucleotides. Such structures can include, but are not limited to, duplexes, hairpins, cruciforms, bends, and triplexes. Oligonucleotides may be generated in any manner, including chemical synthesis, DNA replication, reverse transcription, PCR, or a combination thereof. In some embodiments, oligonucleotides that form invasive cleavage structures are generated in a reaction (e.g., by extension of a primer in an enzymatic extension reaction).Because mononucleotides are reacted to make oligonucleotides in a manner such that the 5' phosphate of one mononucleotide pentose ring is attached to the 3' oxygen of its neighbor in one direction via a phosphodiester linkage, an end of an oligonucleotide is referred to as the “5' terminus” or 5' end if its 5' phosphate is not linked to the 3' oxygen of a mononucleotide pentose ring and as the “3' terminus or 3' end” if its 3' oxygen is not linkedto a 5' phosphate of a subsequent mononucleotide pentose ring. As used herein, a nucleic acid sequence, even if internal to a larger oligonucleotide, also may be said to have 5' and 3' ends. As distinct from the 3' and 5' ends of a nucleic acid molecule, the 3' and 5' ends of a nucleic acid sequence need not be at the 3' and 5' termini of the molecule strand. A first region along a nucleic acid strand is said to be upstream of another region if the 3' end of the first region is before the 5' end of the second region when moving along a strand of nucleic acid in a 5' to 3' direction.When two different, non-overlapping oligonucleotides anneal to different regions of the same linear complementary nucleic acid sequence, and the 3' end of one oligonucleotide points towards the 5' end of the other, the former may be called the “upstream” oligonucleotide and the latter the “downstream” oligonucleotide.As used herein, “base pairing rules” refers to the rules for the pairing of nucleotide bases established by Watson and Crick. In standard Watson-Crick geometry, large purines (A and G bases) base pair with a small pyrimidine bases (T and C), with A pairing with T (or U) and G pairing with C, so that the A-T base pair is essentially the same size as the G-C base pair. This means that the rungs of a DNA ladder, formed from either A-T (or A-U) or G-C base pairs, all have the same length across the helix. Under the standard Watson-Crick rules for base pairing, A-T and A-U base pairs form two hydrogen bonds between the base moieties, and G-C base pairs form three hydrogen bonds.Nucleotides may pair in non-standard ways, such as in “wobble” base pairing, in which one or more hydrogen bonds are formed but the geometry of the base pair does not fit within the standard Watson-Crick geometry. Among the most common of THE wobble base pairs are G-T (or U) base pairing, in which a single hydrogen bond is formed between G and T (or G and U). Non-Watson-Crick base pairing also includes base pairs formed by inosine (I) with C, T, or A, purine base pairs A-G (or I), A-A, and G- C, and the reverse Watson- Crick base pair A-T, in which the T ring is rotated 180° from the normal Watson-Crick pair. See, e.g., Hyone-Myong Eun, Enzymology Primer for Recombinant DNA Technology, Academic Press, (1996),As used herein, “standard base pair / base pairing” refers to base pairs having standard Watson-Crick geometry, and encompassing A-T, A-U, and G-C base pairs. The term encompasses base pairing of modified analogs of these nucleotides in which the modification (e.g., an attached dye or other moiety) does not substantially alter the hydrogen bonding between the bases (e.g., the number, positions, or acceptor / donor pairs of the hydrogen bonds. )As used herein, “non-standard base pair / base pairing” refers to pairing betweennucleotides e.g., natural nucleotides, nucleotide analogs, that does not fall within standard base pairing having standard Watson-Crick geometry.As used herein, the terms “complementary” or “complementarity” are used in reference to polynucleotides (e.g., a sequence of two or more nucleotides (e.g., an oligonucleotide or a target nucleic acid)) related by the base-pairing rules. For example, the sequence “5'-A-G-T-3',” is complementary to the sequence “3 -T-C-A-5'.” Complementarity may be “partial,” in which only some of the nucleic acid bases are matched according to the base pairing rules. Or, there may be “complete” or “total” complementarity between the nucleic acid bases. The degree of complementarity between nucleic acid strands has significant effects on the efficiency and strength of hybridization between nucleic acid strands. This is of particular importance in amplification reactions, as well as detection methods that depend upon the association of two or more nucleic acid strands. Either term may also be used in reference to individual nucleotides, especially within the context of polynucleotides. For example, a particular nucleotide within an oligonucleotide may be noted for its complementarity, or lack thereof, to a nucleotide within another nucleic acid sequence (e.g., a target sequence), in contrast or comparison to the complementarity between the rest of the oligonucleotide and the nucleic acid sequence.The complement of a nucleic acid sequence as used herein refers to an oligonucleotide which, when aligned with the nucleic acid sequence such that the 5' end of one sequence is paired with the 3' end of the other, is in “antiparallel association.” Nucleotide analogs, as discussed above, may be included in the nucleic acids of the present technology, and include, for example, inosine, 7-deazaguanine, iso-C, and iso-G. Complementarity need not be perfect; stable duplexes may contain mismatched base pairs or unmatched bases. Those skilled in the art of nucleic acid technology can determine duplex stability empirically considering a number of variables including, for example, the length of the oligonucleotide, base composition and sequence of the oligonucleotide, ionic strength, and incidence of mismatched base pairs.As used herein, the term “label” refers to any moiety (e.g., chemical species) that can be detected or can lead to a detectable response. In some preferred embodiments, detection of a label provides quantifiable information. Labels can be any known detectable moiety, such as, for example, a radioactive label (e.g., radionuclides), a ligand (e.g., biotin or avidin), a chromophore (e.g., a dye or particle that imparts a detectable color), a hapten (e.g., digoxygenin), a mass label, latex beads, metal particles, a paramagnetic label, a luminescent compound (e.g., bioluminescent, phosphorescent, or chemiluminescent labels) or afluorescent compound. A label may also comprise a phosphor particle for detection. In particularly preferred embodiments, the phosphor particle is an up-converting phosphor particle (see, e.g., Ostermayer, F.W. Preparation and properties of infrared-to-visible conversion phosphors. Metall. Trans. 752, 747-755

[1971] ). In some embodiments, rare earth- doped ceramic particles are used as phosphor particles. Phosphor particles may be detected by any suitable method, including but not limited to up-converting phosphor technology (UPT), in which up-converting phosphors transfer low energy infrared (IR) radiation to high- energy visible light. While the present invention is not limited to any particular mechanism, in some embodiments the UPT up-converts infrared light to visible light by multi-photon absorption and subsequent emission of dopant-dependent phosphorescence. See, e.g., U.S. Patent No. 6,399,397, Issued June 4, 2002 to Zarling, et al. van De Rijke, et cd.. Nature Biotechnol. 19(3):273-6

[2001] ; Corstjens, etal., lEE Proc. Nanobiotechnol. 152(2):64

[2005] , each incorporated by reference herein in its entirety.As used herein, the term “distinct” in reference to signals, e.g., from different labels, refers to signals that can be differentiated one from another, e.g, by spectral properties such as fluorescence emission wavelength, color, absorbance, mass, size, fluorescence polarization properties, charge, etc., or by capability of interaction with another moiety, such as with a chemical reagent, an enzyme, an antibody, etc.A label may be joined, directly or indirectly, to an oligonucleotide or other biological molecule. Direct labeling can occur through bonds or interactions that link the label to the oligonucleotide, including covalent bonds or non-covalent interactions such as hydrogen bonding, hydrophobic and ionic interactions, or through formation of chelates or coordination complexes. Indirect labeling can occur through use of a bridging moiety or “linker”, such as an antibody or additional oligonucleotide(s), which is / are either directly or indirectly labeled.Labels can be used alone or in combination with moi eties that can suppress (e.g, quench), excite, or transfer (e.g., shift) emission spectra (e.g., fluorescence resonance energy transfer (FRET)) of a label (e.g., a luminescent label).A “polymerase” is an enzyme generally for joining 3'-OH 5 '-triphosphate nucleotides, oligomers, and their analogs. Polymerases include, but are not limited to, template-dependent DNA-dependent DNA polymerases, DNA-dependent RNA polymerases, RNA-dependent DNA polymerases, and RNA-dependent RNA polymerases. Polymerases include but are not limited to T7 DNA polymerase, T3 DNA polymerase, T4 DNA polymerase, T7 RNA polymerase, T3 RNA polymerase, SP6 RNA polymerase, DNA polymerase 1, KI enow fragment, Thermophilus aquaticus DNA polymerase, Tth DNA polymerase, Vent DNApolymerase (New England Biolabs), Deep Vent DNA polymerase (New England Biolabs), Bst DNA Polymerase Large Fragment, Stoeffel Fragment, 9°N DNA Polymerase, Pfu DNA Polymerase, Tfl DNA Polymerase, RepliPHI Phi29 Polymerase, TH DNA polymerase, eukaryotic DNA polymerase beta, telomerase, Therminator polymerase (New England Biolabs), KOD HiFi DNA polymerase (Novagen), K0D1 DNA polymerase, Q-beta replicase, terminal transferase, AMV reverse transcriptase, M-MLV reverse transcriptase, Phi6 reverse transcriptase, HIV-1 reverse transcriptase, novel polymerases discovered by bioprospecting, and polymerases cited in US 2007 / 0048748, U.S. Pat. Nos. 6,329,178;6,602,695; and 6,395,524 (incorporated by reference). These polymerases include wild-type, mutant isoforms, and genetically engineered variants.A “DNA polymerase” is a polymerase that produces DNA from deoxynucleotide monomers (dNTPs). “Eubacterial DNA polymerase” as used herein refers to the Pol A type DNA polymerases (repair polymerases) from Eubacteria, including but not limited to DNA Polymerase I from E. coli. Taq DNA polymerase from Thermus aquaticus and DNA Pol I enzymes from other members of genus Thermus, and other eubacterial species etc.As used herein, the term “target,” as used in reference to a nucleic acid strand or site on a nucleic acid strand, refers to a nucleic acid species or nucleic acid sequence or structure sought to be sorted out from other nucleic acids, e.g., by probe binding, amplification, isolation, capture, etc. For example, when used in reference to the polymerase chain reaction, “target” refers to the region of nucleic acid bounded by the primers used for polymerase chain reaction, while when used in an assay in which target DNA is not amplified, e.g., in some embodiments of an invasive cleavage assay, a target comprises the site at which a probe and invasive oligonucleotides (e.g., INVADER oligonucleotide) bind to form an invasive cleavage structure, such that the presence of the target nucleic acid can be detected. A “segment” is defined as a region of nucleic acid within the target sequence. As used in reference to a double-stranded nucleic acid, the term “target” is not limited to a particular strand of the duplexed target, e.g., a coding strand, but may be used in reference to either one or both strands of, for example, a double-stranded gene or reference DNA.Accordingly, as used herein, “non-target”, e.g., as it is used to describe a nucleic acid such as a DNA, refers to nucleic acid that may be present in a reaction, but that is not the subject of detection or characterization by the reaction. In some embodiments, non-target nucleic acid may refer to nucleic acid present in a sample that does not, e.g., contain a target sequence, while in some embodiments, non-target may refer to exogenous nucleic acid, i.e.,nucleic acid that does not originate from a sample containing or suspected of containing a target nucleic acid, and that is added to a reaction, e.g., to normalize the activity of an enzyme (e.g., polymerase) to reduce variability in the performance of the enzyme in the reaction.As used herein, the terms “cell-free” and “circulating cell-free” as used in reference to nucleic acids from blood are used interchangeably and refer to nucleic acids, e.g., DNA and RNA species, that are found in blood but that are not within cells in the blood. The terms as used herein with respect to nucleic acid extracted from blood refer to the nature and location of the nucleic acid prior to collection of the sample from the subject and prior to extraction of the nucleic acid from the blood sample.The term “marker”, as used herein, refers to a substance (e.g., a nucleic acid, or a region of a nucleic acid, or a protein) that may be used to distinguish non-normal cells (e.g., cancer cells, cells carrying a mutation) from normal cells (e.g., non-cancerous cells or cells lacking a mutation), e.g., based on presence, absence, or status (e.g, methylation state, mutation) of the marker substance. As used herein “normal” methylation of a marker refers to a degree of methylation typically found in normal cells, e.g., in non-cancerous cells.As used herein, the term “amplification reagents” refers to those reagents (deoxyribonucleoside triphosphates, buffer, etc.), needed for amplification except for primers, nucleic acid template, and the amplification enzyme. Typically, amplification reagents along with other reaction components are placed and contained in a reaction vessel.A “reaction mixture” is a mixture of reagents (e.g., oligonucleotides, target nucleic acids, enzymes, etc.) in a combination and / or locus in which a reaction can occur, e.g., a mixture of reagents in a single reaction vessel, at a locus in a fluidic device, at locus on a surface, etc.As used herein, a “multiplex” reaction is a reaction (e.g., RCA, ligation, PCR, PCR- flap assay) that operates on multiple targets (2, 3, 4, 5, 6, 7, 8, . . . . 20, 30, 100, 1000, 100,000, etc.) in a single reaction mixture. Multiplexed reactions are distinguished from reactions that operate on one target analyte per reaction mixture. As used herein, the term “highly multiplexed” refers to reactions that operate on at least 6, preferably at least 10, more preferably at least 20 or more different targets (e.g., different genes, or regions of genes) in a single reaction mixture.As used herein, the term “control” when used in reference to nucleic acid detection or analysis refers to a nucleic acid having known features (e.g., known sequence, known copy-number per cell), for use in comparison to an experimental target (e.g., a nucleic acid of unknown concentration). A control may be an endogenous, preferably invariant gene against which a test or target nucleic acid in an assay can be normalized. Such normalizing controls for sample-to-sample variations that may occur in, for example, sample processing, assay efficiency, etc., and allows accurate sample-to-sample data comparison. Controls may also be external. For example, in quantitative assays such as qPCR, QuARTS, etc., a “calibrator” or “calibration control” is a nucleic acid of known sequence, e.g., having the same sequence as a portion of an experimental target nucleic acid, and a known concentration or series of concentrations (e.g., a serially diluted control target for generation of calibration curved in quantitative PCR). Typically, calibration controls are analyzed using the same reagents and reaction conditions as are used on an experimental DNA. In certain embodiments, the measurement of the calibrators is done at the same time, e.g., in the same thermal cycler, as the experimental assay. In preferred embodiments, multiple calibrators may be included in a single plasmid, such that the different calibrator sequences are easily provided in equimolar amounts. In particularly preferred embodiments, plasmid calibrators are digested, e.g., with one or more restriction enzymes, to release calibrator portion from the plasmid vector. See, e.g., WO 2015 / 066695, which is included herein by reference.As used herein the term “fish DNA” refers to bulk (e.g., genomic) DNA isolated from fish, e.g., as described in U.S. Patent No.9, 212, 392. Bulk purified fish DNA is commercially available, e.g., provided in the form of cod and / or herring sperm DNA (Roche Applied Science, Mannheim, Germany) or salmon DNA (USB / Affymetrix). “Fish DNA” is distinct from any particular gene from a fish that is in isolated form, e.g., that has been separately synthesized or that has been separated from the other DNA of the fish genome.As used herein, the “sensitivity” of a given marker (or set of markers used together) refers to the percentage of samples that report a DNA methylation value above a threshold value that distinguishes between neoplastic and non-neoplastic samples. In some embodiments, a positive is defined as a histology-confirmed neoplasia that reports a DNA methylation value above a threshold value (e.g., the range associated with disease), and a false negative is defined as a histology-confirmed neoplasia that reports a DNA methylation value below the threshold value (e.g., the range associated with no disease). The value of sensitivity, therefore, reflects the probability that a DNA methylation measurement for a given marker obtained from a known diseased sample will be in the range of disease- associated measurements. As defined here, the clinical relevance of the calculated sensitivityvalue represents an estimation of the probability that a given marker would detect the presence of a clinical condition when applied to a subject with that condition.As used herein, the “specificity” of a given marker (or set of markers used together) refers to the percentage of non-neoplastic samples that report a DNA methylation value below a threshold value that distinguishes between neoplastic and non-neoplastic samples. In some embodiments, a negative is defined as a histology-confirmed non-neoplastic sample that reports a DNA methylation value below the threshold value (e.g., the range associated with no disease) and a false positive is defined as a histology-confirmed non-neoplastic sample that reports a DNA methylation value above the threshold value (e.g., the range associated with disease). The value of specificity, therefore, reflects the probability that a DNA methylation measurement for a given marker obtained from a known non-neoplastic sample will be in the range of non-disease associated measurements. As defined here, the clinical relevance of the calculated specificity value represents an estimation of the probability that a given marker would detect the absence of a clinical condition when applied to a patient without that condition.As used herein, a “selected nucleotide” refers to one nucleotide of the four typically occurring nucleotides in a nucleic acid molecule (C, G, T, and A for DNA and C, G, U, and A for RNA), and can include methylated derivatives of the typically occurring nucleotides (e.g., when C is the selected nucleotide, both methylated and unmethylated C are included within the meaning of a selected nucleotide), whereas a methylated selected nucleotide refers specifically to a nucleotide that is typically methylated and an unmethylated selected nucleotides refers specifically to a nucleotide that typically occurs in unmethylated form.The term “methylation-specific restriction enzyme” refers to a restriction enzyme that selectively digests a nucleic acid dependent on the methylation state of its recognition site. In the case of a restriction enzyme that specifically cuts if the recognition site is not methylated or is hemi-methylated (a methylation-sensitive enzyme), the cut will not take place (or will take place with a significantly reduced efficiency) if the recognition site is methylated on one or both strands. In the case of a restriction enzyme that specifically cuts only if the recognition site is methylated (a methylation-dependent enzyme), the cut will not take place (or will take place with a significantly reduced efficiency) if the recognition site is not methylated. Preferred are methylation-specific restriction enzymes, the recognition sequence of which contains a CG dinucleotide (for instance a recognition sequence such as CGCG orCCCGGG). Further preferred for some embodiments are restriction enzymes that do not cut if the cytosine in this dinucleotide is methylated at the carbon atom C5.The terms “selectively binds” and “specifically binds” (or selectively or specifically binding, bound, hybridizes, hybridizing, anneals, annealing, etc.) as used in reference to interaction between oligonucleotides or other nucleic acids are used herein interchangeably to refer to hybridization or base-pairing that is sufficiently sequence-selective that the oligonucleotide or nucleic acid will preferentially hybridize to a particular nucleic acid (e.g., a target nucleic acid having a particular nucleotide sequence) and will not substantially bind to non-target nucleic acids (e.g., having slightly or completely different nucleotide sequences than the target nucleic acid), under conditions in which selective or specific binding is conducted.The term “probe” refers to an oligonucleotide (e.g., a sequence of nucleotides), whether occurring naturally as in a purified restriction digest or produced synthetically, recombinantly, or by PCR amplification, that is capable of hybridizing to another oligonucleotide of interest. A probe may be single-stranded or double-stranded. Probes are useful in the detection, identification, and isolation of particular gene sequences (e.g., a “capture probe”). It is contemplated that any probe used in the present invention may, in some embodiments, be labeled with any “reporter molecule,” so that it is detectable in any detection system, including, but not limited to enzyme (e.g., ELISA, as well as enzyme-based histochemical assays), fluorescent, radioactive, and luminescent systems. It is not intended that the present invention be limited to any particular detection system or label. Probe oligonucleotides include, e.g., “flap oligonucleotides” that, when used in reference to a flap assay, refers to an oligonucleotide that interacts with a target nucleic acid to form a cleavage structure in the presence of an invasive oligonucleotide, and padlock probes that interact with a nucleic acid strand to form an invasive cleavage structure. In some embodiments, flap oligonucleotides comprise a portion, typically a 5' portion that forms a single-stranded 5' flap of several nucleotides in the invasive cleavage structure. As used herein in reference to invasive cleavage structures, “probe oligonucleotide” is used interchangeably with “flap oligonucleotide” and refer to the oligonucleotide that forms the downstream duplex and is cleaved in an invasive cleavage structure.The term “invasive oligonucleotide” refers to an oligonucleotide that hybridizes to a target nucleic acid at a location adjacent to the region of hybridization between a probe and the target nucleic acid, wherein the 3' end of the invasive oligonucleotide comprises a portion(e.g., a chemical moiety, or one or more nucleotides) that overlaps with the region of hybridization between the probe and target. The 3' terminal nucleotide of the invasive oligonucleotide may or may not base pair with a nucleotide in the target. In some embodiments, the invasive oligonucleotide contains sequences at its 3' end that are substantially the same as sequences located at the 5' end of a portion of the probe oligonucleotide that anneals to the target strand. In some embodiments, e.g., in a PCR-flap assay, a primer used for amplification may also serve as an invasive oligonucleotide with probe.As used herein, the term “kit” refers to any delivery system for delivering materials. In the context of nucleic acid purification systems and reaction assays, such delivery systems include systems that allow for the storage, transport, or delivery of reagents and devices (e.g., chaotropic salts, particles, buffers, denaturants, oligonucleotides, filters etc. in the appropriate containers) and / or supporting materials (e.g., sample processing or sample storage vessels, written instructions for performing a procedure, etc.) from one location to another. For example, kits include one or more enclosures (e.g., boxes) containing the relevant reaction reagents and / or supporting materials. As used herein, the term “fragmented kit” refers to a delivery system comprising two or more separate containers that each contains a subportion of the total kit components. The containers may be delivered to the intended recipient together or separately. For example, a first container may contain materials for sample collection and a buffer, while a second container contains capture oligonucleotides and denaturant. The term “fragmented kit” is intended to encompass kits containing Analyte specific reagents (ASR's) regulated under section 520(e) of the Federal Food, Drug, and Cosmetic Act, but are not limited thereto. Indeed, any delivery system comprising two or more separate containers that each contains a subportion of the total kit components are included in the term “fragmented kit.” In contrast, a “combined kit” refers to a delivery system containing all of the components of a reaction assay in a single container (e.g., in a single box housing each of the desired components). The term “kit” includes both fragmented and combined kits.The term “system” as used herein refers to a collection of articles for use for a particular purpose. In some embodiments, the articles comprise instructions for use, as information supplied on e.g., an article, on paper, online (e.g., at a website or web address) on recordable media (e.g., diskette, CD, DVD, flash drive, etc.). In some embodiments, instructions direct a user to an online location, e.g., a website for viewing, hearing, and / or downloading instructions.In some embodiments, instructions or other information are provided as an application (“app”), e.g., for a computer or for a mobile device, such as a smart phone.BRIEF DESCRIPTION OF THE DRAWINGSFigs. 1A and IB provide schematic diagrams of invasive cleavage structures formed from three nucleic acid strands. In Fig. 1 A, an invasive oligonucleotide is hybridized with a nucleic acid strand, e.g., a target DNA, to form an upstream duplex, with a single nucleotide unpaired at the 3' end of the invasive oligonucleotide. A downstream oligonucleotide is hybridized to the nucleic acid strand to form a downstream duplex. The downstream oligonucleotide comprises an unpaired 5' flap. As illustrated, the upstream and downstream duplexes are immediately adjacent to each other, such that the target strand has no gap in base pairing between the upstream and downstream duplexes. Fig. IB is similar, but the downstream oligonucleotide does not comprise an unpaired 5' flap. The structures shown in Figs. 1 A and IB are both substrates for cleavage by FEN-1 endonucleases, with cleavage typically occurring after the first paired nucleotide (z.e., the 5'-most paired nucleotide) of the oligonucleotide in the downstream duplex, as illustrated.Figs. 2A, 2B, and 2C together illustrate embodiment of the technology. In Fig. 2A, an unligatable padlock probe is shown hybridized to a nucleic acid strand. The padlock probe comprises a target-specific 3' portion that forms an upstream duplex with the target strand, and a target-specific 5' end that forms an adjacent downstream duplex with the target strand. The grey line indicates a connecting backbone, e.g., a sequence of nucleotides that joins the two target-specific regions of the probe. The 5' terminus of the probe is configured to be unligatable. For example, in some embodiments, the 5' terminal nucleotide lacks an accessible 5' phosphate for ligation, as indicated by the “xx” in this figure. The “xx” may be, e.g., a hexane, a non-standard nucleotide, an abasic nucleotide e.g., a ribose or deoxyribose), a dye tag, a flap nucleic acid sequence, a hairpin flap, or any other group or modification that prevents ligation of a phosphate at the 5' end of the downstream duplexed portion of the probe to the hydroxyl at the 3' end. In preferred embodiments, the “xx” moiety is attached to the 5' nucleotide of the duplexed portion of the probe sequence via a phosphate, e.g., a phosphodiester bond, as shown schematically in Fig. 2A and 2B.Fig. 2B illustrates a structure formed when the Fig. 2A structure is treated with a FEN endonuclease that cleaves the 5' portion of the probe after the first base pair of the downstream duplex, thereby releasing at least one nucleotide, preferably only one nucleotide, and the 5' blocking group. Cleavage by a FEN endonuclease leaves a phosphate on the 5'terminus, resulting in a nick that is ligatable using, e.g., a DNA ligase. After cleavage of the 5' end of the padlock probe, the 3' terminal nucleotide of the probe can hybridize to the target strand to form a ligatable nick with the cleaved 5' end of the padlock probe.Fig. 2C illustrates the structure formed when the Fig. 2B structure is treated with ligase to covalently link the ends of the cleaved padlock probe, whereby the probe is circularized.Figs. 3A-3C show an embodiment in which the hybridized padlock probe does not comprise an unpaired 5' flap and in which the 5' terminal nucleotide of the probe has a 5' OH group. As illustrated in Fig. 3B, cleavage of the structure by a FEN endonuclease removes the 5' terminal nucleotide and leaves a 5' terminus having a 5' phosphate. After cleavage of the 5' end, the 3' terminal nucleotide can hybridize to the target strand to form a ligatable nick. Fig. 3C illustrates the structure formed when the Fig. 3B structure is treated with ligase to covalently link the ends of the cleaved padlock probe, whereby the probe is circularized.Figs. 4A-4C show an embodiment in which the hybridized padlock probe comprises an unpaired 5' flap. As illustrated in Fig. 4B, cleavage of the structure by a FEN endonuclease removes the 5' flap and one nucleotide of the downstream duplex and leaves a 5' terminus having a 5' phosphate. After cleavage of the 5' flap, the 3' terminal nucleotide of the probe can hybridize to the target strand to form a ligatable nick. Fig. 4C illustrates the structure formed when the Fig. 4B structure is treated with ligase to covalently link the ends of the cleaved padlock probe, whereby the probe is circularized.Figs 5 A and 5B illustrate an embodiment for detecting a methylated C in a strand of bisulfite-treated DNA in which unmethylated cytosines have been converted to deoxy uracil (dU). Fig. 5A shows a padlock probe hybridized to methylated DNA to form upstream and downstream duplexes, with the methylated C nucleotide (shown in bold) hybridizing to the probe to form the first base pair of a downstream duplex. In the manner illustrated in Figs. 2A-2C, the structure in Fig. 5A is cleavable with a FEN endonuclease to form a ligatable nick. Fig. 5B shows the same padlock probe hybridized to unmethylated DNA that has been converted with bisulfite as described above. There is a gap between the upstream and downstream duplexes, and the downstream duplex is mismatched to the target DNA. The structure shown in Fig. 5B is not readily cleavable by a strict FEN-1 endonuclease to form a ligatable nick.Figs 6A and 6B illustrate an embodiment for detecting an unmethylated C in a strand of bisulfite-treated DNA in which unmethylated cytosines have been converted to dU. Fig. 6A shows a padlock probe hybridized to unmethylated bisulfite-treated DNA to formupstream and downstream duplexes, with the converted dU nucleotide (shown in bold) hybridizing to the probe to form the first base pair of a downstream duplex. In the manner illustrated in Figs. 2A-2C, the structure in Fig. 6A is cleavable with a FEN endonuclease to form a ligatable nick. Fig. 6B shows the same padlock probe hybridized to methylated DNA that has been treated with bisulfite as described above, but in which methylation blocks conversion of C nucleotides in CpG loci. There is a gap between the upstream and downstream duplexes, and the downstream duplex is also mismatched to the target DNA. The structure shown in Fig. 6B is not readily cleavable by a strict FEN-1 endonuclease to form a ligatable nick.Figure 7 illustrates an invasive cleavage structure formed on a target strand by hybridization of a padlock probe oligonucleotide having a 5' hairpin-forming region and an upstream invasive 3' portion. When the 5' hairpin is present, e.g., at temperatures below the melting temperature of the hairpin stem, cleavage at the indicated site is suppressed. At flap assay reaction temperatures, typically above the observed melting temperature of the 5' hairpin, flap endonuclease cleavage at the indicated cleavage site is not suppressed, and cleavage can occur to release the 5' flap arm from the padlock probe oligonucleotide to produce a ligatable nick. See, e.g., WO 2021 / 055508 Al to Gagrat, et al., which is incorporated herein by reference in its entirety.DETAILED DESCRIPTION OF THE INVENTIONThe technology relates to using padlock probes to form structures cleavable by flap endonucleases. In particular, the technology relates to methods of using flap endonucleases, in particular thermostable FEN-1 endonucleases, to cleave a probe hybridized to a target nucleic acid in a cleavage structure, wherein the hybridized cleaved probe becomes circularizable by a ligase.Target nucleic acidIn preferred embodiments, target nucleic acids comprise DNA, including but not limited to cDNA, amplified DNA, genomic DNA, circulating cell-free DNA, etc. In some embodiments the DNA is treated with a methylation-specific reagent, as defined hereinabove. For example, in the embodiments illustrated in Figs. 2A-6B, the target strand show represents DNA that has been treated with a bisulfite reagent such that unmethylated cytosines are converted to deoxyuracils (shown as U). In some embodiments, target nucleic acid strandsare replicated or amplified, e.g., by PCR, prior to hybridization of the padlock probe. In such embodiments, the Us in the converted target DNA sequence are replaced by Ts in the replicate strands. The probe embodiments illustrated in Figs. 2A-6B are equally applicable to such replicated target DNA. Similarly, sequence variations not related to methylation, e.g., single nucleotide polymorphisms (SNPs) and mutations may be distinguished from reference nucleic acids using similar design strategies.Padlock probesIn preferred embodiments, a padlock probe of the technology comprises a 3' portion serving as an invasive oligonucleotide and a 5' portion serving as a probe oligonucleotide, wherein the 3' portion and 5' portion are linked, preferably covalently linked, via a backbone portion of the molecule. The lengths and sequences of the 5' and 3' portions may be selected to selectively hybridize to particular target strands to form an invasive cleavage structure under conditions of an assay to be performed. For example, in some embodiments, the sequences are selected to form an invasive cleavage structure with a target nucleic acid under conditions useful or optimal for cleavage by a thermostable FEN-1 endonuclease, preferably a strict FEN-1 endonuclease, e.g., at a temperature above 60°C, and in the presence of a high Mg++buffer (e.g., preferably greater than 4 mM Mg++, more preferably at least about 5, 6, 7, 8, 9, 10, or 11 mM Mg++.) In certain preferred embodiments, the 5' portion forms a downstream duplex that is at least 6 bp, preferably at least 7, preferably at least 8, 9, 10, 11, 12, 13, or 14 bp. In certain preferred embodiments, the 3' portion forms an upstream duplex that is at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 bp. See, e.g., Kaiser M.W., et al. (1999) J. Biol. Chem., 274:21387.In preferred embodiments, the backbone is composed of nucleic acid. In addition to the 3' and 5' portions, the padlock probe may comprise one or more regions useful in detection (e.g., in amplification, sequencing, individual identification, etc.), including but not limited to primer binding sites, detection tags, identifying sequences, promoter sites (e.g., for transcription), or secondary targets for additional probe binding. See, e.g., U.S. Pat. No. 6,316,229 to Lizardi; and U.S. Pat. Pub. 2014 / 0342354, to Evans, each of which is incorporated herein by reference in its entirety.Hybridization of padlock probe and FEN cleavageIn preferred embodiments, padlock probes are hybridized to target nucleic acid, preferably single-stranded nucleic acid, under suitable conditions, e.g., in a buffer suitable for sequence-specific hybridization of DNA, e.g., in the presence of mono- or divalent cations. In some embodiments the buffer is also suitable for flap cleavage by a FEN enzyme, while in some embodiments, components of a flap cleavage buffer are added after hybridization. In some embodiments, hybridization is done in the presence of a FEN enzyme, preferably a strict thermostable FEN-1 enzyme, such that cleavage occurs when hybridization to the target nucleic acid forms an invasive structure.An aspect of the technology is use of FEN endonuclease under conditions in which the enzyme exhibits maximum specificity for an invasive cleavage structure, e.g., as illustrated in Figs. 1 A and IB, as compared to, e.g., a gapped structure (having a singlestranded gap between the upstream and downstream duplexes), Y-structure (having a 5' flap but no overlap between the upstream and downstream duplexes), pseudo-Y structure (having a 5' flap and an un-duplexed template strand upstream of the downstream duplex), etc. Thus, in preferred embodiments, cleavage of hybridized padlock probes is done in a Mg++flap assay buffer. As used herein, the term “Mg++flap assay buffer” refers to a buffer solution for a flap endonuclease that includes Mg++as the predominant or essentially only divalent cation in the buffer, such that cleavage reactions performed in the buffer, e.g., cleavage of hybridized padlock probe that forms an invasive cleavage structure, exhibits characteristics of the presence of Mg++, and does not exhibit characteristics associated with the presence of other divalent cations e.g., alternative cleavage activities of flap endonucleases associated with use of Mn++with or in place of Mg++), as described, e.g., by Kaiser, et al., supra.In some embodiments the flap assay buffer has very low or no KC1 and comprises elevated Mg++as compared to standard PCR buffer, (e.g., a flap assay buffer of the technology comprises >6 mM, preferably > 7mM, more preferably 7.5mM Mg++, while PCR buffers typically comprise about 1.5 to 2.5 mM Mg++.) In preferred embodiments, cleavage is conducted in a flap assay buffer comprising 7.5 mM MgCh, 10 mM MOPS, 0.3 mM Tris- HC1, pH 8.0, 0.8 mM KC1, 0.1 pg / pL BSA, 0.0001% Tween-20, and 0. 0001% IGEPAL CA- 630. While not limiting the technology to any particular FEN endonuclease, in preferred embodiments, cleavage reactions comprise a thermostable FEN-1 endonuclease selected from CLEAVASE 2.0 nuclease (Hologic, Inc.), Archaeoglobus fulgidus (Afu) FEN-1, Pyrococcus furiosus (Pfu) FEN-1, and Archaeoglobus veneficus (Ave) FEN-1 (see, e.g., WO 02 / 070755).LigationLigation of a 5' end of nucleic acid to a 3' end of nucleic acid can occur when the two ends are base paired to adjacent nucleotides of a complementary sequence, and wherein the 5' terminal nucleotide has a 5' phosphate and the 3' terminal nucleotide has a 3' hydroxyl group. Base pairing of the respective end nucleotides to the adjacent nucleotides in a template strand forms a nucleic acid duplex containing a nick. Ligation of the two ends at the nick can be catalyzed by DNA ligase. Providing conditions for ligation will therefore usually comprise providing a DNA ligase enzyme and reaction conditions under which the DNA ligase ligates the two ends to form a continuous nucleic acid strand, closing the nick. Preferably, a high- fidelity ligase is used under conditions that maximize mismatch discrimination. For example, an amount of HiFi Taq DNA Ligase (New England Biolabs) is used, preferably in a buffer of 20 mM Tris-HCl; 150 mM KC1; 10 mM MgCh; 10 mM DTT; 1 mM NAD; 0.1% Triton® X- 100; (pH 8.5 @ 25°C) and is incubated at between 37-75°C for 10-60 min, or by thermal cycling at 95°C for 30 s to 1 min, followed by annealing / ligation for 1 - 5 min at a ligation temperature selected for a particular probe used. A number of other ligase enzymes are commercially available, including Ampligase (Epicentre), for which suitable conditions are to add 1 U enzyme and incubate at 55° C. for 1 hour in ligase buffer. In some embodiments, padlock probes are ligated in 10 mM Tris-acetate pH 7.5, 10 mM magnesium acetate, 50 mM NaCl, 1 mM ATP, 1 pg / mL BSA and 0.2 U / pL T4 DNA ligase (Amersham Pharmacia Biotech, Uppsala, Sweden) at 37°C for 30 min in the presence of the target DNA. See, e.g., Nilsson, et al., Nucl. Acids Res. 30(14):e66 (2002) and Lohman, G. et al. (2016). Nucleic Acids Res. 44(2):el4-el4 DOI: 0.1093 / nar / gkv898, each of which is incorporated herein by reference in its entirety.In some embodiments, a cleaved padlock probe is ligated in the same buffer in which FEN cleavage occurs, while in some embodiments, the cleavage reaction mixture is desalted or diluted prior to ligation, e.g., to provide buffer conditions more suitable for a selected ligase. In some embodiments, reaction conditions for cleavage are selected for compatibility with a selected ligase, whereby the cleaved probes may be used in a ligation reaction without dilution or desalting.For example, in some embodiments, cleavage and ligation are performed in a buffer comprising MgCh, and ATP (e.g., 10 mM MOPS, pH 7.5, 2 mM MgCh, 20 pg / ml tRNA, 1 mM ATP; see Kaiser et al., supra).In some embodiments, , a reaction mixture comprises target nucleic acids, padlock probes, FEN endonuclease, and a ligase enzyme. It has been shown that HiFi Taq DNA Ligase can be used in alternative buffers, e.g., Taq DNA Ligase buffer with reduced activity and fidelity. HiFi Taq DNA Ligase can also be used in a variety of polymerase buffers when supplemented with 1 mM NAD, with reduced activity and modestly reduced fidelity:(see www<dot>neb<dot>com / faqs / 2016 / 03 / 18 / can-hifi-taq-dna-ligase-be-used-in-other- buffers)1Supplemented with 1 mM NAD• IX HiF i T aq DNA Ligase buffer:20 mM Tris-HCl; 150 mM KC1; 10 mM MgCh; 10 mM DTT; 1 mM NAD; 0.1% Triton® X-100; (pH 8.5 @ 25°C)• Phi29 Reaction buffer:50 mM Tris-HCI pH 7.5, 10 mM MgC12, 10 mM (NH4)SO4, 4 mM dithiothreitol (pH 7.5 @ 25°C)• Standard Taq reaction buffer conditions:10 mM Tris-HCl; 50 mM KC1; andl.5 mM MgCh (pH 8.3 @ 25°C).• Exemplary flap assay reaction buffer:7.5 mM MgCh, 10 mM MOPS, 0.3 mM Tris-HCl, pH 8.0, 0.8 mM KC1, 0.1 pg / pL BSA, 0.0001% Tween-20, and 0. 0001% IGEPAL CA-630It is contemplated that the similarities of these buffers are indicative that this and other ligases are compatible with conditions suitable for DNA polymerase and FEN-1 endonuclease activities. For example, as noted above, Kaiser performed cleavage and ligation in a buffer comprising 10 mM MOPS, pH 7.5, 2 mM MgCh, 20 pg / ml tRNA, and 1 mM ATP (see Kaiser et al., supra).Enrichment of ligated productsA method may include enriching the ligation products before detection. Products may be enriched by amplification and / or by solid phase chemistry. Circular nucleic acid products may be selectively enriched by treating the sample with exonuclease (e.g., Lambda exonuclease) to digest linear nucleic acid products, e.g., target nucleic acid fragments, unligated padlock probes, etc. In general, exonuclease degradation may be used to enrich for ligation products when the ligation products are protected from exonuclease degradation. Exonuclease should then be deactivated (e.g. by heat) before any subsequent step involving polymerization, e.g. before rolling circle amplification. See, e.g., US Pat. No. 10,526,643, supra, and U.S. Patent No. 11,186,863.Exonuclease may be added to remove non-reacted probes and fragments. Suitable conditions are incubation at 37°C for 1 hour in corresponding exonuclease buffer, followed by enzyme inactivation at 80°C for 20 minutes. Where capture / detect methods are used, ligation products may be enriched by capturing the products on a solid phase via the capture moiety. For example, as illustrated in example 1 of US Pat. No. 10526643B2, a solution containing ligation products may be mixed with 10 mL M-280 streptavidin coated magnetic beads (Invitrogen) in Tris-HCI (pH 7.5), 3.5 mM EDTA and 0.07% Tween-20 in a final volume of 200 ml, and incubated at room temperature for 15 minutes. After incubation, the beads are collected using a magnet and supernatant is removed. See also Example 3 in U.S. Patent No. 11,186,863. Other ways of enriching for ligation products include specifically size-selecting ligation products.Amplification of ligated productsCircularization of padlock probes is readily detected by a number of different nucleic acid assays, e.g., PCR (e.g., across the ligated junction), ligase chain reaction, reporter probe hybridization, rolling circle amplification, etc. Suitable amplification techniques include but are not limited to rolling circle amplification (see below), bridge PCR (Adessi C, et al.,Nucleic Acids Res. 2000 Oct 15;28(20):E87), emulsion PCR (digital PCR in emulsions was described by Dressman et al., Proc Natl Acad Sci U S A. 2003 Jul 22;100(15):8817-22. Epub 2003 Jul 1 1 ) and digital PCR (Vogelstein & Kinzler, Proc Natl Acad Sci U S A. 1999 Aug 3;96(16):9236-41). Clonal localized amplification in gels was described by Mitra & Church, Nucleic Acids Res. 1999 December 15; 27(24): e34. An embodiment of the present method may comprise amplifying the ligation products and obtaining a cumulative signal which is a combination of individual signals from the amplified products. Preferably, ligation products are amplified across the ligation junction.In preferred embodiments, amplification may comprise providing conditions for rolling circle replication of the circles of nucleic acid and detecting the products of rolling circle replication. Rolling circle replication is described in, for example, US 5,854,033 (Lizardi); Fire & Xu, Proc Natl Acad Sci U S A. 1995 May 9;92(10):4641 -5, and M. Ali, et al. “Rolling circle amplification: a versatile tool for chemical biology, materials science and medicine”. Chemical Society Reviews. 43 (10): 3324-3341, each of which is incorporated herein by reference in its entirety, for all purposes.Rolling circle replication is an amplification of a circular nucleic acid molecule using a hybridized primer that is extended using strand-displacing DNA polymerase, resulting in large DNA molecules containing tens to hundreds of tandem repeats complementary to the circular template.(See, e.g., M. Ali, et al. supra, see also US Pat. No. 10,526,643B2, which is incorporated herein by reference in its entirety, for all purposes.) Polymerases typically used in RCA are Phi29 ( 29), Bst, and Vent exo-DNA polymerases, with Phi29 DNA polymerase being preferred in view of its superior processivity and strand displacement ability. Additional amplification can be obtained using a cascade of strand displacement reactions. For example, rolling circle replication may be hyper-branched rolling circle replication, as described by Lizardi et al., Nat Genet. 1998 Jul;19(3):225-32.In some embodiments, the ligation reaction mixture is desalted or diluted prior to detection, e.g., to provide buffer conditions more suitable for a selected polymerase. In some embodiments, reaction conditions for cleavage and / or ligation are selected for compatibility with a selected polymerase, whereby the ligated material may be used in an amplification reaction without dilution or desalting.By way of non-limiting example, in some embodiments, polymerization reactions are performed using a primer hybridized to the ligated padlock probe in 50 mM Tris-HCI pH 7.5,10 mM MgC12, 20 mM (NH4)SO4, 10 mM dithiothreitol, 0.2 pg / mL BSA, 0.25 mM dNTPs, and 2 ng / mL Phi29 DNA polymerase at 37°C. In some embodiments, primers for RCA are immobilized on a support, e.g., a bead or other surface. See, e.g., U.S., Pat. No. 6,316,229 to Lizardi and US Pat. No. 10,526,643B2.In some embodiments, RCA reactions may be monitored in real time, during the extension reaction. For example, the RCA may be performed in the presence of 100 nM of a molecular beacon probe that is complementary to the concatemer product and 300 nM ROX dye. Molecular beacon probes typically comprise a FRET system comprising a fluorophore that is quenched in the unhybridized probe and that is unquenched when the probe hybridizes to a target sequence. The reactions are followed in real time by measuring fluorescence, e.g., in an ABI 7700 real-time PCR instrument. Fluorescence values are typically given as a ratio between the fluorescence emitted by the molecular beacon (e.g., labeled with FAM, HEX or other fluorphore) and the ROX reference dye.Numerous methods have been developed for detecting the products of RCA, e.g., in solution, in emulsions, bound to beads or surfaces, etc. See, e.g., US 7,862,999 to U.S. Pat. No. 6,316,229 to Lizardi, et al.; M. Ali, et al. “Rolling circle amplification: a versatile tool for chemical biology, materials science and medicine”. Chemical Society Reviews. 43 (10): 3324-3341, and U.S. Patent No. 11,186,863, each of which is incorporated herein by reference in its entirety, for all purposes.In some embodiments, RCA is performed in the same buffer in which FEN cleavage and / or ligation occurs. In certain embodiments, a reaction mixture comprises target nucleic acids, padlock probes, FEN endonuclease, a ligase enzyme, and a DNA polymerase.Identifying elements in padlock probesIn some embodiments, a padlock probe may comprise sequence elements that enable downstream analysis of the circularized molecule or the RCA product. For example, in some embodiments, padlock probes comprise tag sequences, identifying sequences, , one or more primer binding sites, restriction sites, etc., which are generally useful for capturing, identifying, sequencing, and / or counting individual probes or families of probes that have been circularized by ligation in the presence of target nucleic acid. For example, in some embodiments, counting detected codes and estimating target quantity -based counts of detected codes, e.g., finds use in estimating target quantity based on a correlation of counts ofdetected codes and target quantity in the sample analyzed. See, e.g., U.S. Pat. No. 6,316,229 to Lizardi and U.S. Pat. Pub. 2014 / 0342354, to Evans, each of which is incorporated herein by reference in its entirety for all purposes. In particularly preferred embodiments, the ligated padlock probes are used as template for a DNA polymerase, e.g., in a rolling circle amplification reaction or a variant thereof.Additional materials incorporated herein by reference1. Kaiser M, et al. J Biol Chem. 1999. Vol 274, No 30, 21387-21394.2. Dorjsuren D, et al. Nucleic Acids Research. 2011. Vol 39, No 2 el 13. Lyamichev V, et al. Biochemistry. 2000. 39, 9523-9532.4. Tsutakawa S, et al. Cell. 2011. 145, 198-211.5. Tumey N, et al. Bioorg & Med Chem Let. 2004. 15, 277-281.6. Hall JG, et al. Proc Natl Acad Sci USA 2000; 97: 8272-8277. Sensitive detection of DNA polymorphisms by the serial invasive signal amplification reaction.7. Allawi HT, et al. J of Clin Microbio 2006; vol 44, no. 9: 3443-3447. Invader Plus Method Detects Herpes Simplex Virus in Cerebrospinal Fluid and Simultaneously Differentiates Types 1 and 2.8. M. Ali, et al. “Rolling circle amplification: a versatile tool for chemical biology, materials science and medicine.” Chemical Society Reviews. 43 (10): 3324-3341.9. M. Nilsson, et al. “Padlock probes: circularizing oligonucleotides for localized DNA detection”. Science. 265 (5181): 2085-2088 (1994).10. J. Baner, et al., Signal amplification of padlock probes by rolling circle replication Nucleic Acids Research 26 (22): 5073-5078 (1998).11. P.-J. J. Huang, and J. Liu, “Molecular Beacon Lighting up on Grapheme Oxide, ’’Anal. Chem. 84:4192-4198 (2012)12. US 10,526,643 B2 “Multiplex Detection of Nucleic Acids”13. U.S. Pat. Pub. US 2015 / 0284786 Al “Compositions and methods for molecular inversion probe assays.14. Hardenbol, et al., Multiplexed genotyping with sequence-tagged molecular inversion probes. Nature Biotechnology v21(6):673 (2003)15. Mohsen, M. et al., The Discovery of Rolling Circle Amplification and Rolling Circle Transcription, Acc Chem Res. 2016 November 15; 49(11): 2540-2550. doi : 10.1021 / acs . accounts .6b00417All literature and similar materials cited in this application, including but not limited to, patents, patent applications, articles, books, treatises, and internet web pages are expressly incorporated by reference in their entirety for any purpose. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of ordinary skill in the art to which the various embodiments described herein belongs. When definitions of terms in incorporated references appear to differ from the definitions provided in the present teachings, the definition provided in the present teachings shall control.Various modifications and variations of the described compositions, methods, and uses of the technology will be apparent to those skilled in the art without departing from the scope and spirit of the technology as described. Although the technology has been described in connection with specific exemplary embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention that are obvious to those skilled in molecular biology, nucleic acid chemistry, biochemistry, medical science, or related fields are intended to be within the scope of the following claims.Features, materials, characteristics, or groups described in conjunction with a particular aspect, embodiment, or example are to be understood to be applicable to any other aspect, embodiment or example described in this section or elsewhere in this specification unless incompatible therewith. All of the features disclosed in this specification (including any accompanying claims, abstract and drawings), and / or all of the steps of any method or process so disclosed, may be combined in any combination, except combinations where at least some of such features and / or steps are mutually exclusive. The protection is not restricted to the details of any foregoing embodiments. The protection extends to any novel one, or any novel combination, of the features disclosed in this specification (including any accompanying claims, abstract and drawings), or to any novel one, or any novel combination, of the steps of any method or process so disclosed.Furthermore, certain features that are described in this disclosure in the context of separate implementations can also be implemented in combination in a single implementation. Conversely, various features that are described in the context of a single implementation can also be implemented in multiple implementations separately or in anysuitable subcombination. Moreover, although features may be described above as acting in certain combinations, one or more features from a claimed combination can, in some cases, be excised from the combination, and the combination may be claimed as a subcombination or variation of a subcombination.

Claims

CLAIMSWhat is claimed is:

1. A method, comprising: a) providing a padlock probe hybridized to a strand of target nucleic acid to form a FEN-1 cleavage substrate complex comprising a downstream duplex and an upstream duplex that define an invasive cleavage structure cleavable at a cleavage site in the padlock probe on a 3' side of a first paired nucleotide of the downstream duplex by a strict FEN-1 endonuclease in a Mg++flap assay buffer, wherein the padlock probe comprises a 5' blocking group; b) cleaving the cleavage substrate complex with a strict FEN-1 endonuclease in conditions wherein cleavage of the invasive cleavage structure at the cleavage site produces: i) a cleaved substrate complex comprising a nick that is ligatable by a DNA ligase, and ii) a 5' cleavage product consisting of the 5' blocking group and the first paired nucleotide from the downstream duplex of the cleavage substrate complex; and c) treating the cleaved substrate complex with a ligase to form a circularized padlock probe.

2. The method of claim 1, wherein in the cleavage substrate complex, the 5' blocking group is attached to a 5' position of the first paired nucleotide in the downstream duplex.

3. The method of claim 1 or claim 2, wherein the 5' blocking group comprises a moiety selected from a hexane, a non-standard nucleotide, an abasic nucleotide, a fhiorophore, a flap nucleic acid sequence, a hairpin flap nucleic acid sequence, and an hydroxyl group.

4. The method of claim 3, wherein the 5' blocking group is attached to the first paired nucleotide by a phosphodiester bond.

5. The method of any one of claims 1-4, wherein the 5' blocking group comprises a non-standard nucleotide.

6. The method of any one of claims 1-5, wherein the 5' blocking group does not comprise a standard nucleotide.

7. The method of any one of claims 1-6, wherein the strict FEN-1 endonuclease is selected from the group consisting of Archaeoglobus fulgidus (Afu) FEN-1, Pyrococcus furiosus (Pfu) FEN-1, and Archaeoglobus veneficus (Ave) FEN-1, and CLEAVASE 2.0 nuclease.

8. The method of any one of claims 1-7, further comprising detecting the circularized padlock probe in a nucleic acid detection assay.

9. The method of claim 8, wherein the nucleic acid detection assay comprises rolling circle amplification.

10. The method of any one of claims 1-9, wherein the target nucleic acid is extracted from a sample.

11. The method of claim 10, wherein the target nucleic acid is treated with a methylation-specific reagent.

12. The method of claim 11, wherein the methylation-specific reagent modifies a methylated nucleotide to produce a different nucleotide.

13. The method of claim 12, wherein the methylation-specific reagent modifies a methylated cytosine to produce a dihydrouracil.

14. The method of claim 11, wherein the methylation-specific reagent modifies an unmethylated nucleotide to produce a different nucleotide.

15. The method of claim 14, wherein the methylation-specific reagent modifies an unmethylated cytosine to produce a uracil.

16. The method of any one of claims 1-9, wherein the target nucleic acid is amplified nucleic acid.

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