Antioxidant composition and use thereof in nucleic acid detectionTechnical FieldThe present invention relates to the field of nucleic acid detection, to an antioxidant composition, to a reagent comprising said composition, to a kit comprising said composition or reagent, to the use of said composition or reagent as a nucleic acid protecting agent in nucleic acid detection, and to a method of nucleic acid detection.
BackgroundIn many nucleic acid detection methods (e.g., DNA sequencing, sequencing by synthesis, sequencing by ligation, etc.), it is necessary to excite a fluorescent group carried on a nucleic acid to be detected (or a complementary nucleic acid thereof) by light irradiation such as laser irradiation, and obtain information of the nucleic acid to be detected by an optical system, base recognition software, etc. The continuous strong irradiation of the excitation light source and the active oxygen groups in the solution can influence the nucleic acid, so that the nucleic acid is damaged or degraded, the strength of fluorescence detection signals is lost, and the number of detection cycles is reduced or the detection accuracy is reduced.
The buffer solution used in the base recognition process is called a scanning reagent or a photographing reagent. Laser damage to the template nucleotide may 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 oxidation and photodamage resistant nucleic acid protective agent, is applied to nucleic acid sequencing, can support long-reading and long-double-ended sequencing, and has lower error rate. However, during long storage, L-ascorbate is oxidized and then undergoes discoloration, which affects the quality of the scan and, in turn, the sequencing of the polynucleotide. Furthermore, in the prior art, the concentration of nucleic acid protecting agent components in some scanning reagents is relatively high. The use of high concentration nucleic acid protecting agents makes the scanning reagents susceptible to salt precipitation during low temperature storage or during long-term sequencing. In addition, the use of a nucleic acid protecting agent at a high concentration may increase the cost of the product.
Disclosure of Invention
The notoginsenoside R1 can be used as a nucleic acid protecting agent, and can also protect template nucleotide under a very low working concentration. In addition, the notoginsenoside R1 is not absorbed in the ultraviolet and visible light region, has no interference on base recognition signals, and can not generate a color change phenomenon even if oxidized in the long-term storage process.
In practical application, the inventor finds that notoginsenoside R1 is relatively suitable for short-reading long single-ended sequencing as a nucleic acid protective agent, and has low sequencing error rate, but when the number of sequencing cycles is large (especially double-ended sequencing), the signal of two chains is back-up and the quality is poor, so that the accuracy and the sequencing quality are required to be improved.
In order to solve the problems, the application provides an antioxidant composition, which comprises notoginsenoside R1 or other saponin compounds, glycyrrhizic acid or derivatives thereof, and 5' -adenosine monophosphate or salts thereof or carnosine, and optionally, other antioxidants. The antioxidant composition achieves the effects of better protecting nucleic acid and reducing photoinduced damage through the combined action of a plurality of antioxidants.
Antioxidant composition
In one aspect, the present application provides a composition comprising:
(1) The saponin compounds are selected from one or more of Notoginseng radix saponin R1, ginsenoside Rg1, ginsenoside Rd, ginsenoside Rb2, ginsenoside Rb3, ginsenoside Rc, ginsenoside Rf, and ginsenoside Re;
(2) Glycyrrhizic acid, its salt or its hydrate, and
(3) Adenosine 5' -monophosphate or a salt thereof, or carnosine;
optionally, the composition further comprises other antioxidants.
In the application, the structural formula of each saponin compound is shown as follows:
in some embodiments, the saponin compound is notoginsenoside R1.
Glycyrrhizic acid is also called glycyrrhizin triterpenoid saponin and glycyrrhizin, belongs to triterpenoid compounds, is named Glycyrrhizic Acid in English, has CAS number of 1405-86-3, has molecular formula of C42H62O16, has molecular weight of 822.93, and has chemical structural formula as follows:
glycyrrhizic acid is easily soluble in hot water and ethanol, has low solubility in cold water, and can be used in the form of salt or salt hydrate to increase water solubility.
In the present application, the salt of glycyrrhizic acid refers to a salt of 1,2 or 3 carboxyl groups in glycyrrhizic acid with a suitable inorganic or organic cation (base), and includes, but is not limited to, alkali metal salts such as sodium salt, 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 base salts such as ammonium salt, organic base salts such as t-octylamine salt, dibenzylamine salt, morpholinium salt, glucosamine salt, phenylglycine alkyl ester salt, ethylenediamine salt, N-methylglucamine salt, guanidine salt, diethylamine salt, triethylamine salt, dicyclohexylamine salt, N' -dibenzylethylenediamine salt, chloroprocaine salt, procaine salt, diethanolamine salt, N-benzyl-phenethylamine salt, piperazine salt, tetramethylamine salt, tris (hydroxymethyl) aminomethane salt.
In some embodiments, the salt of glycyrrhizic acid is selected from alkali metal or ammonium salts, such as monopotassium, monosodium, monoammonium, dipotassium, disodium, diammonium, tripotassium, trisodium, trismmonium salts.
In the present application, the hydrate of glycyrrhizic acid or the hydrate of glycyrrhizic acid salt refers to a substance formed by associating glycyrrhizic acid or glycyrrhizic acid salt with one or more water molecules. The glycyrrhetate may be any of the salts described above.
In some embodiments, the hydrate of the salt of glycyrrhizic acid is selected from the group consisting of hydrates of alkali metal or ammonium salts of glycyrrhizic acid, such as hydrates of sodium or potassium salts.
In some embodiments, the salt of glycyrrhizic acid or hydrate of a salt of glycyrrhizic acid is selected from the group consisting of:
monoammonium glycyrrhetate, an exemplary structure of which is as follows:
trisodium glycyrrhetate hydrate having the following exemplary structure:
a monopotassium glycyrrhizinate, an exemplary structure of which is as follows:
diammonium glycyrrhizinate, an exemplary structure of which is as follows:
dipotassium glycyrrhizinate hydrate, an exemplary structure of which is as follows:
Adenosine 5'-monophosphate (Adenosine' -monophosphate) CAS number 61-19-8, molecular formula C10H14N5O7 P, molecular weight 347.201, chemical structural formula:
salts of adenosine 5' -monophosphate are salts of the phosphate groups in adenosine 5' -monophosphate with 1 or 2 suitable inorganic or organic cations (bases) or salts of the amino groups in adenosine 5' -monophosphate with 1 or 2 suitable inorganic or organic anions (acids).
Salts of the phosphate groups with 1 or 2 suitable inorganic or organic cations (bases) include, but are not limited to, alkali metal salts such as sodium, potassium, lithium, etc., alkaline earth metal salts such as calcium, magnesium, etc., other metal salts such as aluminum, iron, zinc, copper, nickel, cobalt, etc., inorganic base salts such as ammonium salts, organic base salts such as tertiary octylamine salts, dibenzylamine salts, morpholine salts, glucosamine salts, phenylglycine alkyl ester salts, ethylenediamine salts, N-methylglucamine salts, guanidine salts, diethylamine salts, triethylamine salts, dicyclohexylamine salts, N' -dibenzylethylenediamine salts, chloroprocaine salts, procaine salts, diethanolamine salts, N-benzyl-phenethylamine salts, piperazine salts, tetramethylamine salts, tris (hydroxymethyl) aminomethane salts.
Salts of the amino group with 1 or 2 suitable inorganic or organic anions (acids) include, but are not limited to, hydrohalates such as hydrofluoric acid, hydrochloride, hydrobromide, hydroiodide, and the like, inorganic acid salts such as nitrate, perchlorate, sulfate, phosphate, and the like, lower alkane sulfonates such as methanesulfonate, trifluoromethanesulfonate, ethanesulfonate, and the like, aryl sulfonates such as benzenesulfonate, p-benzenesulfonate, and the like, organic acid salts such as acetate, malate, fumarate, succinate, citrate, tartrate, oxalate, maleate, and the like, amino acid salts such as glycinate, trimethylglycinate, arginate, ornithine, glutamate, aspartate, and the like.
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 monosodium adenosine 5' -monophosphate having the chemical formula:
Carnosine (L-Carnosine), the academic name beta-alanyl-L-histidine, is a dipeptide consisting of two amino acids, namely beta-alanine and L-histidine, with CAS number 305-84-0, molecular formula C9H14N4O3, molecular weight 226.24, and chemical structural formula as follows:
In some embodiments, the other antioxidant may be Dithiothreitol (DTT) or Trolox (water-soluble vitamin E).
In some embodiments, the molar ratio of the saponins to (glycyrrhizic acid, salt of glycyrrhizic acid, or hydrate of salt of glycyrrhizic acid) in the composition is 4:1-1:10 (e.g., 4:1-3:1 (e.g., 1.67:0.5), 3:1-2:1, 2:1-1:1 (e.g., 1.67:1.5), 1:1-1:2 (e.g., 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, the molar ratio of the saponins to the adenosine 5' -monophosphate or salt thereof to the carnosine in the composition is 1:1-1:100 (e.g., 1:1-1:5 (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 the saponins to other antioxidants (e.g., DTT, trolox, etc.) in the composition is 1:1-1:50 (e.g., 1:1-1:5 (e.g., 1.67:8), 1:5-1:10, 1:10-1:20 (e.g., 1.67:20), 1:20-1:30, 1:30-1:40, 1:40-1:50).
In some embodiments, the molar ratio of (glycyrrhizic acid, salt of glycyrrhizic acid, or hydrate of salt of glycyrrhizic acid) (5' -adenosine monophosphate or a salt thereof, or carnosine) in the composition is 1:1-1:15 (e.g., 1:1-1:3, 1:3-1:5, 1:5-1:10, or 1:10-1:15).
The proportions of the components in the compositions of the invention may be adjusted as desired (e.g., the proportions described in the embodiments above may be employed) to achieve a suitable oxidation resistance to avoid damage to dntps or DNBs due to excessive oxidation resistance and to reduce sequencing quality.
Reagent, kit and use thereof
In one aspect, the application provides an agent comprising a composition of the application and a buffer solution (e.g., tris buffer solution).
In the present invention, tris buffer means a buffer using Tris (hydroxymethyl) aminomethane (Tris) as a buffer system.
Tris is widely used in the preparation of buffer solutions for biochemistry and molecular biology. Tris is weak base, the pH of the aqueous solution of the Tris is about 10.5, and hydrochloric acid is added to adjust the pH value to a required value, so that the buffer solution with the pH value can be obtained. The buffer solution may also be formulated using Tris and its hydrochloride salt (Tris-HCl).
Thus, in some embodiments, the agent comprises water, the composition of the invention, tris (hydroxymethyl) aminomethane (Tris Base), tris-HCl, optionally together with ethylenediamine tetraacetic acid and/or a solubilizing agent.
In some embodiments, the pH of the reagent is 6.0 to 9.0 (e.g., 6.0 to 7.0, 7.0 to 8.0, or 8.0 to 9.0).
In some embodiments, the concentration of glycyrrhizic acid, a salt of glycyrrhizic acid, or a hydrate of a salt of glycyrrhizic acid is 0.1 to 10mM, for example 0.1 to 0.5mM, 0.5 to 1mM, 1 to 1.5mM, 1.5 to 2.5mM, 2.5 to 3mM, 3 to 5mM, 5 to 7mM, or 7 to 10mM.
In some embodiments, the concentration of the saponins is 0.1 to 5mM, such as 0.1 to 0.5mM, 0.5 to 1mM, 1 to 1.5mM, 1.5 to 2.0mM (e.g., 1.67 mM), 2.0 to 2.5mM, 2.5 to 3mM, 3 to 4mM, or 4 to 5mM. Saponins are limited by their own solubility in the reagent, and excessive amounts are difficult to dissolve and even insoluble. The concentration ranges are selected so that the saponins are completely dissolved in the reagent.
In some embodiments, the concentration of the adenosine 5' -monophosphate or salt thereof or carnosine is 0.1 to 200mm, e.g., 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 to 200mm.
In some embodiments, the concentration of the additional antioxidant is 0.1 to 50mM, such as 0.1 to 0.5mM, 0.5 to 1mM, 1 to 1.5mM, 1.5 to 2.5mM, 2.5 to 3mM, 3 to 5mM, 5 to 7.5mM, 7.5 to 10mM, 10 to 30mM, or 30 to 50mM.
In some embodiments, the reagent is a scanning reagent.
In some embodiments, the scanning reagent comprises 1-2M Tris buffer, 1-2 mM notoginsenoside R1, 0.5-2.5 mM glycyrrhizic acid, salts of glycyrrhizic acid or hydrates of salts of glycyrrhizic acid, 7-8 mM adenosine 5' -monophosphate or salts thereof or carnosine, 8~9mM Trolox,9~10mM ethylenediamine tetraacetic acid, and 10-20 mM DTT.
In certain embodiments, the agent further comprises sodium chloride and/or a stabilizer for DNA (e.g., tween-20). The sodium chloride serves to provide a saline background and protect the primers in the assay.
In another aspect, the application provides the use of a composition or reagent of the application as a nucleic acid protecting agent in nucleic acid detection.
In certain embodiments, the nucleic acid protecting agent is used to protect nucleic acids, reduce or avoid photodamage (photoinduced damage) or oxidative damage.
In certain embodiments, the nucleic acid detection involves detecting a fluorescent signal. In certain embodiments, the fluorescent signal may be generated by a photoreaction or a non-photoreaction (e.g., a bioluminescence reaction). In certain embodiments, the bioluminescent reaction refers to a reaction in which luciferase catalyzes its substrate to generate a fluorescent signal.
In certain embodiments, the nucleic acid detection involves a photoreaction or a non-photoreaction (e.g., bioluminescence reaction).
The term "photoreaction" as used herein refers to a reaction that is exposed to an optical energy source. Typically in such reactions, an optical energy source (light) is provided to observe the production and/or consumption of reactants or products having particular optical characteristics indicative of their presence, such as changes in absorption spectrum and/or emission spectrum (changes in intensity, wavelength, etc.) of the reaction mixture or components thereof.
The term "non-photoreactive reaction" as used herein refers to a reaction that can be detected without the aid of an optical energy source. In a non-illuminated reaction, the generation and/or consumption of a reactant or product may be detected by generating an optical signal by means such as bioluminescence or by a change in an electrical signal.
In certain embodiments, the photoreaction includes an optical signal initiated by base extension or an optical signal initiated by hybridization of a probe, which base extension may cause the generation of an optical signal (e.g., a fluorescent signal).
In certain embodiments, the optical signal in the photoreaction according to the present invention is preferably fluorescence.
In certain embodiments, the nucleic acid detection is nucleic acid sequencing (sequencing) or other detection, such as high throughput sequencing, e.g., sequencing-by-synthesis (SBS sequencing), sequencing-by-ligation, sequencing-by-hybridization, nanopore sequencing, or composite probe-anchored 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 application.
In certain embodiments, the kits of the invention may further comprise one or more additional reagents required for detection of nucleic acids, e.g., primers, polymerase, buffer solution, wash solution, or any combination thereof.
In certain embodiments, the kits of the invention are used for nucleic acid sequence determination.
In certain embodiments, the kits of the invention may further comprise reagents for immobilizing (e.g., by covalent or non-covalent attachment) the nucleic acid molecule to be sequenced to a support, primers for initiating nucleotide polymerization, a polymerase for conducting nucleotide polymerization, one or more buffer solutions, one or more wash solutions, or any combination thereof.
In certain embodiments, the kits of the invention may further comprise reagents and/or devices for extracting nucleic acid molecules from a sample. Methods for extracting nucleic acid molecules from a sample are well known in the art. Thus, various reagents and/or devices for extracting nucleic acid molecules, such as reagents for disrupting cells, reagents for precipitating DNA, reagents for washing DNA, reagents for dissolving DNA, reagents for precipitating RNA, reagents for washing RNA, reagents for dissolving RNA, reagents for removing proteins, reagents for removing DNA (e.g., when the nucleic acid molecule of interest is RNA), reagents for removing RNA (e.g., when the nucleic acid molecule of interest is DNA), and any combination thereof, may be configured in the kits of the invention as desired.
In certain embodiments, the kits of the invention further comprise reagents for pre-treating the nucleic acid molecules. In the kit of the present invention, the reagent for pretreating a nucleic acid molecule is not additionally limited and may be selected according to actual needs. The reagents for pre-treating the nucleic acid molecule include, for example, reagents for fragmenting the nucleic acid molecule (e.g., dnase I), reagents for replenishing the ends of the nucleic acid molecule (e.g., DNA polymerase, e.g., T4DNA polymerase, pfu DNA polymerase, klenow DNA polymerase), linker molecules, tag molecules, reagents for ligating the linker molecules to the nucleic acid molecule of interest (e.g., a ligase such as T4DNA ligase), reagents for repairing the ends of the nucleic acid (e.g., a DNA polymerase that loses 3'-5' exonuclease activity but exhibits 5'-3' exonuclease activity), reagents for amplifying the nucleic acid molecule (e.g., DNA polymerase, primers, dntps), reagents for separating and purifying the nucleic acid molecule (e.g., a chromatographic column), and any combination thereof.
In certain embodiments, the kits of the invention further comprise a support for immobilizing the nucleic acid molecule to be sequenced. In general, the support for immobilizing the nucleic acid molecule to be sequenced is in a solid phase for ease of handling. Thus, in the present disclosure, a "support" is sometimes also referred to as a "solid support" or "solid support". However, it should be understood that references herein to a "support" are not limited to solids, but may also be semi-solids (e.g., gels).
As used herein, the terms "loaded," "immobilized," and "attached," when used in reference to a nucleic acid, mean directly or indirectly attached to a solid support via covalent or non-covalent bonds. In certain embodiments of the present disclosure, the methods of the invention comprise immobilizing a nucleic acid on a solid support via covalent attachment. But in general, it is only necessary that the nucleic acid remain immobilized or attached to the solid support under conditions where it is desired to use the solid support (e.g., in applications requiring nucleic acid amplification and/or sequencing). In certain embodiments, immobilizing the nucleic acid on the solid support may include immobilizing an 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 a portion of the primer sequence is capable of hybridizing to a complementary nucleic acid sequence, and then hybridizing the nucleic acid to be immobilized to the oligonucleotide, in which case the immobilized oligonucleotide or polynucleotide may be in the 3' -5' orientation. In certain embodiments, immobilizing the nucleic acid on the solid support may include binding the 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 other means than base pairing hybridization, such as covalent attachment as described above. Non-limiting examples of attachment of nucleic acids to solid supports include nucleic acid hybridization, biotin streptavidin binding, sulfhydryl binding, photoactivated binding, covalent binding, antibody-antigen, physical confinement via hydrogels or other porous polymers, and the like. Various exemplary methods for immobilizing nucleic acids on a solid support can be found, for example, in G.Steinberg-Tatman et al, bioconjugate Chemistry 2006,17,841-848; xu X. Et al Journal of THE AMERIC 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, all of which are incorporated herein by reference in their entirety, especially for all purposes in connection with the preparation of solid supports having nucleic acids immobilized thereon.
In the present invention, the support may be made of various suitable materials. Such materials include, for example, inorganic substances, natural polymers, synthetic polymers, and any combination thereof. Specific examples include, but are not limited to, cellulose derivatives (e.g., nitrocellulose), acrylic resins, glass, silica gel, silica, polystyrene, gelatin, polyvinylpyrrolidone, copolymers of vinyl and acrylamide, polystyrene connective crosslinked with divinylbenzene, etc. (see, e.g., MERRIFIELD BIOCHEMISTRY 1964,3,1385-1390), polyacrylamide, latex, dextran, rubber, silicon, plastics, natural sponges, metal plastics, crosslinked dextran (e.g., sephadexTM), sepharose (SepharoseTM), and other supports known to those skilled in the art.
In certain preferred embodiments, the support for immobilizing the nucleic acid molecule to be sequenced may be a solid support comprising an inert substrate or matrix (e.g., slide, polymer bead, etc.) that has been functionalized, for example, by the application of an intermediate material 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, particularly polyacrylamide hydrogels described in WO 2005/065814 and US 2008/0280773, the contents of which are incorporated herein by reference in their entirety. In such embodiments, the biomolecule (e.g., polynucleotide) may be directly covalently linked to the intermediate material (e.g., hydrogel), and the intermediate material itself may be non-covalently linked to the substrate or matrix (e.g., glass substrate). In certain preferred embodiments, the support is a glass slide or silicon wafer surface modified with a layer of avidin, amino, acrylamidosilane, or aldehyde-based 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 for immobilizing the nucleic acid molecules to be sequenced is an array of beads or wells (which are also referred to as a chip). The array may be prepared using any of the materials outlined herein for the preparation of solid supports, and preferably the surface of the beads or wells on the array are 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 comprise 10-102、102-103、103-104、104-105、105-106、106-107、107-108、108-109、1010-1011、1011-1012 or more beads or wells. In certain exemplary embodiments, the surface of each bead or well may immobilize one or more nucleic acid molecules. Accordingly, each array may immobilize 10-102、102-103、103-104、104-105、105-106、106-107、107-108、108-109、1010-1011、1011-1012 or more nucleic acid molecules. Thus, such arrays may be particularly advantageous for high throughput sequencing of nucleic acid molecules.
In certain preferred embodiments, the kits of the invention further comprise reagents for immobilizing (e.g., by covalent or non-covalent attachment) the nucleic acid molecule to be sequenced to a support. Such agents include, for example, agents that activate or modify a nucleic acid molecule (e.g., at its 5' end), such as phosphoric acid, thiols, amines, carboxylic acids, or aldehydes, agents that activate or modify the surface of a support, such as amino-alkoxysilane (e.g., aminopropyl trimethoxysilane, aminopropyl triethoxysilane, 4-aminobutyl triethoxysilane, etc.), cross-linking agents, such as succinic anhydride, phenyldiisoisothiocyanate (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-gamma-maleimidyloxy-succinimidyl ester (GMBS), 4- (p-maleimidophenyl) butanoic acid Succinimide (SMPB), and any combination thereof.
In certain preferred embodiments, the kits of the invention further comprise primers for initiating nucleotide polymerization. In the present invention, the primer is not additionally limited as long as it can specifically anneal to a region of the target nucleic acid molecule. In some exemplary embodiments, the primers may be 5-50bp in length, e.g., 5-10, 10-15, 15-20, 20-25, 25-30, 30-35, 35-40, 40-45, 45-50bp. In some exemplary embodiments, the primer may comprise naturally occurring or non-naturally occurring nucleotides. In some exemplary embodiments, the primer comprises or consists of naturally occurring nucleotides. In some exemplary embodiments, the primer comprises a modified nucleotide, such as a Locked Nucleic Acid (LNA). In certain preferred embodiments, the primer comprises a universal primer sequence.
In certain preferred embodiments, the kits of the invention further comprise a polymerase for performing nucleotide polymerization reactions. In the present invention, polymerization may be carried out using various suitable polymerases. In some exemplary embodiments, the polymerase is capable of synthesizing a new DNA strand (e.g., a DNA polymerase) using DNA as a template. In some exemplary embodiments, the polymerase is capable of synthesizing a new DNA strand (e.g., reverse transcriptase) using RNA as a template. In some exemplary embodiments, the polymerase is capable of synthesizing a new RNA strand (e.g., RNA polymerase) using DNA or RNA as a template. 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, buffers for DNase I, buffers for DNA polymerase, buffers for ligase, buffers for eluting nucleic acid molecules, buffers for dissolving nucleic acid molecules, buffers for performing nucleotide polymerization reactions (e.g., PCR), and buffers for performing ligation reactions. Kits of the invention may comprise any one or more of the buffer solutions described above.
In the present invention, "buffer" and "buffer solution" have the same meaning and may be used interchangeably.
In certain embodiments, the buffer solution for DNA polymerase comprises monovalent salt ions (e.g., sodium ions, chloride ions) and/or divalent salt ions (e.g., magnesium ions, sulfate ions, manganese ions). In certain embodiments, the monovalent or divalent salt ion is present in the buffer solution at a concentration of 10. Mu.M to 200mM, e.g., 10. Mu.M, 50. Mu.M, 100. Mu.M, 200. Mu.M, 500. Mu.M, 1mM, 3mM, 10mM, 20mM, 50mM, 100mM, 150mM, or 200mM.
In certain embodiments, the buffer solution for DNA polymerase comprises Tris (hydroxymethyl aminomethane). In certain embodiments, the concentration of Tris in the buffer solution is 10mM-200mM, e.g., 10mM, 20mM, 50mM, 100mM, 150mM, or 200mM.
In certain embodiments, the buffer solution for DNA polymerase comprises an organic solvent, such as DMSO or glycerol (glycerin). In certain embodiments, the organic solvent is present in the buffer solution in an amount of 0.01% -10% by mass, 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, e.g., 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 a DNA polymerase comprises monovalent salt ions (e.g., sodium ions, chloride ions), divalent salt ions (e.g., magnesium ions, sulfate ions, manganese ions), tris, and an organic solvent (e.g., DMSO or glycerol). In certain embodiments, the buffer solution phase has a pH of 8.8.
In certain preferred embodiments, the kits of the invention further comprise one or more wash solutions. Examples of such wash solutions include, but are not limited to, phosphate buffers, citrate buffers, tris-HCl buffers, acetate buffers, carbonate buffers, and the like. Kits of the invention may comprise any one or more of the wash solutions described above.
In another aspect, the application provides the use of a composition, reagent or kit of the application for nucleic acid detection. In certain embodiments, the nucleic acid detection involves detecting a fluorescent signal. In certain embodiments, the fluorescent signal may be generated by a reaction that is photoreactive or non-photoreactive (e.g., bioluminescent). In certain embodiments, the bioluminescence refers to a reaction of a luciferase enzyme that catalyzes a substrate thereof to produce a fluorescent signal. In certain embodiments, the nucleic acid detection involves a photoreactive non-photoreactive (e.g., bioluminescent reaction). In certain embodiments, the photoreaction includes base extension that can cause the generation of an optical signal (e.g., a fluorescent signal). . In certain embodiments, the optical characteristic of the illumination reaction described herein is preferably fluorescence. In certain embodiments, the nucleic acid detection can be used for nucleic acid sequencing (sequencing) or other scenarios (e.g., quantitative PCR). In certain embodiments, the nucleic acid detection is a nucleic acid sequence determination, such as high throughput sequencing, e.g., 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 application, comprising dissolving each component of the reagent in ultrapure water to prepare a transparent uniform solution, and then filtering the solution to obtain the reagent of the present application. Ultrasound-assisted dissolution may be used.
In another aspect, the application provides a method of inhibiting degradation of a nucleic acid comprising using a composition or agent of the application as a nucleic acid protecting agent. In some embodiments, the nucleic acid degradation is photo-induced nucleic acid degradation or oxidation-induced nucleic acid degradation. In some embodiments, the method comprises subjecting a reaction mixture comprising nucleic acids to a photoreaction or a non-photoreaction (e.g., bioluminescence reaction) in the presence of the composition or reagent.
In another aspect, the application provides a method of detecting a target nucleic acid molecule comprising using a composition or reagent of the application as a nucleic acid protecting agent. In some embodiments, the methods comprise subjecting a reaction mixture comprising a target nucleic acid molecule to a photoreaction or a non-photoreaction (e.g., bioluminescence reaction) in the presence of the composition or reagent.
In certain embodiments, the method comprises signal acquisition and detection of a fluorescent signal from the reaction mixture, wherein the reaction mixture comprises a reagent that generates a fluorescent signal (e.g., a fluorescent-labeled reagent), a target nucleic acid molecule, and a buffer comprising a composition of the invention.
In certain embodiments, the fluorescent signal is generated by an illumination reaction or a bioluminescence reaction.
In certain embodiments, the method comprises subjecting the reaction mixture to light irradiation to detect a fluorescent signal from the light irradiation reaction, wherein the reaction mixture comprises a fluorescent labeling reactant, a target nucleic acid molecule, and a buffer comprising a composition of the invention.
In certain embodiments, the reaction mixture in which the photoreaction is performed comprises a composition or reagent of the present 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, sequencing by ligation, sequencing by hybridization, nanopore sequencing, or cPAL sequencing.
In certain embodiments, the fluorescent signal-producing reactant comprises labeled or unlabeled nucleotides (e.g., dNTPs), optionally further comprising other reagents that allow the reactant to produce a fluorescent signal. In certain embodiments, each reactant that can generate a fluorescent signal corresponds to a nucleotide type, and each reactant can generate a signal that is distinguishable from the other to identify incorporation of a particular nucleotide. For example, four reactants, each containing adenine, guanine, cytosine and thymine to be incorporated, can generate different fluorescent signals, making them readily distinguishable from one another.
In certain embodiments, the nucleotides in the fluorescent signal-producing reactant (e.g., fluorescently labeled nucleotides) also bear a blocking group (e.g., a 3' blocking group) to reversibly prevent further base extension.
In certain embodiments, the nucleotide (e.g., fluorescently labeled nucleotide) in the fluorescent signal-producing reactant is selected from nucleoside polyphosphates (or analogs thereof), such as dntps.
In certain embodiments, the reaction mixture further comprises an enzyme, such as a polymerase, helicase, exonuclease, or ligase, preferably the reaction mixture comprises a polymerase, such as a DNA polymerase.
In certain embodiments, the reaction mixture further comprises a primer.
In certain embodiments, the method comprises incorporating nucleotides (e.g., fluorescently labeled nucleotides) in the fluorescent signal-producing reactant into the complementary strand of a target nucleic acid molecule, administering conditions that allow the reactant to produce a fluorescent signal in the presence of a composition or reagent of the present invention and detecting the fluorescent signal of the reaction mixture (e.g., illuminating the reaction mixture), and determining the identity of the incorporated nucleotides. In certain embodiments, the determining the identity of the incorporated nucleotide comprises detecting (e.g., photographing) a fluorescent signal (e.g., fluorescent label) associated with the incorporated nucleotide.
In certain embodiments, the method further comprises removing a portion (e.g., a fluorescent label) directly or indirectly attached thereto that can generate a fluorescent signal from the incorporated nucleotide and/or washing to remove unincorporated nucleotides. In certain embodiments, the method further comprises removing blocking groups from the incorporated nucleotide to allow further extension.
In certain embodiments, the method comprises multiple incorporation and determination of the identity of the bases present in each incorporated nucleotide to determine the sequence of the target nucleic acid molecule.
In certain embodiments, the target nucleic acid molecule is 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 to a solid support, such as a chip.
In another aspect, the application provides a method of detecting a nucleic acid sequence comprising incorporating one or more modified nucleotides with a label into a nucleic acid strand complementary to a nucleic acid template strand, determining the type of the one or more incorporated nucleotides by detecting the label, wherein the step of determining the type of incorporated nucleotide is performed in a buffer comprising a composition of the application.
In certain embodiments, the label is a label that can generate a fluorescent signal. In certain embodiments, the fluorescent signal is generated by an illumination reaction or a bioluminescence reaction. In certain embodiments, the methods are used for nucleic acid sequencing. In certain embodiments, the labeled modified nucleotide is (1) a fluorescently labeled nucleotide (e.g., dNTP), or (2) a nucleotide bearing a tag (e.g., dNTP) that is capable of specifically binding to luciferase.
In certain embodiments, the step of determining the type of nucleotide incorporated comprises administering conditions that allow the label to generate a fluorescent signal and detecting the fluorescent signal of the buffer solution in the presence of the composition of the invention and determining the identity of the nucleotide incorporated. Optionally, the method further comprises removing the portion directly or indirectly attached thereto that can generate a fluorescent signal from the incorporated nucleotide and/or washing to remove unincorporated nucleotides. In certain embodiments, the method comprises multiple incorporation and determination of the identity of the bases present in each incorporated nucleotide to determine the sequence of the target nucleic acid molecule.
In certain embodiments, the buffer solution is Tris buffer solution. In certain embodiments, the pH of the buffer solution is 6.0 to 9.0. In some embodiments, the concentration of glycyrrhizic acid, a salt of glycyrrhizic acid or a hydrate of a salt of glycyrrhizic acid in the buffer solution is 0.1-10 mM. In certain embodiments, the concentration of the 5' -monophosphate adenosine or a salt thereof or carnosine in the buffer solution is 0.1 to 200mm. In certain embodiments, the concentration of the additional antioxidant in the buffer solution is 0.1 to 50mM. 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, sequencing by ligation, sequencing by hybridization, nanopore sequencing, or cPAL sequencing.
Sequencing based on photoreaction
In certain embodiments, the fluorescent signal is generated by a photoreaction. In such embodiments, the label is preferably a fluorescent label. In certain embodiments, the method comprises irradiating the reaction mixture with light, detecting a fluorescent signal from the light reaction, wherein the reaction mixture comprises a reagent that generates a fluorescent signal, a target nucleic acid molecule, and a buffer comprising a composition of the invention. In certain embodiments, the reaction mixture in which the photoreaction is performed comprises a composition or reagent of the present invention.
In certain embodiments, the reagent that can generate a fluorescent signal comprises a fluorescently labeled nucleotide (e.g., dNTP). In certain embodiments, the fluorescent label may be attached to the nucleotide (e.g., its base) via a linker. The linker may be acid labile, photolabile or contain disulfide bonds.
In certain embodiments, the method comprises incorporating fluorescent-labeled nucleotides into the 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 identity of the incorporated nucleotides. In certain embodiments, the determining the identity of the incorporated nucleotide comprises detecting (e.g., photographing) a fluorescent label of the incorporated nucleotide. In certain embodiments, the method further comprises removing the fluorescent label attached thereto from the incorporated nucleotide and/or washing to remove unincorporated nucleotide. In certain embodiments, the method further comprises removing blocking groups from the incorporated nucleotide to allow further extension.
In certain embodiments, the reagents that can generate a fluorescent signal include unlabeled nucleotides (e.g., dNTPs) and a fluorescently labeled affinity reagent (e.g., an antibody) that is capable of specifically binding to the unlabeled nucleotides. Such an embodiment may be referred to as Cool MPS sequencing, the detailed teachings of which may be found in PCT international application WO2018129214A1. In such embodiments, the fluorescent groups are not directly labeled on the incorporated nucleotide, but are labeled on an affinity reagent (e.g., antibody, aptamer, affimer, knottin, etc.) that can specifically bind to the base, sugar, cleavable blocking group, or combination of these components incorporated into the nucleotide based on SBS sequencing principles, and thus the type of nucleotide incorporated can be identified by the affinity reagent.
In certain embodiments, the method comprises incorporating unlabeled nucleotides into the complementary strand of the target nucleic acid molecule, providing a fluorescently labeled affinity reagent and indirectly attaching the fluorescent label to the incorporated nucleotides via specific binding between the affinity reagent and the nucleotides, irradiating the reaction mixture in the presence of a composition or reagent of the present invention, and determining the identity of the incorporated nucleotides. In certain embodiments, the determining the identity of the incorporated nucleotide comprises detecting (e.g., photographing) a fluorescent label of an affinity reagent to which the incorporated nucleotide is attached. In certain embodiments, the method further comprises removing the affinity reagent attached thereto from the incorporated nucleotide and/or washing to remove unincorporated nucleotide. In certain embodiments, the method further comprises removing blocking groups from the incorporated nucleotide to allow further extension.
Sequencing based on biological self-luminous reaction
In certain embodiments, the fluorescent signal is generated by a bioluminescence reaction. The detection principle of bioluminescence includes that a fluorescent signal is not directly marked on a nucleotide to be incorporated, but an affinity substance such as biotin or digoxin is marked, after polymerization, a pairing member of the affinity substance with luciferase is added, thereby binding the luciferase to the incorporated nucleotide, and then a reaction substrate is added to generate a light signal to identify the identity of the incorporated nucleotide, and the process does not require excitation light irradiation. Detailed teachings about bioluminescent reactions can be found in, for example, PCT International application WO2020227953A1.
In certain embodiments, the reagents that generate a fluorescent signal include a nucleotide bearing a tag (e.g., dNTP), a luciferase that is capable of specifically binding the tag, and a substrate for the luciferase.
In certain embodiments, the nucleotide carries a tag that is a member of any pair of molecules that are capable of specifically binding to each other. Specific binding between the members of the pairing effects ligation of the nucleotide to the luciferase. Exemplary pairing members include, but are not limited to, (a) hapten or antigenic compound such as digoxin-digoxin antibodies, N3G-N3G antibodies, FITC-FITC antibodies in combination with a corresponding antibody or binding portion or fragment thereof, (b) nucleic acid aptamers and proteins, (c) non-immune binding pairs (e.g., biotin-avidin, biotin-streptavidin, biotin-neutravidin), (d) hormone-hormone binding protein, (e) receptor-agonists or antagonists, (f) lectin-carbohydrate, (G) enzyme-enzyme cofactors, (h) enzyme-enzyme inhibitors, and (i) complementary oligonucleotide or polynucleotide pairs capable of forming a nucleic acid duplex.
In certain embodiments, the nucleotide carries a label that is a small molecule label selected from biotin, digoxin, N3G, or FITC, and the luciferase carries a pairing member that corresponds to the small molecule label. For example, in one embodiment, the nucleotide carries a label that is biotin, then the luciferase may be streptavidin-labeled luciferase, and the nucleotide carries a label that is digoxin, then the luciferase may be digoxin antibody-labeled luciferase. Such sources of luciferase include, but are not limited to, firefly, gaussia, renilla et al organisms. For example, the Streptavidin-labeled luciferase may be SA-Gluc from Adivity, streptavidin-Gaussia princeps luciferase. The digoxin antibody labeled luciferase may be digoxin antibody-Gluc or digoxin antibody-Nluc.
In certain embodiments, the method comprises incorporating a nucleotide bearing a tag into a complementary strand of a target nucleic acid molecule, providing a luciferase enzyme linked to a pair member capable of specifically binding the tag and indirectly linking the luciferase enzyme to the incorporated nucleotide by specific binding between the pair members, and providing a substrate for the luciferase enzyme in the presence of a composition or reagent of the invention to generate a fluorescent signal to thereby determine the identity of the incorporated nucleotide. In certain embodiments, the method further comprises removing the luciferase linked thereto from the incorporated nucleotide and/or washing to remove unincorporated nucleotide. In certain embodiments, the method further comprises removing blocking groups from the incorporated nucleotide to allow further extension.
In certain embodiments, the methods may also be used for quantitative PCR. In certain embodiments, the reagent that can generate a fluorescent signal is a fluorescent probe. In certain embodiments, the reaction mixture further comprises an enzyme, such as a polymerase, helicase, exonuclease, or ligase, preferably the reaction mixture comprises a polymerase, such as a DNA polymerase. In certain embodiments, the reaction mixture further comprises a primer.
Synthetic sequencing will be discussed below as an example, which is not meant to be limiting. All uses and methods of 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 application provides a method of nucleic acid sequencing comprising using the composition, reagent or kit of the application. In certain embodiments, the sequencing methods of the application comprise performing a scanning photographic detection while synthesizing a growing polynucleotide complementary to the single stranded polynucleotide of interest.
In certain embodiments, the method of determining the sequence of a single stranded polynucleotide of interest comprises:
(a) Providing a duplex comprising a growing nucleic acid strand and a nucleic acid molecule to be sequenced;
(b) Performing a reaction cycle comprising the following steps (i), (ii) and (iii):
Step (i) incorporating the nucleotide into the growing nucleic acid strand using a polymerase to form a nucleic acid intermediate comprising 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 invention;
step (iii) removing blocking groups on the nucleic acid intermediate using a cleavage reagent.
In certain embodiments, the reaction cycle further comprises step (iv) of removing the detectable label on the nucleic acid intermediate using a cleavage reagent.
In certain embodiments, the method comprises the steps of:
firstly, loading DNA nanospheres (DNA nanoball, DNB for short) onto a prepared sequencing chip;
Step two, pumping the prepared dNTP molecular mixed solution into a chip, and adding dNTP onto a complementary strand of the DNA to be detected by using DNA polymerase;
Thirdly, photographing and scanning, wherein dNTPs are modified molecules with fluorescent groups, and photographing is carried out by using laser as excitation wavelength; since the laser has a photodamage effect on DNA, a scanning reagent containing a nucleic acid protecting agent is added in the photographing step to photograph;
Fourthly, cutting off and eluting the base end fluorescent group and the 3 'through a cutting reagent, so that the 3' -OH is exposed to carry out the next round of reaction;
And fifthly, analyzing the photographing result to determine the base sequence of the nucleic acid molecule to be detected.
In the present invention, the nucleic acid may include a nucleotide or a nucleotide analog. Nucleotides generally contain a sugar, a nucleobase and at least one phosphate group. The nucleotides include deoxyribonucleotides, modified deoxyribonucleotides, ribonucleotides, modified ribonucleotides, peptide nucleotides, modified phosphate sugar backbone nucleotides, and mixtures thereof. Examples of nucleotides include, for example, adenosine Monophosphate (AMP), adenosine Diphosphate (ADP), adenosine Triphosphate (ATP), thymine Monophosphate (TMP), thymine Diphosphate (TDP), thymine Triphosphate (TTP), cytidine diphosphate (CMP), cytidine triphosphate (CDP), cytidine Triphosphate (CTP), guanosine Monophosphate (GMP), guanosine Diphosphate (GDP), guanosine Triphosphate (GTP), uridine Monophosphate (UMP), uridine Diphosphate (UDP), uridine Triphosphate (UTP), deoxyadenylate (dabp), deoxyadenosine diphosphate (dADP), deoxyadenosine triphosphate (dATP), deoxythymidine diphosphate (dTTP), deoxycytidine triphosphate (dCTP), deoxyguanosine triphosphate (dGMP), deoxyguanosine diphosphate (dGDP), deoxyuridine triphosphate (dGTP), deoxyuridine monophosphate (dGTP), deoxyuridine diphosphate (dGTP), and deoxyuridine diphosphate (dGTP). Nucleotide analogs comprising modified nucleobases can also be used in the methods described herein. Exemplary modified nucleobases that may be included in polynucleotides, whether having a natural backbone or similar structure, include, for example, inosine, xanthine, hypoxanthine, isocytosine, isoguanine, 2-amino-purine, 5-methylcytosine, 5-hydroxymethylcytosine, 2-amino-adenine, 6-methyladenine, 6-methylguanine, 2-propylguanine, 2-propyladenine, 2-thiouracil, 2-thiothymine, 2-thiocytosine, 15-halouracil, 15-halocytosine, 5-propynyluracil, 5-propynylcytosine, 6-azouracil, 6-azocytosine, 6-azothymine, 5-uracil, 4-thiouracil, 8-haloadenine or guanine, 8-amino-adenine or guanine, 8-thioadenine or guanine, 8-hydroxy-adenine or guanine, 5-halo-substituted uracil or cytosine, 7-methylguanine, 7-methyladenine, 8-azaadenine, 7-azaadenine, 3-azaadenine, and the like. As known in the art, certain nucleotide analogs cannot be incorporated into polynucleotides, for example, nucleotide analogs such as adenosine 5' -phosphosulfate.
In the method of the present invention, the nucleic acid molecule to be sequenced is not limited by its length. In certain preferred embodiments, the nucleic acid molecule to be sequenced may be at least 10bp, at least 20bp, at least 30bp, at least 40bp, at least 50bp, at least 100bp, at least 200bp, at least 300bp, at least 400bp, at least 500bp, at least 1000bp, or at least 2000bp in length. In certain preferred embodiments, the nucleic acid molecule to be sequenced may 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 length. In certain preferred embodiments, the nucleic acid molecules to be sequenced may have a length of 10-1000bp to facilitate high throughput sequencing.
In certain preferred embodiments, the nucleic acid molecule may be pre-treated prior to immobilization of the nucleic acid molecule on the support. Such pretreatments include, but are not limited to, fragmentation of nucleic acid molecules, end-to-end ligation, addition of linkers, addition of tags, amplification of nucleic acid molecules, isolation and purification of nucleic acid molecules, and any combination thereof.
The term "nanospheres" as used herein generally refers to macromolecules or complexes having a compact, e.g., a (near) spherical shape, typically having an inner diameter ranging between about 1nm and about 1000nm, preferably between about 50nm and about 500nm.
The term "nucleic acid nanospheres" as used herein is generally concatemers comprising multiple copies of a target nucleic acid molecule. These nucleic acid copies are typically arranged one after the other 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, causes a nanosphere folding (folding) configuration. In general, multiple copies of a target nucleic acid molecule in a nucleic acid nanosphere each comprise a linker sequence of known sequence to facilitate amplification or sequencing thereof. The linker sequences of the target nucleic acid molecules are typically identical, but may also be different. Nucleic acid nanospheres typically comprise DNA nanospheres, also referred to herein as DNB (DNA nanoball).
Nucleic acid nanospheres can be produced using, for example, rolling circle Replication (RCA). The RCA process has been used to prepare multiple sequential copies of the M13 genome (Blanco et al, (1989) J Biol Chem 264:8935-8940). In this method, the nucleic acid is replicated via linear concatamerization. Guidance regarding the selection of conditions and reagents for RCA reactions can be found by those skilled in the art in a number of references, including U.S. Pat. nos. 5,426,180,854,033, US6,143,495 and US5,871,921, which are incorporated herein by reference in their entirety for all purposes, particularly for the full teachings regarding the preparation of nucleic acid nanospheres using RCA or other methods.
The nucleic acid nanospheres may be loaded onto the surface of a solid support as described herein. The nanospheres may be attached to the surface of the solid support by any suitable method, non-limiting examples of which include nucleic acid hybridization, biotin streptavidin binding, sulfhydryl binding, photoactivated binding, covalent binding, antibody-antigen, physical confinement via hydrogels or other porous polymers, and the like, or combinations thereof. In some cases, the nanospheres may be digested with nucleases (e.g., DNA nucleases) to produce smaller nanospheres or fragments from the nanospheres.
In certain embodiments, the solid support surface may bear reactive functional groups that react with complementary functional groups on the polynucleotide molecules to form covalent bonds, for example, in the same manner as the techniques used to attach cDNA to microarrays, see, e.g., smirnov et al (2004), genes, chromosomes & Cancer, 4:72-77, 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 (e.g. -OH groups). The attachment via covalent bonds formed between the polynucleotide molecule and the reactive functional groups on the surface is also referred to herein as "chemical attachment".
In other embodiments, the polynucleotide molecule may be adsorbed onto a surface. In such embodiments, the polynucleotide is immobilized by non-specific interactions with the surface, or by non-covalent interactions such as hydrogen bonding, van der Waals forces, and the like.
In other embodiments, the nucleic acid library may be a double-stranded nucleic acid fragment immobilized on the surface of a solid support by ligation with oligonucleotides immobilized on the surface of the solid support, followed by a bridge amplification reaction to prepare the sequencing library.
Advantageous effectsThe invention can realize the following beneficial effects:
The antioxidant composition can be used as a nucleic acid protective agent in nucleic acid detection, especially for double-end long-read long sequencing, can simultaneously improve the two-chain signal return value and the Q30 value, and reduces the error rate.
Embodiments of the present invention will be described in detail below with reference to the accompanying drawings and examples, but it will be understood by those skilled in the art that the following drawings and examples are only for illustrating the present invention and are not to be construed as limiting 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 following detailed description of the preferred embodiments and the accompanying drawings.
DrawingsFIG. 1 is a graph showing the Q30 (%) of the sample obtained by adding glycyrrhizic acid and monosodium 5' -monophosphate to the scanning reagent for notoginsenoside R1 and sequencing the resulting sample in example 1.
FIG. 2 is a Q30 (%) curve of the addition of glycyrrhizic acid and carnosine to the scanning reagent for notoginsenoside R1 in example 2 and sequencing.
Detailed DescriptionEmbodiments of the present invention will be described in detail below with reference to examples, but it will be understood by those skilled in the art that the following examples are only for illustrating the present invention and should not be construed as limiting the scope of the present invention. The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.
The sequencing slide was loaded with single-stranded DNA Nanospheres (DNBs) comprising e.coli genomic DNA. In examples 1 and 2 below, imaging tests were performed using MGI sequencing slides (MGISEQ-2000 RS sequencing slides) with a pitch size of 900nm and a binding area of about 200nm, the kit was from DNBSEQ-G400HMTM high throughput sequencing kit (FCL PE 100), and the library was from standard library reagent V3.0 (26 ng/branch, E320, barcode 97-104).
Example 1 the effect of notoginsenoside R1+glycyrrhizic acid+5' -monophosphate monosodium on the decrease in two-chain Q30, error rate and signal return was evaluated.
1. Experimental material (Main reagent and consumable)
TABLE 1 reagent, consumable information
2. Instrument for measuring and controlling the intensity of light
Table 2 instrument information
| Instrument for measuring and controlling the intensity of light | Model number | Instrument numbering |
| Sequencer | MGISEQ-2000RS | R10040100210039 |
| Gene amplification instrument | 6337 | 180005432N00099 |
| Qubit instrument | Qubit4.0 | 180005432N00006 |
| Analytical balance | MS204TS | 180005436N00082 |
3. Experimental sample
Table 3 sample information
4. Design of experiment
Different concentrations of monosodium 5 '-monophosphate and glycyrrhizic acid were added to the notoginsenoside R1 scanning reagent (containing 1M Tris buffer, 1.67mM notoginsenoside R1,8mM Trolox,10mM ethylenediamine tetraacetic acid, 20mM DTT), respectively, while a control scanning reagent was prepared without adding monosodium 5' -monophosphate and glycyrrhizic acid. PE150 sequencing is carried out on the same MGISEQ-2000RS sequencer, and the sequencing quality of each scanning reagent is compared after the sequencing is carried out. The data of several scan reagents, especially two-strand Q30 (%), two-strand Q30 decrease (%), error rate (%) and signal return value are compared.
5. Evaluation index and Standard
Table 4 evaluation criteria
| Evaluation index | Qualification standard |
| Two-chain Q30 (%) | The higher the better or the equivalent value |
| Two-chain Q30 drop value (%) | The lower the better or the equivalent value |
| Error Rate (%) | The lower the better or the equivalent value |
| Signal rise-back value | The higher the better or the equivalent value |
Q30 refers to the ratio of base with estimated error rate lower than 0.001 (accuracy higher than 99.9%) in basecall results, error rate refers to the average value of mismatch at each position in MAPPEDREADS, and the index of signal rise value is only for PE sequencing part and reflects two-chain signal rise condition. When the gain effect of the scanning reagent is evaluated, the three indexes are generally selected as judging indexes, the three indexes of the experimental group and the control group are compared with each other, and taking Q30 as an example, as long as the Q30 of the experimental group is higher than the control group in the next report, the added compound combination has the gain effect on sequencing, and the higher the experimental group Q30 is than the test group, the stronger the gain effect is.
6. Experimental procedure
A. Preparation of DNB
1) Taking out DNB preparation buffer solution, DNB polymerase mixed solution I, TE buffer solution and DNB termination buffer solution, placing on an ice box for about 0.5h, shaking and uniformly mixing for 5s by using a vortex oscillator, and centrifuging briefly and placing on the ice box for standby.
2) A reaction mixture was prepared on ice using 0.2mL octal or PCR tube as follows:
| Component (A) | Addition amount (mu L) |
| Library ssDNA | 3 |
| TE buffer | 17 |
| DNB preparation buffer | 20 |
| Total volume of | 40 |
3) The reaction mixture is evenly mixed by shaking by a vortex oscillator, centrifuged by a mini centrifuge for 5s, and placed in a PCR instrument for primer hybridization, and the reaction conditions are shown in the following table:
| temperature (temperature) | Time of |
| Thermal cover (105℃) | On |
| 95°C | 1min |
| 65°C | 1min |
| 40°C | 1min |
| 4°C | Hold |
4) The DNB polymerase mixture II (LC) was removed and placed on an ice bin and centrifuged briefly for 5s and placed on an ice bin for further use.
5) After the PCR instrument reached 4 ℃, the PCR tube was removed and centrifuged in a mini centrifuge for 5s, the following components were added on ice:
| Component (A) | 100 ΜL System addition (μL) |
| DNB polymerase Mixed solution I | 40 |
| DNB polymerase Mixed solution II (LC) | 4 |
6) The reaction mixture is evenly mixed by a vortex oscillator, and is centrifuged for 5s by a mini centrifuge and is immediately placed in a PCR instrument, and the reaction conditions are as follows:
| temperature (temperature) | Time of |
| Thermal cover (35℃) | On |
| 30°C | 25min |
| 4°C | Hold |
7) After the PCR instrument reaches 4 ℃, the PCR tube is taken out, 20 mu L of stop buffer solution is added, and the mixture is slowly and uniformly mixed for 5 to 8 times by using a flaring suction head.
8) After DNB preparation was completed, 2. Mu.L of DNB was taken and usedSSDNA ASSAY KIT and its useThe Fluorometer instrument performs concentration detection at 15.3 ng/. Mu.L.
B. preparation of slides and sequencing reagent tanks
1) The slide is taken out from a refrigerator with the temperature of minus 25 ℃ to minus 15 ℃ and is placed for at least 60min (not more than 24 h) under the room temperature environment.
2) Taking out DNB loading buffer II, placing on an ice box for about 0.5h to melt, shaking and uniformly mixing for 5s by using a vortex oscillator, and placing on the ice box for standby after short centrifugation.
3) A new 1.5mL centrifuge tube was removed and a DNB loading system was formulated as shown in the following table:
| Component (A) | FCL addition amount (μL) |
| DNB Loading buffer II | 33 |
| DNB | 99 |
| Total volume of | 132 |
4) The DNB loading system is slowly and evenly mixed for 5-8 times by using a wide-mouth suction head.
5) The MGIDL-200H portable sample injector is taken out, and the sealing gasket is installed in the sealing gasket groove. The chips are mounted in the applicators, respectively.
6) 27 Mu L DNB is respectively loaded on L01-L04 by using a flaring gun head, and the mixture is kept at room temperature for more than half an hour.
7) Preparing a PE150 sequencing reagent tank, taking out the HotMPS dNTP mixed solution and the HotMPS dNTP mixed solution II required by the preparation in advance for 1h, and melting at room temperature for later use.
8) And taking Adv Seqenzyme II out before using, and placing on ice for standby.
9) Sample addition was performed according to the following table:
| Reagent(s) | Hole 1 | No. 2 hole | 15 # Hole |
| HotMP SdNTP mixed solution | 1500μL | | / |
| HotMPS dNTP Mixed liquor II | | 1500μL | / |
| Adv Seqenzyme II | 1000μL | 1000μL | / |
| MDA REAGENT + MDA enzyme | / | / | 3875μL+125μL |
10 Mixing the reagents uniformly for later use.
C. sequencing on machine
The reagent tank was placed in the instrument, the script was edited, and PE150 sequencing was started.
Table 5 experimental design
| Experiment group number | Conditions (conditions) |
| 1 | Notoginseng radix saponin R1 scanning reagent |
| 2 | Notoginseng radix saponin R1 scanning reagent +0.5mM glycyrrhizic acid +7.5mM adenosine 5' -monophosphate monosodium salt |
| 3 | Notoginseng radix saponin R1 scanning reagent +1.5mM glycyrrhizic acid +7.5mM adenosine 5' -monophosphate monosodium salt |
| 4 | Notoginseng radix saponin R1 scanning reagent +2.5mM glycyrrhizic acid +7.5mM adenosine 5' -monophosphate monosodium salt |
PE150 sequencing was performed using DNB templates and fluorescent-labeled nucleotides with reversible terminators. The reversible terminator nucleotide has a structure that can be cleaved, which is located at the base-linked fluorescent group and at the ribose 3' -OH position. In each cycle, DNA polymerase is used to add the above-mentioned nucleotide and the scanning reagent is pumped in before shooting, the imaging instrument used in shooting process is MGISEQ-2000RS, the excitation wavelength of irradiated fluorescent dye is about 532nm and 660nm, the exposure time is 1-100 cycles 40 ms, 100-200 cycles 40 ms and 200-300 cycles 50 ms. After each cycle, the fluorophores and protecting groups were removed with 10mM THPP.
FIG. 1 shows Q30 (%) curves for each group sequenced. As shown in the figure, compared with the notoginsenoside R1 scanning reagent, the two-chain Q30 (%) reduction value of the glycyrrhizic acid and the 5' -monophosphate adenosine monosodium added with different concentrations is in the range of 7-11.3 and is lower than 13.
Table 6 shows the detection results of the respective evaluation indexes. And adding glycyrrhizic acid and monosodium 5' -monophosphate to the notoginsenoside R1 scanning reagent and performing PE150 sequencing on the obtained product. Compared with the scanning reagent of notoginsenoside R1, two chains Q30 (%) added with glycyrrhizic acid and 5' -adenosine monophosphate with different concentrations are about 94 and higher than 92, the two chains Q30 decrease values (%) are in the range of 7-11.3 and are lower than 13, the two chains error rates (%) are 0.18-0.35 and are lower than 0.58, and the signal return values are 2.14-2.32 and are higher than 2.
Table 6 influence of addition of glycyrrhizic acid and monosodium 5' -monophosphate on two-chain Q30 (%), two-chain Q30 decrease (%), error rate (%) and signal return value on the basis of the scanning reagent for notoginsenoside R1.
These results indicate that inclusion of varying concentrations of glycyrrhizic acid and monosodium 5' -monophosphate in the notoginsenoside R1 scanning reagent can reduce the degradation of sequencing quality due to the effect of photodamage on the DNA template, the extension strand, or both.
Example 2 the effect of adding glycyrrhizic acid and carnosine on the two-chain Q30 (%), the two-chain Q30 decrease (%), the error rate (%) and the signal return value on the addition of the notoginsenoside R1 scanning reagent was examined and evaluated.
The protocol is the same as in example 1 except that the test group scan reagent is replaced.
| Experiment group number | Conditions (conditions) |
| 1 | Notoginseng radix saponin R1 scanning reagent |
| 2 | Notoginseng radix saponin R1 scanning reagent +0.5mM glycyrrhizic acid +7.5mM carnosine |
| 3 | Notoginseng radix saponin R1 scanning reagent +1mM glycyrrhizic acid +7.5mM carnosine |
FIG. 2 shows Q30 (%) curves for each group sequenced. As shown in the figure, the scanning reagent of notoginsenoside R1 added with glycyrrhizic acid and carnosine two chains with different concentrations can reduce the Q30 by about 9-12 and below 13.
Table 7 shows the sequencing results of PE150 sequencing with the addition of glycyrrhizic acid and carnosine to the notoginsenoside R1 scanning reagent. Compared with the scanning reagent of the notoginsenoside R1, the two-chain Q30 (%) added with the glycyrrhizic acid and the carnosine with different concentrations is about 93 and higher than 92, the two-chain Q30 decrease values (%) are about 9-12 and lower than 13, the two-chain error rates (%) are about 0.35 and lower than 0.58, and the signal return values are 2.17-2.44 and higher than 2.
Table 7 influence of addition of glycyrrhizic acid and carnosine on two-chain Q30 (%), two-chain Q30 decrease (%), error rate (%) and signal return value on the basis of the scanning reagent for notoginsenoside R1.
These results indicate that inclusion of varying concentrations of glycyrrhizic acid and carnosine in the notoginsenoside R1 scanning reagent can reduce the degradation of sequencing quality due to the effect of photodamage on the DNA template, the extension strand, or both.
The above embodiments are only for illustrating the technical solution of the present invention, but not for limiting, and the technical solution of the present invention is modified or replaced equivalently, without departing from the spirit and scope of the technical solution of the present invention, and should be covered in the protection scope of the present invention.