Reagents and methods for amplification reactions

Abstract

This disclosure relates to method for preparing a mixture of at least one Hot-Start-Like Reagent and a DNA polymerase prior to assembly of a complete master-mix for amplification of double-stranded amplicons in a closed-reaction vessel, the method comprising combining at least one Hot-Start-Like (HSL) Reagent and at least one DNA polymerase at an operative ratio of units of said reagent to units of said DNA polymerase. The HSL Reagent being comprised of one or two non-amplifiable single-stranded oligonucleotides and said operative ratio being determined by quantitative end-point melt-curve analysis of a fluorescent dye that binds to double-stranded DNA molecules in said closed-reaction vessel.

Description

[0001] REAGENTSAND METHODS FOR AMPLIFICATION REACTIONS

[0002] RELATED APPLICATIONS

[0003] This application claims priority to U.S. Ser. No. 63 / 664,672 filed on 26 June 2024, which is hereby incorporated into this disclosure in its entirety.

[0004] SEQUENCE LISTING

[0005] The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created 25 June 2025, is named ECOL002PCT_st26.XML and is 30,000 bytes in size.

[0006] FIELD OF THE DISCLOSURE

[0007] This disclosure relates to reagents and methods for preparing mixtures useful for hot- start polymerase chain reaction amplification systems.

[0008] BACKGROUND INFORMATION

[0009] PCR amplification and isothermal amplification are methods that depend on the use of short oligonucleotide primers, dNTP precursors, monovalent and divalent salts, buffers, additional additives such as sugars and / or proteins, and one or more enzymes. The type of enzyme that carries out PCR amplification is often referred to as Taq DNA polymerase. As a class, these enzymes are heat-stable insofar as they can be heated to nearly the boiling temperature of water and then cooled, without causing them to lose functionality. However, since they are irreversibly denatured by freezing, these enzymes are typically dissolved in glycerol for shipping or storing below 4°C. They are typically used in the temperature range of 60-80°C to synthesize new DNA strands by addition of dNTP’s to the 3’ end of a primer that is hybridized to a DNA template strand.

[0010] In contrast, rolling circle amplification and loop-mediated isothermal amplification are two methods of isothermal amplification. These reactions use DNA polymerases that are temperature-sensitive.

[0011] One-step RT-PCR uses a reverse transcription for synthesis of a DNA template from an RNA template, followed by amplification of the DNA template using a DNA polymerase in the same closed-vessel master-mix. Most commercial reverse transcriptases are active in the range of 45-65 °C and are rapidly and irreversibly denatured by higher temperatures. Thus, incubation at about 45-65 °C for several minutes is often used to carry out reverse transcription before the temperature of the reaction is raised to about 95 °C to inactivate the reverse transcriptase and separate the product strands prior to beginning the process of PCR amplification when the temperature is lowered again.

[0012] Standard PCR protocols typically use an initial 95-99°C heating step of 2-15 minutes to irreversibly denature the “hot-start” activity which is included in most commercial hot-start “Taq” type DNA Polymerases. A hot-start reagent is added to the DNA polymerase in an effort to prevent primer extension when the reaction components are being mixed at room temperature or even on ice. In the absence of a hot-start reagent, polymerase activities at these times can result in a large class of artifacts due to “mis-priming”. The products of mis-priming include primer-dimers and primer oligomers. These artifacts consume useful primers and degrade the accuracy and sensitivity of PCR amplification (Rice et al., 2007, Nature Protocols, 2(10): 2429-2438; Chou et al., 1992, Nucleic Acids Res., 20(7): 1717-23). Formation of primerdimers and primer-oligomers becomes more and more likely as the number of primer pairs is increased in multiplexed and highly multiplexed reactions. These types of artifacts are particularly troublesome in whole-genome amplification reactions initiated with small amounts of genomic DNA.

[0013] Mis-priming by “Taq” polymerases during reaction set-up is also manifest as artifactual single-nucleotide polymorphisms (SNPs) due to incorporation of an incorrect nucleotide during primer extension. This type of artifact frequently occurs at low temperatures when perfectly matched and mismatched primers are used to differentially amplify particular genetic alleles. Amplification of artifactual SNP’s degrades the accuracy of many types of protocols aimed at early detection of cancer-causing mutations. There is a need in the art for reagents and methods that provide robust “hot-start” activity at the start of PCR amplification. Solutions for such problems are provided by this disclosure.

[0014] SUMMARY OF THE DISCLOSURE

[0015] The present disclosure relates to, in some preferred embodiments, the preparation and usage of Hot-Start-Like (HSL) reagents for an amplification reaction, which involves placing, concentrating, and storing the HSL reagent(s) in a reaction vessel designed to amplify a nucleic acid target sequence, either in the absence or presence of any other component(s) of an amplification reaction master-mix. In some aspects, the present disclosure provides a process of preparing and using at least one HSL Reagent for an amplification reaction. Such a process may comprise the following steps:

[0016] (a) placing a solution containing a concentrate of the at least one HSL Reagent in a reaction vessel, wherein the at least one HSL Reagent is present in the reaction vessel at an amount sufficient to suppress mis-priming before, during, or after the amplification reaction is carried out in the reaction vessel;

[0017] (b) rehydrating the concentrate of the at least one HSL Reagent in the reaction vessel; and

[0018] (c) placing the reaction vessel in a device suitable for the amplification reaction.

[0019] In some preferred embodiments, this disclosure provides methods for preparing a mixture of at least one Hot-Start-Like Reagent and a DNA polymerase, prior to assembly of a complete master-mix for amplification of double- stranded amplicons in a closed-reaction vessel, the method comprising combining at least one Hot-Start-Like (HSL) reagent and at least one DNA polymerase at an operative ratio of units of said at least one HSL reagent to units of said at least one DNA polymerase wherein: the at least one HSL reagent comprises at least one DNA oligonucleotide modified at its 3’ and 5’ terminal nucleotides such that the at least one single-stranded oligonucleotide is non-amplifiable by said DNA polymerase; and, either the at least one single-stranded oligonucleotide has a temperature-dependent reversible doublestranded hairpin conformation, or he at least one single-stranded DNA oligonucleotide hybridizes to a second non-amplifiable single-stranded DNA oligonucleotide generating a double-stranded DNA molecule in a temperature-dependent manner; and, said operative ratio is determined by quantitative end-point melt-curve analysis of a fluorescent dye that binds to double-stranded DNA molecules in said closed-reaction vessel.

[0020] In some embodiments, the concentrate in step (a) may be further dehydrated to produce a hyper-concentrate or a dried concentrate in a reaction vessel prior to step (b).

[0021] In some embodiments, the at least one HSL Reagent may comprise a non-amplifiable temperature-dependent intramolecular-hybridizing single-stranded DNA oligomer, having covalently bound moieties on both its 5’ and 3’ ends.

[0022] In some embodiments, the at least one HSL Reagent may comprise two non-amplifiable temperature-dependent intermolecular-hybridizing single-stranded DNA oligomers, having one to four covalently bound moieties on their 5’ or 3’ ends. In some embodiments, the at least one HSL Reagent may comprise a closed circular double-stranded DNA.

[0023] In some examples, the at least one HSL Reagent comprises a hairpin-shaped singlestranded oligonucleotide modified at both the 3 ’ and 5 ’ ends. In some specific examples, hairpin shaped single stranded oligonucleotides are modified by addition of dabcyl moieties, Black Hole Quencher™ moieties, and / or 2’ O-methyl nucleotides at the end of the stem.

[0024] In some examples, the at least one HSL Reagent comprises a pair of complementary or partially complementary oligonucleotides that form a hybrid 6-50 nucleotides long, wherein the oligonucleotides are modified on one or both ends by addition of polycyclic moieties. In some specific examples, the oligonucleotides are modified by addition of dabcyl or coumarin moieties that do not have bulky portions that are non-planar.

[0025] In some embodiments, the at least one HSL Reagent may comprise three non- amplifiable temperature-dependent intermolecular-hybridizing single-stranded DNA oligomers, having one-to-six covalently bound moieties on their 5’ or 3’ ends.

[0026] In some examples, the at least one HSL Reagent comprises a pair of oligonucleotide strands that form a hybrid at least six nucleotides long with a calculated Tm of at least 32°C, wherein strands in the terminal region of the hybrid contain interacting label moieties, which comprises a fluorophore on one strand and either a fluorophore or a non-fluorescent quencher on the other strand.

[0027] In some embodiments, the at least one HSL Reagent may include from one to four single-stranded overhangs. Alternatively, or in addition, the at least one HSL Reagent forms a stem-loop structure when not hybridized.

[0028] In some embodiments, the solution in step (a) may comprise two HSL Reagents, which may consist of three strands.

[0029] In some embodiments, the number of units of the at least one HSL Reagent placed in the reaction vessel has a ratio in the range of 0.1 :1 to 10:1 relative to the number of units of DNA binding enzyme placed in the reaction vessel.

[0030] In some embodiments, the concentration of the at least one Hot-Start-Like Reagent in its mixture is at least 1.5 times greater than its nano-molar concentration in the complete amplification master-mix in a closed-vessel.

[0031] In some embodiments, the reaction vessel suitable for the process disclosed above and herein may have rigid walls and may be made of plastic, polymer, glass, carbon, or metal. Alternatively, the reaction vessel may be a pliable film. Still alternatively, the reaction vessel may have walls made of a non-aqueous fluid, e.g., an oil or a lipid. In some examples, the reaction vessel is a plastic tube, a multi-welled plastic plate, a multi-welled film, an array of small chambers, a microfluidic device, a self-contained cassette, or a nano-chip printed with hydrophobic and hydrophilic surface arrays.

[0032] In some embodiments, the further dehydration step may be accomplished by using an overlay, which is in contact with the concentrate in the reaction vessel, and / or wherein the overlay is made of a non-aqueous material that absorbs water. In some embodiments, the concentration of the at least one HSL Reagent is a concentrate, a hyper-concentrate, or a dried- concentrate.

[0033] In some embodiments, the reaction vessel containing the concentrate, the hyperconcentrate, or the dried-concentrate of the at least one HSL Reagent may be sealed with a cap. In some examples, the cap is made of plastic, polymer, glass, carbon, or metal.

[0034] In some embodiments, the concentrate, the hyper-concentrate, or the dried-concentrate of the at least one HSL Reagent may further comprise at least one enzyme having a binding pocket for at least one double- stranded oligonucleotide. In some examples, the at least one enzyme is a DNA polymerase (e.g., a Type A DNA polymerase, a Type B polymerase, or a Type C DNA polymerase). In some examples, the at least one enzyme is an exonuclease (e.g. , the 5’-to-3’ exonuclease of Taq polymerase, or the 3’-to-5’ proofreading exonuclease of Taq polymerase). In some examples, the at least one enzyme is a DNA ligase (e.g., Bacteriophage T4 ligase, or E. Coli ligase).

[0035] In some embodiments, the rehydrating step (b) may comprise diluting the concentrate, the hyper-concentrate, or the dried-concentrate of the at least one HSL Reagent in the reaction vessel by addition of a measured volume of an aqueous solution containing one or more additional components of a complete amplification reaction master-mix, one or more nucleic acid amplification template molecules for generation of at least one amplicon, or a combination thereof. In some examples, the one or more additional components of a complete amplification reaction master-mix include a monovalent cationic salt, a pH buffer, a divalent cationic salt, a dNTP, glycerol, dimethyl sulfoxide, a sugar, a heat denaturable protein, a polypeptide, a nonionic detergent, and / or other organic compound(s). Exemplary non-ionic detergents include, but are not limited to, Tween 20, NP-40, and Triton X-100. Exemplary organic compounds include, but are not limited to, betaine, TMAC, formamide, 7-deaza-2'-deoxyguanosine, and PEG.

[0036] In some aspects, the present disclosure also provides a reaction vessel that is pre-loaded with a concentrate, a hyper-concentrate, or a dried -concentrate of at least one HSL Reagent for an amplification reaction. Such a reaction vessel is suitable for the any of the process described above and herein.

[0037] In some embodiments, the at least one HSL Reagent may comprise a non-amplifiable temperature-dependent intramolecular-hybridizing single-stranded DNA oligomer, having covalently bound moieties on both its 5’ and 3’ ends.

[0038] In some embodiments, the at least one HSL Reagent may comprise two non-amplifiable temperature-dependent intermolecular-hybridizing single-stranded DNA oligomers, having one to four covalently bound moieties on their 5’ or 3’ ends.

[0039] In some embodiments, the at least one HSL Reagent may comprise three non- amplifiable temperature-dependent intermolecular-hybridizing single-stranded DNA oligomers, having one-to-six covalently bound moieties on their 5’ or 3’ ends.

[0040] In some embodiments, the at least one HSL Reagent may comprise a closed circular double-stranded DNA.

[0041] In some examples, the at least one HSL Reagent comprises a hairpin-shaped singlestranded oligonucleotides modified at both the 3’ and 5’ ends. In some specific examples, hairpin shaped single stranded oligonucleotides are modified by addition of dabcyl moieties, Black Hole Quencher™ moieties, and / or 2’ O-methyl nucleotides at the end of the stem.

[0042] In some examples, the at least one HSL Reagent comprises a pair of complementary or partially complementary oligonucleotides that form a hybrid 6-50 nucleotides long, wherein the oligonucleotides are modified on one or both ends by addition of polycyclic moieties. In some specific examples, the oligonucleotides are modified by addition of dabcyl or coumarin moieties that do not have bulky portions that are non-planar.

[0043] In some examples, the at least one HSL Reagent comprises a pair of oligonucleotide strands that form a hybrid at least six nucleotides long with a calculated Tm of at least 32°C, wherein strands in the terminal region of the hybrid contain interacting label moieties, which comprises a fluorophore on one strand and either a fluorophore or a non-fluorescent quencher on the other strand.

[0044] In some embodiments, the at least one HSL Reagent may include from one to four single- stranded overhangs. Alternatively, or in addition, the at least one HSL Reagent forms a stem-loop structure when not hybridized.

[0045] In some embodiments, the reaction vessel may be pre-loaded with two HSL Reagents, which may consist of three strands.

[0046] In some embodiments, the number of units of the at least one HSL Reagent placed in the reaction vessel has an operative ratio in the range 0.1 :1 to 10:1 relative to the number of units of DNA binding enzyme placed in the reaction vessel. In some embodiments, the concentration of the at least one Hot-Start-Like Reagent in its mixture is at least 1.5 times greater than its nano-molar concentration in the complete amplification master-mix in a closed-vessel.

[0047] In some embodiments, the reaction vessel suitable for the process disclosed above and herein may have rigid walls and may be made of plastic, polymer, glass, carbon, or metal. Alternatively, the reaction vessel may be a pliable film. Still alternatively, the reaction vessel may have walls made of a non-aqueous fluid, e.g., an oil or a lipid.

[0048] In some examples, the reaction vessel is a plastic tube, a multi-welled plastic plate, a multi-welled film, an array of small chambers, a microfluidic device, a self-contained cassette, or a nano-chip printed with hydrophobic and hydrophilic surface arrays.

[0049] In some embodiments, the further dehydration step may be accomplished by using an overlay, which is in contact with the concentrate in the reaction vessel, and / or wherein the overlay is made of a non-aqueous material that absorbs water. In some embodiments, the at least one HSL Reagent is a concentrate, a hyper-concentrate, or a dried-concentrate.

[0050] In some embodiments, the reaction vessel containing the concentrate, the hyperconcentrate, or the dried-concentrate of the at least one HSL Reagent may be sealed with a cap. In some examples, the cap is made of plastic, polymer, glass, carbon, or metal.

[0051] In some embodiments, the concentrate, the hyper-concentrate, or the dried-concentrate of the at least one HSL Reagent may further comprise at least one enzyme having a binding pocket for at least one double- stranded oligonucleotide. In some examples, the at least one enzyme is a DNA polymerase (e.g., a Type A DNA polymerase, a Type B polymerase, or a Type C DNA polymerase). In some cases, the DNA polymerase is combined with a reverse transcriptase (e.g., avian myeloblastosis virus reverse transcriptase or Moloney murine leukemia virus reverse transcriptase). In some examples, the at least one enzyme is an exonuclease (e.g. , the 5’-to-3’ exonuclease of Taq polymerase, or the 3’-to-5’ proofreading exonuclease of Taq polymerase). In some cases, the at least one enzyme is a DNA ligase (e.g. , Bacteriophage T4 ligase, or E. Coli ligase).

[0052] In some embodiments, the concentrate, the hyper-concentrate, or the dried-concentrate of the at least one HSL Reagent may be diluted in the reaction vessel by addition of a measured volume of an aqueous solution containing one or more additional components of a complete amplification reaction master-mix, one or more nucleic acid amplification template molecules for generation of at least one amplicon, or a combination thereof, before an amplification reaction. In some examples, the one or more additional components of a complete amplification reaction master-mix include a monovalent cationic salt, a pH buffer, a divalent cationic salt, a dNTP, glycerol, dimethyl sulfoxide, a sugar, a heat denaturable protein, a polypeptide, a nonionic detergent, and / or other organic compound(s). Exemplary non-ionic detergents include, but are not limited to, Tween 20, NP-40, and Triton X-100. Exemplary organic compounds include, but are not limited to, betaine, TMAC, formamide, 7-deaza-2'-deoxyguanosine, and PEG.

[0053] The details of one or more embodiments of the invention are set forth in the description below. Other features or advantages of the present invention will be apparent from the following drawings and detailed description of several embodiments, and also from the appended claims.

[0054] BRIEF DESCRIPTION OF THE FIGURES AND DRAWINGS

[0055] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present disclosure, which can be better understood by reference to the drawings in combination with the detailed description of specific embodiments presented herein.

[0056] FIG. 1 gel electrophoretic analysis of the impact of HSL Reagent#! on PCR amplification of individual pairs of symmetric primers (Table 2) used in construction of a 10- Plex reaction (FIG 3).

[0057] FIG. 2 gel electrophoretic analysis demonstrating that HSL Reagent#! enables 10-plex PCR amplification using the ten pairs of symmetric primers described in Table 2 and FIG. 1.

[0058] FIG. 3A shows the three double-stranded DNA amplicons and their lengths in base pairs that correspond to Curve 305. FIG. 3B shows the SYBR Green melt curve analysis in a titration series of separate reactions that were set up in separate vessels containing the HSL Reagent#! and Taq in unit-to-unit ratios of 0.44: 1 (Curve 301), 1:1 (Curve 302), 3:1 (Curve 303), 5:1 (Curve 304), and 10:1 (Curve 305). Each reaction vessel was sealed with a cap, stored for later use, and subsequently diluted by the addition of aqueous solutions containing the remaining components of a complete multiplex amplification reaction master mix and genomic DNA targets to enable the multiplex amplification of specific products once the reaction vessels were placed in a PCR cycler.

[0059] FIG. 4 shows the results from the no-DNA controls for the samples in FIG. 3.

[0060] FIG. 5 compares the efficacy of dried HSL Reagent#! (Curves 501) to non-dried controls of concentrated HSL Reagent#l (Curves 502). In both cases, all reactions contained genomic DNA, and the HSL Reagent#l and the DNA polymerase were used in a 5:1 ratio of units-to-units. The set of reaction vessels shown in (Curves 501) were dried, sealed with a cap, stored for later use, and subsequently rehydrated and diluted by the addition of aqueous solutions containing the remaining components of a complete multiplex amplification reaction master mix and genomic DNA targets to enable the multiplex amplification of specific products once the reaction vessels were placed in a PCR cycler.

[0061] FIG. 6 shows the results from the no-DNA controls for the samples in FIG. 5.

[0062] FIG. 7 compares the efficacy of dried HSL Reagent#! plus DNA polymerase (Curves

[0063] 701) to non-dried controls of concentrated HSL Reagent# 1 plus DNA polymerase (Curves

[0064] 702). In both cases, all reactions contained genomic DNA. Curves 701 shows that concentrated HSL Reagent#! and DNA polymerase can be prepared in PCR buffer, MgCh, and dNTPs at a 5:1 ratio, units-to-units, and can be dried in separate reaction vessels, sealed with a cap, stored for later use, and subsequently rehydrated and diluted by the addition of aqueous solutions containing the remaining components of a complete multiplex amplification reaction master mix, as well as genomic DNA targets to enable the multiplex amplification of specific products once the reaction vessels are placed in a PCR cycler.

[0065] FIG. 8 shows -dF / dT melt curve analysis for the no-DNA controls for the samples in FIG. 7

[0066] FIG. 9 shows -dF / dT melt curve analysis for the four cases in which amplification was tested using dried EconoTaq prepared in PCR buffer, MgCh, and dNTPs and subsequently rehydrated and diluted with aqueous solutions containing either only the remaining components of a complete multiplex amplification reaction master mix and genomic DNA targets (including the HSL Reagent#! at a 5:1 reagent-to-DNA polymerase ratio) to enable the multiplex amplification of specific products once the reaction vessels are placed in a PCR cycler.

[0067] FIG.10 shows -dF / dT melt curve analysis of the no-DNA-control for the experiment in FIG. 9

[0068] FIG.ll shows the SYBR Green melt curve analysis in a titration series of separate reactions that were set up in separate vessels containing the HSL Reagent#2 and Taq in unit- to-unit ratios of 1 : 1 (Curve 1101), 3:1 (Curve 1102), 5: 1 (Curve 1103), 10: 1 (Curve 1104). Each reaction vessel was sealed with a cap, stored for later use, and subsequently diluted by the addition of aqueous solutions containing the remaining components of a complete multiplex amplification reaction master mix and genomic DNA targets to enable the multiplex amplification of specific products once the reaction vessels were placed in a PCR cycler.

[0069] FIG.12 shows -dF / dT melt curve analysis of amplified products generated in separate reactions that were set up in vessels containing the HSL Reagent#3 and the DNA polymerase in unit-to-unit ratios of 0.055: 1 (Curve 1201), 0.111 :1 (Curve 1202), 0.222:1 (Curve 1203), and 0.444: 1 (Curve 1204). Each reaction vessel was sealed with a cap, stored for later use, and subsequently diluted by the addition of aqueous solutions containing the remaining components of a complete multiplex amplification reaction master mix and genomic DNA targets to enable the multiplex amplification of specific products once the reaction vessels were placed in a PCR cycler.

[0070] FIG.13 shows -dF / dT melt curve analysis of amplified products generated in separate reactions that were set up in vessels containing the HSL Reagent#4 and the DNA polymerase in unit-to-unit ratios of 0.055: 1 (Curve 1301), 0.111:1 (Curve 1302), 0.222: 1 (Curve 1303), and 0.444:1 (Curve 1304). Each reaction vessel was sealed with a cap, stored for later use, and subsequently diluted by the addition of aqueous solutions containing the remaining components of a complete multiplex amplification reaction master mix and genomic DNA targets to enable the multiplex amplification of specific products once the reaction vessels were placed in a PCR cycler.

[0071] FIG.14 shows -dF / dT melt curve analysis of amplified products generated in separate reactions that were set up in vessels containing the HSL Reagent#4 and the DNA polymerase in unit-to-unit ratios of 0.055:1 (Curve 1401), 0.111:1 (Curve 1402), 0.222:1 (Curve 1403), and 0.444:1 (Curve 1404). Each reaction vessel was sealed with a cap, stored for later use, and subsequently diluted by the addition of aqueous solutions containing the remaining components of a complete multiplex amplification reaction master mix and genomic DNA targets to enable the multiplex amplification of specific products once the reaction vessels were placed in a PCR cycler.

[0072] FIG.15 shows -dF / dT melt curve analysis of amplified products generated in separate reactions that were set up in vessels containing either dried HSL Reagent#3 (Curve 1501 and 1502) or dried HSL Reagent#4 (Curve 1503 and 1504) and either not dried EconoTaq (Curve 1501 and 1503), or dried EconoTaq (Curve 1502 and 1504). In all cases, thereagent-to-enzyme ratio was 0.222:1.

[0073] FIG.16A shows -dF / dT melt Curve 1601 over the temperature range of 42.5-75°C for a reaction containing HSL R.#6 and Taq at a ratio of 0.042: 1. The arrow marks the temperature at which the two arms of the reagent melt apart. The reagent hairpin is fully closed below about 68°C and fully opened above about 73°C. FIG. 16B shows -dF / dT melt curve analysis of amplified products generated in separate reactions over the temperature range of 72.5-95°C. These reactions were set up in vessels containing either HSL Reagent#6 to Taq at a ratio of 0.042:1 (Curve 1601), or HSL Reagent#6 to Taq at a ratio of 0.083:1 (Curve 1602), or HSL Reagent#6 to Taq at a ratio of 0.25:1 (Curve 1603), or HSL Reagent#6 to Taq at a ratio of 0.5 : 1 (Curve 1604), or HSL Reagent#6 to Taq at a ratio of 1:1 (Curve 1605).

[0074] FIG.17A shows -dF / dT melt Curve 1706 over the temperature range of 46-96°C for a reaction containing HSL R.#7 and Taq at a ratio of 1 :1. The arrow marks the temperature at which the two arms of the reagent melt apart. The reagent hairpin is fully closed below about 68 °C and fully opened above about 73 °C. FIG. 17B shows -dF / dT melt Curve analysis of amplified products generated in separate reactions over the temperature range of 72-95°C. These reactions were set up in vessels containing either HSL Reagent#7 to Taq at a ratio of 0.0: 1 (Curve 1701), or HSL Reagent#7 to Taq at a ratio of 0.042: 1 (Curve 1702), or HSL Reagent#7 to Taq at a ratio of 0.083:1 (Curve 1703), or HSL Reagent#7 to Taq at a ratio of 0.25: 1 (Curve 1704), or HSL Reagent#7 to Taq at a ratio of 0.5: 1 (Curve 1705), or HSL Reagent#7 to Taq at a ratio of 1 : 1 (Curve 1706).

[0075] FIG.18 shows -dF / dT melt curve analysis of amplified products generated in separate reactions over the temperature range of 72-96°C. These reactions were set up in vessels containing either HSL Reagent#8 to Taq at a ratio of 0:1 (Curve 1801), or HSL Reagent#8 to Taq at a ratio of 0.33:1 (Curve 1802), or HSL Reagent#8 to Taq at a ratio of 1: 1 (Curve 1803), or HSL Reagent#8 to Taq at a ratio of 1.33: 1 (Curve 1804), or HSL Reagent#8 to Taq at a ratio of 1.66: 1 (Curve 1805).

[0076] FIG.19 shows -dF / dT melt curve analysis of amplified products generated in three types of reactions over the temperature range of 75-97° C. All three types of reactions were carried out in replicates. The ratio of HSL R.#8 to Taq was 1.66:1 in all cases. The top panel shows the result when neither the HSL R.#8 nor the Taq was dried. It is similar to curve 1805. The middle panel shows the result when the HSL R.#8 was dried and then resuspended using Taq that was not dried. The bottom panel shows the results when the HSL R.#8 and the Taq were incubated together and dried.

[0077] FIG.20 shows -dF / dT melt curve analysis of amplified products generated in separate replicate reactions over the temperature range of 75-95°C. Each of these reactions were set up in vessels containing 1 unit of HSL R.#l, plus either no HSL R. #9 (Curve 2001), or plus 0.5 units of HSL R.#9 (Curve 2002), or plus 1.0 units of HSL R.#9 (Curve 2003), or plus 2.0 units of HSL R.#9 (Curve 2004), or plus 4.0 units of HSL R.#9 (Curve 2005).

[0078] FIG.21 first derivative melt curve analysis of the fluorescent readings as a function of temperature, over three temperature ranges, for the samples containing genomic DNA, in which each reaction used either HSL Reagents#l,2,3,4, or 8 at its optimal reagent-to-Taq ratio. Left Panel - temperature range 42-70°C; Middle Panel - temperature range 40-95°C; Right Panel - temperature range 70-94°C.

[0079] FIG.22A, Model illustrating the structural domains of Taq Polymerase; FIG.22B, Model illustrating the position of HSL Reagent#l superimposed on Taq Polymerase; FIG.22C, Model illustrating the position of HSL Reagent#8 superimposed on Taq Polymerase.

[0080] DETAILED DESCRIPTION

[0081] The present disclosure provides a process of preparing and using HSL Reagent(s). In particular, the present disclosure describes how measured amounts of one or more such reagent(s) can be added directly into a reaction vessel or other container prior to initiation of an amplification reaction, under conditions in which the concentrate of such reagent(s) either remains in solution or dries out. In some situations, such reagent(s) can also be combined in solution with one or more reactants in a mixture-of -reactants prior to being placed in a reaction vessel or other container. Because such reagents are known to bind in the binding pockets of enzymes used in various methods of nucleic acid amplification, they can be mixed with those enzymes in measured proportions when both the enzymes and the reagents are in solution in a buffer with salts and MgCh, thereby making a mixed-concentrate, prior to being added to a reaction vessel or other container. Such reagents are chemically and thermally stable; as such, they can be stored for long periods of time, with or without additional reactants, prior to addition of additional components of an amplification master-mix. Such reagents can be added to one or more reaction vessels or other containers manually, or by automated high-throughput robotic systems under sterile conditions common to the vessel manufacturing industry.

[0082] In some aspects, the present disclosure provides a process of preparing and using at least one HSL Reagent for an amplification reaction.

[0083] In some embodiments, the process may involve placing a volume of a solution containing a concentrate of at least one HSL Reagent required for a single amplification reaction into a reaction vessel. The concentrate may be stored in the reaction vessel that is sealed with a cap for use at a later time, or may be first rendered into a hyper-concentrate or a dried-concentrate via a dehydration process in the reaction vessel prior to being sealed and stored. The concentrate, hyper-concentrate, or dried-concentrate may then be re-dissolved and diluted by addition of a measured volume of an aqueous solution, or an aqueous solution containing one or more additional components of a complete reaction master-mix, or by addition of an aqueous solution containing one or more nucleic acid amplification template molecules for generation of at least one amplicon. Following addition of all amplification reaction components, the reaction vessel is placed in a device having the capacity for PCR or isothermal amplification.

[0084] In some embodiments, the process may involve placing a volume of a solution containing a concentrate of at least one HSL Reagent required for a single amplification reaction and at least one enzyme having a nucleic acid binding site into a reaction vessel. The concentrate may be stored in the reaction vessel that is sealed with a cap for use at a later time, or may be first rendered into a hyper-concentrate or a dried-concentrate via a dehydration process in the reaction vessel prior to being sealed and stored. The concentrate, hyperconcentrate, or dried-concentrate may then be re-dissolved and diluted by addition of a measured volume of an aqueous solution, or an aqueous solution containing one or more additional components of a complete reaction master-mix, or by addition of an aqueous solution containing one or more nucleic acid amplification template molecules for generation of at least one amplicon. Following addition of all amplification reaction components, the reaction vessel is placed in a device having the capacity for PCR or isothermal amplification.

[0085] In some embodiments, the process may involve placing a volume of a solution containing a concentrate of at least one HSL Reagent required for a single amplification reaction into a reaction vessel. The concentrate may be stored in the reaction vessel that is sealed with a cap for use at a later time, or may be first rendered into a hyper-concentrate or a dried-concentrate via a dehydration process in the reaction vessel prior to being sealed and stored. The concentrate, hyper-concentrate, or dried-concentrate, and any or all components of an amplification master-mix may then be re-dissolved and diluted by addition of a measured volume of an aqueous solution or by addition of an aqueous solution containing one or more nucleic acid amplification template molecules for generation of at least one amplicon. Subsequently, the reaction vessel is placed in a device having the capacity for PCR or isothermal amplification.

[0086] In some embodiments, the process may involve placing a volume of a solution containing a concentrate of at least one HSL Reagent required for a single amplification reaction into a container other than a reaction vessel. The concentrate may be stored in the container that is sealed with a cap for use at a later time, or may be first rendered into a hyperconcentrate or a dried-concentrate via a dehydration process in the container prior to being sealed and stored. The concentrate, hyper-concentrate, or dried-concentrate, and any or all components of an amplification master-mix may then be re-dissolved and diluted by addition of a measured volume of an aqueous solution or by addition of an aqueous solution containing one or more nucleic acid amplification template molecules for generation of at least one amplicon. The resulting complete master mix may then be redistributed in multiple reaction vessels, which may be placed in a device having the capacity for PCR or isothermal amplification.

[0087] In other aspects, the present disclosure provides a reaction vessel pre-loaded with a concentrate, a hyper-concentrate, or a dried-concentrate of at least one HSL Reagent for an amplification reaction. Suitable reaction vessels or containers include, but are not limited to, plastic tubes; multi-welled plastic plates; multi-welled films; arrays of small chambers; microfluidic devices; self-contained cassettes, nano-chips printed with hydrophobic and hydrophilic surface arrays.

[0088] A. HSL Reagent(s)

[0089] HSL Reagent(s) are a class of DNA-based oligonucleotide reagents having covalently attached modifying groups, which are used as additives in a mixture-of-reactants that interact to amplify one or more nucleic acid sequences in a temperature-dependent protocol (Rice et al., 2007, Nature Protocols, 2(10): 2429-2438; Chou et al., 1992, Nucleic Acids Res., 20(7): 1717-23). When used alone or in combination, these reagents serve to prevent errors in amplification prior to, during, and after amplification of at least one target sequence. Reagent(s) having these properties are described in U.S. Patent Nos. 7,517,977B2; 9,034,605B2; 9,758, 813A; and 10,240, 178B2. Table 1 provides a summary of some properties of the particular HSL Reagents used in the Examples below. As one versed in the art will appreciate, the HSL Regents #1-9 listed in Table 1 are merely those that have thus far been tested for their relative potencies; many other designs of each type could be compared using the same tests.

[0090] Table 1

[0091] Docket No.: ECOL002.PCT

[0092] Table 1 Legend: * Potency is measured as the specific reagent-to-DNA Polymerase unit ratio needed to achieve optimum amplification of all three amplicons in the DNA triplex assay and is then normalized relative to the optimum reagent-to-DNA Polymerase unit ratio using HSL Reagent #1. Very Inhibitory; Partially Inhibitory, Poor (incomplete), n.d. Not Determined.5Comparison of the HSL Reagent #1 Curves 302-305, FIG.3B, and HSL Reagent #2 Curves 1101-1104, FIG.ll, establish that HSL Reagent #2 is less potent than HSL Reagent #1.

[0093] Table 2

[0094] Suitable HSL Reagents may include one or more of the following: (a) a non-amplifiable temperature-dependent intramolecular-hybridizing single-stranded DNA oligomer, having covalently bound moieties on both its 5’ and 3’ ends; (b) two non-amplifiable temperaturedependent intermolecular-hybridizing single-stranded DNA oligomers, having one to four covalently bound moieties on their 5’ or 3’ ends; (c) three non-amplifiable temperaturedependent intermolecular-hybridizing single-stranded DNA oligomers, having one-to-six covalently bound moieties on their 5 ’ or 3 ’ ends; or (d) a closed circular double-stranded DNA.

[0095] In some examples, the HSL Reagent comprises a hairpin- shaped single-stranded oligonucleotides modified at both the 3’ and 5’ ends. In some specific examples, hairpin shaped single stranded oligonucleotides are modified by addition of dabcyl moieties, Black Hole Quencher™ moieties, and / or 2’ O-methyl nucleotides at the end of the stem. In some examples, the HSL Reagent comprises a pair of complementary or partially complementary oligonucleotides that form a hybrid 6-50 nucleotides long, wherein the oligonucleotides are modified on one or both ends by addition of polycyclic moieties. In some specific examples, the oligonucleotides are modified by addition of dabcyl or coumarin moieties that do not have bulky portions that are non-planar. In some examples, the HSL Reagent comprises a pair of oligonucleotide strands that form a hybrid at least six nucleotides long with a calculated Tm of at least 32°C, wherein strands in the terminal region of the hybrid contain interacting label moieties, which comprises a fhiorophore on one strand and either a fluorophore or a non-fluorescent quencher on the other strand.

[0096] In some embodiments, the HSL Reagent may include from one to four single-stranded overhangs. Alternatively, or in addition, the HSL Reagent forms a stem-loop structure when not hybridized.

[0097] HSL Reagents are temperature-dependent, concentration-dependent, ion-dependent inhibitors of at least one type of enzyme activity exhibited by DNA polymerases, Reverse Transcriptases, DNA Ligases, DNA Exonucleases, and DNA Endonucleases. HSL Reagents are competitive inhibitors of enzymes because they bind within the substrate-binding pockets of enzymes. Taq DNA polymerase molecules are comprised of a polymerase domain and an exonuclease domain, both of which can bind DNA.

[0098] In contrast to protein and chemical conventional hot-start agents, HSL Reagents are not consumed, irreversibly denatured, or destroyed by heating up to 99°C. HSL Reagents are readily adjusted in terms of their nucleotide lengths, composition, hybridization temperatures, shape, and covalent modification. Such reagents can be manufactured and purified in either small quantities or very large bulk quantities using readily available methods for oligonucleotide synthesis and modification.

[0099] Although one does not admit to being bound by any theory, our working hypothesis is that in their double-stranded conformation, HSL Reagents act as competitive inhibitors of enzymes that process nucleic acids. As such, the functionality of each particular reagent depends on the following variables, at least: i) the size of the particular reagent; ii) the stability or relatively stability of the double- stranded conformation of the particular reagent; iii) the temperature at which a particular reagent adopts a double- stranded conformation; iv) the particular structure of moieties that may be covalently attached to either, or both, the 3’ and 5’ nucleotides of a particular reagent; v) the interactions of these moieties with each other that may influence the stability of the double- stranded conformation of a particular reagent; vi) the interactions between a particular HSL Reagent and its target particular enzyme; vii) the interactions among target molecules that have a bound HSL Reagent; viii) the interactions of a HSL Reagent with additional ionic salts in a reaction mixture; ix) the rates at which particular reagents change their conformation; x) the rates and ease with which particular reagents interact with, bind to, remain bound to a particular enzyme; xi) the concentrations of particular reagents, as well as the concentrations of particular enzymes; xii) the extent and the rate of temperature fluctuations of a reaction in which all reaction components interact.

[0100] In light of all the above variables, the relative ranking of the potency of individual HSL Reagents to suppress mis-priming during reaction set-up and amplification requires the invention of a versatile, quantitative test. A test of this nature is described below. This test is also useful for analysis of whether HSL Reagents are fully functional after they have been dried in a reaction vessel, with or without the particular enzyme with which they will be used.

[0101] B. HSL Reagent(s) can be used to Enhance Multiplexing

[0102] HSL Reagent can be used to suppress mis-priming during assembly of a master-mix for a multiplexed amplification reaction. Indeed, this is a rigorous test for the efficacy of an HSL Reagent because the risks of mis-priming are greatly increased by combining multiple pairs of primers in the same reaction vessel. By way of example, FIGS. 1 and 2 illustrate the benefits of adding HSL Reagent#l during assembly of a 10-plex reaction. HSL Reagent#l is comprised of a single- stranded oligonucleotide of the type described in U.S. Patent 10,240,178 B2. It can reversibly change its conformation from an extended linear single-strand to a stem-loop structure, in a temperature-dependent manner.

[0103] First, two large sets of symmetric primer pairs were chosen from two unrelated papers in the literature, strictly on the basis of amplicon product size. Six pairs of primers for sequences in the lambda phage genome (Le, T and Hidalgo- Ashrafi, E., 2009, BioTechniques 47, 972-973) were chosen, plus an additional four pairs of primers for sequences in the mouse genome (Shum, J and Paul, N., 2009, Analytical Biochemistry, 388, 266-272). None of the twenty primers were checked for compatibility with each other, and some primer pairs had mismatched melting temperatures (Table 3).

[0104] Table 3

[0105] Initially, each pair of primers was individually tested without and with HSL Reagent#! using two different non-hot-start Type A DNA polymerases in the absence of target DNA. The results with the two enzymes were similar. This disclosure refers to an operative ratio of units of said at least one HSL reagent to units of said at least one DNA polymerase. In some preferred embodiments, the operative ratio is relative to a ratio of 10: 1 defined by the nano-molar amount of HSL Reagent 1 to one-unit of a DNA polymerase (said one-unit being given by the enzyme manufacturer) in a mixture, prior to assembly of a complete master-mix in which primer-dimer synthesis is inhibited to the maximal possible extent in an amplification reaction in a closed-vessel. A nano-molar (nm) amount of an HSL reagent can he determined using standard techniques that are well-known and routine to those of ordinary skill in the art. The unit of a polymerase is typically defined by the manufacturer. For instance, one unit of EconoTaq DNA Polymerase is defined by its manufacturer (Biosearch Techs.) as the amount of enzyme that catalyzes the incorporation of 10 nmoles of dNTP into acid-insoluble material in 30 minutes at 70°C in 50 mM Tris-HCl (pH

[0106] 9.0), 50 mM NaCl, 5 mM MgCh, 200 pM dGTP, dATP, dTTP, dCTP (a mix of un-labelled and [33P]dCTP), 10 pg Activated Calf Thymus DNA, and 0.1 mg / ml BSA. This definition can also include the absence of endonuclease or nicking activity (as determined by incubation of 10 U of EconoTaq DNA Polymerase with 1 pg of supercoiled pBR322 DNA for 16 hours at 70 °C results in no detectable conversion to relaxed or linear forms detectable by agarose gel electrophoresis), absence of exonuclease activity (as determined by incubation of 10 U of EconoTaq DNA Polymerase with 1 pg of Hindlll-cut lambda DNA for 16 hours at 70°C resulted in no smearing of bands on agarose gels), and / or purity (e.g., 99% pure as determined by SDS PAGE with no detectable DNA contamination). Similar definitions can be utilized with respect to other polymerases as would be understood by those of ordinary skill in the art. The nucleotides in the master mixes disclosed herein are preferably certified free of nucleases and phosphatases. In preferred embodiments, these definitions of units of HSL reagents and units of polymerases are used in determining an operative ratio of this disclosure.

[0107] In FIG. 1, each 25pl reaction without added DNA contained IX EconoTaq PCR buffer (EconoTaq Lucigen, Middleton, Wisconsin), 3 mM MgCh, 400 nM dNTPs, and 200 nM of all 10 individual primer pairs listed in Table 3. These each no-DNA reaction (FIG. 2A and B) also received a volume of lOmM Tris-Cl, pH 8.3, or an equivalent volume containing Hot- Start-Like Reagent#l, and 2.5 units of EconoTaq DNA polymerase. Each 25p 1 reaction with DNA (FIG. 2C and D) received 500 genomes of lambda DNA (Thermo Scientific™ Lambda DNA, Waltham, MA) plus 500 genomes of mouse DNA (EMD Millipore™ Novagen™ Mouse Genomic DNA, Burlington, MA), as well as IX EconoTaq PCR buffer containing 3mM Mg2+, 400nM dNTPs, and 200nM of all 10 individual primer-pairs listed in Table 3, plus either an equivalent volume of lOmM Tris-Cl, pH 8.3 or an equivalent volume containing Hot-Start- Like Reagent#l and 2.5 units of EconoTaq DNA polymerase. Thus, the number of units of Hot- Start-Like Reagent#l to units of EconoTaq enzyme was 1 :1 in these monoplex reactions. The thermocycling conditions for all reactions were: 1 cycle at 95°C for 3 min; 40 cycles at 95°C, 10 sec; 60°C, 15 sec; 72°C, 30 sec; then extension at 72°C for 5 min in an Agilent Aria Mx Real Time PCR Cycler.

[0108] As shown in FIG. 1A each pair of primers generated primer-dimers in the absence of both target DNA and HSL Reagent#l. In contrast, as shown in FIG. IB, there was a complete absence of primer-dimer formation in the presence of HSL Reagent#l. Each pair of primers was then individually tested with 500 genomic copies of lambda DNA and 500 genomic copies of mouse DNA. In the absence of HSL Reagent# 1 , each pair of primers generated some amount of its expected product, but also generated primer-dimers and additional non-specific products, FIG. 1C. Non-specific amplification was particularly prevalent for primer pairs 4 and 6. In contrast, in the presence of HSL Reagent#!, all reactions were virtually free of spurious side products, FIG. ID. As a result, the collection of amplification products formed a diagonal ladder of size fragments from 139 to 962 base pairs in length, as intended. Lane M in quadrants of FIG. 1 corresponds to a standard ladder of DNA size markers.

[0109] FIG. 2 shows the results of a complete 10-plex reaction was assembled in the absence or presence of HSL Reagent#!, as well as in the absence or presence of 500 copies of lambda DNA plus 500 copies of mouse DNA. Each 25 pl reaction contained EconoTaq IX PCR buffer, 3mM Mg2+, 400nM dNTPs, and 200nM of all 10 primer-pairs listed in Table 3, either 3.75 units HSL Reagent#! (lanes “With”) or an equivalent volume of lOmM Tris-Cl, PH 8.3 (lanes labeled “No”), and 1.25 units of EconoTaq DNA polymerase. Thus, the number of units of HSL Reagent#l to units of EconoTaq enzyme was 3:1 in the 10-plex reaction. Lanes labeled NTC received no DNA, but did receive an equivalent volume of lOmM Tris-Cl, PH 8.3. Lanes labeled -i-Target receive 500 genomes of lambda DNA plus 500 genomes of mouse DNA. Cycling conditions were 95°C for 3 mini 40 cycles at 95°C, 10 sec; 60°C, 15 sec; 72°C, 30 sec; then a final extension at 72°C for 5 min. in an Agilent Aria Mx Real Time PCR Cycler.

[0110] The results show that in the absence of DNA and HSL Reagent#!, the 10-plex reaction generated a very pronounced smear of primer-dimers. In the absence of DNA and presence of HSL Reagent#!, the 10-plex reaction generates a faint primer-dimer band at the bottom of the gel. In the presence of DNA but absence of HSL Reagent#!, the 10-plex reaction again had a smear of primer dimers and only one higher band of unknown composition. In contrast, in the presence of DNA and the presence of HSL Reagent#!, the 10-plex reaction generated all ten of the expected double-stranded DNA products. These results show that HSL Reagent#! inhibits mis-priming errors that occurred in its absence and thereby enhances assembly of multiplex reactions.

[0111] C. Construction of a Convenient Triplex Assay for Quantitative Comparison of Different Types of HSL Reagents, Table 4.

[0112] The results shown in FIGS. 1 and 2 used end-point gel electrophoresis to analyze the doubled- stranded DNA molecules generated during symmetric PCR amplification. End-point gel analysis, however, is neither a convenient nor a quantitative method for measuring the efficacy with which different types of HSL Reagents suppress mis-priming. As a person versed in the art can appreciate, there are potentially many possible ways to construct better assays for rapid, quantitative, and convenient testing of the interactions of HSL Reagents and various enzymes. We chose to design and use end-point SYBR Green melt curve analysis, which can be carried out in the same tube in which double-stranded DNA molecules have accumulated. The multiplex reaction described in FIGS 1 -2 served as the basis for constructing the desired testing system.

[0113] Table 4 lists preferred key parameters of the three primer pairs used to build a triplex reaction. The lengths of the three double-stranded DNA products were 293, 431 , and 515 base pairs, and their melting temperature were predicted melting peaks of these amplicons were determined using the uMelt application at dna-utah.org / umelt / quartz / um.php with the thermodynamic parameters disclosed in Blake and Delcourt (1998, Nucleic Acids Res. 26: 3323-3332).

[0114] Table 4

[0115] The triplex reaction described above proved very convenient, reproducible, and 5 informative for quantitative testing and comparison of various HSL Reagents under various conditions, as described in the Examples below. PCR amplification reaction was carried out in replicate tubes manufactured by Bio Molecular Systems for use in a MIC Thermocycler, with overlay oil removed. Each tube was assembled using 13 pL of 1.92X EconoTaq PCR buffer without MgCh (LGC BioSearch, Lucigen Division, Middleton, WI), 5.76 mM MgCh 0 (ThermoFisher Scientific, Waltham, MA), 768 pM dNTPs (Meridian Biosciences, Memphis, Tennessee), 1.15X SYBR-Green I (ThermoFisher Scientific, Waltham, MA), and 1.25 units non-hot start, MgCh-free EconoTaq DNA polymerase (LGC BioSearch, Lucigen Division, Middleton, WI), with or without a particular HSL Reagent being tested. Each HSL Reagent, or an equivalent volume of 10 mM Tris-Cl pH 8.3 buffer, was mixed with the Taq DNA polymerase and incubated at room temperature for 5 minutes at the start of each test.

[0116] The contents of the tubes designated for DNA samples were subsequently further diluted by adding an aqueous solution consisting of 9.5 pL with 500 copies mouse genomic DNA, 500 copies lambda virus DNA (Thomas Scientific, Chadds Ford Township, PA), and 526 nM of each of three primer pairs (IDT, Newark, NJ) designed for amplification of two mouse DNA products and one lambda DNA product. The contents of the no-DNA control tubes were further diluted by adding an aqueous solution consisting of 9.5 pL with 526 nM each of two mouse primer pairs and a lambda primer pair. The resulting 25 pL amplification reactions consisted of IX EconoTaq PCR buffer, 3mM MgCl2, 400 pM dNTPs, 0.6X SYBR Green I, 200 nM each primer, 1.25 units EconoTaq DNA polymerase, and no, or, 0.05 units / pL- 0.5 units / pL HSL Reagent#l (1.25 - 12.5 total units), with or without 500 copies mouse genomic DNA and 500 copies Lambda virus DNA.

[0117] Once the amplification reactions were assembled, 8pl of oil was added to each MIC PCR tube before placing the tubes in a MIC PCR Cycler (Biomolecular Systems, Upper Coomera QLD, Australia). The PCR thermocycling profile consisted of 95°C for 3 minutes, 40 cycles of 95°C for 10 seconds, 60°C for 15 seconds, 72°C for 30 seconds, followed by a 5 min extension at 72°C and high- resolution melting analysis of the amplification products at 0.1°C / sec from 72°C to 95 °C with fluorescence acquisition in the FAM channel. The MIC PCR Cycler Standard Taq (V.3) setting was used for temperature control.

[0118] The resulting amplicons were visualized by end-point melt curve analysis in the FAM channel, FIG. 3A. Each of the resulting amplicons is a separate double-stranded DNA molecule, but their melt profiles appear to fuse into a single profile comprised of three peaks because they are all stained with SYBR Green. The melt temperatures of the three peaks do not reflect the base-pair lengths of the amplicons, because the amplicons have different nucleotide compositions (see Table 4). The heights of the three peaks do not reflect the amount of each amplicon because SYBR Green is known to shift from shorter to longer amplicons during melting from low to high temperature. Nevertheless, as shown in the examples below, the test shown here proved to be a valuable tool for measuring and comparing the quantitative potencies of different HSL- Reagents. D. Processing of HSL Reagent(s)

[0119] In some aspects, the present disclosure provides a process of preparing and using at least one HSL Reagent for an amplification reaction. Such a process may comprise the following steps:

[0120] (a) placing a solution containing a concentrate of the at least one HSL Reagent in a reaction vessel, wherein the at least one HSL Reagent is present in the reaction vessel at an amount sufficient to suppress mis-priming before, during, or after the amplification reaction is carried out in the reaction vessel;

[0121] (b) rehydrating the concentrate of the at least one HSL Reagent in the reaction vessel; and

[0122] (c) placing the reaction vessel in a device suitable for the amplification reaction.

[0123] In some embodiments, the concentrate in step (a) may be further dehydrated to produce a hyper-concentrate or a dried concentrate in a reaction vessel prior to step (b).

[0124] As used herein, the term “a solution” refers to a mixture comprised of a liquid at room temperature, as well as all ions, buffers, molecules, macromolecules, nucleic acids, or other substances dissolved in it.

[0125] As used herein, the term “an aqueous solution” refers to a mixture comprised of liquid water at room temperature, as well as all ions, buffers, molecules, macromolecules, nucleic acids, or other substances dissolved in it.

[0126] As used herein, the term “a concentrate” refers to a solution containing a higher concentration of one or more molecules than the concentration of the same molecule(s) in an amplification master-mix at the start of an amplification reaction.

[0127] As used herein, the term “a hyper-concentrate” refers to a concentrate whose concentration of one or more molecules is increased by a dehydration process which removes some but not all of the water molecules in an aqueous solution.

[0128] As used herein, the term “a dried-concentrate” refers to a concentrate comprising one or more molecules from which water molecules have been removed by a dehydration process.

[0129] As used herein, the term “a dehydration process” refers to a process of removing water from an aqueous solution, regardless of whether the process involves turning liquid water into gas at a temperature above or below the freezing point of liquid water, or the process occurs at a pressure above or below atmospheric pressure at sea level. Dehydration process may or may not involve formation of ice and sublimation of ice. In some embodiments, the solution in step (a) may comprise two HSL Reagents, which may consist of three strands.

[0130] In some embodiments, the number of units of the at least one HSL Reagent placed in the reaction vessel has a ratio in the range of 0.1 :1 to 10:1 relative to the number of units of DNA binding enzyme placed in the reaction vessel.

[0131] In some embodiments, the concentration of the at least one Hot-Start-Like Reagent in its mixture is at least 1.5 times greater than its nano-molar concentration in the complete amplification master-mix in a closed-vessel.

[0132] In some embodiments, the further dehydration step may be accomplished by using an overlay, which is in contact with the concentrate in the reaction vessel, and / or wherein the overlay is made of a non-aqueous material that absorbs water. In some embodiments, the concentration of the at least one HSL Reagent is a concentrate, a hyper-concentrate, or a the dried-concentrate.

[0133] As used herein, the term “an overlay” refers to a layer of a substance that is in contact with the aqueous solution in a reaction vessel or other container and, at least temporarily, in contact with the atmosphere. The composition, thickness, and volume of the overlay can be adjusted to enhance or reduce the transfer of water molecules comprising the aqueous solution through the overlay to the atmosphere.

[0134] In some embodiments, the concentrate of the at least one HSL Reagent may be rehydrated / diluted in the reaction vessel by addition of a measured volume of an aqueous solution containing one or more additional components of a complete amplification reaction master-mix, one or more nucleic acid amplification template molecules for generation of at least one amplicon, or a combination thereof, before an amplification reaction. In some examples, the one or more additional components of a complete amplification reaction mastermix include a monovalent cationic salt, a pH buffer, a divalent cationic salt, a dNTP, glycerol, dimethyl sulfoxide, a sugar, a heat denaturable protein, a polypeptide, a non-ionic detergent, and / or other organic compound(s). Exemplary non-ionic detergents include, but are not limited to, Tween 20, NP-40, and Triton X-100. Exemplary organic compounds include, but are not limited to, betaine, TMAC, formamide, 7-deaza-2'-deoxyguanosine, and PEG.

[0135] E. Reaction Vessel

[0136] In some aspects, the present disclosure also provides a reaction vessel that is pre-loaded with a concentrate, a hyper-concentrate, or a dried -concentrate of at least one HSL Reagent for an amplification reaction. Such a reaction vessel is suitable for the any of the processes described above and herein.

[0137] In some embodiments, the reaction vessel may be pre-loaded with two HSL Reagents, which may consist of three strands. In some embodiments, the number of units of the at least one HSL Reagent placed in the reaction vessel has a ratio in the range of 0.1 : 1 to 10: 1 relative to the number of units of DNA binding enzyme placed in the reaction vessel. In some embodiments, the concentration of the at least one Hot-Start-Like Reagent in its mixture is at least 1.5 times greater than its nano-molar concentration in the complete amplification mastermix in a closed-vessel.

[0138] As used herein, the term “a reaction vessel” refers to an object capable of containing an aqueous solution having a volume equal to or greater than 1 picoliter and a design that permits it to be used in a device for amplification. In many instances, a reaction vessel is made of a plastic or a polymer, but a reaction vessel can also be a container made of glass, or on a surface made of glass, or can be made of carbon or metal. In many instances, a reaction vessel has rigid walls, but a reaction vessel can also be a pliable film or have walls made of a non-aqueous fluid, such as an oil or a lipid.

[0139] As used herein, the term “a cap” refers to an object used to close or seal a reaction vessel. In many instances, a cap is made of a plastic or polymer, but a cap can also be made of glass, carbon, or metal.

[0140] As used herein, the atmosphere outside of a reaction vessel or other container is a gas having a pressure above, equal to, or below the pressure of air at sea level. Depending on its atomic composition, the atmosphere at room temperature can be a mixture of cases, such as air, or can be an atomically homogeneous gas, such a Nitrogen gas, Helium gas, Hydrogen gas, Carbon Dioxide, or Methane. The atmosphere can also be a mixture of gases or a homogeneous gas that can be dry or moist because it also contains an amount of water, H2O gas.

[0141] In some examples, the reaction vessel is a plastic tube, a multi-welled plastic plate, a multi-welled film, an array of small chambers, a microfluidic device, a self-contained cassette, or a nano-chip printed with hydrophobic and hydrophilic surface arrays.

[0142] F. General Techniques

[0143] The practice of the present disclosure will employ, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry, and immunology, which are within the skill of the art. Such techniques are explained fully in the literature, such as Molecular Cloning: A Laboratory Manual, second edition (Sambrook, et al., 1989) Cold Spring Harbor Press; Oligonucleotide Synthesis (M. J. Gait, ed. 1984); Methods in Molecular Biology, Humana Press; Cell Biology: A Laboratory Notebook (J. E. Cellis, ed., 1989) Academic Press; Animal Cell Culture (R. I. Freshney, ed. 1987); Introduction to Cell and Tissue Culture (J. P. Mather and P. E. Roberts, 1998) Plenum Press; Cell and Tissue Culture: Laboratory Procedures (A. Doyle, J. B. Griffiths, and D. G. Newell, eds. 1993-8) J. Wiley and Sons; Methods in Enzymology (Academic Press, Inc.); Handbook of Experimental Immunology (D. M. Weir and C. C. Blackwell, eds.): Gene Transfer Vectors for Mammalian Cells (J. M. Miller and M. P. Calos, eds., 1987); Current Protocols in Molecular Biology (F. M. Ausubel, et al. eds. 1987); PCR: The Polymerase Chain Reaction, (Mullis, et al., eds. 1994); Current Protocols in Immunology (J. E. Coligan et al., eds., 1991); Short Protocols in Molecular Biology (Wiley and Sons, 1999); Immunobiology (C. A. Janeway and P. Travers, 1997); Antibodies (P. Finch, 1997); Antibodies: a practice approach (D. Catty., ed., IRL Press, 1988-1989); Monoclonal antibodies: a practical approach (P. Shepherd and C. Dean, eds., Oxford University Press, 2000); Using antibodies: a laboratory manual (E. Harlow and D. Lane (Cold Spring Harbor Laboratory Press, 1999); The Antibodies (M. Zanetti and J. D. Capra, eds. Harwood Academic Publishers, 1995); DNA Cloning: A practical Approach, Volumes I and II (D.N. Glover, ed. 1985); Nucleic Acid Hybridization (B.D. Hames & S.J. Higgins eds.)1985»; Transcription and Translation (B.D. Hames & S.J. Higgins, eds. (1984»; Animal Cell Culture (R.I. Freshney, ed. (1986»; Immobilized Cells and Enzymes (IRL Press, (1986» ; and B. Perbal, A Practical Guide To Molecular Cloning (1984); F.M. Ausubel et al. (eds.).

[0144] As discussed herein and in the Examples below, this disclosure provides preferred embodiments 1-53 below. Other embodiments are also disclosed herein as will be understood by those of ordinary skill in the art.

[0145] 1. A method for preparing a mixture of at least one Hot-Start- Like Reagent and a DNA polymerase, prior to assembly of a complete master-mix for amplification of doublestranded amplicons in a closed-reaction vessel, the method comprising combining at least one Hot-Start-Like (HSL) reagent and at least one DNA polymerase at an operative ratio of units of said at least one HSL reagent to units of said at least one DNA polymerase wherein: the at least one HSL reagent comprises at least one DNA oligonucleotide modified at its 3’ and 5’ terminal nucleotides such that the at least one single-stranded oligonucleotide is non-amplifiable by said DNA polymerase; and, either the at least one single-stranded oligonucleotide has a temperature-dependent reversible double-stranded hairpin conformation, or the at least one single-stranded DNA oligonucleotide hybridizes to a second non-amplifiable single-stranded DNA oligonucleotide generating a doublestranded DNA molecule in a temperature-dependent manner; and, said operative ratio is determined by quantitative end-point melt-curve analysis of a fluorescent dye that binds to double-stranded DNA molecules in said closed- reaction vessel. The method of embodiment 1, wherein the double- stranded DNA is selected from the group consisting of: i) temperature-dependent double- stranded conformations of said at least one HSL reagent; ii) primer-dimers; iii) primer-oligomers; iv) two or more distinguishable sequence-specific amplicons; and v) one or more non-specific amplicons. The method of embodiment 1, wherein the at least one DNA oligonucleotide is singlestranded. The method of embodiment 1 wherein the complete master-mix comprises the mixture and at least one component selected from the group consisting of a monovalent cationic salt, a pH buffer, a divalent cationic salt, a dNTP, glycerol, dimethyl sulfoxide, a sugar, a heat denaturable protein, a polypeptide, a non-ionic detergent, optionally wherein the nonionic detergent is Tween-20, NP-40, or Triton X-100, and an organic compound selected from the group consisting of betaine, TMAC, formamide, 7-deaza-2'-deoxy uanosine, and polyethylene glycol (PEG). The method of embodiment 1, wherein the operative ratio is relative to a ratio of 10:1 defined by the nano-molar amount of HSL Reagent 1 to one-unit of a DNA polymerase (said one-unit being given by the enzyme manufacturer) in a mixture, prior to assembly of a complete master-mix in which primer-dimer synthesis is inhibited to the maximal possible extent in an amplification reaction in a closed- vessel. 6. The method of embodiment 5, wherein the amplification reaction is in the absence of a DNA template.

[0146] 7. The method of embodiment 1, wherein said mixture is prepared in a buffered solution, MgCh, and dNTPs.

[0147] 8. The method of embodiment 1, wherein an optimal operative ratio is based on: the lowest level of primer-dimers and primer-oligomers, as determined in an amplification reaction containing no template DNA; the highest level of two or more distinguishable sequence-specific amplicons, as determined in an amplification reaction containing template DNA; and, the lowest level of non-specific amplicons produced in an amplification reaction containing template DNA.

[0148] 9. The method of any preceding embodiment, wherein the at least one HSL reagent is selected from the group consisting of:

[0149] (a) a non-amplifiable temperature-dependent intramolecular-hybridizing single-stranded DNA oligomer, having covalently bound moieties on both its 5' and 3’ ends;

[0150] (b) two non-amplifiable temperature-dependent intermolecular-hybridizing singlestranded DNA oligomers, having two to six covalently bound moieties on their 5’ or 3’ ends;

[0151] (c) three non-amplifiable temperature-dependent intermolecular-hybridizing singlestranded DNA oligomers, having two to six covalently bound moieties on their 5’ or 3‘ ends;

[0152] (d ) a closed-circular double- stranded DNA;

[0153] (e) a hairpin-shaped, single- stranded oligonucleotides modified at both the 3’ and 5’ ends, optionally by addition of dabcyl moieties, Black Hole Quencher™ moieties, and / or 2’ O-methyl nucleotides at the end of the stem;

[0154] (f ) a pair of complementary or partially complementary oligonucleotides that form a hybrid 6-50 nucleotides long, wherein the oligonucleotides are modified on one or both ends by addition of polycyclic moieties, optionally by addition of dabcyl or coumarin moieties that do not have bulky portions that are non-planar; (g) a pair of oligonucleotide strands that form a hybrid at least six nucleotides long with a calculated Tm of at least 32°C, wherein strands in the terminal region of the hybrid contain interacting label moieties, which comprises a fluorophore on one strand and either a fluorophore or a non-fluorescent quencher on the other strand;

[0155] (h) a double-stranded polynucleotide comprising from one to four single-stranded overhangs; and

[0156] (i) an oligonucleotide comprising a stem-loop structure when not hybridized. The method of any preceding embodiment wherein the HSL reagent is selected from the group consisting of:

[0157] 5’B2-GAATAATATAGCCCCCCCCCCCCCCCCCCCCCCCCCCCCCTATATTATTC-BB (SEQ ID NO: 1 (HSL Reagent 1));

[0158] 5’D-GAATAATATAGCCCCCCCCCCCCCCCCCCCCCCCCCCCCCTATATTATTC-D (SEQ ID NO: 2 (HSL Reagent 2);

[0159] 5’D-CATTATAACAGGCATATAGGGACAGTTATAATG-D (SEQ ID NO: 3 (HSL Reagent 3));

[0160] 5’D-CTTAATTATAATGAAATTATAATTAAG-D (SEQ ID NO: 4 (HSL Reagent 4);

[0161] 5’D-CATTATAATGAAATTATAATG-D (SEQ ID NO: 5 (HSL Reagent 5);

[0162] 5’M-CGGCGTCATATAGACGCCG-M 3-Carbon Spacer (SEQ ID NO: 6 (HSL Reagent 6));

[0163] 5’B1-CGCGGCGTCATATAGACGCCGCG-B1 (SEQ ID NO: 7 (HSL Reagent 7)); the double stranded construct: 5’-GAAATAAAATAAAAATAAAATA-D

[0164] 3 ’ D-CTTTATTTT ATTTTTATTTT AT-D

[0165] (SEQ ID NO: 8 (HSL Reagent 8)); and, the double stranded construct:

[0166] 5’CAGCTGGCCTGGGAAGCGTGCAGTCGGACC-BB 3’BB-GTCGACCGGACCCTTCGCACGTCAGCCTGG-B2

[0167] (SEQ ID NO: 9 (HSL Reagent 9); wherein Bl is Black Hole Quencher 1, B2 is Black Hole Quencher 2, BB is Bioscarch Blue, D is dabcyl, and M is [2’-OMeC][2’OMeG]. The method of embodiment 1, wherein the mixture comprises at least two HSL reagents. The method of embodiment 10, wherein the at least two HSL reagents are:

[0168] 5’B2-GAATAATATAGCCCCCCCCCCCCCCCCCCCCCCCCCCCCCTATATTATTC-BB (SEQ ID NO: 1 (HSL reagent 1)); and, 5’D-GAATAATATAGCCCCCCCCCCCCCCCCCCCCCCCCCCCCCTATATTATTC-D (SEQ ID NO: 2 (HSL reagent 2)).

[0169] 13. The method of any one of embodiments 1- 11, wherein the mixture comprises two HSL reagents, and optionally comprises three oligonucleotides.

[0170] 14. The method of any one of embodiments 1-12, wherein the number of units of the at least one HSL reagent placed in the reaction vessel has a ratio in the range of 0.1: 1 to 10:1 relative to the number of units of DNA-binding enzyme placed in the reaction vessel.

[0171] 15. The method of embodiment 1, wherein the concentration of the at least one HSL reagent in the mixture is at least 1.5 times greater than its nano-molar concentration in the complete amplification master-mix in a closed- vessel.

[0172] 16. The method of embodiment 1 , wherein the operational ratio of units of HSL reagent to units DNA polymerase is from 0.1:1 to 10: 1.

[0173] 17. The method of embodiment 14, wherein the ratio is selected from the group consisting of 0.1:1, 1 :1, 3:1, 5: 1, and 10: 1.

[0174] 18. The method of any one of embodiments 1-15, wherein the closed-reaction vessel: has rigid walls comprised of a material selected from the group consisting of plastic, polymer, glass, carbon, and metal; is a pliable fdm; or has walls comprised of a non-aqueous fluid, optionally wherein the fluid is oil or lipid.

[0175] 19. The method of any preceding embodiment, wherein the closed-reaction vessel is a plastic tube, a multi-welled plastic plate, a multi-welled film, an array of small chambers, a microfluidic device, a self-contained cassette, or a nano-chip printed with hydrophobic and hydrophilic surface arrays.

[0176] 20. The method of embodiment 1 wherein the mixture comprises of a dried concentrate of a HSL reagent and a DNA polymerase in solution.

[0177] 21. The method of embodiment 1, wherein the mixture is a dried concentrate.

[0178] 22. The method of embodiment 19, further comprising resuspending the mixture into an aqueous solution from the dried concentrate. 23. The method of embodiment 1, wherein the at least one thermostable DNA polymerase has been reconstituted from a dried concentrate.

[0179] 24. The method of any one of embodiments 20-23, wherein the dried concentrate comprises an overlay in contact with the concentrate in the reaction vessel, and / or the overlay is made of a non-aqueous material that absorbs water.

[0180] 25. The method of embodiment 24, wherein the overlay is an oil or a polymer.

[0181] 26. The method of any one of embodiments 20-25, wherein the dried concentrate is prepared using a vacuum, at least one drying agent, and / or warming to a temperature of 40-60 °C.

[0182] 27. The reaction vessel of any one of embodiments 20-26, wherein the nano-molar concentration of the concentrate of the at least one HSL reagent prior to drying is at least 1.5 times greater than its nano-molar concentration in the amplification master-mix in a closed-vessel.

[0183] 28. The method of any preceding embodiment, wherein the closed reaction vessel is sealed with a cap.

[0184] 29. The method of embodiment 28, wherein the cap is made of plastic, polymer, glass, carbon, or metal.

[0185] 30. The method of any one of embodiments 20-29, wherein the dried concentrate further comprises at least one enzyme having a binding pocket for at least one double-stranded oligonucleotide.

[0186] 31. The method of embodiment 30, wherein the at least one enzyme is selected from the group consisting of:

[0187] (a) a DNA polymerase, which is optionally a Type A DNA polymerase, a Type B polymerase, or a Type C DNA polymerase;

[0188] (b) a reverse transcriptase, which is optionally avian myeloblastosis virus reverse transcriptase or Moloney murine leukemia virus reverse transcriptase;

[0189] (c) an exonuclease, which is optionally the 5’-to-3’ exonuclease of Tag polymerase, or the 3’-to-5’ proofreading exonuclease of Taq polymerase; and

[0190] (d) a DNA ligase, which is optionally Bacteriophage T4 ligase, or E. Coli ligase. The method of any one of embodiments 20-30, further comprising diluting the concentrate or the dried concentrate of the at least one HSL reagent in the reaction vessel by addition of a measured volume of an aqueous solution containing one or more additional components of a complete amplification reaction master-mix, one or more nucleic-acid amplification template molecules for generation of at least one amplicon, or a combination thereof. The method of embodiment 32, wherein the one or more additional components of a complete amplification reaction master-mix are selected from the group consisting of a monovalent cationic salt, a pH buffer, a divalent cationic salt, a dNTP, glycerol, dimethyl sulfoxide, a sugar, a heat denaturable protein, a polypeptide, a non-ionic detergent, optionally wherein the non-ionic detergent is Tween-20, NP-40, or Triton X-100, and an organic compound selected from the group consisting of betaine, TMAC, formamide, 7-deaza-2'-deoxyguanosine, and polyethylene glycol (PEG). The method of embodiment 1 wherein the identity and the amount of each possible type of double-stranded molecule present in said closed-vessel at end-point, is determined from the peak or valley in a first derivative plot of fluorescence intensity (-dF / dT) indicative of the binding or release of said fluorescent dye bound-to or released- from double- stranded DNA changing its conformation is a temperature-dependent manner. The method of embodiment 1 wherein the identity and amount of each possible type of double-stranded molecule in a set of amplifications reactions, over a range of operative ratios of units of said reagent to units of said DNA polymerase, can be used to compare different HSL reagents in terms of their capacities to increase or inhibit amplification of primer-dimers, primer-oligomers, sequence-specific amplicons, and / or non-specific amplicons. The method of embodiment 35 wherein two or more batches of a master-mix comprised of the same operative ratios of units of the same HSL reagent(s) and the same units of the same enzyme(s) from the same manufacturer(s), but have been produced separately in time or location or equipment and are then compared at different times or locations or equipment, in order to establish the similarities or differences among said batches in terms of their capacities to amplify the same products in the same test assays. The method of 35 wherein the identity and magnitude of each possible type of doublestranded molecule in a set of amplifications reactions, over a range of operative ratios of units of said reagent to units of said DNA polymerase, can be used to determine when, during the course of an amplification reaction, a particular type of HSL reagent acts to increase or inhibit amplification of primer-dimers, primer-oligomers, sequence-specific amplicons, and non-specific amplicons.

[0191] 38. The method of any one of embodiments 34-37 wherein the identity and the magnitude of two or more peaks or valleys in two or more -dF / dT plots of two or more HSL reagents is used to determine the relative potencies of those reagents to increase or inhibit amplification of primer-dimers, primer-oligomers, sequence-specific amplicons, nonspecific amplicons over a range of operative ratios of units of said reagent to units of said DNA polymerase.

[0192] 39. A reaction vessel comprising a concentrate or a dried concentrate of at least one HSL reagent for an amplification reaction.

[0193] 40. The reaction vessel of embodiment 39 prepared by the method of any one of embodiments 1-38.

[0194] 41. The reaction vessel of embodiment 39 or 40, wherein the at least one HSL reagent is selected from the group consisting of:

[0195] (a) a non-amplifiable temperature-dependent intramolecular-hybridizing single-stranded DNA oligomer, having covalently bound moieties on both its 5’ and 3’ ends;

[0196] (b) two non-amplifiable temperature-dependent intermolecular-hybridizing singlestranded DNA oligomers, having two to six covalently bound moieties on their 5’ or 3' ends;

[0197] (c) three non-amplifiable temperature-dependent intermolecular-hybridizing singlestranded DNA oligomers, having two to six covalently bound moieties on their 5’ or 3’ ends;

[0198] (d ) a closed-circular double- stranded DNA;

[0199] (e) a hairpin shaped single stranded oligonucleotides modified at both the 3’ and 5’ ends, optionally by addition of dabcyl moieties, Black Hole Quencher™ moieties, and / or 2’ O-methyl nucleotides at the end of the stem;

[0200] (f ) a pair of complementary or partially complementary oligonucleotides that form a hybrid 6-50 nucleotides long, wherein the oligonucleotides are modified on one or both ends by addition of polycyclic moieties, optionally by addition of dabcyl or coumarin moieties that do not have bulky portions that are non-planar;

[0201] (g) a pair of oligonucleotide strands that form a hybrid at least six nucleotides long with a calculated Tm of at least 32 °C, wherein strands in the terminal region of the hybrid contain interacting label moieties, which comprises a fluorophore on one strand and either a fluorophore or a non-fluorescent quencher on the other strand;

[0202] (h) a double-stranded polynucleotide comprising from one to four single-stranded overhangs; and

[0203] (i) an oligonucleotide comprising a stem-loop structure when not hybridized.

[0204] 42. The reaction vessel of embodiment 39 or 40 wherein the HSL reagent is selected from the group consisting of:

[0205] 5’B2-GAATAATATAGCCCCCCCCCCCCCCCCCCCCCCCCCCCCCTATATTATTC-BB (SEQ ID NO: 1 (HSL Reagent 1));

[0206] 5’D-GAATAATATAGCCCCCCCCCCCCCCCCCCCCCCCCCCCCCTATATTATTC-D (SEQ ID NO: 2 (HSL Reagent 2);

[0207] 5’D-CATTATAACAGGCATATAGGGACAGTTATAATG-D (SEQ ID NO: 3 (HSL Reagent 3));

[0208] 5’D-CTTAATTATAATGAAATTATAATTAAG-D (SEQ ID NO: 4 (HSL Reagent 4);

[0209] 5’D-CATTATAATGAAATTATAATG-D (SEQ ID NO: 5 (HSL Reagent 5);

[0210] 5’M-CGGCGTCATATAGACGCCG-M 3-Carbon Spacer (SEQ ID NO: 6 (HSL Reagent 6));

[0211] 5’B1-CGCGGCGTCATATAGACGCCGCG-B1 (SEQ ID NO: 7 (HSL Reagent 7)); the double-stranded construct: 5’D-GAAATAAAATAAAAATAAAATA-D 3’D-CTTTATTTTATTTTTATTTTAT-D

[0212] (SEQ ID NO: 8 (HSL Reagent 8)); and, the double-stranded construct:

[0213] 5 CAGCTGGCCTGGGAAGCGTGCAGTCGGACC-BB

[0214] 3 ’ BB-GTCGACCGGACCCTTCGC ACGTC AGCCTGG-B2 (SEQ ID NO: 9 (HSL Reagent 9); wherein Bl is Black Hole Quencher 1; B2 is Black Hole Quencher 2, BB is Biosearch Blue, D is dabcyl, and M is [2’-OMeC][2’OMeG].

[0215] 43. The reaction vessel of any one of embodiments 39-42, wherein the mixture comprises at least two HSL reagents. The reaction vessel of embodiment 43, wherein the at least two HSL reagents are:

[0216] 5’B2-GAATAATATAGCCCCCCCCCCCCCCCCCCCCCCCCCCCCCTATATTATTC-BB (SEQ ID NO: 1 (HSL Reagent 1)); and,

[0217] 5’D-GAATAATATAGCCCCCCCCCCCCCCCCCCCCCCCCCCCCCTATATTATTC-D (SEQ ID NO: 2 (HSL Reagent 2). The reaction vessel of embodiment 43 or 44, wherein the mixture comprises two HSL reagents, and optionally comprises three oligonucleotides. The reaction vessel of any one of embodiments 39-45, wherein the number of units of the at least one HSL reagent placed in the reaction vessel has a ratio in the range of 1 : 1 to 10:1 relative to the number of units of DNA-binding enzyme placed in the reaction vessel. The reaction vessel of any one of embodiments 39-46, wherein: the reaction vessel has rigid walls comprised of a material selected from the group consisting of plastic, polymer, glass, carbon, and metal; the reaction vessel is a pliable film; or the reaction vessel has walls comprised of a non-aqueous fluid, optionally wherein the fluid is oil or lipid. The reaction vessel of any one of embodiments 39-47, wherein the reaction vessel is a plastic tube, a multi-welled plastic plate, a multi-welled film, an array of small chambers, a microfluidic device, a self-contained cassette, or a nano-chip printed with hydrophobic and hydrophilic surface arrays. The reaction vessel of any one of embodiments 39-48, wherein the reaction vessel is sealed with a cap. The reaction vessel of embodiment 49, wherein the cap is made of plastic, polymer, glass, carbon, or metal. An open reaction vessel of any one of embodiments 39-50 comprising an aqueous solution containing one or more additional components of a complete amplification reaction mastermix, one or more nucleic acid amplification template molecules for generation of at least one amplicon, or a combination thereof, before an amplification reaction. The open reaction vessel of embodiment 51, wherein the one or more additional components of a complete amplification reaction master-mix include at least one of a monovalent cationic salt, a pH buffer, a divalent cationic salt, a dNTP, glycerol, dimethyl sulfoxide, a sugar, a heat denaturable protein, a polypeptide, a non-ionic detergent, and / or other organic compound(s), optionally wherein the non-ionic detergent is Tween-20, NP-40, or Triton X-100, and wherein the other organic compound is selected from the group consisting of betaine, TMAC, formamide, 7-deaza-2'-deoxy guanosine, and PEG.

[0218] 53. A reaction vessel of any one of embodiments 39-52 comprising a DNA amplification master-mix contained therein.

[0219] Without further elaboration, it is believed that one skilled in the art can, based on the above description, utilize the present invention to its fullest extent. The following specific embodiments are, therefore, to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever. All publications cited herein are incorporated by reference for the purposes or subject matter referenced herein.

[0220] Example 1: Placement and Testing of Concentrate of HSL Reagent#! in Reaction Vessel, FIGS. 3B-4

[0221] HSL Reagents# 1, Table 1, is comprised of a single-stranded oligonucleotide of the type described in U.S. Patent No. 10,240,178. The results shown in FIGS. 3B-4 demonstrate that concentrates of HSL Reagent#! designed to form ratios of HSL Reagent#l-to-DNA polymerase units in the final amplification reactions of 0:l, 1 :1, 3:1, 5: 1, and 10: 1 can be placed in separate reaction vessels, sealed with a cap, stored for later use, and subsequently diluted by the addition of aqueous solutions containing the remaining components of a complete multiplex amplification reaction master mix to enable the multiplex amplification of specific products once the reaction vessels are placed in a PCR cycler. The HSL Reagent#! comprised a non- amplifiable single-stranded modified oligonucleotide having covalently bound moieties on both its 5’ and 3’ ends. The modified oligonucleotide was able to form either a linear structure or a closed-hairpin structure in a temperature-dependent manner. A unit of the HSL Reagent#! corresponded to the amount of reagent required to suppress mis-priming in a control monoplex reaction by a unit of Taq DNA polymerase.

[0222] The various concentrates of HSL Reagent#! were prepared in nuclease- free water at 0 units / pL (no HSL Reagent#! control), 0.5 units / uL, 1.5 units / pL, 3 units / pL, and 5 units / pL to generate ratios of HSL Reagent#! units-to-Taq DNA polymerase units in the final amplification reaction of 1: 1 (1.25 reagent units to 1.25 Taq DNA polymerase units), 3: 1 (3.75 reagent units to 1.25 Taq DNA polymerase units), 5:1 (6.25 reagent units to 1.25 Taq DNA polymerase units), and 10:1 (12.5 reagent units to 1.25 Taq DNA polymerase units). A fixed volume of each of these concentrates (i.e., 2.5 pL), corresponding to 0, 1.25, 3.75, 6.25, and 12.5 units total, respectively, was placed into each of eight separate reaction vessels for each concentrate (four vessels for replicate reactions with genomic DNA and four vessels for replicate no-DNA control reactions). The reaction vessels consisted of MIC PCR cycler tubes (Biomolecular Systems, Upper Coomera QLD, Australia) with the oil removed - MIC PCR tubes contain oil to prevent evaporation of the amplification reactions during thermocycling. The MIC PCR tubes with the HSL Reagent# 1 concentrates were then capped and set aside for later use.

[0223] FIG. 3B shows the plot of the average first derivative of the resulting fluorescent readings as a function of temperature from replicate amplification reactions containing mouse and Lambda virus genomic DNA with and without the various HSL Reagent#l concentrates. In the absence of the HSL Reagent#l, the reactions generated broad melting peaks from 77°C to 91°C corresponding to non-specific amplification products (no HSLReagent#l, Curve 301). In contrast, in the presence of the HSL Reagent#l, the reactions with genomic DNA generated the expected melting peaks of 85°C for the mouse 515 bp amplicon, 88.7°C for the lambda 431 bp amplicon, and 92.4°C for the mouse 293 bp amplicon (1:1 reagent-to-Taq DNA polymerase ratio, Curve 302; 3: 1 reagent-to-Taq DNA polymerase ratio, Curve 303; 5: 1 reagent-to-Taq DNA polymerase ratio, Curve 304; 10: 1 reagent-to-Taq DNA polymerase ratio, Curve 305). The melting profile also showed minor non-specific melting peaks at 80.7°C and 91 .2°C, with the latter decreasing in magnitude proportionally with the number of HSL Reagent# 1 units in the reactions. It was noted that the relative height of the different melting peaks from multiplex reactions in the SYBR Green melting profile was not typically proportional to the product yield (Giglio S, et al., 2003, Nucleic Acids Res. 31(22): el36. doi: 10.1093 / nar / gngl35. PMID: 14602929; PMCID: PMC275573). From this data, it was determined that a 5: 1 reagent-to-Taq DNA polymerase ratio was adequate to generate the specific products for the multiplex amplification reaction.

[0224] FIG. 4 shows the plot of the average first derivative of the resulting fluorescent readings as a function of temperature from replicate no-DNA control reactions with and without the various HSL Reagent concentrates. In the absence of HSL Reagent#l, the no-DNA control reactions generated broad melting peaks from 77°C to 91 °C corresponding to non-specific primer dimer products (no HSL Reagent# 1, Curve 401). The presence of the HSL Reagent# 1 narrowed the temperature range of the melting profile of the non-specific primer dimer products and decreased their amounts proportionally to the number of units of the reagent (1:1 reagent-to-Taq DNA polymerase ratio, Curve 402; 3: 1 reagent-to-Taq DNA polymerase ratio, Curve 403; 5: 1 reagent-to- Taq DN A polymerase ratio, Curve 404; 10: 1 reagent-to- Taq DNA polymerase ratio, Curve 405). These results show that the HSL Reagent#l suppressed the formation of primer dimers in a dose-dependent manner.

[0225] Taken together, the data in FIGS. 3-4 demonstrate the dose-dependent suppression of non-specific primer dimers and primer oligomers achieved in the presence and absence of target DNA when the ratio of the HSL Reagent#! to the DNA polymerase, units-to-units, was increased from 0:1 to 1 :1, to 3:1, to 5:1, and to 10:1. Based on these data, a ratio of 5:1 for this particular HSL Reagent and this particular DNA polymerase was designated as adequate for use in further testing. However, close examination of the data in FIGS. 3-4 reveals that the three amplicons are even more precise non-specific shoulders are even lower when the HSL Reagent# 1 to Taq ratio is 10:1.

[0226] The results shown in Example 1 suggest that a concentrate of the HSL Reagent# 1 prepared in nuclease-free water can be placed into a reaction vessel in an amount required for a single amplification reaction, stored in a reaction vessel sealed with a cap for use at a later time, diluted by the addition of aqueous solutions containing the additional components of a complete reaction master mix, nucleic acid amplification template molecules and primers, and placed in a PCR cycler to support amplification of the intended amplicon products. These results also show that the ratio of HSL Reagent#! to Taq DNA polymerase for mis-priming suppression and specific product amplification ranges from 1 :1 to at least 10: 1 .

[0227] Example 2: Production and Testing of Dried HSL Reagent#!, FIGS 5-6.

[0228] The results presented in Example 2 demonstrate that a concentrate of an HSL Reagent, in this case HSL Reagent#l, can be dried in a reaction vessel in an amount required for an individual multiplex amplification reaction, stored sealed with a cap, and subsequently rehydrated and diluted by the addition of an aqueous solutions containing the remaining components of a complete amplification reaction master mix to support specific PCR amplification once the reaction vessel is placed in a PCR thermocycler.

[0229] A concentrate of the HSL Reagent# 1 was prepared in nuclease-free water at a concentration of 3.125 units / pL to generate a 5:1 ratio of HSL Reagent#l units to Taq DNA polymerase units in the final amplification reaction (6.25 reagent units to 1.25 Taq units). A fixed volume of the concentrate corresponding to 6.25 reagent units total (two microliters) was placed into each of eight separate reaction vessels (four vessels for replicate no-DNA control reactions and four vessels for replicate reactions with genomic DNA). The reaction vessels consisted of MIC PCR cycler tubes (Biomolecular Systems, Upper Coomera QLD, Australia) with the oil previously removed (MIC PCR tubes contain oil to prevent evaporation of the amplification reactions during thermocycling). The HSL Reagent#! concentrate in each of the tubes was then dried by placing the tubes uncapped in a MIC PCR Cycler and running the instrument at a constant temperature of 60°C for 60 minutes. After drying, the tubes were sealed and stored for use later the same day. The remaining stock of HSL Reagent#! at 3. 125 units / p L was stored on ice for use in the non-dried reagent controls.

[0230] For PCR amplification, the dried HSL Reagent#l in each MIC reaction vessel was first rehydrated and diluted by adding an aqueous solution of concentrated PCR reaction components consisting of 13.5 pL 1.70X EconoTaq PCR buffer without MgCh (LGC BioSearch, Lucigen Division, Middleton, WI), 5.1 mM MgC12 (ThermoFisher Scientific, Waltham, MA), 680 pM dNTPs (Meridian Biosciences, Memphis, Tennessee), IX SYBR- Green I (ThermoFisher Scientific, Waltham, MA) WI) plus 2 pL nuclease free water to account for the initial volume of the dried HSL Reagent#l concentrate. Each tube was then incubated for 5 minutes at room temperature to allow for rehydration of the dried concentrate before the samples were mixed by pipetting twelve times. Each tube was subsequently incubated for 5 minutes at room temperature to allow binding of the HSL Reagent#l to Taq DNA polymerase. For the non-dried HSL Reagent#l controls, 2 pL of the reserved HSL Reagent#! concentrate at 3.125 units / pL were added into each of 8 empty MIC PCR tubes without oil, followed by the addition of the same 13.5 pL of aqueous solution of concentrated PCR reaction components described above into each tube and incubation for 5 minutes at room temperature to allow binding of the HSL Reagent# 1 to Taq DNA polymerase.

[0231] The contents of the tubes designated for DNA samples were subsequently further diluted by adding an aqueous solution consisting of 9.5 pL with 500 copies mouse genomic DNA, 500 copies lambda virus DNA (Thomas Scientific, Chadds Ford Township, PA) and 526 nM of each of three primer pairs (IDT, Newark, NJ) designed for amplification of two mouse DNA products and one lambda DNA product. The contents of the no-DNA control tubes were further diluted by adding an aqueous solution consisting of 9.5 pL with 526 nM each of two mouse primer pairs and a lambda primer pair. The resulting 25 pL amplification reactions consisted of IX EconoTaq PCR buffer, 3 mM MgCh, 400 pM dNTPs, 0.6X SYBR Green I, 200 nM each primer, 1.25 units EconoTaq DNA polymerase, 0.25 units / pL HSL Reagent#! (6.25 units total, a 5:1 reagent-to-Taq ratio), with or without 500 copies mouse genomic DNA and 500 copies Lambda virus DNA.

[0232] The same primer pairs from Example 1 above (i.e., the primer pair of SEQ ID NOs: 1 and 2 for amplification of a 293 bp product from the mouse genome, the primer pair of SEQ ID NOs: 3 and 4 for amplification of a 515 bp product from the mouse genome, and the primer pair of SEQ ID NOs: 5 and 6 for amplification of a 413 bp product from the Lambda DNA genome) were also used in this example. These primer pairs were selected solely on the basis that the predicted melting temperature of their specific product should generate melting peaks that can be resolved by SYBR Green high-resolution melting Curve analysis following multiplex amplification. The predicted melting temperatures of amplicons for the three primer pairs are 86°C for the mouse 515 bp amplicon (observed 85°C), 89.5°C (observed 88.7°C )for the lambda 431 bp amplicon, and 92.4°C (observed 92.2°C) for the mouse 293 bp amplicon. Melting peak predictions were determined using the uMelt application at dna- utah.org / umelt / quartz / um.php with the thermodynamic parameters disclosed in Blake and Delcourt (1998, Nucleic Acids Res. 26: 3323-3332). The primers were not evaluated for primer dimer formation in the multiplex amplification reaction.

[0233] Once the amplification reactions were assembled, oil was added to each MIC PCR tube before placing the tubes in a MIC PCR Cycler (Biomolecular Systems, Upper Coomera QLD, Australia). The PCR thermocycling profile consisted of 95 °C for 3 minutes, 40 cycles of 95°C for 10 seconds, 60°C for 15 seconds, 72°C for 30 seconds, followed by a 5 min extension at 72°C and high- resolution melting analysis of the amplification products at 0. l°C / sec from 72°C to 95 °C with fluorescence acquisition in the FAM channel. The MIC PCR Cycler Standard Taq (V.3) setting was used for temperature control.

[0234] FIG. 5 shows the first derivative Curve of the fluorescent readings as a function of temperature for the tubes containing genomic DNA. The tubes with rehydrated dried HSL Reagent# 1 (Curve 501) generated the same melting profile as the control tubes with non-dried HSL Reagent# 1 (Curve 502).

[0235] FIG. 6 shows the first derivative Curve of the fluorescent readings as a function of temperature for the no-DNA control tubes. The tubes with rehydrated dried HSL Reagent#l (Curve 601) generated the same melting profile as the control tubes with non-dried HSL Reagent#! (Curve 602).

[0236] Taken together, the data in FIGS. 5-6 demonstrate that the rehydrated dried HSL Reagent#l suppressed the formation of non-specific primer dimers and oligomers with the same efficiency as non-dried HSL Reagent# 1.

[0237] These results suggest that a concentrate of the HSL Reagent# 1 with the number of units required to generate specific products in an individual amplification reaction can be placed into a reaction vessel, dried, and stored in the reaction vessel sealed with a cap for use at a later time. The concentrate can subsequently be rehydrated and diluted by the addition of aqueous solutions containing the additional components of a complete reaction master-mix, nucleic acid amplification template molecules, and primers, and placed in a PCR cycler to support amplification of the intended amplicon products.

[0238] Example 3: Production and Testing of Dried HSL Reagent#! in the Absence or Presence of DNA Polymerase FIGS 7-8.

[0239] This example demonstrates that a concentrate of a HSL Reagent#! required for an individual multiplex amplification reaction can be either be mixed with a DNA polymerase in an aqueous solution containing PCR buffer, MgCh, and dNTPs, dried in a reaction vessel, stored sealed with a cap, and subsequently rehydrated and diluted by the addition of aqueous solutions containing the remaining components of a complete amplification reaction master mix to support specific PCR amplification once the reaction vessel is placed in a PCR thermocycler. The HSL Reagent#! comprised a non-amplifiable single-stranded modified oligonucleotide having covalently bound moieties on both its 5’ and 3’ ends. The modified oligonucleotide was able to form either a linear structure or a closed-hairpin structure in a temperature-dependent manner. A unit of the HSL Reagent#! corresponded to the amount of reagent required to suppress mis-priming in a control monoplex reaction by a unit of Taq DNA polymerase.

[0240] In this example, a concentrate of the HSL Reagent#! mixed with the Taq DNA polymerase containing 3.125 units / pL HSL Reagent#! and 0.625 units / pL EconoTaq DNA polymerase (5: 1 reagent-to-Taq ratio) was prepared in IX EconoTaq PCR Buffer, 3 mM MgCL. and 400 pM dNTP and incubated for 5 minutes at room temperature to allow binding of the HSL Reagent# 1 to Taq DNA polymerase. A fixed volume of the concentrate corresponding to 6.25 units HSL Reagent#! and 1.25 units EconoTaq DNA polymerase (two microliters) was placed into each of eight separate reaction vessels (four vessels for replicate no-DNA control reactions and four vessels for replicate reactions with genomic DNA). The reaction vessels consisted of MIC PCR cycler tubes (Biomolecular Systems, Upper Coomera QLD, Australia) with the oil previously removed (MIC PCR tubes contain oil to prevent evaporation of the amplification reactions during thermocycling). The HSL Reagent#! -enzyme concentrate in each of the tubes was then dried by placing the tubes uncapped in a MIC PCR Cycler and running the instrument at a constant temperature of 60°C for 60 minutes. After drying, the tubes were sealed and stored for use later the same day. The remaining stock of HSL Reagent#l- enzyme concentrate was stored on ice for use for the non-dried controls. For PCR amplification, the dried HSL Reagent#! -enzyme concentrate in each MIC reaction vessel was first rehydrated and diluted by adding an aqueous solution of concentrated PCR reaction components consisting of 13.5 ,uL 1.70X EconoTaq PCR buffer without MgCh (LGC BioSearch, Lucigen Division, Middleton, WI), 5.1 mM MgCh (ThermoFisher Scientific, Waltham, MA), 680 pM dNTPs (Meridian Biosciences, Memphis, Tennessee), IX SYBR- Green I (ThermoFisher Scientific, Waltham, MA) WI) plus 2 L nuclease free water to account for the initial volume of the dried hot-start-enzyme concentrate. Each tube was then incubated for 5 minutes at room temperature to allow for rehydration of the dried concentrate before the samples were mixed by pipetting twelve times. Each tube was subsequently incubated for 5 minutes at room temperature to allow binding of the HSL Reagent#! to Taq DNA polymerase, even though the HSL Reagent# 1 was already bound to the enzyme prior to drying. For the nondried HSL Reagent#l -enzyme mix controls, 2 pL of the reserved HSL Reagent#! -enzyme concentrate was added into each of 8 empty MIC PCR tubes without oil, followed by the addition of the same 13.5 pL of aqueous solution of concentrated PCR reaction components described above into each tube. No subsequent incubation for 5 minutes at room temperature to allow binding of the HSL Reagent#! to Taq DNA polymerase was required at this step because the HSL Reagent# 1 was already bound to the enzyme.

[0241] The contents of the tubes designated for DNA samples were subsequently further diluted by adding an aqueous solution consisting of 9.5 pL with 500 copies mouse genomic DNA, 500 copies lambda virus DNA (Thomas Scientific, Chadds Ford Township, PA) and 526 nM of each of three primer pairs (IDT, Newark, NJ) designed for amplification of two mouse DNA products and one lambda DNA product. The contents of the no-DNA control tubes were further diluted by adding an aqueous solution consisting of 9.5 pL with 526 nM each of two mouse primer pairs and a lambda primer pair. The resulting 25 pL amplification reactions consisted of IX EconoTaq PCR buffer, 3 mM MgCh, 400 pM dNTPs, 0.6X SYBR Green I, 200 nM each primer, 1.25 units EconoTaq DNA polymerase, 0.25 units / pL HSL Reagent#! (6.25 units total, a 5: 1 reagent-to-Taq ratio), with or without 500 copies mouse genomic DNA and 500 copies Lambda virus DNA.

[0242] As in Example 1 and Example 2 before, the same primer pairs (i.e., the primer pair of SEQ ID NOs: 1 and 2 for amplification of a 293 bp product from the mouse genome, the primer pair of SEQ ID NOs: 3 and 4 for amplification of a 515 bp product from the mouse genome, and the primer pair of SEQ ID NOs: 5 and 6 for amplification of a 413 bp product from the Lambda DNA genome) were also used in this example. These primer pairs were selected solely on the basis that the predicted melting temperature of their specific product should generate melting peaks that can be resolved by SYBR Green high-resolution melting curve analysis following multiplex amplification. The predicted melting temperatures of amplicons for the three primer pairs are 86°C for the mouse 515 bp amplicon (observed 85°C), 89.75°C (observed 88.7°C) for the lambda 431 bp amplicon, and 92.4°C (observed 92.2°C) for the mouse 293 bp amplicon. Melting peak predictions were determined using the uMelt application at https: / / www.dna-utah.org / umelt / quartz / um.php with the thermodynamic parameters disclosed in Blake and Delcourt (1998). The primers were not evaluated for primer dimer formation in the multiplex amplification reaction.

[0243] Once the amplification reactions were assembled, oil was added to each MIC PCR tube before placing the tubes in a MIC PCR Cycler (Biomolecular Systems, Upper Coomera QLD, Australia). The PCR thermocycling profile consisted of 95 °C for 3 minutes, 40 cycles of 95°C for 10 seconds, 60°C for 15 seconds, 72°C for 30 seconds, followed by a 5 min extension at 72°C and high- resolution melting analysis of the amplification products at 0.1°C / sec from 72°C to 95 °C with fluorescence acquisition in the FAM channel. The MIC PCR Cycler Standard Taq (V.3) setting was used for temperature control.

[0244] FIG. 7 shows the first derivative curve of the fluorescent readings as a function of temperature for the tubes containing genomic DNA. The tubes with rehydrated dried HSL Reagent#! -enzyme mix (Curve 701) generated the same melting profile as the control tubes with non-dried HSL Reagent#! -enzyme mix (Curve 702).

[0245] FIG. 8 shows the first derivative Curve of the fluorescent readings as a function of temperature for the no-DNA controls. The tubes with rehydrated dried HSL Reagent#! -enzyme mix (Curve 801) generated the same melting profile as the control tubes with non-dried HSL Reagent#! -enzyme mix (Curve 802).

[0246] Taken together, the data in FIGS. 7-8 demonstrate that amplification reactions with rehydrated dried HSL Reagent#! plus DNA polymerase generated the same specific products, at the same efficiency, as control amplification reactions using non-dried reaction components. In addition, the HSL Reagent#! was equally effective at suppressing primer dimers and oligomers when it was dried with the enzyme, as when the components were not dried.

[0247] These results suggest that a concentrate of the HSL Reagent# 1 mixed with a DNA polymerase with the number of units required for an individual amplification reaction prepared in an aqueous solution containing PCR buffer, MgC12, and dNTPs can be placed into a reaction vessel, dried, stored in the reaction vessel sealed with a cap for use at a later time. The concentrate can subsequently be rehydrated and diluted by the addition of aqueous solutions containing the additional components of a complete reaction master mix, nucleic acid amplification template molecules, and primers, and placed in a PCR cycler to support amplification of the intended amplicon products.

[0248] Example 4: Production of Dried DNA Polymerase and Testing in the Absence or Presence of HSL Reagent#!, FIGS 9-10.

[0249] This example demonstrates that a concentrate of a DNA polymerase in the absence of an added HSL Reagent can be dried in a vessel without loss of DNA synthesis activity. This example also demonstrates that following resuspension of the dried DNA polymerase in a final aqueous solution containing all the additional components required for multiplex amplification of double- stranded DNA products, amplification of the correct products free of artifacts of mispriming requires the inclusion of HSL Reagent#l in the aqueous solution.

[0250] Four test cases were examined in this example:

[0251] TEST CASE 1 : DNA polymerase activity with drying - HSL Reagent#l included in the PCR master mix used to resuspend and dilute the dried DNA polymerase.

[0252] TEST CASE 2: DNA polymerase activity with drying - No HSLReagent#l included in the PCR master mix used to resuspend and dilute the dried DNA polymerase.

[0253] TEST CASE 3: DNA polymerase activity without drying - HSL Reagent#! included in the PCR master mix used to dilute the DNA polymerase.

[0254] TEST CASE 4: DNA polymerase activity without drying - No HSL Reagent#l included in the PCR master mix used to dilute the DNA polymerase.

[0255] A concentrate of a DNA polymerase was prepared in a solution comprised of a PCR buffer, MgCh, and dNTPs. This mixture of components was either added to separate oil-free reaction vessels in the amount required for a single multiplex amplification reaction, and each reaction vessel was then either dried (Test Cases 1 and 2) or not dried (Test Cases 3 and 4). All reaction vessels were then sealed with a cap and stored. Following storage, the dried DNA polymerase in all the vessels was either reconstituted with an aqueous solution containing PCR buffer, MgCh, and dNTPs, and HSL Reagent#! (Test Case 1), or was reconstituted with a similar solution lacking the HSL Reagent#! (Test Case 2). In Test Cases 3 and 4, the non-dried DNA polymerase concentrate was then either diluted with an aqueous solution containing PCR buffer, MgCh, and dNTPs, and the HSL Reagent#! (Test Case 3), or was diluted with a similar solution lacking the HSL Reagent#! (Test Case 4). One half of the vessels in each of the four Test Cases were then either further diluted by the addition of genomic DNA and primers (Plus- DNA samples), or were further diluted with only primers (no-DNA controls). All reaction vessels were MIC PCR cycler tubes (Biomolecular Systems, Upper Coomera QLD, Australia) from which the oil that comes inside the tubes had been removed. The HSL Reagent comprised a non-amplifiable single-stranded modified oligonucleotide having covalently bound moieties on both its 5’ and 3’ ends. The modified oligonucleotide was able to form either a linear structure or a closed-hairpin structure in a temperature-dependent manner. In reactions containing HSL Reagent#!, the ratio of the reagent to the DNA polymerase was 5:1 units of reagent to units of enzyme. As demonstrated in Examples 1-3 above, this ratio of reagent-to-enzyme units is sufficient to suppress all forms of mis-priming in this multiplex reaction. A unit of the HSL Reagent#! corresponds to the amount of reagent required to suppress mis-priming in a control monoplex reaction by a unit of Taq DNA polymerase.

[0256] As in Examples 1-3 before, the same primer pairs (z.e. , the primer pair of SEQ ID NOs: 1 and 2 for amplification of a 293 bp product from the mouse genome, the primer pair of SEQ ID NOs: 3 and 4 for amplification of a 515 bp product from the mouse genome, and the primer pair of SEQ ID NOs: 5 and 6 for amplification of a 413 bp product from the Lambda DNA genome) were also used in this example. These primer pairs were selected solely on the basis that the predicted melting temperature of their specific product should generate melting peaks that can be resolved by SYBR Green high-resolution melting Curve analysis following multiplex amplification. The predicted melting temperatures of amplicons for the three primer pairs are 86°C for the mouse 515 bp amplicon (observed 85°C), 89.75°C (observed 88.7°C) for the lambda 431 bp amplicon, and 92.4°C (observed 92.2°C) for the mouse 293 bp amplicon. Melting peak predictions were determined using the uMelt application at dna- utah.org / umelt / quartz / um.php with the thermodynamic parameters disclosed in Blake and Delcourt (1998, Nucleic Acids Res. 26: 3323-3332). The primers were not evaluated for primer dimer formation in the multiplex amplification reaction.

[0257] For each of the test cases of DNA polymerase activity following drying (Test Cases 1 and 2):

[0258] 1. Eight replicate reactions were prepared by adding 2 pl of a concentrate containing 1.25 units EconoTaq DNA polymerase (LGC BioSearch, Lucigen Division, Middleton, WI), IX EconoTaq PCR Buffer without MgCh (LGC BioSearch, Lucigen Division, Middleton, WI), 3 mM MgCh (ThermoFisher Scientific, Waltham, MA), and 400 pM dNTPs (Meridian Biosciences, Memphis, Tennessee) into separate MIC tubes without oil. 2. The enzyme concentrate in each MIC tube was then dried by placing the MIC tubes uncapped in a MIC PCR Cycler (Biomolecular Systems, Upper Coomera QLD, Australia) and running the instrument at a constant temperature of 60°C for 60 minutes. After drying, the MIC tubes were sealed with a cap and stored at room temperature for later use.

[0259] 3. Following storage, the eight replicates for Test Case 1 were reconstituted with an aqueous solution comprised of 13.5 pL 1.7X EconoTaq PCR buffer without MgCh, 5.1 mM MgCh, 680 ,uM dNTPs, IX SYBR-Green I (ThermoFisher Scientific, Waltham, MA) WI), 10.62 units HSL Reagent#! plus 2pL nuclease-free water to account for the initial volume of the dried enzyme concentrate. The eight replicates for Test Case 2 were reconstituted with a similar aqueous solution without the HSL Reagent# 1.

[0260] 4. The samples were then incubated for 5 minutes at room temperature to allow rehydration of the dried DNA polymerase, mixed by pipetting up and down, and incubated for an additional 5 minutes at room temperature to allow binding of the HSL Reagent#! to the polymerase, when present.

[0261] 5. For each test case, four of the eight replicates were then supplemented with 9.5 pL with 500 copies mouse genomic DNA, 500 copies lambda virus DNA (both reagents from Thomas Scientific, Chadds Ford Township, PA) and 526 nM of each of three primer pairs (IDT, Newark, NJ) (Plus-DNA samples). The remaining four replicates were supplemented with 9.5 uL TE buffer and 526 nM primers (no-DNA controls).

[0262] 6. The resulting 25 pL amplification reactions consisted of IX EconoTaq PCR buffer, 3 mM MgCh, 400 pM dNTPs, 0.6X SYBR Green I, 200 nM each primer, 1.25 units reconstituted dried EconoTaq DNA polymerase, with either 0.25 units / pL HSL Reagent#! (6.25 units total, a 5:1 reagent-to-Taq ratio, Test Case 1), or not (Test Case 2), and either mouse genomic DNA and Lambda virus DNA (500 copies each; (Plus-DNA samples) or not (no-DNA controls)).

[0263] For each of the test cases of DNA polymerase activity without drying (Test Cases 3 and 4):

[0264] 1. Eight replicate reactions were prepared by adding 2 pl of a concentrate containing 1.25 units EconoTaq DNA polymerase, IX EconoTaq PCR Buffer without MgCh, 3 mM MgCh, and 400 pM dNTPs into separate MIC tubes without oil. 2. The enzyme concentrate in the MIC tubes was not dried. Instead, the MIC tubes were just sealed with a cap and stored on ice for later use.

[0265] 3. Following storage, the eight replicates for Test Case 3 were reconstituted with an aqueous solution comprised of 13.5 pL 1.7X EconoTaq PCR buffer without MgCh, 5.1 mM MgCh, 680 pM dNTPs, IX SYBR-Green I, 10.62 units HSL Reagent#! plus 2pL nuclease-free water to account for the initial volume of the dried enzyme concentrate. The eight replicates for Test Case 4 were reconstituted with a similar aqueous solution without the HSL Reagent# 1.

[0266] 4. The samples were then incubated for 5 minutes at room temperature to allow rehydration of the dried DNA polymerase, mixed by pipetting up and down, and incubated for an additional 5 minutes at room temperature to allow binding of the HSL Reagent#l to the polymerase, when present.

[0267] 5. For each test case, four replicates were then supplemented with 9.5 pL of 500 copies mouse genomic DNA, 500 copies lambda virus DNA, and 526 nM of each of three primer pairs (Plus-DNA samples). The remaining four replicates were supplemented with 9.5 uL TE buffer and 526 nM primers (no-DNA Controls).

[0268] 6. The resulting 25 pL amplification reactions consisted of IX EconoTaq PCR buffer, 3 mM MgCh, 400 pM dNTPs, 0.6X SYBR Green I, 200 nM each primer, 1.25 units never-dried EconoTaq DNA polymerase, with either 0.25 units / pL HSL Reagent#l (6.25 units total, a 5:1 reagent-to-Taq ratio, Test Case 3), or not (Test Case 4), and either mouse genomic DNA and Lambda virus DNA (500 copies each; (Plus-DNA samples) or not (no-DNA controls).

[0269] Once the amplification reactions were assembled, 8 pl of Light Molecular Biology Reagent grade mineral oil (MP Biomedicals, Irvine, CA, USA) was added to each MIC PCR tube before placing the tubes in a MIC PCR Cycler. The PCR thermocycling profile consisted of 95°C for 3 minutes, 40 cycles of 95°C for 10 seconds, 60°C for 15 seconds, 72°C for 30 seconds, followed by a 5 min extension at 72°C and high-resolution melting analysis of the amplification products at 0.1°C / sec from 72°C to 95°C with fluorescence acquisition in the FAM channel. The MIC PCR Cycler Standard Taq (V.3) setting was used for temperature control.

[0270] FIG. 9 shows the first derivative melt curve analysis of the fluorescent readings as a function of temperature for the samples containing genomic DNA for Test Cases 1-4. Cases 1 and 2 used dried DNA polymerase, while Cases 3 and 4 used undried DNA polymerase. Case 1 with DNA and HSL Reagent#! added during rehydration (Curve 901) generated the same melting peaks as Case 3 (Curve 903), for the amplicon products (85°C for the mouse 515 bp amplicon, 88.7°C for the lambda 431 bp amplicon, and 92.4°C for the mouse 293 bp amplicon. In fact, as discussed below, the amplicon peaks generated in Case 1 (Curve 901) had mispriming, while the amplicon peaks in Case 3 (Curve 903) exhibited very low levels of misprimed products. In contrast, Case 2 and Case 4, rehydrated with added DNA but no HSL Reagent#! (Curves 902 and 904), generated almost only non-specific peaks.

[0271] The results demonstrate that a concentrate of a EconoTaq prepared in PCR buffer, MgCh, and dNTPs can be placed in separate reaction vessels, dried, sealed with a cap, stored for later use, and subsequently rehydrated and diluted with aqueous solutions containing the remaining components of a complete multiplex amplification reaction master mix and genomic DNA targets (including the HSL Reagent#! at a 5: 1 reagent-to-DNA polymerase ratio) to enable the multiplex amplification of specific products once the reaction vessels are placed in a PCR cycler. These findings supplement evidence above that demonstrates that amplification reactions with rehydrated dried HSL Reagent#! generated the same specific products as control amplification reactions with non-dried HSL Reagent#!. In the absence of the HSL Reagent#!, both the test and control amplification reactions generated non-specific products.

[0272] FIG. 10 shows the first derivative curve of the fluorescent readings as a function of temperature for the no-DNA control samples for Test Cases 1-4. In these no-DNA cases, consisting of both dried EconoTaq and non-dried EconoTaq supplemented with the HSL Reagent#! upon rehydration, Test Case 1 using dried Econo Taq and HSL Reagent#! (Curve 1001) mis-primed less than Test Case 3 (Curve 1003) using non-dried Econo Taq and HSL Reagent#!. Similarly, Test Case 2 (Curve 1002) using dried Econo Taq without the Reagent mis-primed less than Test Case 4 (Curve 1004) using non-dried Econo Taq without Reagent. Case 4 (Curve 1004) mis-primed most extensively and generated primer-dimers and oligomers, because it used non-dried Econo Taq without HSL Reagent#!, but Case 3 (Curve 1003) also made primer-dimers and oligomers because the non-dried Econo Taq acted very quickly before it interacted with added HLS Reagent# 1.

[0273] Table 5 below summarizes the findings based on the above tests.

[0274] Table 5

[0275] Summary of Test Cases 1-4

[0276] As shown here, when HSL Reagent#l was added prior to amplification, the Plus- DNA samples in Curve 901 exhibited no mis-priming. These results were generated using dried DNA Polymerase that was reconstituted using the HSL Reagent#! and were then amplified without additional storage. In contrast, Curve 903 samples show slight levels of mis-priming (note the width of the 293 base-pair peak), likely due to the fact that they were generated using not-dried DNA Polymerase that was stored for a period of time prior to the addition of the HSL Reagent# 1. Similarly, the no-DNA controls shown in Curve 1001 were distinctly cleaner than those shown in Curve 1003.

[0277] Also, as shown here, when HSL Reagent#! was not added prior to amplification, both sets of plus-DNA samples, Curves 902 and 904, demonstrate that the DNA polymerase misprimed in the absence of added HSL Reagent#l, but the extent of mis-priming in Curve 904 was greater than the extent of mis-priming in Curve 902. Similarly, both sets of no-DNA controls, Curves 1002 and 1004, demonstrate that the DNA polymerase mis-primed in the absence of added HSL Reagent#!, but the extent of mis-priming in Curve 1004 was greater than the extent of mis-priming in Curve 1002.

[0278] The primary conclusions that can be drawn from these results are that EconoTaq DNA Polymerase retains full activity when dried under the conditions described here, and that dried- reconstituted EconoTaq DNA Polymerase makes mis-priming errors in the absence of the added HSL Reagent#!. These conclusions are in accordance with the results presented in Examples 1-3 above. However, these results also support a subtle conclusion about the kinetics of mis-priming, i.e., mis-priming is initiated very rapidly. Thus, when dried Econo Taq is used, it first interacted with HSL Reagent #1 prior to interacting with primers and templates. In contrast, when non-dried enzyme was used, it initiated mis-priming immediately upon addition of primers and templates. Comparison of Curves 901 and 903 with Curves 304 and 305, FIG. 3B, reveals that Curve 901 closely resembles Curve 305 generated with a 10:1 ratio of HSL Reagent#l to Econo Taq, while Curve 903 closely resembles Curve 304 generated with a 5:1 ratio of HSL Reagent#! to Econo Taq. Overall, the comparison of the data in this example and the examples above demonstrate that combination of 10 units of HSL Reagent#! per unit of Econo Taq in a concentrated solution, prior to addition of the other components of a master mix, is the optimum combination of reaction components for prevention of mis-priming, regardless of whether or not the enzyme-reagent concentrate is dried.

[0279] Example 5; Testing of Concentrate of HSL Reagent#2 in a Reaction Vessel, FIGS.ll.

[0280] HSL Reagent#2 is comprised of a single- stranded oligonucleotide of the type described in U.S. Patent No. 7,517,977, Table 1. HSL Reagent#2 has the same oligonucleotide sequence as HSL Reagent#! and therefore can fold into the same stem-loop structure as HSL Reagent#! . However, HSL Reagent#2 has dabcyl moieties covalently linked to its 5’ and 3’ ends, while HSL Reagent#! has a Back Hole Quencher 2 covalently linked to its 5’ and a Biosearch Blue moiety covalently linked to its 3’ end. The structures of these moieties are shown in Table 2, and it is clear that dabcyl groups extend a long distance away from the ends of the oligonucleotide to which they are linked. This may explain why the melting temperature of closed HSL Reagent#2 stem-loop hairpin is 3°C higher than the melting temperature of the HSL closed Reagent#! stem- loop hairpin (see Table 1 and FIG. 21). Nevertheless, comparison of the HSL Reagent#! curves in FIG.3B and the HSL Reagent#2 curves in FIG.ll reveals that HSL Reagent#2 is less potent than HSL Reagent#!, despite its higher melting temperature, see discussion in Examples 11-13 below.

[0281] Example 6: Placement and Testing of Concentrate of HSL Reagent#3-5 in a Reaction Vessel, FIGS.12-14.

[0282] Each of HSL Reagents #s 3-5 is comprised of a single-stranded oligonucleotide of the type described in U.S. Patent No. 7,517,977, Table 1. Each of these HSL Reagents can reversibly change its conformation from an extended linear single-strand to a stem-loop structure, in a temperature-dependent manner. Each of these HSL Reagents was first tested for its activity without drying over a range of concentrations, using the same experimental test as described in Example 1, and then was tested with drying, either using the Reagent alone or the Reagent and the DNA Polymerase, using the protocols describe in Examples 2 and 3. The only difference between the tests using HSL Reagents #s 3-5 and those using HSL Reagent#! was that the ratio of units of each HSL Reagents #s 3-5-to-units of EconoTaq was in the range to 0.055: 1 - to - 0.444: 1 because the potency of HSL Reagents #s 3-5 was already thought to be greater than the potency of HSL Reagent#! (Rice et al., 2007, Nature Protocols, 2(10): 2429-2438).

[0283] The data shown in FIG. 12 demonstrate that the addition of HSL Reagent#3 to EconoTaq at a ratio of 0.222 units to 1 unit of enzyme reproducibly resulted in the accumulation of all three double-stranded amplicons. Neither lower nor higher concentrations of HSL Reagent#3 were as good. At a ratio of 0.111 : 1 , some primer-dimers were still present, while at a ratio of 0.444: 1, some partial products accumulated, indicating that this higher ratio of reagents partially inhibited amplification.

[0284] The data shown in FIG. 13 demonstrate that the addition of HSL Reagent#4 to EconoTaq at a ratio of 0.222 units to 1 unit of enzyme reproducibly resulted in the accumulation of all three double-stranded amplicons. Both lower and higher concentrations of HSL Reagent#4 were not as good. At a ratio of 0.111 : 1 , some primer-dimers were still present, while at a ratio of 0.444:1, some partial products accumulated, indicating that the reagent inhibited amplification.

[0285] The data shown in FIG. 14 demonstrate that addition of HSL Reagent#5 to EconoTaq at a ratio of 0.222 units-to 1 unit enzyme was not sufficient to assure clean amplification of all three double- stranded products, and began to become inhibitory at a ratio of 0.444 units-to 1 unit enzyme. Higher concentrations of HSL Reagent#5 were not tested.

[0286] Example 7: Production and Testing of Dried HSL Reagent#3-4 in the Absence or Presences of DNA Polymerase, FIG. 15.

[0287] HSL Reagents#3-4 were next evaluated by drying in the absence or presence of a DNA polymerase using the protocol described in Examples 2 and 3. Based on the results of Example 5, drying was carried out using a ratio of 0.222 units HSL Reagents#3 or 4 to 1 unit EconoTaq.

[0288] The data shown in FIG. 15 demonstrate that dried HSL Reagent#3-4 recover full activity once they are rehydrated, regardless of whether they are dried in the absence or presence of EconoTaq. In addition, the data demonstrate that dried HSL Reagent#4, like not- dried HSL Reagent#4, is more potent than dried HSL Reagent#3, regardless of whether the DNA polymerase is or is not also dried along with the reagent.

[0289] Example 8: HSL Reagents 6-7 Having High Tms are Inhibitory. HSL Reagents #6 and #7 are comprised of a single- stranded oligonucleotide of the type described in U.S. Patent No. 7,517,977. Both ends of HSL Reagent #6 had two 2’0-methyl nucleotides, such that the oligonucleotide could close into a highly stable hairpin that only opened at about 69°C, black aiTow FIG.16A. Both ends of HSL Reagent #7 had a covalently linked Black Hole Quencher 1 moiety, such that the oligonucleotide could close into a highly stable hairpin that only opened at about 72°C, black arrow FIG. 17A.

[0290] Addition of increasing concentrations of HSL Reagent #6 to the triplex assay, FIG. 16B, generated a confusing array of peaks. Overall, there was a diminution in the amplitude of the non-specific lower-temperature peaks and a shift toward double- stranded products that melted at high temperatures, but increasing levels of specific products did not accumulate and higher concentrations of HSL Reagent #6 seem to inhibit amplification.

[0291] In contrast, the addition of increasing concentrations of HSL Reagent #7 to the triplex assay, FIG. 17B, generated a reproducible accumulation of the 43 Ibp and the 293bp products, but the 515bp product was never amplified. This is consistent with the high melting temperature of HSL Reagent #7 significantly reducing the number of DNA polymerase molecules that were active at 60°C and 72°C, the temperatures used for primer annealing and extension. Under these conditions, the long 515bp amplicon cannot be synthesized, while the shorter amplicons can be synthesized. Primer-dimer amplification persists under these conditions because primer-dimers are very short and the highest ratio of HSL Reagent #7 to Enzyme tested was 1: 1.

[0292] Example 9: Placement and Testing of Concentrate of HSL Reagent#8 Vessel, FIG. 18.

[0293] HSL Reagent #8, Table 1, is comprised of two complementary, anti -parallel singlestranded DNA oligonucleotides of the type described in U.S. Patent Nos. 9,034,605 B2 and 9,758,813 B2. Each end of each strand of HSL Reagents#8 is modified by a covalently attached dabcyl moiety. The two strands were used at equal concentrations. The ratio of Reagent to Enzyme used in this example ranged from 0.33: 1 up to 1.66:1.

[0294] HSL Reagent#8 was first tested for its activity without drying over a range of concentrations, using the same experimental test as described in Example 1, and then was tested with drying, either using the Reagent alone or the Reagent and the DNA Polymerase, using the protocols described in Examples 2 and 3. The only difference between the tests using HSL Reagents#8 and those using HSL Reagent# 1 was that the ratio of units of HSL Reagents #8-to-units of EconoTaq was in the range to 0.33: 1 - to - 1.66: 1 because the potency of HSL Reagent#8 was already thought to be greater than the potency of HSL Reagent#!.

[0295] The data shown in FIG. 18 demonstrate that the addition of HSL Reagent#8 to EconoTaq at a ratio of 1.66 units to 1 unit of enzyme reproducibly resulted in the accumulation of all three double-stranded amplicons. Both lower concentrations of HSL Reagent#8 were not as good, and higher concentrations were not tested. At a ratio of 1.33: 1, some primer-dimers were still present, suggesting that 1.66:1 is the optimal ratio of this reagent to EconoTaq.

[0296] Example 10; Production and Testing of Dried HSL Reagent #8 in the Absence or Presence of DNA Polymerase, FIG. 19.

[0297] HSL Reagent#8 was next evaluated by drying in the absence or presence of a DNA polymerase using the protocol described in Examples 2 and 3. Based on the results of Example 9, drying was carried out using a ratio of 1.66 units HSL Reagents#8 to 1 unit EconoTaq.

[0298] The data shown in FIG. 19 demonstrate that dried HSL Reagent#8 dried in the absence of EconoTaq, recovered full activity once rehydrated. However, the data in FIG. 19 also shows that 1 of 3 replicates of HSL Reagent#6 dried with EconoTaq, still showed amplification of non-specific product. This suggests that the true optimum of HSL Reagent#8-to-EconoTaq is likely to be slightly lower than 1.66: 1.

[0299] Example 11 - Testing of Concentrate of HSL Reagent#9 in a Reaction Vessel, FIGS.20.

[0300] HSL Reagent #9, Table 1, is comprised of two complementary, anti -parallel singlestranded DNA oligonucleotides of the type described in U.S. Patent Nos. 9,034,605 B2 and 9,758,813 B2. However, one strand of Reagent#9 has a modified 5’ end and a 3’ end with a covalently linked Biosearch Blue moiety. The complementary strand has a covalently linked Biosearch Blue on its 3’ end and a covalently linked Black Hole Quencher 2 on its 5’ end. The two strands were used at equal concentrations. The ratio of Reagent#9 to Enzyme used in this example ranged from 0: 1 to 4: 1. The melting temperature of HSL Reagent#9 is 86°C.

[0301] HSL Reagent#! was also present in all reactions at 1 :1 ratio with Econo Taq. The experiment was designed this way to establish whether addition of HSL Reagent#2 enhanced or inhibited amplification of specific products when added at Reagent#2 to Taq ratios of 0.5:1, 1:1, 2:1, and 4:1.

[0302] The results shown in FIG.20 demonstrate that, as expected, the low level of HSL Reagent# 1 present in all reactions was not enough to generate specific products, but did result in amplification of products, which likely included some of the specific amplicons, Curve 2001. Non-specific products were not generated when HSL Reagent#2 was present at 0.5: 1, and all three specific amplicons were generated, albeit not at their maximum possible extent, Curve

[0303] 2002. Addition of HSL Reagent#2 at a ratio of 1 : 1 entirely inhibited amplification of the 515 bp amplicon and partially inhibited amplification of the 431 bp and 293 bp amplicons, Curve

[0304] 2003. Addition of HSL Reagent#2 at a ratio of 2:1 inhibited all amplification of the 515 bp product and virtually all amplification of the 431 bp and 293 bp amplicons, Curve 2004. Addition of HSL Reagent#2 at a ratio of 4: 1 inhibited all amplification of all three specific amplicons, but allowed for amplification of very clean primer-dimers, Curve 2005. The significance of these results is discussed below in Example 13.

[0305] Example 12 - Conclusions Drawn from Comparison of HSL Reagents# 1-4& 8, FIG. 21.

[0306] FIG.21 - Middle Panels show first derivative melt curve analysis of the fluorescent readings as a function of temperature, over the temperature range of 40-95 °C, for the samples containing genomic DNA, in which each reaction used either HSL Reagents#! ,2,3,4, or 8 at its optimal reagent-to-Taq ratio (see Examples above). The left-half of each middle panel, between 40-70°C, reveals the first derivative melt curve for each of the five reagents, while the right-half of each middle panel, between 70-95 °C, reveals the first derivative melt curve of the primer-dimer peak and the three specific-product peaks for each of the five reagents.

[0307] FIG.21 - Left Panels show the first derivative melt curve for each of the reagents expanded over the temperature range of 42 — 70°C on the X-axis and expanded on the Y-axis to cover just the portion of the melt curve containing the valley of each reagent. The valley for each melt curve is the temperature at which 50% of the arms of the single-stranded reagent (#sl-4), or 50% of the complementary strands of a two-strand reagent (#8), are doublestranded. The arms or complementary strands are fully double-stranded at temperatures below the valley and are fully single- stranded at temperatures above the valley. The fact that HSL Reagents#3&4 are more potent than HSL Reagents#l&2 (Table 1) is reflected in the fact that the melt curve valleys for #s3&4 are actually much shallower than the melt curve valleys for #1&2 (Left-half of Middle panels). FIG.21 - Right Panels show the first derivative melt curve for each of the reagents expanded over the temperature range of 70-94°C on the X-axis and expanded on the Y-axis to cover just the portion of the melt curve containing the product peaks. The diagonal lines between the Right Panels and the right half of the Middle Panels mark the location of the 0.00 value on each of the Y-axes.

[0308] HSL Reagents #1-4 have Tm’s between 50-65°C and can form hairpins with doublestranded arms that are 9-12 base pairs long and intervening loops that are 3-29 bases long, Table 1. HSL Reagent #8 also has a Tm in the 50-65 °C range but can only form a doublestranded molecule that is 22 bases long. In contrast, the temperature range of HSL Reagents #6, 7, & 9 Tm’s is 72-86°C, and these reagents become more and more inhibitory to product amplification as their concentrations are raised.

[0309] Only HSL Reagent#5 has a Tm (56°C) in the range of most potent HSL Reagents, but HSL Reagent#5 is a poor reagent in terms of amplification specificity. This is probably because the loop of the hairpin is only three nucleotides long, while the arms of the hairpin are only 9 base pairs long and are AT-rich. Thus, the closed HSL Reagent#5 hairpin structurally resembles a short, rather unstable, double-stranded molecule. Comparison of Curve 1404 and Curve 1403 indicates that raising the concentration of HSL Reagent#5::Taq above 0.444:1 would only further inhibit amplification of all three specific amplicons.

[0310] Discussion of Mechanisms of Reagent and Taq Interactions:

[0311] PCR amplification is a dynamic, error-prone process. Mis-priming events, particularly primer-dimer formation, can occur at low temperatures before thermal cycling begins, and multiplexing using many pairs of primers can increase the chances of errors at this stage. Simultaneous amplification of many product strands can also increase the chances of mis- priming during thermal cycling at higher temperatures. Efficient initiation of PCR amplification therefore requires thorough inhibition of Taq polymerase activity before the start of thermal cycling, followed by sequence- specific amplification once the polymerase is released from the inhibitor during thermal cycling. Finally, if a reaction is temporarily cooled to a low temperature during an amplification protocol and is then thermal cycled again for additional rounds of amplification, mis-priming may occur during the cooling period and must be inhibited. HSL Reagents are hot-start-like reagents that are not irreversibly denatured by heating. These reagents can be added to a PCR master mix to prevent mis-priming before, during, and after PCR amplification, and their addition significantly improves the efficiency and accuracy of multiplexed reactions [Rice et al., 2007, Nature Protocols, 2(10): 2429-2438]. All formulations of HSL Reagents are modified oligonucleotides that can reversibly transition between double- stranded and single- stranded conformations in a temperaturedependent manner. In their double- stranded form, HSL Reagents can reversibly inhibit DNA polymerase by binding to and releasing from the DNA-binding pocket of the enzyme. The challenge in designing and using HSL Reagents stems from the fact that they must function in different ways during different stages of PCR amplification.

[0312] In the triplex assay described here, SYBR green fluorescent analysis was used for quantitative end-point melt curve analysis of double- stranded DNA molecules including primer-dimers, primer-oligomers when no DNA templates were present; and. non-specific and specific amplicons that were 515, 431, and 293 base pairs long when low concentration of appropriate DNA templates were added to the reaction. Under these circumstances, SYBR Green is detecting the levels of product generated as a function of the particular formulation and concentration of the HSL Reagent used.

[0313] All types of amplification reactions suffer from a fundamental dichotomy that must be resolved in order to achieve efficient product generation; prior to amplification of products during master-mix assembly, when low temperatures and long incubation times favor mispriming, the enzymes involved must be completely inhibited. But, during the thermal cycling phase of the reaction, the enzymes involved must be highly active at their optimal temperatures. The triplex PCR amplification reaction described here was used to test the capacity of various HSL Reagents, in combination with a Taq polymerase, to resolve this dichotomy. The results demonstrate that optimal amplification of the correct amplicons depends on multiple features of the HSL Reagents.

[0314] For instance, despite the fact that HSL Reagents# 1&2 have the same oligonucleotide sequences with an overall length of 50 nucleotides, comparison of Curves 302 and 1101 reveals that HSL Reagent#l is more efficient than HSL Reagent#2 at generating correct products when added to the master-mix at a 1: 1 ratio of reagent units-to-Taq units. We conclude from this observation that the presence of the two dabcyl moieties linked to the 5’ and 3’ ends HSL Regent#2 must slightly decrease binding of reagent to the polymerase, as compared to the BHQ2 and Biosearch Blue moieties linked to the 5 ’ and 3 ’ ends of HSL Reagent# 1. In contrast, comparison of Curves 305 and 1104 reveals that both reagents are equally efficient at generating correct products when added to the master-mix at a 10:1 ratio of reagent units-to- Taq units. We conclude from this observation that the discrepancy of reagent-to-enzyme binding can be overcome by mass action. Both HSL Reagent#l and HSL Reagent#2 at a 10:1 ratio with Taq Polymerase work very well at preventing primer-dimer formation, a mis-priming event that occurs prior to amplification (see Curves 305, 405, and 1104). Equally extensive suppression of primer-dimer synthesis is not observed when the ratio HSL Reagent# 1&2 is lower than 10: 1 (see Curves 301- 304, 401-404, 1101-1103). We conclude from this that inhibition of primer-dimer synthesis requires binding of a reagent molecule to virtually every Taq molecule.

[0315] High-specificity amplification of just amplicons 515 base pairs, 431 base pairs, and 293 base pairs also occurs when HSL Reagent#! &2 are used at a 10: 1 ratio with Taq Polymerase. This is because the melting temperatures of these two reagents are 5O-53°C (see Left Panels in FIG. 21), assuring that virtually no closed-hairpins are present at the primer annealing and extension temperatures of 60°-72°C.

[0316] The above conclusions about HSL Reagents# 1&2 are supported by the results shown in FIG.20. In this experiment, all reactions contained HSL Reagent#l at a 1: 1 ratio with Taq polymerase. At this low reagent-to-enzyme ratio, all reactions amplified a mixture of specific and non-specific products (see Curves 2001 and 302). Curve 2002 shows that addition of a low concentration of HSL Reagent#9 (0.5:1 ratio) eliminated amplification of the non-specific products and increased the amplification of all three specific amplicons. Addition of increasingly higher concentrations of HSL Reagent#9 (1: 1, 2:1, 4:1 ratios) had dramatic effects. First, synthesis of the 515 base pair amplicon was inhibited, and then synthesis of the 431 base pair and 239 base pair amplicons was inhibited. But, as the synthesis of each of the correct amplicons decreased, the accumulation of primer-dimers increased.

[0317] The above results are explained by the fact that HSL Reagent#9 is comprised of two 30-base-pair-long complementary oligonucleotides that melt apart at 86°C. Thus, unlike HSL Reagents#l&2, HSLReagent#9 binds to Taq polymerase and remains bound to the polymerase throughout thermal cycling. As more and more HSL Reagent#9 is added to the reaction, fewer and fewer uninhibited Taq molecules are available during thermal cycling. For this reason, synthesis of all three correct products increases and then decreases. However, even at 4: 1 , the highest ratio of HSL Reagent#9 to Taq used in this experiment, the sum total of HSL Reagent#9&l molecules only reaches 5:1, which is not sufficient to inhibit amplification of primer-dimers prior to the start of amplification. The resulting primer-dimers are very “clean” (see Curves 2004 and 2005) because primer oligomerization during thermal cycling is also inhibited.

[0318] The titrations tests of HSL Reagents #3, 4, 8 shown in FIGS.12, 13, 18 demonstrate for each of these formulations that specific reagent-to-Taq ratio exists at which a reaction generates all three specific amplicons in amounts similar to those seen using 10: 1 ratios of HSL Reagents#l&2 (compare Curves 305, 1104, 1203, 1303, 1805). These experiments also demonstrate that the concentrations of reagents HSL Reagents #3, 4, 8 used in the successful reactions are all significantly lower than the optimal concentration of HSL Reagents# 1&2. For this reason, HSL Reagents #3,4,8 are listed in Table 1 as having potencies that are 6-45 times greater than those of HSL Reagents #1&2. However, a closer look at the results in FIGS.12, 13, 18 & 19 reveals that the optimum concentrations of HSL Reagents #3,4,8 are close to becoming inhibitory, and, if in fact, HSL Reagents #3, 4, 8 would be highly inhibitory if their concentrations were raised to the 10: 1 ratio used for HSL Reagents#l&2.

[0319] Discussion of Models of Reagent and Taq Interactions:

[0320] The answer to the above conundrum can be found in the structures of HSL Reagents #3,4,8 compared to the structures of HSL Reagents#l&2. Both HSL Reagents#l&2 have a loop that is 28 nucleotides long and arms of 11 nucleotides. All oligos that form a stem-loop structure must “zipper up” from the base of the arms outward to release the stress inherent in forming a double helix. Thus, the 11 -base-long arms of HSL Reagents#l&2 “flail” to a greater extent than arms of equivalent length attached to shorter loops. This accounts for why the HSL Reagent #1&2 hairpin has a Tm of 50-53°C, while all of the other stem-loop HSL Reagents listed in Table 1 have higher Tm’s. Thus, on the basis of Tm alone, a Taq polymerase molecule at the primer annealing temperature is more likely to encounter a double-stranded molecule of HSL Reagent#3-9, rather than an HSL Reagent#l&2 molecule in the closed-hairpin conformation. Only HSL Reagent#5 has a Tm almost as low as HSL Reagents# 1&2, because its arms are only 9 nucleotides long, its loop is only 3 nucleotides long, and its entire sequence is AT-rich. FIG.14 shows that HSL Reagent#5 is a relatively poor HSL Reagent.

[0321] But, there is more to the story. The Tm of each reagent in Table 1 is a measure of double-strand opening and closing for reagents in solution. It is not a direct measure of doublestrand binding and release from the polymerase. The structure of the Taq Polymerase protein is shown in FIG.22A. The single amino acid chain is 832 residues long and is folded into separate domains that resemble a right-handed glove comprised of a “fingers” and an opposing “thumb” domains. In the presence of dNTP’s, the fingers and thump domains open and close while carrying out DNA synthesis by 3 ’to 5’ addition of the appropriate nucleotide to the primed double-stranded DNA, which traverses the “palm” domain of the polymerase and exits the enzyme behind the ‘5 exonuclease domain, FIG.22A [Kim Y, Eom SH, Wang J, Lee D, Suh SW, Steitz TA. Crystal Structure of Themms aquaticus DNA polymerase. Nature 1995; 376: 612-616], In light of this mechanism of action, FIG.22B proposes that the 11-base pair “arm” of HSL Reagents# 1 or #2 is inhibited from advancing all the way into the DNA binding groove of the enzyme because it is anchored to the long 28-base loop of the reagent. In contrast, this would not be the case for HSL Reagents#8-9, which have double-stranded structures but no loops. HSL Reagents#3-7 that have small loops that may well fit snugly into the DNA binding groove, where they would be gripped between the closed “fingers and thumb” domains. They would be unable to advance or retreat because of their covalently-linked modifications, FIG.22C. (Note the HSL Reagents shown in FIGS.22B&C are superimposed on the Taq Polymerase molecule to emphasize their length difference, rather than their precise locations within the enzyme). Based on the above models, it is likely that Taq polymerase would release an HSL Reagents#l or #2 molecule more rapidly above 50°C than any other HSL formulation. This would functionally increase the inhibitory power of all HSL molecules except those of HSL Reagents# 1&2. These structural differences, in addition to the low Tm’s of HSL Reagents#l in solution, would guarantee that the vast majority of these molecules are non- inhibitory random-coiled oligonucleotides above 60°C. In contrast, all of the other formulations of HSL Reagents would be both more potent and more inhibitory.

[0322] Conclusions:

[0323] The results presented here demonstrate that a triplex end-point assay can be used for several purposes: A) to provide an easy-to-use, rapid test to quantitatively determine the optimal concentration of each HSL Reagent formulation required to achieve efficient amplification of a set of specific products; B) to provide a test that can used for quality control analysis of different batches of each HSL Reagent formulation; C) to provide a quantitative test that can be used to compare the activity of HSL Reagents in a complete master-mix, depending on whether the reagent is used in solution, or is first concentrated or dried in a reaction vessel, or is first mixed with or dried with a DNA polymerase prio22r to being tested in a complete master-mix; D) to provide a quantitative test that can be used to compare the potency of different HSL Reagent formulations under conditions A-C above; E) when synthesis and accumulation of a particular product takes place during the course of the closed-tube PCR reaction.

[0324] HSL Reagents#l&2 are the most useful formulations of hot-start-like reagents tested because they can be used at high reagent-to-enzyme ratios to inhibit primer-dimer synthesis at low temperature, while not inhibiting polymerase activity during thermal cycling. In addition, HSL Reagents#! &2 can be used in combination with low concentrations of double- stranded hot-start-like reagents, like HSL Reagents#8&9, to preferentially inhibit mis-priming during thermal cycling.

[0325] OTHER EMBODIMENTS

[0326] All of the features disclosed in this specification may be combined in any combination. Each feature disclosed in this specification may be replaced by an alternative feature serving the same, equivalent, or similar purpose. Thus, unless expressly stated otherwise, each feature disclosed is only an example of a generic series of equivalent or similar features.

[0327] From the above description, one skilled in the art can easily ascertain the essential characteristics of the present invention, and without departing from the spirit and scope thereof, can make various changes and modifications of the invention to adapt it to various usages and conditions. Thus, other embodiments are also captured within the claims of this dislcosure.

[0328] EQUIVALENTS

[0329] While several inventive embodiments have been described and illustrated herein, those of ordinary skill in the art will readily envision a variety of other means and / or structures for performing the function and / or obtaining the results and / or one or more of the advantages described herein, and each of such variations and / or modifications is deemed to be within the scope of the inventive embodiments described herein. More generally, those skilled in the art will readily appreciate that all parameters, dimensions, materials, and configurations described herein are meant to be exemplary and that the actual parameters, dimensions, materials, and / or configurations will depend upon the specific application or applications for which the inventive teachings is / are used. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific inventive embodiments described herein. It is, therefore, to be understood that the foregoing embodiments are presented by way of example only and that, within the scope of the appended claims and equivalents thereto, inventive embodiments may be practiced otherwise than as specifically described and claimed. Inventive embodiments of the present disclosure are directed to each individual feature, system, article, material, kit, and / or method described herein. In addition, any combination of two or more such features, systems, articles, materials, kits, and / or methods, if such features, systems, articles, materials, kits, and / or methods are not mutually inconsistent, is included within the inventive scope of the present disclosure. All definitions, as defined and used herein, should be understood to control over dictionary definitions, definitions in documents incorporated by reference, and / or ordinary meanings of the defined terms.

[0330] All references, patents, and patent applications disclosed herein are incorporated by reference with respect to the subject matter for which each is cited, which in some cases may encompass the entirety of the document.

[0331] The indefinite articles “a” and “an,” as used herein in the specification and in the claims, unless clearly indicated to the contrary, should be understood to mean “at least one.”

[0332] The phrase “and / or,” as used herein in the specification and in the claims, should be understood to mean “either or both” of the elements so conjoined, i.e.. elements that are conjunctively present in some cases and disjunctively present in other cases. Multiple elements listed with “and / or” should be construed in the same fashion, i.e. , “one or more” of the elements so conjoined. Other elements may optionally be present other than the elements specifically identified by the “and / or” clause, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, a reference to “A and / or B”, when used in conjunction with open-ended language such as “comprising” can refer, in one embodiment, to A only (optionally including elements other than B); in another embodiment, to B only (optionally including elements other than A); in yet another embodiment, to both A and B (optionally including other elements); etc.

[0333] As used herein in the specification and in the claims, “or” should be understood to have the same meaning as “and / or” as defined above. For example, when separating items in a list, “or” or “and / or” shall be interpreted as being inclusive, i.e., the inclusion of at least one, but also including more than one, of a number or list of elements, and, optionally, additional unlisted items. Only terms clearly indicated to the contrary, such as “only one of’ or “exactly one of,” or, when used in the claims, “consisting of,” will refer to the inclusion of exactly one element of a number or list of elements. In general, the term “or” as used herein shall only be interpreted as indicating exclusive alternatives i.e. “one or the other but not both”) when preceded by terms of exclusivity, such as “either,” “one of,” “only one of,” or “exactly one of.” “Consisting essentially of,” when used in the claims, shall have its ordinary meaning as used in the field of patent law.

[0334] As used herein in the specification and in the claims, the phrase “at least one,” in reference to a list of one or more elements, should be understood to mean at least one element selected from any one or more of the elements in the list of elements, but not necessarily including at least one of each and every element specifically listed within the list of elements and not excluding any combinations of elements in the list of elements. This definition also allows that elements may optionally be present other than the elements specifically identified within the list of elements to which the phrase “at least one” refers, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, “at least one of A and B” (or, equivalently, “at least one of A or B,” or, equivalently “at least one of A and / or B”) can refer, in one embodiment, to at least one, optionally including more than one, A, with no B present (and optionally including elements other than B); in another embodiment, to at least one, optionally including more than one, B, with no A present (and optionally including elements other than A); in yet another embodiment, to at least one, optionally including more than one, A, and at least one, optionally including more than one, B (and optionally including other elements); etc.

[0335] It should also be understood that, unless clearly indicated to the contrary, in any methods claimed herein that include more than one step or act, the order of the steps or acts of the method is not necessarily limited to the order in which the steps or acts of the method are recited.

Claims

1. CLAIMSWhat is claimed is:

1. A method for preparing a mixture of at least one Hot-Start- Like Reagent and a DNA polymerase, prior to assembly of a complete master-mix for amplification of doublestranded amplicons in a closed-reaction vessel, the method comprising combining at least one Hot-Start-Like (HSL) reagent and at least one DNA polymerase at an operative ratio of units of said at least one HSL reagent to units of said at least one DNA polymerase wherein: the at least one HSL reagent comprises at least one DNA oligonucleotide modified at its 3’ and 5’ terminal nucleotides such that the at least one single-stranded oligonucleotide is non-amplifiable by said DNA polymerase; and, either the at least one single-stranded oligonucleotide has a temperature-dependent reversible double-stranded hairpin conformation, or the at least one single- stranded DNA oligonucleotide hybridizes to a second non-amplifiable single-stranded DNA oligonucleotide generating a doublestranded DNA molecule in a temperature-dependent manner; and, said operative ratio is determined by quantitative end-point melt-curve analysis of a fluorescent dye that binds to double-stranded DNA molecules in said closed- reaction vessel.

2. The method of claim 1 , wherein the double-stranded DNA is selected from the group consisting of: i) temperature-dependent double-stranded conformations of said at least one HSL reagent; ii) primer-dimers; iii) primer-oligomers; iv) two or more distinguishable sequence-specific amplicons; and v) one or more non-specific amplicons.

3. The method of claim 1 , wherein the at least one DNA oligonucleotide is single-stranded.

4. The method of claim 1 wherein the complete master-mix comprises the mixture and at least one component selected from the group consisting of a monovalent cationic salt, a pH buffer, a divalent cationic salt, a dNTP, glycerol, dimethyl sulfoxide, a sugar, a heat denaturable protein, a polypeptide, a non-ionic detergent, optionally wherein the non-ionic detergent is Tween-20, NP-40, or Triton X-100, and an organic compound selected from the groupconsisting of betaine, TMAC, formamide, 7-deaza-2'-deoxy uanosine, and polyethylene glycol (PEG).

5. The method of claim 1 , wherein the operative ratio is relative to a ratio of 10: 1 defined by the nano-molar amount of HSL Reagent 1 to one-unit of a DNA polymerase (said one- unit being given by the enzyme manufacturer) in a mixture, prior to assembly of a complete master-mix in which primer-dimer synthesis is inhibited to the maximal possible extent in an amplification reaction in a closed- vessel.

6. The method of claim 5, wherein the amplification reaction is in the absence of a DNA template.

7. The method of claim 1, wherein said mixture is prepared in a buffered solution, MgCL, and dNTPs.

8. The method of claim 1 , wherein an optimal operative ratio is based on: the lowest level of primer-dimers and primer-oligomers, as determined in an amplification reaction containing no template DNA; the highest level of two or more distinguishable sequence-specific amplicons, as determined in an amplification reaction containing template DNA; and, the lowest level of non-specific amplicons produced in an amplification reaction containing template DNA.

9. The method of any preceding claim, wherein the at least one HSL reagent is selected from the group consisting of:(a) a non-amplifiable temperature-dependent intramolecular-hybridizing single-stranded DNA oligomer, having covalently bound moi eties on both its 5‘ and 3’ ends;(b) two non-amplifiable temperature-dependent intermolecular-hybridizing singlestranded DNA oligomers, having two to six covalently bound moieties on their 5’ or 3' ends;(c) three non-amplifiable temperature-dependent intermolecular-hybridizing singlestranded DNA oligomers, having two to six covalently bound moieties on their 5’ or 3‘ ends;(d ) a closed-circular double-stranded DNA;(e) a hairpin-shaped, single- stranded oligonucleotides modified at both the 3’ and 5’ ends, optionally by addition of dabcyl moieties, Black Hole Quencher™ moieties, and / or 2’ O-methyl nucleotides at the end of the stem;(f ) a pair of complementary or partially complementary oligonucleotides that form a hybrid 6-50 nucleotides long, wherein the oligonucleotides are modified on one or both ends by addition of polycyclic moieties, optionally by addition of dabcyl or coumarin moieties that do not have bulky portions that are non-planar;(g) a pair of oligonucleotide strands that form a hybrid at least six nucleotides long with a calculated Tm of at least 32°C, wherein strands in the terminal region of the hybrid contain interacting label moieties, which comprises a fluorophore on one strand and either a fluorophore or a non-fluorescent quencher on the other strand;(h) a double-stranded polynucleotide comprising from one to four single-stranded overhangs; and(i) an oligonucleotide comprising a stem-loop structure when not hybridized.

10. The method of any preceding claim wherein the HSL reagent is selected from the group consisting of:5’B2-GAATAATATAGCCCCCCCCCCCCCCCCCCCCCCCCCCCCCTATATTATTC-BB (SEQ ID NO: 1 (HSL Reagent 1));5’D-GAATAATATAGCCCCCCCCCCCCCCCCCCCCCCCCCCCCCTATATTATTC-D (SEQ ID NO: 2 (HSL Reagent 2);5’D-CATTATAACAGGCATATAGGGACAGTTATAATG-D (SEQ ID NO: 3 (HSL Reagent 3));5’D-CTTAATTATAATGAAATTATAATTAAG-D (SEQ ID NO: 4 (HSL Reagent 4);5’D-CATTATAATGAAATTATAATG-D (SEQ ID NO: 5 (HSL Reagent 5);5’M-CGGCGTCATATAGACGCCG-M 3-Carbon Spacer (SEQ ID NO: 6 (HSL Reagent 6));5’B1-CGCGGCGTCATATAGACGCCGCG-B1 (SEQ ID NO: 7 (HSL Reagent 7)); the double stranded construct: 5’-GAAATAAAATAAAAATAAAATA-D 3’D-CTTTATTTTATTTTTATTTTAT-D(SEQ ID NO: 8 (HSL Reagent 8)); and, the double stranded construct:5’CAGCTGGCCTGGGAAGCGTGCAGTCGGACC-BB3 ’ BB-GTCGACCGGACCCTTCGC ACGTC AGCCTGG-B2 (SEQ ID NO: 9 (HSL Reagent 9); wherein B 1 is Black Hole Quencher 1 , B2 is Black Hole Quencher 2, BB is Biosearch Blue, D is dabcyl, and M is [2’-OMeC][2’OMeG],11. The method of claim 1 , wherein the mixture comprises at least two HSL reagents.

12. The method of claim 10, wherein the at least two HSL reagents are:5’B2-GAATAATATAGCCCCCCCCCCCCCCCCCCCCCCCCCCCCCTATATTATTC-BB (SEQ ID NO: 1 (HSL reagent 1)); and,5’D-GAATAATATAGCCCCCCCCCCCCCCCCCCCCCCCCCCCCCTATATTATTC-D (SEQ ID NO: 2 (HSL reagent 2)).

13. The method of any one of claims 1-11, wherein the mixture comprises two HSL reagents, and optionally comprises three oligonucleotides.

14. The method of any one of claims 1-12, wherein the number of units of the at least one HSL reagent placed in the reaction vessel has a ratio in the range of 0.1 : 1 to 10:1 relative to the number of units of DNA-binding enzyme placed in the reaction vessel.

15. The method of claim 1, wherein the concentration of the at least one HSL reagent in the mixture is at least 1.5 times greater than its nano-molar concentration in the complete amplification master-mix in a closed- vessel.

16. The method of claim 1 , wherein the operational ratio of units of HSL reagent to units DNA polymerase is from 0.1:1 to 10: 1.

17. The method of claim 14, wherein the ratio is selected from the group consisting of 0.1 :1, 1: 1, 3: 1, 5:1, and 10: 1.

18. The method of any one of claims 1-15, wherein the closed-reaction vessel: has rigid walls comprised of a material selected from the group consisting of plastic, polymer, glass, carbon, and metal; is a pliable film; or has walls comprised of a non-aqueous fluid, optionally wherein the fluid is oil or lipid.

19. The method of any preceding claim, wherein the closed-reaction vessel is a plastic tube, a multi-welled plastic plate, a multi -welled film, an array of small chambers, a microfluidic device, a self-contained cassette, or a nano-chip printed with hydrophobic and hydrophilic surface arrays.

20. The method of claim 1 wherein the mixture comprises of a dried concentrate of a HSL reagent and a DNA polymerase in solution.

21. The method of claim 1 , wherein the mixture is a dried concentrate.

22. The method of claim 19, further comprising resuspending the mixture into an aqueous solution from the dried concentrate.

23. The method of claim 1, wherein the at least one thermostable DNA polymerase has been reconstituted from a dried concentrate.

24. The method of any one of claims 20-23, wherein the dried concentrate comprises an overlay in contact with the concentrate in the reaction vessel, and / or the overlay is made of a nonaqueous material that absorbs water.

25. The method of claim 24, wherein the overlay is an oil or a polymer.

26. The method of any one of claims 20-25, wherein the dried concentrate is prepared using a vacuum, at least one drying agent, and / or warming to a temperature of 40-60 °C.

27. The reaction vessel of any one of claims 20-26, wherein the nano-molar concentration of the concentrate of the at least one HSL reagent prior to drying is at least 1.5 times greater than its nano-molar concentration in the amplification master-mix in a closed- vessel.

28. The method of any preceding claim, wherein the closed reaction vessel is sealed with a cap.

29. The method of claim 28, wherein the cap is made of plastic, polymer, glass, carbon, or metal.

30. The method of any one of claims 20-29, wherein the dried concentrate further comprises at least one enzyme having a binding pocket for at least one double- stranded oligonucleotide.

31. The method of claim 30, wherein the at least one enzyme is selected from the group consisting of:(a) a DNA polymerase, which is optionally a Type A DNA polymerase, a Type B polymerase, or a Type C DNA polymerase;(b) a reverse transcriptase, which is optionally avian myeloblastosis virus reverse transcriptase or Moloney murine leukemia virus reverse transcriptase;(c) an exonuclease, which is optionally the 5’-to-3’ exonuclease of Tag polymerase, or the 3’-to-5’ proofreading exonuclease of Taq polymerase; and(d) a DNA ligase, which is optionally Bacteriophage T4 ligase, or E. Coli ligase.

32. The method of any one of claims 20-30, further comprising diluting the concentrate or the dried concentrate of the at least one HSL reagent in the reaction vessel by addition of a measured volume of an aqueous solution containing one or more additional components of a complete amplification reaction master-mix, one or more nucleic-acid amplification template molecules for generation of at least one amplicon, or a combination thereof.

33. The method of claim 32, wherein the one or more additional components of a complete amplification reaction master-mix are selected from the group consisting of a monovalent cationic salt, a pH buffer, a divalent cationic salt, a dNTP, glycerol, dimethyl sulfoxide, a sugar, a heat denaturable protein, a polypeptide, a non-ionic detergent, optionally wherein the non-ionic detergent is Tween-20, NP-40, or Triton X-100, and an organic compound selected from the group consisting of betaine, TMAC, formamide, 7-deaza-2'-deoxyguanosine, and polyethylene glycol (PEG).

34. The method of claim 1 wherein the identity and the amount of each possible type of doublestranded molecule present in said closed-vessel at end-point, is determined from the peak or valley in a first derivative plot of fluorescence intensity (-dF / dT) indicative of the binding or release of said fluorescent dye bound-to or released- from double- stranded DNA changing its conformation is a temperature-dependent manner.

35. The method of claim 1 wherein the identity and amount of each possible type of doublestranded molecule in a set of amplifications reactions, over a range of operative ratios of units of said reagent to units of said DNA polymerase, can be used to compare different HSL reagents in terms of their capacities to increase or inhibit amplification of primerdimers, primer-oligomers, sequence-specific amplicons, and / or non-specific amplicons.

36. The method of claim 35 wherein two or more batches of a master-mix comprised of the same operative ratios of units of the same HSL reagent(s) and the same units of the same enzyme(s) from the same manufacturer(s), but have been produced separately in time or location or equipment and are then compared at different times or locations or equipment,in order to establish the similarities or differences among said batches in terms of their capacities to amplify the same products in the same test assays.

37. The method of 35 wherein the identity and magnitude of each possible type of doublestranded molecule in a set of amplifications reactions, over a range of operative ratios of units of said reagent to units of said DNA polymerase, can be used to determine when, during the course of an amplification reaction, a particular type of HSL reagent acts to increase or inhibit amplification of primer-dimers, primer-oligomers, sequence-specific amplicons, and non-specific amplicons.

38. The method of any one of claims 34-37 wherein the identity and the magnitude of two or more peaks or valleys in two or more -dF / dT plots of two or more HSL reagents is used to determine the relative potencies of those reagents to increase or inhibit amplification of primer-dimers, primer-oligomers, sequence-specific amplicons, non-specific amplicons over a range of operative ratios of units of said reagent to units of said DNA polymerase.

39. A reaction vessel comprising a concentrate or a dried concentrate of at least one HSL reagent for an amplification reaction.

40. The reaction vessel of claim 39 prepared by the method of any one of claims 1-38.

41. The reaction vessel of claim 39 or 40, wherein the at least one HSL reagent is selected from the group consisting of:(a) a non-amplifiable temperature-dependent intramolecular-hybridizing single-stranded DNA oligomer, having covalently bound moieties on both its 5’ and 3’ ends;(b) two non-amplifiable temperature-dependent intermolecular-hybridizing singlestranded DNA oligomers, having two to six covalently bound moieties on their 5’ or 3' ends;(c) three non-amplifiable temperature-dependent intermolecular-hybridizing singlestranded DNA oligomers, having two to six covalently bound moieties on their 5’ or 3‘ ends;(d ) a closed-circular double-stranded DNA;(e) a hairpin shaped single stranded oligonucleotides modified at both the 3’ and 5’ ends, optionally by addition of dabcyl moieties, Black Hole Quencher™ moieties, and / or 2’ O-methyl nucleotides at the end of the stem;(f ) a pair of complementary or partially complementary oligonucleotides that form a hybrid 6-50 nucleotides long, wherein the oligonucleotides are modified on one or both ends by addition of polycyclic moieties, optionally by addition of dabcyl or coumarin moieties that do not have bulky portions that are non-planar;(g) a pair of oligonucleotide strands that form a hybrid at least six nucleotides long with a calculated Tm of at least 32 °C, wherein strands in the terminal region of the hybrid contain interacting label moieties, which comprises a fluorophore on one strand and either a fluorophore or a non-fluorescent quencher on the other strand;(h) a double-stranded polynucleotide comprising from one to four single-stranded overhangs; and(i) an oligonucleotide comprising a stem-loop structure when not hybridized.

42. The reaction vessel of claim 39 or 40 wherein the HSL reagent is selected from the group consisting of:5’B2-GAATAATATAGCCCCCCCCCCCCCCCCCCCCCCCCCCCCCTATATTATTC-BB (SEQ ID NO: 1 (HSL Reagent 1));5’D-GAATAATATAGCCCCCCCCCCCCCCCCCCCCCCCCCCCCCTATATTATTC-D (SEQ ID NO: 2 (HSL Reagent 2);5’D-CATTATAACAGGCATATAGGGACAGTTATAATG-D (SEQ ID NO: 3 (HSL Reagent 3));5’D-CTTAATTATAATGAAATTATAATTAAG-D (SEQ ID NO: 4 (HSL Reagent 4);5’D-CATTATAATGAAATTATAATG-D (SEQ ID NO: 5 (HSL Reagent 5);5’M-CGGCGTCATATAGACGCCG-M 3-Carbon Spacer (SEQ ID NO: 6 (HSL Reagent 6));5’B1-CGCGGCGTCATATAGACGCCGCG-B1 (SEQ ID NO: 7 (HSL Reagent 7)); the double-stranded construct: 5 ’ D-GAAAT A A AATA A A AATA A A ATA-D3 ’ D-CTTTATTTTATTTTTATTTTAT-D(SEQ ID NO: 8 (HSL Reagent 8)); and, the double-stranded construct:5 ’ CAGCTGGCCTGGGAAGCGTGCAGTCGGACC-BB3 ’ BB-GTCGACCGGACCCTTCGC ACGTC AGCCTGG-B2 (SEQ ID NO: 9 (HSL Reagent 9); wherein Bl is Black Hole Quencher 1; B2 is Black Hole Quencher 2, BB is Biosearch Blue, D is dabcyl, and M is [2’-OMeC][2’OMeG],43. The reaction vessel of any one of claims 39-42, wherein the mixture comprises at least two HSL reagents.

44. The reaction vessel of claim 43, wherein the at least two HSL reagents are:5’B2-GAATAATATAGCCCCCCCCCCCCCCCCCCCCCCCCCCCCCTATATTATTC-BB (SEQ ID NO: 1 (HSL Reagent 1)); and,5’D-GAATAATATAGCCCCCCCCCCCCCCCCCCCCCCCCCCCCCTATATTATTC-D (SEQ ID NO: 2 (HSL Reagent 2).

45. The reaction vessel of claim 43 or 44, wherein the mixture comprises two HSL reagents, and optionally comprises three oligonucleotides.

46. The reaction vessel of any one of claims 39-45, wherein the number of units of the at least one HSL reagent placed in the reaction vessel has a ratio in the range of 1 : 1 to 10: 1 relative to the number of units of DNA-binding enzyme placed in the reaction vessel.

47. The reaction vessel of any one of claims 39-46, wherein: the reaction vessel has rigid walls comprised of a material selected from the group consisting of plastic, polymer, glass, carbon, and metal; the reaction vessel is a pliable film; or the reaction vessel has walls comprised of a non-aqueous fluid, optionally wherein the fluid is oil or lipid.

48. The reaction vessel of any one of claims 39-47, wherein the reaction vessel is a plastic tube, a multi- welled plastic plate, a multi-welled film, an array of small chambers, a microfluidic device, a self-contained cassette, or a nano-chip printed with hydrophobic and hydrophilic surface arrays.

49. The reaction vessel of any one of claims 39-48, wherein the reaction vessel is sealed with a cap.

50. The reaction vessel of claim 49, wherein the cap is made of plastic, polymer, glass, carbon, or metal.

51. An open reaction vessel of any one of claims 39-50 comprising an aqueous solution containing one or more additional components of a complete amplification reaction mastermix, one or more nucleic acid amplification template molecules for generation of at least one amplicon, or a combination thereof, before an amplification reaction.

52. The open reaction vessel of claim 51, wherein the one or more additional components of a complete amplification reaction master-mix include at least one of a monovalent cationic salt, a pH buffer, a divalent cationic salt, a dNTP, glycerol, dimethyl sulfoxide, a sugar, a heat denaturable protein, a polypeptide, a non-ionic detergent, and / or other organic compound(s), optionally wherein the non-ionic detergent is Tween- 20, NP-40, or Triton X-100, and wherein the other organic compound is selected from the group consisting of betaine, TMAC, formamide, 7-deaza-2'-deoxyguanosine, and PEG.

53. A reaction vessel of any one of claims 39-52 comprising a DNA amplification master-mix contained therein.

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