WO2024050817A1 - 甘草酸或其衍生物在核酸检测中的用途 - Google Patents

甘草酸或其衍生物在核酸检测中的用途 Download PDF

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WO2024050817A1
WO2024050817A1 PCT/CN2022/118117 CN2022118117W WO2024050817A1 WO 2024050817 A1 WO2024050817 A1 WO 2024050817A1 CN 2022118117 W CN2022118117 W CN 2022118117W WO 2024050817 A1 WO2024050817 A1 WO 2024050817A1
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salt
glycyrrhizic acid
nucleic acid
hydrate
glycyrrhizic
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PCT/CN2022/118117
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English (en)
French (fr)
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贾曼
张桢
王静静
徐崇钧
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深圳华大智造科技股份有限公司
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Priority to PCT/CN2022/118117 priority Critical patent/WO2024050817A1/zh
Publication of WO2024050817A1 publication Critical patent/WO2024050817A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay

Definitions

  • the present invention relates to the field of nucleic acid detection and the use of glycyrrhizic acid or its derivatives in nucleic acid detection.
  • the present invention relates to reagents containing glycyrrhizic acid or its derivatives, the use of glycyrrhizic acid or its derivatives as nucleic acid protective agents in nucleic acid detection, and a nucleic acid detection method.
  • Sequencing by synthesis is the theoretical basis of polynucleic acid sequencing, and it is crucial for base identification during the sequencing process.
  • base identification is determined by identifying the type of fluorescent dye labeled on different bases. The process includes: exposing the nucleotide to ultraviolet-visible light, and the chromophore in the fluorescent dye emits Fluorescence, the fluorescent signal is then detected and photographed, and the type of fluorescent dye can be determined.
  • Lasers with wavelengths in the UV-visible range can be used as light sources.
  • the continuous strong irradiation of the excitation light source and the reactive oxygen groups in the solution will cause damage to nucleic acids, resulting in the loss of fluorescence detection signal intensity and high error rates, seriously affecting the quality of nucleic acid 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.
  • concentration of the nucleic acid protecting agent component in the scanning reagent is relatively high.
  • the use of high-concentration nucleic acid protective agents causes salt precipitation of scanning reagents when stored at low temperatures or during long-term sequencing processes.
  • some nucleic acid protective components may become discolored after being oxidized, thereby affecting the scanning quality and thus the sequencing quality of polynucleotides.
  • the use of high concentrations of nucleic acid protective agents will increase product costs.
  • the present invention uses glycyrrhizic acid or its derivatives as a nucleic acid protecting agent, and can also protect the template nucleotide at a very low concentration.
  • Glycyrrhizic acid or its derivatives have no absorption in the ultraviolet-visible light region and do not interfere with base recognition signals. Even if they are oxidized during long-term storage, there will be no discoloration.
  • 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.
  • the present application provides a reagent comprising glycyrrhizic acid, a salt of glycyrrhizic acid, or a hydrate of a salt of glycyrrhizic acid, and a Tris buffer solution.
  • 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'-
  • 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.
  • 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.
  • 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.
  • 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.
  • 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:
  • 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.
  • Tris buffer solution can be used as a solvent to dissolve nucleic acids.
  • glycyrrhizic acid or its derivatives can also protect the template nucleotide at a very low concentration.
  • the concentration of glycyrrhizic acid, a salt of glycyrrhizic acid or a hydrate of a salt of glycyrrhizic acid is 0.5-3mM, such as 0.5-0.6mM, 0.6-0.7mM, 0.7-0.8mM, 0.8-0.9 mM, 0.9-1.0mM, 1-2mM or 2-3mM.
  • the reagent is a scanning reagent.
  • the reagents further comprise sodium chloride and/or a stabilizing agent for DNA (eg, Tween-20).
  • 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.
  • the Tris buffer solution includes water, tris-hydroxymethylaminomethane (Tris Base), sodium chloride, Tween-20, and tris-hydroxymethylaminomethane hydrochloride (Tris-HCl).
  • the Tris buffer solution is prepared by the following method: Dissolving Tris Base (powder), sodium chloride (powder), Tween-20, Tris-HCl (powder) in a certain ratio in ultrasonic acid pure water.
  • the application provides the use of glycyrrhizic acid, a salt of glycyrrhizic acid or a hydrate of a salt of glycyrrhizic acid as a nucleic acid protective agent in nucleic acid detection, the salt of glycyrrhizic acid or the hydrate of a salt of glycyrrhizic acid being as above defined by the text.
  • the nucleic acid protecting agent is used to protect nucleic acids and reduce or avoid photodamage or oxidative damage.
  • the nucleic acid detection involves detecting a fluorescent signal.
  • the fluorescent signal can be generated by an illumination reaction or a non-illumination reaction (eg, a bioautoluminescence reaction).
  • the bioautoluminescence reaction refers to a reaction in which luciferase catalyzes its substrate to produce a fluorescent signal.
  • the nucleic acid detection involves an illuminated reaction or a non-illuminated reaction (eg, a bioluminescence reaction).
  • 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.).
  • non-illuminated reaction refers to a reaction that can be detected without the aid of optical energy.
  • 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.
  • 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.
  • an optical signal eg, a fluorescent signal
  • the light signal in the illumination reaction of the present invention is preferably fluorescence.
  • the nucleic acid detection can be used for nucleic acid sequence determination (sequencing) or other detection (eg, quantitative PCR).
  • the nucleic acid detection is nucleic acid sequence determination (sequencing), such as high-throughput sequencing, such as sequencing by synthesis (SBS sequencing), ligation sequencing, hybridization sequencing, nanopore sequencing, or composite probe- Anchor molecule ligation (Combinatorial probe-anchor ligation, cPAL) sequencing.
  • SBS sequencing sequencing by synthesis
  • ligation sequencing hybridization sequencing
  • nanopore sequencing nanopore sequencing
  • composite probe- Anchor molecule ligation Combinatorial probe-anchor ligation, cPAL
  • the nucleic acid detection is quantitative PCR.
  • the application provides a kit comprising a reagent of the invention.
  • kits of the invention may also contain one or more other reagents required for nucleic acid detection, such as primers, polymerases, buffer solutions, wash solutions, or any combination thereof.
  • kits of the invention are used for nucleic acid sequence determination.
  • 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 reagent for immobilizing the nucleic acid molecule to be sequenced to a support for example, immobilization by covalent or non-covalent linkage
  • a primer for nucleotide polymerization a polymerase for nucleotide polymerization
  • one or more buffer solutions for example, one or more wash solutions; or any combination thereof.
  • 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.
  • kits of the invention further comprise reagents for pretreating nucleic acid molecules.
  • 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
  • kits of the invention further comprise a support for immobilizing the nucleic acid molecules to be sequenced.
  • 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 phase support.”
  • a “support” is sometimes also referred to as a “solid support” or “solid phase support.”
  • the "support” mentioned herein is not limited to solid , which can also be semi-solid (such as a gel).
  • 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.
  • 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 (eg, in applications requiring nucleic acid amplification and/or sequencing).
  • 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.
  • 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.
  • loading may occur by means other than base pairing hybridization, such as covalent attachment as described above.
  • 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.
  • the support may be made of various suitable materials.
  • 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.
  • 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.
  • inert substrate or matrix e.g., glass slide, polymer beads, etc.
  • intermediate materials containing reactive groups that allow covalent attachment of biomolecules such as polynucleotides.
  • 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.
  • a biomolecule e.g., a polynucleotide
  • an intermediate material e.g., a hydrogel
  • 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.
  • the support or solid support is not limited to its size, shape and configuration.
  • 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.
  • 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.
  • 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.
  • one or more nucleic acid molecules can be immobilized on the surface of each bead or well.
  • 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.
  • 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).
  • 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-d
  • kits of the invention further comprise primers for initiating nucleotide polymerization reactions.
  • the primer is not subject to additional restrictions as long as it can specifically anneal to a region of the target nucleic acid molecule.
  • 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.
  • the primers may comprise naturally occurring or non-naturally occurring nucleotides.
  • the primers comprise or consist of naturally occurring nucleotides.
  • the primers comprise modified nucleotides, such as locked nucleic acids (LNA).
  • the primers comprise universal primer sequences.
  • kits of the invention further comprise a polymerase for performing nucleotide polymerization reactions.
  • various suitable polymerases may be used to carry out the polymerization reaction.
  • the polymerase is capable of synthesizing new DNA strands using DNA as a template (eg, DNA polymerase).
  • the polymerase is capable of synthesizing new DNA strands using RNA as a template (eg, reverse transcriptase).
  • the polymerase is capable of synthesizing new RNA strands using DNA or RNA as a template (eg, RNA polymerase).
  • the polymerase is selected from the group consisting of DNA polymerase, RNA polymerase, and reverse transcriptase.
  • kits of the invention further comprise one or more excision reagents.
  • the excision reagent is selected from the group consisting of endonuclease IV and alkaline phosphatase.
  • kits of the invention further comprise one or more buffer solutions.
  • buffer solutions 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
  • buffer solutions for ligation reactions may contain any one or more of the above buffer solutions.
  • buffer and “buffer solution” have the same meaning and can be used interchangeably.
  • 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).
  • the concentration of the monovalent salt ion or divalent salt ion in the buffer solution is 10 ⁇ M-200 mM, such as 10 ⁇ M, 50 ⁇ M, 100 ⁇ M, 200 ⁇ M, 500 ⁇ M, 1 mM, 3mM, 10mM, 20mM, 50mM, 100mM, 150mM or 200mM.
  • the buffer solution for DNA polymerase comprises tris(hydroxymethylaminomethane) (Tris).
  • Tris tris(hydroxymethylaminomethane)
  • the concentration of Tris in the buffer solution is 10mM-200mM, such as 10mM, 20mM, 50mM, 100mM, 150mM or 200mM.
  • the buffer solution for DNA polymerase contains an organic solvent, such as DMSO or glycerol (glycerol).
  • 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%.
  • 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.
  • 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).
  • monovalent salt ions such as sodium ions, chloride ions
  • divalent salt ions such as magnesium ions, sulfate ions, manganese ions
  • Tris such as DMSO or glycerol
  • the pH of the buffer solution phase is 8.8.
  • kits of the invention further comprise one or more washing solutions.
  • 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.
  • the application provides the use of the reagent or kit of the invention for nucleic acid detection.
  • the nucleic acid detection involves detecting a fluorescent signal.
  • the fluorescent signal can be generated by an illumination reaction or a non-illumination reaction (eg, a bioautoluminescence reaction).
  • the bioautoluminescence refers to a reaction in which luciferase catalyzes its substrate to produce a fluorescent signal.
  • the nucleic acid detection involves an illuminated reaction or a non-illuminated reaction (eg, a bioluminescence reaction).
  • the illumination reaction includes a light signal triggered by base extension that can cause the generation of an optical signal (eg, a fluorescent signal).
  • the optical characteristic in the illumination reaction of the present invention is preferably fluorescence.
  • the nucleic acid detection can be used for nucleic acid sequence determination (sequencing) or other detection (eg, quantitative PCR).
  • 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.
  • the nucleic acid detection is quantitative PCR.
  • 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.
  • the present application provides a method for inhibiting nucleic acid degradation, which includes using glycyrrhizic acid, a salt of glycyrrhizic acid, or a hydrate of a salt of glycyrrhizic acid as a nucleic acid protecting agent.
  • the nucleic acid degradation is light-induced nucleic acid degradation or oxidation-induced nucleic acid degradation.
  • the method includes subjecting a reaction mixture comprising a nucleic acid to an illumination reaction or a non-illumination reaction (e.g., bioluminescence) in the presence of glycyrrhizic acid, a salt of glycyrrhizic acid, or a hydrate of a salt of glycyrrhizic acid. reaction).
  • an illumination reaction or a non-illumination reaction e.g., bioluminescence
  • the present application provides a method for detecting a target nucleic acid molecule, which includes using glycyrrhizic acid, a salt of glycyrrhizic acid, or a hydrate of a salt of glycyrrhizic acid as a nucleic acid protecting agent.
  • the method includes subjecting a reaction mixture comprising a target nucleic acid molecule to an illumination reaction or a non-illumination reaction (e.g., biological self-luminous reaction).
  • 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 glycyrrhizic acid, a salt of glycyrrhizic acid, or a hydrate of a salt of glycyrrhizic acid.
  • a reactant that can generate a fluorescent signal such as a fluorescent labeling reactant
  • a target nucleic acid molecule and a buffer comprising glycyrrhizic acid, a salt of glycyrrhizic acid, or a hydrate of a salt of glycyrrhizic acid.
  • the fluorescent signal is generated by a light reaction or a bioluminescence reaction.
  • 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 glycyrrhizic acid, a salt of glycyrrhizic acid, or a hydrate of a salt of glycyrrhizic acid.
  • the reaction mixture in which the light reaction is performed includes glycyrrhizic acid, a salt of glycyrrhizic acid, or a hydrate of a salt of glycyrrhizic acid.
  • the methods are used for nucleic acid sequence determination.
  • the sequencing is high-throughput sequencing.
  • the sequencing is SBS sequencing, ligation sequencing, hybridization sequencing, nanopore sequencing, or cPAL sequencing.
  • 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.
  • 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.
  • 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.
  • a blocking group e.g., a 3' blocking group
  • the nucleotides (eg, fluorescently labeled nucleotides) in the fluorescent signal-generating reactant are selected from nucleoside polyphosphates (or analogs thereof), such as dNTPs.
  • 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.
  • an enzyme such as a polymerase, helicase, exonuclease, or ligase; preferably, the reaction mixture includes a polymerase, such as a DNA polymerase.
  • the reaction mixture further includes primers.
  • 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 a target nucleic acid molecule; In the presence of a buffer solution of a salt of glycyrrhizic acid or a hydrate of a salt of glycyrrhizic acid, conditions allowing the reactant to generate a fluorescent signal are provided and the fluorescent signal of the reaction mixture is detected (for example, the reaction mixture is irradiated), and the doped mixture is determined.
  • the identity of the incoming nucleotide includes detecting (eg, photographing) a fluorescent signal (eg, fluorescent label) associated with the incorporated nucleotide.
  • 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.
  • the method further includes removing a blocking group from the incorporated nucleotide to allow further extension.
  • 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.
  • the target nucleic acid molecules are present in a nucleic acid array.
  • each site on the array can include multiple copies of a single target nucleic acid molecule.
  • the nucleic acid array is immobilized on a solid support, such as a chip.
  • 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 Determining the type of the one or more incorporated nucleotides, wherein the step of determining the type of the incorporated nucleotide is in a buffer comprising glycyrrhizic acid, a salt of glycyrrhizic acid or a hydrate of a salt of glycyrrhizic acid in progress.
  • the label is a label that generates a fluorescent signal.
  • the fluorescent signal is generated by a light reaction or a bioluminescence reaction.
  • the methods are used for nucleic acid sequence determination (sequencing).
  • 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.
  • the step of determining the type of incorporated nucleotide includes administering a label that allows the label to generate a fluorescent signal in the presence of glycyrrhizic acid, a salt of glycyrrhizic acid, or a hydrate of a salt of glycyrrhizic acid.
  • a label that allows the label to generate a fluorescent signal in the presence of glycyrrhizic acid, a salt of glycyrrhizic acid, or a hydrate of a salt of glycyrrhizic acid.
  • 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.
  • 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.
  • the salt of glycyrrhizic acid is selected from alkali metal salts or ammonium salts.
  • the hydrate of a salt of glycyrrhizic acid is selected from a hydrate of an alkali metal or ammonium salt of glycyrrhizic acid.
  • the salt of glycyrrhizic acid or the hydrate of the salt of glycyrrhizic acid is selected from: monoammonium glycyrrhizinate, trisodium glycyrrhizinate hydrate, monopotassium glycyrrhizinate, diammonium glycyrrhizinate, glycyrrhizinate Dipotassium acid hydrate.
  • the concentration of glycyrrhizic acid, a salt of glycyrrhizic acid or a hydrate of a salt of glycyrrhizic acid in the buffer solution is 0.5-3mM.
  • the buffer solution also contains sodium chloride and/or a stabilizing agent for DNA (eg, Tween-20).
  • the buffer solution is a scanning reagent.
  • the methods are used for nucleic acid sequence determination.
  • the sequencing is high-throughput sequencing.
  • the sequencing is SBS sequencing, ligation sequencing, hybridization sequencing, nanopore sequencing, or cPAL sequencing.
  • the fluorescent signal is generated by a reaction with light.
  • the label is preferably a fluorescent label.
  • 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 contains glycyrrhizic acid, a salt of glycyrrhizic acid or a hydrate of a salt of glycyrrhizic acid.
  • the reaction mixture in which the light reaction is performed includes glycyrrhizic acid, a salt of glycyrrhizic acid, or a hydrate of a salt of glycyrrhizic acid.
  • the reactant that generates a fluorescent signal includes fluorescently labeled nucleotides (eg, dNTPs).
  • 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.
  • the method includes: incorporating a fluorescently labeled nucleotide into a complementary strand of a target nucleic acid molecule; irradiating the reaction in the presence of glycyrrhizic acid, a salt of glycyrrhizic acid, or a hydrate of a salt of glycyrrhizic acid mixture and determine the identity of the incorporated nucleotides.
  • determining the identity of the incorporated nucleotide includes detecting (eg, photographing) a fluorescent label incorporated into the nucleotide.
  • 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.
  • 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.
  • unlabeled nucleotides such as dNTPs
  • fluorescently labeled affinity reagents such as antibodies
  • 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.
  • the affinity reagent such as antibody, aptamer, Affimer, Knottin, etc.
  • 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 Conjugation indirectly attaches a fluorescent label to the incorporated nucleotide; the reaction mixture is irradiated in the presence of glycyrrhizic acid, a salt of glycyrrhizic acid, or a hydrate of a salt of glycyrrhizic acid and the identity of the incorporated nucleotide is determined.
  • 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.
  • 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.
  • bioautoluminescent reactions can be found in, for example, PCT International Application WO2020227953A1.
  • 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 .
  • a tagged nucleotide e.g., dNTP
  • a luciferase capable of specifically binding the tag
  • a substrate for the luciferase a substrate for the luciferase
  • 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
  • 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.
  • 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.
  • 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.
  • 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; providing a substrate for said luciferase in the presence of glycyrrhizic acid, a salt of glycyrrhizic acid or a hydrate of a salt of glycyrrhizic acid to produce a fluorescent signal that determines the identity of the incorporated nucleotide.
  • 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.
  • the methods can also be used for quantitative PCR.
  • the reactant that generates a fluorescent signal is a fluorescent probe.
  • 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 salt of glycyrrhizic acid or the hydrate of the salt of glycyrrhizic acid may be selected from any salt or hydrate defined above.
  • the salt of glycyrrhizic acid is selected from alkali metal salts or ammonium salts.
  • the hydrate of a salt of glycyrrhizic acid is selected from a hydrate of an alkali metal or ammonium salt of glycyrrhizic acid.
  • the salt of glycyrrhizic acid or the hydrate of the salt of glycyrrhizic acid is selected from: monoammonium glycyrrhizinate, trisodium glycyrrhizinate hydrate, monopotassium glycyrrhizinate, diammonium glycyrrhizinate, glycyrrhizinate Dipotassium acid hydrate.
  • the glycyrrhizic acid, a salt of glycyrrhizic acid, or a hydrate of a salt of glycyrrhizic acid is present in the buffer at a concentration of 0.5-3mM.
  • the buffer also contains sodium chloride and/or a stabilizing agent for DNA (eg, Tween-20).
  • the present application provides a method for nucleic acid sequencing, which method includes using the reagent of the present invention.
  • the sequencing method of the present invention includes synthesizing a growing polynucleotide complementary to the target single-stranded polynucleotide, and then performing scanning and photographing detection.
  • the method of determining the sequence of a single-stranded polynucleotide of interest includes:
  • duplex includes a growing nucleic acid strand and a nucleic acid molecule to be sequenced
  • the reaction cycle further includes the step (iv) of removing the detectable label on the nucleic acid intermediate using an excision reagent.
  • the method includes the steps of:
  • the first step is to load the DNA nanoball (DNB) onto the prepared sequencing chip;
  • 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;
  • 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.
  • 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.
  • 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), deoxythym
  • 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-a
  • the nucleic acid molecules to be sequenced are not limited by their length.
  • 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.
  • 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.
  • the nucleic acid molecules to be sequenced may have a length of 10-1000 bp to facilitate high-throughput sequencing.
  • the nucleic acid molecules can be pre-treated before 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.
  • nanosphere 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.
  • nucleic acid nanospheres 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.
  • 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).
  • RCA rolling circle replication
  • 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.
  • nucleases eg, DNA nucleases
  • 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.”
  • polynucleotide molecules can be adsorbed to surfaces.
  • 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.
  • 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.
  • the present invention uses glycyrrhizic acid or its derivatives as a nucleic acid protecting agent, which can protect the template nucleotide even at a very low concentration.
  • the glycyrrhizic acid or its derivatives used in the present invention have no absorption in the ultraviolet and visible light regions. Even after multiple rounds of sequencing reactions, the data quality is still very high, there is no interference with the base recognition signal, and it will not be oxidized during long-term storage. Discoloration occurs.
  • the optimized scanning reagent of the present invention the quality of nucleic acid sequencing, especially polynucleotide sequencing, can be greatly improved.
  • Figure 1 shows the effects of different concentrations of glycyrrhizic acid on sequenced Q30 in Example 1.
  • Figure 2 shows the effects of different concentrations of glycyrrhizic acid on sequencing lag in Example 1.
  • MGISEQ-2000RS sequencer MGIDL-200H loader, MGISEQ-2000RS sequencing slide, MGISEQ-2000RS high-throughput sequencing reagent set FCLPE150, the excitation wavelengths of the instruments are: 532nm and 650nm respectively.
  • Tris Base (powder), sodium chloride (powder), Tween-20, Tris-HCl (powder), and glycyrrhizic acid were all purchased from compliant chemical reagent supply companies.
  • MGISEQ-2000RS high-throughput sequencing kit, product number is 1000012536, branded by MGI.
  • Escherichia coli single-stranded circular DNA is used as the template, which is the standard library reagent V3.0.
  • DNA polymerase (Cpas DNA Polymerase) is from MGI.
  • DNB nanospheres are derived from MGI, dATP-1, which refers to adenine nucleotides with both reversible blocking group modification and Cy5 fluorescence modification; dTTP-1, which refers to both reversible blocking group modification and ROX Fluorescently modified thymine nucleotides; dGTP-1, which refers to guanine nucleotides that have both reversible blocking group modifications and Cy3 fluorescence modifications; and dCTP-1, which refers to having both reversible blocking groups Both modified and EF700 fluorescently modified cytosine nucleotides were obtained from MGI.
  • HotMPS Scanning Reagent V2.0 (HotMPS Image Reagent V2.0), add a certain amount of glycyrrhizic acid powder and dissolve it with ultrasonic for 20 minutes to prepare the required concentration.
  • the specific amount is shown in Table 1. Dissolve the above reagent until it is transparent and uniform. The solution was filtered through a 0.22 micron filter membrane and set aside for later use.
  • the blank control is the commercial HotMPS scanning reagent V2.0.
  • Sequencing process In the first step, load DNB nanospheres onto the prepared sequencing chip.
  • the prepared mixed solution of dNTP molecules is pumped into the chip and DNA polymerase is used to add dNTP to the complementary strand of the DNA parent strand.
  • the third step is to take pictures and scan. Since dNTPs are modified molecules with fluorescent groups, laser is used as the excitation wavelength to take pictures. Since laser light damages DNA, a scanning reagent is used as a protective agent during the photo-taking step. After taking the photo, the base type is confirmed.
  • the base-terminal fluorescent group and the 3’ blocker are excised and eluted using an excision reagent, leaving the 3’-OH exposed for the next round of reaction.
  • the #10 well reagent in the kit is used as a reference for the scanning reagent.
  • PE100+70 sequencing is performed on the MGISEQ-2000RS sequencing platform according to the above experimental process.
  • Figure 2 shows the effect of different concentrations of glycyrrhizic acid on sequencing lag.
  • 1.00mM glycyrrhizic acid solution can effectively reduce the lag of the second strand of sequencing.
  • the possible reason is: during first-strand sequencing, the antioxidant glycyrrhizic acid affects DNB nanospheres. And a chain plays a very good protective role.
  • the experimental conditions of the blank control are shown in Table 1, and the working concentrations of glycyrrhizic acid are shown in Table 1.

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Abstract

一种甘草酸或其衍生物在核酸检测中的用途。特别的，涉及包含甘草酸或其衍生物的试剂，甘草酸或其衍生物在核酸检测中作为核酸保护剂的用途，以及一种核酸检测方法。

Description

甘草酸或其衍生物在核酸检测中的用途 技术领域
本发明涉及核酸检测领域，涉及甘草酸或其衍生物在核酸检测中的用途。特别地，本发明涉及包含甘草酸或其衍生物的试剂，甘草酸或其衍生物在核酸检测中作为核酸保护剂的用途，以及一种核酸检测方法。
背景技术
边合成边测序是多核酸测序的理论基础，在测序的过程中对于碱基的识别至关重要。一般情况下，碱基的识别是通过识别标记在不同碱基上的荧光染料类型来确定的，过程包括：使核苷酸暴露于紫外-可见光的照射之下，荧光染料中的生色团发出荧光，之后荧光信号被检测并拍照，进而可以确定荧光染料的类型。
可以使用波长在紫外-可见光范围内的激光作为光源。但是，激发光源的持续强照射以及溶液中的活性氧基团都会对核酸产生损伤，导致荧光检测信号强度的损失和高错误率等，严重影响核酸检测的质量。
在碱基识别过程中所使用的缓冲溶液被称为扫描试剂或拍照试剂。可以通过向扫描试剂中添加核酸保护剂来阻止激光对模板核苷酸的损伤。然而，现有技术中，扫描试剂中核酸保护剂成分的浓度较高。高浓度核酸保护剂的使用使得扫描试剂在低温保存时或者在长时间的测序过程出现盐析出的现象。此外，在长时间储放过程中，有些核酸保护成分被氧化后会出现变色现象，从而影响扫描质量，进而影响多核苷酸的测序质量。另外，高浓度的核酸保护剂的使用会使得产品成本增加。
发明内容
本发明使用甘草酸或其衍生物作为核酸保护剂，在很低的使用浓度下，也可以达到对模板核苷酸的保护。甘草酸或其衍生物在紫外可见光区没有吸收，不存在对碱基识别信号干扰，即使长期储存过程中被氧化也不会出现变色现象。
甘草酸又称甘草三萜皂苷、甘草甜素，属于三萜化合物，英文名为Glycyrrhizic Acid，CAS编号为1405-86-3，分子式是C 42H 62O 16，分子量为822.93，化学结构式如下：
Figure PCTCN2022118117-appb-000001
甘草酸易溶于热水及乙醇，在冷水中的溶解度较低，可以盐或盐的水合物形式使用，以增加水溶性。
本申请提供了以下发明：
在一个方面，本申请提供了一种试剂，其包含甘草酸、甘草酸的盐或甘草酸的盐的水合物，还包含Tris缓冲溶液。
本申请中，甘草酸的盐指的是甘草酸中的1个、2个或3个羧基与适当的无机或者有机阳离子(碱)形成的盐，包括但不限于：碱金属盐，如钠盐、钾盐、锂盐等；碱土金属盐，如钙盐、镁盐等；其他金属盐，如铝盐、铁盐、锌盐、铜盐、镍盐、钴盐等；无机碱盐，如铵盐；有机碱盐，如叔辛基胺盐、二苄基胺盐、吗啉盐、葡糖胺盐、苯基甘氨酸烷基酯盐、乙二胺盐、N-甲基葡糖胺盐、胍盐、二乙胺盐、三乙胺盐、二环己基胺盐、N,N’-二苄基乙二胺盐、氯普鲁卡因盐、普鲁卡因盐、二乙醇胺盐、N-苄基-苯乙基胺盐、哌嗪盐、四甲基胺盐、三(羟甲基)氨基甲烷盐。
在一些实施方案中，所述甘草酸的盐选自碱金属盐或铵盐，例如单钾盐、单钠盐、单铵盐、二钾盐、二钠盐、二铵盐、三钾盐、三钠盐、三铵盐。
本申请中，甘草酸的水合物或甘草酸的盐的水合物指的是甘草酸或甘草酸盐与一个或多个水分子缔合形成的物质。所述甘草酸盐可以是上述任一种盐。
在一些实施方案中，所述甘草酸的盐的水合物选自甘草酸碱金属盐或铵盐的水合物，例如钠盐或钾盐的水合物。
在一些实施方案中，所述甘草酸的盐或甘草酸的盐的水合物选自：
甘草酸单铵盐，其示例性的结构如下：
Figure PCTCN2022118117-appb-000002
甘草酸三钠水合物，其示例性的结构如下：
Figure PCTCN2022118117-appb-000003
甘草酸单钾盐，其示例性的结构如下：
Figure PCTCN2022118117-appb-000004
甘草酸二铵盐，其示例性的结构如下：
Figure PCTCN2022118117-appb-000005
甘草酸二钾水合物，其示例性的结构如下：
Figure PCTCN2022118117-appb-000006
本发明中，Tris缓冲溶液指的是以三羟甲基氨基甲烷(Tris)作为缓冲体系的缓冲溶液。
Tris被广泛用于生物化学和分子生物学的缓冲溶液的制备。Tris为弱碱，其碱的水溶液pH在10.5左右，加入盐酸调节pH值至所需值，即可获得该pH值的缓冲液。也可使用Tris与其盐酸盐(Tris-HCl)配制缓冲溶液。
Tris缓冲溶液的有效缓冲范围在pH7.0到9.2之间。Tris缓冲溶液可用作溶解核酸的溶剂。
本发明中，甘草酸或其衍生物在很低的使用浓度下，也可以达到对模板核苷酸的保护。在某些实施方案中，所述甘草酸、甘草酸的盐或甘草酸的盐的水合物的浓度为0.5-3mM，例如0.5-0.6mM、0.6-0.7mM、0.7-0.8mM、0.8-0.9mM、0.9-1.0mM、1-2mM或2-3mM。
在一些实施方案中，所述试剂是扫描试剂。
在某些实施方案中，所述试剂还包含氯化钠和/或DNA的稳定剂(例如吐温-20)。氯化钠的作用是提供盐溶液背景，保护检测中的引物。
在某些实施方案中，所述Tris缓冲溶液包含水，三羟甲基氨基甲烷(Tris Base)，氯化钠，吐温-20和三羟甲基氨基甲烷盐酸盐(Tris-HCl)。
在某些实施方案中，所述Tris缓冲溶液通过以下方法制得：将Tris Base(粉末)，氯化钠(粉末)，Tween-20，Tris-HCl(粉末)按一定的配比溶解在超纯水中。
在另一个方面，本申请提供了甘草酸、甘草酸的盐或甘草酸的盐的水合物在核酸检测中作为核酸保护剂的用途，所述甘草酸的盐或甘草酸的盐的水合物如上文所定义。
在某些实施方案中，所述核酸保护剂被用来保护核酸，减轻或避免光损伤或氧化损伤。
在某些实施方案中，所述核酸检测涉及检测荧光信号。在某些实施方案中，所述荧光信号可以通过光照反应或非光照反应(例如生物自发光反应)产生。在某些实施方案中，所述生物自发光反应是指荧光素酶催化其底物以产生荧光信号的反应。
在某些实施方案中，所述核酸检测涉及光照反应或非光照反应(例如生物自发光反应)。
本文所用的术语“光照反应”是指暴露于光学能源的反应。通常在所述反应中，提供光学能源(光照)以观察反应物或产物的产生和/或消耗，反应物或产物具有特别的指示其存在的光学特征，例如反应混合物或其组分的吸收光谱和/或发射光谱的变化(强度、波长等方面的变化)。
本文所用的术语“非光照反应”是指不借助光学能源而可以检测的反应。在非光照反应中，可以通过例如生物自发光等途径产生光学信号或者通过电信号的改变，由此可以检测反应物或产物的产生和/或消耗。
在某些实施方案中，所述光照反应包括碱基延伸引发的光信号或探针杂交引发的光信号，所述碱基延伸可引起光学信号(例如荧光信号)的产生。在某些实施方案中，本发明所述的光照反应中的光信号优选是荧光。
在某些实施方案中，所述核酸检测可用于核酸序列测定(测序)或其他检测(例如定量PCR)。
在某些实施方案中，所述核酸检测是核酸序列测定(测序)，例如高通量测序，例如边合成边测序(SBS测序)、连接测序、杂交测序、纳米孔测序、或复合探针-锚定分子连接(Combinatorial probe-anchor ligation,cPAL)测序。
在某些实施方案中，所述核酸检测是定量PCR。
在另一个方面，本申请提供了一种试剂盒，其包含本发明的试剂。
在某些实施方案中，本发明的试剂盒还可以包含一种或多种核酸检测所需的其他试剂，例如，引物、聚合酶、缓冲溶液、洗涤溶液，或其任何组合。
在某些实施方案中，本发明的试剂盒用于核酸序列测定。
在某些实施方案中，本发明的试剂盒还可以包含：用于将待测序的核酸分子与支持物固定(例如，通过共价或非共价连接进行固定)的试剂；用于起始核苷酸聚合反应的引物；用于进行核苷酸聚合反应的聚合酶；一种或多种缓冲溶液；一种或多种洗涤溶液；或其任何组合。
在某些实施方案中，本发明的试剂盒还可以包含，用于从样品中提取核酸分子的试剂和/或装置。用于从样品中提取核酸分子的方法是本领域熟知的。因此，可根据需要，在本发明的试剂盒中配置各种用于提取核酸分子的试剂和/或装置，例如用于破碎细胞的试剂，用于沉淀DNA的试剂，用于洗涤DNA的试剂，用于溶解DNA的试剂，用于沉淀RNA的试剂，用于洗涤RNA的试剂，用于溶解RNA的试剂，用于去除蛋白的试剂，用于去除 DNA的试剂(例如当目的核酸分子为RNA时)，用于去除RNA的试剂(例如当目的核酸分子为DNA时)，及其任何组合。
在某些实施方案中，本发明的试剂盒还包含，用于预处理核酸分子的试剂。在本发明的试剂盒中，用于预处理核酸分子的试剂不受额外限制，并且可根据实际需要选择。所述用于预处理核酸分子的试剂包括例如，用于核酸分子片段化的试剂(例如DNA酶I)，用于补齐核酸分子末端的试剂(例如DNA聚合酶，例如T4DNA聚合酶，Pfu DNA聚合酶，Klenow DNA聚合酶)，接头分子，标签分子，用于将接头分子与目的核酸分子相连接的试剂(例如连接酶，例如T4DNA连接酶)，用于修复核酸末端的试剂(例如，丧失3'-5'核酸外切酶活性但显示5'-3'核酸外切酶活性的DNA聚合酶)，用于扩增核酸分子的试剂(例如，DNA聚合酶，引物，dNTP)，用于分离和纯化核酸分子的试剂(例如层析柱)，以及其任何组合。
在某些实施方案中，本发明的试剂盒还包含用于固定待测序的核酸分子的支持物。在通常情况下，用于固定待测序的核酸分子的支持物呈固相，以便于操作。因此，在本公开内容中，“支持物”有时也被称为“固体支持物”或“固相支持物。”然而，应当理解的是，本文所提及的“支持物”并不限于固体，其还可以是半固体(例如凝胶)。
如本文所用的，术语“装载、”“固定”和“附着”当提及核酸使用时，意指经由共价键或非共价键直接或间接附接至固体支持物。在本公开内容的某些实施方案中，本发明的方法包括经由共价附接将核酸固定在固体支持物上。但是通常地，仅需要的是在期望使用固体支持物的条件下(例如在需要核酸扩增和/或测序的应用中)，核酸保持固定或附接至固体支持物。在某些实施方案中，将核酸固定在固体支持物上可以包括将待用作捕获引物或扩增引物的寡核苷酸固定在固体支持物上，使得3'末端对于酶促延伸是可利用的并且该引物序列的至少一部分能够杂交至互补核酸序列；然后将待固定的核酸杂交至所述寡核苷酸，在这种情况下固定的寡核苷酸或多核苷酸可以为3'-5'方向。在某些实施方案中，将核酸固定在固体支持物上可以包括通过氨基化修饰将核酸结合蛋白质结合在固体支持物上，并通过核酸结合蛋白质捕获核酸分子。可选地，装载可以通过除碱基配对杂交之外的其他方式发生，例如上文描述的共价附接。核酸与固体支持物附接方式的非限制性示例包括核酸杂交、生物素链霉亲和素结合、巯基结合、光活化结合、共价结合、抗体-抗原、经由水凝胶或其他多孔聚合物的物理限制等。用于将核酸固定在固体支持物上的各种示例性方法可参见例如G.Steinberg-Tatman等人,Bioconjugate Chemistry 2006,17,841-848；Xu X.等人Journal of the Americ  an Chemical Society 128(2006)9286-9287；美国专利申请US 5639603、US 5641658、US2010248991；国际专利申请WO 2001062982、WO 2001012862、WO 2007111937、WO0006770，为了所有目的，特别是为了与制备形成其上固定有核酸的固体支持物有关的全部教导，以上文献均通过引用全文并入本文。
在本发明中，所述支持物可以由各种合适的材料制成。此类材料包括例如：无机物、天然聚合物、合成聚合物，以及其任何组合。具体的例子包括但不限于：纤维素、纤维素衍生物(例如硝化纤维素)、丙烯酸树脂、玻璃、硅胶、二氧化硅、聚苯乙烯、明胶、聚乙烯吡咯烷酮、乙烯基和丙烯酰胺的共聚物、与二乙烯基苯等交联的聚苯乙缔(参见例如，Merrifield Biochemistry 1964,3,1385-1390)、聚丙烯酰胺、乳胶、葡聚糖、橡胶、硅、塑料、天然海绵、金属塑料、交联的葡聚糖(例如，Sephadex TM)、琼脂糖凝胶(Sepharose TM)，以及本领域技术人员已知的其他支持物。
在某些优选的实施方案中，用于固定待测序的核酸分子的支持物可以是包括惰性基底或基质(例如，载玻片、聚合物珠等)的固体支持物，所述惰性基底或基质已例如通过应用含有活性基团的中间材料而被功能化，所述活性基团允许共价连接诸如多核苷酸的生物分子。此类支持物的实例包括但不限于，负载于诸如玻璃的惰性基底上的聚丙酰胺水凝胶，特别是WO 2005/065814和US 2008/0280773中描述的聚丙烯酰胺水凝胶，其中，所述专利申请的内容通过引用以其全文并入本文。在此类实施方案中，生物分子(例如多核苷酸)可被直接地共价地连接至中间材料(例如水凝胶)，而中间材料其自身可被非共价地连接至基底或基质(例如，玻璃基底)。在某些优选的实施方案中，所述支持物为表面修饰了一层亲和素、氨基、丙烯酰胺硅烷或醛基化学基团的玻片或硅片。
在本发明中，支持物或固体支持物不受限于其大小、形状和构造。在一些实施方案中，支持物或固体支持物是平面结构，例如载片、芯片、微芯片和/或阵列。此类支持物的表面可以是平面层的形式。
在某些优选的实施方案中，用于固定待测序的核酸分子的支持物为珠或孔的阵列(其也被称为芯片)。所述阵列可以使用本文概述的用于制备固体支持物的任何材料来制备，并且优选地，阵列上的珠或孔的表面进行了官能化，以利于核酸分子的固定。阵列上的珠或孔的数目不受限制。例如，每一个阵列可包含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或更多个珠或孔。在某些示例性实施方案中，每个珠或孔的表面可固定一个或多个核酸分子。相应地，每一个阵列可固定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或 更多个核酸分子。因此，此类阵列可特别有利地用于核酸分子的高通量测序。
在某些优选的实施方案中，本发明的试剂盒还包含用于将待测序的核酸分子与支持物固定(例如，通过共价或非共价连接进行固定)的试剂。此类试剂包括例如对核酸分子(例如其5'端)进行活化或修饰的试剂，例如磷酸、硫醇、胺、羧酸或醛；对支持物的表面进行活化或修饰的试剂，例如氨基-烷氧基硅烷(例如氨基丙基三甲氧基硅烷、氨基丙基三乙氧基硅烷、4-氨基丁基三乙氧基硅烷等)；交联剂，例如琥珀酰酐、苯基二异硫氰酸盐(Guo等人，1994)、马来酸酐(Yang等人，1998)、1-乙基-3-(3-二甲基氨基丙基)-碳二亚胺盐酸盐(EDC)、间-马来酰亚胺基苯甲酸-N-羟基琥珀酰亚胺酯(MBS)、N-琥珀酰亚胺基[4-碘代乙酰基]氨基苯甲酸(SIAB)、4-(N-马来酰亚胺基甲基)环己烷-1-羧酸琥珀酰亚胺(SMCC)、N-γ-马来酰亚胺基丁酰氧基-琥珀酰亚胺酯(GMBS)、4-(p-马来酰亚胺基苯基)丁酸琥珀酰亚胺(SMPB)；以及其任何组合。
在某些优选的实施方案中，本发明的试剂盒还包含用于起始核苷酸聚合反应的引物。在本发明中，引物不受额外的限制，只要它能够特异性地退火到目标核酸分子的一个区域上。在一些示例性实施方案中，所述引物的长度可以为5-50bp，例如5-10、10-15、15-20、20-25、25-30、30-35、35-40、40-45、45-50bp。在一些示例性实施方案中，所述引物可包含天然存在或非天然存在的核苷酸。在一些示例性实施方案中，所述引物包含天然存在的核苷酸或者由天然存在的核苷酸组成。在一些示例性实施方案中，所述引物包含经修饰的核苷酸，例如锁核酸(LNA)。在某些优选的实施方案中，所述引物包含通用引物序列。
在某些优选的实施方案中，本发明的试剂盒还包含用于进行核苷酸聚合反应的聚合酶。在本发明中，可使用各种合适的聚合酶进行聚合反应。在一些示例性实施方案中，所述聚合酶能够以DNA为模板合成新的DNA链(例如DNA聚合酶)。在一些示例性实施方案中，所述聚合酶能够以RNA为模板合成新的DNA链(例如反转录酶)。在一些示例性实施方案中，所述聚合酶能够以DNA或RNA为模板合成新的RNA链(例如RNA聚合酶)。因此，在某些优选的实施方案中，所述聚合酶选自DNA聚合酶，RNA聚合酶，和反转录酶。
在某些优选的实施方案中，本发明的试剂盒还包含一种或多种切除试剂。在某些实施方案中，所述切除试剂选自内切酶IV和碱性磷酸酶。
在某些优选的实施方案中，本发明的试剂盒还包含一种或多种缓冲溶液。此类缓冲液包括但不限于，用于DNA酶I的缓冲溶液，用于DNA聚合酶的缓冲溶液，用于连接酶的缓冲溶液，用于洗脱核酸分子的缓冲溶液，用于溶解核酸分子的缓冲溶液，用于进行核苷 酸聚合反应(例如PCR)的缓冲溶液，和用于进行连接反应的缓冲溶液。本发明的试剂盒可包含上述缓冲溶液的任一种或多种。
本发明中，“缓冲液”和“缓冲溶液”具有相同的含义，并且可以互换使用。
在某些实施方案中，所述用于DNA聚合酶的缓冲溶液包含一价盐离子(例如钠离子、氯离子)和/或二价盐离子(例如镁离子、硫酸根离子，锰离子)。在某些实施方案中，所述一价盐离子或二价盐离子在所述缓冲溶液中的浓度为10μM-200mM，例如10μM、50μM、100μM、200μM、500μM、1mM、3mM、10mM、20mM、50mM、100mM、150mM或200mM。
在某些实施方案中，所述用于DNA聚合酶的缓冲溶液包含三羟甲基氨基甲烷(Tris)。在某些实施方案中，Tris在所述缓冲溶液中的浓度为10mM-200mM，例如10mM、20mM、50mM、100mM、150mM或200mM。
在某些实施方案中，所述用于DNA聚合酶的缓冲溶液包含有机溶剂，例如DMSO或丙三醇(甘油)。在某些实施方案中，所述有机溶剂在所述缓冲溶液中的质量含量为0.01％-10％，例如0.01％、0.02％、0.05％、1％、2％、5％或10％。
在某些实施方案中，所述用于DNA聚合酶的缓冲溶液的pH为7.0-9.0，例如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或9.0。
在某些实施方案中，所述用于DNA聚合酶的缓冲溶液包含：一价盐离子(例如钠离子、氯离子)、二价盐离子(例如镁离子、硫酸根离子、锰离子)、Tris和有机溶剂(例如DMSO或甘油)。在某些实施方案中，所述缓冲溶液相的pH为8.8。
在某些优选的实施方案中，本发明的试剂盒还包含一种或多种洗涤溶液。此类洗涤溶液的实例包括但不限于，磷酸盐缓冲液，柠檬酸盐缓冲液，Tris-HCl缓冲液，醋酸盐缓冲液，碳酸盐缓冲液等。本发明的试剂盒可包含上述洗涤溶液的任一种或多种。
在另一个方面，本申请提供了本发明的试剂或试剂盒用于核酸检测的用途。在某些实施方案中，所述核酸检测涉及检测荧光信号。在某些实施方案中，所述荧光信号可以通过光照反应或非光照反应(例如生物自发光反应)产生。在某些实施方案中，所述生物自发光是指荧光素酶催化其底物以产生荧光信号的反应。在某些实施方案中，所述核酸检测涉及光照反应或非光照反应(例如生物自发光反应)。在某些实施方案中，所述光照反应包括碱基延伸引发的光信号，所述碱基延伸可引起光学信号(例如荧光信号)的产生。在某些实施方案中，本发明所述的光照反应中的光学特征优选是荧光。在某些实施方案中，所述核酸检测可用于核酸序列测定(测序)或其他检测(例如定量PCR)。在某些实施方案中， 所述核酸检测是核酸序列测定，例如高通量测序，例如SBS测序、连接测序、杂交测序、纳米孔测序、或cPAL测序。在某些实施方案中，所述核酸检测是定量PCR。
在另一个方面，本申请提供了制备本发明的试剂的方法，包括将所述试剂的各成分溶解在超纯水中，制成透明均一的溶液，之后对溶液进行过滤，得到本发明的试剂。可使用超声辅助溶解。
在另一个方面，本申请提供了一种抑制核酸降解的方法，其包括使用甘草酸、甘草酸的盐或甘草酸的盐的水合物作为核酸保护剂。在一些实施方案中，所述核酸降解为光诱导的核酸降解或氧化引起的核酸降解。在一些实施方案中，所述方法包括：在存在甘草酸、甘草酸的盐或甘草酸的盐的水合物的条件下，对包含核酸的反应混合物进行光照反应或非光照反应(例如生物自发光反应)。
在另一个方面，本申请提供了一种检测靶核酸分子的方法，其包括在使用甘草酸、甘草酸的盐或甘草酸的盐的水合物作为核酸保护剂。在一些实施方案中，所述方法包括：在存在甘草酸、甘草酸的盐或甘草酸的盐的水合物的条件下，对包含靶核酸分子的反应混合物进行光照反应或非光照反应(例如生物自发光反应)。在某些实施方案中，所述方法包括：对所述反应混合物进行信号采集和检测荧光信号；其中，所述反应混合物包含可产生荧光信号的反应物(例如荧光标记反应物)、靶核酸分子以及缓冲液，所述缓冲液包含甘草酸、甘草酸的盐或甘草酸的盐的水合物。
在某些实施方案中，所述荧光信号通过光照反应或生物自发光反应产生。
在某些实施方案中，所述方法包括：对所述反应混合物进行光照射，检测来自光照反应的荧光信号；其中，所述反应混合物包含荧光标记反应物、靶核酸分子以及缓冲液，所述缓冲液包含甘草酸、甘草酸的盐或甘草酸的盐的水合物。
在某些实施方案中，进行光照反应的反应混合物包含甘草酸、甘草酸的盐或甘草酸的盐的水合物。
在某些实施方案中，所述方法用于核酸序列测定。在某些实施方案中，所述测序是高通量测序。在某些实施方案中，所述测序是SBS测序、连接测序、杂交测序、纳米孔测序、或cPAL测序。
在某些实施方案中，所述可产生荧光信号的反应物包括：标记或未标记的核苷酸(例如dNTP)，任选地还包含允许所述反应物产生荧光信号的其他试剂。在某些实施方案中，每种可产生荧光信号的反应物对应一种核苷酸类型，并且每种反应物可以产生相互区分的信号，以识别特定核苷酸的掺入。例如，分别包含待掺入的腺嘌呤、鸟嘌呤、胞嘧啶和胸 腺嘧啶的四种反应物可产生不同的荧光信号，使它们易于相互区分。
在某些实施方案中，所述可产生荧光信号的反应物中的核苷酸(例如荧光标记的核苷酸)还带有阻断基团(例如3’阻断基团)以可逆阻止进一步的碱基延伸。
在某些实施方案中，所述可产生荧光信号的反应物中的核苷酸(例如荧光标记的核苷酸)选自核苷多磷酸盐(或其类似物)，例如dNTP。
在某些实施方案中，所述反应混合物还包含酶，例如聚合酶、解旋酶、外切核酸酶、或连接酶；优选地，所述反应混合物包含聚合酶，例如DNA聚合酶。
在某些实施方案中，所述反应混合物还包含引物。
在某些实施方案中，所述方法包括：掺入所述可产生荧光信号的反应物中的核苷酸(例如荧光标记的核苷酸)至靶核酸分子的互补链；在包含甘草酸、甘草酸的盐或甘草酸的盐的水合物的缓冲液存在下，给予允许所述反应物产生荧光信号的条件并检测所述反应混合物的荧光信号(例如照射所述反应混合物)，并确定掺入的核苷酸的身份。在某些实施方案中，所述确定掺入的核苷酸的身份包括对与被掺入核苷酸的关联的荧光信号(例如荧光标记)进行检测(例如拍照)。
在某些实施方案中，所述方法还包括：从掺入的核苷酸中去除与其直接或间接连接的可产生荧光信号的部分(例如荧光标记)；和/或，洗涤以去除未掺入的核苷酸。在某些实施方案中，所述方法还包括：从掺入的核苷酸中去除阻断基团以允许进一步延伸。
在某些实施方案中，所述方法包括多次掺入并确定存在于每个掺入的核苷酸中的碱基的身份，以确定靶核酸分子的序列。
在某些实施方案中，所述靶核酸分子存在于核酸阵列。在某些实施方案中，所述阵列上的每个位点可以包括单个靶核酸分子的多个拷贝。在某些实施方案中，所述核酸阵列固定于固相支持物，例如芯片。
在另一个方面，本申请提供了一种核酸序列的检测方法，包括：将一种或多种带标记修饰的核苷酸掺入到与核酸模板链互补的核酸链中，通过检测所述标记确定所述一种或多种掺入的核苷酸的类型，其中，确定掺入的核苷酸的类型的步骤在包含甘草酸、甘草酸的盐或甘草酸的盐的水合物的缓冲液中进行。
在某些实施方案中，所述标记为可产生荧光信号的标记。在某些实施方案中，所述荧光信号通过光照反应或生物自发光反应产生。在某些实施方案中，所述方法用于核酸序列测定(测序)。在某些实施方案中，所述带标记修饰的核苷酸为：(1)荧光标记的核苷酸(例如dNTP)；或(2)带有标签的核苷酸(例如dNTP)，所述标签能够特异性结合荧光 素酶。
在某些实施方案中，所述确定掺入的核苷酸的类型的步骤包括：在甘草酸、甘草酸的盐或甘草酸的盐的水合物存在下，给予允许所述标记产生荧光信号的条件并检测所述缓冲溶液的荧光信号，并确定掺入的核苷酸的身份。任选地，所述方法还包括：从掺入的核苷酸中去除与其直接或间接连接的可产生荧光信号的部分；和/或，洗涤以去除未掺入的核苷酸。在某些实施方案中，所述方法包括多次掺入并确定存在于每个掺入的核苷酸中的碱基的身份，以确定靶核酸分子的序列。
在某些实施方案中，所述甘草酸的盐选自碱金属盐或铵盐。在某些实施方案中，所述甘草酸的盐的水合物选自甘草酸碱金属盐或铵盐的水合物。在某些实施方案中，所述甘草酸的盐或甘草酸的盐的水合物选自：甘草酸单铵盐、甘草酸三钠水合物、甘草酸单钾盐、甘草酸二铵盐、甘草酸二钾水合物。在某些实施方案中，所述缓冲溶液中，甘草酸、甘草酸的盐或甘草酸的盐的水合物的浓度为0.5-3mM。在某些实施方案中，所述缓冲溶液还包含氯化钠和/或DNA的稳定剂(例如吐温-20)。
在某些实施方案中，所述缓冲溶液为扫描试剂。
在某些实施方案中，所述方法用于核酸序列测定。在某些实施方案中，所述测序是高通量测序。在某些实施方案中，所述测序是SBS测序、连接测序、杂交测序、纳米孔测序、或cPAL测序。
基于光照反应的测序
在某些实施方案中，所述荧光信号由光照反应产生。在此类实施方案中，所述标记优选是荧光标记。在某些实施方案中，所述方法包括：对所述反应混合物进行光照射，检测来自光照反应的荧光信号；其中，所述反应混合物包含可产生荧光信号的反应物、靶核酸分子以及缓冲液，所述缓冲液包含甘草酸、甘草酸的盐或甘草酸的盐的水合物。在某些实施方案中，进行光照反应的反应混合物包含甘草酸、甘草酸的盐或甘草酸的盐的水合物。
在某些实施方案中，所述可产生荧光信号的反应物包括：荧光标记的核苷酸(例如dNTP)。在某些实施方案中，所述荧光标记可以通过接头与核苷酸(例如其碱基)连接。接头可以是酸不稳定的、光不稳定的或含有二硫键。
在某些实施方案中，所述方法包括：掺入荧光标记的核苷酸至靶核酸分子的互补链；在甘草酸、甘草酸的盐或甘草酸的盐的水合物存在下照射所述反应混合物，并确定掺入的核苷酸的身份。在某些实施方案中，所述确定掺入的核苷酸的身份包括对被掺入核苷酸的荧光标记进行检测(例如拍照)。在某些实施方案中，所述方法还包括：从掺入的核苷酸中 去除与其连接的荧光标记；和/或，洗涤以去除未掺入的核苷酸。在某些实施方案中，所述方法还包括：从掺入的核苷酸中去除阻断基团以允许进一步延伸。
在某些实施方案中，所述可产生荧光信号的反应物包括：未标记的核苷酸(例如dNTP)以及荧光标记的亲和试剂(例如抗体)，所述亲和试剂能够特异性结合所述未标记的核苷酸。此类实施方案可以被称为Cool MPS测序，其详细教导可参考PCT国际申请WO2018129214A1。在此类实施方案中，基于SBS测序原理，荧光基团不是直接标记在掺入核苷酸上，而是标记在亲和试剂(例如抗体、适体、Affimer、Knottin等)上，亲和试剂可以特异性结合掺入核苷酸中的碱基、糖、可裂解的阻断基团或这些组分的组合，因此可以通过亲和试剂来鉴定被掺入的核苷酸类型。
在某些实施方案中，所述方法包括：掺入未标记的核苷酸至靶核酸分子的互补链；提供荧光标记的亲和试剂，并通过亲和试剂与核苷酸之间的特异性结合将荧光标记间接连接至掺入的核苷酸上；在甘草酸、甘草酸的盐或甘草酸的盐的水合物存在下照射所述反应混合物，并确定掺入的核苷酸的身份。在某些实施方案中，所述确定掺入的核苷酸的身份包括对被掺入核苷酸所连接的亲和试剂的荧光标记进行检测(例如拍照)。在某些实施方案中，所述方法还包括：从掺入的核苷酸中去除与其连接的亲和试剂；和/或，洗涤以去除未掺入的核苷酸。在某些实施方案中，所述方法还包括：从掺入的核苷酸中去除阻断基团以允许进一步延伸。
基于生物自发光反应的测序
在某些实施方案中，所述荧光信号由生物自发光反应产生。生物自发光的检测原理包括，待掺入核苷酸上不直接标记荧光信号，而是标记生物素或地高辛等亲和物质，聚合反应之后，加入带有荧光素酶的上述亲和物质的配对成员，从而将荧光素酶结合至被掺入的核苷酸上，然后加入反应底物产生光信号，以识别被掺入核苷酸的身份，该过程不需要激发光照射。关于生物自发光反应的详细教导可参加例如PCT国际申请WO2020227953A1。
在某些实施方案中，所述可产生荧光信号的反应物包括：带有标签的核苷酸(例如dNTP)、能够特异性结合所述标签的荧光素酶以及所述荧光素酶的底物。
在某些实施方案中，所述核苷酸带有的标签是任何能够彼此特异性结合的分子配对的成员。配对成员之间的特异性结合实现核苷酸与荧光素酶的连接。示例性的配对成员包括但不限于：(a)与相应抗体或其结合部分或片段组合的半抗原或抗原性化合物，例如地高辛-地高辛抗体，N3G-N3G抗体，FITC-FITC抗体；(b)核酸适配体和蛋白质；(c)非免疫结合对(例如生物素-抗生物素蛋白、生物素-链霉亲和素、生物素-中性抗生蛋白)；(d)激素-激 素结合蛋白；(e)受体-受体激动剂或拮抗剂；(f)凝集素-碳水化合物；(g)酶-酶辅因子；(h)酶-酶抑制剂；和(i)能够形成核酸双链体的互补的寡核苷酸或多核苷酸对。
在某些实施方案中，所述核苷酸带有的标记是小分子标记物，其选自生物素、地高辛、N3G或FITC，荧光素酶带有与所述小分子标记物对应的配对成员。例如，在一个具体实施方案中，所述核苷酸带有的标记是生物素，则荧光素酶可以是经链霉亲和素标记的荧光素酶；所述核苷酸带有的标记是地高辛，则荧光素酶可以是经地高辛抗体标记的荧光素酶。所述荧光素酶来源包括但不限于firefly，gaussia，Renilla等生物。例如，经链霉亲和素标记的荧光素酶可以是Adivity公司的SA-Gluc:Streptavidin-Gaussia princeps luciferase。经地高辛抗体标记的荧光素酶可以是digoxin抗体-Gluc或digoxin抗体-Nluc。
在某些实施方案中，所述方法包括：掺入带有标签的核苷酸至靶核酸分子的互补链；提供连接有能够特异性结合所述标签的配对成员的荧光素酶，并通过配对成员之间的特异性结合将荧光素酶间接连接至掺入的核苷酸上；在甘草酸、甘草酸的盐或甘草酸的盐的水合物存在下，提供所述荧光素酶的底物以产生荧光信号，从而确定掺入的核苷酸的身份。在某些实施方案中，所述方法还包括：从掺入的核苷酸中去除与其连接的荧光素酶；和/或，洗涤以去除未掺入的核苷酸。在某些实施方案中，所述方法还包括：从掺入的核苷酸中去除阻断基团以允许进一步延伸。
在某些实施方案中，所述方法也可以用于定量PCR。在某些实施方案中，所述可产生荧光信号的反应物是荧光探针。在某些实施方案中，所述反应混合物还包含酶，例如聚合酶、解旋酶、外切核酸酶、或连接酶；优选地，所述反应混合物包含聚合酶，例如DNA聚合酶。在某些实施方案中，所述反应混合物还包含引物。
本发明的抑制核酸降解的方法或检测靶核酸分子的方法中，所述甘草酸的盐或甘草酸的盐的水合物可以选自上文定义的任意盐或水合物。在某些实施方案中，所述甘草酸的盐选自碱金属盐或铵盐。在某些实施方案中，所述甘草酸的盐的水合物选自甘草酸碱金属盐或铵盐的水合物。在某些实施方案中，所述甘草酸的盐或甘草酸的盐的水合物选自：甘草酸单铵盐、甘草酸三钠水合物、甘草酸单钾盐、甘草酸二铵盐、甘草酸二钾水合物。在某些实施方案中，所述甘草酸、甘草酸的盐或甘草酸的盐的水合物存在于缓冲液中，且浓度为0.5-3mM。在某些实施方案中，所述缓冲液还包含氯化钠和/或DNA的稳定剂(例如吐温-20)。
以下将以合成测序为例进行讨论，这并不意味着限制。在涉及光照反应的核酸检测步骤中使用本发明的试剂的所有用途和方法都包括在本发明的范围内。
在另一个方面，本申请提供了一种核酸测序的方法，所述方法包括使用本发明的试剂。在某些实施方案中，本发明的测序方法包括合成目标单链多核苷酸互补的生长的多核苷酸，然后进行扫描拍照检测。
在某些实施方案中，所述测定目标单链多核苷酸的序列的方法包括：
(a)提供双链体、核苷酸、聚合酶和切除试剂；所述双链体包含生长的核酸链以及待测序的核酸分子；
(b)进行包含以下步骤(i)、(ii)和(iii)的反应循环：
步骤(i)：使用聚合酶，使核苷酸并入生长的核酸链，形成包含阻断基团和可检测标记的核酸中间体；
步骤(ii)：在存在本发明的试剂的条件下对所述核酸中间体上的可检测标记进行检测；
步骤(iii)：使用切除试剂将核酸中间体上的阻断基团除去。
在某些实施方案中，所述反应循环还包括步骤(iv)：使用切除试剂将核酸中间体上的可检测标记除去。
在某些实施方案中，所述方法包括以下步骤：
第一步，将DNA纳米球(DNA nanoball，简称DNB)加载到准备好的测序芯片上；
第二步，将准备好的dNTP分子混合溶液泵入芯片用DNA聚合酶将dNTP加入到待测DNA的互补链上；
第三步，拍照扫描，由于dNTP是被修饰的带有荧光基团分子，用激光作为激发波长进行拍照；由于激光对于DNA有光损伤作用，因此在拍照这一步骤加入包含核酸保护剂的扫描试剂进行拍照；
第四步，通过切除试剂将碱基端荧光基团和3’的阻断切除并洗脱干净，使得3’-OH裸露从而进行下一轮反应；
第五步，通过对拍照结果进行分析，确定待测核酸分子的碱基序列。
本发明中，核酸可以包括核苷酸或核苷酸类似物。核苷酸通常含有糖、核碱基和至少一个磷酸基。核苷酸包括脱氧核糖核苷酸、修饰的脱氧核糖核苷酸、核糖核苷酸、修饰的核糖核苷酸、肽核苷酸、修饰的肽核苷酸、修饰磷酸盐糖主链核苷酸及其混合物。核苷酸的实例包括(例如)腺苷一磷酸(AMP)、腺苷二磷酸(ADP)、腺苷三磷酸(ATP)、胸苷一磷酸(TMP)、胸苷二磷酸(TDP)、胸苷三磷酸(TTP)、胞苷酸(CMP)、胞苷二磷酸(CDP)、胞苷三磷酸(CTP)、鸟苷一磷酸(GMP)、鸟苷二磷酸(GDP)、鸟苷三磷酸(GTP)、 尿苷一磷酸(UMP)、尿苷二磷酸(UDP)、尿苷三磷酸(UTP)、脱氧腺苷酸(dAMP)、脱氧腺苷二磷酸(dADP)、脱氧腺苷三磷酸(dATP)、脱氧胸腺嘧啶核苷一磷酸(dTMP)、脱氧胸腺嘧啶核苷二磷酸(dTDP)、脱氧胸苷三磷酸(dTTP)、去氧胞二磷(dCDP)、脱氧胞苷三磷酸(dCTP)、脱氧鸟苷一磷酸(dGMP)、脱氧鸟苷二磷酸(dGDP)、脱氧鸟苷三磷酸(dGTP)、脱氧尿苷一磷酸(dUMP)、脱氧尿苷二磷酸(dUDP)和脱氧尿苷三磷酸(dUTP)。还可以在本文所述的方法中使用包含修饰的核碱基的核苷酸类似物。无论是具有天然主链还是类似结构，可以包含在多核苷酸中的示例性修饰的核碱基包括(例如)肌苷、黄嘌呤、次黄嘌呤、异胞嘧啶、异鸟嘌呤、2-氨基嘌呤、5-甲基胞嘧啶、5-羟甲基胞嘧啶、2-氨基腺嘌呤、6-甲基腺嘌呤、6-甲基鸟嘌呤、2-丙基鸟嘌呤、2-丙基腺嘌呤、2-硫脲嘧啶、2-硫胸腺嘧啶、2-硫胞嘧啶、15-卤代脲嘧啶、15-卤代胞嘧啶、5-丙炔基尿嘧啶、5-丙炔基胞嘧啶、6-偶氮尿嘧啶、6-偶氮胞嘧啶、6-偶氮胸腺嘧啶、5-尿嘧啶、4-硫尿嘧啶、8-卤代腺嘌呤或鸟嘌呤、8-氨基腺嘌呤或鸟嘌呤、8-硫腺嘌呤或鸟嘌呤、8-硫烷基腺嘌呤或鸟嘌呤、8-羟基腺嘌呤或鸟嘌呤、5-卤素取代的尿嘧啶或胞嘧啶、7-甲基鸟嘌呤、7-甲基腺嘌呤、8-氮杂鸟嘌呤、8-氮杂腺嘌呤、7-去氮鸟嘌呤、7-去氮腺嘌呤、3-去氮鸟嘌呤、3-去氮腺嘌呤等。如本领域中已知的，某些核苷酸类似物不能引入多核苷酸，例如，核苷酸类似物，如腺苷5′-磷酰硫酸。
在本发明的方法中，待测序的核酸分子不受其长度的限制。在某些优选的实施方案中，待测序的核酸分子的长度可以为至少10bp，至少20bp，至少30bp，至少40bp，至少50bp，至少100bp，至少200bp，至少300bp，至少400bp，至少500bp，至少1000bp，或者至少2000bp。在某些优选的实施方案中，待测序的核酸分子的长度可以为10-20bp，20-30bp，30-40bp，40-50bp，50-100bp，100-200bp，200-300bp，300-400bp，400-500bp，500-1000bp，1000-2000bp，或者超过2000bp。在某些优选的实施方案中，待测序的核酸分子可具有10-1000bp的长度，以利于进行高通量测序。
在某些优选的实施方案中，在将核酸分子固定于支持物之前，可以对核酸分子进行预处理。此类预处理包括但不限于，核酸分子的片段化，末端的补齐，接头的添加，标签的添加，核酸分子的扩增，核酸分子的分离和纯化，以及其任何组合。
如本文所用的术语“纳米球”一般表示大分子或复合体，其具有紧凑的，例如内径范围典型地在大约1nm与大约1000nm之间，优选地大约50nm与大约500nm之间的(近似)球形形状。
如本文所用的术语“核酸纳米球”通常是包含多拷贝的靶核酸分子的多联体。这些 核酸拷贝典型地一个接一个地布置在核苷酸的连续线型链中，但是本发明的核酸纳米球还可以利用本文描述的方法由任何核酸分子制成。该串联重复结构连同DNA的单链性质引起纳米球折叠(folding)配置。一般而言，核酸纳米球中的多拷贝的靶核酸分子各自包含序列已知的接头序列，以便于对其进行扩增或测序。各靶核酸分子的接头序列通常是相同的，但也可以不同。核酸纳米球通常包括DNA纳米球，在本文中也称为DNB(DNA nanoball)。
核酸纳米球可以使用例如滚环复制(RCA)来产生。RCA过程曾被用于制备多个连续拷贝的M13基因组(Blanco等人,(1989)J Biol Chem 264:8935-8940)。在这种方法中，核酸经线性多联体化复制。本领域技术人员可以在许多参考文献中找到关于选择RCA反应的条件和试剂的指南，包括美国专利US5,426,180、US5,854,033、US6,143,495和US5,871,921，为了所有目的，特别是为了与利用RCA或其他方法制备核酸纳米球有关的全部教导，这些文献均通过引用全文并入本文。
核酸纳米球可以装载在如本文所述的固体支持物的表面上。纳米球可以通过任何合适的方法附着到固体支持物的表面，这样的方法的非限制性示例包括核酸杂交、生物素链霉亲和素结合、巯基结合、光活化结合、共价结合、抗体-抗原、经由水凝胶或其他多孔聚合物的物理限制等，或它们的组合。在一些情况下，纳米球可以用核酸酶(例如，DNA核酸酶)消化，以便从纳米球产生较小的纳米球或片段。
某些实施方案中，固体支持物表面可能带有反应性官能团，所述反应性官能团与多核苷酸分子上的互补官能团反应形成共价键，例如采用与附着cDNA到微阵列上所用的技术相同的方式进行，例如参见Smirnov等人(2004),Genes,Chromosomes&Cancer,4 0:72-77和Beaucage(2001),Current Medicinal Chemistry,8:1213_1244，这两份文献通过引用并入本文。DNB还可以有效地附着到疏水性表面，例如带有低浓度的各种反应官能团(例如-OH基团)的干净的玻璃表面。经由多核苷酸分子和表面上的反应性官能团之间形成的共价键的附着在本文中又称为“化学附着”。
在其他实施方案中，多核苷酸分子可以吸附到表面上。在这种实施方式中，多核苷酸通过与表面的非特异性相互作用，或者通过诸如氢键、范德华力等的非共价相互作用被固定。
在其它实施方案中，核酸文库可以是双链核酸片段，通过与固定在固体支持物表面的寡核苷酸发生连接反应固定在固体支持物表面，然后进行桥式扩增反应制备测序文库。
有益效果
本发明可以实现以下有益效果：
本发明使用甘草酸或其衍生物作为核酸保护剂，即使用很低的浓度也可以达到对模板核苷酸的保护。本发明使用的甘草酸或其衍生物在紫外可见光区没有吸收，即使在多轮测序反应之后，数据质量依然很高，不存在对碱基识别信号干扰，而且长期储存过程中被氧化也不会出现变色现象。通过使用本发明优化的扫描试剂可以大大提高核酸测序，特别是多核苷酸测序的质量。
下面将结合附图和实施例对本发明的实施方案进行详细描述，但是，本领域技术人员将理解，下列附图和实施例仅用于说明本发明，而不是对本发明的范围的限定。根据附图和优选实施方案的下列详细描述，本发明的各种目的和有利方面对于本领域技术人员来说将变得显然。
附图说明
图1显示了实施例1中不同浓度甘草酸对测序的Q30的影响。
图2显示了实施例1中不同浓度甘草酸对测序lag的影响。
具体实施方式
下面将结合实施例对本发明的实施方案进行详细描述，但是本领域技术人员将会理解，下列实施例仅用于说明本发明，而不应视为限定本发明的范围。实施例中未注明具体条件者，按照常规条件或制造商建议的条件进行。所用试剂或仪器未注明生产厂商者，均为可以通过市购获得的常规产品。
实施例
1.实验器材
MGISEQ-2000RS测序仪，MGIDL-200H加载仪，MGISEQ-2000RS测序载片，MGISEQ-2000RS高通量测序试剂套装FCLPE150,仪器得激发波长：分别为532nm,650nm。
2.实验所用试剂和原料
Tris Base(粉末)，氯化钠(粉末)，Tween-20，Tris-HCl(粉末)、甘草酸均购于合规化学试剂供应公司。MGISEQ-2000RS高通量测序试剂盒，货号为1000012536，品牌为华大智 造。大肠杆菌单链环DNA为模板即标准文库试剂V3.0。DNA聚合酶(Cpas DNA Polymerase)来自MGI。DNB纳米球来源于MGI，dATP-1，其是指同时具有可逆阻断基团修饰和Cy5荧光修饰的腺嘌呤核苷酸；dTTP-1，其是指同时具有可逆阻断基团修饰和ROX荧光修饰的胸腺嘧啶核苷酸；dGTP-1，其是指同时具有可逆阻断基团修饰和Cy3荧光修饰的鸟嘌呤核苷酸；以及dCTP-1，其是指同时具有可逆阻断基团修饰和EF700荧光修饰的胞嘧啶核苷酸均来自MGI。
3.实验方法：
(a)扫描试剂的配制
取HotMPS扫描试剂V2.0(HotMPS Image Reagent V2.0)，将一定量的甘草酸粉末加入其中超声溶解20分钟配制成需要的浓度，具体量如表1所示，将上述试剂溶解成透明均一的溶液经过0.22微米的滤膜过滤以待备用。
表1：甘草酸工作液实验条件
空白对照中添加甘草酸的量 甘草酸最终的工作液浓度
0.822g 1mM
1.644g 2mM
2.466g 3mM
备注：空白对照即商品化的HotMPS扫描试剂V2.0。
(b)DNA测序方法：
测序流程：第一步，将DNB纳米球加载到准备好的测序芯片上。
第二步，将准备好的dNTP分子混合溶液泵入芯片用DNA聚合酶将dNTP加入到DNA母链的互补链上。
第三步，拍照扫描，由于dNTP是被修饰的带有荧光基团分子，用激光作为激发波长进行拍照。由于激光对于DNA有光损伤作用，因此在拍照这一步骤应用扫描试剂作为保护剂进行拍照，拍照结束后进行碱基类型的确认。
第四步，通过切除试剂将碱基端荧光基团和3’的阻断切除并洗脱干净，使得3’-OH裸露从而进行下一轮反应。
使用MGISEQ-2000RS高通量测序试剂盒，试剂盒中#10号孔试剂作为扫描试剂的对照品，按照上述的实验流程在MGISEQ-2000RS测序平台进行PE100+70测序，简言之，每次将配制好的扫描试剂作为实验组将试剂盒中#10号的扫描试剂替换作为考察对象，然后统计每个循环的Q30下降幅度，每个循环的测序错误率曲线来评估测序质量。
实施例1评价甘草酸浓度对测序质量的影响
按照上述实验方法将空白对照与不同浓度的甘草酸进行PE150测序，其结果如图1所示，结果表明1.00mM的甘草酸溶液能有效提高二连测序的Q30数值，其最后一个循环的提高率达6.8％。由于浓度过大，抗氧化剂本身会自氧化，不利于测序质量提高，如2.00，3.00mM的甘草酸溶液测试结果所示。
图2显示了不同浓度甘草酸对测序lag的影响，其中1.00mM的甘草酸溶液能有效降低测序二链的lag，可能的原因是：在进行一链测序时，抗氧化剂甘草酸对DNB纳米球以及一链起到了很好的保护作用。其中空白对照的实验条件如表1所示，甘草酸的工作也浓度如表1所示。
以上实施例仅用以说明本发明的技术方案而非限制，对本发明的技术方案进行修改或者等同替换，而不脱离本发明技术方案的宗旨和范围的，其均应涵盖在本发明的保护范围之内。

Claims (9)

  1. 一种试剂，其包含甘草酸、甘草酸的盐或甘草酸的盐的水合物，还包含Tris缓冲溶液；
    优选地，所述甘草酸的盐选自碱金属盐或铵盐；
    优选地，所述甘草酸的盐的水合物选自甘草酸碱金属盐或铵盐的水合物；
    优选地，所述甘草酸的盐或甘草酸的盐的水合物选自：甘草酸单铵盐、甘草酸三钠水合物、甘草酸单钾盐、甘草酸二铵盐、甘草酸二钾水合物；
    优选地，所述试剂中，甘草酸、甘草酸的盐或甘草酸的盐的水合物的浓度为0.5-3mM；
    优选地，所述试剂还包含氯化钠和/或DNA的稳定剂(例如吐温-20)。
  2. 甘草酸、甘草酸的盐或甘草酸的盐的水合物在核酸检测中作为核酸保护剂的用途；
    优选地，所述核酸检测涉及光照反应或非光照反应；
    优选地，所述核酸检测涉及测序反应；
    优选地，所述甘草酸的盐选自碱金属盐或铵盐；
    优选地，所述甘草酸的盐的水合物选自甘草酸碱金属盐或铵盐的水合物；
    优选地，所述甘草酸的盐或甘草酸的盐的水合物选自：甘草酸单铵盐、甘草酸三钠水合物、甘草酸单钾盐、甘草酸二铵盐、甘草酸二钾水合物。
  3. 权利要求2所述的用途，其中，所述核酸检测是核酸序列测定，例如高通量测序；例如SBS测序、连接测序、杂交测序、纳米孔测序、或cPAL测序。
  4. 权利要求2所述的用途，其中，所述核酸检测是定量PCR。
  5. 一种试剂盒，所述试剂盒包含权利要求1的试剂；
    优选地，所述试剂盒还包含一种或多种核酸检测所需的其他试剂，例如引物、聚合酶、缓冲溶液、洗涤溶液，或其任何组合。
  6. 一种核酸序列的检测方法，包括：将一种或多种带标记修饰的核苷酸掺入到与核酸模板链互补的核酸链中，通过检测所述标记确定所述一种或多种掺入的核苷酸的类型，其 中，确定掺入的核苷酸的类型的步骤在包含甘草酸、甘草酸的盐或甘草酸的盐的水合物的缓冲溶液中进行。
  7. 权利要求6的方法，所述标记为可产生荧光信号的标记；
    优选地，所述荧光信号通过光照反应或生物自发光反应产生；
    优选地，所述带标记修饰的核苷酸为：(1)荧光标记的核苷酸(例如dNTP)；或(2)带有标签的核苷酸(例如dNTP)，所述标签能够特异性结合荧光素酶。
  8. 权利要求7所述的方法，其中，所述确定掺入的核苷酸的类型的步骤包括：在甘草酸、甘草酸的盐或甘草酸的盐的水合物存在下，给予允许所述标记产生荧光信号的条件并检测所述缓冲溶液的荧光信号，并确定掺入的核苷酸的身份；
    任选地，所述方法还包括：从掺入的核苷酸中去除与其直接或间接连接的可产生荧光信号的部分；和/或，洗涤以去除未掺入的核苷酸；
    优选地，所述方法包括多次掺入并确定存在于每个掺入的核苷酸中的碱基的身份，以确定靶核酸分子的序列。
  9. 权利要求6-8任一项所述的方法，其中，所述甘草酸的盐选自碱金属盐或铵盐；
    优选地，所述甘草酸的盐的水合物选自甘草酸碱金属盐或铵盐的水合物；
    优选地，所述甘草酸的盐或甘草酸的盐的水合物选自：甘草酸单铵盐、甘草酸三钠水合物、甘草酸单钾盐、甘草酸二铵盐、甘草酸二钾水合物；
    优选地，所述缓冲溶液中，甘草酸、甘草酸的盐或甘草酸的盐的水合物的浓度为0.5-3mM；
    优选地，所述缓冲溶液还包含氯化钠和/或DNA的稳定剂(例如吐温-20)；
    优选地，所述缓冲溶液为扫描试剂。
PCT/CN2022/118117 2022-09-09 2022-09-09 甘草酸或其衍生物在核酸检测中的用途 WO2024050817A1 (zh)

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