Method and device for measuring double-chain body unwinding or chain exchange efficiency

Abstract

Provided herein are quantitative assay methods for evaluating duplex unwinding efficiency or chain exchange efficiency during isothermal amplification.

Description

[0001] Priority claim

[0002] This application claims priority to U.S. patent application No. 17 / 863,110 filed on July 12, 2022, the entire contents of which are incorporated herein by reference. Background Art

[0003] Nucleic acid amplification tests (NAATs) are highly sensitive and critical for detecting pathogens that pose one of the major threats to global public health. However, most current NAAT assays are PCR-based, requiring instrumentation that is incompatible with resource-limited settings. As an alternative to PCR-based assays, isothermal amplification-based assays are easy-to-use processes that rapidly amplify nucleic acids at a constant temperature, suitable for use in point-of-care settings or at-home testing. These methods have been widely accepted in clinical applications during the SARS-CoV-2 pandemic, and the FDA has approved multiple emergency use authorization (EUA) kits. Summary of the invention

[0004] The present disclosure is based, at least in part, on the development of a quantitative assay that utilizes fragment analysis to assess duplex unwinding efficiency or strand exchange efficiency during isothermal amplification.

[0005] Isothermal techniques are based on a group of synergistic enzymes, such as HDA (helicase-dependent amplification) and RPA (recombinase polymerase amplification), which open DNA duplexes without thermal cycling. In HDA and RPA techniques, rapid isothermal amplification depends on the synchronization of multiple events (duplex unwinding, primer binding, polymerase extension) with multiple enzymes (enzymes for duplex unwinding or strand exchange, polymerases and auxiliary proteins). It is of interest to develop new assays for quantitatively analyzing duplex unwinding or strand exchange efficiency to further implement and improve assays based on isothermal amplification. Such methods and assays are described herein.

[0006] Thus, aspects of the present disclosure provide methods for detecting unwinding or strand exchange of a double-stranded nucleic acid, the method comprising: providing a sample comprising a pre-annealed duplex, wherein the pre-annealed duplex comprises a target nucleic acid and a synthetic nucleic acid comprising an optional first detectable label; a primer comprising a second detectable label; an amplifying enzyme for amplifying the nucleic acid; and an unwinding enzyme for unwinding or strand exchange of the double-stranded nucleic acid; incubating the sample under isothermal amplification conditions and for a time sufficient for nucleic acid amplification; and detecting the second detectable label and optionally, if present, the first detectable label.

[0007] In some embodiments, the synthetic nucleic acid comprises an initial primer, and wherein the initial primer comprises the first detectable label.

[0008] In some embodiments, the incubation further comprises extending the initial primer, thereby providing an extension product comprising the first detectable label; unwinding the extension product; annealing a primer comprising the second detectable label to a portion of the target nucleic acid; and extending the primer, thereby providing a further extension product comprising the second detectable label.

[0009] In some embodiments, the synthetic nucleic acid comprises an extension product of an initial primer, and wherein the initial primer comprises the first detectable label. In some embodiments, the synthetic nucleic acid comprises an extended nucleic acid containing about 20 or more nucleotides. In some embodiments, the extended nucleic acid comprises the first detectable label.

[0010] In some embodiments, incubating further comprises unwinding the duplex; annealing a primer comprising the second detectable label to a portion of the target nucleic acid; and extending the primer, thereby providing a further extension product comprising the second detectable label.

[0011] In some embodiments, the method further comprises, prior to said providing, annealing said target nucleic acid and said synthetic nucleic acid, thereby forming said pre-annealed duplex; and optionally cooling said pre-annealed duplex.

[0012] In some embodiments, the method further comprises, prior to said providing, annealing said target nucleic acid and said synthetic nucleic acid, wherein said synthetic nucleic acid is configured as an initial primer; and extending said initial primer under polymerase primer extension conditions, thereby forming said pre-annealed duplex.

[0013] In some embodiments, the method further comprises, after the incubation, inactivating the sample using heat inactivation (e.g., about 45-100° C.) and / or chemical inactivation (e.g., chelating agents such as ethylenediaminetetraacetic acid (EDTA), proteases such as proteinase K, etc.).

[0014] In some embodiments, the method further comprises, prior to said detecting, purifying said sample to remove one or more chemical or biological components.

[0015] In some embodiments, the synthetic nucleic acid and the primer comprise the same nucleotide sequence. In some embodiments, the synthetic nucleic acid is shorter than the primer or is the same length as the primer. In some embodiments, the synthetic nucleic acid is longer than the primer. In some embodiments, the synthetic nucleic acid and the primer independently have a length of about 5 to 100 nucleotides.

[0016] In some embodiments, the pre-annealed duplex comprises a 3'-tail end and / or a 5' tail end. In some embodiments, the pre-annealed duplex comprises at least one blunt end. In some embodiments, the pre-annealed duplex has a length of about 15 to 500 base pairs.

[0017] In some embodiments, the ratio of the primer to the pre-annealed duplex is from 1:1 to 50:1.

[0018] In some embodiments, the first and second detectable labels are the same or different. In some embodiments, the first and second detectable labels are independently provided at the 5'-end or internally.

[0019] In some embodiments, the synthetic nucleic acid comprises a plurality of first detectable labels, and / or wherein the primer comprises a plurality of second detectable labels. In some embodiments, the first and second detectable labels are selected from fluorescent labels, radioactive labels, chemiluminescent labels, or dyes.

[0020] In some embodiments, the sample further comprises 3'-amino-2',3'-dideoxyribonucleotide 5'-triphosphate (nNTP), divalent ions, denaturants, buffers, and / or salts.

[0021] In some embodiments, the amplification enzyme and / or the helicase is selected from a helicase, a recombinase, a polymerase, a reverse transcriptase, a thermophilic form thereof, a thermostable form thereof, and a recombinant form thereof.

[0022] In some embodiments, the isothermal amplification conditions comprise a temperature of about 20 to 75°C.

[0023] In some embodiments, detection includes electrophoretic analysis, optionally on a microfluidic device. In some embodiments, the electrophoretic analysis includes analyzing the fragment size of the amplicon comprising the second detectable label. In some embodiments, the electrophoretic analysis further includes comparing the fragment size of the amplicon with a labeled size standard (e.g., a fluorescently labeled size standard).

[0024] Aspects of the disclosure provide kits comprising: a pre-annealed duplex, a primer comprising a second detectable label, an amplification enzyme, a helicase, and instructions for performing the methods described herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figures 1A-1D Schematic diagram illustrating a non-limiting method for detecting unwinding of a double-stranded nucleic acid during isothermal amplification using a fluorescently labeled primer (Design 1). Such a method may include a fluorescently labeled primer that is added in excess in a reaction mixture and used to form a pre-annealed duplex that includes a target strand and the fluorescently labeled primer. Figure 1A is a non-limiting schematic diagram showing a reaction mixture for detecting duplex unwinding during isothermal amplification. Figure 1B is a non-limiting schematic diagram showing an electropherogram of fluorescent fragments produced in an analytical reaction mixture. Figure 1C is a graph showing the theoretical percentage of extension product for reaction mixtures with different primer:duplex ratios. Figure 1D is a non-limiting schematic diagram showing different pre-annealed duplexes that can be used in the methods described herein.

[0026] Figures 2A-2D Schematic diagram illustrating an additional non-limiting method for detecting unwinding of a double-stranded nucleic acid during isothermal amplification using a fluorescently labeled primer (Design 2). Such a method may include a fluorescently labeled primer that is added to a reaction mixture and a longer fluorescently labeled synthetic nucleic acid that is used to form a pre-annealed duplex comprising a target strand and a fluorescently labeled complementary strand. Figure 2A is a non-limiting schematic diagram showing a reaction mixture for detecting duplex unwinding during isothermal amplification. Figure 2B is a non-limiting schematic diagram showing an electropherogram of fluorescent fragments produced in an analytical reaction mixture. Figure 2C is a graph showing the theoretical percentage of extension product for reaction mixtures with different primer:duplex ratios. Figure 2D is a non-limiting schematic diagram showing different pre-annealed duplexes that can be used in the methods described herein.

[0027] Figure 3A-3B are images of electropherograms from analysis of tHDA unwinding efficiency using Design 1. Schematic diagrams of the pre-annealed duplex structures used in the reactions are shown above each panel. Left panel: The reaction mixture includes a pre-annealed duplex with a blunt end (at the 3'-end of the template) and a 5'-tail end. Right panel: The reaction mixture includes a pre-annealed duplex with a 5'-tail end and a 3'-tail end. The reaction mixture includes different ratios of pre-annealed duplexes and fluorescently labeled primers (see Table 2). The reaction mixture includes tHDA ( Figure 3A) or BstLF( Figure 3B ).

[0028] Figure 4 are images of electropherograms from analysis of tHDA unwinding efficiency using Design 2. Left: 1:1 primer to duplex ratio. Right: 2:1 primer to duplex ratio.

[0029] Figure 5 are images of electropherograms from analysis of duplex strand exchange efficiency of RPA using Design 2. Left: 1:1 primer to duplex ratio. Right: 2:1 primer to duplex ratio. DETAILED DESCRIPTION

[0030] Provided herein are methods for detecting unwinding or strand exchange of double-stranded nucleic acids. Such methods can employ labeled nucleic acids in pre-annealed duplexes and / or primers. By using such labeled nucleic acids, unwinding and extension events can be monitored during isothermal amplification. For example, unwinding can be detected by using a pre-annealed duplex, the pre-annealed duplex comprising (i) a target strand (or template), the target strand (or template) being bound to (ii) a synthetic nucleic acid, the synthetic nucleic acid being labeled with a detectable label. Unwinding will release the synthetic nucleic acid, and the nucleic acid (or an extension product comprising the nucleic acid) can be detected by labeling.

[0031] In addition, unwinding will release the target strand to allow further binding by other primers. If the other primers include detectable labels, the extension products of such primers will also include detectable labels. In this way, using labeled pre-annealed duplexes and / or labeled primers can allow detection of repeated unwinding, primer annealing and primer extension events. In some cases, primer extension events can be considered as effective duplex unwinding events for isothermal amplification. Therefore, monitoring primer extension products rather than unwinding oligonucleotides can indicate the efficiency of duplex unwinding under isothermal amplification.

[0032] In one non-limiting example, the method includes a labeled primer, which can be added to the reaction mixture in any useful amount (e.g., excess or not excess) and can be used to form a pre-annealed duplex including a target strand and a fluorescently labeled primer. Figures 1A-1D Such an approach is described, also referred to as Design 1.

[0033] like Figure 1AAs shown in (below), when the reaction mixture contains a polymerase and a buffer, only the fluorescently labeled primers in the pre-annealed duplex can be extended by the polymerase. Under some non-restrictive isothermal conditions, no excess fluorescently labeled primers in the reaction mixture are extended because the duplex can undergo limited or no unwinding to release the target strand (or template) of the duplex. Therefore, the binding of excess fluorescently labeled primers to the target strand is limited, and a limited level of extension products will be formed with such excess primers.

[0034] The amount of extension product and fluorescently labeled primer (unextended fluorescently labeled primer) in the reaction mixture can then be detected by size separation and fluorescence detection, for example, using a microfluidic platform such as GXTouch TM Nucleic acid analyzer. Under conditions containing a polymerase without unwinding activity, only the labeled primer in the pre-annealed duplex can be extended by the polymerase, and the detectable signal from the label in the reaction mixture will depend on the concentration of the labeled pre-annealed duplex and labeled primer in the reaction mixture. Figure 1B As shown, where the initial reaction mixture includes a primer-to-duplex ratio of approximately 1:1, using only the polymerase, approximately 50% of the detected fluorescence comes from the extension product (solid black peak), and approximately 50% comes from the fluorescently labeled primer (open gray peak).

[0035] Also like Figure 1A As shown in (above), when the reaction mixture contains a helicase and a polymerase (isothermal amplification mixture), the duplex is unwound by the helicase, thereby releasing the target strand (or template) from the duplex to allow it to bind to the excess fluorescently labeled primer in the reaction mixture. The fluorescently labeled primer in the newly formed duplex can then be extended by the polymerase to produce a fluorescently labeled extension fragment. The amount of fluorescently labeled extension product formed can indicate the duplex unwinding efficiency of the helicase. As shown in Figure 1B As shown, under some non-restrictive isothermal amplification conditions, approximately 100% of the detected fluorescence comes from the extension product (solid black peak), indicating that the helicase efficiently unwinds the duplex to allow the fluorescently labeled primer to anneal in the reaction mixture, where it can be extended by the polymerase.

[0036] Figure 1CThe theoretical percentage of extension products for reaction mixtures containing multiple primers to duplex ratios is shown in Figure 3. When the reaction mixture contains only polymerase, increasing the primer:duplex ratio reduces the amount of extension products formed and detected, because unwinding does not occur, thereby limiting the formation of new duplexes with fluorescently labeled primers from the reaction mixture. When the reaction mixture contains polymerase and helicase, fluorescently labeled primers can be extended by the duplex via the cooperative unwinding of the helicase and the cooperative extension of the polymerase in the newly formed duplex. The reaction mixture lacking an excess of fluorescently labeled primers can be used as a positive control for detecting extension products under isothermal amplification conditions.

[0037] like Figure 1D As shown, the pre-annealed duplex may include a target nucleic acid chain that is longer than the fluorescently labeled primer. A portion of the target nucleic acid may be fully complementary to the fluorescently labeled primer to allow the target nucleic acid to hybridize to a duplex with the fluorescently labeled primer. Depending on the position of the complementary portion within the target chain, the duplex may include a flat end on one side (e.g., at the 3'-end of the target) and a 5'-tail end; or it may include a 3'-tail end and a 5'-tail end. In addition, although a fluorescent label is shown, any detectable label may be used in such a construct or any method herein.

[0038] Provided herein are methods for detecting unwinding of double-stranded nucleic acids using short fluorescently labeled primers and pre-annealed duplexes in which one strand is fluorescently labeled that are added to a reaction mixture. Figures 2A-2D Describe such an approach, also called Design 2.

[0039] like Figure 2A As shown in (bottom), without protein for duplex unwinding in the reaction mixture, the fluorescently labeled primer cannot bind to the internal sequence of the target strand and thus only longer fragments will be detected. In contrast, in the case where protein is used for duplex unwinding in the reaction mixture ( Figure 2A In the upper middle panel), there will be recycling of duplex unwinding and primer binding / extension, which leads to accumulation of extended fragments, which are detected together with longer fragments.

[0040] Then you can use Figure 2B The amount of extension products and fluorescently labeled primers (unextended fluorescently labeled primers) in the reaction mixture is detected by size separation and fluorescence detection as shown in . As an indicator of double-strand unwinding efficiency in isothermal amplification, the extended fragments can be analyzed in two ways: 1) the peak area ratio of the extended fragment to the initial double-stranded fragment (ratio of extended fragment to initial fragment, shown in Example 3); and / or 2) the percentage of the extended fragment, which is calculated as the ratio of the peak area of ​​the extended fragment to the total fragment including the extended fragment and the initial long fragment (shown in Examples 2 and 3).

[0041] Figure 2C The theoretical percentages of extension products for reaction mixtures containing various primer-to-duplex ratios are shown in FIG. When the primer-to-duplex ratio is 1:1, there is no double-strand unwinding process, and the proportion of extension products is about 0%, while the initial duplex fragment accounts for 100% (or the ratio of extended fragments to initial fragments is 0). For the strand unwinding process, depending on the additional amount of primers and the unwinding efficiency, the proportion of extension products can be about 50% (or the ratio of extended fragments to initial fragments is about 1) or more.

[0042] like Figure 2D As shown in , the pre-annealed duplex may include a target nucleic acid strand and a fluorescently labeled strand. The target strand and the fluorescently labeled strand may be approximately the same length; or the fluorescently labeled strand may be shorter than the target strand. Thus, the duplex may include a blunt end on one or both sides, or it may include a 3'-tail end and / or a 5'-tail end. Furthermore, although a fluorescent label is shown, any detectable label may be used in such a construct or any method herein.

[0043] I. Components for use in unwinding or strand exchange assays

[0044] The methods and devices disclosed herein relate to: (i) a pre-annealed duplex comprising a target nucleic acid and a synthetic nucleic acid, the synthetic nucleic acid optionally being conjugated to a detectable label; (ii) a primer; (iii) an amplification enzyme; and (iv) an enzyme for unwinding or strand exchange of the double-stranded nucleic acid. The primer, and optionally the synthetic nucleic acid, is conjugated to a detectable label.

[0045] (i) Pre-annealed duplex

[0046] A pre-annealed duplex refers to a double-stranded nucleic acid formed by annealing or hybridizing a target nucleic acid with a synthetic nucleic acid (each of which is a single-stranded nucleic acid). The duplex may include at least one double-stranded region that may or may not extend along the entire target nucleic acid. For example, the duplex may include one or more regions characterized as single-stranded, such as at the tail end.

[0047] The target nucleic acid and the synthetic nucleic acid can be any length suitable for hybridization and duplex formation. The target nucleic acid and the synthetic nucleic acid can be the same length or different lengths.

[0048] In some embodiments, the target nucleic acid has a length of about 15 to about 500 nucleotides, e.g., about 25 to about 500 nucleotides, about 50 to about 500 nucleotides, about 75 to about 500 nucleotides, about 100 to about 500 nucleotides, about 150 to about 500 nucleotides, about 200 to about 500 nucleotides, about 250 to about 500 nucleotides, about 300 to about 500 nucleotides, about 350 to about 500 nucleotides, about 400 to about 500 nucleotides, about 450 to about 500 nucleotides, about 15 to about 450 nucleotides, about 15 to about 400 nucleotides, about 15 to about 350 nucleotides, about 15 to about 300 nucleotides, about 15 to about 250 nucleotides, about 15 to about 200 nucleotides, about 15 to about 150 nucleotides, about 15 to about 100 nucleotides, about 15 to about 50 nucleotides, or about 15 to about 25 nucleotides.

[0049] In some embodiments, the synthetic nucleic acid has a length of about 10 to about 100 nucleotides, e.g., about 15 to about 100 nucleotides, about 20 to about 100 nucleotides, about 30 to about 100 nucleotides, about 40 to about 100 nucleotides, about 50 to about 100 nucleotides, about 60 to about 100 nucleotides, about 70 to about 100 nucleotides, about 80 to about 100 nucleotides, about 90 to about 100 nucleotides, about 10 to about 90 nucleotides, about 10 to about 80 nucleotides, about 10 to about 70 nucleotides, about 10 to about 60 nucleotides, about 10 to about 50 nucleotides, about 10 to about 40 nucleotides, about 10 to about 30 nucleotides, about 10 to about 20 nucleotides, or about 10 to about 15 nucleotides.

[0050] The target nucleic acid and the synthetic nucleic acid may comprise any sequence, such as a naturally occurring sequence or a modified sequence. The target nucleic acid and the synthetic nucleic acid may be obtained from one or more sources, such as isolated from a biological sample or obtained from a commercial source.

[0051] All or part of the synthetic nucleic acid may be complementary or substantially complementary to the target nucleic acid. Substantially complementary or substantially complementary refers to nucleotide sequences that hybridize to each other. For example, the target nucleic acid and the synthetic nucleic acid may be complementary to each other at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more in any useful region (e.g., in a region of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50 or more nucleotides, or refer to two nucleic acids that hybridize under stringent conditions). Stringent conditions may refer to conditions under which a nucleic acid that has complementarity or substantial complementarity with a target sequence hybridizes primarily with the target sequence, and does not substantially hybridize with a non-target sequence. Stringent conditions are typically sequence-dependent and vary depending on many factors. In general, the longer the sequence, the higher the temperature at which the sequence specifically hybridizes with its target sequence. Non-limiting examples of stringent conditions are described in detail in Tijssen (1993), Laboratory Techniques In Biochemistry And Molecular Biology—Hybridization With Nucleic Acid Probes Part 1, Chapter 2 “Overview of principles of hybridization and the strategy of Nucleic Acid Probe Assay”, Elsevier, NY.

[0052] Depending on the length and complementarity of the target nucleic acid and the synthetic nucleic acid, the pre-annealed duplex may include a 3'-tail end, a 5'-tail end, a blunt end, or a combination thereof. In some embodiments, the pre-annealed duplex includes a blunt end and a 3'-tail end or a 5'-tail end. In some embodiments, the pre-annealed duplex includes two blunt ends.

[0053] The target nucleic acid and synthetic nucleic acid may include DNA, RNA or a combination thereof (e.g., DNA / RNA hybrids). The target nucleic acid and / or synthetic nucleic acid may include one or more modified nucleotides. Any modified nucleotide known in the art may be included in the target nucleic acid and / or the synthetic nucleic acid for use in the methods described herein. The one or more modified nucleotides may be included anywhere in the target nucleic acid and / or the synthetic nucleic acid, e.g., 3'-end of nucleic acid, 5'-end of nucleic acid, inside or a combination thereof.

[0054] The synthetic nucleic acid may include the same sequence as the primer, or the synthetic nucleic acid may include a different sequence. The synthetic nucleic acid may be shorter, longer, or the same length as the primer. In some instances, the synthetic nucleic acid includes a primer. In some instances, the synthetic nucleic acid includes an extension product of a primer.

[0055] Synthetic nucleic acid can include one or more (one or more) detectable labels, such as detectable labels known in the art or described herein. The one or more (one or more) detectable labels can be included in any place in the synthetic nucleic acid, such as, 3'-end of nucleic acid, 5'-end of nucleic acid, inside or its combination. In some non-limiting examples, multiple detectable labels can be present. In other non-limiting examples, the label is not present at the 3'-end.

[0056] Pre-annealed duplexes can be formed using any method known in the art or described herein. For example, pre-annealed duplexes can be formed by mixing the target nucleic acid and the synthetic nucleic acid, heating the mixture (e.g., 95° C. for 5 minutes), and allowing the mixture to cool to room temperature (e.g., about 60 minutes). In some examples, pre-annealed duplexes can be formed by mixing the target nucleic acid and the primer, optionally the extension product of the primer, heating the mixture, and allowing the mixture to cool to room temperature.

[0057] (ii) Primers

[0058] A primer refers to an oligonucleotide comprising a nucleotide sequence that can hybridize or anneal to a target nucleic acid. The primer hybridizes or anneals at or near a specific target region (also referred to as a target sequence) in the target nucleic acid.

[0059] Primer can be any length suitable for hybridizing with target nucleic acid.Described primer can be identical with the length of described target nucleic acid or described primer can be shorter than described target nucleic acid in length.In some embodiments, described primer has the length of about 10 to about 100 nucleotides, for example, about 15 to about 100 nucleotides, about 20 to about 100 nucleotides, about 30 to about 100 nucleotides, about 40 to about 100 nucleotides, about 50 to about 100 nucleotides, about 60 to about 100 nucleotides, about 70 to about 100 nucleotides, about 80 to about 100 nucleotides, about 90 to about 100 nucleotides, about 10 to about 90 nucleotides, about 10 to about 80 nucleotides, about 10 to about 70 nucleotides, about 10 to about 60 nucleotides, about 10 to about 50 nucleotides, about 10 to about 40 nucleotides, about 10 to about 30 nucleotides, about 10 to about 20 nucleotides or about 10 to about 15 nucleotides.

[0060] All or a portion of the primers may be complementary or substantially complementary to the target nucleic acid. Substantially complementary refers to nucleotide sequences that hybridize to each other. For example, the primer and the target nucleic acid may be complementary to each other at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more in any useful region (e.g., in a region of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50 or more nucleotides, or refer to two nucleic acids that hybridize under stringent conditions).

[0061] The primer may comprise any sequence, such as a naturally occurring sequence or a modified sequence. The primer may be obtained from one or more sources, such as, for example, isolated from a biological sample or obtained from a commercial source.

[0062] Primers can include DNA, RNA, or a combination thereof (e.g., DNA / RNA hybrids). Primers can include one or more modified nucleotides. Any modified nucleotides known in the art can be included in the primers for the methods described herein. The one or more modified nucleotides can be included anywhere in the primer, e.g., at the 3'-end of the primer, the 5'-end of the primer, inside, or a combination thereof.

[0063] Primers may include one or more (one or more) detectable labels, such as detectable labels known in the art or described herein. The one or more (one or more) detectable labels may be included anywhere in the primer, such as, the 3'-end of the primer, the 5'-end of the primer, inside, or a combination thereof.

[0064] (iii) Amplification enzyme

[0065] Amplification enzyme refers to any enzyme of a polymer of a synthetic nucleic acid. Amplification enzymes include, but are not limited to, polymerases (e.g., DNA polymerases, RNA polymerases). DNA polymerases include, but are not limited to, DNA-dependent DNA polymerases and RNA-dependent DNA polymerases, including reverse transcriptases. RNA polymerases include, but are not limited to, DNA-dependent RNA polymerases and RNA-dependent RNA polymerases. Amplification enzymes used in the methods described herein may be naturally occurring or genetically modified.

[0066] (iv) Helicase

[0067] Helicase refers to any enzyme that unwinds and / or separates two nucleic acid chains (e.g., DNA, RNA, RNA-DNA hybrids). Helicases include enzymes with unwinding and / or strand exchange activity. Examples of helicases include, but are not limited to, helicases (e.g., DNA helicases, RNA helicases), polymerases with strand displacement activity (e.g., phi29, Bst DNA polymerase, BcaBEST DNA polymerase, Vent (exo-) DNA polymerase, MS-2 bacteriophage DNA polymerase, z-Taq DNA polymerase, Taq polymerase, Bsm DNA polymerase (Bsm), and variants thereof, such as Bst 2.0 or Bst 2.0 WarmStart TM DNA polymerase (New England Biolabs, Ipswich, Mass.) and combinations thereof (e.g., a blend of strand displacement polymerase and Taq), recombinase (e.g., Cre recombinase, Hin recombinase, Tre recombinase, FLP recombinase), and auxiliary proteins for unwinding (e.g., uvsY, a recombinase loading factor). The helicase used in the methods described herein may be a naturally occurring form or a thermophilic form thereof, a thermostable form thereof, or a recombinant form thereof.

[0068] (v) Detectable label

[0069] Primers and optionally synthetic nucleic acids used in the methods and devices described herein are conjugated to detectable labels. When both the primers and the synthetic nucleic acids are conjugated to detectable labels, the detectable labels may be the same or different.

[0070] As used herein, "detectable label" refers to any molecule that can release a detectable signal directly or indirectly. In some embodiments, the detectable label can be a fluorophore (e.g., CY5). As used herein, the term "fluorophore" (also called "fluorescent marker" or "fluorescent dye") refers to a portion that absorbs light energy at a defined excitation wavelength and emits light energy at a different wavelength.

[0071] Examples of fluorophores include, but are not limited to, cyanine derivatives (e.g., cyanine, indocarbocyanine, oxacarbocyanine, thiacarbocyanine, and merocyanine), xanthene derivatives (e.g., fluorescein, rhodamine, Oregon Green, eosin, and Texas Red), naphthalene derivatives (e.g., dansyl and prodan derivatives), pyrene derivatives (e.g., Cascade Blue), oxadiazole derivatives (e.g., pyridyloxazole, nitrobenzoxadiazole, and benzoxadiazole), oxazine derivatives (e.g., Nile Red, Nile Blue, cresyl violet, and oxazine 70), acridine derivatives (e.g., proflavine, acridine orange, and acridine yellow), arylmethine derivatives (e.g., auramine, crystal violet, and malachite green), tetrapyrrole derivatives (e.g., porphyrin, phthalocyanine, and bilirubin), coumarin derivatives, or fluorescent proteins (e.g., green fluorescent protein).

[0072] In some embodiments, the detectable label is a radioactive label (e.g., 35 S. 125 I, or radioactive phosphates such as 32 P or 33 P). In some embodiments, the detectable label is a chemiluminescent label (e.g., an acridinium ester or a ruthenium ester). Detectable labels include other labels, such as biotin, avidin, streptavidin, digoxigenin, haptens, quantum dots, nanoparticles, etc.

[0073] II. Assays for detecting unwinding or strand exchange efficiency

[0074] Aspects of the present disclosure provide methods for detecting the unwinding of double-stranded nucleic acids. In order to perform the test methods described herein, a sample is incubated under isothermal amplification conditions and for a time sufficient for nucleic acid amplification, the sample comprising (i) a pre-annealed duplex comprising a target nucleic acid and a synthetic nucleic acid, the synthetic nucleic acid optionally conjugated to a detectable label; (ii) a primer conjugated to a detectable label; (iii) an amplification enzyme; and (iv) an enzyme for unwinding or strand exchange of double-stranded nucleic acids. The amplification product in the sample can be detected by size and by detecting the signal released from the detectable label.

[0075] The methods described herein encompass incubating the sample for any period of time sufficient for nucleic acid amplification. In some embodiments, the sample is incubated for about 1 to 30 minutes, such as about 5 to 30 minutes, about 10 to 30 minutes, about 15 to 30 minutes, about 20 to 30 minutes, about 25 to 30 minutes, about 1 to 25 minutes, about 1 to 20 minutes, about 1 to 15 minutes, about 1 to 10 minutes, about 1 to 5 minutes, or about 1 to 2 minutes.

[0076] The methods described herein encompass incubating the sample at any temperature sufficient for nucleic acid amplification. In some embodiments, the sample is incubated at a temperature of about 20 to 75°C, such as about 25 to 75°C, about 30 to 75°C, about 35 to 75°C, about 40 to 75°C, about 45 to 75°C, about 50 to 75°C, about 55 to 75°C, about 60 to 75°C, about 65 to 75°C, about 70 to 75°C, about 20 to 70°C, about 20 to 65°C, about 20 to 60°C, about 20 to 55°C, about 20 to 50°C, about 20 to 45°C, about 20 to 40°C, about 20 to 35°C, about 20 to 30°C, or about 20 to 25°C.

[0077] Any amount of pre-annealed duplexes suitable for nucleic acid amplification can be used in the methods described herein. For example, the sample can include pre-annealed duplexes at a concentration of 0.1 to 10 μM. In some embodiments, the sample can include pre-annealed duplexes at a concentration of 0.25 to 10 μM, 0.5 to 10 μM, 1 to 10 μM, 2.5 to 10 μM, 5 to 10 μM, 7.5 to 10 μM, 0.1 to 7.5 μM, 0.1 to 5 μM, 0.1 to 2.5 μM, 0.1 to 1 μM, 0.1 to 0.5 μM, or 0.1 to 0.25 μM.

[0078] Any amount of primers suitable for nucleic acid amplification can be used in the methods described herein. For example, the sample may include primers at a concentration of 0.1 to 10 μM. In some embodiments, the sample may include primers at a concentration of 0.25 to 10 μM, 0.5 to 10 μM, 1 to 10 μM, 2.5 to 10 μM, 5 to 10 μM, 7.5 to 10 μM, 0.1 to 7.5 μM, 0.1 to 5 μM, 0.1 to 2.5 μM, 0.1 to 1 μM, 0.1 to 0.5 μM, or 0.1 to 0.25 μM.

[0079] Any ratio of primers to pre-annealed duplexes suitable for nucleic acid amplification can be used in the methods described herein. For example, the sample can include a ratio of primers to pre-annealed duplexes of 1:1 to 50:1. In some embodiments, the sample can include a ratio of primers to pre-annealed duplexes of 1:1 to 45:1, 1:1 to 40:1, 1:1 to 35:1, 1:1 to 30:1, 1:1 to 25:1, 1:1 to 20:1, 1:1 to 15:1, 1:1 to 10:1, 1:1 to 5:1, 1:1 to 4:1, 1:1 to 3:1, or 1:1 to 2:1.

[0080] The sample may include one or more amplification enzymes and / or one or more enzymes for unwinding or strand exchange. The sample may include additional components including, but not limited to, deoxyribonucleotide triphosphates (dNTPs, including ATP), buffers, water, salts, divalent ions (e.g., divalent cations such as Mg, ++ ), detergents, denaturants, crowding agents, or combinations thereof. Additional components may include one or more of the following: betaine, dimethyl sulfoxide, ethylene glycol, glycerol, formamide, 7-deaza-2'-deoxyguanosine 5'-triphosphate, 2'-deoxyinosine 5'-triphosphate, or 1,2-propylene glycol.

[0081] The methods provided herein may include inactivating the sample prior to detecting nucleic acid amplification. The sample may be inactivated via heat inactivation (e.g., incubation at about 45-100° C.), chemical inactivation (e.g., incubation in the presence of a chelating agent such as EDTA or a protease such as proteinase K), or a combination thereof.

[0082] The methods provided herein may include purifying the sample before detecting nucleic acid amplification. The purification step may be used to remove one or more chemical components (e.g., crowding agents, high salt concentrations) and / or one or more biological components (e.g., proteins, such as serum proteins, albumins, or globulins, including bovine serum albumin (BSA) as an example). The sample may be purified using any method known in the art or described herein. In some instances, the purified sample includes a concentrated sample. In some instances, the purified sample is purified using a commercially available kit (e.g., ZYMO RESEARCH ZR-96 Oligo Clean & Concentrator TM Kit; Catalog No. D4062).

[0083] The methods provided herein encompass detection of nucleic acid amplification, which can be achieved by detecting extension products. Various methods known in the art or described herein can be used to detect the size of extension products and the signal generated by the detectable label on the extension product. In some embodiments, electrophoresis (e.g., capillary electrophoresis, polyacrylamide gel electrophoresis) can be used, optionally on a microfluidic device, to detect extension products. In some embodiments, the size of the extension product is determined using labeled size standards (e.g., fluorescently labeled size standards).

[0084] In a non-limiting example, the product obtained after isothermal amplification can be subjected to fragment analysis. Such products may include extension products, primers, pre-annealed duplexes, and labeled forms of one or more of these. Fragment analysis may include electrophoretic separation of sample products, detection of signals generated by detectable labels of sample products, and analysis of such signals, which may include determining fragment size and / or concentration using calibration standards (e.g., ladder or labeled standards).

[0085] Examples of microfluidic devices and assays suitable for use in the methods provided herein are described below and in, e.g., U.S. Patent Nos. 5,976,336, 7,419,784, 7,276,330, 7,081,190, 5,948,227, 6,042,710, and 6,440,284, each of which is incorporated herein by reference in its entirety.

[0086] III. Microfluidic Devices

[0087] Also provided herein is a microfluidic device for detecting the unwinding or strand exchange of double-stranded nucleic acids.Any method described herein can be carried out on the microfluidic device described herein.In a non-limiting example, microfluid or micro means having at least one dimension less than 1mm.For example, a microfluidic structure (for example, any structure described herein) can have a length, width, height, cross-sectional dimension, perimeter, radius (for example, outer radius or inner radius) or diameter less than 1mm.

[0088] The microfluidic device can be configured to receive one or more inlets, one or more outlets, and an area for providing fluid communication between the one or more inlets and one or more outlets for receiving a sample (e.g., any described herein). In some cases, fluid communication refers to any duct, channel, tube, pipe, chamber, or passage through which a substance, such as a liquid, gas, or solid, can pass substantially without restriction when the passage is open. When the passage is closed, the substance is substantially restricted from passing. Typically, limited diffusion of a substance through a plate, substrate, and / or substrate material does not constitute fluid communication, and the limited diffusion may or may not occur depending on the composition of the substance and the material.

[0089] Such regions may include channels, chambers, etc. In addition, regions may be configured to perform any of the methods described herein. In one embodiment, the microfluidic device may include a channel configured to transport a sample (e.g., including pre-annealed duplexes, primers or labeled forms thereof, amplification enzymes for amplifying nucleic acids, helicases for unwinding double-stranded nucleic acids, detectable labels, extension products or labeled forms thereof, amplicons or labeled forms thereof, and combinations thereof).

[0090] The microfluidic device may include a detection zone in fluid communication with the channel, wherein the detection zone is configured to detect any detectable label (e.g., a first and / or second detectable label, as described herein). Such a device may be used, for example, for fragment separation and / or fragment analysis. Isothermal amplification may or may not be used in such a device.

[0091] Optionally, the microfluidic device can be configured to perform on-chip isothermal amplification. Such a device can include an incubation zone that is fluidly connected to the channel and / or detection zone. In a non-limiting embodiment, the incubation zone is configured to incubate the sample under isothermal amplification conditions for a time sufficient for nucleic acid amplification. A description of amplification conditions is provided herein.

[0092] IV. Kit

[0093] The present disclosure also provides a kit for detecting unwinding or strand exchange of double-stranded nucleic acids. Such a kit may include a pre-annealed duplex and / or a target nucleic acid and a synthetic nucleic acid for forming a pre-annealed duplex; a primer; an amplification enzyme; and an enzyme for unwinding or strand exchange of double-stranded nucleic acids. The primer, and optionally the synthetic nucleic acid, may be conjugated to a detectable label.

[0094] The kits may also include instructions for practicing any of the methods described herein.The instructions provided in the kits of the present disclosure are typically written instructions on a label or package insert.

[0095] The kits provided herein are in suitable packaging. Suitable packaging includes, but is not limited to, containers, bottles, vials, and flexible packaging. The kits may include additional components such as buffers and explanatory information.

[0096] The present disclosure also covers the following embodiments:

[0097] 1. A method for detecting unwinding or strand exchange of a double-stranded nucleic acid, the method comprising: providing a sample comprising a pre-annealed duplex, wherein the pre-annealed duplex comprises a target nucleic acid and a synthetic nucleic acid comprising an optional first detectable label; a primer comprising a second detectable label; an amplification enzyme for amplifying the nucleic acid; and a helicase for unwinding or strand exchange of the double-stranded nucleic acid; incubating the sample under isothermal amplification conditions and for a time sufficient for nucleic acid amplification; and detecting the second detectable label and, optionally, if present, the first detectable label.

[0098] 2. The method of embodiment 1, wherein the synthetic nucleic acid comprises an initial primer, and wherein the initial primer comprises the first detectable label.

[0099] 3. A method as described in embodiment 2, wherein the incubation further includes: extending the initial primer to provide an extension product comprising the first detectable label; unwinding the extension product; annealing a primer comprising the second detectable label to a portion of the target nucleic acid; and extending the primer to provide a further extension product comprising the second detectable label.

[0100] 4. A method as described in embodiment 1, wherein the synthetic nucleic acid comprises an extension product of an initial primer, and wherein the initial primer comprises the first detectable label.

[0101] 5. A method as described in embodiment 1, wherein the synthetic nucleic acid comprises an extended nucleic acid containing about 20 or more nucleotides.

[0102] 6. A method as described in embodiment 5, wherein the extended nucleic acid comprises the first detectable label.

[0103] 7. A method as described in embodiments 1-6, wherein the incubation further comprises: unwinding the duplex; annealing a primer comprising the second detectable label to a portion of the target nucleic acid; and extending the primer to provide a further extension product comprising the second detectable label.

[0104] 8. The method of embodiments 1-7, further comprising, prior to said providing, annealing said target nucleic acid and said synthetic nucleic acid to form a pre-annealed duplex; and optionally cooling said pre-annealed duplex.

[0105] 9. The method of embodiments 1-7, further comprising, prior to said providing, annealing said target nucleic acid and said synthetic nucleic acid, wherein said synthetic nucleic acid is configured as an initial primer; and

[0106] The initial primer is extended under polymerase primer extension conditions, thereby forming the pre-annealed duplex.

[0107] 10. The method as described in embodiments 1-9 further includes, after the incubation: inactivating the sample using heat inactivation (e.g., about 45-100° C.) and / or chemical inactivation (e.g., chelating agents such as EDTA, proteases such as proteinase K, etc.).

[0108] 11. The method as described in embodiments 1-10 further includes, before the detection: purifying the sample to remove one or more chemical or biological components.

[0109] 12. A method as described in embodiment 1, wherein the synthetic nucleic acid and the primer contain the same nucleic acid sequence.

[0110] 13. A method as described in embodiment 1, wherein the synthetic nucleic acid is shorter than the primer or has the same length as the primer.

[0111] 14. A method as described in embodiment 1, wherein the synthetic nucleic acid is longer than the primer.

[0112] 15. A method as described in embodiment 1, wherein the synthetic nucleic acid and the primer independently have a length of about 5 to 100 nucleotides.

[0113] 16. The method of embodiments 1-15, wherein the pre-annealed duplex comprises a 3'-tail and / or a 5'-tail.

[0114] 17. A method as described in embodiments 1-16, wherein the pre-annealed duplex includes at least one blunt end.

[0115] 18. The method of embodiments 1-17, wherein the pre-annealed duplex has a length of about 15 to 500 base pairs.

[0116] 19. The method of embodiments 1-18, wherein the ratio of the primer to the pre-annealed duplex is 1:1 to 50:1.

[0117] 20. The method of embodiments 1-19, wherein the first and second detectable labels are the same or different.

[0118] 21. The method of embodiment 20, wherein the first and second detectable labels are independently provided at the 5'-end or internally.

[0119] 22. A method as described in embodiments 20-21, wherein the synthetic nucleic acid comprises multiple first detectable labels, and / or wherein the primer comprises multiple second detectable labels.

[0120] 23. A method as described in embodiment 20, wherein the first and second detectable labels are selected from fluorescent labels, radioactive labels, chemiluminescent labels, or dyes.

[0121] 24. A method as described in embodiments 1-23, wherein the sample further contains 3'-amino-2',3'-dideoxyribonucleotide 5'-triphosphate (nNTP), divalent ions, denaturants, buffers, and / or salts.

[0122] 25. A method as described in embodiments 1-24, wherein the amplification enzyme and / or the helicase is selected from a helicase, a recombinase, a polymerase, a reverse transcriptase, a thermophilic form thereof, a thermostable form thereof, and a recombinant form thereof.

[0123] 26. A method as described in embodiments 1-25, wherein the isothermal amplification conditions include a temperature of about 20 to 75°C.

[0124] 27. A method as described in embodiments 1-26, wherein the detection comprises electrophoresis analysis, optionally on a microfluidic device.

[0125] 28. A method as described in embodiment 27, wherein the electrophoresis analysis includes analyzing the fragment size of the amplicon containing the second detectable label.

[0126] 29. The method of embodiment 28, wherein the electrophoretic analysis further comprises comparing the fragment size of the amplicons with a labeled size standard (e.g., a fluorescently labeled size standard).

[0127] 30. A kit comprising: a pre-annealed duplex, a primer comprising a second detectable label, an amplification enzyme, a helicase, and instructions for performing the method of embodiments 1-29.

[0128] Without further elaboration, it is believed that those skilled in the art will be able to utilize the present invention to the greatest extent based on the above description. Therefore, the following specific embodiments are to be interpreted as illustrative only and not to limit the remainder of the present disclosure in any way. For the purpose or subject matter cited herein, all publications cited herein are incorporated by reference.

[0129] Example

[0130] In order that the described invention may be more fully understood, the following examples are set forth.The examples described in this application are provided to illustrate the methods and compositions provided herein, and should not be construed in any way as limiting the scope thereof.

[0131] Example 1: Detection of unwinding of double-stranded nucleic acid by DNA helicase using fluorescently labeled primers (Design 1)

[0132] This example describes the detection of unwinding of double-stranded nucleic acids by DNA helicase using fluorescently labeled primers that are added in excess in the reaction mixture and used to form a pre-annealed duplex comprising the target strand and the fluorescently labeled primer (Design 1).

[0133] Helicase-dependent amplification (HDA) relies on the double-stranded DNA (dsDNA) unwinding activity of helicases to generate single-stranded templates for primer annealing and extension by strand-displacing DNA polymerases. Higher dsDNA unwinding activity of helicases promotes more template-primer binding and contributes to higher amplification efficiency and faster reactions. Thermophilic helicase-dependent amplification (tHDA) utilizes the thermostable UvrD helicase (TteUvrD) to selectively amplify target sequences at 60-65°C, which shows improved amplification sensitivity without the need for the MutL accessory protein. This study aims to use a microfluidic device, specifically GX Touch TM Nucleic acid analyzer system, evaluating the dsDNA unwinding efficiency of the tHDA system. Although such a device is used in this example, other devices, systems and analysis methods can be implemented.

[0134] Two different pre-annealed duplexes were formed and the unwinding efficiency of the tHDA system was analyzed. Annealing primer_80 and target_80 provided a blunt-ended, pre-annealed duplex product, while annealing primer_80 and target_110 provided a 3' tail-ended, pre-annealed duplex product. Prior to forming the duplexes, the primers were internally labeled with a fluorophore. Oligonucleotides were obtained from IDT or Eurofin. The sequences of the target strands and primers are shown in Table 1.

[0135] Table 1. Oligonucleotide sequences.

[0136]

[0137] The primer and target strand were mixed at a molar ratio of approximately 1:1 in the tHDA kit ( Pre-annealed duplexes were prepared by mixing in 1X Annealing Buffer II of II Universal tHDA Kit, Catalog # HOI IOS. The final duplex concentration was 250 nM. The annealing mixture was heated at 95°C for 2 min and then slowly cooled to 25°C at a ramp rate of 0.1°C / sec.

[0138] The isothermal amplification reaction mixture was prepared by mixing the following components in 1X Annealing Buffer II (total volume of 20 μL): 4 mM MgSO4, 40 mM NaCl, 1.4 μL of dNTP solution, 1.4 μL enzyme mix, and various amounts of pre-annealed duplexes and primers.

[0139] Four conditions of pre-annealed duplex and primer mixture were evaluated: 100 nM pre-annealed duplex only (A5, B4); 50 nM pre-annealed duplex + 50 nM primer (primer_80) (A4, B3); 20 nM pre-annealed duplex + 80 nM primer (primer_80) (A3, B2); and 100 nM primer (primer_80) only (Al, A2, Bl).

[0140] Will The enzyme mixture (conditions A3-A5) was replaced with Bst DNA polymerase large fragment (B2-B4) to test the baseline polymerase extension activity from Bst DNA polymerase without unwinding the duplex by the helicase. In addition, a reaction mixture was prepared with 100 nM of a primer (primer_80) without enzyme and a pre-annealed template to test the effect of enzyme and buffer on the migration of primer (A2). Each reaction mixture was incubated at 65°C for 20 minutes and then inactivated at 95°C for 5 minutes. Use GX Touch TM The products were analyzed using a PerkinElmer ELISA instrument (PerkinElmer, part # CLS137031) and PerkinElmer's Fluorescence Fragmentation Analysis Evaluation Kit. Data analysis was performed using GX Reviewer software (perkinelmer.com / lab-products-and-services / resources / labchip-and-optimizer-software-downloads.html).

[0141] like Figure 3A-3B As shown in Table 2, the initial primer size was about 10-12 nt (observed size), and the fully extended product size was about 77 nt (observed size). Partial extension products between 15 and 77 nt were detected. The results are summarized as follows:

[0142] A1, B1: no duplex control, complete extension product is 0%.

[0143] A2: No enzyme control, complete extension product is 0%.

[0144] • A3 and B2: No additional primer controls, fully extended product is approximately 100%.

[0145] • A4-A5: For all primer:duplex ratios evaluated, duplexes with blunt ends have higher unwinding efficiency (more fully extended products) than duplexes without blunt ends.

[0146] • B3-B4: For all primer:duplex ratios evaluated, duplexes with blunt ends had higher unwinding efficiency (more fully extended products) than duplexes without shuffled ends.

[0147] B2-B4: The duplex can be unwound for polymerase extension in the absence of helicase and in the presence of BstLF DNA polymerase. This may be due to "thermal breathing" of the duplex DNA or denaturation of the duplex DNA at the reaction temperature (65°C). Since the duplex includes 20mers, which are short double-stranded nucleic acids, it may be more sensitive to elevated temperatures.

[0148] Table 2. Experimental details and results summary

[0149]

[0150] *Tested with flat end template only.

[0151] a: Total peak area includes all peak size areas (initial primer, partially extended and fully extended products). The percentage of fully extended peak was calculated as the fully extended peak area divided by the total peak area.

[0152] In summary, the experimental results described herein indicate that the unwinding efficiency of DNA helicases under isothermal amplification conditions can be detected using fluorescently labeled primers, which are added in excess in the reaction mixture and used to form a pre-annealed duplex comprising a target strand and a fluorescently labeled primer (Design 1). In some non-limiting examples, the extension product (partial or complete) can be an indicator of the success of a multi-enzyme synchronization event.

[0153] Example 2: Detection of unwinding of double-stranded nucleic acid by DNA helicase using fluorescently labeled primers (Design 2)

[0154] This example describes the detection of unwinding of double-stranded nucleic acids by DNA helicase using a short fluorescently labeled primer added to the reaction mixture and a pre-annealed duplex in which one strand is fluorescently labeled (Design 2). The oligonucleotide sequences used in this study are shown in Table 3.

[0155] Table 3. Oligonucleotide sequences

[0156]

[0157] This study was designed to use a microfluidic device, specifically GX Touch TM The nucleic acid analyzer system was used to evaluate the dsDNA unwinding efficiency of the tHDA system.

[0158] First, 110mer pre-annealed duplexes were prepared by polymerase extension reaction using 2X Phire Hot Start II PCR Master (Thermofisher, catalog # F125S) with 3 μM primer (primer_110) and 2.5 μM target strand (target_110mer) in a 20 μL reaction under the following thermal cycle program: 98°C / 30 sec, 30 cycles (98°C / 5 sec, 55°C / 5 sec, 72°C / 10 sec), 72°C / 1 min according to the user manual. GX Touch TM The polymerase extension products were tested on a nucleic acid analyzer (PerkinElmer, catalog #CLS137031). A slightly excessive amount of primers was used to ensure that there was no free complementary strand (110 nt) in the reaction product. Otherwise, primer-80 in the isothermal amplification reaction could bind to the free complementary strand to generate a short fragment (80 nt), thereby generating a false positive fragment.

[0159] By mixing 4mM MgSO4, 40mM NaCl, 1.4μL dNTP solution and 1.4 μL The enzyme mix was mixed with the following two different primer / duplex preparations in a The tHDA-based isothermal amplification reaction was performed in 20 μL reaction mixtures of the PCR product of the ELISA kit (NEB, catalog # H0110S): 300 nM primer (primer_80) and 300 nM 110mer duplex; and 300 nM primer (primer_80) and 150 nM duplex. The reaction mixture was incubated at 65°C for 20 minutes and then inactivated at 95°C for 5 minutes. The enzyme mix was replaced with water as a negative control, and Bst DNA polymerase Large Fragment (BstLF) (NEB, catalog #M0275S) was used as a polymerase control. GX Touch TM The products were analyzed using the PerkinElmer ELISA kit (PerkinElmer, part #CLS137031). Data analysis was performed using GX Reviewer software (perkinelmer.com / lab-products-and-services / resources / labchip-and-optimizer-software-downloads.html).

[0160] Compared with BstLF, tHDA unwinds dsDNA duplexes with higher efficiency and induces more primer extension products. Figure 4 As shown, two peaks were detected when the tHDA reaction system did not contain an enzyme (conditions 5, 6): the initial primer size was observed at about 12nt, and a 110nt fragment of the dsDNA duplex was observed at about 105nt. Using the tHDA enzyme present in the reaction system, an extension product (80nt) from a dsDNA unwinding event can be detected at about 78nt. When the primer-to-duplex ratio was 1:1 (condition 1), the extended fragment was about 50% of the total fragment (extended fragment and original duplex fragment), indicating that most of the 300nM duplex template was unwound, and most of the 300nM primer could anneal to the target chain and extend to 80nt. When the primer-to-duplex ratio was 2:1 (condition 2), the percentage of extended fragments increased to 65%, indicating that more duplex structures (from the initial template or extension products) could be unwound and then annealed / extended with additional primers.

[0161] In contrast, when only BstLF was present in the reaction system (conditions 3, 4), no extension product (~80nt peak) was visible under both conditions. For this duplex structure of 110 bp with 41.8% GC and Tm (melting temperature) above 70°C, there was no effective unwinding event or "thermal breathing" event. Compared with the shorter duplex used in Design 1, the longer duplex was less affected by high temperature.

[0162] A summary of the experimental details and results is provided in Table 4.

[0163] Table 4. Experimental details and results summary.

[0164]

[0165] a: Total peak area includes the initial 110mer peak area and the extended product 80mer peak area. The extended peak percentage is calculated as the extended peak area divided by the total peak area.

[0166] Taken together, these data indicate that, in some non-limiting examples, under defined tHDA isothermal amplification conditions, the tHDA enzyme mixture can effectively unwind blunt-ended dsDNA duplexes and synchronize duplex unwinding, primer binding, and polymerase extension in only 20 minutes. In other non-limiting examples, the design may be less affected by "thermal breathing" or effects associated with thermal denaturation. Therefore, such a design (or a modified form thereof) may be suitable for characterizing isothermal amplification unwinding efficiency over a wide temperature range, including at elevated temperatures, such as greater than about 45°C.

[0167] Example 3: Detection of duplex strand exchange efficiency during recombinase polymerase amplification (RPA)

[0168] This example describes the detection of strand exchange during recombinase polymerase amplification (RPA) using short fluorescently labeled primers added to the reaction mixture and pre-annealed duplexes where one strand is fluorescently labeled (Design 2). The oligonucleotide sequences used in this study are shown in Table 3.

[0169] The RPA process operates at 37-42°C and begins when the recombinase protein uvsX binds to the primer to form a recombinase-primer complex, which then initiates the strand exchange / invasion process to open the duplex. Primers of 30-35 nucleotides in length are used in the RPA reaction, although primers as short as 18 nt can work. This study was designed using PerkinElmer's GX Touch TM The strand exchange / invasion efficiency of the RPA system with two different primer designs was evaluated by the Nucleic Acid Analyzer System.

[0170] Pre-annealed duplexes were formed as described in Example 2. 25 μL of 2x reaction buffer, 5 μL of 10x basic E-Mix, 14 mM MgOAc, 1.8 mM (total) dNTPs and 2.5 μL of 20xCore reaction mixture were mixed with the following two different primer / duplex preparations in a 5% flask with RPA-based isothermal amplification reactions were performed in 50 μL reaction mixtures of a liquid-based kit (TwistDx, catalog # TALQAS01): 300 nM primer (primer_80_v2, 35 nt) and 300 nM 110mer duplex; and 300 nM primer (primer_80_v2) and 150 nM 110mer duplex. The reaction mixture was incubated at 37 ° C for 20 minutes and then inactivated at 95 ° C for 5 minutes. As a control, water was used to replace the 20xCore reaction mixture (RPA enzyme mixture) as a negative control, and Bst DNA polymerase large fragment was used as a polymerase control because it can have some activity at 37 ° C. In addition, the primer length preference of RPA-based amplification was tested by replacing primer_80_v2 with a 20 nt primer (primer_80) in the reaction mixture.

[0171] After isothermal amplification, nucleic acid purification was performed using the ZR-96 Oligo Clean & Concentrator Kit (ZYMORESEARCH, catalog # D4062). First, 100 μL of oligo binding buffer was added to 50 μL of amplified product, which was then mixed with 400 μL of ethanol (Sigma, catalog # 459828-1L) and transferred to a Zymo-Spin mounted on a collection plate. TMI-96 plate. After centrifugation at 2500xg for 5 minutes at room temperature, the column was washed once with 750 μL of DNA wash buffer and centrifuged twice at 2500xg for 5 minutes at room temperature to ensure complete removal of the wash buffer. The plate was then transferred to an elution plate and 25 μL of water was added directly to the matrix of each well. Finally, the plate was centrifuged at 2500xg for 5 minutes at room temperature to collect the eluate. 3 μL of the product was analyzed by the PerkinElmer Fluorescence Fragment Analysis Evaluation Kit according to the evaluation user guide. Using LabChip Data analysis was performed using GX Reviewer software (perkinelmer.com / lab-products-and-services / resources / labchip-and-optimizer-software-downloads.html).

[0172] Compared to the polymerase control, for longer primers, the RPA enzyme mix was more efficient at opening the dsDNA duplex and extending the primer using the unwound duplex template. Figure 5 As shown in , when there is no enzyme in the RPA system, two peaks can be mainly detected: primers (35nt primers are observed at 41nt in conditions 7 and 8, and 20nt primers are observed at 13nt (data not shown)) and 110nt initial fragments from dsDNA duplexes (observed at 111nt). Using the RPA enzyme mixture, an extension product 80nt can be detected at about 87nt. However, under the 20nt primer condition and in the case of multiple small fragments less than 80nt, the ratio of the extended fragment to the initial fragment is only 0.7. Under the 35nt primer condition, when the primer-to-duplex ratio is 1:1, the ratio of the extended fragment to the initial fragment is 0.8, and increases to 2.7 when the primer-to-duplex ratio is 2:1. In comparison, when BstLF polymerase is used in the absence of a recombinase mixture, the amount of polymerase extension products is significantly reduced, and the ratio of the extended fragment to the initial fragment is 0.3, which does not increase even when the primer-to-duplex ratio is increased to 2:1. A summary of the experimental details and results is provided in Table 5.

[0173] Table 5. Experimental details and results summary.

[0174]

[0175] a: Total peak area includes the initial 110mer peak area and the extended product 80mer peak area. The extended peak percentage is calculated as the extended peak area divided by the total peak area.

[0176] These results show that the strand exchange activity of the RPA enzyme mixture provides higher amplification efficiency compared to BstLF polymerase, and that the RPA system with longer primers (35nt) has higher strand exchange efficiency than the short primers (20nt). These results also show that, in some non-limiting cases, amplification can occur in only 20 minutes under the RPA isothermal amplification conditions described herein.

[0177] Other Implementations

[0178] It is understood that although the invention has been described in conjunction with the detailed description of the invention, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages and modifications are within the scope of the following claims.

Claims

1. A method for detecting unwinding or strand exchange of a double-stranded nucleic acid, the method comprising: providing a sample comprising a pre-annealed duplex, wherein the pre-annealed duplex comprises a target nucleic acid and a synthetic nucleic acid comprising an optional first detectable label; a primer comprising a second detectable label; Amplification enzymes for amplifying nucleic acids; and helicases for unwinding or strand exchanging double-stranded nucleic acids; incubating the sample under isothermal amplification conditions and for a time sufficient for nucleic acid amplification; and The second detectable label and optionally, if present, the first detectable label are detected.

2. The method of claim 1, wherein the synthetic nucleic acid comprises an initial primer, and wherein the initial primer comprises the first detectable label.

3. The method of claim 2, wherein the incubation further comprises: extending the initial primer to thereby provide an extension product comprising the first detectable label; unwinding the extension product; annealing a primer comprising the second detectable label to a portion of the target nucleic acid; and The primer is extended to provide a further extension product comprising the second detectable label.

4. The method of claim 1, wherein the synthetic nucleic acid comprises an extension product of an initial primer, and wherein the initial primer comprises the first detectable label.

5. The method of claim 1, wherein the synthetic nucleic acid comprises an extended nucleic acid comprising about 20 or more nucleotides.

6. The method of claim 5, wherein the extended nucleic acid comprises the first detectable label.

7. The method of claim 1, wherein the incubating further comprises: unwinding the duplex; annealing a primer comprising the second detectable label to a portion of the target nucleic acid; and The primer is extended to provide a further extension product comprising the second detectable label.

8. The method of claim 1, further comprising before said providing: annealing the target nucleic acid and the synthetic nucleic acid to form the pre-annealed duplex; and The pre-annealed duplex is optionally cooled.

9. The method of claim 1, further comprising before said providing: annealing the target nucleic acid and the synthetic nucleic acid, wherein the synthetic nucleic acid is configured as an initial primer; and The initial primer is extended under polymerase primer extension conditions, thereby forming the pre-annealed duplex.

10. The method of claim 1, further comprising after said incubation: The samples are inactivated using heat inactivation and / or chemical inactivation.

11. The method of claim 1, further comprising before the detection: The sample is purified to remove one or more chemical or biological components.

12. The method of claim 1, wherein the synthetic nucleic acid and the primer comprise the same nucleic acid sequence.

13. The method of claim 1, wherein the synthetic nucleic acid is shorter than the primer or is the same length as the primer.

14. The method of claim 1, wherein the synthetic nucleic acid is longer than the primer.

15. The method of claim 1, wherein the synthetic nucleic acid and the primer independently have a length of about 5 to 100 nucleotides.

16. The method of claim 1, wherein the pre-annealed duplex comprises a 3'-tail and / or a 5'-tail.

17. The method of claim 1, wherein the pre-annealed duplex comprises at least one blunt end.

18. The method of claim 1, wherein the pre-annealed duplex has a length of about 15 to 500 base pairs.

19. The method of claim 1, wherein the ratio of the primer to the pre-annealed duplex is 1:1 to 50:

1.

20. The method of claim 1, wherein the first and second detectable labels are the same or different.

21. The method of claim 20, wherein the first and second detectable labels are independently provided at the 5'-end or internally.

22. The method of claim 20, wherein the synthetic nucleic acid comprises a plurality of first detectable labels, and / or wherein the primer comprises a plurality of second detectable labels.

23. The method of claim 20, wherein the first and second detectable labels are selected from fluorescent labels, radioactive labels, chemiluminescent labels, or dyes.

24. The method of claim 1, wherein the sample further comprises 3'-amino-2',3'-dideoxyribonucleotide 5'-triphosphates (nNTPs), divalent ions, denaturants, buffers, and / or salts.

25. The method of claim 1, wherein the amplification enzyme and / or the helicase is selected from a helicase, a recombinase, a polymerase, a reverse transcriptase, a thermophilic form thereof, a thermostable form thereof, and a recombinant form thereof.

26. The method of claim 1, wherein the isothermal amplification conditions comprise a temperature of about 20 to 75°C.

27. The method of claim 1, wherein the detecting comprises electrophoretic analysis, optionally on a microfluidic device.

28. The method of claim 27, wherein the electrophoretic analysis comprises analyzing fragment sizes of amplicons comprising the second detectable label.

29. The method of claim 28, wherein the electrophoretic analysis further comprises comparing the fragment size of the amplicons to a labeled size standard (eg, a fluorescently labeled size standard).

30. A kit comprising: a pre-annealed duplex, a primer comprising a second detectable label, an amplification enzyme, a helicase, and instructions for performing the method of claim 1.

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