WO2024050820A1 - Antioxidant composition and use thereof in nucleic acid detection - Google Patents

Abstract

An antioxidant composition and the use thereof in nucleic acid detection. The present invention relates to an antioxidant composition, a reagent containing the composition, a kit containing the composition or reagent, the use of the composition or reagent as a nucleic acid protection agent in nucleic acid detection, and a method for detecting a nucleic acid. The antioxidant composition contains: a saponin compound, which is selected from one or more of notoginsenoside R1, ginsenoside Rg1, ginsenoside Rd, ginsenoside Rb2, ginsenoside Rb3, ginsenoside Rc, ginsenoside Rf and ginsenoside Re; glycyrrhizic acid, or a salt of glycyrrhizic acid, or a hydrate of a salt of glycyrrhizic acid; and adenosine 5'-monophosphate or a salt thereof, or carnosine.

Description

Antioxidant composition and its use in nucleic acid detection Technical field

The present invention relates to the field of nucleic acid detection, and relates to an antioxidant composition, a reagent containing the composition, a kit containing the composition or reagent, and the use of the composition or reagent as a nucleic acid protective agent in nucleic acid detection. , and a nucleic acid detection method.

Background technique

In many nucleic acid detection methods (such as DNA sequencing methods, synthesis sequencing or ligation sequencing, etc.), it is necessary to irradiate the fluorescent group on the nucleic acid to be detected (or its complementary nucleic acid) through light irradiation, such as laser irradiation. Group excitation is used, and the information of the nucleic acid to be detected is obtained through optical systems and base recognition software. The continuous strong irradiation of the excitation light source and the reactive oxygen groups in the solution will affect the nucleic acid, causing damage or degradation of the nucleic acid, resulting in the loss of fluorescence detection signal intensity, thereby reducing the number of detection cycles or reducing the accuracy of the detection.

The buffer solution used in the base calling process is called a scanning reagent or a photographing reagent. Laser damage to template nucleotides can be prevented by adding a nucleic acid protecting agent to the scanning reagent. The commercialized L-ascorbate combination formula can be used as an effective antioxidant and anti-photodamage nucleic acid protectant for nucleic acid sequencing, and can support long-read, paired-end sequencing, and has a low error rate. However, during long-term storage, L-ascorbate will discolor after being oxidized, thus affecting the scanning quality and thus the sequencing quality of polynucleotides. In addition, in the prior art, some scanning reagents contain a relatively high concentration of nucleic acid protecting agent components. The use of high-concentration nucleic acid protective agents makes the scanning reagent prone to salt precipitation when stored at low temperatures or during long-term sequencing processes. In addition, the use of high concentrations of nucleic acid protective agents will increase product costs.

Contents of the invention

Notoginseng saponin R1 can be used as a nucleic acid protective agent, and can also protect template nucleotides at very low working concentrations. In addition, Panax notoginseng saponin R1 has no absorption in the ultraviolet and visible light regions, does not interfere with base recognition signals, and will not change color even if it is oxidized during long-term storage.

The inventor found in practical applications that Panax notoginseng saponin R1 as a nucleic acid protectant is more suitable for short-read single-end sequencing. The error rate of sequencing is low, but when the number of sequencing cycles is large (especially paired-end sequencing) , the signal recovery and quality of the second strand are poor, so the accuracy and sequencing quality need to be improved.

In order to solve the above problems, the present application provides an antioxidant composition, which contains: notoginseng saponin R1 or other saponin compounds, glycyrrhizic acid or its derivatives, and 5'-adenosine monophosphate or its salt or carnosine; Optionally, the composition also contains other antioxidants. The antioxidant composition works together with multiple antioxidants to better protect nucleic acids and reduce light-induced damage.

Antioxidant composition

In one aspect, the application provides a composition comprising:

(1) Saponin compounds, selected from one or more of notoginsenoside R1, ginsenoside Rg1, ginsenoside Rd, ginsenoside Rb2, ginsenoside Rb3, ginsenoside Rc, ginsenoside Rf, and ginsenoside Re;

(2) Glycyrrhizic acid, a salt of glycyrrhizic acid or a hydrate of a salt of glycyrrhizic acid; and

(3) 5'-adenosine monophosphate or its salt, or carnosine;

Optionally, the composition also contains other antioxidants.

In this application, the structural formulas of each saponin compound are as follows:

In some embodiments, the saponin compound is notoginsenoside R1.

Glycyrrhizic acid, also known as licorice triterpene saponin and glycyrrhizin, is a triterpene compound. Its English name is Glycyrrhizic Acid. Its CAS number is 1405-86-3. Its molecular formula is C ₄₂ H ₆₂ O ₁₆ and its molecular weight is 822.93. Its chemical structural formula is as follows:

Glycyrrhizic acid is easily soluble in hot water and ethanol, but has low solubility in cold water. It can be used in the form of salt or salt hydrate to increase water solubility.

In this application, salts of glycyrrhizic acid refer to salts formed by 1, 2 or 3 carboxyl groups in glycyrrhizic acid and appropriate inorganic or organic cations (bases), including but not limited to: alkali metal salts, such as sodium salts , potassium salt, lithium salt, etc.; alkaline earth metal salts, such as calcium salt, magnesium salt, etc.; other metal salts, such as aluminum salt, iron salt, zinc salt, copper salt, nickel salt, cobalt salt, etc.; inorganic alkali salts, such as ammonium salt Salt; organic base salt, such as tert-octylamine salt, dibenzylamine salt, morpholine salt, glucosamine salt, phenylglycine alkyl ester salt, ethylenediamine salt, N-methylglucamine salt, Guanidine salt, diethylamine salt, triethylamine salt, dicyclohexylamine salt, N,N'-dibenzylethylenediamine salt, chloroprocaine salt, procaine salt, diethanolamine salt, N - Benzyl-phenylethylamine salt, piperazine salt, tetramethylamine salt, tris(hydroxymethyl)aminomethane salt.

In some embodiments, the salt of glycyrrhizic acid is selected from an alkali metal salt or an ammonium salt, such as a monopotassium salt, a monosodium salt, a monoammonium salt, a dipotassium salt, a disodium salt, a diammonium salt, a tripotassium salt, Trisodium salt, triammonium salt.

In this application, glycyrrhizic acid hydrate or glycyrrhizic acid salt hydrate refers to a substance formed by the association of glycyrrhizic acid or glycyrrhizic acid salt with one or more water molecules. The glycyrrhizic acid salt may be any of the above salts.

In some embodiments, the hydrate of a salt of glycyrrhizic acid is selected from a hydrate of an alkali metal or ammonium salt of glycyrrhizic acid, such as a hydrate of a sodium or potassium salt.

In some embodiments, the salt of glycyrrhizic acid or the hydrate of a salt of glycyrrhizic acid is selected from:

Glycyrrhizic acid monoammonium salt, its exemplary structure is as follows:

Trisodium glycyrrhizinate hydrate has an exemplary structure as follows:

Glycyrrhizic acid monopotassium salt, its exemplary structure is as follows:

Diammonium glycyrrhizinate, its exemplary structure is as follows:

Dipotassium glycyrrhizinate hydrate has an exemplary structure as follows:

The CAS number of Adenosine 5'-monophosphate is 61-19-8, the molecular formula is C ₁₀ H ₁₄ N ₅ O ₇ P, the molecular weight is 347.201, and the chemical structural formula is as follows:

The salt of 5'-adenosine monophosphate is a salt formed by the phosphate group in 5'-adenosine monophosphate and 1 or 2 appropriate inorganic or organic cations (bases), or it is a salt of 5'-adenosine monophosphate A salt formed between the amino group and one or two appropriate inorganic or organic anions (acids).

The salts formed by the phosphate group and one or two appropriate inorganic or organic cations (bases) include but are not limited to: alkali metal salts, such as sodium salts, potassium salts, lithium salts, etc.; alkaline earth metal salts, such as calcium salts , magnesium salt, etc.; other metal salts, such as aluminum salt, iron salt, zinc salt, copper salt, nickel salt, cobalt salt, etc.; inorganic alkali salts, such as ammonium salt; organic alkali salts, such as tert-octylamine salt, dibenzyl Ammonium salt, morpholine salt, glucosamine salt, phenylglycine alkyl ester salt, ethylenediamine salt, N-methylglucamine salt, guanidine salt, diethylamine salt, triethylamine salt, bicyclic Hexylamine salt, N,N'-dibenzylethylenediamine salt, chloroprocaine salt, procaine salt, diethanolamine salt, N-benzyl-phenylethylamine salt, piperazine salt, tetrazine salt Methylamine salt, tris(hydroxymethyl)aminomethane salt.

The salts formed by the amino group and 1 or 2 appropriate inorganic or organic anions (acids) include, but are not limited to: hydrohalides, such as hydrofluorates, hydrochlorides, hydrobromides, and hydroiodates. etc.; inorganic acid salts, such as nitrates, perchlorates, sulfates, phosphates, etc.; lower alkane sulfonates, such as methanesulfonate, trifluoromethanesulfonate, ethanesulfonate, etc.; aryl sulfonates Acid salts, such as benzenesulfonate, p-benzenesulfonate, etc.; organic acid salts, such as acetate, malate, fumarate, succinate, citrate, tartrate, oxalate, horsenate, etc. acid salts, etc.; amino acid salts, such as glycinate, trimethylglycinate, arginate, ornithine, glutamate, aspartate, etc.

In some embodiments, the salt of adenosine 5'-monophosphate is selected from alkali metal salts, such as sodium salts.

In some embodiments, the salt of adenosine 5'-monophosphate is adenosine 5'-monophosphate monosodium, and the chemical structural formula is as follows:

Carnosine (L-Carnosine), scientific name β-alanyl-L-histidine, is a dipeptide composed of two amino acids: β-alanine and L-histidine, CAS number is 305-84- 0, the molecular formula is C ₉ H ₁₄ N ₄ O ₃ , the molecular weight is 226.24, and the chemical structural formula is as follows:

In some embodiments, the other antioxidant may be dithiothreitol (DTT) or Trolox (water-soluble vitamin E).

In some embodiments, in the composition, the molar ratio of the saponin compound to (glycyrrhizic acid, a salt of glycyrrhizic acid or a hydrate of a salt of glycyrrhizic acid) is 4:1 to 1:10 (for example, 4:1 to 1:10). 3:1 (for example, 1.67:0.5), 3:1～2:1, 2:1～1:1 (for example, 1.67:1.5), 1:1～1:2 (for example, 1.67:2.5), 1:2～ 1:3, 1:3～1:4, 1:4～1:5, 1:5～1:6, 1:6～1:7, 1:7～1:8, 1:8～1: 9 or 1:9~1:10.

In some embodiments, in the composition, the molar ratio of the saponin compound to adenosine 5'-monophosphate or its salt and carnosine is 1:1 to 1:100 (e.g., 1:1 to 1:5 (e.g. 1.67:7.5), 1:5～1:10, 1:10～1:20, 1:20～1:30, 1:30～1:40, 1:40～1:50, 1:50～1 :60, 1:60～1:70, 1:70～1:80, 1:80～1:90 or 1:90～1:100).

In some embodiments, the molar ratio of saponins to other antioxidants (such as DTT, Trolox, etc.) in the composition is 1:1 to 1:50 (for example, 1:1 to 1:5 (for example, 1.67) :8), 1:5～1:10, 1:10～1:20 (for example, 1.67:20), 1:20～1:30, 1:30～1:40, 1:40～1:50) .

In some embodiments, in the composition, the molar ratio of (glycyrrhizic acid, a salt of glycyrrhizic acid or a hydrate of a salt of glycyrrhizic acid): (adenosine 5'-monophosphate or a salt thereof or carnosine) is 1: 1～1:15 (for example, 1:1～1:3, 1:3～1:5, 1:5～1:10 or 1:10～1:15).

The proportion of each component in the composition of the present invention can be adjusted as needed (for example, the proportion in the above embodiment can be used) to obtain appropriate antioxidant properties and avoid damage to dNTP or DNB due to excessive antioxidant properties. Decrease sequencing quality.

Reagents, kits and their uses

In one aspect, the application provides a reagent comprising a composition of the invention and a buffer solution (eg, Tris buffer solution).

In the present invention, Tris buffer solution refers to a buffer solution using trishydroxymethylaminomethane (Tris) as a buffer system.

Tris is widely used in the preparation of buffer solutions in biochemistry and molecular biology. Tris is a weak base, and the pH of its alkali aqueous solution is around 10.5. Add hydrochloric acid to adjust the pH to the desired value, and a buffer at that pH can be obtained. Tris and its hydrochloride salt (Tris-HCl) can also be used to prepare buffer solutions.

Accordingly, in some embodiments, the reagents comprise: water, a composition of the invention, Tris Base, Tris-HCl, and optionally ethylenediaminetetraacetic acid and/or Solvent.

In some embodiments, the pH of the reagent is between 6.0 and 9.0 (eg, 6.0-7.0, 7.0-8.0, or 8.0-9.0).

In some embodiments, the concentration of the glycyrrhizic acid, the salt of glycyrrhizic acid or the hydrate of the salt of glycyrrhizic acid is 0.1~10mM, such as 0.1~0.5mM, 0.5~1mM, 1~1.5mM, 1.5~2.5mM, 2.5～3mM, 3～5mM, 5～7mM or 7～10mM.

In some embodiments, the concentration of the saponin compound is 0.1~5mM, such as 0.1~0.5mM, 0.5~1mM, 1~1.5mM, 1.5~2.0mM (such as 1.67mM), 2.0~2.5mM, 2.5~ 3mM, 3～4mM or 4～5mM. The saponin compounds are limited by their own solubility in the reagent. If the amount is excessive, it will be difficult to dissolve completely or even become insoluble. The above concentration range is selected so that the saponin compounds can be completely dissolved in the reagent.

In some embodiments, the concentration of adenosine 5'-monophosphate or a salt thereof or carnosine is 0.1~200mM, such as 0.1~0.5mM, 0.5~1mM, 1~1.5mM, 1.5~2.5mM, 2.5~3mM , 3～5mM, 5～7.5mM, 7.5～10mM, 10～30mM, 30～50mM, 50～75mM, 75～100mM, 100～120mM, 120～150mM, 150～180mM or 180～200mM.

In some embodiments, the concentration of the other antioxidants is 0.1-50mM, such as 0.1-0.5mM, 0.5-1mM, 1-1.5mM, 1.5-2.5mM, 2.5-3mM, 3-5mM, 5-7.5mM , 7.5～10mM, 10～30mM or 30～50mM.

In some embodiments, the reagent is a scanning reagent.

In some embodiments, the scanning reagent includes 1-2M Tris buffer, 1-2mM notoginsenoside R1, 0.5-2.5mM glycyrrhizic acid, a salt of glycyrrhizic acid or a hydrate of a salt of glycyrrhizic acid, 7-8mM 5 '-Adenosine monophosphate or its salt or carnosine, 8~9mM Trolox, 9~10mM ethylenediaminetetraacetic acid, and 10~20mM DTT.

In certain embodiments, the reagents further comprise sodium chloride and/or a stabilizing agent for DNA (eg, Tween-20). The function of sodium chloride is to provide a salt solution background and protect the primers in the detection.

In another aspect, the present application provides the use of the composition or reagent of the present application as a nucleic acid protecting agent in nucleic acid detection.

In certain embodiments, the nucleic acid protective agent is used to protect nucleic acids and reduce or avoid photodamage (light-induced damage) or oxidative damage.

In certain embodiments, the nucleic acid detection involves detecting a fluorescent signal. In certain embodiments, the fluorescent signal can be generated by an illumination reaction or a non-illumination reaction (eg, a bioautoluminescence reaction). In certain embodiments, the bioautoluminescence reaction refers to a reaction in which luciferase catalyzes its substrate to produce a fluorescent signal.

In certain embodiments, the nucleic acid detection involves an illuminated reaction or a non-illuminated reaction (eg, a bioluminescence reaction).

As used herein, the term "illumination reaction" refers to a reaction upon exposure to optical energy. Typically in the reaction, optical energy (illumination) is provided to observe the production and/or consumption of reactants or products that have specific optical characteristics indicating their presence, such as the absorption spectrum of the reaction mixture or its components. and/or changes in the emission spectrum (changes in intensity, wavelength, etc.).

As used herein, the term "non-illuminated reaction" refers to a reaction that can be detected without the aid of optical energy. In non-illuminated reactions, an optical signal can be generated through pathways such as bioautoluminescence or through changes in electrical signals, whereby the production and/or consumption of reactants or products can be detected.

In certain embodiments, the illumination reaction includes a light signal triggered by base extension, which can cause the generation of an optical signal (eg, a fluorescent signal), or a light signal triggered by probe hybridization.

In certain embodiments, the light signal in the illumination reaction of the present invention is preferably fluorescence.

In certain embodiments, the nucleic acid detection is nucleic acid sequence determination (sequencing) or other detection, such as high-throughput sequencing, such as sequencing by synthesis (SBS sequencing), ligation sequencing, hybridization sequencing, nanopore sequencing, or multiplex sequencing. Combinatorial probe-anchor ligation (cPAL) sequencing.

In certain embodiments, the nucleic acid detection is quantitative PCR.

In another aspect, the application provides a kit comprising a composition or agent of the invention.

In certain embodiments, the kits of the present invention may also contain one or more other reagents required for nucleic acid detection, such as primers, polymerases, buffer solutions, washing solutions, or any combination thereof.

In certain embodiments, kits of the invention are used for nucleic acid sequence determination.

In certain embodiments, the kit of the present invention may also include: a reagent for immobilizing the nucleic acid molecule to be sequenced to a support (for example, immobilization by covalent or non-covalent linkage); for initiating nucleic acid molecules. A primer for nucleotide polymerization; a polymerase for nucleotide polymerization; one or more buffer solutions; one or more wash solutions; or any combination thereof.

In certain embodiments, kits of the present invention may also include reagents and/or devices for extracting nucleic acid molecules from a sample. Methods for extracting nucleic acid molecules from samples are well known in the art. Therefore, various reagents and/or devices for extracting nucleic acid molecules can be configured in the kit of the present invention as needed, such as reagents for disrupting cells, reagents for precipitating DNA, and reagents for washing DNA. Reagents used to dissolve DNA, reagents used to precipitate RNA, reagents used to wash RNA, reagents used to dissolve RNA, reagents used to remove proteins, reagents used to remove DNA (for example, when the target nucleic acid molecule is RNA ), reagents for removing RNA (for example, when the nucleic acid molecule of interest is DNA), and any combination thereof.

In certain embodiments, kits of the invention further comprise reagents for pretreating nucleic acid molecules. In the kit of the present invention, the reagents used to pretreat nucleic acid molecules are not subject to additional restrictions and can be selected according to actual needs. The reagents used to pretreat nucleic acid molecules include, for example, reagents used to fragment nucleic acid molecules (such as DNase I), reagents used to complete the ends of nucleic acid molecules (such as DNA polymerase, such as T4 DNA polymerase, Pfu DNA Polymerase, Klenow DNA polymerase), adapter molecules, label molecules, reagents used to connect adapter molecules to nucleic acid molecules of interest (such as ligases, such as T4 DNA ligase), reagents used to repair nucleic acid ends (such as loss DNA polymerases that exhibit 3'-5' exonuclease activity but exhibit 5'-3' exonuclease activity), reagents used to amplify nucleic acid molecules (e.g., DNA polymerases, primers, dNTPs), for Reagents for isolating and purifying nucleic acid molecules (eg, chromatography columns), and any combination thereof.

In certain embodiments, the kits of the invention further comprise a support for immobilizing the nucleic acid molecules to be sequenced. Under normal circumstances, the support used to immobilize the nucleic acid molecules to be sequenced is in a solid phase to facilitate handling. Therefore, in this disclosure, a "support" is sometimes also referred to as a "solid support" or "solid support." However, it should be understood that the "support" mentioned herein is not limited to a solid, and may also be a semi-solid (eg, a gel).

As used herein, the terms "loaded," "immobilized," and "attached" when used with reference to a nucleic acid, mean direct or indirect attachment to a solid support via covalent or non-covalent bonds. In certain embodiments of the present disclosure, methods of the invention include immobilizing a nucleic acid on a solid support via covalent attachment. Typically, however, all that is required is that the nucleic acid remains immobilized or attached to the solid support under the conditions in which use of the solid support is desired (e.g., in applications requiring nucleic acid amplification and/or sequencing). In certain embodiments, immobilizing the nucleic acid on the solid support can include immobilizing the oligonucleotide to be used as a capture primer or amplification primer on the solid support such that the 3' end is available for enzymatic extension. and at least part of the primer sequence is capable of hybridizing to a complementary nucleic acid sequence; the nucleic acid to be immobilized is then hybridized to the oligonucleotide, in which case the immobilized oligonucleotide or polynucleotide can be 3'- 5' direction. In certain embodiments, immobilizing a nucleic acid on a solid support may include binding a nucleic acid binding protein to the solid support via amination modification, and capturing the nucleic acid molecule via the nucleic acid binding protein. Alternatively, loading may occur by means other than base pairing hybridization, such as covalent attachment as described above. Non-limiting examples of means of attachment of nucleic acids to solid supports include nucleic acid hybridization, biotin-streptavidin conjugation, sulfhydryl conjugation, photoactivated conjugation, covalent conjugation, antibody-antigen, via hydrogels or other porous polymers physical limitations, etc. Various exemplary methods for immobilizing nucleic acids on solid supports can be found, for example, in G. Steinberg-Tatman et al., Bioconjugate Chemistry 2006, 17, 841-848; Xu X. et al. Journal of the American an Chemical Society 128 (2006 )9286-9287; U.S. Patent Applications US 5639603, US 5641658, US2010248991; International Patent Applications WO 2001062982, WO 2001012862, WO 2007111937, WO0006770, for all purposes, in particular in connection with the preparation of solid supports having nucleic acids immobilized thereon The entire teachings of the above documents are incorporated herein by reference in their entirety.

In the present invention, the support may be made of various suitable materials. Such materials include, for example, inorganics, natural polymers, synthetic polymers, and any combination thereof. Specific examples include, but are not limited to: cellulose, cellulose derivatives (such as nitrocellulose), acrylic resins, glass, silica gel, silica, polystyrene, gelatin, polyvinylpyrrolidone, copolymers of vinyl and acrylamide materials, polystyrene cross-linked with divinylbenzene, etc. (see, e.g., Merrifield Biochemistry 1964, 3, 1385-1390), polyacrylamide, latex, dextran, rubber, silicon, plastic, natural sponge, metal Plastic, cross-linked dextran (eg, Sephadex ^™ ), agarose gel (Sepharose ^™ ), and other supports known to those skilled in the art.

In certain preferred embodiments, the support for immobilizing nucleic acid molecules to be sequenced can be a solid support including an inert substrate or matrix (e.g., glass slide, polymer beads, etc.), which have been functionalized, for example, by the application of intermediate materials containing reactive groups that allow covalent attachment of biomolecules such as polynucleotides. Examples of such supports include, but are not limited to, polyacrylamide hydrogels supported on an inert substrate such as glass, in particular those described in WO 2005/065814 and US 2008/0280773, wherein The contents of the aforementioned patent applications are incorporated herein by reference in their entirety. In such embodiments, a biomolecule (e.g., a polynucleotide) can be directly covalently linked to an intermediate material (e.g., a hydrogel), which itself can be non-covalently linked to a substrate or matrix ( For example, glass substrate). In certain preferred embodiments, the support is a glass slide or silicon chip whose surface is modified with a layer of avidin, amino, acrylamide silane or aldehyde chemical groups.

In the present invention, the support or solid support is not limited to its size, shape and configuration. In some embodiments, the support or solid support is a planar structure, such as a slide, chip, microchip, and/or array. The surface of such a support may be in the form of a planar layer.

In certain preferred embodiments, the support used to immobilize the nucleic acid molecules to be sequenced is an array of beads or wells (which is also referred to as a chip). The array may be prepared using any of the materials outlined herein for preparing solid supports, and preferably the surface of the beads or wells on the array is functionalized to facilitate the immobilization of nucleic acid molecules. The number of beads or wells on the array is not limited. For example, each array may contain 10-10 ² , 10 ² -10 ³ , 10 3 -10 ⁴ , 10 ⁴ -10 ⁵ , 10 ^{5 -10 6} , 10 ⁶ -10 ⁷ , 10 ⁷ -10 ⁸ , 10 ⁸ -10 ⁹ , 10 ¹⁰ -10 ¹¹ , 10 ¹¹ -10 ¹² or more beads or holes. In certain exemplary embodiments, one or more nucleic acid molecules can be immobilized on the surface of each bead or well. Correspondingly, each array can be fixed with 10-10 ² , 10 ² -10 ³ , 10 3 -10 ⁴ , 10 ^{4 -10 5} , 10 ⁵ -10 ⁶ , 10 ⁶ -10 ⁷ , 10 ⁷ -10 ⁸ , 10 ⁸ -10 ⁹ , 10 ¹⁰ -10 ¹¹ , 10 ¹¹ -10 ¹² or more nucleic acid molecules. Therefore, such arrays may be particularly advantageously used for high-throughput sequencing of nucleic acid molecules.

In certain preferred embodiments, the kits of the invention further comprise reagents for immobilizing the nucleic acid molecule to be sequenced to the support (eg, immobilization via covalent or non-covalent linkage). Such reagents include, for example, reagents that activate or modify the nucleic acid molecule (eg, its 5' end), such as phosphates, thiols, amines, carboxylic acids, or aldehydes; reagents that activate or modify the surface of the support, such as amino- Alkoxysilanes (such as aminopropyltrimethoxysilane, aminopropyltriethoxysilane, 4-aminobutyltriethoxysilane, etc.); cross-linking agents, such as succinic anhydride, phenyldiisosulfide Cyanate (Guo et al., 1994), maleic anhydride (Yang et al., 1998), 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) , m-maleimidobenzoic acid-N-hydroxysuccinimide ester (MBS), N-succinimidyl [4-iodoacetyl] aminobenzoic acid (SIAB), 4-(N -Maleimidomethyl)cyclohexane-1-carboxylic acid succinimide (SMCC), N-γ-maleimidobutyryloxy-succinimide ester (GMBS), 4-(p-maleimidophenyl)butyric acid succinimide (SMPB); and any combination thereof.

In certain preferred embodiments, the kits of the invention further comprise primers for initiating nucleotide polymerization reactions. In the present invention, the primer is not subject to additional restrictions as long as it can specifically anneal to a region of the target nucleic acid molecule. In some exemplary embodiments, the length of the primer may be 5-50 bp, such as 5-10, 10-15, 15-20, 20-25, 25-30, 30-35, 35-40, 40- 45, 45-50bp. In some exemplary embodiments, the primers may comprise naturally occurring or non-naturally occurring nucleotides. In some exemplary embodiments, the primers comprise or consist of naturally occurring nucleotides. In some exemplary embodiments, the primers comprise modified nucleotides, such as locked nucleic acids (LNA). In certain preferred embodiments, the primers comprise universal primer sequences.

In certain preferred embodiments, the kits of the invention further comprise a polymerase for performing nucleotide polymerization reactions. In the present invention, various suitable polymerases may be used to carry out the polymerization reaction. In some exemplary embodiments, the polymerase is capable of synthesizing new DNA strands using DNA as a template (eg, DNA polymerase). In some exemplary embodiments, the polymerase is capable of synthesizing new DNA strands using RNA as a template (eg, reverse transcriptase). In some exemplary embodiments, the polymerase is capable of synthesizing new RNA strands using DNA or RNA as a template (eg, RNA polymerase). Thus, in certain preferred embodiments, the polymerase is selected from the group consisting of DNA polymerase, RNA polymerase, and reverse transcriptase.

In certain preferred embodiments, the kits of the invention further comprise one or more excision reagents. In certain embodiments, the excision reagent is selected from the group consisting of endonuclease IV and alkaline phosphatase.

In certain preferred embodiments, the kits of the invention further comprise one or more buffer solutions. Such buffers include, but are not limited to, buffer solutions for DNase I, buffer solutions for DNA polymerase, buffer solutions for ligase, buffer solutions for elution of nucleic acid molecules, and buffer solutions for dissolving nucleic acid molecules. Buffer solutions for nucleotide polymerization reactions (such as PCR), and buffer solutions for ligation reactions. The kit of the present invention may contain any one or more of the above buffer solutions.

In the present invention, "buffer" and "buffer solution" have the same meaning and can be used interchangeably.

In certain embodiments, the buffer solution for DNA polymerase contains monovalent salt ions (eg, sodium ions, chloride ions) and/or divalent salt ions (eg, magnesium ions, sulfate ions, manganese ions). In certain embodiments, the concentration of the monovalent salt ion or divalent salt ion in the buffer solution is 10 μM-200 mM, such as 10 μM, 50 μM, 100 μM, 200 μM, 500 μM, 1 mM, 3mM, 10mM, 20mM, 50mM, 100mM, 150mM or 200mM.

In certain embodiments, the buffer solution for DNA polymerase comprises tris(hydroxymethylaminomethane) (Tris). In certain embodiments, the concentration of Tris in the buffer solution is 10mM-200mM, such as 10mM, 20mM, 50mM, 100mM, 150mM or 200mM.

In certain embodiments, the buffer solution for DNA polymerase contains an organic solvent, such as DMSO or glycerol (glycerol). In certain embodiments, the mass content of the organic solvent in the buffer solution is 0.01%-10%, such as 0.01%, 0.02%, 0.05%, 1%, 2%, 5% or 10%.

In certain embodiments, the pH of the buffer solution for DNA polymerase is 7.0-9.0, such as 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2 , 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9 or 9.0.

In certain embodiments, the buffer solution for DNA polymerase includes: monovalent salt ions (such as sodium ions, chloride ions), divalent salt ions (such as magnesium ions, sulfate ions, manganese ions), Tris and organic solvents (such as DMSO or glycerol). In certain embodiments, the pH of the buffer solution phase is 8.8.

In certain preferred embodiments, the kits of the invention further comprise one or more washing solutions. Examples of such wash solutions include, but are not limited to, phosphate buffer, citrate buffer, Tris-HCl buffer, acetate buffer, carbonate buffer, and the like. The kit of the present invention may contain any one or more of the above-mentioned washing solutions.

In another aspect, the application provides the use of the composition, reagent or kit of the invention for nucleic acid detection. In certain embodiments, the nucleic acid detection involves detecting a fluorescent signal. In certain embodiments, the fluorescent signal can be generated by an illumination reaction or a non-illumination reaction (eg, a bioautoluminescence reaction). In certain embodiments, the bioautoluminescence refers to a reaction in which luciferase catalyzes its substrate to produce a fluorescent signal. In certain embodiments, the nucleic acid detection involves an illumination reaction other than an illumination reaction (eg, a bioluminescence reaction). In certain embodiments, the illumination reaction includes base extension that results in the generation of an optical signal (eg, a fluorescent signal). . In certain embodiments, the optical characteristic in the illumination reaction of the present invention is preferably fluorescence. In certain embodiments, the nucleic acid detection can be used for nucleic acid sequence determination (sequencing) or other scenarios (eg, quantitative PCR). In certain embodiments, the nucleic acid detection is nucleic acid sequence determination, such as high-throughput sequencing, such as SBS sequencing, ligation sequencing, hybridization sequencing, nanopore sequencing, or cPAL sequencing. In certain embodiments, the nucleic acid detection is quantitative PCR.

In another aspect, the present application provides a method for preparing the reagent of the present invention, which includes dissolving each component of the reagent in ultrapure water to form a transparent and uniform solution, and then filtering the solution to obtain the reagent of the present invention. . Ultrasound can be used to assist dissolution.

In another aspect, the present application provides a method of inhibiting nucleic acid degradation, comprising using a composition or agent of the invention as a nucleic acid protecting agent. In some embodiments, the nucleic acid degradation is light-induced nucleic acid degradation or oxidation-induced nucleic acid degradation. In some embodiments, the method includes subjecting a reaction mixture comprising a nucleic acid to an illuminated reaction or a non-illuminated reaction (eg, a bioluminescence reaction) in the presence of the composition or reagent.

In another aspect, the present application provides a method of detecting a target nucleic acid molecule, which includes using a composition or reagent of the invention as a nucleic acid protecting agent. In some embodiments, the method includes subjecting a reaction mixture comprising a target nucleic acid molecule to an illuminated reaction or a non-illuminated reaction (eg, a bioluminescent reaction) in the presence of the composition or reagent.

In certain embodiments, the method includes: performing signal collection and detecting a fluorescent signal on the reaction mixture; wherein the reaction mixture includes a reactant that can generate a fluorescent signal (such as a fluorescent labeling reactant), a target nucleic acid molecule and a buffer comprising a composition of the invention.

In certain embodiments, the fluorescent signal is generated by a light reaction or a bioluminescence reaction.

In certain embodiments, the method includes: irradiating the reaction mixture with light and detecting a fluorescent signal from the illumination reaction; wherein the reaction mixture includes a fluorescently labeled reactant, a target nucleic acid molecule, and a buffer, and the The buffer contains the composition of the invention.

In certain embodiments, the reaction mixture in which the illumination reaction is performed includes a composition or reagent of the invention.

In certain embodiments, the methods are used for nucleic acid sequence determination. In certain embodiments, the sequencing is high-throughput sequencing. In certain embodiments, the sequencing is SBS sequencing, ligation sequencing, hybridization sequencing, nanopore sequencing, or cPAL sequencing.

In certain embodiments, the reactant that can generate a fluorescent signal includes labeled or unlabeled nucleotides (eg, dNTPs), optionally including other reagents that allow the reactant to generate a fluorescent signal. In certain embodiments, each reagent that can generate a fluorescent signal corresponds to one nucleotide type, and each reagent can generate a signal that is distinguishable from one another to identify incorporation of a specific nucleotide. For example, four reagents each containing adenine, guanine, cytosine, and thymine to be incorporated can produce different fluorescent signals, making them easily distinguishable from each other.

In certain embodiments, the nucleotides (e.g., fluorescently labeled nucleotides) in the reactant that can generate a fluorescent signal also carry a blocking group (e.g., a 3' blocking group) to reversibly prevent further base extension.

In certain embodiments, the nucleotides (eg, fluorescently labeled nucleotides) in the fluorescent signal-generating reactant are selected from nucleoside polyphosphates (or analogs thereof), such as dNTPs.

In certain embodiments, the reaction mixture further includes an enzyme, such as a polymerase, helicase, exonuclease, or ligase; preferably, the reaction mixture includes a polymerase, such as a DNA polymerase.

In certain embodiments, the reaction mixture further includes primers.

In certain embodiments, the method includes: incorporating a nucleotide (eg, a fluorescently labeled nucleotide) in the reactant that can generate a fluorescent signal to a complementary strand of the target nucleic acid molecule; in the composition of the invention or in the presence of a reagent, applying conditions that allow the reactant to generate a fluorescent signal and detecting the fluorescent signal of the reaction mixture (eg, irradiating the reaction mixture), and determining the identity of the incorporated nucleotide. In certain embodiments, determining the identity of the incorporated nucleotide includes detecting (eg, photographing) a fluorescent signal (eg, fluorescent label) associated with the incorporated nucleotide.

In certain embodiments, the method further includes: removing from the incorporated nucleotide a moiety that can generate a fluorescent signal (e.g., a fluorescent label) directly or indirectly linked thereto; and/or washing to remove unincorporated nucleotides. of nucleotides. In certain embodiments, the method further includes removing a blocking group from the incorporated nucleotide to allow further extension.

In certain embodiments, the method includes multiple incorporations and determining the identity of the base present in each incorporated nucleotide to determine the sequence of the target nucleic acid molecule.

In certain embodiments, the target nucleic acid molecules are present in a nucleic acid array. In certain embodiments, each site on the array can include multiple copies of a single target nucleic acid molecule. In certain embodiments, the nucleic acid array is immobilized on a solid support, such as a chip.

In another aspect, the present application provides a method for detecting nucleic acid sequences, including: incorporating one or more labeled modified nucleotides into a nucleic acid strand complementary to the nucleic acid template strand, and detecting the label The type of the one or more incorporated nucleotides is determined, wherein the step of determining the type of incorporated nucleotide is performed in a buffer comprising a composition of the invention.

In certain embodiments, the label is a label that generates a fluorescent signal. In certain embodiments, the fluorescent signal is generated by a light reaction or a bioluminescence reaction. In certain embodiments, the methods are used for nucleic acid sequence determination (sequencing). In certain embodiments, the labeled modified nucleotides are: (1) fluorescently labeled nucleotides (e.g., dNTPs); or (2) tagged nucleotides (e.g., dNTPs), said The tag specifically binds luciferase.

In certain embodiments, the step of determining the type of incorporated nucleotide includes: in the presence of the composition of the invention, providing conditions that allow the label to generate a fluorescent signal and detecting the fluorescent signal of the buffer solution , and determine the identity of the incorporated nucleotide. Optionally, the method further includes: removing from the incorporated nucleotides a portion directly or indirectly connected thereto that can generate a fluorescent signal; and/or washing to remove unincorporated nucleotides. In certain embodiments, the method includes multiple incorporations and determining the identity of the base present in each incorporated nucleotide to determine the sequence of the target nucleic acid molecule.

In certain embodiments, the buffer solution is a Tris buffer solution. In certain embodiments, the pH of the buffer solution is between 6.0 and 9.0. In certain embodiments, the concentration of the glycyrrhizic acid, the salt of glycyrrhizic acid or the hydrate of the salt of glycyrrhizic acid in the buffer solution is 0.1 to 10 mM. In certain embodiments, the concentration of adenosine 5'-monophosphate or its salt or carnosine in the buffer solution is 0.1-200 mM. In certain embodiments, the concentration of the other antioxidants in the buffer solution is 0.1-50 mM. In certain embodiments, the buffer solution is a scanning reagent.

In certain embodiments, the methods are used for nucleic acid sequence determination. In certain embodiments, the sequencing is high-throughput sequencing. In certain embodiments, the sequencing is SBS sequencing, ligation sequencing, hybridization sequencing, nanopore sequencing, or cPAL sequencing.

Light reaction-based sequencing

In certain embodiments, the fluorescent signal is generated by a reaction with light. In such embodiments, the label is preferably a fluorescent label. In certain embodiments, the method includes: irradiating the reaction mixture with light and detecting a fluorescent signal from the illumination reaction; wherein the reaction mixture includes a reactant that can generate a fluorescent signal, a target nucleic acid molecule, and a buffer. , the buffer solution contains the composition of the invention. In certain embodiments, the reaction mixture in which the illumination reaction is performed includes a composition or reagent of the invention.

In certain embodiments, the reactant that generates a fluorescent signal includes fluorescently labeled nucleotides (e.g., dNTPs). In certain embodiments, the fluorescent label can be linked to a nucleotide (eg, its base) via a linker. Linkers can be acid-labile, photolabile, or contain disulfide bonds.

In certain embodiments, the method includes: incorporating a fluorescently labeled nucleotide into a complementary strand of a target nucleic acid molecule; irradiating the reaction mixture in the presence of a composition or reagent of the invention, and determining the incorporated nucleoside Acid identity. In certain embodiments, determining the identity of the incorporated nucleotide includes detecting (eg, photographing) a fluorescent label incorporated into the nucleotide. In certain embodiments, the method further includes removing a fluorescent label attached thereto from the incorporated nucleotides; and/or washing to remove unincorporated nucleotides. In certain embodiments, the method further includes removing a blocking group from the incorporated nucleotide to allow further extension.

In certain embodiments, the reactant that can generate a fluorescent signal includes: unlabeled nucleotides (such as dNTPs) and fluorescently labeled affinity reagents (such as antibodies) that can specifically bind to the unlabeled nucleotides. Such an implementation may be referred to as Cool MPS sequencing, the detailed teachings of which may be referred to PCT International Application WO2018129214A1. In such embodiments, based on the principle of SBS sequencing, the fluorescent group is not directly labeled on the incorporated nucleotide, but is labeled on the affinity reagent (such as antibody, aptamer, Affimer, Knottin, etc.), and the affinity reagent Affinity reagents can specifically bind to bases, sugars, cleavable blocking groups, or combinations of these components incorporated into nucleotides, so the type of nucleotide being incorporated can be identified by affinity reagents.

In certain embodiments, the method includes: incorporating an unlabeled nucleotide into a complementary strand of a target nucleic acid molecule; providing a fluorescently labeled affinity reagent, and passing specificity between the affinity reagent and the nucleotide Binding indirectly attaches a fluorescent label to the incorporated nucleotide; the reaction mixture is illuminated in the presence of a composition or reagent of the invention and the identity of the incorporated nucleotide is determined. In certain embodiments, determining the identity of the incorporated nucleotide includes detecting (eg, photographing) a fluorescent label of an affinity reagent to which the incorporated nucleotide is attached. In certain embodiments, the method further includes removing an affinity reagent attached thereto from the incorporated nucleotides; and/or washing to remove unincorporated nucleotides. In certain embodiments, the method further includes removing a blocking group from the incorporated nucleotide to allow further extension.

Sequencing based on bioluminescent reactions

In certain embodiments, the fluorescent signal is generated by a bioautoluminescent reaction. The detection principle of bioautoluminescence includes not directly labeling the fluorescent signal on the nucleotide to be incorporated, but labeling it with an affinity substance such as biotin or digoxigenin. After the polymerization reaction, the above affinity substance with luciferase is added. Pairing members, thereby binding luciferase to the incorporated nucleotide, and then adding a reaction substrate to generate a light signal to identify the identity of the incorporated nucleotide. This process does not require excitation light irradiation. Detailed teachings on bioautoluminescent reactions can be found in, for example, PCT International Application WO2020227953A1.

In certain embodiments, the reactant that can generate a fluorescent signal includes: a tagged nucleotide (e.g., dNTP), a luciferase capable of specifically binding the tag, and a substrate for the luciferase .

In certain embodiments, the nucleotides are tagged as members of any molecular pair capable of specifically binding to each other. Specific binding between pairing members enables the linkage of nucleotides to luciferase. Exemplary pair members include, but are not limited to: (a) haptens or antigenic compounds in combination with corresponding antibodies or binding portions or fragments thereof, such as digoxin-digoxin antibodies, N3G-N3G antibodies, FITC-FITC antibodies; ; (b) Nucleic acid aptamers and proteins; (c) Non-immune binding pairs (such as biotin-avidin, biotin-streptavidin, biotin-neutral avidin); (d) hormones - Hormone binding proteins; (e) receptors - receptor agonists or antagonists; (f) lectins - carbohydrates; (g) enzymes - enzyme cofactors; (h) enzymes - enzyme inhibitors; and (i) Complementary pairs of oligonucleotides or polynucleotides capable of forming nucleic acid duplexes.

In certain embodiments, the label carried by the nucleotide is a small molecule label selected from biotin, digoxigenin, N3G or FITC, and the luciferase carries a label corresponding to the small molecule label Pair members. For example, in a specific embodiment, the label carried by the nucleotide is biotin, then the luciferase may be a luciferase labeled with streptavidin; the label carried by the nucleotide is digoxigenin, the luciferase may be luciferase labeled with a digoxigenin antibody. The sources of luciferase include but are not limited to firefly, gaussia, Renilla and other organisms. For example, the streptavidin-labeled luciferase can be Adivity's SA-Gluc: Streptavidin-Gaussia princeps luciferase. The luciferase labeled with digoxin antibody can be digoxin antibody-Gluc or digoxin antibody-Nluc.

In certain embodiments, the method includes: incorporating a tagged nucleotide into a complementary strand of a target nucleic acid molecule; providing a luciferase linked to a pairing member capable of specifically binding the tag, and by pairing Specific binding between members indirectly links luciferase to the incorporated nucleotide; in the presence of a composition or reagent of the invention, a substrate for said luciferase is provided to generate a fluorescent signal thereby determining incorporation The identity of the nucleotide. In certain embodiments, the method further includes removing luciferase linked thereto from the incorporated nucleotides; and/or washing to remove unincorporated nucleotides. In certain embodiments, the method further includes removing a blocking group from the incorporated nucleotide to allow further extension.

In certain embodiments, the methods can also be used for quantitative PCR. In certain embodiments, the reactant that generates a fluorescent signal is a fluorescent probe. In certain embodiments, the reaction mixture further includes an enzyme, such as a polymerase, helicase, exonuclease, or ligase; preferably, the reaction mixture includes a polymerase, such as a DNA polymerase. In certain embodiments, the reaction mixture further includes primers.

The following discussion will take sequencing-by-synthesis as an example, but this is not meant to be a limitation. All uses and methods using the reagents of the invention in nucleic acid detection steps involving illumination reactions are included within the scope of the invention.

In another aspect, the present application provides a method for nucleic acid sequencing, which method includes using the composition, reagent or kit of the present invention. In certain embodiments, the sequencing method of the present invention includes synthesizing a growing polynucleotide complementary to the target single-stranded polynucleotide while performing scanning and photographing detection.

In certain embodiments, the method of determining the sequence of a single-stranded polynucleotide of interest includes:

(a) providing a duplex, a nucleotide, a polymerase, and an excision reagent; the duplex includes a growing nucleic acid strand and a nucleic acid molecule to be sequenced;

(b) Carry out a reaction cycle comprising the following steps (i), (ii) and (iii):

Step (i): using a polymerase to incorporate nucleotides into the growing nucleic acid chain to form a nucleic acid intermediate containing a blocking group and a detectable label;

Step (ii): detecting the detectable label on the nucleic acid intermediate in the presence of the composition or reagent of the present invention;

Step (iii): Use an excision reagent to remove the blocking group on the nucleic acid intermediate.

In certain embodiments, the reaction cycle further includes the step (iv) of removing the detectable label on the nucleic acid intermediate using an excision reagent.

In certain embodiments, the method includes the steps of:

The first step is to load the DNA nanoball (DNB) onto the prepared sequencing chip;

In the second step, pump the prepared mixed solution of dNTP molecules into the chip and use DNA polymerase to add dNTP to the complementary strand of the DNA to be tested;

The third step is to take photos and scans. Since dNTPs are modified molecules with fluorescent groups, lasers are used as the excitation wavelength to take photos. Since lasers can cause photodamage to DNA, a scan containing a nucleic acid protective agent is added to the step of taking photos. Reagents are photographed;

In the fourth step, the base-terminal fluorescent group and the 3’ blocker are removed and eluted through the excision reagent, leaving the 3’-OH exposed for the next round of reaction;

The fifth step is to determine the base sequence of the nucleic acid molecule to be tested by analyzing the photo results.

In the present invention, nucleic acids may include nucleotides or nucleotide analogs. Nucleotides usually contain a sugar, a nucleobase, and at least one phosphate group. Nucleotides include deoxyribonucleotides, modified deoxyribonucleotides, ribonucleotides, modified ribonucleotides, peptide nucleotides, modified peptide nucleotides, modified phosphate sugar backbone nucleosides Acids and their mixtures. Examples of nucleotides include, for example, adenosine monophosphate (AMP), adenosine diphosphate (ADP), adenosine triphosphate (ATP), thymidine monophosphate (TMP), thymidine diphosphate (TDP), thymidine diphosphate (TDP), Triphosphate (TTP), cytidine monophosphate (CMP), cytidine diphosphate (CDP), cytidine triphosphate (CTP), guanosine monophosphate (GMP), guanosine diphosphate (GDP), guanosine triphosphate (GTP), uridine monophosphate (UMP), uridine diphosphate (UDP), uridine triphosphate (UTP), deoxyadenosine monophosphate (dAMP), deoxyadenosine diphosphate (dADP), deoxyadenosine triphosphate (dATP), deoxythymidine monophosphate (dTMP), deoxythymidine diphosphate (dTDP), deoxythymidine triphosphate (dTTP), deoxycytidine triphosphate (dCDP), deoxycytidine triphosphate ( dCTP), deoxyguanosine monophosphate (dGMP), deoxyguanosine diphosphate (dGDP), deoxyguanosine triphosphate (dGTP), deoxyuridine monophosphate (dUMP), deoxyuridine diphosphate (dUDP) and deoxyuridine glycoside triphosphate (dUTP). Nucleotide analogs containing modified nucleobases may also be used in the methods described herein. Exemplary modified nucleobases that may be included in polynucleotides, whether with native backbones or similar structures, include, for example, inosine, xanthine, hypoxanthine, isocytosine, isoguanine, 2-amino Purine, 5-methylcytosine, 5-hydroxymethylcytosine, 2-aminoadenine, 6-methyladenine, 6-methylguanine, 2-propylguanine, 2-propyladenine , 2-thiouracil, 2-thiothymine, 2-thiocytosine, 15-halogenated uracil, 15-halogenated cytosine, 5-propynyluracil, 5-propynylcytosine, 6 -Azouracil, 6-azocytosine, 6-azothymine, 5-uracil, 4-thiouracil, 8-haloadenine or guanine, 8-aminoadenine or guanine, 8-Thioadenine or guanine, 8-Thioalkyladenine or guanine, 8-hydroxyadenine or guanine, 5-halogen substituted uracil or cytosine, 7-methylguanine, 7-methyl Adenine, 8-azaguanine, 8-azaadenine, 7-deazaguanine, 7-deazaadenine, 3-deazaguanine, 3-deazaadenine, etc. As is known in the art, certain nucleotide analogs cannot be incorporated into polynucleotides, for example, nucleotide analogs such as adenosine 5'-phosphoryl sulfate.

In the method of the present invention, the nucleic acid molecules to be sequenced are not limited by their length. In certain preferred embodiments, the length of the nucleic acid molecule to be sequenced can be at least 10 bp, at least 20 bp, at least 30 bp, at least 40 bp, at least 50 bp, at least 100 bp, at least 200 bp, at least 300 bp, at least 400 bp, at least 500 bp, at least 1000 bp. , or at least 2000bp. In certain preferred embodiments, the length of the nucleic acid molecules to be sequenced can be 10-20bp, 20-30bp, 30-40bp, 40-50bp, 50-100bp, 100-200bp, 200-300bp, 300-400bp, 400-500bp, 500-1000bp, 1000-2000bp, or more than 2000bp. In certain preferred embodiments, the nucleic acid molecules to be sequenced may have a length of 10-1000 bp to facilitate high-throughput sequencing.

In certain preferred embodiments, the nucleic acid molecules may be pre-treated prior to immobilizing the nucleic acid molecules on the support. Such preprocessing includes, but is not limited to, fragmentation of nucleic acid molecules, end filling, addition of adapters, addition of tags, amplification of nucleic acid molecules, isolation and purification of nucleic acid molecules, and any combination thereof.

The term "nanosphere" as used herein generally refers to a macromolecule or complex having a compact, e.g. (approximately) spherical shape with an inner diameter typically ranging between about 1 nm and about 1000 nm, preferably between about 50 nm and about 500 nm. shape.

The term "nucleic acid nanospheres" as used herein is generally a concatemer containing multiple copies of a target nucleic acid molecule. These nucleic acid copies are typically arranged one after another in a continuous linear chain of nucleotides, but the nucleic acid nanospheres of the invention can also be made from any nucleic acid molecule using the methods described herein. This tandem repeat structure, along with the single-stranded nature of DNA, results in a folding configuration of the nanospheres. Generally, multiple copies of target nucleic acid molecules in nucleic acid nanospheres each contain a linker sequence with a known sequence to facilitate their amplification or sequencing. The linker sequences for each target nucleic acid molecule are usually the same, but can also be different. Nucleic acid nanoballs usually include DNA nanoballs, also referred to as DNB (DNA nanoballs) in this article.

Nucleic acid nanospheres can be produced using, for example, rolling circle replication (RCA). The RCA process has been used to prepare multiple contiguous copies of the M13 genome (Blanco et al. (1989) J Biol Chem 264:8935-8940). In this method, nucleic acids are replicated in linear concatemers. Those skilled in the art can find guidance on the selection of conditions and reagents for the RCA reaction in a number of references, including U.S. Patent Nos. 5,426,180, 5,854,033, 6,143,495, and 5,871,921, for all purposes, particularly for those utilizing All teachings related to the preparation of nucleic acid nanospheres by RCA or other methods are incorporated herein by reference in their entirety.

Nucleic acid nanospheres can be loaded on the surface of a solid support as described herein. Nanospheres may be attached to the surface of the solid support by any suitable method, non-limiting examples of such methods include nucleic acid hybridization, biotin-streptavidin conjugation, sulfhydryl conjugation, photoactivated conjugation, covalent conjugation, antibody- Antigens, physical confinement via hydrogels or other porous polymers, etc., or combinations thereof. In some cases, nanospheres can be digested with nucleases (eg, DNA nucleases) to produce smaller nanospheres or fragments from the nanospheres.

In certain embodiments, the surface of the solid support may bear reactive functional groups that react with complementary functional groups on the polynucleotide molecules to form covalent bonds, e.g., using the same techniques used to attach cDNA to microarrays. (2004), Genes, Chromosomes & Cancer, 4 0:72-77 and Beaucage (2001), Current Medicinal Chemistry, 8:1213_1244, both of which are incorporated herein by reference. DNB can also be effectively attached to hydrophobic surfaces, such as clean glass surfaces with low concentrations of various reactive functional groups (such as -OH groups). Attachment via covalent bonds formed between the polynucleotide molecule and reactive functional groups on the surface is also referred to herein as "chemical attachment."

In other embodiments, polynucleotide molecules can be adsorbed to surfaces. In this embodiment, the polynucleotide is immobilized through non-specific interactions with the surface, or through non-covalent interactions such as hydrogen bonding, van der Waals forces, and the like.

In other embodiments, the nucleic acid library can be a double-stranded nucleic acid fragment, which is immobilized on the surface of the solid support through a ligation reaction with an oligonucleotide immobilized on the surface of the solid support, and then a bridge amplification reaction is performed to prepare a sequencing library.

beneficial effects

The present invention can achieve the following beneficial effects:

The antioxidant composition of the present invention can be used as a nucleic acid protective agent in nucleic acid detection, especially for paired-end long-read sequencing. It can simultaneously increase the second-strand signal recovery value and Q30 value, and reduce the error rate.

The embodiments of the present invention will be described in detail below with reference to the accompanying drawings and examples. However, those skilled in the art will understand that the following drawings and examples are only used to illustrate the present invention and do not limit the scope of the present invention. Various objects and advantageous aspects of the present invention will become apparent to those skilled in the art from the accompanying drawings and the following detailed description of preferred embodiments.

Description of the drawings

Figure 1 is the Q30 (%) curve of Example 1, in which glycyrrhizic acid and 5'-adenosine monophosphate monosodium were added to the notoginsenoside R1 scanning reagent and sequenced.

Figure 2 is the Q30 (%) curve of Example 2, in which glycyrrhizic acid and carnosine were added to the notoginsenoside R1 scanning reagent and sequenced.

Detailed ways

The embodiments of the present invention will be described in detail below with reference to examples, but those skilled in the art will understand that the following examples are only used to illustrate the present invention and should not be regarded as limiting the scope of the present invention. If the specific conditions are not specified in the examples, the conditions should be carried out according to the conventional conditions or the conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not indicated, they are all conventional products that can be purchased commercially.

The sequencing slide is loaded with single-stranded DNA nanospheres (DNBs), which contain E. coli genomic DNA. In the following Examples 1 and 2, MGI's sequencing slides were used for imaging tests (MGISEQ-2000RS sequencing slides), in which the pitch size was 900nm and the binding area was about 200nm; the kit was from DNBSEQ-G400HM ^TM Qualcomm Quantitative sequencing kit (FCL PE100); library comes from standard library reagent V3.0 (26ng/tube, E320, Barcode 97-104).

Example 1 Detection and evaluation of the effects of notoginseng saponin R1 + glycyrrhizic acid + 5'-adenosine monophosphate monosodium on the decrease value, error rate and signal recovery value of second-chain Q30.

1. Experimental materials (main reagents and consumables)

Table 1 Reagents and consumables information

2.Instruments

Table 2 Instrument information

instrument	model	device number
sequencer	MGISEQ-2000RS	R10040100210039
Gene cycler	6337	180005432N00099
Qubit instrument	Qubit4.0	180005432N00006
Analytical Balances	MS204TS	180005436N00082

3. Experimental samples

Table 3 sample information

4.Experimental design

Add different concentrations of 5'-adenosine monophosphate monosodium and notoginsenoside R1 scanning reagent (including 1M Tris buffer, 1.67mM notoginsenoside R1, 8mM Trolox, 10mM ethylenediaminetetraacetic acid, 20mM DTT) respectively. Glycyrrhizic acid, and prepare a control scanning reagent without adding adenosine 5'-monophosphate monosodium and glycyrrhizic acid. Perform PE150 sequencing on the same MGISEQ-2000RS sequencer, and compare the sequencing quality of each scanning reagent after getting off the machine. Compare the data of several scanning reagents, especially indicators such as second-strand Q30 (%), second-strand Q30 drop value (%), error rate (%), and signal recovery value.

5. Evaluation indicators and standards

Table 4 Evaluation Criteria

Evaluation index	Eligibility criteria
Second chain Q30(%)	The higher the better or the values are equivalent
Second chain Q30 drop value (%)	The lower the better or the values are equivalent

Error rate(%)	The lower the better or the values are equivalent
Signal recovery value	The higher the better or the values are equivalent

Q30 refers to the proportion of bases in the basecall results with an estimated error rate lower than 0.001 (accuracy higher than 99.9%); the error rate refers to the average mismatch of each site in MappedReads; the signal recovery value is only for PE sequencing In part, it reflects the rebound of the second chain signal. When evaluating the gain effect of scanning reagents, these three indicators are generally selected as evaluation indicators, and the three indicators of the experimental group and the control group are compared with each other. Taking Q30 as an example, as long as the experimental group is included in the disembarkation report If the Q30 of the experimental group is higher than that of the control group, it means that the added compound combination has a gain effect on sequencing. The higher the Q30 of the experimental group is than the test group, the stronger the gain effect.

6. Experimental steps

a. Preparation of DNB

1) Take out the DNB preparation buffer, DNB polymerase mixture I, TE buffer and DNB stop buffer, and place them on an ice box for about 0.5 hours. After melting, use a vortex shaker to shake and mix for 5 seconds, and then centrifuge briefly. Set aside on ice.

2) Take a 0.2mL eight-tube tube or PCR tube and prepare the reaction mixture on ice according to the following system:

Components	Adding volume (μL)
Library ssDNA	3
TE buffer	17
DNB preparation buffer	20
total capacity	40

3) Mix the reaction mixture with a vortex shaker, centrifuge it in a mini centrifuge for 5 seconds, and place it in a PCR machine for primer hybridization. The reaction conditions are shown in the table below:

temperature	time
Hot lid (105℃)	On
95℃		1min
65℃	1min
40℃	1min
4℃	Hold

4) Take out the DNB polymerase mixture II (LC) and place it on an ice box, centrifuge briefly for 5 seconds, and place it on an ice box for later use.

5) When the PCR machine reaches 4°C, take out the PCR tube, centrifuge it in a mini centrifuge for 5 seconds, and add the following components on ice:

Components	Adding volume of 100μL system (μL)
DNB polymerase mix I	40
DNB polymerase mix II (LC)	4

6) The reaction mixture was vortexed and mixed evenly, centrifuged in a mini centrifuge for 5 seconds, and immediately placed in a PCR machine. The reaction conditions were as follows:

temperature	time
Hot lid (35℃)	On
30℃	25min
4℃	Hold

7) When the PCR machine reaches 4°C, take out the PCR tube, add 20 μL of stop buffer, and mix slowly 5-8 times with an expanded pipette.

8) After DNB preparation is completed, take 2 μL DNB and use

ssDNA Assay Kit and

The Fluorometer instrument performs concentration detection, and the concentration is 15.3ng/μL.

b. Prepare slides and sequencing reagent tanks

1) Take out the slide from the -25℃~-15℃ refrigerator and place it at room temperature for at least 60min (not more than 24h).

2) Take out the DNB loading buffer II and place it on an ice box for about 0.5 hours until it melts. Use a vortex shaker to mix evenly for 5 seconds. Centrifuge briefly and place it on an ice box for later use.

3) Take out a new 1.5mL centrifuge tube and prepare the DNB loading system as shown in the following table:

Components	Amount of FCL added (μL)
DNB loading buffer II	33
DNB	99
total capacity	132

4) The DNB loading system is slowly mixed 5-8 times with a wide-mouth pipette.

5) Take out the MGIDL-200H portable sampler and install the sealing gasket into the gasket groove. The chips are installed into the sampler respectively.

6) Load 27 μL DNB into L01-L04 with an enlarged pipe tip, and let stand at room temperature for more than half an hour.

7) Prepare the PE150 sequencing reagent tank and take out the required HotMPS dNTP Mixture and HotMPS dNTP Mixture II 1 hour in advance, melt at room temperature and set aside.

8) Take out Adv Seqenzyme II before use and place it on ice for later use.

9) Add samples according to the following table:

Reagents	Hole No. 1	Hole 2	Hole 15
HotMP SdNTP Mixture	1500μL	​	/
HotMPS dNTP Mix II	​	1500μL	/
Adv Seqenzyme II	1000μL	1000μL	/
MDA Reagent+MDA enzyme	/	/	3875μL+125μL

10) Mix the reagents and set aside.

c. On-machine sequencing

Put the reagent tank into the instrument, edit the script, and start PE150 sequencing.

Table 5 Experimental design

Experimental group number	condition
1	Notoginsenoside R1 scanning reagent
2	Notoginsenoside R1 scanning reagent + 0.5mM glycyrrhizic acid + 7.5mM 5'-adenosine monophosphate monosodium
3	Panax notoginseng saponin R1 scanning reagent + 1.5mM glycyrrhizic acid + 7.5mM 5'-adenosine monophosphate monosodium
4	Notoginsenoside R1 scanning reagent + 2.5mM glycyrrhizic acid + 7.5mM 5'-adenosine monophosphate monosodium

PE150 sequencing was performed using DNB templates and fluorescently labeled nucleotides with reversible terminators. The reversible terminator nucleotide has a cleavable structure located at the base-linked fluorescent group and the ribose 3'-OH position. In each cycle, use DNA polymerase to add the above nucleotides, and pump in the scanning reagent before taking pictures; the imaging instrument used during the picture taking process is MGISEQ-2000RS, and the excitation wavelength of the fluorescent dye is approximately 532nm and 660nm, and the exposure The time is 40 milliseconds for 1-100 cycles, 40 milliseconds for 100-200 cycles, and 50 milliseconds for 200-300 cycles. After each cycle, fluorophores and protecting groups were removed with 10mM THPP.

Figure 1 shows the Q30 (%) curves of each group of sequencing. As shown in the figure, compared with the Panax notoginseng saponin R1 scanning reagent, the decrease value of the second-chain Q30 (%) added with different concentrations of glycyrrhizic acid and 5'-adenosine monophosphate monosodium is in the range of 7-11.3, all lower than 13.

Table 6 shows the detection results of each evaluation index. The sequencing results of PE150 sequencing using the Panax notoginseng saponin R1 scanning reagent and adding glycyrrhizic acid and 5'-adenosine monophosphate monosodium. Compared with the notoginsenoside R1 scanning reagent, the second-chain Q30 (%) of the second-chain Q30 (%) added with different concentrations of glycyrrhizic acid and 5'-adenosine monophosphate monosodium is about 94, which is higher than 92; the second-chain Q30 decrease value (% ) in the range of 7-11.3, all lower than 13; the second chain error rate (%) is 0.18-0.35, both lower than 0.58; the signal recovery value is 2.14-2.32, both higher than 2.

Table 6 Adding glycyrrhizic acid and 5'-adenosine monophosphate monosodium on the basis of Panax notoginseng saponin R1 scanning reagent has effects on second-chain Q30 (%), second-chain Q30 decrease value (%), error rate (%) and signal recovery value Impact.

These results indicate that including different concentrations of glycyrrhizic acid and adenosine 5'-monophosphate monosodium in the Panax notoginseng saponin R1 scanning reagent can reduce the degradation of sequencing quality due to the impact of photodamage on DNA templates, extended strands, or both. .

Example 2 detects and evaluates the effects of adding glycyrrhizic acid and carnosine on the basis of Panax notoginseng saponin R1 scanning reagent on second-chain Q30 (%), second-chain Q30 decrease value (%), error rate (%) and signal recovery value.

The experimental protocol is the same as Example 1, except that the test group scanning reagent is replaced.

Experimental group number	condition
1	Notoginsenoside R1 scanning reagent
2	Notoginsenoside R1 scanning reagent + 0.5mM glycyrrhizic acid + 7.5mM carnosine
3	Notoginsenoside R1 scanning reagent + 1mM glycyrrhizic acid + 7.5mM carnosine

Figure 2 shows the Q30 (%) curves of each group of sequencing. As shown in the figure, compared with the notoginsenoside R1 scanning reagent, adding the notoginsenoside R1 scanning reagent with different concentrations of glycyrrhizic acid and carnosine second chain can reduce the Q30 value (%) by about 9-12, which is lower than 13.

Table 7 shows the sequencing results of PE150 sequencing using the Panax notoginsenoside R1 scanning reagent and adding glycyrrhizic acid and carnosine. Compared with the notoginsenoside R1 scanning reagent, the second-chain Q30 (%) of the second-chain Q30 (%) added with different concentrations of glycyrrhizic acid and carnosine is about 93, which is higher than 92; the second-chain Q30 decrease value (%) is about 9-12, both Lower than 13; its second chain error rate (%) is about 0.35, both lower than 0.58; signal rebound value is 2.17-2.44, both higher than 2.

Table 7 Effects of adding glycyrrhizic acid and carnosine on the second chain Q30 (%), second chain Q30 decrease value (%), error rate (%) and signal recovery value based on the Panax notoginseng saponin R1 scanning reagent.

These results indicate that including different concentrations of glycyrrhizic acid and carnosine in the Panax notoginseng saponin R1 scanning reagent can reduce the degradation of sequencing quality due to the impact of photodamage on DNA templates, extended strands, or both.

The above embodiments are only used to illustrate the technical solutions of the present invention but not to limit them. Any modification or equivalent replacement of the technical solutions of the present invention without departing from the purpose and scope of the technical solutions of the present invention shall be covered by the protection scope of the present invention. within.

Claims (10)

1. A composition containing:

   (1) Saponin compounds, selected from one or more of notoginsenoside R1, ginsenoside Rg1, ginsenoside Rd, ginsenoside Rb2, ginsenoside Rb3, ginsenoside Rc, ginsenoside Rf, and ginsenoside Re;

   (2) Glycyrrhizic acid, a salt of glycyrrhizic acid or a hydrate of a salt of glycyrrhizic acid; and

   (3) 5'-adenosine monophosphate or its salt, or carnosine;

   Optionally, the composition may also contain other antioxidants.
2. The composition of claim 1, wherein the saponin compound is notoginseng saponin R1, and/or the salt of 5'-adenosine monophosphate is a sodium salt of 5'-adenosine monophosphate (for example 5'-adenosine monophosphate monosodium).
3. The composition according to claim 1 or 2, wherein the other antioxidants are selected from the group consisting of dithiothreitol (DTT) and Trolox (water-soluble vitamin E).
4. A reagent comprising the composition of any one of claims 1-3 and a buffer solution (such as Tris buffer solution);

   Preferably, the pH of the reagent is 6.0 to 9.0;

   Preferably, in the reagent, the concentration of the glycyrrhizic acid, the salt of glycyrrhizic acid or the hydrate of the salt of glycyrrhizic acid is 0.1 to 10mM;

   Preferably, in the reagent, the concentration of adenosine 5'-monophosphate or its salt or carnosine is 0.1-200mM;

   Preferably, in the reagent, the concentration of the other antioxidants is 0.1-50mM;

   Preferably, the reagent is a scanning reagent.
5. The use of the composition of any one of claims 1 to 3 or the reagent of claim 4 as a nucleic acid protecting agent in nucleic acid detection;

   Preferably, the nucleic acid detection involves an illumination reaction or a non-illumination reaction (such as a bioautoluminescence reaction);

   Preferably, the nucleic acid detection involves a sequencing reaction;

   Preferably, the nucleic acid detection is nucleic acid sequence determination, such as high-throughput sequencing, such as SBS sequencing, ligation sequencing, hybridization sequencing, nanopore sequencing, or cPAL sequencing;

   Preferably, the nucleic acid detection is quantitative PCR.
6. A kit comprising the composition of any one of claims 1-3 or the reagent of claim 4;

   Preferably, the kit contains one or more other reagents required for nucleic acid detection, such as primers, polymerases, buffer solutions, wash solutions, or any combination thereof.
7. A method for detecting nucleic acid sequences, including: incorporating one or more labeled modified nucleotides into a nucleic acid strand complementary to the nucleic acid template strand, and determining the one or more incorporated nucleotides by detecting the label. The type of incorporated nucleotide, wherein the step of determining the type of incorporated nucleotide is performed in a buffer solution containing the composition of any one of claims 1-3.
8. The method of claim 7, wherein the label is a label that can generate a fluorescent signal;

   Preferably, the fluorescent signal is generated by a light reaction or a bioluminescence reaction;

   Preferably, the labeled modified nucleotides are: (1) fluorescently labeled nucleotides (such as dNTPs); or (2) tagged nucleotides (such as dNTPs), and the labels can specifically Conjugate luciferase.
9. The method of claim 8, wherein the step of determining the type of incorporated nucleotide comprises: in the presence of the composition or reagent, providing conditions that allow the label to generate a fluorescent signal and detecting the buffer Fluorescent signal from the solution and determine the identity of the incorporated nucleotide;

   Optionally, the method further includes: removing from the incorporated nucleotides a portion directly or indirectly connected thereto that can generate a fluorescent signal; and/or washing to remove unincorporated nucleotides;

   Preferably, the method involves multiple incorporations and determining the identity of the base present in each incorporated nucleotide to determine the sequence of the target nucleic acid molecule.
10. The method according to any one of claims 7-9, wherein the buffer solution is a Tris buffer solution;

    Preferably, the pH of the buffer solution is 6.0 to 9.0;

    Preferably, in the buffer solution, the concentration of the glycyrrhizic acid, the salt of glycyrrhizic acid or the hydrate of the salt of glycyrrhizic acid is 0.1 to 10mM;

    Preferably, in the buffer solution, the concentration of adenosine 5'-monophosphate or its salt or carnosine is 0.1-200mM;

    Preferably, the concentration of the other antioxidants in the buffer solution is 0.1-50mM;

    Preferably, the buffer solution is a scanning reagent.

Images